l i steriu,

Listeriosis,
and
Food Safety
 FOOD SCIENCE AND TECHNOLOGY

A Series of Monographs, Textbooks, and Reference Books

EDITORIAL BOARD

Owen R. Fennema University of Wisconsin-Madison

Marcus Karel Rutgers University
Gary W. Sanderson Universal Foods Corporation
Steven R. Tannenbaum Massachusetts Institute of Technology
Pieter Walstra Wageningen Agricultural University
John R. Whitaker University of California-Davis

1. Flavor Research: Principles and Techniques, R. Teranishi, I, Hornstein, P. Is-

senberg, and E. L. Wick
2. Principles of Enzymology for the Food Sciences, John R, Whitaker
3. Low-Temperature Preservation of Foods and Living Matter, Owen R. Fenne-
ma, William D. Powrie, and Elmer H. Marth
4. Principles of Food Science
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13. Handbook of Vitamins: Nutritional, Biochemical, and Clinical Aspects, edited
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14. Starch Conversion Technology, edited by G. M. A. van Beynum and J. A.
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15. Food Chemistry: Second Edition, Revised and Expanded, edited by Owen R.
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18. Citrus Fruits and Their Products: Analysis and Technology, S. V. Ting and
Russell L. Rouseff
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Moskowitz
23. Seafoods and Fish Oils in Human Health and Disease, John E. Kinsella
24. Postharvest Physiology of Vegetables, edited by J. Weichmann
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26. Food Toxicology, Parts A and 6, Jose M. Concon
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29. Protein Quality and the Effects of Processing, edited by R. Dixon Phillips and
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30. Adulteration of Fruit Juice Beverages, edited by Steven Nagy, John A. Atta-
way, and Martha E. Rhodes
31. Foodborne Bacterial Pathogens, edited by Michael P, Doyle
32. Legumes: Chemistry, Technology, and Human Nutrition, edited by Ruth H.
Ma tth ews
33. Industrialization of Indigenous Fermented Foods, edited by Keith H. Steinkraus
34. International Food Regulation Handbook: Policy Science Law, edited by
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35. Food Additives, edited by A. Larry Branen, P. Michael Davidson, and Seppo
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36. Safety of Irradiated Foods, J, F. Diehl
37. Omega-3 Fatty Acids in Health and Disease, edited by Robert S. Lees and
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38. Food Emulsions: Second Edition, Revised and Expanded, edited by Ksre Lar-
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39. Seafood: Effects of Technology on Nutrition, George M, Pigott and Barbee
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40. Handbook of Vitamins: Second Edition, Revised and Expanded, edited by
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46. Listeria, Listeriosis, and Food Safety, Elliot T. Ryser and Elmer H. Marth
47. Acesulfame-K, edited by D. G. Mayer and F. H. Kemper
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49. Food Extrusion Science and Technology, edited by Jozef L. Kokini, Chi-Tang
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52. Food Analysis by HPLC, edited by Leo M. L. Nollet
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54. Clostridium botulinum: Ecology and Control in Foods, edited by Andreas H. W.
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56. Low-Calorie Foods Handbook, edited by Aaron M. Altschul
57. Antimicrobials in Foods: Second Edition, Revised and Expanded, edited by P.
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58. Lactic Acid Bacteria, edited by Seppo Salminen and Atte von Wright
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81. Food Emulsions: Third Edition, Revised and Expanded, edited by Stig E, Fri-
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83. Milk and Dairy Product Technology, Edgar Spreer
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85. Lactic Acid Bacteria: Microbiology and Functional Aspects, Second Edition,
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92. Listeria, Listeriosis, and Food Safety, edited by Elliot 7: Ryser and Elmer U.
Marth

Additional Volumes in Preparation

Complex Carbohydrates in Foods, edited by Susan Sungsoo Cho, Leon

Prosky, and Mark Dreher

Handbook of Food Preservation, edited by M. Shafiur Rahman

Food Safety: Science, International Regulation, and Control, edited by C. A.

van der Heeden, Sanford Miller, Maged Younes, and Lawrence Fishbein
This page intentionally left blank
 Listeriu,
Listeriosis,
and
Food Safety
Second Edition, Revised and Expanded

edited by

EIIiot T. Ryser
Department of Food Science and Human Nutrition
Michigan State University
East Lansing, Michigan

EImer H. Marth
Department of Food Science
University of Wisconsin-Ma dison
Madison, Wisconsin

M A R C E L

MARCEL
DEKKER,
INC. NEWYORK BASEL
D E K K E R
Library of Congress Cataloging-in-Publication Data

Listeria, listeriosis, and food safety / edited by Elliot T. Ryser,

Elmer Marth. -- 2nd ed., rev. and expanded.
p. cm. -- (Foodscienceand technology ; 92)
Includes bibliographical references and index.
ISBN: 0-8247-0235-2 (alk. paper)
1. Listeriosis. 2. Listeria monocytogenes. 3. Foodborne diseases.
4. Food-Microbiology. 1. Ryser, Elliot T. 11. Marth, Elmer H.
111. Series: Food science and technology (Marcel Dekker, Inc.) ; 92
QR201 .L7R9 1999
615.9 5 2 9 3 7 4 ~ 2 1 98-50963
CIP

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Preface to the Second Edition

Listeriosis and Listeria monocytogenes continue to be of worldwide interest to the food

industry and regulatory agencies, to scientists in various disciplines, and to consumers of
food. Such interest is prompted by the occasional appearance of L. monocytogenes in
ready-to-eat foods leading to the removal of such products from the marketplace. Further-
more, sporadic cases of listeriosis continue to occur and there have been several food-
associated outbreaks of the disease since the first edition of this book was published.
Scientists in several disciplines have studied and are still studying different aspects
of the listeriosis problem. Their efforts have resulted in the development of much new
information that has appeared in hundreds, if not thousands, of papers published since
writing of the first edition of this book was completed in 1990. This explosion of informa-
tion warranted publication of a second edition.
The second edition differs markedly from the first, published in 1991. Whereas we
were the sole authors of the earlier edition, chapters in this edition have been prepared
by various experts in the field. We now serve as editors, although one of us (ETR) has
revised several chapters. We feel that the contributions by the new authors have resulted
in an improved book that has appeared in a timely fashion.
Each of the chapters in the first edition has been retained, but each has been revised
and expanded with new information added where approprhte. Two additional chapters
not found in the first edition, dealing with typing methods and pathogenesis, have also
been included. Thus, this book contains 17 chapters which address the following topics:
(a) description of L. monocytogenes; (b) occurrence and behavior of this pathogen in vari-
ous natural environments; (c) animal and human listeriosis; (d) pathogenesis of L. mono-
cytogenes; (e) characteristics of L. monocytogenes that are kmportant to food processors;
(f) conventional and rapid methods to isolate, detect, and identify L. monocytogenes;
(g) strain-specific typing of L. monocytogenes; (h) foodborne listeriosis; (i) incidence of
behavior of L. monocytogenes in unfermented and fermented dairy products, meat, poultry
(including eggs), fish and seafood, and products of plant origin; and (j) incidence and
control of this pathogen within various types of food-processing facilities.

iii
iv Preface to the Second Edition

This book is useful to advanced undergraduate students, graduate students, and prac-
titioners in fooddairy microbiology, fooddairy science, bacteriology/microbiology, pub-
lic health, dietetics, meat science, poultry science, and veterinary medicine. It also will
be helpful to personnel in the fooddairy industry and in regulatory agencies and to re-
searchers in industrial, governmental, and university laboratories.

Elliot T.Ryser
Elmer H. Marth
Preface to the First Edition

Interest in the occurrence of Listeria in food, particularly Listeria monocytogenes, esca-

lated rapidly during the 1980s and continues unabated as a result of several major out-
breaks of foodborne listeriosis. The first of these occurred during 1981 and involved con-
sumption of contaminated coleslaw. In 1983, the reputation of the American dairy industry
for producing safe products suffered when epidemiological evidence showed that 14 of
49 people in Massachusetts died after consuming pasteurized milk that was supposedly
contaminated with L. monocytogenes. Two years later, consumption of contaminated
Mexican-style cheese manufactured in California was directly linked to more than 142
cases of listeriosis, including at least 40 deaths.
Heightened public concern regarding the prevalence of L. monocytogenes in food
prompted the United States Food and Drug Administration to initiate a series of Listeria
surveillance programs. Subsequent discovery of this pathogen in many varieties of domes-
tic and imported cheese, in ice cream, and in other dairy products prompted numerous
product recalls, which in turn have led to staggering financ.ia1 losses for the industry,
including several lawsuits. These listeriosis outbreaks, together with a subsequent epi-
demic in Switzerland involving consumption of Vacherin Mont d' Or soft-ripened cheese
and discovery of L. monocytogenes in raw and ready-to-eat meat, poultry, seafood, and
vegetables, have underscored the need for additional information concerning foodborne
listeriosis.
In 1961 Professor H. P. R. Seeliger, now retired from the University of Wiirzburg,
published his time-honored book entitled Listeriosis. While his monograph has provided
scientists, veterinarians, and the medical profession with much-needed information regard-
ing Listeria and humadanimal listeriosis as well as pathological, bacteriological, and sero-
logical methods to diagnose this disease, documented cases of foodborne listeriosis were
virtually unknown 30 years ago. Although much information in his book is still valid
today, some of the knowledge regarding media andor methods used to isolate, detect,
and identify L. monocytogenes in clinical and, particularly, nonclinical specimens is now
largely out of date. The emergence of L. monocytogenes as a serious foodborne pathogen

V
wi Preface t o the First Edition

together with the virtual flood of Listeria-related papers that have appeared in scientific
journals, trade journals, and numerous conference proceedings prompted us to review
and summarize the current information so that food industry personnel, public health and
regulatory officials, food microbiologists, veterinarians, and academicians have a ready
source of information regarding this now fully emerged foodborne pathogen.
This book consists of 15 chapters which address the following topics: (a) L. mono-
cytogenes as the causative agent of listeriosis; (b) occurrence and survival of this pathogen
in various natural environments; (c) human and animal listeriosis; (d) characteristics of
L. monocytogenes that are important to food processors; (e) conventional and rapid meth-
ods for isolating, detecting, and identifying L. monocytogenes in food; (f ) recognition of
cases and outbreaks of foodborne listeriosis; (g) incidence and behavior of L. monocyto-
genes in fermented and unfennented dairy products, meat, poultry (including eggs), sea-
food and products of plant origin; and finally (h) incidence and control of this pathogen
within various types of food-processing facilities. It is evident that major emphasis has
been given to information that is directly applicable to food processors. Since information
concerning the bacterium and the disease has been admirably reviewed by Professor See-
liger and others, our discussion of these topics should not be considered exhaustive. Thus
the first four chapters of this book supply only pertinent background information to com-
plement our discussion of foodborne listeriosis.
While many in the scientific community must be commended for the extraordinary
progress made since 1985 toward understanding foodborne listeriosis, the continuing ex-
plosion of information concerning Listeria and foodborne listeriosis has made the 3-
year task of compiling an up-to-date review of this subject quite difficult. Therefore, to
produce as current a document as possible, we have included a bibliography of references
that have appeared since the writing of the book was completed.
We acknowledge with gratitude the many investigators whose findings made this
book both necessary and possible. Special thanks go to those individuals who shared
unpublished information with us so that we could make the book as up to date as possible.
Our thanks also go to those scientists who provided photographs or drawings; each person
is acknowledged where the appropriate figure appears in the book. We thank Barbara
Kamp, Pat Gustafson, Beverly Scullion, and Judy Grudzina for typing various parts of
the manuscript. Illustrations were prepared by Jennifer Blitz and Suzanne Smith-their
help is acknowledged and appreciated. Special thanks go to Dr. Ralston B. Read, Jr.,
formerly director of the Microbiology Division of the Food and Drug Administration and
now deceased, who in 1984 encouraged development of a research program on foodborne
Listeria at the University of Wisconsin-Madison, and to Dr. Joseph A. ODonnell, for-
merly with Dairy Research, Inc. and now director of the California Dairy Foods Research
Center, for his early interest in and support of research on behavior of L. monocytogenes
in dairy foods.
Research done in the Department of Food Science at the University of Wisconsin-
Madison and described in this book was supported by the U.S. Food and Drug Administra-
tion; National Cheese Institute; the National Dairy Promotion and Research Board; the
Wisconsin Milk Marketing Board; Kraft, Inc.; Carlin Foods; Chr. Hansens Laboratory,
Inc.; the Aristotelian University of Thessaloniki, Greece; the Cultural and Educational
Bureau of the Egyptian Embassy in the U.S.; the Malaysian Agricultural Research and
Development Institute; the Korean Professors Fund; and the College of Agricultural and
Life Sciences, the Center for Dairy Research, and the Food Research Institute, all of the
Preface to the First Edition vii

University of Wisconsin. We thank all of these agencies for their interest in and support
of research on L. rnonocytogenes.
Our book is dedicated to all persons who have contributed to a better understanding
of foodborne listeriosis so that control of this disease is facilitated.

Elliot T. Ryser
Elmer H. Marth
This page intentionally left blank
Contents

Preface to the Second Edition iii

Preface to the First Edition V
Contributors xi

1. The Genus Listeria and Listeria monocytogenes: Phylogenetic Position,

Taxonomy, and Identification 1
Jocelyne Rocourt
2. Listeria monocytogenes in the Natural Environment 21
David R. Fenlon
3. Listeriosis in Animals 39
Irene V. Wesley
4. Listeriosis in Humans 75
Laurence Slutsker and Anne Schuchat
5. Pathogenesis of Listeria monocytogenes 97
Michuel Kuhn and Werner Goebel
6. Characteristics of Listeria monocytogenes Important to Food Processors 131
Yuqian Lou and Ahmed E. Yousef
7. Conventional Methods to Detect and Isolate Listeria monocytogenes 225
Catherine W. Donnelly
8. Rapid Methods for Detection of Listeria 26 1
Car1 A. Batt
9. Subtyping Listeria rnonocytogenes 279
Lewis M. Graves, Bala Swaminathan, and Susan B. Hunter

ix
X Contents

10. Foodborne Listeriosis 299

Elliot T. Ryser
11. Incidence and Behavior of Listeria monocytogenes in Unfermented Dairy
Products 359
Elliot T. Ryser
12. Incidence and Behavior of Listeria monocytogenes in Cheese and Other
Fermented Dairy Products 41 1
Elliot T. Ryser
13. Incidence and Behavior of Listeria monocytogenes in Meat Products 505
Jeflrey M. Farber and Pearl I. Peterkin
14. Incidence and Behavior of Listeria monocytogenes in Poultry and Egg
Products 565
Nelson A. Cox, J. Stan Bailey, and Elliot T. Ryser
15. Incidence and Behavior of Listeria monocytogenes in Fish and Seafood
Products 60 1
Karen C. Jinneman, Marleen M. Wekell, and Me1 W. Eklund
16. Incidence and Behavior of Listeria monocytogenes in Products of Plant
Origin 63 1
Robert E. Brackett
17. Incidence and Control of Listeria in Food-Processing Facilities 657
Robert Gravani

Appendix 711

Index 719
Contributors

J. Stan Bailey Russell Research Center, Agricultural Research Service, U.S. Department
of Agriculture, Athens, Georgia
Car1 A. Batt Department of Food Science, Cornell University, Ithaca, New York
Robert E. Brackett Center for Food Safety and Quality Enhancement, The University
of Georgia, Griffin, Georgia
Nelson A. Cox Russell Research Center, Agricultural Research Service, U.S. ,Depart-
ment of Agriculture, Athens, Georgia
Catherine W. Donnelly University of Vermont, Burlington, Vermont
Me1 W. Eklund" U.S. National Marine Fisheries Service, Northwest Fisheries Science
Center, Seattle, Washington
Jeffrey M. Farber Bureau of Microbial Hazards, Food Directorate, Health Canada, Ot-
tawa, Ontario, Canada
David R. Fenlon Animal Biology Division, Scottish Agricultural College, Aberdeen,
Scotland
Werner Goebel Department of Biology, University of Wiirzburg, Wurzburg, Germany
Robert Gravani Department of Food Science, Cornell University, Ithaca, New York
Lewis M. Graves Foodborne Diseases Laboratory Section, Centers for Disease Control
and Prevention, Atlanta, Georgia
Susan B. Hunter Foodborne Diseases Laboratory Section, Centers for Disease Control
and Prevention, Atlanta, Georgia

* Retired.
xi
xii Contributors

Karen C. Jinneman Seafood Products Research Center, U.S. Food and Drug Adminis-
tration, Bothell, Washington
Michael Kuhn Department of Biology, University of Wurzburg, Wurzburg, Gerrnany
Yuqian Lou Bil Mar Foods, Inc., Zeeland, Michigan
Pearl I. Peterkin Bureau of Microbial Hazards, Food Directorate, Health Canada,
Ottawa, Ontario, Canada
Jocelyne Rocourt Listeria Laboratory, Institut Pasteur, Paris, France
Elliot T. Ryser Department of Food Science and Human Nutrition, Michigan State
University, East Laming, Michigan
Anne Schuchat Respiratory Diseases Branch, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta, Georgia
Laurence Slutsker Foodborne and Diarrheal Diseases Branch, National Center for In-
fectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Bala Swaminathan Centers for Disease Control and Prevention, Atlanta, Georgia
Marleen M. Wekell Seafood Products Research Center, U.S. Food and Drug Adminis-
tration, Bothell, Washington
Irene V. Wesley National Animal Disease Center, U.S. Department of Agriculture,
Ames, Iowa
Ahmed E. Yousef Department of Food Science and Technology and Department of
Microbiology, The Ohio State University, Columbus, Ohio
The Genus Listeria and Listeria
monocytogenes: Bhylogenetic
Position, Taxonomy, and
Identification

JOCELYNE
ROCOURT
lnstitut Pasteur, Paris, France

HISTORY
The first published description of Listeria monocytogenes, which rapidly became the refer-
ence, was written by Murray et al. in 1926 [ ZOO]. A few earlier.reports may have described
Listeria isolation [53,141], the most plausible of which is certainly that by Hulphers [64].
However, the authors of these reports did not deposit their isolates in a permanent collec-
tion, so no subsequent investigations or comparisons with further strains were possible.
Murray et al. [loo] observed six cases of rather sudden death of young rabbits in
1924 in the animal breeding establishment of the Department of Pathology of Cambridge
University, and many more such cases occurred in the succeeding 15 months. The interest-
ing characteristics presented by the disease and the increasing mortality prompted an inves-
tigation. The authors wrote at that time [loo]:
Both the natural and the experimental disease have interesting and characteristic features and
their consideration has forced us to the conclusion that the causative organism either has not
been described previously, or has been inadequately described and so cannot be traced in the
literature. In either case, we feel justified in naming it. Its salient character is the production
2 Rocount

of a large mononuclear leucocytosis. This is far the most important and most striking character
we have discovered and we name the microorganism we shall describe in this paper Bacte-
rium monocytogenes. The question of the generic name is more difficult and we have not
succeeded in associating our organism with many other genera proposed in Bergey sManual
ofDeterminative Bacteriology ( 1925). We propose for the present to use the undefined Bacte-
rium ([. . .], for, if the present chaos is to be resolved and if the classification adopted by
the American Society of Bacteriologists is to be improved, it will be achieved only by co-
operation and with this end in view we cannot use the term Bacillus).
In 1927, during investigations of unusual deaths observed in gerbils near Johannesburg,
South Africa, Pirie [ 1 161 discovered a new microorganism, agent of what he called the
Tiger River disease. He named this new agent Listerella hepatolytica for the following
reasons:
The causative organism is a Gram-positive bacillus for which, from its most striking patho-
genic effect, I propose the specific name hepatolytica, and the generic name Listerella,
dedicating it in honour of Lord Lister, one of the most distinguished of those concerned with
bacteriology whose name has not been commemorated in bacteriological nomenclature.2
Both discoverers, Murray and Pirie, sent their strains to the National Type Collection at
the Lister Institute in London. Dr. Leningham, the director, was struck by the similarity
of the two microorganisms and put Murray and Pirie into contact. As the identity was
clear, they decided to call this bacterium Listerella monocytogenes [99,117].
However, in 1939, the Judicial Commission of the International Committee on Sys-
tematic Bacteriology rejected the generic name Listerella because it had been previ-
ously used for a mycetozoan (a slime mold) in 1906 in honor of Arthur Lister (young
brother of Lord Lister) and for a species of foraminifer (a marine protozoan) in 1933 in
honor of Joseph Jackson Lister (father of Lord Lister). As noted by Gibbons in 1972 [48],
it is certainly unique that the same name was chosen for three quite different groups of
microorganisms to honor the contributions of a father and his two sons. The next year,
in 1940, Pirie proposed the name Listeria [ 1171.
Before, and even after this date, numerous names were used to designate L. monocy-
togenes: Bacterium rnonocytogenes hominis and later Listerella hominis by Nyfeldt,
who considered that it was the agent of infectious mononucleosis [ 104,1051; Corynebac-
terium pawulum by Schultz et al. in 1934 [ 1401; Listerella ovis by Gill in 1937
[49]; Listerella bovina, L. gallinaria, L. cunniculi, and L. gerbilli by Nyfeldt
[105,106]; Erysipelothrix monocytogenes by Wilson and Miles in 1946 [ 1731; and
Corynebacterium infantisepticurn by Potel in 1951 during his first observations of fetal
and neonatal listeriosis in Germany [ 1191.
Unlike some pathogenic agents responsible for large outbreaks which have marked
the history of humans for centuries, for example, Vibrio cholerae or Yersinia pestis, the
history of L. monocytogenes and listeriosis is recent: It began officially in 1924. The first
confirmed diagnosis in a human was that of a soldier suffering from meningitis at the end

Names of species within quotes are no longer valid.

Were Listerella, and later Listeria, named in honor of Lord (Joseph) Lister, the father of antiseptic surgery,
or in honor of Sir Frederick Spencer Lister, Director of the South African Institute of Medical Research from
1926 to 1939? Gibbons, in 1972, tried to elucidate this nomenclatural point and came to the conclusion, together
with other authors, that Pirie chose Listerella to honor Lord Joseph Lister [48,99,142].
The Genus Listeria and Listeria monocytogenes 3

of World War I (retrospective identification of the strain [24]), and before this case, there
are no validated observations. Interestingly, however, a historian has suggested that L.
monocytogenes could have been the cause of Queen Ams 17 unsuccessful pregnancies
( 17th century) [ 1371.

PHYLOGENETIC POSITION OF THE GENUS LISTERIA

The relationship of Listeria to other bacteria remained obscure until the 1970s. Absent
from the first three editions of Bergey sManual of Determinative Bacteriology published
in 1923, 1925, and 1930, the genus Listeria was included in the tribe Kurthia of the
Corynebacteriaceae family in the next edition in 1934. In the sixth and seventh editions
(published in 1948 and 1957, respectively), Listeria was still a member of the Corynebact-
eriaceae, whereas in the next edition (1974), Listeria was considered as a genus of uncer-
tain affiliation and was placed with Erysipelothrix and Caryophanon after the family of
Lactobacillaceae [3-61. Finally, Listeria was classified with Lactobacillus, Erysipelothrix,
Brochothrix, Renibacterium, Kurthia, and Caryophanon in the section of regular, non-
sporing, gram-positive rods in Bergey s Manual of Systematic Bacteriology [7]. How
can these repeated reclassifications be explained?
On the basis of morphological resemblances (gram-positive, non-spore-forming
rod), Listerin has long been associated with the coryneform group of bacteria. However,
with the successive introduction and development of numerical taxonomy, chemotaxon-
omy, DNA/I)NA hybridation, and more recently rRNA (ribosomal RNA) sequencing, the
phylogenetic position of Listeria has been far more clearly defined.

Numerical Taxonomy
With development of computers for handling large amounts of data, numerical taxonomy
provided the first attempts to investigate in depth the phylogenetic position of Listeria
among gram-positive bacteria. In the first studies, Listeria was included among coryne-
form bacteria and actinomycetes and, consequently, was located either with the coryne-
bacteria [13,28] or in an indefinite position [16,62]. In contrast, since 1969, more natural
relationships were described when Listeria was compared with various representatives of
lactic acid bacteria [29,159,160]. The close relatedness with these microorganisms was
clearly demonstrated in 1975 by the broader numerical taxonomic survey of Jones, who
studied 173 characteristics of 233 strains of various genera, including both coryneform
and lactic acid bacteria 1701. The refined position of Listeria was later investigated by
Wilkinson and Jones in 1977 and Feresu and Jones in 1988 137, 1721. From these works,
it became clear that Listeria is distinct from other known genera, including Erysipelothrix
and Brochothrix thermosphacta (formerly Microbacterium ,thermosphactum), and that it
is closely related to Lactobacillus and Streptococcus. Consequently, Wilkinson and Jones
[ 1721 suggested that Listeria, Gemella, Brochothrix, Streptococcus, and Lactobacillus be
classified in the family Lactobacillaceae. Despite some imprecision concerning the exact
position of higher taxonomic relationships, especially of Brochothrix, certain lactobacilli,
and Carnobacterium [37,172], conclusions based on numerical analysis of data for large
numbers of phenotypic features were the precursors of the current phylogenetic classifica-
tion of the genus Listeria.
4 Rocourt

Chemotaxonomy
Several chemotaxonomic markers have been especially useful for solving the phylogenetic
position of the genus Listeria, reinforcing its distinctness from coryneform bacteria and
its relatedness to the lactic acid bacteria as evidenced by numerical taxonomic studies. The
G+C % DNA content of L. monocytogenes isolates ranges from 36 to 42% [37,125,160],
indicating that Listeria belongs to the low G+C % DNA content (<55%) group of gram-
positive bacteria.
Lipoteichoic acids (amphilic polymers of the cytoplasmic membranes) have been
isolated from L. monocytogenes [59,135,164]. These acids consist of hydrophilic poly-
glycerophosphate chains covalently attached to glyco- or phosphatidylglycolipids, the hy-
drophilic moieties of the molecules, and exhibit structural analogies with lipoteichoic acids
from other bacteria. Although lipoteichoic acids show a distinct structural diversity in
their hydrophilic and lipophilic portions, a given lipoteichoic acid is known to be a fairly
stable characteristic and so may be used as a taxonomic marker. For Listeria, the presence
of particular lipoteichoic acids provides further evidence that the genus is a biochemically
coherent taxon [ 1351. Furthermore, lipoteichoic acids are absent from coryneform bacteria
but are found in Bacillus, Staphylococcus, Streptococcus, and Lactobacillus, indicating
that Listeria should be grouped with these latter microorganisms [ 1351. With the exception
of one report [94], free mycolic acids, which are specific for high G+C % DNA content
gram-positive bacteria, have not been detected in Listeria [37,73]. The presence of respira-
tory menaquinones (seven isoprene units), of predominantly methyl-branched cellular fatty
acids and meso-DAP as the major peptidoglycan diamino acid support the close relat-
edness of Listeria and Brochothrix and the greater distance from lactobacilli
[2 1,22,37,40,41,12I]. Analysis of low molecular weight RNA profiles also supports the
independent identity of L. monocytogenes among other gram-positive taxa [ 1571.

rRNA Sequencing
Recent analysis of the 16s and 23s rRNA of L. monocytogenes has further clarified the
position of Listeria with regard to other genera of gram-positive bacteria. In 1986, Ludwig
et al. [9 11 unambiguously demonstrated, using the 16s rRNA cataloguing approach (partial
sequencing), that Listeria forms with Brochothrix thermosphacta one of several sublines
within the Clostridium subdivision. They did not detect any relationship with coryne-
form bacteria (except for universal and highly conserved 16s rRNA sequences that are
common to all eubacteria and those of gram-positive bacteria, respectively). Reverse tran-
scriptase sequencing of 16s rRNA data confirmed this phylogenetic position of Listeria
and indicated that the Listeria-Brochothrix subline is approximately equidistant from the
Bacillus and Enterococcus-Carnobacteriumsublines [22]. On the basis of these data and
chemotaxonomic properties, Collins et al. 1221 considered that (a) Listeria is phylogeneti-
cally remote from Lactobacillus and should not be included in the family Lactobacillaceae
and (b) the Listeria-Brochothrix subline probably merits a separate family, the Liste-
riaceae. This great distance between Lactobacillus and Listeria has been recently con-
firmed by sequencing 23s rRNA, with Listeria to be most similar to Bacillus and Staphylo-
coccus [ 1361.

Conclusion
Data accumulated during the last three decades clearly demonstrate that Listeria is a well-
defined taxon that possesses a number of features distinguishing it from neighboring taxa.
The Genus Listeria and Listeria monocytogenes 5

It is not a coryneform bacterium as evidenced by numerical phenetic studies, chemotaxo-

nomic properties (low G+C % DNA content, lack of mycolic acids, and presence of
lipoteichoic acid) and various rRNA sequencing analyses. However, the exact phyloge-
netic position of this genus remains controversial. Although it is generally agreed that
Listerias nearest neighbor is Brochothrix, its relationship to other low G+C % DNA
content gram-positive bacteria, especially Luctobacillus, needs further clarification.

TAXONOMY OF THE GENUS LlSTERIA

For many years, the genus Listeria was monospecific containing only the type species,
L. monocytogenes. L. denitriJcicans(because of its ability to reduce nitrates) was added in
1948 [158], 1,. grayi (in honor of M.L. Gray, an American microbiologist) in 1966 [89],
L. murrayi (in honor of E.G.D. Murray, a Canadian microbiologist) in 1971 [171], L.
innocua (because of its innocuousness or harmlessness) in 1981 [143], L. ivanovii (in
honor of I. Ivanov, a Bulgarian microbiologist) in 1985 [147], L. welshimeri (in honor of
H.J. Welshimer, an American microbiologist) in 1983, and L. seeligeri (in honor of H.P.R.
Seeliger, a German microbiologist) in 1983 [ 1251.
As for the phylogenetic analysis of Listeria, introduction of molecular biology meth-
ods allowed a better appreciation of the diversity within the genus Listeria which now
contains only six species: L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L.
seeligeri, and L. grayi.

L. monocytogenes, L. ivanovii, L. welshimeri, and L.

seeligeri
Among methods used to compare strains, serotyping was the first and crucial approach
to elucidate the infrageneric structure of the genus Listeria. Until 1960, Listeria was nearly
exclusively isolated from pathological samples, thus isolation of virtually no species but
L. monocytogenes. The first antigenic scheme was devised by Paterson [ 1 10,111], who
described the first four serovars. This scheme was later exl~endedby Donker-Voet and
Seeliger with the addition of new serovars [32,145]. In 1962, Ivanov observed atypical
L. rnonocytogenes strains isolated from sheep abortions and proposed to allocate them to
a new species L. bulgarica on the basis of their strong hemolytic activity and their new
antigenic structure (serovar 5) [66,67]. Years later, with development of selective media,
many additional strains were isolated from various environmental sources. Seeliger col-
lected and serotyped hundreds of strains between 1965 and 1980 and observed that many
were nonhemolytic, characterized by particular antigenic factors (serovars 6a and 6b [for-
merly 4f and 4g] and undesignated serovars) and apparently nonpathogenic. He proposed
to name these strains L. innocua in 1981 [ 1431. Thus, simple phenotypic methods, serotyp-
ing and hemolysis, led to the demonstration that the species .L. monocytogenes, as defined
in the eighth edition of the Bergeys Manual of Determinative Bacteriology [6], was het-
erogeneous, covering a number of different species.
Early DNA/DNA hybridizations (filter study), by Stuart and Welshimer in 1974,
showed L. monocytogenes to be heterogeneous [161]. However, the number of DNA hy-
bridization groups in their collection of strains could not be ascertained, as only one DNA
from L. monocytogenes was labeled; moreover, serovars were not indicated. Further DNA/
DNA hybridization studies (S 1 method), aimed at resolving the genomic heterogeneity
of the so-called L. monocytogenes and evaluating the validity of the new species L. bulgar-
6 Rocourt

ica and L. innocua (not officially validated at that time), were undertaken in 1982 with
many strains of various origins [ 1291. Five DNA relatedness groups were found among
strains formerly identified as L. monocytogenes:

Genomic group 1 contained the type strain of L. monocytogenes,thus corresponding

to L. monocytogenes sensu stricto; it included strains belonging to serovars 1/2a,
1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7.
Genomic group 2 consisted of strongly hemolytic strains, all belonging to serovar
5, confirming Ivanovs proposal that these strains, for which he suggested the
name L. bulgarica, deserved species rank [66]; they were named L. ivanovii
in 1985 [147]. Further investigations of strains of this species using multilocus
enzyme electrophoresis, DNA/DNA hybridizations, and rRNA gene restriction
patterns resolved to describe two subspecies, L. ivanovii subsp. ivanovii (ribose
positive) and L. ivanovii subsp. londoniensis (ribose negative) [ 101.
Genomic group 3 contained strains of serovars 4ab, 6a, 6b, and undesignated sero-
vars that were nonhemolytic and nonpathogenic for mice, including the two
strains that Seeliger previously proposed as reference strains for L. innocua [ 1431.
This group of strains corresponded to L. innocua, with this species being officially
validated in 1983 [165].
Genomic group 4 contained nonhemolytic strains of serovar 6a and 6b. These strains
produced acid from D-xylose and were nonpathogenic for mice [127,128], and
therefore corresponded to a group of strains previously described by Groves and
Welshimer in 1977 [56].The group was given species status and called L. wel-
shimeri in 1983 [125].
Genomic group 5, an unexpected group, included hemolytic and nonpathogenic
strains of various serovars (1/2c, 4c, 4d, 6b, and undesignated serovars) [ 127,1281
and was subsequently named L. seeligeri [ 1251.

DNA/DNA homology experiments (optical method) in 1993 supported these results [58].
Numerical taxonomic surveys confirmed that L. monocytogenes, as defined in the eighth
edition of Bergeys Manual of Determinative Bacteriology, was not a single taxon. How-
ever, this method is of limited sensitivity for bacteria which differ by few characteristics,
with these studies also being of little help in resolving the heterogeneity [37,72]. Interest-
ingly, this new genomic classification was tested using multilocus enzyme electrophoresis
( 18 enzyme loci analyzed). Matrix cluster analysis of the genetic distances between paired
electrophoretic types revealed that L. monocytogenes, L. ivanovii, L. welshimeri, and L.
seeligeri each corresponded to a single cluster with no overlaps between them [lO].

L. grayi (and L. murrayi)

The long controversy about the taxonomic position of L. grayi and L. murrayi started
when DNA/DNA homology studies of Stuart and Welshimer in 1974 demonstrated (a) a
low DNA relatedness between L. monocytogenes and L. grayi and L. murrayi and (b) a
high genomic homology between L. grayi and L. murrayi; these data were supported by
numerical phenetic analyses [ 160,1611. The authors proposed to transfer L. grayi and L.
murrayi to a new genus, Murraya with M. grayi as the type species, divided into
two subspecies M.grayi subsp. grayi and M. grayi subsp. murrayi. Two
questions arose from this proposal which was not officially validated in the Approved
The Genus Listeria and Listeria monocytogenes 7

Lists of Bacterial Names: (a) do L. grayi and L. murrayi belong to the genus Listeria?;
(b) do L. grayi and L. murrayi belong to a single species?
L. grayi, L. murrayi, and L. monocytogenes share several similarities: They cluster
in all numerical taxonomic studies [70,159,16 I , 1721, possess lipoteichoic acid [ 1351, tei-
choic acid of the polyribitol phosphate type [41], peptidoglycan of the A1 gamma variation
[40], nonhydrogenated menaquinones of the MK-7 type [21,37], and the same cyto-
chromes (albdo) [37]. However, other features support the distinctness of the L. grayi
and L. murrayi pair within the genus Listeria: A slight difference in G+C % DNA content
(42 vs 36-38) 137,1611, several biochemical reactions [146], the nature of substitution of
lipoteichoic acids [ 1351, slight differences in protein electrophoregrams [88], cellular fatty
acid composition [ 1021, antigenic structure [ 1461, and low DNA homology values [ 1601.
Finally, the 16s rRNA oligonucleotide cataloging of L. murrayi placed it close to L. mono-
cytogenes. These data, together with the substantial phenotypic similarity with L. monocy-
togenes, provided no support for exclusion of L. murrayi (and the closely related species
L. grayi) from the genus Listeria [ 1301.
The close relationship between L. grayi and L. murrayi was evidenced by various
investigations: These two species comprise a single distinct cluster in numerical taxonomic
analysis [37,172], in multilocus enzyme electrophoresis analysis [ 101, and in DNA/DNA
hybridization studies [ 1601. In addition, they share a number of chemotaxonomic proper-
ties which distinguish them from the other Listeria species: same DNA base composition
values [37,160], same substitution of lipoteichoic acids [ 1351, same cellular fatty acid and
fatty aldehyde patterns [75], and a common antigenic structure despite small differences
[145,168]. They have been distinguished from each other only on the basis of nitrate
reduction [171]. Finally, recent reexamination of the genomic relatedness of L. grayi and
L. murrayi using DNA/DNA hybridizations and multilocus enzyme electrophoresis indi-
cated that they should be considered to be members of a single species, L. grayi [132].
These data are consistent with 16s and 23s rRNA sequencing and cellular protein electro-
phoretic pattern data [22,78,136].

L, denitrificans/Jonesia denitrificans
Although only a single isolate is currently known for this species, there have been an
amazing number of papers dealing with its taxonomic position. As early as 1966, a numeri-
cal taxonomic study showed that L. denitrificans clustered with certain coryneform bacte-
ria, and this was later confirmed [ 16,70,159,160]. Results of chemotaxonomic studies and
DNA/DNA hybridization further emphasized the phenotypic differences between L. deni-
trijicans and other members of the genus Listeria [20,21,40,41,135,161]. In 1987, 16s
rRNA cataloging confirmed that this species is not a member of the genus Listeria and
belongs to the coryneform group of bacteria [ 13I]. Consequently, this species was trans-
ferred to the newly formed genus, Jonesia, and is now officially recognized as Jonesia
denitrijicans .

Present State of the Taxonomy of the Genus Listeria

The genus Listeria currently contains six species: L. monocytogenes, L. ivanovii, L. innoc-
ua, L. welshimeri, L. seeligeri, and L. grayi, as evidenced by DNA homology values,
16s rRNA sequencing homology, chemotaxonomic properties, and multilocus enzyme
analysis. Based on DNA/DNA hybridization, 16s rRNA cataloging and reverse tran-
scriptase sequencing of 16s and 23s rRNA, the genus embraces two closely related but
8 Rocourt

obviously distinct lines of descent: One contains L. grayi and the other L. monocytogenes,
L. ivanovii, L. innocua, L. welshimeri, and L. seeligeri. The species within this line can
be divided into two groups, (a) L. monocytogenes and L. innocua and (b) L. ivanovii, L.
seeligeri, and L. welshimeri [22,129,130,136].

IDENTIFICATION OF BACTERIA OF THE GENUS LISTERIA

One of the most practical goals of bacterial taxonomy is generation of information for
construction of reliable identification schemes so that newly isolated bacteria can be identi-
fied and their role in a particular environment can be assessed.

Genus Characteristics
Morphology
Listeria is a small (0.5 pm in diameter and 1-2 pm in length), regular gram-positive rod
with rounded ends. Cells are found singly, or in short chains, or may be arranged in V
and Y forms or in palisades. Sometimes cells are coccoid, averaging about 0.5 pm in
diameter and may be confused with streptococci. In old cultures, some cells lose the ability
to retain Gram stain and may be occasionally mistaken for Haernophilus. Long, thin,
filamentous cells appear in old and rough cultures and also after osmotic shock [57,74,84].
Listeria does not produce spores and capsules are not formed [144].
Listeria is motile because of its few peritrichous flagella when cultured at 20-25C
(not or very weakly motile at 37C) [45]. Hanging-drop preparations of fresh cultures in
tryptose phosphate broth incubated at 20C show characteristic tumbling motility: cells
start with twisting and wriggling movements which increase to fast, eccentric rotations
before they suddenly move quickly in various directions. Stab cultures in semisolid motil-
ity medium produce a typical picture of umbrella or inverted pine tree growth about
one half centimeter below the surface because of the microaerophilic nature of the organ-
ism. A recent report indicates that L. rnonocytogenes and L. innocua differ markedly in
motility and flagellin production at 37C: L. monocytogenes strains are virtually nonmotile
and produce little or no detectable flagellin, whereas strains of L. innocua are frequently
motile and produce substantial amounts of flagellin [8 11.
Cu Iture
On nutrient agar, colonies are 0.2-0.8 mm in diameter, smooth, punctiform, bluish gray,
translucent, and slightly raised with a fine surface texture and entire margin after 24 h of
incubation. After 5-10 days, well-separated colonies may be 5 mm or more in diameter.
When cultures of Listeria grown for 18-24 h at 37C on a clear medium are examined
with a binocular microscope under obliquely transmitted light, the smooth colonies exhibit
a typical blue-green iridescence [52,85,97]. Even when the population of contaminants is
rather high and that of Listeria low, Listeria can still be recognized because of this charac-
teristic [52,90], Rough colonies may occasionally be observed [54]. Conversion of smooth
colonies to rough colonies is not reversible [141]. Differences in virulence between rough
and smooth colonies have been observed [61,84,87]. Petite colony formation by strains
grown on esculin-containing agar has been described [ 1531.
Listeria usually grows well on most commonly used bacteriological media. The
growth rate is increased by the presence of fermentable sugar, particularly glucose. On
plate culture, Listeria has a particularly penetrating acid odor which may be caused by
The Genus Listeria and Listeria monocytogenes 9

formation of carboxylic acids, hydroxy acids, and alcohols [27]. In broth, the medium
becomes turbid after 8-24 h of incubation at 37C. Profuse growth is always observed
slightly below the clear area near the surface of the medium, indicating the propensity
for Listeria to grow better at oxygen tensions lower than that of air [141].
The normal temperature limits for growth are + 1-2C to 45C [76,144]; however,
some multiplication has been reported to occur in chicken broth and pasteurized milk
during extended incubation at -0.1 to 0.4"C [ 1691. Growth is slow at refrigeration temper-
atures, with generation times of 30-40 h at +4"C in skim milk, for example [134]. This
property was first used by Gray [55] for selective cold enrichment of a contaminated
sample. In broth, Listeria normally grows from pH 4.4-9.6, optimally at pH 7
[ 15,47,107,112]. Growth can occur in media containing 10% (w/v) NaCl with survival at
higher concentrations [ 146,1491. Survival at low pH and high salt concentration is strongly
temperature-dependent [ 191. Listeria is one of the few foodborne pathogens that can grow
at an a, value below 0.93 [35,112].
Nutritional Requirements
According to published data, growth factors for Listeria include cystine, leucine, isoleu-
cine, arginine, methionine, valine, cysteine, riboflavin, biotin, thiamine, and thioctic acid
[ 120,151,1701. Growth is stimulated by Fe3 and phenylalanine [ 120,15I]. In some experi-
+

ments, virulent strains grew faster in the presence of iron than did avirulent strains 1251.
Glucose and glutamine are required as primary sources of carbon and nitrogen [ 146,1201.
Chemically defined media have been described for Listeria [ 120,122,1501.
Metabolism and Biochemical Characters
Listeria is aerobic, microaerophilic, facultatively anaerobic, catalase-positive (rare cata-
lase-negative strains have been observed) and oxidase-neg,ative. Although Feresu and
Jones [37] found cytochrome a,bdo, the presence of cytochrome is controversial [ 109,1631.
Listeria is homofermentative and oxidizes glycolytic intermediate compounds [27]. It pos-
sesses glucose oxidase and NADH oxidase activities [ 1091. All strains grow on glucose
forming lactate, acetate, and acetoin as main endproducts under aerobic conditions
[27,113,133]. Acetoin is not produced under anaerobic conditions. Anaerobically, only
hexoses and pentoses support growth; aerobically, maltose and lactose support growth of
some strains, but sucrose does not [ 1 131. Catabolism of glucose proceeds by the Embden-
Meyerhof pathway both aerobically and anaerobically [ 1461. L. monocytogenes imports
glucose by a high-affinity phosphoenolpyruvate-dependentphosphotransferase system and
a low-affinity proton motive force-mediated system [ 17,1081. All strains are methyl red
and Voges-Proskauer test positive. Acid is also produced from amygdalin, cellobiose,
fructose, mannose, salicin, maltose, dextrin, alpha-methyl-D-glucoside, and glycerol. Acid
production from galactose, lactose, melezitose, sorbitol, starch, sucrose, and trehalose is
variable. Acid is almost never produced from adonitol, arabinose, dulcitol, erythritol, gly-
cogen, inositol, inulin, melibiose, raffinose, or sorbose. Phenylalanine-deaminase,orni-
thine, lysine, and arginine decarboxylases are not produced. H2S is not produced. Urea is
not hydrolyzed and indole is not produced. Additional information on biochemical tests
can be found in references 37, 79, 126, 146, 148, and 172.

Species Identification
All Listeria species are phenotypically very similar, but they can be distinguished by the
following tests: hemolysis, acid production from D-xylose, L-rhamnose, alpha-methy1-D-
 Rocourt

FIGURE1 Identification o f Listeria species.

mannoside, and mannitol [ 1281 (Fig. 1). The phenotypic similarities are consistent with
the high genomic homologies between the different species [22,129,136].
Hemolysis is a key characteristic for speciation of isolates and clearly is the most
difficult characteristic to detect. During a collaborative study on Listeria identification,
Higgins and Robison [60] noted that a large percentage of errors in identification of L.
seeligeri and L. ivanovii was caused by inaccurate reading of the CAMP3-testand hemoly-
sis. Isolates of L. monocytogenes and L. seeligeri show narrow, slight clearing zones of
beta-hemolysis. L. ivanovii shows wide, clearly delineated zones of beta-hemolysis. In
contrast, L. innocua, L. welshimeri, and L. grayi are not hemolytic. L. innocua can produce
a green zone of hemolysis on certain media [ 118,1561. L. monocytogenes hemolyzes blood
from sheep, horses, cows, guinea pigs, piglets, and humans [ 138,144,154,166l.Various
methods have been developed to determine hemolytic activity, especially for weakly he-
molytic strains (L. seeligeri and some L. monocytogenes isolates) and include examination
of hemolysis underneath the colony, prolonged incubation (48 h), incubation for several
hours at +4"C, use of thin layer blood agar plates [86], several media 1441, tube tests and
microplate techniques with erythrocyte suspensions [30,31,1621, addition of an exosub-
stance from Rhodococcus equi, Staphylococcus aureus, or L. ivanovii to the blood agar
[95,154,155],and the CAMP-test with S. aureus and R. equi [ 14,42,56,65,66,97,101,128].
Positive CAMP-tests are indicated by an enhanced zone of beta-hemolysis at the intersec-
tion of the test strains, L. monocytogenes, L. ivanovii, and L. seeligeri with S. aureus and
L. ivanovii with R. equi. Conflicting readings of the CAMP-test with R. equi have been
reported, some authors considering L. monocytogenes to be positive [39,139,167] and
others negative [128,146]. The typical positive CAMP-test with R. equi, as observed with
L. ivanovii, gives a shovel-like shape. In contrast, when this test is positive with L. rnonocy-

The original CAMP-test was described by Christie, Atkins, and Munch-Peterson, who observed this lytic phe-
nomenon for Srreprococcus in 1944, and the test is named after these authors [ 141.
The Genus Listeria and Listeria monocytogenes 11

togenes, the shape is that of an onion. This could reflect either different abilities of R.
equi strains to interact with L. monocytogenes because of different amounts of listeriolysin
0 secreted by L. monocytogenes strains or variations in the capability of R. equi strains
to secrete cholesterol oxidase [38,167]. However, whether or not it is positive, this test
is not essential, as L. monocytogenes and L. ivanovii can be easily distinguished by acid
production from D-xylose, L-rhamnose, and alpha-methyl-D-mannoside. The L. monocy-
togenes exosubstance involved in the CAMP reaction with S. uureus and R. equi is listerio-
lysin 0 [ 1231. The exosubstances of S. aureus and R. equi are a sphingomyelinase C and
a cholesterol oxidase, respectively [38,96,123].
Hemolysin is a major virulence factor of L. monocytogenes. Three species, L. mono-
cytogenes, L. ivanovii, and L. seeligeri, are hemolytic and possess the virulence gene
cluster as recently demonstrated [50]; however, only two species, L. monocytogenes and
L. ivanovii, are naturally and experimentally pathogenic [93,127], L. ivanovii being mainly
responsible for abortion in animals. Therefore, pathogenicity should not be presumed on
the observation of hemolysis alone. Few nonhemolytic L. monocytogenes isolates have
been observed, with the best known example being the type strain of this species
[6 1,71,801. Dissociation between hemolytic and nonhemolytic colonies is rarely observed
[ 1 151. Nonhemolytic and several weakly hemolytic strains are non- or weakly pathogenic
[ 12,23,30,36,61,114,1621.Despite these atypical strains, routine pathogenicity testing for
L. monocytogenes is generally unnecessary [90].
Additional tests, especially to distinguish L. monocytogenes from L. iiznocua, have
been proposed and include detection of phospolipase C activity [ 18,1031, hydrolysis of
D-alanine-p-nitroanilide[79], and hydrolysis of a naphthylamide substrate (API Listeria
[81)*
Various commercial miniaturized culture or enzyme multitest assays are now used
for Listeria identification, since conventional culture procedures for identification are te-
dious and time consuming. They include API 50 CH [83,126], API-ZYM [127], API 20
STREP [92], API Listeria [8,9,44], API Coryne [82], Micro-ID Listeria [2,8,60,124], Mast
ID [83], RAPID CORYNE [46], RAPID ID 32 Strep [43], and a microtiter plate method
[ 1521. Information provided by API ZYM, API 20 STREP, and RAPID ID 32 Strep distin-
guishes isolates at the genus level, whereas API Listeria, Mast-ID, API Coryne, and API
50 CH are more appropriate for both genus and species identification.
Phenotypic markers are used for routine identification of Listeria isolates. More
sophisticated methods have been described and some can help speciate atypical isolates.
These methods include 16s rRNA sequencing [26], sequence analysis of the 16s-23s
internal transcribed spacer loci [33,5 11, ribotyping 1681, random amplification of polymor-
phic DNA [34], repetitive element sequence-based PCR 1691, multilocus enzyme electro-
phoresis [ 10I, cellular protein electrophoretic pattern analysis [78], enzymatic profiling
using fluorogenic substrates [77], analysis of fermentation products by frequency-pulsed
electon-capture gas-liquid chromatography [27], Fourier transform infrared spectroscopy
analysis [63], and thermogram determination [ 11.

CONCLUSION
Studies on the phylogenetic position of Listeria started when numerical phenetic studies
were applied to gram-positive bacteria. These first studies were of primary importance in
demonstrating that Listeria was not a coryneform bacterium. These data were confirmed
by 16s rRNA cataloging. The refined location of Listeria within the low C + C % DNA
12 Rocourt

content gram-positive bacteria was later determined by reverse transcriptase 16s and 23s
sequencing data. Numerical taxonomic studies revealed a certain heterogeneity within this
genus with the exact species content determined by DNA/DNA hybridization and rRNA
sequencing. Based on this genomic dissection, the genus contains six species which are
divided into two sublines of descent. The present state of Listeria taxonomy is the result
of more than 20 years of work done in various laboratories in different countries, using
as many methods as imaginable. Most of this work was done during the last two decades,
and the number of publications in this field is now decreasing. Fortunately, the distinction
between L. rnonocytogenes and nonpathogenic species was already defined when food-
borne transmission of listeriosis became a public health problem with a major economic
impact on the food industry. This allowed efforts to be restricted to food contaminated
with L. rnonocytogenes, since food contaminated by other Listeria species is of no public
health concern. Now, both introduction of molecular biology methods and the need to
develop new tools to understand listeriosis epidemiology are generating renewed interest
in classification of L. rnonocytogenes strains (see Chap. 9).

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18 Rocourt

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Listeria monocytogenes in the
Natural Environment

DAVIDR. FENLON
Scottish Agricultural College, Aberdeen, Scotland

INTRODUCTION
Listeria monocytogenes is commonly found in soil and water and on plant material, partic-
ularly that undergoing decay, with these environments being re,gardedas the natural habitat
of the organism [86]. Decayed vegetation, such as aerobically spoiled silage, supports
development of high numbers of L. monocytogenes, and has been cited as the source
of infection in numerous cases of listeriosis in farm animals, and may be the origin of
contamination capable of spreading along the food chain. The organism can survive longer
under adverse environmental conditions than many other non-spore-forming bacteria of
importance in foodbome disease. This resistance, together with the ability to colonize,
multiply, and persist on processing equipment makes L. monocytogenes a particular threat
to the food industry.
Table 1 shows the persistence of L. monocytogenes in various natural and farm
environments and summarizes results from some of the studies mentioned in this chapter.
This ability to survive for long periods may explain why the natural environment can act
as a reservoir of contamination capable of spreading to animal and plant food products.
It is only relatively recently, with the introduction of improve:d molecular typing methods
such as multilocus enzyme electrophoresis (MEE), random fragment length polymorphism
(RFLP), random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis
(PFGE), that the full story of listeriosis epidemiology is emerging.

21
22 Fenlon

TABLE
1 Survival of L. monocytogenes in Various Environmental Samples
Storage temperature
Sample ("C)
Soil
sterile soil (I)a Outside-winter/spring 154
clay soil (I) 24-26 225
sealed tubes 24-26 67
fertile soil (I)
sealed tubes 24-26 295
cotton-plugged tubes 24-26 67
top soil (I)
exposed to sunlight NGh 12
not exposed to sunlight NG 182
moist soil NG -497
dry soil NG >730
soil 4-12 240-3 1 I
soil 18-20 201-271
Fecal material
cattle feces (NC)' 5 182-2 190
moist horse/sheep feces (I) Outside 347
dry horse/sheep feces (I) Outside 730
sheep feces Outside 242
liquid manure Summer 36
liquid manure Winter 106
sewage
sewage sludge cake (NC)
surface 28-32 35
interior 48-56 49
sprayed on field Outside >56
Water
sterilized pond water (I) Outside 7
unsterilized pond water (I) Outside <7-63
pond water 35-37 346
pond water 15-20 299
pond water/ice 2-8 790-928
pond/river water 37 325
pond/river water 2-5 750
water Outside 140-300
distilled water (I) 4 <9
Animal feed
silage (NC) 4 450
silage (NC) 5 180-2 190
mixed feed (I) Outside 188-275
oats (I) Outside 150-300
hay (1) Outside 145-189
straw (NC/I) ca.22 365
straw (I) Outside 47-207
straw Outside-summer 23
straw Outside-winter 135
aInoculated.
Not given.
Naturally contaminated.
Source: Adapted from Refs. 3, 4, and 75.
Listeria monocytogenes in the Natural Environment 23

ISOLATION OF LISTERIA MONOCYTOGENES FROM

ENVIRONMENTAL SAMPLES
Although L. monocytogenes is widely distributed, the numbers of the organism present
in most environmental habitats are very low. Isolation methods have therefore required
that relatively large samples be selectively enriched to increase numbers to detectable
levels and suppress the competing background microflora of thc: sample. Such procedures
have ranged from the original cold enrichment at 4C over many weeks or months [ 51]
to the present widely used and relatively rapid enrichment broths of the Food and Drug
Administration (FDA) [66] and UVM [70] methods with selectivity based on acriflavine
and nalidixic acid. Plating media also have improved; PALCAM agar [93] is less prone
to overgrowth with competitive organisms than Oxford agar [53]and is sensitive enough
for direct plating of samples for enumeration of L. morzocytogenes, particularly when high
numbers of Listeria are present [36]. It does, however, require incubation at 30C for
48 h, whereas with less contaminated samples, Oxford agar gives similar results in a
shorter time at 37C. In the United States, lithium chloride phenylethanol moxolactam
agar (LPM) is a popular medium [29]. In many highly contaminated environmental sam-
ples, such as feces or decayed plant material, where L. monocytogenes numbers may be
low and high numbers of competing microorganisms are present, a two-stage enrichment
procedure is often necessary. This is particularly so when Enterococcus spp. (941 or B a d -
lus spp. are present, as some strains of these species can initially have a colonial morphol-
ogy similar to L. monocytogenes. These two-stage enrichment methods, such as the US
Department of Agriculture (USDA) procedure, consist of a first stage with lower acrifla-
vine concentration (12 mg/L) from which, if necessary, a subculture can be made after
24 h to a second broth containing a higher acriflavine concentration (25 mg/L). This is
the standard procedure using UVM and Fraser enrichment broths; a similar technique was
described for fecal and other contaminated environmental samples by Fenlon et al. [36].
A draft international standard (DIS 1 1290- 1) based on a two-stage enrichment has recently
been proposed by the International Organization for Standardization [5].
Enrichment methods can be adapted to provide quantitative most probable num-
ber (MPN) estimates of L. monocytogenes populations in environmental samples [36,981.
Since L. monocytogenes is widely distributed, the pathogen must be enumerated so that
the relative risks posed by each habitat to the food chain and the circumstances that allow
survival and proliferation of the organism can be determined [28].

Soil and Vegetation

Soil is often referred to as the source of Listeria contamination particularly for silage [33].
Fertile agricultural soil receives decaying plant material, animal waste, and sewage sludge,
all of which are well-documented sources of L. monocytogenes. The study of Weiss and
Seeliger [ l o l l showed that L. monocytogenes was present in plant samples from 9.7% of
cornfields, 13.3% of grainfields, 12.5% of cultivated fields, 44% of uncultivated fields,
15.5% of meadows and pastures, 21.3% of forests, and 23.1% of wildlife feeding areas
examined in southern Germany. Surface soils had similar levels, but analysis of soil sam-
ples taken at a depth of 10 cm gave significantly fewer positive samples, indicating that
vegetation is a principal component in Listeria contamination of the soil. Welshimer and
Donker-Voet [ 1041 were unable to isolate L. monocytogenes from soil or dead vegetation
in early autumn, yet soil and decayed vegetation taken in the following spring were nearly
24 Fenlon

all positive for the organism. Fenlon et al. [36] examined five 25-g samples each of grass,
leaves, stems, and roots/stems from two crops of growing sward before harvesting. No
L. monocytogenes were detected in any samples. Only L. innocua and L. seeligeri were
isolated from 3 of 10 samples from the root/stem area. However, L. monocytogenes was
detected in 9 of 10 samples of cut grass from the same crops after wilting for 24 h before
ensiling. Other vegetable crops such as lettuce and carrots postharvest did not carry the
organism to the same extent. Farber [27] similarly found little Listeria contamination of
unprocessed vegetables. Other workers [46] have reported higher levels, with the degree
of processing and packaging having a significant effect on Listeria levels [10,16].
The higher incidence of L. monocytogenes associated with harvested (processed)
grass compared with other plant products has been attributed to the presence of a sheath
of decaying plant material at the base of the plant which might act as an inoculum at
harvest [36]. Whittenbury [ 1061 demonstrated the importance of the sheath area as a source
of lactic acid bacteria, which have a similar natural habitat to Listeria in ensiling of grass.
This may be a model for contamination of grass with Listeria during the ensiling process.
Other potential sources of L. monocytogenes in soils are from natural deposition of feces
by animals and spreading of animal waste and sewage sludge as fertilizer.
Survival of L. monocytogenes in soil depends on soil type and its moisture content.
Welshimer [ 1031, using cotton wool-plugged tubes containing either clay or fertile soils
inoculated with L. monocytogenes and stored for 67 days at 24-26"C, showed numbers
decreased in both by seven orders of magnitude as the soils dried out. Repeating the
experiment with sealed tubes to prevent water loss, the decrease was similar for the clay
soil but only two orders of magnitude for fertile soil. Watkins and Sleath [98] demonstrated
that in soil on land to which sewage sludge had been applied, there was little decrease in
Listeria spp. numbers (approximately 170 cfu per 100 g soil) over an 8-week period,
whereas salmonellae, applied at a similar rate, decreased to undetectable levels in less
than 4 weeks. This difference in rate of survival may be a reflection on the original habitat
of each species. Fenlon et al. [36] did not find L. monocytogenes in the few soils examined
that were associated with vegetable crops, but soil from fields where cattle or sheep had
been kept on silage diets did contain L. monocytogenes. In a study using autoclaved soils
inoculated with L. monocytogenes (approximately 5 X 102cfu/g) and stored at ambient
winter temperatures ranging from -15" to +18"C, Botzler [14] found an increase to 1
X 107cfu/g over a 154-day period. However, it is not known what effect the autoclaving
process had on Listeria growth by increasing availability of nutrients and eliminating
competing organisms.
From evidence in the literature to date, it does not appear that soil is a natural
reservoir in which L. monocytogenes multiplies. The widespread presence of the organism
in soil probably results from contamination by decaying plant and fecal material, with
damp surface soil providing a cool, moist protective environment and decaying vegetation
the substrate, which together enable L. monocytogenes to survive from season to season.

Fecal Material
L. monocytogenes has been found in the feces of a wide variety of healthy animal species.
Gray and Killinger [49] listed 37 mammals from whose feces the organism had been
isolated. Given that vegetation is the natural habitat of the organism, and that most reports
have involved cultivation of L. monocytogenes solely by enrichment techniques with few
quantitative data, it is not surprising that carriage in grazing animals such as healthy sheep
Listeria monocytogenes in the Natural Environment 25

[52,68,90], goats [65,96], and cattle [36,56,96] is well documented. However, L. rnonocy-
togenes excretion also has been reported in pigs [25,83], chickens [7,47,77], turkeys
[9,55,77], pheasants [89], gulls [30], rooks [30], pigeons [82], fish [6], and crustaceans [6].
As L. rnonocytogenes appears to be a food-related pathogen [73] with naturally occurring
listeriosis being recorded in many other animals, including mice [92], voles [79], rats [90],
rabbits [79,90,99], guinea pigs [84], chinchillas [38], lemmings [82], hyraxes [82], mink
[63], skunks [84], horses [102], dogs, [63,92], cats [79], foxes [79], deer [26,79], buffalo
[25], giraffes [ 181, bats [57], ducks [82], partridges [82], eagles [82], parrots [82], canaries
[82], starlings [63], frogs [13], turtles [13J,ticks [4], and flies [4], it is reasonable to suspect
these animals to also excrete the organism in their feces. Humans, both symptomatic and
asymptomatic carriers, excrete L. monocytogenes . Ralovich [841 summarized data, primar-
ily of European origin, indicating that 1.8-9.0% of healthy individuals excrete L. rnonocy-
togenes in their feces.
The influence of diet on excretion of L. monocytogenes has recently been studied,
particularly in ruminants. Low et al. [68] reported a low incidence of excretion in a flock
of 100 grazing sheep; this increased significantly ( P < .OOOOl) to between 10 and 33%
once silage feeding commenced, confirming the finding of Husu [58] that the tendency
is for excretion rates to be lower in grazing animals. Fenlon et al. [36] monitored two
groups of cattle. While grazing, none of those tested (n = 10 and 13) excreted L. monocyto-
genes. When tested after silage feeding commenced, 4 of 14 [28.6%) in one group and
4 of 13 (30.8%) in the other excreted L. rnonocytogenes.Numbers of Listeria in the excreta
were low, ranging from present in 25 g to 11 cfu/g. In the same study, examination of
feces of sheep on a hay diet showed no detectable Listeria, presumably because the mois-
ture level in hay is too low to support Listeria growth. Formulated diets for intensively
reared pigs and poultry also were free of the organism. Furthermore, of 9 samples of
broiler poultry litter tested, only 1 was positive and all 10 swabs of feces taken at the
processing plant from crates used to transport the birds were negative. Similarly, only 1
of 47 fecal samples from pigs and piglets was positive. Genigeorgis et al. [41], in a study
at a poultry processing plant, found the incidence of Listeria on carcasses increased as
processing progressed. L. monocytogenes was not isolated from composite feather samples
(n = 16) from live hanging birds or their hind gut contents (n = 16). Husu et al. [59]
noted that most 2-day-old chicks dosed orally with L. rnonocytogenes had eliminated the
organism within 9 days, indicating that chickens are unlikely reservoirs of the organism
with any carriage probably being transient. Dijkstra [21J examined the intestines of 2373
broilers from 146 farms, and showed that 4.1 % were contaminated with Listeria. Increased
excretion because of stress, as associated with salmonellosis, has not been well docu-
mented for listeriosis. Ralovich [84] observed increased excretion of L. rnonocytogenes
by sheep housed under stressful conditions. Fenlon [36] reported that in animals traveling
to three abattoirs, the level of L. rnonocytogenes in feces from cattle traveling a long
distance (>loo km) was greater than in those traveling to nearby abattoirs (<25 km)
(P = .003). 'The highest excretion rate found for L. rnonocytogenes was 800 cfu/g feces,
although L. ivanovii was found at a level of 6.4 X 104cfu/g in sheep feces. Numbers of
L. monocytogenes transferred to red meat carcasses tended to be low and rarely were
detected by swabbing, usually requiring enrichment of meat samples taken from the lower
parts of hanging carcasses. In abattoirs with a good hygienic standard, feces are only
responsible for intermittent low-level direct contamination. This may be sufficient to pro-
vide an inoculum for colonization of equipment used for further processing of the meat
and therefore may be a source of indirect contamination of foods, especially if hygiene
26 Fenlon

standards are poor. Studies have shown that more highly processed meat products are
more consistently contaminated with L. rnonocytogenes [36,87].

Sewage
One of the earliest definitive quantitative studies on L. monocytogenes was that of Watkins
and Sleath [98]. They reported levels >18,000 cfu/L in trade effluents associated with
animals and sewage sludges from treatment plants in northeast England. Levels of the
organism in primary tank settled raw sewage ranged from 700 to 18,000 cfu/L. Much
higher levels of Listeria spp, were reported by Geuenich and Muller [42] in a West German
sewage treatment plant. Both untreated waste and filtered effluent had 103- 10scfu Listeria
spp./mL. The fact that there was only a 10-fold reduction in numbers between untreated
and treated waste suggests that biological oxidation may not be an effective method for
eliminating viable Listeria in sewage. Studies in northeastern Scotland [36] showed L.
monocytogenes numbers in untreated sewage to be 120 cfu/mL and in treated effluent 2-
21 cfu/mL. Anaerobic digestion reduced numbers to 1. I cfu/g, and lime-treated sludge
had no detectable Listeria.
In Iraq, Al-Ghazali and Al-Azawi [2,3] studied survival of L. monocytogenes during
sewage treatment and in stored sewage sludge cake. A decrease of 85-97% in viable
Listeria numbers occurred during activation and digestion stages of the sewage treatment
process [2], although all steps were detrimental to the organisms survival. They recovered
<3-15 L. monocytogenes cfu/mL from final effluent and <3-7 cfu/mL from sludge cake.
The latter is frequently dried and used as a fertilizer in developing countries, although
treated sludge is increasingly subjected to land disposal in European countries as restric-
tions on sea disposal come into force. These same authors [3] demonstrated that the liste-
riae were inactivated when sludges were stored in direct sunlight for at least 8 weeks.
Inactivation was slower in the interior of the sludge piles. As reported earlier [98], studies
in the United Kingdom showed that when L. monocytogenes-contaminated sewage sludge
was spread on land, numbers of the pathogen failed to decrease over an 8-week period.
Since fecal contamination has been linked to one foodborne outbreak of human listeriosis
associated with coleslaw [SS], potentially contaminated sludges and animal wastes should
be plowed into the soil and not spread on crops which are eaten raw.

Water
The ubiquitous nature of L. monocytogenes and use of surface waterways for discharge
of sewage effluents will inevitably result in the presence of the organism in a wide range
of lakes, rivers, and streams. Dijkstra [22] found the organism in 21% of the surface
water samples in northern Netherlands, and noted that even though the lakes were used
by swimmers, no human cases were linked to this activity. A study of the course of the
River Don in northeastern Scotland [36] showed 42% of 19 samples (100 mL) were posi-
tive for L. monocytogenes in May and 53% 6 months later; numbers ranged from 10 to
350 cfu/L. Highest numbers were found at a sampling point below a sewage plant, but
this was not consistent for both sampling occasions. No factor, seasonal or otherwise,
could be related to the presence or numbers of L. monocytogenes which occurred over
the entire course of the river. In another study in the United Kingdom [39], 30 samples
(100 mL) from 21 sites showed 8 sites positive for L. seeligeri (27%), 1 for L. innocua,
Listeria monocytogenes in the Natural Environment 27

and 1 for L. welshirneri. Soil may be the source of contamination at these sites, since
McGowan [69] reported that L. seeligeri was more frequently isolated from soils than
either L. innocua or L. rnonocytogenes.
No waterborne cases of human listeriosis have been reported; however, water may
act as a source of contamination for certain foods. Soonthoranant and Garland [91] found
L. rnonocytogenes in 35- 100%of discharges from a sewage treatment pond and fish pro-
cessing factory effluents, which also contained sewage. Inshore marine waters, which
eventually received these discharges, contained L. monocytogenes in 6.6% of samples
with the contamination rate of Pacific oysters and blue mussels being 15.4%, which proba-
bly reflected their filter feeding habits. These findings confirm and extend the California
study of Colburn et al. [17] on fresh and low-salinity waters in which 81 and 62%, respec-
tively, harbored Listeria spp. including L. rnonocytogenes. One of three bay water samples
contained L. rnonocytogenes, and L. innocua was found in one of 35 oysters sampled.
Furthermore, Motes [76] isolated L. rnonocytogenes from shrimp caught off the U.S. Gulf
Coast, and Destro et al. [ 191 showed that some strains associated with shrimp can persist
in processing plants and enter the final product.
Gray et al. [50] successfully infected two sheep, four goats, and one cow by supply-
ing them with L. rnonocytogenes-contaminated water. Althoug,h current evidence on direct
infection of humans and animals with Listeria via water remains sparse, there is a legiti-
mate risk. A greater risk would appear to be contamination of foods such as marine and
freshwater fish with polluted water, especially those going for further processing [76], as
it is known that certain strains of L. rnonocytogenes can colonize equipment and contami-
nate the final product [72].

Animal Feeds
Most formulated animal feeds have low levels of available water which restricts multipli-
cation of L. monocytogenes. The same is true of hay and cereal grains, so although L.
rnonocytogenes has been reported in such materials 1401, the numbers are unlikely to reach
levels which present a serious risk to animals. Many formulated feeds are sold in a pelleted
form and will have received a degree of heat treatment capable of killing a high proportion,
if not all, Listeria present. The animal feed most closely linked with animal listeriosis
is silage, and this association is well documented. Olafson I811 noted the link between
Listerella encephalitis in ruminants and silage in 1940, and in 1960, Gray [48] reported
isolating L. monocytogenes from the fetus of a pregnant mouse fed poor-quality L. rnonocy-
togenes-contaminated silage implicated in death and abortion in cattle. Identical serotypes
of L. rnonocytogenes were isolated postmortem from the mice and cattle. Today, there
are numerous reports linking the feeding of silage with listeriosis outbreaks in sheep and
cows [32,37,44,45,52,68,78,100]. Much of this problem can be attributed to the high num-
bers of L. monocytogenes present in contaminated silage [31,321 as compared with other
animal feeds. In good-quality silage prepared from grass, maize, whole crop cereals, or
leguminous plants, which may or may not be wilted (dried to optimum moisture content)
in the field before e n d i n g , the onset of anaerobic Conditions stimulates the indigenous
or inoculated lactic acid bacteria to multiply rapidly; generally reaching a maximum of
around 10 cfu/g within 48 h. These bacteria convert the plant sugars to lactic acid, causing
a rapid decrease in pH [7 11 with well-preserved silages generally having a pH 5 4 . 5 . These
acidic conditions inhibit growth of both spoilage microorganisms and Listeria as long as
anaerobic conditions are maintained. Higher dry matter silages tend to have a higher pH,
28 Fenlon

but the lower level of available water compensates for any lessening in the preservative
effect caused by reduced acidity. Grass silages in cooler, wetter climates tend to have
lower sugar levels and higher moisture contents resulting in a poorer, slower fermentation,
and so are more susceptible to L. rnonocytogenes contamination than grass and maize
silages grown in countries with warmer climates.
L. monocytogenes Contamination is most frequently associated with poor-quality
silage. In 1979, Gr@ntsol[52] analyzed 291 grass silages from 113 farms and isolated L.
rnonocytogenes from 22% of samples with a pH <4.0; 37% with a pH of 4.0-5.0, and
56% with a pH of >5.0. In a study of clamp silage implicated in an outbreak of listeriosis
in cattle [32], levels of L. rnonocytogenes in excess of 12,000 cfu/g were found in the
surface layer (pH 8.3-8.5), whereas no Listeria spp. were detected 15 cm into the silage
mass and the pH was 4.5.
Gitter [44] noted that from 1975 to 1985, the incidence of listeriosis in sheep in
Great Britain increased from less than 50 incidents per annum to over 250. During the
same period, the rise in cattle listeriosis was much lower. He also reported that the pattern
changed from isolated single incidents to much larger flock outbreaks. This was attributed
to a change in the conserved forage used to feed sheep. In Great Britain, sheep are mainly
kept on upland areas and before this time were traditionally fed hay. The wet climate of
these hill farms is not conducive to the making of good-quality hay, and silage was not
an economic option before the mid 197Os, as it required expensive capital outlay for silos.
The invention of the big baler and the half-ton round bale made silage feeding feasible
by baling grass and sealing it in large plastic bags to make silage. Unfortunately, some
of the early attempts to make silage in this way were not very successful, and baled silage
was of poorer quality than clamp silage [30]. As L. monocytogenes is a surface problem
(Figure 1) and big bales have a much greater surface area than clamp or silo silage, the
potential for contamination to develop is much greater in bale silage if it is not made
correctly and aerobic deterioration takes place.
Fenlon [31] reproduced the problem in 500-g laboratory bales ensiled in plastic
bags. After 2-3 weeks, L. rnonocytogenes could be detected at levels of 2 1.1 X 106cfu/g
silage in moldy areas near the tie end of the bag where air infiltrated. In the center of
bags, the pH remained at 3.8, silage appeared to be of good visual quality, and it was
free of Listeria. The association between L. monocytogenes growth in silage and pH is
shown in Figure 2. The relationship between oxygen tension, pH, and L. rnonocytogenes
contamination was demonstrated by Donald et al. [24], who infused laboratory silos with
gas mixtures containing from 0.1 to 5.0% oxygen and demonstrated that the greater the
oxygen level, the more rapidly the pH rose with subsequent multiplication of Listeria.
Listeria die in well-fermented silage; however, if the pH increases before all cells
are killed, then surviving Listeria will multiply. Dijkstra [20] showed that L. rnonocyto-
genes can survive 4-6 years in naturally contaminated silage. Fenlon et al. [36] noted
that the organism could survive over 1 year in bags which had been used to wrap big
bales and stored for reuse. Fortunately, much of the L. monocytogenes contamination in
silage occurs in visibly moldy areas, and if these are removed and discarded before feeding,
the challenge to the animal is considerably reduced [33].
Inflammation of the iris (iritis) caused by L. rnonocytogenes has been increasingly
reported [97], particularly in cattle. This condition occurs when cattle and sheep burrow
their heads into bale silage in self-feeding systems. The eye becomes infected via abrasions
caused by stems of grass contaminated with the organism. Modifying feeding practices
to prevent eye contact with silage is the best preventative measure [67]. More obscure
Listeria monocytogenes in the Natural Environment 29

Big Bale Silage air

air

f air
tie
Bagged bale Wrapped bale
1 1 1 - 1 1 1 1 1 1 1 1 1 1 D D 1 1 1 1 1

plastic sheet
Clamp Silage 1

air
good quality silage pH4.0

Silage showing aerobic deterioration and

therefore high risk of L. monocytogenes
contamination

FIGURE1 Effect of air on development of Listeria rnonocytogenes contamination in

clamp and bale silage.

6 7.5
7
5
6.5
4
6
3 5.5 PH
5
2
4.5
1
4
0.1 3.5
0 5 10 15 20 25 30 35
Days
Center bale: 43 Lmon + pH;
Tie end: Q L.mon + pH

FIGURE 2 Effect of aerobic deterioration o n pH and survival and growth of Listeria

rnonocytogenes in laboratory bale silage. (From Ref. 33.)
30 Fenlon

causes of listeriosis have been reported, including silages made from orange peel and
artichokes [95], several outbreaks in Canada and the northern United States have been
caused by cattle feeding on ponderosa pine needles [ I].

TRANSMISSION
Being so widely distributed in the environment, animals and humans frequently come in
contact with L. monocytogenes through a variety of sources. What is also apparent is the
relatively low incidence of clinical listeriosis in both animals and humans. Traditionally,
the disease in both animals and humans has occurred as individual sporadic cases, and
although this is probably still the predominant form of the disease in humans [73], the
organism has since emerged as a serious foodborne pathogen, with well-documented out-
breaks being associated with processed foods, such as coleslaw [%], soft cheese [11,61],
pat6 [43], pork tongue [60], and pork rilletts [61]. These larger outbreaks have almost all
been attributable to serovar 4 strains principally serovar 4b, which, when typed by several
methods, have been shown to be closely related [15,60]. The origin of these outbreak
strains is unclear. Boerlin and Piffaretti [ 121 used MEE and showed that the electrophoretic
type (ET) which caused the soft cheese outbreak in Switzerland was also widely distributed
in bovine milk, bovine feces, minced meat, silage, and soil as well as in human and animal
clinical cases, thus suggesting an environmental origin. However, relying on one typing
method can be misleading. MEE reportedly has good discrimination for serotype 1/2 iso-
lates, which can be subdivided into a large number of ETs. However, it is less effective
for serotype 4 strains, most of which fall into relatively few ETs. Donachie et al. [23]
subjected serotype 4 isolates of the same ET from a variety of sources to analysis using
PFGE and found that all but one of the human isolates could be grouped into exclusive
human PFGE types. Multiple isolates from other sources, such as silage, also fell into a
single type. This study confirmed the need to use a combination of typing methods to
obtain maximum discrimination of strains. Further evidence for an environmental origin
for outbreak-related strains was obtained by Wesley and Ashton [ 1051, who in a retrospec-
tive subtyping study subjected clinical, environmental, and factory isolates from the Mexi-
can-style soft cheese outbreak to restriction enzyme analysis. This study showed that the
causative strain was recovered from samples of curd, the pasteurizer, cooler water, a floor
drain, and insects caught in the factory, with the widespread presence being related to
poor hygiene in the plant. McLauchlin and Nichols [74] demonstrated a direct relationship
between poor hygiene, as measured by total viable count, and the presence of Listeria
spp. in 4405 samples of seafood. A similar relationship between food processing hygiene
and Listeria was shown when pit6 samples were tested in the recent UK outbreak [43].
Sporadic and small outbreaks (<10 cases) tend to be caused by a much more diverse
range of strains than the larger outbreaks 1721, and although serovar 4b predominates over
others, serovar 1/2b is often found. In one study [72], isolates of L. monocytogenes from
cases of human listeriosis and foods were collected in the United Kingdom over a 30-year
period, and were sent to the Public Health Laboratory Service Food Hygiene Laboratory in
London for serotyping. Isolates from human listeriosis cases were of serovars 4b (60%),
1/2a (17%), 1/2b (1 l%), and 1/2c (4%) and from foods 4b (22%), 1/2a (32%), 1/2b
(15%) and 1/2c (21%), although this predominance of serovar 4b in human cases may
be the result of more virulent qualities of this serovar, the greater preponderance of serovar
112 in food isolates may be explained by the ability of these strains to adapt better to
ecological niches in the food processing environment. In a recent 12-month survey of raw
Listeria monocytogenes in the Natural Environment 31

milk from 160 farm bulk tanks, L. monocytogenes contamination was low, 4.3-9.3%, all
were serovar 1, and the maximum level was 35 cfu/mL. Most contamination was sporadic
with a diverse array of ETs present [35]. Harvey and Gilmour [54], using MEE and RFLP
analysis, compared L. monocytogenes isolates from four milk processing centers and two
dairy farms in Northern Ireland with food and clinical isolates and found the dairy-related
isolates to be quite distinct. Recurrent strains, specific to each dairy processor, colonized
plants over long periods. When isolates from a poultry processing environment were exam-
ined with RAPD analysis, Lawrence and Gilmour [64] showeld that a single RAPD type
was predominant in the raw processing environment over the 6-month period of the study,
surviving the clean-in-place schedules. In a follow-up study 12 months later, the same
RAPD type was again isolated from final cooked products.
Norrung and Skovgard [SO] found the genetic diversity of ETs isolated from cooked
meat products to be lower (0.439) than those from raw meat (0.903). They suggested that
certain clones may be better adapted to processing. An alternative explanation might be
that the diverse range of strains found on raw meat has been eliminated during heating,
and that strains on cooked products represent a more restricted range of strains present
in the postcooking environment. The extremely diverse range of strains on raw meat and
poultry products was shown by Ryser et al. [87], who tested by ribotyping up to 10 isolates
per sample from enriched samples of ground beef, pork sausage, ground turkey, and
chicken. They demonstrated that the strain selected was influenced by the enrichment
medium, but, more importantly, over half of all positive saniples had more than one L.
monocytogeizes ribotype (RT), some with as many as three. In some instances, detection
of certain clinically important ribotypes of L. monncytogenes, which were apparently over-
grown by other RTs, was only possible when 10 isolates from a sample were typed.
In animal listeriosis, where the change from hay to silage feeding for sheep has
resulted in outbreaks involving whole flocks, a similar pattern of diversity is emerging. A
temporal difficulty also exists in linking contaminated silage with cases of clinical disease,
particularly the encephalitic form, because of the long incubation period 1681. Identical
strains have been recovered from animals with clinical disease and silage fed to them
[68,105]. However, silage may contain a diverse array of L. monocytogenes strains, re-
sulting in flocks being exposed to and infected by [8] multiple strains. Nonetheless, within
a single sheep, one strain dominates during infection. Low et al. [68] noted that 6 distinct
phage types were present among 45 isolates of L. monocytogenes from baled silage. The
67 positive fecal isolates from 100 sheep being fed the silage were even more diverse
with 10 phage types other than those present in the silage being identified. The authors
commented that several strains present in feces were absent from silage. They noted that
since sheep consumed 100- 1000 times more silage than was analyzed, Listeria strains
present in excreta were more likely to be representative of the total silage population.
Two of the isolates recovered from the three clinical cases during the trial period were
serovar 4b and one was 1/2a. Identical phage types were isolated from silage and fecal
samples taken before the onset of disease, but were not necessarily the dominant strain
present, thus confirming the necessity to examine and type rriultiple isolates from individ-
ual samples. In another study 1341, the extent of Listeria contamination in silage was
directly related to its hygienic quality, as measured by the numbers of Enterobacteriaeceae
present, illustrating that management of postharvest processing of the grass can markedly
affect numbers of Listeria present.
The natural environment appears to be the initial reservoir for virulent strains of L.
monocytogenes which can enter and pass along the food chain, but this contamination is
32 Fenlon

usually of a low level and sporadic. It is significant that poultry products are more contami-
nated than beef, yet the environment in which beef cattle are reared presents a greater
risk of contact with the organism than that of the intensively reared broiler chicken. How-
ever, in processing, the latter is exposed to greater risk of contamination from other car-
casses and mechanical equipment than is in the beef carcass [85].It is at the processing
stage of food and feedstuffs that amplification of numbers and persistent contamination
occur, which in turn present a potentially more serious challenge to human and animal
health (Fig. 3).
Although discriminating power of typing methods is improving, a standardized sys-
tem is not yet in place, and it appears that a significant number of isolates must be typed
per sample, particularly with raw meats and silages, to ensure detection of the most impor-
tant strains. What is clear is that the natural environment harbors a highly diverse range
of L. monocytogenes strains, including some with the potential to cause clinical listeriosis
in humans and animals. This contamination is usually minimal and sporadic. However,
in the absence of good manufacturing and hygiene practices in both human and animal
food production, the food processing environment can become a ready source of virulent
strains with the ability to colonize equipment and contaminate food products.

0
Soil 1
f
Plants

Water
Manure Ruminants
Other
meat producing
animals and

Equipment and
Sewage ~ Environmental
Sources

0 Areas of greatest potential risk of

L. monocytogenes multiplication.
4 Consumer at risk
Direct consumption of minimally
processed products i.e. whole fresh
vegetables, cooked carcass cuts of
meat and fish and effectively
pasteurised milk presents a low risk.

FIGURE3 Spread of Listeria monocytogenes to the food chain from the natural envi-
ronment.
Listeria monocytogenes in the Natural Environment 33

ACKNOWLEDGMENTS
The author gratefully acknowledges the contribution of Professors E. T. Ryser and E. H.
Marth on which this revised chapter is based, and also the assistance and critical comments
of colleagues within and outside the Scottish Agricultural College (SAC). SAC receives
financial support from The Scottish Office Agriculture Environment and Fisheries Depart-
ment.

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Listeriosis in Animals

IRENE
V. WESLEY
National Animal Disease Center, U.S. Department of Agriculture,
Ames, Iowa

INTRODUCTION
Numerous animal species are susceptible to listerial infection, with a large proportion of
healthy asymptomatic animals shedding Listeria monocytogenes in their feces. Although
most infections are subclinical, listeriosis in animals can occur either sporadically or as
epidemics and often leads to fatal forms of encephalitis. Virtually all domestic animals
are susceptible to listeriosis [ 1781, with sheep [7,109,205,207,239,285,302], cattle [ 109,
205,209,2 17,226,255,2991, goats [ 169,170,2431, and less frequently chickens [ 106,200,
205,2231 succumbing to infection. Several comprehensive reviews have detailed the distri-
bution and pathology of L. mnnocytrogenes in food animals [7,108,127,160,176,2191.

INCIDENCE
Listeriosis in domestic livestock is being recognized with increasing frequency around
the world [107,279]. Since listeriosis is not a reportable disease in animals, the exact
incidence of listerial infections in domestic livestock remains unknown. According to
Ralovich [221], the annual number of cases in animals has increased substantially since
1966 with about 2200, 1000, and 900 cases being reported in Bulgaria, eastern Ger-
many, and Hungary in 1976, 1972, and 1980, respectively. Reports of listeriosis in do-
mestic animals also have increased in New Zealand, Germany, Greece, and England.
This may reflect a natural emergence, increased awareness, and/or improved detection
methods.

39
40 Wesley

Livestock losses attributed to L. monocytogenes may be substantial. During the early

1970s, the agricultural economies of Australia and Norway were adversely affected by
the loss of approximately I million and 2000-2500 sheep, respectively, from listerial
infection. Before a vaccine became available and lowered the incidence of listeriosis,
infections in Norwegian livestock remained relatively constant with approximately 1900-
2300 sheep herds, 90-160 goat herds, and 3-17 cattle herds being affected during the
10-year period between 1977 and 1986 [301]. In the United Kingdom, approximately
30,000 ovine abortions occur annually with 0.05-0.13% attributed to listeriosis [S]. In
The Netherlands between 1970 and 1985, the annual percentage of bovine abortions attrib-
uted to L. monocytogenes in cattle ranged between 0.7 and 8.7% (average of 3.2%), which
represented 234-928 cases [70].
In addition to relatively small numbers of acutely infected sheep, goats, and cattle,
substantially larger proportions of animals within a herd may be asymptomatic carriers
of L. monocytogenes and shed the organism in feces and milk [221,239]. Although the
humoral immune response may not have a major role in acquired resistance against listeri-
osis, serum antibody levels are useful for serodiagnosis of Listeria carriage in healthy
animals [29]. Sindoni et al. [247] conducted a serological survey in Italy on the frequency
of Listeria antibodies in apparently healthy sheep, cows, goats, and swine. Based on an
agglutination test, sheep ( I U%), cows ( 1 0.4%), goats (17.3%), and swine ( 1 3.3%) tested
positive, suggesting previous exposure to L. monocytogenes. However, these data should
be interpreted with some caution, since antibodies to several gram-positive bacteria can
cross react with Listeria antigens.
In a 1995 German survey of domestic and companion animals [288], L. monocyto-
genes was found in fecal samples from healthy bovines (33%), sheep (8%), birds (8%),
pigs (5.9%), horses (4.8%), and dogs (0.9%). The role of the symptomless carrier was
clearly demonstrated in another report in which 30 of 44 listeriosis outbreaks on sheep
farms involved introduction of clinically healthy animals from known infected herds [239].
Seasonal variation in the number of cases of animal listeriosis has often been observed.
In the Northern Hemisphere (England, Bulgaria, Hungary, United States, France, and Ger-
many), cases in domestic animals generally occur from late November to early May with
the greatest incidence during February and March [ 1081. Numbers of listeriosis cases
increased when animals were fed silage during periods of extreme cold, whereas sharp
decreases in numbers of reported cases were observed as soon as pasture was available.
Data from The Netherlands [70] indicate that most cases of listerial abortion in cattle
occurred between December and May. Approximately 40% of these cases were linked to
consumption of contaminated silage. Although in Norway listeriosis can be diagnosed
year-round in sheep and goats, the illness is far more prevalent from October to June and
also appears to be influenced by housing and feeding conditions [221]. Recent changes
in production methods have reduced levels of L. monocytogenes in silage, which in turn
has led to a considerable decrease in the incidence of listeriosis in silage-fed animals. Yet
an increase in ovine listeriosis with conversion to big bale silage production has been
reported in the United Kingdom [297]. The quality of the silage, including pH and micro-
nutrient composition, may predispose livestock to infection. To illustrate, sheep fed silage
had a lower number of lymphocytes, lower total serum protein levels, and elevated serum
iron compared with sheep fed hay [7].
Transmission of L. monocytogenes from livestock to humans occurs by (a) direct
contact with infected animals, especially during calving or lambing, and (b) consumption
of contaminated raw milk. Primary cutaneous listeriosis is regarded as an occupational
Listeriosis in Animals 41

disease of veterinarians and farmers who have attended deliveries of stillborn or aborting
bovine fetuses [4,44,191,207,211,2751. A case of septicemia and ultimately fatal meningi-
tis was reported in a Dutch farmer who developed cutaneous lesions after assisting in the
delivery of a stillborn calf [199].
Apart from human infections acquired from contact with infected animals or from
food directly contaminated by an infected animal, the connections, if any, between human
and animal listeriosis are unclear. The springtime peaks of animal listeriosis and the au-
tumn seasonality of human cases suggest that cases are not causally related. The source
of contamination for human food and animal feed is usually environmental [ 176,1901.
The rise in cases of human listeriosis is probably the result, in part, of changes in food
manufacturing and postprocessing contamination [ 1761. The World Health Organization
(WHO) [294] concluded that foodborne listeriosis is predominantly transmitted by non-
zoonotic means, and that L. rnonocytogenes is an environmental organism whose primary
route of transmission to humans is via foods contaminated during production. Several
strain-specific typing methods such as multilocus enzyme electrophoresis and pulsed field
gel electrophoresis have shown that L. rnonocytogenes strains isolated from meat or raw
milk mainly originated from the processing environment rather than from animals
[32,119]. Although an animal origin of contamination was inferred for the three major
epidemics of listeriosis occurring in North America, in the absence of documented evi-
dence, the role of direct animal involvement in human foodborne outbreaks of the disease
remains speculative [290].

Sheep
Ovine listeriosis is commonly caused by L. rnonocytogenes serotypes 1/2,3, and 4 as well
as by L. ivanovii [ 1741. Although listeric-like infections were previously observed in
sheep [239], Gill is credited with the first isolation of L. rnonocytogenes from domestic
farm animals [loo]. In 1929, he observed an illness in sheep in New Zealand which he
called circling disease. This name is still used today to describe listerial encephalitis,
encephalomyelitis, and meningioencephalitis [239].
Clinical manifestations of ovine listeriosis are (a) encephalitis, (b) placentitis with
abortions occurring in the last trimester [ 109,I8 1,2801, and (c) gastrointestinal septicemia
with hepatitis, splenitis, and pneumonitis [ 1541. Encephalitis is the most common form
diagnosed in sheep [154]. Lambs as young as 5 weeks of age may develop septicemia
with older feedlot lambs (4-8 months) manifesting encephalitis. All sheep are probably
exposed to the same contaminated feed, indicating a high natural resistance with 5-10%
of exposed animals exhibiting clinical signs [ 1541.
L. rnonocytogenes usually enters the animal via ingestion. Following entry into the
intestinal epithelium via either M cells in Peyers patches or epithelia1 cells [229], a bacter-
emic or septicemic phase or latent infection may develop, depending on the immune status
of the host. L. monocytogenes subsequently colonizes the viscera, gravid uterus, or medulla
oblongata [ 1541. In pregnant animals, the organism can localize in the placentomes and
enter the amniotic fluid. The fetus aspirates the pathogen, which multiplies and kills the
fetus late in gestation [267]. A single flock may experience abortion, septicemia, and
encephalitis [ 1791.
Direct entry via abrasions or lesions of the buccal mucosa, lips and nostrils, or
conjunctiva may lead to encephalitis. Because entry into the dental terminals of the trigem-
inal nerve in sheep can cause an ascending neuritis and encephalitis [49,50], listerial en-
42 Wesley

cephalitis is most common in winter and early spring in sheep that are losing and cutting
teeth [ 171. L. monocytogenes penetrates the buccal epithelium, accesses the endings of
the trigeminal (V) and hypoglossal (XII) cranial nerves, and enters the brain stem where
replication and dissemination to the medulla and pons occur. In severe cases, respiratory
failure and death follow within 1 month of infection [174,154]. Abrasions of the eye by
contaminated silage leads to ophthalmitis without any other clinical manifestations [283].
Meningoencephalitis caused by L. monocytogenes is the most common bacterial
infection of the central nervous system of adult sheep [237]. After an incubation period
of not more than 3 weeks, clinical symptoms of ovine encephalitis appear. These include
elevated temperature and refusal to eat or drink followed by neurological disturbances,
which include grinding of teeth, paralysis of masticatory muscles, and a stiff walk. At
this point, the animal moves in circles to the right or left, depending on the direction in
which the head is bent. This characteristic movement accounts for the name circling dis-
ease. Excessive salivation often occurs because of the animal's inability to swallow. In
advanced stages, muscular incoordination develops and is followed by inability of the
animal to walk. Death usually occurs within 2-3 days after onset of clinical symptoms,
with the illness seldom lingering beyond 10 days [239]. In the brain stem, Listeria antigens
are characteristically variable but always sparse. Perivascular microabscesses with L. mon-
ocytogenes and microgranulomas in histopathological specimens of brain stems are char-
acteristic. T lymphocytes (CD8' and CD4') and B lymphocytes contribute to the inflam-
matory process [ 1591.
After ingestion and hematogenous spread to the gravid uterus, L. monocytogenes
appears in the amniotic fluid and fetus within 48 h [ 1741. Listerial infections in pregnant
sheep often result in premature birth and infectious abortions [ 109,I 8 1,2801. This illness
seldom occurs concurrently with encephalitis [ 1351. Initially, pregnant ewes contract puru-
lent metritis, from which most recover. Intrauterine transmission of L. monocytogenes via
the placenta leads to a septic infection of the fetus, which in turn gives rise to abortion
or premature delivery with most fatalities occurring as stillbirths. Clinical symptoms are
resolved following expulsion of the fetus, after which the ewes recover. Morbidity in ewes
ranges from 1 to 20% [272] with mortality of lambs usually being high [154]. Injecting
L. monocytogenes into pregnant ewes caused 10% of those animals to abort. Significant
retardation of bone growth is seen in lambs born to experimentally infected ewes [ 1041.
Septicemia is most frequent in neonates and lambs and appears 2-3 days after oral
infection, although congenital and navel infection can also occur [ 1741. Septicemia is
characterized by an elevated temperature, loss of appetite, and diarrhea. Although death
may eventually occur as a result of extensive liver damage and focal pneumonia, the
mortality rate is much lower for the septicemic than for the encephalitic form of listeriosis.
Listeriosis is not a reportable disease in animals, thus precluding comparisons be-
tween countries. In addition, most infections in livestock are subclinical and therefore go
undiagnosed [ 1541. In Hungary, 14% of sheep excreted Listeria [222], but the distinction
between L. monocytogenes and L. ivanovii was not made. Although the incidence of L.
monocytogenes in sheep and cattle has decreased in The Netherlands, which has been
attributed in part to the change of silage production, in Great Britain modifications in
silage production may have caused an increase in ovine listeriosis [ 1021. To illustrate, in
1975, listeriosis was reported in a modest number of cattle (n = 37) and sheep (n = 44).
By 1984, sheep listeriosis cases had increased (n = 269) with little change in the number
of bovine listeriosis cases [ 1021. In parallel with the rise in the incidence in the United
Listeriosis in Animals 43

Kingdom, there was also a change in the disease pattern with both encephalitis and abor-
tion occurring in the same flock 102,179j.
Listeriosis occurs most frequently in late autumn, winter, and early spring. Stress
factors, such as abrupt changes in feed, concurrent disease, changes in dentition, and physi-
cal or viral damage to the epithelia] lining of the digestive tract may predispose to infection
11541. Introduction of new animals into the flock and overcrowding also are contributing
fdctors [ 1531. Climatic changes, such as heavy rains [302], especially following a drought,
which may spoil feed [228], or periods of extremely cold weather, which may cause ani-
mals to be housed indoors resulting in overcrowding [ 193,2851, are also determinants. A
prospective study conducted on early lambing flocks in southwest England ( 1989- 1991)
monitored 44 1 3 lambs from birth to slaughter for listerial meningoencephalitis. Two of
three flocks developed clinical disease attributed to L. monocytogenes serotype 1/2. Six
weeks before lambing, ewes on the two affected premises were bedded in straw; ewes on
the farm without clinical listeriosis were on softwood slats [ I 101. In the single flock with
no cases, preventive measures after lambing included replacing bedding and silage daily
and regular cleaning of the silage feeders which were on concrete floors. In the affected
flocks, silage was replaced on alternate days and the silage feeders were on soil-based
floors and thus easily contaminated. Before weaning, lambs were eating more silage, since
ewes were producing less milk. Although all lambs were exposed to these risk factors,
only an estimated 1.3% developed clinical infection with death occurring in 0.56% (21
of 4413) lambs from 4 to 32 days postweaning [109].
Quality of silage, as measured by digestibility ( D value), may influence the incidence
of ovine listeriosis [88]. In an outbreak of ovine listeriosis in the United Kingdom, the D
value of silage was below the optimum of 65-70% 12971. In Scotland, listeriosis caused
by L. monocytogenes serotype 1/2 occurred in sheep which were reluctant to eat poor-
quality silage [ 1791. Silage analysis indicated pH > 4 and a high ash content, reflecting
soil contamination. Despite antibiotic treatment, 19 ewes died, more than 60 developed
vaginal discharges, and 94 were barren at lambing [ 179).
The role of silage feeding in an epizootic of encephalitic listeriosis has been investi-
gated. A British study found a significant association between silage feeding and develop-
ment of ovine listerial encephalitis (relative risk of 3.8). In another report, excretion of
L. monocytogenes by sheep was linked to diet, with animals being fed entirely on hay or
manufactured diets not excreting detectable levels of L. monocytogenes. However, animals
fed on silage commonly excreted the organism [297]. The feeding of poor-quality silage
(pH 7.8) which was highly contaminated (10' L. monocytogeneslg) was the cause of an
outbreak in Spain [272]. In this outbreak, the flock consisted of 450 animals (attack rate
11.8%) with a case fatality rate of 94.3%.
Multiple strains of L. monocytogenes of the same or different serotype may be in-
volved in an outbreak in the same flock. When isolates of the same serovar are recovered
from a single outbreak, they may be further differentiated by DNA fingerprinting, phage
typing [ 101, or pyrolysis mass spectrometry [ 1751. In the Spanish outbreak just mentioned,
the serovar and phagovar of the L. monocytogenes strains isolated from two silage samples
and the brains of 3 of the 53 affected sheep were indistinguishable [272]. In addition,
DNA fingerprinting by random amplified polymorphic DNA (RAPD) analysis and ribotyp-
ing have been used to affirm that L. monocytogenes strains from silage, farm equipment,
and sheep brains were identical [295,296]. In contrast, examination of outbreaks of ovine
listeriosis in Scotland indicated multiple strains of L. monocytogenes serovar I /2 could
44 Wesley

be recovered from the silage incriminated in the outbreak. By multilocus enzyme electro-
phoresis (MEE), the L. rnonocytogenes strains for each of the affected animals in an out-
break were identical. Yet by MEE, none of the strains from the silage matched those
recovered from the brains. This could reflect bias during sampling from the bales, thus
missing a small virulent population of L. monocytogenes in the vegetation which, upon
entry into the ovine host, preferentially replicated to become the dominant strain and thus
cause disease [20].
Reports on the incidence of L. monocytogenes in ewes milk are limited. In Spain,
L. monocytogenes was present in 2.2% of 1052 ewes milk samples representing 283
farms. Yet, L. monocytogenes was recovered from 18% of milk tanker samples in Spain.
Tests of farm ewes milk samples indicated contamination by L. rnonocytogenes was sig-
nificantly higher on premises where cows were also reared than on farms where only ewes
were maintained [230]. The data suggest environmental contamination on farms resulting
from either L. monocytogenes excretion in cows feces or common exposure to contami-
nated ensilage on premises shared by sheep and cattle [230]. Interestingly, no seasonal
variation in milk contamination rates was evident.
Although not widely practiced in the United States, vaccination with live attenuated
strains of L. rnonocytogenes can effectively reduce ovine listeriosis [ 116,168,202,2821.In
Norway, immunization of sheep with a commercially available live attenuated vaccine of
serovars 1/2a and 4b reduced the incidence of listeriosis and abortions when compared
with unvaccinated control farms [115,116]. In this study [115], half of the sheep in 70
flocks (total of 3 130 sheep) with a history of listeriosis received two attenuated strains of L.
rnonocytogenes serotypes 12 and 4b, whereas the remaining sheep served as unvaccinated
controls. Both groups of animals were then housed together in the same pens. Results of
this study showed listeriosis incidences of 1 and 3% in vaccinated and unvaccinated sheep,
respectively. In 1984, a special license was issued to allow limited use of this vaccine in
a 2-year field study [116]. After vaccinating approximately 8% of all Norwegian sheep
(about 145,000 head), the incidence of listeriosis decreased from approximately 4% before
introduction of the vaccine to 1.5% after vaccination began. The incidence of abortions
was 0.7% in vaccinated compared with 1.1% in unvaccinated flocks. In another study, an
experimental vaccine, consisting of L. rnonocytogenes serovars 1/2a and 4b which were
attenuated via metabolic drift mutations, was tested in sheep, lambs, and ewes [168]. The
results of field tests indicated that vaccinated ewes delivered more lambs free of listeriosis
(93.4 vs 69.7%) and of higher birth weight (2.2 kg vs 1.8 kg) than lambs from control
unvaccinated ewes. In addition, L. rnonocytogenes was not isolated from milk samples of
vaccinated ewes in contrast to controls in which 32% of milk samples yielded Listeria
[ 1681. Although vaccination produced few adverse side effects, economic constraints sug-
gest that vaccination of sheep should be confined to flocks that have exhibited recurrent
listerial infections.
The epidemiology and economics of clinical listeriosis were described for a flock
of sheep in southern Illinois [202]. In that study, in which consumption of contaminated
silage was a key factor, unvaccinated Rambouillet ewes were more at risk (odds ratio 4.6)
than other ewes and yearling ewes were more at risk than older animals (odds ratio 4.1).
Interestingly, use of a bacterin did not decrease the risk of L. rnonocytogenes in Rambouil-
lets (odds ratio 0.8) but did among ewes of other breeds (odds radio 0.1). This indicates that
the inherent susceptibility of Rambouillet ewes to L. rnonocytogenes cannot be modified by
vaccination [202].
Serosurveys have been used to estimate the occurrence of listeriosis in sheep [250].
Listeriosis in Animals 45

However, antibodies to other gram-positive microbes cross react with Listeria antigens,
thus giving rise to false-positive results. In 1990, Berche and coworkers [26j developed
a more specific test for L. rnonocytogenes based on detection of antibodies to listeriolysin
0 (LLO) an antigenic protein of 58 kD. LLO is a virulence factor unique to L. monocyto-
genes that is required for the organism to escape from vacuoles and grow intracellularly.
It is not found in other Listeria species, including L. ivanovii. Antibodies to LLO which
are specific for L. monocytogenes and do not cross react with other Listeria species have
been detected in lambs experimentally infected with L. monocytogenes [ 13,166,1771.Puri-
fied LLO was evaluated as a specific antigen to detect both humoral and cell-mediated
immune responses of sheep infected with L. rnonocytogenes, L. innocua, and L. ivanovii
[ 131. LLO antibodies were seen only in sheep infected with L. rnonocytogenes. Further-
more, in a blastogenesis assay and skin test, two indicators of cell-mediated immunity,
only L. rnonocytogenes-infected sheep responded to LLO [ 131.
Listeria ivanovii (formerly L. monocytogenes, serotype 5 ; [240]) was first described
in association with ovine abortion and accounts for up to 8% of all animal listeriosis cases
[ 101. Listeria ivanovii is a recognized cause of ovine abortions and stillbirths and unthrifty
lambs [37,63,130,180,1811. Goats are not susceptible to L. ivanovii. To illustrate, L. iva-
novii and L. rrzonocytogeneswere both reported in two migratory flocks of sheep and goats
in Himachal Kadesh, India. Whereas L. monocytogenes was isolated from both sheep and
goats, L. ivanovii was cultured only from sheep [244]. Experimental infections of sheep
with L. ivanovii indicate that, unlike L. rnonocytogenes, abortions occur without encephali-
tis [ 1371. Factors which predispose to L. ivanovii abortions in livestock are similar to
those described for L. monocytogenes: a lowering of ewes resktance to infection by nutri-
tional stress, periods of cold and wet weather, feeding of poor-quality silage, and exposure
to carrier animals [75,101,219].
Outbreaks of listeriosis caused by L. ivanovii have been reported to affect up to
45% of pregnant ewes [ 1371. One outbreak in New South Wales involved a total of 1 10
animals which aborted or died shortly after birth. Heavy grazing by sheep (1 20 sheep per
hectare for 18 days) on pastures which had been cut for hay which had not been baled
and which had spoiled as a result of heavy rains was presumed to be the source of initial
contamination by L. ivanovii [242]. Multiple hepatic foci were seen in aborted lambs. L.
ivanovii was cultured from liver, lung, and stomach contents [242]. A report of bovine
abortions caused by L. ivanovii was associated with the grazing of cattle on pastures previ-
ously used by sheep [3]. Although rarely causing infections in humans [189], L. ivanovii
has been reported as a cause of abortion [75] and septicemia in acquired immunodeficiency
syndrome (AIDS) patients [56,164]. Even though L. monocytogenes is regarded as being
more pathogenic, both L. ivanovii and L. monocytogenes invade mammalian cells in tissue
culture, use actin filaments for intercellular spread, induce myometrial contractions in an
in vitro uterine strip model, and elicit conjunctivitis after ocular inoculation into rabbits
[ 1621.

The manifestations of clinical listeriosis in goats and sheep are essentially the same: en-
cephalitis, septicemia, and abortion. As is true for other livestock species, asymptomatic
infections also have been noted in goats [239].
After ingestion, L. rnonocytogenes penetrates the intestinal tract and sets up a tran-
sient bacteremia, which leads to dissemination to the central nervous system, viscera, or
46 Wesley

placenta. Depression, loss of appetite, a drop in milk yield, and elevated body temperature
(up to 41C) are the first indications of septicemia. The animals also may have diarrhea
[ 1131. In the pregnant doe, L. rnonocytogenes may penetrate the placenta, enter the fetus
where it replicates, and cause late-term abortion. In experimental studies with pregnant
goats, localization of L. monocytogenes in the placenta led to an elevation in prostaglandin
F2 and a decrease in progesterone levels. A slight decrease in secretion of estrone sulfate
by the fetal-placental unit prompted myometrial contraction and abortion [80].
Meningoencephalitis is the most frequently reported form of listeriosis in goats with
fatalities reaching 60% [ 1 131. The early signs of listerial encephalitis mirror the lesions
to the respective cranial nerves indicated by roman numerals in parenthesis: drooped ears
(VII), marked drooling (IX and X), protrusion of the tongue (XII), and cud retention from
difficulty in swallowing (IX and X). Goats may be more susceptible than sheep to listerial
encephalitis based on the severity of brain damage in goats [ 161. In an outbreak of encepha-
litis and abortion in a mixed herd of goats and sheep in Iraq, the morbidity (30 vs 17%)
and mortality (21 vs 15%) was higher in goats than in sheep [302]. Heavy rains, cold
weather, and susceptibility of pregnant animals may have contributed to the high numbers
of encephalitis cases [302]. More frequent recovery of L. monocytogenes serotype 1/2b
from goats than from sheep also has been documented in Sudan [246].
Listeriosis is linked to feeding silage [ 170,1741. However, in a study of 355 goat
herds in Missouri, encephalitic listeriosis was correlated with browsing on woody plants
and location of the herd in areas with a preponderance of alkaline soil [ 1441. Heavy browse
consumption may have led to oral lesions followed by penetration by L. monocytogenes
into the dental pulp or buccal cavity that may have led to encephalitis [ 1441.
Although not practiced in the United States, vaccination has limited the number of
goat listeriosis cases [66,93,202]. Before vaccinating goats in Norway with live attenuated
strains, the abortion rate in the test herd was 20-25%. However, after vaccination, the
incidence rate of abortions decreased to 3% [157]. A live attenuated L. monocytogenes
strain (strain Aer), obtained by three successive mutations in regard to streptomycin and
erythromycin resistance, afforded some protection against abortion in vaccinated goats
[93]. The optimal time for vaccinating goats and sheep may be shortly before the mating
season [ 1571.
As with other species of livestock, goats excrete Listeria in feces and milk during
and after septicemia and may contaminate the environment. Thus newborn kids housed
with the does may be infected through the navel or through sucking on soiled teats [ 1141.
Few studies describe the distribution of L. monocytogenes in raw goats milk. In one
report [l 1 I], L. monocytogenes was detected in 3.6% of raw milk samples from cows
with considerably lower recovery of L. monocytogenes from samples from goats (1%)
and ewes (2%) milk.
Serum antibody titers to L. monocytogenes may not indicate the immune status of
the host [ 1951. Animals with septicemia develop high antibody titers, whereas animals
with encephalitis have low titers, presumably because the brain is an immunologically
privileged site. More recently, antibodies to LLO have been proposed as a specific gauge
of antibody status. In experimentally infected goats, an increase in LLO antibodies is
correlated with rapid clearance of L. rnonocytogenes from the gastrointestinal tract and
thus predicts a favorable course of clinical infection [ 1961.
DNA fingerprinting methods are clarifying the role of animals in human infection.
To illustrate, an isolate of L. monocytogenes serovar 1/2b from the brain of a goat with
listeriosis exhibited the identical DNA profile by ribotyping as an isolate from cheese
Listeriosis in Animals 47

made from that goats milk and an isolate from the refrigerator in which the cheese was
kept. It is suggested that the cheese made from the infected goats milk may have contami-
nated the refrigerator shelves, thus serving as a reservoir for L. monocytogenes (741. In
contrast, a human endocarditis fatality with a recent history of exposure to goats showed
that the isolates from humans and animals were of the same serogroup. However, the
goat and human strains were clearly different via DNA analysis, thus making zoonotic
transmission less likely [%I.

The distribution of L. monocytogenes undoubtedly reflects the available pool of susceptible

animals. In North America, most listeriosis cases occur in cattle (82%) with a smaller
percentage in sheep (17%) and fewer still in pigs [21]. From 1993 to 1997, listeriosis
cases (n = 253) submitted to the Iowa State University Veterinary Diagnostic Laboratory
[161] showed a similar distribution and were from cattle (87%), sheep (9%), goats (2%),
and a llama and a horse (0.4% each). In Iowa, 90% of listeriosis cases were diagnosed
as encephalitis [161]. In marked contrast from 1975 to 1984, listeriosis cases in Great
Britain were from sheep (63%), cattle (32%), and with pigs, goats, fowls and other species
constituting less than 1 % each of the total submissions [297 I.
The symptoms of bovine listeriosis include encephalitis, abortion, and septicemia
with miliary abscesses [ 1471. In 1928, Matthews [ 1841 detailed an outbreak of encephalitis
of unknown origin in cattle which, in retrospect, was probably bovine listeriosis. Listerial
encephalitis has since been well documented [60,67,146,22 1,226,2391. However, even in
acute outbreaks, generally no more than 8- 10% of a herd succumbs to infection. In Swit-
zerland, after bovine spongiform encephalopathy, bovine listeriosis is the most frequently
diagnosed neurological disease [ 1241, and thus may be the most common cause of bacterial
infection of the central nervous system in adult cattle [226]. In Missouri [ 1431, from 1986
through 1994, encephalitic listeriosis cases were diagnosed in cattle (67%), goats (30%),
and sheep (13%).
Foodborne transmission is the main mode of infection in naturally occurring listeri-
osis in cattle with silage being most frequently implicated. As in sheep, following inges-
tion, Listeria is disseminated via hematogenous spread to the viscera, brain, and gravid
uterus. In addition, since L. monocytogenes is present in soil, fecal material, and vegeta-
tion, it may enter via abrasions of the nostrils or the conjunctiva while grazing or via the
teat of a lactating cow [ 1421. Direct injection of the conjunctiva, resulting in keratocon-
junctivitis [215], has occurred as a result of contaminated silage particles falling into the
faces of browsing cattle [ 1971.
By travel along peripheral nerves (indicated by Roman numerals), especially the
hypoglossal (XII) and trigeminal (V) cranial nerves innervating the buccal cavity, L. mono-
cytogenes enters the central nervous system and localizes in the pons and medulla. Damage
to the cranial nerves underlies the clinical presentation. For example, lesions of the fifth
(V) cranial and mandibular nerves lead to inability to eat or drink or to retain food in the
mouth. Excessive salivation from difficulty in swallowing (IX and X) and protrusion of
the tongue (XII); ataxia or circling (VIII); facial paralysis, including unilateral drooping
of the lip, ear, and eyelid (VII); and strabismus (VI) reflect damage to the respective
cranial nerves [225]. In the advanced stage, as vision and locomotion are impaired and
the animal becomes increasingly irritable, the illness may be confused with rabies or lead
poisoning. Finally, the animal lapses into a coma and generally dies within 1-2 days
48 Wesley

[239]. Histopathological lesions of the brain stem consist of foci of necrosis infiltrated
with neutrophils, macrophages, and bacteria [ 1421. Perivascular cuffing with mononuclear
cells is evident [267]. Unlike listerial encephalitis in sheep and goats, most cattle survive
at least 4-14 days after the initial onset of symptoms, with a few reports of spontaneous
recovery [22 11.
Listeriosis in cattle is frequently associated with abortion [ 105,209,221,2391,which
generally occurs during the last trimester of pregnancy. However, as demonstrated by
Dora, an 1l-year-old cow with atypical mastitis, healthy calves can be born to chronic
carriers that shed the pathogen in milk [72]. As was true for sheep, L. monocytogenes is
transmitted to the placenta, and then into the fetus. Meningitis in neonates may follow
intrauterine infection with the septicemic young animal dying shortly after birth [241].
In the US Midwest, Listeria spp. are the third most frequently encountered bacteria
from bovine late-trimester abortion cases. L. rnonocytogenes accounted for 1.35% of bo-
vine abortion and stillbirth submissions received by the South Dakota Animal Disease
Research and Diagnostic Laboratory from 1980 to 1989 [ 1551. Ten years earlier, of 2544
bovine abortion cases examined in the Northern Plains region of the United States, 2.2%
were attributed to listeriosis [ 1561. Similarly, in Germany, from 1984 to 1989, L. monocy-
togenes serotype 1/2 was recovered from 1.2% (122 of 993 1) and 1.8% (122 of 993 1) of
bovine abortions [31] in the Erfurt and Cottbus regions, respectively.
L. ivanovii is most often associated with sheep [20,37,63,130,137,180,181,2421 and
is sometimes recovered from cattle [3,101]. In California, during a 3-year period, five cases
of listerial bovine abortion were diagnosed among 243 fetuses submitted for evaluation. L.
ivanovii was recovered from four cases, whereas L. monocytogenes was isolated from
only a single bovine abortion. The pathological findings in these five listeriosis cases were
similar [3].
L. rnonocytogenes may enter a herd through contaminated feeds, introduction of
new stock, and rodents. Bovine abortions and stillbirths occur shortly after contaminated
silage is fed [5]. Improvement in silage production and hay making resulted in a decrease
(from 8.7 to 1.2%) in the number of listerial bovine abortion cases in The Netherlands
[64]. A change in silage production also was linked to a reduction in the percentage of
carrier animals (from 15 to 0.8%) based on fecal sampling [69]. An outbreak in Nigeria
resulted in 35 cases of bovine encephalitis and four abortions in the same herd. Although
the source of infection was unknown, the authors proposed that L. monocytogenes was
introduced into the herd by introducing new animals [2]. As expected, the number of
healthy carriers is lower on farms without overt disease than on farms with clinical listeri-
osis. To illustrate, L. monocytogenes was cultured from 2.0% of cows on farms without
L. monocytogenes and from 6.7% of healthy animals on farms on which listeriosis had
occurred [64]. This may indicate exposure to a common source of infection. Rodents are
known carriers of L. monocytogenes, and fecal contamination of animal feed is a potential
source of contamination [5,107,153].
Although not particularly common, generalized listerial infections can give rise to
mastitis [41,59,60,72,103,148,216,258,281].Beginning in 1938, Schmidt and Nyfeldt pos-
tulated that a small outbreak of human listeriosis in Denmark may have been caused by
drinking milk from mastitic cows. However, the role of Listeria in mastitic infections was
not clearly identified until 1944 when Wramby [300] isolated L. rnonocytogenes from milk
and udders of mastitic cows in Sweden. In 1956, de Vries and Strikwerda [60] described
another case of bovine mastitis in which a penicillin-resistant strain of L. monocytogenes
was cultured from one quarter of a 6-year-old dairy cow. Following acute onset, the condi-
Listeriosis in Animals 49

tion soon became chronic with shedding of L. monocytogenes in milk for 3 months. Pro-
longed excretion of L. monocytogenes in milk [65,66,135,209,2 10,2211, the apparently
normal appearance of the milk, and consumption of raw milk on farms could be important
factors in the transmission and epidemiology of milkborne listerial infections [ 1031. From
a public health aspect, culling of L. monocytogenes-infected cows with clinical mastitis
which do not respond to treatment is recommended [245]. After slaughter, cross contami-
nation of the carcass with bacteria from the infected udder is possible through evisceration,
meat inspection, or other manipulations [274].
L. monocytogenes strain Scott A (serotype 4b), a clinical isolate recovered from
the New England outbreak, has produced experimental mastitis in dairy cows [38,291].
Following repeated intramammary inoculation of 34 Holstein cows with I 03- 107L. mono-
cytogenes cells, 75% of the animals became chronically infected and shed Listeria in milk
(about IO3-1Os L. monocytogenes cfu/mL of milk) intermittently for up to 8 months. The
intramammary route of inoculation was used to simulate infection from contaminated
bedding directly into the teat canal. Interestingly, one of these experimentally infected
cows delivered a normal healthy bull calf. Bourry et al. [4] also induced mastitis via
intramammary inoculation with a single dose of 300 cells of L. monocytogenes serotypes
4b and 112. L. monocytogenes was recovered from the supramammary lymph node but
not from the spleen or liver, indicating clearance in the affected region. DNA profiles
of isolates recovered throughout experimental infection confirmed the persistence of the
inoculated strain [4].
As with sheep [7] and goats [ 1691, L. monocytogenes also is shed in milk by healthy
dairy cattle with no indication of mastitis [64,73,86,103,127,134,135,234,278].Schultz
[234] collected milk samples from 1004 cows and isolated L. monocytogenes from the
milk of 10 animals (0.1%), 7 of which appeared perfectly healthy. Shedding of Listeria
in milk by these animals was intermittent but continued for up to 12 months. Examination
of dairy herds in Yugoslavia [247] also has demonstrated that clinically healthy cows can
act as asymptomatic carriers of L. monocytogenes and secrete the organism in their milk
for months over several lactation periods. In one such survey, L. monocytogenes was
detected in milk from 3.2% of 845 clinically normal cows on seven farms on which listeri-
osis had been previously diagnosed [ 15 81. In addition, Kampelmacher [ 1481 reported that
dairy cattle shed L. monocytogenes at levels of 10,000-20,000 cfu/mL of milk.
Surveys of raw milk prompted by dairy-related outbreaks of human listeriosis in
1983, 1985, and 1987 confirmed that asymptomatic cattle are carriers of L. monocytogenes.
Indirect contamination of bulk milk occurs from unhygienic rnilking practices, if L. mono-
cytogenes is present in feeds, feces, udder surface, or bedding [87], or if an animal is
recovering from a recent infection [ 104,1951. L. rnonocytogenes has been reported in raw
cows milk with a distribution ranging from 0.1 to 45% [22,7 I ,86,87,94,111,122,173,234,
254,260,2651. Recoveries varied depending on whether individual cows, bulk tanks main-
tained on the farm, or milk tankers serving multiple premises were sampled. When avail-
able, data indicate that the incidence of L. monocytogenes from individual farms may be
lower than that reported for processing centers or tanker trucks [120,230]. A 23-year sur-
vey involving 36,200 dairy herds in Denmark indicated that the incidence of L. monocyto-
genes-infected cows varied from 0.01 to 0.1% and of herds with an infected cow from
0.2 to 4.270. However, 8.5% of bulk milk samples (n = 4451 were contaminated with L.
monocytogenes [6]. Regional differences in the recovery of L. rnonocytogenes from milk
have been documented. For example, Dominguez Rodriguez et al. [7 I ] found L. monocyto-
genes in 45.3% of 95 raw milk samples from a single bulk tank (80,000-L capacity). The
50 Wesley

dairy received raw milk from several small farms in western and central Spain over a 16-
month interval. In the northeastern United States, Hayes et al. [122] found L. monocyto-
genes in 12% of raw milk samples. L. monocytogenes was found in 4.4% of raw milk
samples (n = 137) obtained from the Utrecht region of The Netherlands [22] and in 3.8%
of raw milk samples in Scotland 1911. This parallels the recovery of L. monocytogenes
in 4.1% of raw milk samples from Tennessee [231]. Interestingly, in that US study, con-
sumption of raw bulk milk was reported by 35% of dairy producers [231].
A seasonal distribution of L. rnonocytogenes in raw milk, mirroring numerous deter-
minants including a change of diet or weather-related stress, has been observed. Lovett
et al. [ 1733 surveyed raw milk at various times from three different regions in the United
States and found L. monocytogenes in 4.2% of the overall samples. However, recovery
of L. monocytogenes from Massachusetts samples was seasonal with the incidence being
highest during cooler months and lowest in hot weather months. Interestingly, no seasonal
trend was exhibited by samples collected from Ohio, Kentucky, and Indiana [173]. L.
monocytogenes was cultured from 4.9% of raw milk samples obtained from 70 Irish farms
with the incidence being higher in the winter when cows were housed indoors than in the
summer [224]. A study of L. monocytogenes in raw milk in Nebraska indicated a seasonal
distribution with 6% of raw milk samples harboring L. monocytogenes in February; 2%
of samples were positive in July [ 1671. Seasonal variation may be related to silage feeding
during the winter. In Scotland, a seasonal distribution was indicated for L. monocytogenes,
which was present on 25 of the 160 farms surveyed (16%). Contamination was sporadic,
with bacterial titers generally < 1 L. monocytogenes cfu per mL. Although more raw milk
samples were positive for L. monocytogenes in January than at other sampling times
throughout the year, the authors caution that no link to farm management practices was
evident [90]. In Ontario, Canada, some geographical differences were observed, with the
incidence of L. monocytogenes in raw milk being higher in the eastern region [86]. Despite
the modest recovery of L. monocytogenes from raw milk in Ontario (1.3%, 6 of 455
samples), the incidence was lower during the winter and autumn than at other times [86].
In another report, L. monocytogenes was found in 5.14% of raw milk samples screened
monthly in Ontario with no indication of seasonal differences [254].
Normal healthy cattle may intermittently shed Listeria in their feces, with prevalence
rates ranging from a few percent to 52%, with some seasonality [87,131,271,2881. Fecal
shedding may reflect levels of L. monocytogenes in feed. In one study, shedding of L.
monocytogenes (52%) was related to feeding wet feed (e.g., silage of beet tops, oat, and
pea straw); 67% of the samples were contaminated 12521. L. monocytogenes was isolated
from the feces of 8.7% of nearly 4000 randomly selected dairy cows in Finland over a
2-year period [ 1331. Again, L. monocytogenes was recovered more frequently in bovine
feces on farms where L. monocytogenes was in feed than on premises where feed (silage
or pasture grass) was negative for L. monocytogenes [ 13 1,1331. Switching cattle from
grazing to a diet of silage increased fecal shedding of L. monocytogenes. The distribution
was seasonal with L. monocytogenes recovered from feces more frequently during the
indoor season (9.2%) than when animals were on pasture (3.I %). Expectedly, the seasonal
occurrence of L. monocytogenes in milk reflected the frequency of this pathogen in feces
but not in grass silage or pasture grass, thus inferring fecal contamination during milking
[131].
Serosurveys have been used to monitor distribution of L. monocytogenes infections
in dairy cows. Infection rates in cattle can be estimated by measuring antibody levels to
whole cells as well as antibodies specifically targeting LLO [ 12,331. Agglutination titers
Listeriosis in Animals 57

of serum and whey were evaluated in experimentally infected dairy cattle ( n = 34). By
the eighth week postinfection, 80% of the cows exhibited serum titers of > I :20,480.
Whey titers, which reflected the local mucosal immunity to L. monocytogenes in the mam-
mary gland, rarely exceeded I :256, because of the lower immunoglobulin concentration
in milk versus blood [72,293]. Since up to 33% of dairy cattle continued to shed L. monocy-
togenes despite high serum titers, antibody levels did not accurately predict the presence
of L. monocytogenes in milk [72,257,276,293]. As with humans [26], sheep 1 13,166,1771,
and goats [ 1061, antibodies to LLO have been used to evaluate the immune response of
experimentally infected cattle. [ 12,331. A positive response to ILL0 specifically confirmed
previous or current infection with L. rnonocytogenes in dairy cows [ 12,331.
Stress-related immunosuppression associated with change of diet, weather, transpor-
tation [89], pregnancy, parturition, and lactation may lower resistance to bovine listeriosis
[239]. Dexamethasone mimics the stress-related release of glucocorticoids. In cattle, dexa-
methasone elevates total white blood neutrophil counts and decreases eosinophil and lym-
phocyte populations. When administrated to cows experimentally infected with L. monocy-
togenes, dexamethasone increased shedding of the pathogen in milk by up to 100-fold
[291]. Increased levels of L. monocytogenes in milk may reflect impairment of cell-medi-
ated immune mechanisms and phagocytic cell functions that underlie listerial immunity
[269]. Likewise, transport of live animals over long distance!; significantly increased the
level of fecal excretion of L. monocytogenes. However, contamination of the resultant
cattle and sheep carcasses was minimal [92].
A major concern of bovine listeriosis is the potential risk posed to humans. In Den-
mark, a case-control study indicated that human listeriosis was frequently linked to con-
sumption of unpasteurized milk (risk factor of 8.6), although other factors, such as immu-
nosuppression and underlying diseases, were regarded as more significant [ 1401.
Furthermore, a comparison of 33 isolates from bovine mastitis and 27 human clinical
isolates recovered in Denmark during 1993 was made by sero- and ribotyping. Serotyping
showed that all bovine and 63% of human isolates belonged to serogroup 1, whereas 37%
of the human isolates were of serogroup 4. DNA fingerprinting by ribotyping indicated
that a low but constant percentage of Danish dairy herds had cows infected with L. monocy-
togenes strains which were similar to human clinical strains 11411. L. monocytogenes
ribotypes common to both dairy processing and the farm environment (dairy cattle, raw
milk, silage) were also reported in the United States, thus suggesting that the farm may
serve as a reservoir for L. monncytogenes strains capable of entering the dairy processing
facility [9]. The findings also verify the ubiquitous distribution of the pathogen.
Although foodborne listeriosis in humans is more frequently linked to consumption
of contaminated dairy products than to beef, L. monocytogenes was recovered from 3% of
composite fecal samples representing 224 feedlot beef cattle [249]. In a limited study
of experimentally infected Holstein cows (n = 4), L. monocytogenes was cultured from
muscle, organ, and lymphoid tissues at 2 days postinfection; none was recovered at 6 or
54 days after inoculation [ 1451. Thus culled dairy cattle may be an insignificant source
of L. monocytogenes contamination in meat. Epizootics have been observed in both feedlot
and beef cattle herds (5,299). Transport of cattle over long distances increased the level
of fecal excretion of L. monocytogenes, but contamination of carcasses was not high [89].
Yet in this study, L. monocytogenes was detected in 9 1 % (2 1 of 23) of minced beef sam-
ples, demonstrating that processing significantly increases the level of Contamination com-
pared with that of the whole carcass 1891. This hypothesis is further strengthened by
tracking L. monocytogenes strains by multilocus enzyme dectrophoresis. L. monocyto-
52 Wesley

genes strains of electrophoretic type (ET) 1 have been implicated in major human food-
borne epidemics and are found coincidentally in livestock. L. monocytogenes strains of ET
1 predominate in cattle at the beginning of slaughter but are not detected on the carcasses at
the end of processing or in the environment of the abattoir [32]. In contrast, environmental
strains such as ET 19 contaminate the carcass during processing. ET 19 strains were found
on the carcasses of pigs at the end of processing in two slaughterhouses but not on live
animals or at the beginning of slaughter. Overall these findings indicate that contamination
of meat occurs during processing by L. monocytogenes strains which are resident in the
packing plant rather than by strains indigenous to animals [32]

Porcine listeriosis manifests itself primarily as septicemia. Encephalitis is reported less

frequently and abortions are rare [29]. Clinical septicemia is usually observed in the neo-
nate where hepatic necrosis may be a characteristic feature [ 118,1941. Unlike its frequent
occurrence in ruminants (cattle, sheep, and goats), listeriosis is rare in monogastric swine.
Slabospitskii [253] first reported Listeria infection in young swine raised on a Russian
farm and designated the organism as L. suis [29]. The first description of porcine listeriosis
in the United States occurred when Biester and Schwarte [28] reported it in Iowa in swine
with encephalitis. Later, Kerlin and Graham [ 1521 recovered Listeria from the liver of a
pig with no clinical signs of encephalitis. In Norway, Hessen [125] reported listerial septi-
cemia in piglets raised on a farm where sheep had died of listeriosis several weeks earlier.
Whether transmission was from sheep to pigs or the result of common exposure is un-
known.
In natural and experimental infections, listeriosis is more severe in young animals
[30,42,150]. Piglets succumb to infection, whereas adults generally survive. In the neonate,
L. monocytogenes may originate from the tonsils of the sow, penetrate the intestinal tract
of the piglet, and become systemic [267]. Neonatal listeriosis may be seasonal with cases
peaking in early winter [ 1721 and spring.
Listerial encephalitis seldom occurs in pigs. Symptoms of central nervous system
disturbance, including incoordination and progressive weakness followed by death, are
characteristic of listeriosis in the younger animal. Meningoencephalitis in swine begins
with a sudden refusal to eat and is typically followed by various neurological disorders,
including trembling, partial paralysis, incoordination, circling movements, and convul-
sions. Histopathological findings from meningoencephalitis include severe monocytic in-
filtration. Numerous blood vessels, particularly those in the pons, reveal perivascular
cuffing [29,108,219,2391. Although listerial meningoencephalitis in swine is infrequent,
several such outbreaks have been reported, including one in India in which 27 of 75 pigs
died [220].
In England, the Veterinary Investigation Center reported only 14 listeriosis cases
in swine between 1975 and 1982 as compared with 666 cases in sheep and 472 cases in
cattle [ 1021. Listeriosis in pigs is also reported to be uncommon in The Netherlands [201].
Porcine listeriosis comprised 1% of listeriosis cases in western Canada [21]. In Iowa, a
major hog-producing state, of a total of 253 listeriosis submissions to the state veterinary
diagnostic laboratory from 1993 to 1997 none were from pigs. In that same interval, 87%
of listeriosis cases in Iowa were from cattle [161]. Earlier, Blenden reported that cattle
and sheep accounted for 274 of 281 (98%) of listeriosis cases submitted to the Missouri
Diagnostic Laboratory; only 1 case was from pigs [29].
Although few surveys are available describing the prevalence of L. monocytogenes
Listeriosis in Animals 53

in healthy pigs, its distribution can be estimated from surveys of fecal excretors and recov-
eries from tonsils and carcass swabs collected at slaughter. L. monocytogenes was cultured
from 5% of rectal swabs and from 1.9% of hog carcasses in Trinidad [ 11, indicating mini-
mal carcass contamination during processing. L. monocytogenes was present in 13% of
hog tonsils and in 2% of lymph tissues in pigs in Togo, West Africa [ 1291. In Belgium,
16% of fresh pig feces (n = 25 samples) harbored L. monocytogenes [271]; 5.9% of swine
fecal samples were positive in Germany [288]. In Yugoslavia, L. monocytogenes was
cultured more frequently from hog tonsils (25%) than from fecal samples ( 5 % ) taken from
the same animals [277]. In Scotland, L. monocytogenes was not found in either pig feces
before slaughter or on swine carcasses [92]. Likewise, L. monocytogenes was not recov-
ered from hog carcasses in Norway or Sweden [204].
Asymptomatic carriers of Listeria may be more prevalent in eastern Europe. To
illustrate, L. monocytogenes was recovered from 25.6% of swine feces in Hungary [222].
Ralovich [221] reviewed studies in which a fecal recovery rate of 47% in individual ani-
mals and in 11 of 12 among farms was described. Yet in Yugoslavia, 45% of all pigs
examined harbored L. monocytogenes in the tonsils, whereas only 3% were fecal excretors
[40]. A high infection rate in pigs has led to speculation that swine may be important
reservoirs of L. monocytogenes [ 1081.
Husbandry practices such as feeding pigs dry feed or silage, rearing in closed houses,
and maintaining specific pathogen free (SPF) herds as well as differences in sampling
sites (tonsils versus feces) may account for the variation in the incidence of healthy porcine
carriers reported. For example, in Yugoslavia, L. monocytogenes was recovered more
frequently from tonsils of pigs raised on silage (61%) than from animals raised on dry
feed (29%). Interestingly, in that study, L. monocytogenes was also recovered from more
than 19% of pork meat products tested [40]. Although not found in fecal samples in SPF
herds, L. rnonocytogenes was cultured from 2.2% of fecal samples from non-SPF herds
in Denmark [252]. Norrung et al. [206] detected Listeria spp. in 29% of tonsils removed
from market weight hogs and in 75% of pig feed samples tested in Denmark. Unfortu-
nately, data on the specific distribution of L. monocytogenes in this study were not pro-
vided. L. monocytogenes was recovered from the lymph nodes of 5% of slaughtered pigs
and from 8% of pork samples in Bosnia and Hercegovina [ 1711. Of L. monocytogenes
recovered in that survey, 76% were from hog carcasses sampled during the autumn and
winter months [ 1711.
Skovgaard et al. [252] reported that only 1.7% of pig fecal samples yielded L. mono-
cytogenes; however, the pathogen was detected in 12% of ground pork samples tested in
Denmark indicating dissemination of Listeria during processing. In Yugoslavia, where L.
monocytogenes was isolated more frequently from ground pork (69%) than from deep
muscle (O%), contamination occurs during pork processing [40]. In France, an outbreak
involving 279 human cases incriminated pickled pork tongue as a major vehicle of trans-
mission, although other highly processed, ready-to-eat delicatessen items subject to envi-
ronmental contamination were also implicated [ 1381.
Although limited epidemiological data are provided, two cases of human neonatal
listeriosis may have been linked indirectly to contact with pigs [256]. Alternatively, they
may reflect exposure to a common source of contamination.

Avian listeriosis was first described in 1935 [238], 3 years after TenBroeck isolated L.
monocytogenes (then Bacterium monocytogenes) from diseased chickens. Both wild [227]
54 Wesley

and domestic avians, including turkeys [24,12 1,2051, ducks [ 106,2391, geese [ 106,2391,
and pheasants [ 1061, are the largest group of asymptomatic carriers [ 106,1081. A survey
of healthy urban rooks indicated carriage with L. monocytogenes (33%), L. innocua (24%),
and L. seeligeri (8%) [35]. Likewise, L. monocytogenes was detected in 9.5% of fecal
samples from apparently healthy, ring-billed gulls (Larus delawarensis) in Montreal. In
contrast, samples from pigeons in Barcelona were negative [46], whereas L. monocyto-
genes was found in 1% of pigeons examined in Germany [2881.
Up to 33% of all healthy chickens may asymptomatically shed L. monocytogenes
in fecal material [67,68,252]. Birds most likely become infected by pecking Listeria-con-
taminated soil, feces, or dead animals; however, contaminated fecal material also may
pose a hazard to other livestock. Bovine encephalitic listeriosis developed in four cows
which were housed in stables where chicken litter was used as bedding. L. monocytogenes
serogroup 4b was recovered from the bovine brains, litter, and the intestinal contents of
4.1% of the donor birds 1671. In another study, the increased incidence of L. monocyto-
genes in rooks coincided with the nesting season and the peak of ovine listeriosis, which
in turn was linked to consumption of contaminated silage [88]. Thus, although the true
incidence of listeriosis in birds and other forms of domestic livestock is undoubtedly much
higher than published reports, clinical listeriosis is uncommon in domestic fowl.
Despite the many sporadic cases of avian listeriosis that have been documented over
the last 60 years, this disease is far less common in birds than in sheep, goats, and cattle
[ 106,107,108]. For example, listerial infections were discovered in only 13 of more than
38,000 chickens submitted for examination in Pennsylvania between 1960 and 1965 [236].
Furthermore, large-scale outbreaks of listeriosis in chickens appear to be uncommon
[200,212]. In an outbreak in India involving young chicks, death (mortality rate of 60%)
was sudden and usually with no prior symptoms. Some birds exhibited symptoms of weak-
ness and lassitude and a tendency to stand in an isolated dark place [200].
Listeriosis in birds may be a secondary infection associated with viral infections
[57] as well as salmonellosis, Newcastle disease, fowl pest, coryza, coccidiosis, worm
infestations, mites, enteritis, lymphomatosis, ovarian tumors, and other immunocom-
promising conditions [108]. In 1988, an encephalitic form of listeriosis was reported in
broiler chickens in California [54]. Predisposing conditions which may have precipitated
the outbreak included recent debeaking and vaccination with a modified live viral arthritic
vaccine which was given subcutaneously in the neck. L. monocytogenes serotype 4b was
recovered from a liver and multiple brain samples. Three years later, a second outbreak
occurred in breeder replacement birds and affected 0.3% of the 54,000 birds in the flock.
L. monocytogenes was recovered from soil samples collected near an adjacent dairy but
not from other sites on the premises. The stress associated with the unusually cold climate
described in the report coupled with vaccine stress of the 7- to 10-day-old chicks may
have precipitated this outbreak [54].
Septicemia, the most frequent manifestation of listeriosis in chickens and other do-
mestic fowl, is characterized by focal necrosis within the viscera, particularly the liver
and spleen [ 1081. Although not present in all cases [200], cardiac lesions frequently de-
velop, which in turn lead to engorgement of cardiac vessels, pericarditis, and increased
amounts of pericardial fluid [ 108,2231. Other conditions produced by the septicemic form
of avian listeriosis have included splenomeglia, nephritis, peritonitis, enteritis, ulcers in
the ileum and ceca, necrosis of the oviduct, generalized or pulmonary edema, inflammation
of the air sacs, and conjunctivitis. In acute cases, lesions resulting from these conditions
may be partially obscured by congestion and hemorrhages throughout the viscera [ 1081.
Listeriosis in Animals 55

Unfortunately, chickens and other domestic fowl that suffer from listerial septicemia nor-
mally exhibit few overt signs of disease other than progressive emaciation and usually
die within 5-9 days of infection.
Although far less common than the septicemic form of listeriosis, L. monocytogenes
also can produce meningoencephalitis in domestic fowl. Domestic birds suffering from
listerial meningoencephalitis exhibit several striking behavioral changes, including incoor-
dination, tremors, torticollis, unilateraUbilatera1 toe paralysis, and dropped wings, all of
which directly relate to disturbances of the central nervous system [ 181. Such infections
are virtually always fatal. Postmortem examination often reveals congestion and necrotic
foci in the brain [ 181 along with many of the aforementioned conditions that are character-
istic of listerial septicemia. Microscopically, gliosis, and satellitosis in the cerebellum and
microabscesses containing gram-positive bacteria are found in the midbrain and medulla
of birds with encephalitic listeriosis [55].
L. monocytogenes can colonize both chick embryos and young birds with older birds
appearing more resistant [ 108,117]. Following oral challenge of chickens with 102or 106
L. monocytogenes cells, Bailey et al. [ 151 detected the pathogen more frequently in ceca,
spleen, liver, and cloacal swab samples from 1-day-old chicks rather than 14- or 35-day-
old chickens. Diarrhea and emaciation have been noted in experimental infection, thus
facilitating spread via feces and nasal secretions. In a later study, 2-day-old chicks were
experimentally infected with L. monocytogenes. Although most of the inoculated chicks
appeared healthy, depression, ruffled feathers, dullness, and diarrhea followed by death
were noted 2-5 days postinoculation. Milder symptoms such as anorexia and drowsiness
were also observed in several animals. At 5 days postinfection, 100% of the cecal samples
yielded L. monocytogenes. However, the percentage of L. monocytogenes-positive birds
decreased, and by day 28, L. monocytogenes was recovered from the ceca of only 10%
of experimentally infected birds [132]. In another report, with tests on a smaller num-
ber of birds, L. monocytogenes was only found on the first day following infection in 15%
of fecal samples [186]. These data suggest that L. rnonocytogenes is cleared rapidly from
infected birds, indicating that chicks are transiently infected and unlikely reservoirs of L.
monocytogenes. Interestingly, following artificial infection, Pustovaia [2 I 81 found that
Columbiformes (pigeons, doves), Passeriformes (perching birds), and Galliformes (tur-
keys, pheasants) were susceptible, whereas Falconiformes (falcons, hawks) and Strigi-
formes (owls) were resistant [ 1281.
L. monocytogenes has been used to investigate macrophage function in retroviral
infection and the cell-mediated immune response in susceptible and resistant chickens
exposed to Mareks disease virus 145,571. Viral infection depressed the resistance of 10-
day-old chickens to experimental infection with an avian osteopetrosis virus [57]. When
compared with virus-free chickens, the dual infected birds were less efficient in clearing
L. monocytogenes from their spleens [57).
L. monocytogenes can be recovered from infected chicks by inoculation of 1O-day-
old embryos [62]. Thus, egg inoculation has been suggested as an assay to replace the
mouse test to gauge the virulence of L. monocytogenes [ 1 11. For example, L. monocyto-
genes and L. ivanovii are fatal to experimentally inoculated 10-day-old chick embryos.
In contrast, embryos infected with the nonpathogenic species, L. innocua, L. seeligeri,
and L. welshimeri, generally survived [266].
L. monocytogenes is present in 0-33% of healthy birds [67,68,76,99,127,136,288],
with contamination in retail poultry ranging from 17 to 70% [ 14,32,76,92,99,186,208].A
link between transport stress and fecal shedding of L. rnonocytogenes has been suggested.
56 Wesley

In one study, L. monocytogenes was found in 33% of pooled fecal samples collected from
cages suggesting recrudescence of L. monocytogenes because of transport stress. However,
no data on the status of L. monocytogenes in these birds before shipment are provided
[252].
The presence of Listeria on retail poultry probably results from contamination during
processing rather than from the bird. In an effort to trace the source of L. monocytogenes
in retail poultry, low levels of natural carriage ( 5 % ) were reported in cecal samples from
parent flocks providing broilers. L. monocytogenes was not cultured from cecal samples
from over 2000 broilers (90 flocks) in Denmark [208]. However, L. monocytogenes was
found in processed poultry. Comparison of DNA fingerprinting patterns by pulsed-field
gel electrophoresis (PFGE) indicated that live birds contributed little to the total contami-
nation of the product [208].
Once processing is initiated, the numbers (and percentage) of Listeria-positive sam-
ples increase [92]. Studies in poultry slaughterhouses failed to detect L. monocytogenes
in several organs, including the intestinal tract and ceca. Yet it was found in processing
water, in mechanically deboned meats, and on the hands and gloves of 34% of the meat
cutters, indicating cross contamination during processing [99]. Later studies used DNA
profiles of L. monocytogenes collected during processing to indicate significant environ-
mental contamination during processing [32]
Sporadic human cases of listeriosis have been epidemiologically linked to consump-
tion of undercooked poultry products [235]. Analysis of risk factors associated with spo-
radic human listeriosis in the United States indicated that cancer and immunocompromised
patients, in whom 69% of listeriosis cases occur, were more likely than controls to have
eaten undercooked poultry (odds ratio = 3.3) [233].

Minor Species
Listeriosis has been diagnosed in several minor livestock species, such as horses, llamas,
animals raised commercially for pelts, companion animals, deer, and primates. The routes
of transmission and symptoms parallel those of cattle, sheep, and goats.
As in other livestock species, Listeria infection in horses can cause abortion [289],
septicemia [25,51,79,112], and encephalitis [ 1821. In contrast to cattle and sheep, few
cases of equine listeriosis are reported [ 160,182,185,239,263,264,2681.A survey of fecal
samples from 400 German horses indicated a carrier rate of 4.8% for L. monocytogenes,
6% for L. innocua, and 1.5% for L. seeligeri with less than 1% harboring L. welshimeri
[288]. The few surveys describing L. monocytogenes-seropositive horses should be inter-
preted cautiously in the light of possible cross reactivity between antibodies of other bacte-
rial species and Listeria.
Prior contact with cattle and feeding on silage may explain sporadic cases of equine
listeriosis [ 1871. L. monocytogenes was reported from four Welsh and two Shetland ponies
housed together with cattle, one of which was diagnosed with listeriosis, and given poor-
quality silage [79]. At necropsy, L. monocytogenes was cultured from the equine liver,
spleen, heart, kidneys, and lungs [79]. In Tasmania, abortions occurred in two mares which
were allowed to graze on a pasture which had previously been a sheep farm but had most
recently served as a dairy farm. L. monocytogenes serogroup 1 was cultured from the lung
and stomach of one fetus. Following antibiotic therapy, the mare was bred and later gave
birth to a normal live foal, indicating that L. monocytogenes infection does not lead to
permanent infertility [ 1831. Equine abortion, preceded by mild respiratory tract infection
Listeriosis in Animals 57

caused by L. rnonocytogenes serotype 4, was reported for a mare which had wintered with
cattle and had consumed ensilage [289]. L. monocytogenes was cultured from the fetal
liver, lung, spleen, and stomach. Neonatal septicemia was documented in a 3-day-old foal
whose mare was housed indoors and fed poor-quality contaminated hay [126].
As with other species, the origin of infection in equine listeriosis cases may be
unknown. To illustrate, L. monocytogenes was recovered from the brain stem of a 16-
year-old Welsh pony gelding with signs of ataxia, weakness, and deficits of cranial nerves.
No immunological deficit was detected and there was no history of contact with ruminants
or access to silage [182]. A 21-day-old Appaloosa filly was examined because of diarrhea
of 2 weeks duration [284]. As the animals condition deteriorated, gentamicin sulfate and
procaine penicillin G were administered. Septicemia was diagnosed based on the presence
of L. monocyrogenes cultured from the blood. No sources of infection were evident, thus
leading to speculation that L. monocytogenes was transmitted to the foal via contaminated
mares milk.
As is true for humans, listeriosis also occurs more frequently in immunocompro-
mised livestock with defects in both the humoral and cell-mediated immune systems [269].
Listeriosis was described in an Arabian foal with combined irnmunodeficiency [ 5 I]. The
I-month-old foal was ataxic, lethargic, failed to nurse, and spent most of the time with its
head down. Most strikingly, hoofs were dragged when the animal exercised. At necropsy,
widespread lesions were present in the viscera and central nervous system.
In the llama, listeriosis occurs as a septicemia with meningitis in neonates, but more
commonly causes asymmetrical vestibular disease in adults [ 191. Most affected animals
are weaned and grazing or consuming roughage but not silage. Multifocal suppurative
encephalitic listeriosis was diagnosed in two adult llamas, both of which were pregnant.
L. monocytogenes was cultured from one of two animals and was observed in brain stem
lesions of both llamas by fluorescein-conjugatedantibody to L. monocytogenes [43]. In an-
other report, L. monocytogenes caused fatal meningoencephalomyelitis in a 3- to 5-month-
old llama. The animal displayed unilateral peripheral disease progressing to encepha-
litis 12701. The source of infection for cases detailed in these two reports is unknown [ 19,431.
Listeriosis can have an economic impact on commerciall pelt farms. An outbreak of
disseminated visceral listeriosis in chinchillas in Nova Scotia [95] was associated with
consumption of contaminated sugar beet pulp, although L. monocytogenes was not isolated
from the feed. This outbreak occurred in a colony with a 2390 mortality rate of breeding
chinchillas [298]. Approximately 4 days before death, animals were anorexic and hunched
and some had torticollis (twisted necks). However, many animals were found dead without
clinical signs (2981. Hay contaminated with rodent, bird, or ruminant feces has been impli-
cated in previous outbreaks of listeriosis among chinchillas with removal of contaminated
feed often interrupting the cycle of transmission [47,95]. L. monocytogenes could have
been transmitted by coprophagia, since animals defecated in dust bath pans and the pans
were transferred from cage to cage [298]. In an enzootic outbreak of listeriosis in a rab-
bitry, L. monocytogenes 1/2a was cultured from feed samples and from a doe which had
died of septic metritis 12141.
The early literature describes L. monocytogenes in nondomesticated ruminants, in-
cluding reindeer, roe deer, and a Grants gazelle that had previous contact with Listeria-
infected sheep [23,81,83,151,205,2861. L. monocytogenes has been recovered at necropsy
from ruminants housed in zoological parks [8 133,2861. Meningoencephalitis occurred
during the winter and early spring in 42 of 1800 deer in a Danish park. This was preceded,
in the previous spring, by death of six deer which exhibited circling and appeared to be
58 Wesley

blind. The following year, the first sign of illness was a drooping ear, caused by paralysis
of the facial nerve, and a slight inability to follow the herd. No external source of L.
monocytogenes was evident and stress resulting from a poor beech-mast crop, an increased
stocking rate of animals resulting in overcrowding with possible introduction of asymp-
tomatic carriers, and a sudden change in the weather were all potential contributing factors
[811.
Listeriosis is rarely reported in dogs and can cause encephalitis, including circling
[232], and abortion [261]. In a survey of domestic animals, L. monocytogenes was detected
in 1.3% of dog fecal (n = 300) samples and 0.4% of cat fecal (n = 275) samples [288].
The low recovery may indicate that companion animals are not important in the epidemiol-
ogy of listeriosis in humans [287] or that shedding is sporadic. In contrast, serosurveys
which indicate that up to 90% of dogs may be seropositive [48,25 11 should be interpreted
cautiously because of the cross reactivity inherent in agglutination tests. A single report
on possible transmission of L. monocytogenes from humans to dogs [262] warrants reeval-
uation, since the isolates were later identified as L. innocua. Nevertheless, recovery of a
single species of Listeria from both humans and dogs in close proximity could reflect
substantial environmental contamination rather than human-to-dog passage.
Listeriosis also can occur in non-human primates where it manifests itself as septice-
mia [303], meningoencephalitis [61], and stillbirths [ 188,2731, as documented in a large
outdoor breeding colony in California [2 131. Transmission may occur by consumption of
contaminated foods [273]. Attempts have been made to experimentally infect Cynomolgus
monkeys (Macacafascicularis) through feeding [85] and exposure to aerosols [ 1491. Al-
though these monkeys shed L. monocytogenes in their feces for up to 21 days after ingest-
ing I O9 Listeria, neither septicemia nor encephalitis were reported indicating that normal
healthy primates are resistant to L. monocytogenes.

Fish and Crustaceans

Demand for seafood is growing because of its popularity, and the aquaculture industry is
responding by providing fish raised on controlled fish farms rather than depending on the
availability of fresh-caught products. In 1980, in New Zealand, Lennon et al. [163] re-
ported a cluster of 22 perinatal human listeriosis cases. A weak association between these
cases and consumption of contaminated raw fish and shellfish was established. Facinelli
et al. [84] described a case of sporadic listeriosis, in which clinical isolates and those from
the undercooked fish in the patients refrigerator were identical by DNA fingerprinting.
The first report of Listeria in fish came from Romania in 1957 [259]. In that study,
L. monocytogenes was isolated from viscera of pond-reared rainbow trout that presumably
became infected after consuming contaminated donkey meat. The results of current sur-
veys indicate that Listeria is absent from live saltwater fish but is present in live freshwater
species. The bacteria could not be detected in the intestinal tract, skin, and gills of 10 live
salmon [77]. Likewise, L. monocytogenes was not found in either salmon (n = 199) or
in environmental samples taken from a fish farm in Bergen, Norway [78]. In contrast, L.
monocytogenes was recovered from two of the five intestinal tracts of market-purchased
fresh water fish in India [ 1231.
Channel catfish (Zctalurus punctatus) is the most widely cultured species in the
United States with most commercial ponds being located in the southeastern region. A
study of catfish, water, and feed collected from university ponds in Alabama indicated
the presence of L, monocytogenes on skin and viscera (mean presumptive count = 1.99
Listeriosis in Animals 59

log cfu/g wet weight). L. monocytogenes was not detected in the water or feed. Unfortu-
nately, the authors provided no data on the percentage of L. monocytogenes-positive fish,
but they concluded that bacterial concentrations in the viscera suggest cross contamination
is possible during evisceration [ 1651. A survey of three rainbow trout farms in Switzerland
showed that L. monocytogenes was present in the feces (40%) and on the skin (33%; 5
of 15) of fish from one of the farms, yet L. monocytogenes was detected on the finished
product in only 6% of the fish from this farm. In contrast, L. monocytogenes was not
found in feces, skin of fish, or the finished product of two of the farms where fish were
raised in concrete ponds and starved 3-7 days before harvest [ 1391. Similarly, L. monocy-
togenes was not recovered from the skin, gills, intestines, tank water, diet of striped bass
grown in recirculating water tanks [203]. Experimental infection of zebrafish (Bruchy-
dunio rerio) indicated the LDSowas higher in fish than in mice and that L. monocytogenes
did not multiply in fish [ 1921. Experimental infection increased production of granulocytes
and monocytes. In contrast to L. monocytogenes, strains of L. welshimeri, L. innocuu, and
L. seeligeri killed more than 50% of the fish 7 days postinfection [ 1921
Brackett [36] proposed that contamination of fish and shellfish through their ambient
waters may influence distribution of L. monocytogenes. Surface waters, sewage effluents,
and agricultural runoff all may potentially contribute Listeriu spp. to the aquatic environ-
ment. In addition, the presence of L. monocytogenes in sea gulls may be another source
of shellfish contamination [88]. In 1959, L. monocytogenes was detected in crustaceans
gathered from a Russian stream [248]. A more recent survey conducted on the Gulf Coast
of the United States examined shrimp, oysters, and estuarine waters for L. monocytogenes
[198]. The pathogen was detected in 11% of unprocessed shrimp (n = 74) but not in
oysters (n = 7 9 , although some of the oysters were harvested from prohibited shellfish-
growing sites. In a parallel study conducted in freshwater tributaries off the Humboldt-
Arcata Bay in Northern California, L. monocytogenes was detected in freshwater samples
(61%), some of which received runoff from nearby farms. However, L. monocytogenes
was not found in oysters in that study [52] nor in oysters kept in live holding tanks in
seafood markets in Seattle [53]. Albeit a modest number of shellfish were examined, L.
monocytogenes was not detected in fresh shellfish (shrimp, cuttlefish, clams) tested in
Cochin, India [98].
Overall, these data indicate that live fish and shellfish are not likely carriers of L.
monocytogenes. Polymerase chain reaction (PCR) tests have been used to detect L. mono-
cytogenes in experimentally contaminated marinated rainbow trout [82]. With the in-
creased interest in L. monocytogenes in fish and seafoods 1771, this sensitive technique
may be useful for rapid screening of live shellfish and fish for L. monocytogenes. Neverthe-
less, the paucity of reports documenting L. monocytogenes in live freshwater fish and
shellfish suggests that Listeriu spp. detected in the retail product most likely resulted from
postharvest contamination.

TREATMENT
Poor animal husbandry, consumption of contaminated feed, and stress are important fac-
tors in precipating listeriosis. Thus identifying and eliminating these problems are critical
to preventing reoccurrences. In general, since antemortem diagnosis is rarely made, treat-
ment is seldom attempted.
Since listerial encephalitis is a rapidly debilitating disease in ruminants, treatment
must be initiated early during the course of infection if there is to be any reasonable hope
60 Wesley

of a cure. L. monocytogenes is resistant to many drugs but is sensitive to chlortetracycline.

The intravenous injection of chlortetracycline (10 mg/kg body weight per day for 5 days)
is effective in meningoencephalitis of cattle but less so in sheep [219]. If penicillin is
used, high doses are required because of the difficulty of maintaining therapeutic levels
in the brain. Penicillin G should be given at 44,000 U/kg body weight, intramuscularly
daily for 1-2 weeks [96]. If signs of encephalitis are severe, death usually occurs in spite
of treatment. Supportive therapy, which is usually reserved for valuable animals, including
fluid and electrolyte replacement, is indicated for animals having difficulty eating and
drinking as a result of neural damage. Excessive salivation leads to acidosis, which is
remedied by intravenous replacement of bicarbonate ions. Permanent neurological damage
often occurs in ruminants despite proper therapy. In view of the severe economic losses
from listerial encephalitis in sheep, it may be prudent to consider vaccinating animals
against listeriosis, particularly if they are being raised in areas prone to listerial infection
[202].
In birds, tetracyclines (5-10 mg/kg body weight daily for 1 week) are efficacious
in both acute and subacute cases. Treatment of chronic listeriosis is unsuccessful. As
with other livestock species, rigid sanitation and disinfection procedures with culling and
isolation of affected birds may be helpful [97].
Prompt treatment of animals with listeriosis is clearly beneficial, with early diagnosis
dependent on observation of clinical symptoms. In cattle and sheep, appearance of clinical
signs is an indication of neurological damage and thus, of a guarded prognosis for treat-
ment. In all cases, the economics of the attempted treatment must be considered along
with humane euthanasia as an alternative.

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Listeriosis in Humans

LAURENCE SLUTSKER AND ANNESCHUCHAT

Centers for Disease Control and Prevention,
Atlanta, Georgia

INTRODUCTION
Listeria monocytogenes has been recognized as a human pathogen since 1929 [74]. This
organism is found in multiple ecological sites throughout the environment, including soil
[ 1051, water, and decaying vegetation [ 104,1061. Recent studies of epidemic and sporadic
cases of listeriosis have increased our knowledge of important sources of L. monocyto-
genes in human illness. Epidemiological investigations during,the last 15 years have shown
that epidemic listeriosis is a foodborne disease. [ 10,17,23,27,38,44,56,66,80,85]. Simi-
larly, recent studies have suggested that a substantial proportion of sporadic cases of listeri-
osis are also caused by consumption of the organism in foods [72,89,92]. Development
of improved laboratory techniques to detect and subtype L. monocytogenes has also con-
tributed to an improved understanding of human listeriosis [7,9,11,82,83].
Human disease caused by L. monocytogenes usually occurs in certain well-defined
high-risk groups, including pregnant women, neonates, and immunocompromised adults
but may occasionally occur in persons who have no predisposing underlying condition
(Table 1). The ongoing epidemic of acquired immunodeficiency syndrome (AIDS), as
well as widespread use of immunosuppressive medications for treatment of malignancy
and management of organ transplantation, has expanded the immunocompromised popula-
tion at increased risk of listeriosis. Unlike infection with other common foodborne patho-
gens such as Salmonella, which rarely result in fatalities, listeriosis is associated with a
mortality rate of approximately 20% [34]. This high case-fatality rate, along with the
heightened awareness of listeriosis as a foodborne disease and increasing clinical concern

75
76 Slutsker and Schuchat

TABLE
1 Clinical S y n d r o m e s Associated with Infection with L isteria
rnonocytogenes

Predisposing conditions
Population Clinical presentation Diagnosis or circumstances
Pregnant women Fever, _t myalgias, & Blood culture 5
diarrhea Amniotic fluid
Preterm delivery culture
Abortion
Stillbirth
Newborn s
<7 days old Sepsis, pneumonia Blood culture Prematurity
2 7 days old Meningitis, sepsis Cerebrospinal
fluid culture
Nonpregnant adults Sepsis, meningitis, focal Culture of blood, Immunosuppression,
infections cerebrospinal advanced age
fluid, or other
normally sterile
site
Healthy adults Diarrhea and fever Stool culture in se- Possibly large inoc-
lective enrich- ulum
ment broth

about the importance of illness caused by this organism in the expanding population of
highly susceptible persons, has resulted in increased attention to the importance of L.
monocytogenes as a human pathogen.
In this chapter, we will consider various aspects of listeriosis in humans, including
infection and clinical manifestations of disease, epidemiological patterns of disease, diag-
nosis, treatment, and prevention. Information on the microbiology, ecology, pathogenesis,
detection, subtyping, manifestations of infection in other animals, and occurrence of L.
monocytogenes in various foods is presented elsewhere in this book. Although some
foodborne outbreaks of listeriosis will be discussed here as examples of epidemic disease
among humans, a more exhaustive treatment of foodborne listeriosis appears in Chap-
ter 10.

HUMAN INFECTION AND CLINICAL MANIFESTATIONS OF

LlSTERlOSlS
Asymptomatic Carriage
L. monocytogenes is distributed throughout the environment and can be frequently recov-
ered from a broad spectrum of foods [26,78] and from the gastrointestinal tract of healthy
people. Numerous fecal carriage studies of L. monocytogenes among different populations
have been done with widely varying rates reported (Table 2). Some of this variation may be
attributed to differences in populations studied, culture techniques, numbers of specimens
obtained from each individual, specimen handling, and whether results were reported as
a point or cumulative prevalence.
Using cold enrichment culture techniques, investigators in Denmark determined that
Listeriosis in Humans 77

2 Reported Fecal Carriage Rates of Listeria monocytogenes

TABLE
No. % with
Year Population studied L. monocytogenes Method Reference
1972 Slaughterhouse 1147 4.8 Point preva- 13
workers lence
Hospitalized adults 1034 1.2 Point preva-
lence
Hospitalized 195 0 Point preva-
children lence
Hospitalized adults 595 1.0 Point preva-
with diarrhea lence
Household contacts 34 1 26.0 Cumulative
of persons with prevalence
listeriosis over 6
months; up
to 8 cultures
per person
1972 Laboratory workers 26 77.0 Cumulative 52
handling L. mo- prevalence
nocytogenes over 8
weeks; up to
8 cultures
per person
Office workers 26 62.0 Same
with no L. mono-
cytogenes con-
tact
1986 Healthy pregnant 54 2.0 Point preva- 54
women lence
Healthy nonpreg- 60 3.4 Point preva-
nant women lence
1990 Persons with diar- 1000 0.6 Point preva- 68
rhea lence
Healthy food han- 2000 0.8 Point preva-
dlers lence
199 1 Renal transplant pa- 177 5.6 Cumulative 59
tients prevalence
(mean 2.5
specimens
per patient)
Home hemodialy- 80 2.5 Point preva-
sis patients lence
Outpatients with 171 1.8 Point preva-
gastroenteritis lence
1992 Pregnant women 18 5.6 Point preva- 62
with listeriosis lence
Household contacts 60 8.3 Point preva-
of pregnant lence
women with lis-
teriosis
78 Slutsker and Schuchat

TABLE
2 Continued
No. % with
Year Population studied L. monocytogenes Method Reference
Age-, race-, and 7 0 Point preva-
hospital- lence
matched controls
Cheese plant em- 31 9.7 Point preva-
ployees lence
Household contacts 94 10.6 Point preva-
of cheese plant lence
employees
1993 Household contacts 82 21 .o Point preva- 88
of patients with lence
listeriosis (per-
sons)
Households of pa- 28 21.0 Point preva-
tients with listeri- lence
osis with at least
one carrier
1993 Healthy pregnant 147 2.7 Cumulative 40
women prevalence
(mean 2.4
specimens
per patient)

4.8% of 1147 healthy slaughterhouse workers had stool cultures yielding the organism
[ 131. Similar surveys by the same researchers documented L. monocytogenes fecal carriage
rates of 1.2% among 1034 hospitalized adults, none of 195 hospitalized children, and 1%
of 595 hospitalized adults with diarrhea. Kampelmacher et al. reported high rates of fecal
carriage among laboratory workers having daily contact with L. monocytogenes (77% of
26) as well as among office workers who had no contact with the organism (62% of 26).
Because stool cultures were collected weekly for 8 weeks, figures from this study represent
cumulative rather than point prevalence estimates [52]. Subjects had L. monocytogenes
isolated an average of 1.3 times out of the eight serial specimens collected.
Among other populations, a large stool survey conducted in Germany found that 6
of 1000 (0.6%) fecal specimens collected from persons with diarrhea yielded L. monocyto-
genes, giving a point prevalence similar to the 0.8% found in 2000 healthy food handlers
from the same area during the same time period [68]. In 1987, MacGowan et al. surveyed
177 renal transplant recipients with multiple fecal samples obtained over a 1-year period.
Overall, 10 (5.7%) individuals had positive stool cultures, and a positive culture was asso-
ciated with ranitidine use or consumption of three or more types of cheese since the begin-
ning of the year [59]. The same investigators reported fecal carriage rates of 1.8% among
171 patients with gastroenteritis attending a general practice, and 2.5% of 80 home hemo-
dialysis patients. Among all three patient groups, Listeria isolations were highest during
the months of July and August.
Among pregnant women, Lamont and Postlethwaite reported a fecal carriage rate
of L. monocytogenes of 2% among 51 women early in pregnancy (10-16 weeks), similar
to the 3.4% rate observed among 59 nonpregnant women attending the same clinic [54].
Listeriosis in Humans 79

Timing of carriage was examined among 147 women attending an antenatal clinic in the
United Kingdom [40]. One fecal specimen was obtained during each trimester. Among
the four (2.7%) women whose fecal specimens yielded L. monocytogenes, one was in the
first, two in the second, and one in the third trimester. During a large foodborne listeriosis
epidemic in Los Angeles, Mascola et al. compared fecal carriage rates in pregnant women
with listeriosis to age-, sex-, and hospital-matched controls; carriage rates were not sig-
nificantly different between the two groups (1 of 18 vs 0 of 7, respectively, P = NS) [62].
Household contacts of patients with listeriosis also have been surveyed. In Denmark,
fecal specimens from 26% of 34 household contacts of persons with listeriosis yielded L.
monocytogenes [13]. In this study, among 14 households sampled, 5 (36%) had at least
1 household member with a positive stool culture; however, only two family members
had the same serotype of L. monocytogenes as the patient. IJp to eight specimens were
collected from each household contact, suggesting that the carriage rate in this study may
not be directly comparable to others. In the United States, 82 household contacts of 28
patients with invasive listeriosis were identified through active surveillance and investi-
gated [88]. Twenty-one percent of these individuals (and households) were positive for
L. monocytogenes; 88% of 17 isolates were of the same serotype and enzyme type as the
strain from the index patient. The rate of carriage was significantly higher among persons
less than 30 years of age than among older persons. The prevalence rate among 60 house-
hold contacts of 18 pregnant women with listeriosis in Los Angeles was 8.3%, whereas
no Listeria were isolated from 30 household contacts of age-, sex-, and hospital-matched
controls [62].

Invasive Disease in Nonpregnant Adults

Various clinical conditions are reportedly associated with listeriosis in nonpregnant per-
sons; these include malignancy, organ transplants, immunosuppressive therapy, infection
with the human immunodeficiency virus (HIV), and advanced age [4,35,77,91].
The first reported correlation between listeriosis and cancer appeared in 1967, and
it emphasized the severity of manifestations, high fatality rate, and association with lym-
phoreticular malignancies [581. Subsequent reports quickly confirmed this observation
[ 16,951. In a review of 148 adult listeriosis cases reported from 1968 to 1978, malignancy
was the underlying condition in 25% (25 of 102) of cases of meningitis and 33% (15 of
46) of cases of primary bacteremia caused by L. monocytogenes [73]. Similarly, in 261
human listeriosis cases among nonpregnant adults and juveniles reported in Britain from
1967 to 1985, 105 (40%) had a malignant condition. Of these 105 patients, 32% had
leukemia, 29% had lymphoma, and 39% had other types of malignancies [65].
Elderly patients and those who are receiving immunosuppressive therapy may also
account for a substantial proportion of listeriosis cases [65,71,73,96]. For example, in a
series of 84 cases reported in 1994 from Australia, 39% of the 59 cases among nonpregnant
adults were over the age of 60, 46% were on at least 10 mg of prednisone daily, 29%
were on azathioprine or cyclosporin, and 20% had undergone chemotherapy for malig-
nancy [76]. Other disorders accounting for a lower proportion of cases in nonpregnant
adults include alcoholism, diabetes, cirrhosis, chronic renal disease, collagen vascular dis-
eases, sarcoidosis, ulcerative colitis, aplastic anemia, and conditions associated with iron
overload [34,4 1,53,67,72,73,913.
In a study of sporadic listeriosis conducted from 1988 to 1990 in diverse geographi-
cal populations in the United States, 98% of 98 cases that were not pregnancy-associated
80 Slutsker and Schuchat

occurred in persons with at least one underlying illness, although some were conditions
such as heart disease that are not traditionally considered immunosuppressive [89]. The
most frequently identified diseases or conditions were heart disease (33%), corticosteroid
therapy (3 1%), cancer (29%), renal disease (24%), and diabetes (24%); several patients
had more than one underlying condition. Malignancy, corticosteroid use, and HIV infec-
tion or AIDS were the most common immunosuppressive conditions, and at least one of
these three conditions was present in 69% of nonpregnant adult patients. In an earlier
report, 30% of patients with meningitis and 11 % of those with bacteremia caused by L.
rnonocytogenes had no recognized predisposing condition [72]. In this latter study, how-
ever, cases were largely identified through a literature review, and thus may not be compa-
rable to those identified through population-based surveillance.
There have been many reports of patients with HIV infection or AIDS who coun-
tracted listerial meningitis or bacteremia [ 12,22,24,42,611. Although listeriosis does not
appear to be a common opportunistic infection among persons with HIV infection or
AIDS, it nonetheless occurs far more frequently among these persons than among the
general population. In 1989, in a prospective population-based 2-year study in San Fran-
cisco, the incidence of listeriosis among AIDS patients was estimated to be 280 times the
baseline incidence of listeriosis in the general population [89]. Using similar methodology,
a prospective, population-based 2-year study in metropolitan Atlanta estimated that the
annual incidence of listeriosis was 52 and 115 cases per 100,000 patients for those with
HIV infection and AIDS, respectively; these rates were 62 and 145 times the rate among
nonpregnant adults not known to be infected with HIV [51]. A 1995 prospective study
in Los Angeles estimated the annual incidence to be 9 and 96 cases of listeriosis per
100,000 patients in persons with HIV infection and AIDS, respectively, compared with
a rate of 1 per 100,000 in the total population [25]. Differences in risk estimates between
these last two studies likely resulted from use of different methods to estimate the total
numbers of HIV-infected persons in the study areas.
Nonpregnant adults with listeriosis most frequently present with sepsis, meningitis,
or meningoencephalitis. Although meningitis is usually reported to be the most common
form of listeriosis in adults, recent reports have documented bacteremia to be even more
common. In the United States, active surveillance in an aggregate population of 34 million
persons in 1986 found that 66% of 179 nonpregnant adults had bacteremia without menin-
gitis, 19% had meningitis with concurrent bacteremia, and 12% had meningitis without
documented bacteremia [35]. Presenting symptoms in nonpregnant adults with central
nervous system listeriosis may include fever, malaise, ataxia, seizures, and altered mental
status. Listerial brain stem encephalitis (rhombencephalitis) occurs infrequently and is
characterized by asymmetrical cranial nerve deficits, cerebellar signs, and hemiparesis or
hemisensory deficits [5,97,101]. Fever is generally present in patients with bacteremia;
other nonspecific symptoms such as malaise, fatigue, and abdominal pain may also occur.
In meningitis caused by L. monocytogenes, the cerebrospinal fluid may exhibit a pleo-
cytosis; the Gram stain may show gram-positive bacilli but is often unrevealing. Because
the spinal fluid white cell count and differential, glucose, and protein levels can vary
widely, the spinal fluid profile cannot be used to differentiate listerial meningitis from
meningitis caused by other bacteria.
In addition to sepsis, meningitis, and meningoencephalitis, a variety of other clinical
manifestations of infection with L. rnonocytogenes have been described. Endocarditis from
L. monocytogenes occurs primarily in patients with an underlying cardiac lesion, including
prosthetic or porcine valves, and is clinically indistinguishable from other causes of endo-
Listeriosis in Humans 81

carditis [8,33]. Focal infections are rare and usually result from seeding during a preceding
bacteremic phase. Several different sites of involvement have been reported, including
endophthalmitis [6], septic arthritis [70], osteomyelitis [45], pleural infection [63], and
peritonitis [72]. Cutaneous infections without bacteremia have been reported in persons
handling infected animals [7S] and in accidentally exposed laboratory workers [2].

Listeriosis During Pregnancy

Listeriosis may occur at anytime during pregnancy but is most frequently documented
during the third trimester [ 141. Because bacterial cultures are not routinely obtained from
spontaneously aborted fetuses or stillborn neonates, it is diflicult to estimate accurately
the proportion of fetal loss that may be attributable to infection caused by L. monocyto-
genes during pregnancy. In one study, L. monocytogenes was cultured from placental and
fetal samples in 1.6% of spontaneously aborted pregnancies [36]; other estimates have
varied [3]. Women pregnant with multiple gestations may be at increased risk for listeriosis
compared with singleton pregnancies. In Los Angeles from 1985 through 1992, rates of
listeriosis were approximately four times higher among women with multiple rather than
singleton pregnancies (601.
Symptoms associated with listeriosis during pregnancy may be nonspecific, and they
often manifest as only a mild flu-like illness. In a case-control study of sporadic listeriosis,
women who delivered infants with listeriosis were significantly more likely than controls
to report histories of fever, headache, myalgia, or gastrointestinal symptoms, However,
these women were no more likely than controls to report backache or sore throat [87].
Approximately two thirds of pregnant women with listeriosis during pregnancy have this
flu-like prodromal syndrome [ 14,651. These symptoms are associated with the bacteremic
phase of infection and represent the optimal time to obtain diagnostic blood cultures.
Infection of the fetus with L. monocytogenes is thought to result from transplacental
transmission following maternal bacteremia, although some infections could also occur
through ascending spread from vaginal colonization. Intrauterine infection may result in
preterm labor, amnionitis, spontaneous abortion, stillbirth, or early-onset neonatal infec-
tion. Case reports suggest that fetal- or early-onset neonatal infection does not always
follow maternal listeriosis for which treatment has been delayed or not given [30,46,56].
Neonatal infection can be prevented by antibiotic treatment during pregnancy. It is not
recommended that women with a history of pregnancy-associated listeriosis undergo rou-
tine microbiological screening or antimicrobial prophylaxis during subsequent pregnan-
cies. However, dietary counseling should be given on avoiding high-risk foods. Given the
potential adverse consequences of maternal listeriosis and the availability of effective
treatment, it is prudent to evaluate all febrile episodes during pregnancy with blood cul-
tures.

Neonatal Disease: Early Onset

In contrast to the mild clinical illness seen in maternal listeriosis, neonatal infection caused
by L. monocytogenes is a serious and often fatal disease. Neonatal listeriosis is divided
into two clinical forms-early-onset and late-onset listeriosis. These clinical forms parallel
the pattern of the disease seen in neonates with infection from group B streptococci and
as such suggest different modes of transmission for the two forms.
Early-onset neonatal listeriosis occurs in infants infected in utero, and it results in
illness at birth or shortly thereafter, usually defined as occurring within the first week of
82 Slutsker and Schuchat

life. Between 45 and 70% of neonatal listeriosis is of early onset [31,64]; this disease
often presents with sepsis rather than meningitis [35]. Less frequently, infants with early-
onset disease may present with granulomatosis infantiseptica, a syndrome characterized
by disseminated abscesses or granulomas in multiple internal organs, including the liver,
spleen, lungs, kidney, and brain [39]. In this syndrome, evidence of amnionitis or meco-
nium-stained fluid may be present, and the infant may appear obviously ill; in some in-
stances, however, the infant may merely appear weak and may develop respiratory or
circulatory insufficiency. Early-onset disease in the neonate may be complicated by aspira-
tion of meconium fluid with resultant respiratory complications, including cyanosis, apnea,
and pneumonia.

Neonatal Disease: Late Onset

In contrast to early-onset disease, the late-onset type may occur from one to several weeks
after birth [35,102]. Infants are usually born healthy and full term to mothers who have
had uncomplicated pregnancies. Similar to late-onset neonatal disease caused by infection
with group B streptococci, listeriosis in these neonates presents as meningitis more fre-
quently than in early-onset disease. In a review of neonatal listeriosis cases from 1967 to
1985 in Britain, 39 of 42 (93%) infants with late-onset disease had meningitis [64]; simi-
larly, active surveillance for listeriosis in the United States in 1986 documented meningitis
as the presenting syndrome in 88% of late-onset cases [35]. Mortality rates for both early-
and late-onset disease are usually 20-30% [ 15,57,64].
Although transplacental transmission of L. monocytogenes is the presumed source
of infection in early-onset disease, the route of infection in late-onset neonatal disease is
not well understood. Acquisition of infection during passage through the birth canal is
likely, although cases of late-onset disease have been reported following cesarean delivery.
Clusters of late-onset disease have been identified in newborn nurseries, suggesting that
some nosocomial transmission also occurs [48,55,69,94]. In one outbreak of late-onset
disease in Costa Rica, infection was linked to contaminated mineral oil used to bathe
newborns [90].

Noninvasive Disease: Gastrointestinal Illness

It has been postulated that a noninvasive gastrointestinal illness may occur in normal hosts
that consume foods contaminated with an infectious dose of L. monocytogenes, but this
has been difficult to establish. Because the organism is commonly found in many foods,
in an outbreak setting, recovery of L. monocytogenes from implicated foods is seldom
sufficient to verify the source of infection. Moreover, stool specimens from persons with
diarrhea are rarely cultured for L. monocytogenes. When such cultures are obtained, the
frequency of positive cultures among ill persons must be compared with the proportion
of positive stool cultures from well controls, because a substantial minority of persons
are asymptomatic fecal carriers of L. monocytogenes.
Information from several outbreak investigations suggests that L. monocytogenes
may cause a febrile gastroenteritis in normal hosts [86]. The most persuasive evidence
comes from a recent investigation of an outbreak of gastroenteritis and fever linked to
consumption of chocolate milk served at a picnic [23]. Picnic attendees drank chocolate
milk that was later found to be heavily contaminated with L. monocytogenes (109cfu/
mL); 79% of 58 persons who consumed implicated milk from the picnic reported diarrhea,
and 72% reported fever. The median incubation period was 20 h (range 9-32 h). The
Listeriosis in Humans 83

same subtype of L. monocytogenes was isolated from the stools of ill persons and the
chocolate milk. I11 persons were more likely than well persons to have elevated antilisterio-
lysin 0 levels and to have stool cultures that yielded L. monocytogenes. None of the
individuals involved in this outbreak had a chronic illness or immunodeficiency. Of partic-
ular note, three cases of sporadic invasive listeriosis in two other states were also linked
to consumption of the implicated chocolate milk; the same subtype of L. monocytogenes
as the outbreak strain was isolated from these patients.
Other outbreak investigations also support the concept of febrile gastroenteritis being
caused by L. rnonocytogenes. In an outbreak of invasive listeriosis in Philadelphia in 1986
and 1987, case-patients were significantly more likely than controls to have reported fever,
vomiting, or diarrhea in the week before the cases positive culture [93]. In another out-
break investigation, two pregnant women who attended a catered party each delivered
infants infected with the same strain of L. monocytogenes [80]. Diarrhea or fever was
reported by 22% of the 36 party attendees. However, stool cultures were not obtained
until several weeks after the party, and the outbreak strain of L. monocytogenes was iso-
lated from only one other party attendee, so a correlation between L. monocytogenes stool
carriage and gastrointestinal symptoms could not be definitively established. Finally, in
an outbreak of gastroenteritis among immunocompetent adults attending a supper party
in Italy, diarrhea and fever occurred in over 70% of the ill party-goers, and two developed
bacteremia caused by L. rnonocytogenes [84]. The median incubation period from time
of the supper to onset of gastrointestinal symptoms was 18 hours. Although the same
strain of L. rnonocytogenes that was isolated from the patients was also cultured from
several foods leftover from the supper, no stool specimens collected from ill persons
yielded L. rnonocytogenes.
The frequency of febrile gastroenteritis caused by L. monocytogenes remains unde-
termined, as does the infectious dose and characteristics ofthe host that are associated
with this syndrome. Clinicians and public health officials should consider examining stool
cultures for L. rnonocytogenes in outbreaks of illness characterized by fever, diarrhea,
headaches, and myalgia, if stool cultures for other more common enteric pathogens have
been negative. When such an outbreak is suspected, care should be taken to notify the
laboratory that L. monocytogenes is suspected, so that appropriate special culture media
are used.

EPIDEMIOLOGICAL PATTERNS OF LlSTERlOSlS

Our understanding of listeriosis as a foodborne disease and risk factors for illness caused
by infection with L. monocytogenes have increased greatly over the last 15 years through
epidemiological studies of both epidemic (Table 3) and sporadic illness.

Epidemic Listeriosis
The first convincing evidence that listeriosis can be a foodborne disease comes from a
1981 outbreak in Nova Scotia [85]. Thirty-four pregnancy-associated cases and seven
cases in nonpregnant adults occurred over a 6-month period in the Maritime Provinces.
Twenty-seven percent of the infants who were born alive died. No patient had evidence
of underlying immunosuppression. Case-patients were significantly more likely than con-
trols to have consumed locally produced coleslaw in the 3 months before illness onset.
The epidemic strain was subsequently isolated from coleslaw in the refrigerator of one
84 Slutsker and Schuchat

TABLE
3 Foodborne Outbreaks of Invasive Listeriosis
No. of Implicated % of
cases (or likely) perinatal L. monoctogenes
Reference Year Place (deaths) vehicle cases se rotype

44 I979 Massachu- 20 (5) (Raw vegetables) 0 4b

setts, US
85 1981 Nova Scotia, 41 (18) Coleslaw 83 4b
Canada
27 I983 Massachu- 49( 14) Pasteurized milk 14 4b
setts, US
56 1985 California, US 142 (48) Mexican-style 66 4b
cheese
10 1983- I987 Switzerland 122 (34) Soft cheese 53 4b
66 1988- 1989 United - Piit6 - 4b
Kingdom
80 1989 Connecticut, 10 (0) (Shrimp) 33 4b
us
38 1992 France 279 (85) Pork tongue in 0 4b
jelly
84 1993 Italy 18 (0) Rice salad 0 1l2b
23 1994 Illinois, US 48" (0) Pasteurized choc- 0 1 l2b
olate milk
"Includes 45 cases of diarrhea and 3 cases of invasive disease.

patient, and later from two unopened packages of the product. On review of the production
process, it was determined that cabbage used in the coleslaw came from a farm where
cases of listeriosis in sheep had occurred, and that the cabbage fields had been fertilized
with raw sheep manure. Harvested cabbage was stored over the winter and spring in an
unheated shed, potentially enhancing growth of L. monocytogenes. As well as establishing
listeriosis as a foodborne disease, this outbreak also highlighted the potential for uncooked
vegetables to be a source of infection.
Another outbreak of listeriosis, in Massachusetts in 1979, may also have involved
raw produce (441. Twenty patients with serotype 4b infection were hospitalized during a
2-month period during 1979; only nine cases had been detected in the previous 26 months.
Ten of the patients were immunosuppressed adults and five died. Fifteen patients were
thought to have acquired their infection in the hospital. Patients were more likely than
controls to have consumed tuna fish, chicken salad, or cheese, but no one brand was
implicated. It was postulated that raw celery and lettuce, served as a garnish with these
foods, may have been the vehicle of infection. Although a definitive source of infection
was not identified in this outbreak, information suggested that gastrointestinal tract condi-
tions might be important in acquiring infection. Case-patients were significantly more
likely than controls to have taken cimetidine or antacids, raising the possibility that, like
salmonellosis, decreased gastric acidity might increase the chance that L. monocytogenes
could survive passage through the stomach.
Pasteurized milk was identified as the most likely vehicle of infection in another
large outbreak of listeriosis in Massachusetts in 1983 [27]. Forty-nine cases occurred over
a 2-month period, 42 in immunosuppressed adults and 7 in pregnant women; the overall
Listeriosis in Humans 85

case-fatality rate was 29%. Case-patients were more likely than neighborhood-matched
controls to have consumed a specific brand of 2% fat pasteurized milk; other evidence
supporting this milk as the vehicle of infection included a dose-response effect, a protective
effect of drinking low-fat milk (1 % or skim), an association between the implicated brand
of milk and cases of listeriosis in another state, and the linking of a specific phage type
of L. monocytogenes with infection in the 2% milk drinkers. The 2% milk came from
farms where cows were known to have had listeriosis, and multiple serotypes (but not
the epidemic strain) of L. monocytogenes were isolated from raw milk at the implicated
dairy. No defects in the pasteurization process were noted at the dairy. Although this
outbreak initially raised the question of whether pasteurization was adequate to eliminate
L. monocytogenes from milk, subsequent investigations have shown that L. monocytogenes
is inactivated by proper pasteurization [ 181. In this outbreak, contamination likely occurred
during post-pasteurization handling.
The largest North American outbreak of listeriosis occurred in Los Angeles County,
California, in 1985; 142 cases were detected over an 8-month period (561. Pregnant women
accounted for 93 cases, and nonpregnant adults 49 cases; 48 of the nonpregnant adult
cases had predisposing conditions for listeriosis. Among the pregnancy-associated cases,
87% occurred in Hispanic women. The case-fatality rate was 32% among the perinatal
cases (all were fetal or neonatal deaths) and 37% in the nonpregnant adults. A case-control
study implicated a particular brand of Mexican-style soft cheese produced locally in Cali-
fornia as the vehicle, and the epidemic serotypes and phage type were isolated from un-
opened packages of this product. Inadequate pasteurization and mixing of pasteurized and
unpasteurized milk both likely contributed to the contamination.
This outbreak provided valuable data on the incubation period of invasive listeriosis,
since food histories were available for four patients who had a single known exposure to
the implicated cheese. The median incubation period in these patients of 31 days (range
11-70 days) is far longer than that observed for most common foodborne pathogens, and
it highlights the difficulty in obtaining a relevant food history when investigating cases
of sporadic listeriosis.
This outbreak was detected quickly through public health surveillance, because
many infections occurred in an ethnic minority group that sought care primarily at one
medical facility. However, it is likely that the outbreak would not have been as readily
detected had the product been distributed over a larger geographical area or eaten by
a more diverse group of consumers. Detection of outbreaks can be improved by active
surveillance and timely reporting and by serotyping and subtyping L. monocytogenes iso-
lates.
Another soft cheese-related outbreak occurred in Switzerland during 1983 to 1987
with 122 cases of listeriosis affecting 65 pregnant women (and their infants) and 57 non-
pregnant adults [10]. Over one-half of the nonpregnant adult cases had no underlying
predisposirig condition; the case-fatality rate for nonpregnant patients was 32% [ 171. In-
creasing age and clinical presentation with meningoencephalitis were independently asso-
ciated with an increased risk of death. Neurological sequelae were present in 30% of
survivors at follow-up 5 months to 3 years later. Although early case-control studies failed
to incriminate a particular food, in 1987 investigators implicated a locally produced soft
cheese as the vehicle of infection. Two epidemic strains of L. monocytogenes serotype
4b with a particular phage type were isolated from the product and led to an international
recall.
Two recent outbreaks in Europe were associated with ready-to-eat meats. In En-
86 Slutsker and Schuchat

gland, Wales, and Northern Ireland, the annual total of listeriosis cases approximately
doubled from 1987 to 1989 compared with the annual totals for the 3 previous years. Of
the 823 isolates reported during 1987-1989,30-54% were of two subtypes of L. monocy-
togenes serovar 4b (4bX and 4b phage type 6,7) that had occurred less commonly before
and after this period [66]. A microbiological survey showed that L. monocytogenes con-
tamination in meat pit6 from one manufacturer was more frequent (48% of 107 samples
of pit6 from the implicated manufacturer vs 4% of 781 samples of pit6 from other manu-
facturers) and heavy (1 1% of samples of pit6 from manufacturer A with 21000
organismdg vs 0.6% of samples of pit6 from other manufacturers with 2 1000 organisms/
g). Ninety-six percent of pgt6 isolates from manufacturer A were 4bX or 4b phage type
6,7 compared with 19% of isolates from other manufacturers. Patients infected with either
of these two subtypes were significantly more likely to have eaten pit6 than patients in-
fected with other strains. Warning about pit6 consumption and removal of manufacturer
As pit6 from sale in late 1989 resulted in a dramatic decrease in the incidence of listeri-
osis. This investigation illustrated how subtyping can help clarify surveillance data and
ultimately lead to public health action.
In 1992, a different ready-to-eat meat product was implicated in an outbreak of 279
cases of listeriosis in France [38]. Ninety-two cases (33%) were pregnancy related and
187 occurred in nonpregnant adults; 73 (39%) of the nonpregnant adults had no known
predisposing condition for listeriosis. A case-control study implicated one brand of pork
tongue in jelly as the major vehicle in the outbreak; however, other ready-to-eat meats,
cross contaminated by the implicated meat at retail stores where the implicated brand of
pork tongue in jelly was sold, were thought to have been responsible for some infections.
The epidemic strain was isolated from samples of food, and subtyping analysis helped to
confirm the findings of the epidemiological investigation that implicated the pork tongue
in jelly [47].
Heightened surveillance efforts in France have also led to detection of two smaller
outbreaks of listeriosis. In 1993, an outbreak of 39 cases was associated with rilletes (pork
pit&)[37]. In 1995, 20 cases were traced to Brie de Meaux cheese made from raw milk.
Implicated cheese was removed from sale based on results of the epidemiological investi-
gation [37].

Sporadic Disease-Incidence
Although much has been learned about epidemic listeriosis, most cases of human listeriosis
occur sporadically. In the United States, voluntary disease reporting and hospital discharge
data have been used to estimate the number of sporadic listeriosis cases [21]. However,
such methods are generally insensitive and result in underestimates of the true incidence
of listeriosis in the population.
Beginning in 1986, active surveillance for listeriosis in the United States has been
done by the Centers for Disease Control and Prevention (CDC) in several well-defined
populations representing different geographical areas. Surveillance officers systematically
contacted infection control practitioners at all acute-care hospitals and clinical microbiol-
ogy laboratories in the study areas to collect information on all patients from whom L.
monocytogenes was isolated from a normally sterile site [35,89]. The study population
ranged from 19 to 34 million depending on the number of study sites participating each
year [99].
In 1986, in an aggregate population of 34 million persons, the annual incidence of
Listeriosis in Humans 87

listeriosis was 7 cases per million; of the 246 cases that occurred, 67 (27%) were perinatal
and 179 (73%) were nonperinatal [35]. Perinatal listeriosis rates were highest in Los
Angeles (24.3 per 100,000 live births), whereas nonperinatal rates did not vary signifi-
cantly among the study sites. The estimated sensitivity of the surveillance system was
93%.
From 1988 to 1990, in an aggregate population of 19 million, the annualized inci-
dence of listeriosis was 7.4 per million. Incidence rates varied by geographical site, with
the highest rate being observed in San Francisco (9.3 per million) and the lowest rate in
Oklahoma (4.8 per million) [89]. No clear seasonal trends were noted.
In the most recent analysis of combined data from 1989 to 1993 for a study popula-
tion of 19 million, the annualized incidence of listeriosis decreased from 7.9 per million
in 1989 to 4.4 per million in 1993; this decrease was distributed uniformly throughout the
different geographical areas [99]. Based on these data, projected estimates of the number of
cases and deaths from listeriosis in the entire United States population were 1965 cases
and 489 deaths in 1989, decreasing to 1092 cases and 248 deaths in 1993. Case-fatality
rates (23-25%) did not vary significantly by year. Pregnancy-associated cases accounted
for about one third of all cases each year. Both perinatal and nonperinatal case rates
showed similar decreases over the 4-year period. Serotypes 4b (43%), 1/2b (35%), and
1/2a (20%) accounted for almost all infections. The observed decrease in incidence may
have resulted from enhanced listeriosis prevention efforts by the US food industry, includ-
ing enforcement by regulatory agencies of a zero-tolerance policy for processed meat, and
intensified clean-up programs in meat-processing facilities. Published dietary recommen-
dations for consumers may also have contributed to the decreased disease incidence
[20,28,29].
Numerous reports of listeriosis incidence rates in other cities and countries have
been published. For example, based on a search of hospital records, the estimated incidence
of listeriosis in the English city of Bristol from 1983 through 1992 was 3.5 per million
[50]. In England, Wales, and Northern Ireland in 1991, the estimated annual incidence
based on passive case reporting was 1.8 per million [71]. In Denmark, monitoring of
laboratory-diagnosed cases during the 1980s resulted in an estimate of six to seven cases
per million per year [49]. Listeriosis incidence estimates from a number of countries have
been recently summarized [81]. These rates should be interpreted in the context of the
methods used for case ascertainment and reporting in each country.

Sporadic Disease-Dietary Risk Factors

Dietary risk factors for sporadic listeriosis were assessed through case-control studies
conducted in the CDC active surveillance project. Patients identified through surveillance
were enrolled and matched by age, underlying disease, and healthcare provider (as an
indirect measure of geographical location and socioeconomic status). In the 1986- 1987
study, 82 patients and 239 controls were enrolled. Case-patients were significantly more
likely than controls to have eaten uncooked or nonreheated hot dogs (frankfurters) or
undercooked chicken. An estimated 20% of the overall risk of listeriosis was thought to
be attributable to consumption of these foods [92].
The first human case of listeriosis that was microbiologically linked to consumption
of ready-to-eat poultry products occurred in 1989. A strain of L. monocytogenes serotype
1/2a with an identical isoenzyme type was isolated from the blood of a patient with cancer,
an open package of turkey frankfurters and other opened foods in the patients refrigerator,
88 Slutsker and Schuchat

and unopened packages of turkey frankfurters at a retail store [ 191. Subsequent investiga-
tion of the turkey frankfurter production facility found that cultures from a conveyor belt
transporting finished frankfurters yielded the case strain of L. monocytogenes [ 1071. Sys-
tematic culturing at various points in the production process identified likely points where
L. monocytogenes was being introduced into the product and suggested appropriate control
points for reducing contamination in such food processing facilities.
From 1988 to 1990, a larger case-control study of 165 patients and 376 controls
was conducted that included microbiological assessment of foods eaten by patients [89].
Case-patients were significantly more likely than controls to have eaten soft cheeses or
delicatessen counter foods. In a separate analysis examining dietary risks among a subset
of patients defined as highly immunosuppressed (persons with malignancy, AIDS, or organ
transplants or who had received corticosteroids or chemotherapy), consumption of un-
dercooked chicken was associated with a threefold increased risk of listeriosis. Other expo-
sures associated with an increased risk of sporadic disease included recent use of antacids,
laxatives, or H2-blocking agents.
In the microbiological component of this study, foods were collected from the refrig-
erators of 123 patients [78]. L. monocytogenes was isolated from at least one food in the
refrigerators of 64% of patients. Highest contamination rates among the 20 13 food speci-
mens were seen in beef (36%) and poultry (31%) with 7.6% of ready-to-eat foods (pro-
cessed meats, raw vegetables, leftovers, and cheeses) also yielding L. monocytogenes.
One-third of refrigerators contained food isolates of L. monocytogenes that were the same
enzyme type as those isolated from the patient. In multivariate analysis, foods that were
ready-to-eat, foods that contained high numbers of L. monocytogenes, and foods that
yielded serovar 4b were associated with disease.
Dietary risk factors for sporadic listeriosis were also examined in a recent study in
Denmark; drinking unpasteurized milk or eating pgt6 were the only risk factors identified
[49]. However, one-third of cases reported during the study period could not be included
in the risk analysis for sporadic disease, because the ill persons were infected with an
outbreak strain epidemiologically linked to Danish blue-mold cheeses.

Sporadic Disease-Possible Other Sources

Transmission by routes other than food may play a role in a few cases of sporadic listeri-
osis. Sexual transmission of L. monocytogenes has been hypothesized as a possible route
in perinatal listeriosis; however, there is no evidence to support this [79]. Since L. monocy-
togenes can cause asymptomatic bacteremia and survives refrigeration, it is theoretically
possible that transmission through donated blood could occur. Such transmission has been
documented for Yersinia enterocolitica but has not yet been described for L. monocyto-
genes [ 1001.

Diagnosis of listeriosis depends on isolation of L. monocytogenes from a normally sterile

site such as blood or cerebrospinal fluid. Since the organism may be mistaken for a diph-
theroid contaminant on Gram stain, complete bacteriological evaluation should be done.
Recovery of the organism from stool samples is usually not helpful, since asymptomatic
carriage occurs. L. monocytogenes strains isolated from sterile-site specimens usually grow
well in routinely used media. The specimen is directly plated on tryptic soy agar containing
Listeriosis in Humans 89

5% sheep, horse, or rabbit blood. The organism is usually identified within 36 h. Isolation
of the organism from other sources such as stool specimens that contain large numbers
of competing microorganisms is more difficult; these specimens should be selectively
enriched for Listeria spp. before being plated, on Listeria-selective media. Identification
of L. monocytogenes by use of fluorescent antibody methods or approaches that use DNA
probes coupled with PCR technology may prove useful for some specimens. Experimental
assays for antibody to listeriolysin 0 have been useful in some epidemiological investiga-
tions [23] and have been used to support the diagnosis in culture-negative listeriosis of
the central nervous system [32].

TREATMENT
Controlled trials to determine the optimal antibiotic therapy for listeriosis have not been
done. Bacteriostatic drugs such as chloramphenicol or tetracycline have been associated
with high treatment failure rates, and they are not recommended [98]. Generally, ampicillin
or penicillin has been recommended as the drug of choice. However, relapses have been
reported in immunosuppressed patients after 2 weeks of penicillin therapy [ 1031. The
ability of L. monocytogenes to grow and survive within cells probably explains the poor
response to bacteriostatic drugs and the slow response to penicillin [98]. Intracellular con-
centrations of penicillin may be insufficient for complete eradication. Since many immuno-
suppressed patients have a decreased ability to clear infected cells, antibiotic treatment
for 3 to 6 weeks may be prudent [4]. Optimal length of therapy for other groups of patients
has not been established. A prudent treatment course may be 2 weeks for listeriosis in
pregnancy; 2-3 weeks for neonatal listeriosis; 2-4 weeks for nonimmunosuppressed
adults with meningitis and bacteremia; and longer for complicated infections such as endo-
carditis.
Although experimental evidence suggests that aminoglycosides are synergistic with
ampicillin or penicillin in vitro, they penetrate cells poorly and may be ineffective in
the living host. L. monocytogenes continues to grow in cells despite high extracelluar
concentrations of aminoglycosides [43].
Trimethoprim-sulfamethoxazolereadily enters cells and kills L. monocytogenes, and
it may be the most effective treatment. This drug combination has proved effective in
patients with listeriosis who have hypersensitivity to penicillin [ 5 ] .

Since L. monocytogenes is commonly found in the environment, avoiding exposure pre-

sents a difficult challenge. Dietary and food preparation measures have been recommended
to the general public; these should decrease the risk not only of listeriosis but also of
other common foodborne diseases, such as salmonellosis and campylobacteriosis. These
measures include thorough cooking of raw food from animal sources; washing raw vegeta-
bles thoroughly before eating; keeping uncooked meats separate from vegetables, cooked
foods, and ready-to-eat foods; avoiding raw (unpasteurized) milk or foods made from raw
milk; and washing hands, knives, and cutting boards after handling uncooked foods [20].
For persons who are at increased risk for listeriosis, including those who are pregnant
or immunocompromised, there are specific dietary measures that can be taken to decrease
risk. Such persons should avoid foods epidemiologically linked with listeriosis, including
p2t6 and soft cheeses such as Brie, Camembert, blue-veined, or Mexican-style cheese. In
90 Slutsker and Schuchat

addition, ready-to-eat foods such as frankfurters and leftover foods should be cooked until
steaming hot before being eaten. These persons may also choose to avoid delicatessen
foods or thoroughly reheat cold cuts before eating.
In addition to individual advice for consumers, control of listeriosis requires action
from public health agencies and the food industry. Important control strategies from public
health agencies include developing and maintaining timely and effective disease surveil-
lance programs, promptly investigating clusters of listeriosis cases, and enforcing current
regulations designed to minimize L. monocytogenes in foods that are consumed without
further cooking. The food industry should continue to develop and implement hazard
analysis critical control point programs (HACCP) to minimize the presence of L. monocy-
togenes at important points in the processing, distribution, and marketing of processed
foods [ 11.

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Pathogenesis of
Listeria monocytogen es

MICHAEL
KUHNAND WERNER
GOEBEL
University of Wurzburg, Wurzburg, Germany

INTRODUCTION
Studies which aimed to unravel the pathogenicity of Listeria monocytogenrs and its inter-
action with host cells on the cellular, molecular, and genetic levels were initiated only 10
years ago. The early studies used transposon mutagenesis and infection of primary and
established cell lines (epithelia] cell, fibroblast, and macrophage) to obtain insights into
the interaction of L. monocytogenes with eukaryotic host cells (reviewed in refs. 1 1 1 and
174). Development of new genetic tools now allows manipulation of L. monocytogenes,
which has, together with the cell culture models, greatly broadened our understanding of
the molecular and cell biology of L. monocytogenes infections.
Most studies on the cell biology of L. monocytogenes infections used epithelia-like
and macrophage-like cell lines [55,143,194]. Macrophages actively ingest L. monocyto-
genes, but internalization of the bacterium by normally nonphagocytic cells is triggered
by L. moncicytogenes-specific products. Besides the internalization step, the intracellular
life cycle of listeriae in phagocytes or normally nonphagocytic mammalian cells is, how-
ever, very similar. The pathogen first appears in a vacuole, which is subsequently lysed
by most of the ingested bacteria allowing L. monocytogenes to escape into the cytoplasm.
Whereas listeriae begin to replicate in the cytoplasm, cells remaining in the phagosome
are killed and digested. Concomitant with the onset of intracellular replication, L. monocy-
togenes induces nucleation of host actin filaments which form a cloud around the bacterial
cell. The actin filaments are then rearranged to a polar tail which consists of short actin

97
98 Kuhn and Goebel

FIGURE1 The intracellular life cycle o f Listeria monocytogenes. See text for details.
(Adapted from Refs. 14 and 194 and kindly provided by J. Kreft.)

filaments and other host actin binding proteins which stabilize this structure. Formation
of the actin tail at one pole of the bacterial cell produces the propulsive force which moves
the listeriae through the cytoplasm of the host cell. This bacterial movement requires
continuous de novo actin polymerization. Listeriae which reach the surface of the infected
host cell induce formation of pseudopod-like structures with the bacterium at the tip and
the actin tail behind. These pseudopods are taken up by neighboring cells. The bacteria
thus entering the neighboring cells are within a vacuole that is surrounded by a double
membrane which is subsequently lysed to release the listeriae into the cytoplasm of the
new host cell (Fig. 1).
Most of the known virulence genes whose products are involved in the intracellular
life cycle of L. monocytogenes are clustered on the chromosome in the so-called PrfA-
dependent virulence gene cluster. The cluster comprises six well-characterized genes,
p$A, plcA, hly, mpl, actA, and plcB (Fig. 2 and Table 1) and three small open reading
frames (ORFs) of unknown functions downstream of plcB, called X, Y, and Z. The ends
of the gene cluster are defined by genes coding for housekeeping enzymes. Distal from
prfA, defining the left border of the gene cluster, is located the prs gene encoding a
phosphoribosyl-pyrophosphate synthetase [72,115]. The Zdh gene coding for lactate dehy-

FIGURE2 The virulence gene cluster from L. monocytogenes. Black boxes represent
PrfA-boxes and arrows represent transcripts. (Kindly provided b y F. Engelbrecht.)
Pathogenesis of Listeria monocytogenes 99

TABLE
1 Features of the L. rnonocytogenes Virulence Determinantsa
mRNA Sig. PrfA Temp.
Gene Protein ORF bp (kb) AAh MWcal' MWd PI' Seq.' Reg' Reg .g

P@Ah MA 705 0.912.1 235 27.1 27 7.3 +

plcA PI-PLC 95 1 1.112.1 317 36.3 34 10.1 + +
hly' LLO 1617 1.8 529 58.6 58 + +
mplh MPl 1533 1.615.7 5 10 57.4 67/35 6.6 + +
actA ActA 1917 2.915.7 639 67.0 92 - +
plcB' PC-PLC 867 2.915.7 289 32129 + +
inlA' Internalin 2400 2.915 800 86.0 95 4.4 + +
inlB' InlB 1890 5 630 71.1 65 10.1 + +
inlCh InlC 89 1 1 297 29.6 30 6.7 + +
iap P60 1452 1.5 484 50.7 60 10.0 + -

lmaAh LmaA 5 10 170 18.0 21 4.2 -

lmsodh SOD 606 202 22.6 24

cat' Catalase 1464 488 55.9 67
clpc' ClpC 2478 2.5 14.5 826 91.0 7.8 +
a See text for references.
Amino acids (including signal sequence).
Molecular weight (MW) in kD (including signal sequence) and isoelectric point (PI) as calculated from sequence data.
Molecular weight in kDa as calculated from SDS-PAGE
Presence of signal sequence.
M A regulated expression.
g Temperature-regulated expression.

From strain L. monocytogenes EGD.

I From strain L. monocyfogenes L028.
J From L. seeligeri.
100 Kuhn and Goebel

drogenase together with the orfs A and B [72,196] mark the right border of the gene
cluster downstream from plcB and the small orfs X, Y, and Z. The products of these
virulence genes are: listeriolysin (encoded by hly), a phosphatidylinositol-specific phos-
pholipase C (pZcA),a phosphatidylcholine-specificphospholipase C (pZcB),a metallopro-
tease (mpl), ActA, a protein involved in actin polymerization (actA), and the positive
regulatory factor PrfA (prfA). The internalin genes inlA, inlB, and inlC coding for inter-
nalin, InlB, and InlC, respectively, the iap gene coding for p60, and other genes suggested
to play a role in virulence are located outside the virulence gene cluster. Most of these
are, however, connected to the virulence cluster genes, as they are also regulated by the
transcriptional activater PrfA (see below).

MOLECULAR ASPECTS OF ADHESION AND INVASION

Uptake of L. monocytogenes by macrophages of different origin is well documented
[ 113,127,1521.Invasion by L. monocytogenes of different, normally nonphagocytic mam-
malian cell types including murine and human fibroblasts [47,83,113,152], murine and
human epithelial cells [4,47,55,152], murine hepatocytes [40,20 I], and human endothelial
cells [45,108,175,176] also has been described along with invasion and survival of L.
monocytogenes in protozoa of the genera Acanthamoeba and Tetrahymena [ 1261.

Invasion of Nonprofessional Phagocytic Cells

Proteins Internalin (InlA), InlB, and lnlC
Transposon mutagenesis and an appropriate in vitro invasion assay using Caco-2 epithelial
cells resulted in identification of internalin, a surface protein of L. monocytogenes mediat-
ing bacterial invasion into epithelial cells (541. The mutants identified exhibited a lower
invasive capacity than the wild-type strain when tested on different cells. In addition to
reduced invasiveness, mutants also lost the ability to adhere to eukaryotic cells. Trans-
poson insertions were mapped and occur in a chromosomal region, which represents an
operon consisting of the inlA and inlB genes. Expression of inlA in L. innocua, a noninva-
sive Listeria species closely related to L. monocytogenes, renders this species invasive.
This experiment shows that the inlA gene product is necessary and sufficient to mediate
invasion. Internalin is an acidic protein of 800 amino acids [4 1,541 which possesses two
extended repeat domains. Domain A consists of 15 repeats of 22 amino acids each, whereas
domain B consists of 2.5 repeats of about 70 amino acids each. The InlA protein has a
typical N-terminal transport signal sequence, and in the C-terminal part, it has a cell wall-
spanning region followed by a hydrophobic sequence which represents a putative mem-
brane anchor (Fig. 3) [41]. Internalin was originally shown to be a L. monocytogenes
surface protein [54], but substantial amounts of the protein are also found in the superna-
tant liquid [42]. Recently it was shown that surface location is necessary for internalin to
mediate entry of L. monocytogenes into epithelial cells by facilitating direct contact be-
tween the bacterium and the host cell [ 1 181.
The eukaryotic receptor for internalin was identified as E-cadherin by a biochemical
approach using matrix-bound purified internalin to isolate the internalin ligand from epi-
thelial membrane proteins [ 1361. E-cadherin, a member of the cadherin family, is mainly
expressed at the basolateral site of enterocytes [ 191. It binds internalin directly, and its
location on the basolateral membrane of epithelial cells is in line with previous observa-
Pathogenesis of Listeria monocytogenes 101

29 357 462 650 711

SS 8 SR LR
InlB N C*
I I I I 1 I
30 63 238 399 466 559

SS 8 SR
InlC N C
I I I
34 62 234

FIGURE
3 Schematic structure of the members of the internalin family: InlA, InlB, and
InlC. SS, signal peptide; SR, short leucine-rich repeat; LR, long repeat; MA, membrane
anchor.

tions suggesting the basolateral membrane as an entry site for L. monocytogenes [ 1901.
Antibodies directed against the leucine-rich repeat region of internalin block entry of L.
monocytogenes into cells expressing E-cadherin, thereby uriderlining the importance of
the repeat regions of internalin for its function as an invasin [ 1361.
InlB, a 630-amino acid protein, also carries an N-terminal transport signal sequence
and repeat domains, but, in contrast to InlA, has no obvious membrane anchor and no
cell wall-spanning region (Fig. 3) [41]. Nevertheless, InlB is a listerial surface protein,
but the mechanism(s) which target it to the bacterial surface are unknown [40]. Recently
it was shown that inlB mutants still expressing inlA are invasive for human enterocyte-
like Caco-2 cells. InlB is required but is obviously not sufficient to promote entry of L.
monocytogmes into hepatocytes 1401, but results concerning its role in the entry of epithe-
lial cells are controversial [40,125]. Invasion of fibroblasts by L. monocytogenes seems
to be independent of either inlA or inlB and even double mutants are still invasive for
fibroblasts, suggesting that different cell type-specific adhesion and invasion systems are
present in I,. monocytogenes [41,125]. The high sequence sirnilarity of inlA and inlB indi-
cates that the two genes originate from a common ancestral gene and represent members
of a gene family in L. monocytogenes. One additional member of this gene family, called
inlC, was cloned and characterized and encodes a small (297 amino acids), secreted protein
(Fig. 3) which is mainly expressed at later stages of the intracellular life cycle and obvi-
ously not involved in the entry process into epithelia1 cells [48]. InlC was identified inde-
pendently and called Irp (the gene irpA), and it also was found present in the supernatant
liquid of the closely related species L. ivanovii [39,124].
Protein p60
Rough mutants of L. monocytogenes expressing reduced amounts of a 60-kD extracellular
protein, termed p60, show appreciably reduced uptake by 3T6 fibroblast cells [ 1091. These
p60 mutants (also referred to as R-mutants because of their rough colony appearance)
from long cell chains which possess double septa between the individual cells. Treatment
of L. monocytogenes R-mutants with partially purified p60 protein disassociates these cell
chains into normal-sized single bacteria which are again invasive for fibroblasts. Ultrason-
ication, which leads to physical disruption of the cell chains, produces similar single cells
102 Kuhn and Goebel

which are, however, noninvasive. On treatment with wild-type p60, these ultrasonicated
mutant cells are again able to invade fibroblasts [16,109]. Reduced invasiveness of p60
mutants is only observed with certain mammalian host cells. Cell chains of p60 mutants
adhere normally to Caco-2 epithelial cells and are perfectly invasive on disruption of the
bacterial cell chains by ultrasonication without addition of p60 [ 161. Protein p60 is a major
secreted protein of all L. monocytogenes isolates [16,109], but it is also found on the cell
surface of L. monocytogenes [163]. In contrast to other virulence factors, p60 is also an
essential metabolic enzyme of L. rnonocytogenes, since it possesses murein hydrolase
activity which appears to be involved in a late step of cell division [202]. The gene coding
for this obviously bifunctional protein, called iap (invasion associated protein), was cloned
from a L. monocytogenes gene bank using an anti-p60 antiserum and sequenced [103].
Its expression is controlled on the posttranscriptional level by a yet unknown mechanism
[102]. The amino acid sequence of p60 predicts an extremely basic protein of 484 amino
acids with a 27-amino acid signal sequence and an extended repeat domain consisting
of 19 threonine-asparagine units which are separated by a proline-serine-lysine motif. A
single cysteine found in the C-terminal part of p60 is probably essential for its enzymatic
activity [103,202]. A stretch of 50 amino acids in the N-terminal part of the protein which
is also present in p60 proteins of the other Listeria species shows homology to sequences
found in an autolysin of Streptococcusfaecalis (Enterococcusfaecalis). In this species, the
sequence is thought to represent a possible murein binding site. Interestingly, the sequence
motive occurs twice in the p60 protein of L. monocytogenes [202].
Protein ActA
The listerial surface protein ActA (Fig. 4), a major virulence factor primarily involved in
actin-based motility [38,99] (see below for details), was recently suggested also to play
a role in internalin-independent uptake of L. monocytogenes by epithelial cells [3,108].
Analysis of the invasive capacity of an inZA deletion mutant and mutant PKP-1 without
the virulence gene cluster genes [48] complemented with multiple copies of PrfA strongly
suggest that PrfA-dependent proteins from the virulence gene cluster may cause invasion
of Caco-2 cells in the absence of InlA [ 1081. Such an ActA-promoted attachment and
invasion of Chinese hamster ovary (CHO) epithelia-like cells as well as IC-21 murine
macrophages was mediated by interaction of the listerial surface protein ActA with a
heparan-sulfate proteoglycan receptor [3]. Electrostatic interactions between heparan sul-
fate and positively charged residues in the N-terminal part of ActA could presumably
result in low-stringency binding to cell surface proteoglycan receptors which are widely
distributed in mammalian cells [3]. Whether the proposed low-stringency binding of L.
monocytogenes to heparan sulfate proteoglycan receptors triggers uptake directly or results
in adequate presentation of other bacterial factors to the host cell membrane which ulti-
mately lead to phagocytosis remains to be clarified.

29 128 151 263 390 613 630

FIGURE4 Schematic structure of the bifunctional protein ActA. SS, signal peptide;
AP, region critical for actin polymerization; PRR, proline-rich repeat; TA, transmem-
brane domain.
Pathogenesis of Listeria monocytogenes 103

Uptake by Professional Phagocytic Cells

Macrophages of different origin were used in in vitro studies to analyze mechanisms of
L. rnonocytogenes uptake by macrophages, which are generally assumed to take up L.
rnonocytogenes by conventional phagocytosis involving actin-polymerization. Comple-
ment factors C I q and C3 are deposited on the bacterial surface and stimulate L. monocyto-
genes uptake by binding the bacteria to the respective receptors [2,27,43].
The kinetics of uptake and intracellular killing of L. monocytogenes by macrophages
were determined [30,3I , 1551. Macrophages ingest L. rnonocytogenes very rapidly, and
intracellular killing starts shortly after phagocytosis and leads to destruction of most of
the ingested bacteria. In a single macrophage, both killed bacteria inside acidified phago-
somes and phagolysosomes and growing bacteria that have escaped into the cytoplasm
can be detected. These findings suggest competition between phagosome-lysosome fusion
followed by killing of the listeriae and their escape from the acidified phagosome before
phagosome-lysosome fusion occurs. The result of this competition is a population of cyto-
plasmic listeriae able to grow inside the macrophage. The route of uptake by macrophages
may also be important for the fate of invading bacteria. As demonstrated by Drevets et
al. [44], the mode of uptake is critical for subsequent survival, since L. rnonocytogenes
taken up in the presence of complement C3 leads to enhanced killing of the bacterium.
Whether the surface protein InlA contributes substantially to triggering of phagocy-
tosis of L. monocytogenes by macrophages is still under debate. Using bone marrow-
derived macrophages, InlA had only a slight effect on invasion, since an inlAB mutant still
showed more than 60% invasion when compared with the wild-type strain [85]. Uptake of
L. rnonocytogenes by the mouse macrophage-like cell line J774A.1 was inhibited by at
least 50% by the pretreatment of L. rnonocytogenes with anti-InlA antibodies, and recombi-
nant InlA specifically was bound to the macrophages [ 1661. Recently it was suggested
that the listerial protein p60 might enhance phagocytosis by macrophages. Salmonella
typhimuriurn, expressing and secreting p60, seems to be more invasive for phagocytic
cells but not for enterocytes [85]. In line with this assumption is that pretreatment of
L. rnonocytogenes with a polyclonal anti-p60 antiserum inhibits uptake of listeriae by a
macrophage-like cell line [85]. Another factor that could be involved in attachment and
invasion of 1,. rnonocytogenes in macrophages is the listerial cell wall polymer, lipoteichoic
acid. L. rnonocytogenes binds strongly to the macrophage scavenger receptor most likely
via lipoteichoic acid [46,73]. This interaction may also trigger conventional receptor-
mediated phagocytosis of L. rnonocytogenes.

Tissue Tropism and Mechanism of Invasion

The cellular mechanisms of L. rnonocytogenes invasion are still largely unknown. Uptake
of L. rnonocytogenes by macrophages and other mammalian cells is dependent on func-
tional actin microfilaments, since invasion is blocked by treatment with actin-depolymeriz-
ing drugs such as cytochalasins [55,113]. Additionally, entry can be blocked by tyrosine
kinase inhibitors such as genistein [ 160,189,I971 and the tyrosin phosphatase inhibitor
vanadate [ 1071. For epithelia] cell invasion, the signaling events are probably triggered
by binding of internalin to its receptor, E-cadherin. Links between cadherins and signaling
pathways have recently been reported [ 1301. Identification of E-cadherin as the receptor
for internalin and electron microscopic observations strongly support the basolateral mem-
brane of the epithelia] cell as the site for listerial attachment and entry [ 136,1901.However,
104 Kuhn and Goebel

entry by the apical surface of polarized Caco-2 cells was also observed [91]. Attachment
of wild-type L. monocytogenes to these host cells induces structural modification(s) in
microvilli which are not observed with less invasive p$A mutants of L. monocytogenes
(see below) [66,91]. Whether the apical route of infection of polarized cells is also inter-
nalin-dependent is not known, and the overall significance of each of these two proposed
entry sites of epithelial cells is still under debate.
The picture of listerial invasion is becoming more and more complex, since afore-
mentioned data point to different invasion sites on the same cell as well as to different
mechanisms involved in invasion of different cell types. These observations are in line
with the idea of tissue tropism, with different known and unknown bacterial factors like
internalins and p60 being responsible for invasion of different tissues during infection.
Presently the data can be summarized as follows: Protein p60 is clearly not involved in
epithelial cell invasion, but it may play a role in fibroblast invasion [16,109]. SalrnoneZZa
strains expressing p60 are taken up significantly better as the control strains by macro-
phages and hepatocytes, also indicating a role of p60 for invasion of these cell types [85].
Internalin is an important factor in epithelial cell and hepatocyte infection, but the capacity
of InlA alone to promote epithelial cell entry is still under debate, since conflicting results
have been published [40,54,125]. InlB, originally proposed as a specific factor for hepato-
cyte invasion [40], is now suggested also to be critical for epithelial cell invasion, but
both internalins play no role in fibroblast invasion [ 1251. The eukaryotic receptor for InlB
is not known, but the recent report of InlB-dependent stimulation of phosphoinositide-3-
kinase being required for efficient L. monocytogenes invasion of nonprofessional phago-
cytic cells [87] underlines the importance of InlB in the invasion process. High-efficiency
binding to and invasion of human endothelial cells by L. monocytogenes is dependent on
one or both inZAB gene products. However, low-level invasion in the absence of both
internalins was also observed [45]. Internalin-independent invasion was also reported for
the dentritic cell line CB1 [76] and at low levels also for Caco-2 epithelial cells [57]. The
role of the small internalin family member InlC (Irp), if any, in invasion is unclear [48].
L. monocytogenes can spread from macrophages to endothelial cells by direct transfer of
the bacteria from one cell to the other [45]. Because of its expression in the late stages
of the infectious cycle, InlC was thought to be involved in this type of heterologous cell-
to-cell spreading event [48].
The in vivo significance of these listerial proteins is even less clear. InZAB as well
as iap mutants are clearly impaired in virulence in the mouse model [40,85]. However,
an inlAB mutant was only transiently impaired in persistence in the liver and behaved like
the wild-type in spleens and lymph nodes of infected mice [40]. In a different study,
however, inZAB mutants were only rarely found inside hepatocytes, compared with the
wild-type strain, indicating a role for the inlAB locus in hepatocyte invasion in vivo [58].
In contrast, Gregory et al. [74] found that the inlAB operon of L. monocytogenes is not
required for entry into hepatic cells in vivo.

ESCAPE FROM THE PHAGOCYTIC VACUOLE AND

INTRACELLULAR GROWTH
Hemolytic activity detected around colonies of L. monocytogenes growing on blood-agar
plates was long assumed to represent a major virulence determinant, since all clinical
isolates of L. monocytogenes show this hemolytic phenotype. The hemolytic activity re-
Pathogenesis of Listeria monocytogenes 105

sults from the action of a cytolysin, called listeriolysin 0 (LLO). In experimental infec-
tions, all virulent strains were hemolytic, whereas nonhemolytic strains were avirulent.
Nonhemolytic mutants which were obtained after transposon rnutagenesis using the conju-
gative transposons Tn1545 or Tn916 [56,92,152] always proved to be avirulent in the
mouse model. Virulence is restored in hemolytic revertants, which have lost the transposon
insertion or by introduction of the cloned hly gene into a nonhemolytic L. monocytogenes
transposon mutant [25]. Despite the clear correlation between hemolysis and virulence,
the level of hemolysin production in vitro is not directly proportional to virulence of
producing strains in the mouse [94], suggesting that synthesis of LLO under intracellular
conditions is different from that observed under extracellular growth conditions.
Listeriolysin 0, a secreted protein of 58-60 kD, belongs to a family of pore-forming,
sulfhydryl-activated cytolysins for which streptolysin 0 is the prototype [ 1801. All mem-
bers of this family are inhibited by low concentrations of cholesterol and oxygen and
activated by reducing agents like DTT. Cholesterol is considered as a receptor for these
cytolysins, since this component inhibits pore formation and toxicity. On addition to eryth-
rocytes, toxin monomers oligomerise in the target cell membrane to form stable pores
which can be visualized by electron microscopy [ 1481. Listeriolysin 0 has been purified
to homogeneity and its toxicity, as determined by intraperitoneal injection in the mouse,
shows a LDSoof 1.7 pg per mouse. Optimal hemolytic activity is found at pH 5.5, a pH
value which is much lower than that determined for the other SH-activated cytolysins
[60], a property which is in accord with the function of LLO in the acidified phagosome
(see below).
The gene encoding LLO, hly, was cloned from strains of different serovars of L.
monocytogvnes and sequenced [35,132,137]. The deduced amino acid sequence for LLO
yielded 529 amino acids, including a N-terminal signal sequence of 25 amino acids. As
expected, the sequence shows extended homologies with the protein sequences of other
SH-activated cytolysins. The highest homology is observed in the C-terminal part and
includes a highly conserved undecapeptide containing the unique cysteine which was
thought to be essential for cytolytic activity. Site-directed mutagenesis revealed, however,
that cysteine is not essential for hemolytic activity. In contrast, a tryptophan residue, in
close vicinity to cysteine, appears to be required for both hemolytic activity and virulence
[139].
The role of LLO in virulence was determined by injection, intravenous and intraperi-
toneal, of wild-type and nonhemolytic mutants of L. monocytogenes into mice and follow-
ing the fate of the listeriae in liver and spleen. In contrast to the wild-type strain, nonhemo-
lytic mutants are eliminated from these organs within a few hours without eliciting
protective immunity [25,56,92,152]. The role of LLO in intracellular survival was deter-
mined using different mouse and human cell lines. In the human enterocyte-like cell line
Caco-2 [55], mouse 3T6 fibroblasts [ I 131, and mouse CL.7 fibroblasts [152], nonhemolytic
L. monocytogenes mutants were as invasive as isogenic wild-type strains. The nonhemo-
lytic mutants are, however, incapable of intracellular growth and survival within these
host cells and also in mouse peritonea1 macrophages [ 1 131, mouse bone marrow-derived
macrophages [ 1521, and the mouse macrophage-like cell line 5774 [ 1521. Electron micros-
copy of infected macrophages and epithelia1 cells reveals that nonhemolytic L,
monocytogenes mutants which are found inside cells are unable to open the phagosome
to escape into the cytoplasm of the host cells [55,194]. Bafilomycin treatment inhibits
vacuolar acidification and prevents L. monocytogenes from escaping phagosomes of in-
fected Caco-2 cells. These findings further support the importance of the low pH activity
106 Kuhn and Goebel

optimum of LLO for its role as a vacuole opener [24]. Taken together, these results suggest
that hemolytic activity is indispensable for lysis of the phagosomal membrane. Additional
evidence for LLO being essential for lysis of the phagosomal membrane and for intracellu-
lar growth was obtained by infection of macrophages with a Bacillus subtilis strain ex-
pressing LLO [7]. This engineered strain escapes from the phagosome into the cytoplasm,
whereas the nonhemolytic B. subtilis parental strain stays in the phagosome, as do the
nonhemolytic L. monocytogenes mutants.
Recently, Portnoy and coworkers [ 88,891 analyzed the role of LLO by constructing
L. monocytogenes strains which secrete the closely related extracellular cytolysin perfrin-
golysin instead of LLO. Such a strain escaped from the vacuole but damaged the host
cell. Using an elegant selection procedure, mutants were isolated which did not damage
the host cell on perfringolysin expression in the cytoplasm. The mutated perfringolysins
were either less hemolytic at neutral pH, generally less active, or had a shorter half-life
in the cytoplasm. Thus the low activity of LLO at neutral pH values and its short half-life
in the cytoplasm are critical parameters of its suitability as a phagosome opener without
concomitant cytotoxicity. Strains expressing mutated perfringolysins which allow intracel-
Mar growth without cell damage are, however, totally avirulent. This unexpected finding
points to additional functions for LLO in converting the host cell cytoplasm into a suitable
growth compartment [ 64,891.
Fusion of L. monocytogenes-containing phagosomes with endosomes has been ob-
served in electron microscopy studies [ 1941.However, it is not known whether such an
event is necessary for L. monocytogenes to progress through its intracellular life cycle.
The recent description of rab5-regulated fusion of L. manocytogenes-containing phago-
somes with endosomes and the observation that live L. monocytogenes upregulates this
process by recruiting rab5 to the membrane strongly argues for phagogosome-lysosome
fusion as being an important step in the life cycle of L. monocytogenes [l].
Listeriolysin 0-independent escape of L. monocytogenes from primary vacuoles in
human epithelia1 cells [ 1521 is mediated by the phosphatidylcholine-specific phospholip-
ase C (PC-PLC) and a metalloprotease [ 1 291. The phosphatidylinositol-specific phospholi-
pase C (PI-PLC) contributes to vacuole escape in other cells like bone marrow-derived
macrophages [ 181. Phospholipase activity of L. monocytogenes cultures was first observed
as a zone of opacity surrounding colonies on egg yolk agar [53]. Transposon mutants of
L. monocytogenes lacking phospholipase activity were identified by formation of small
plaques on fibroblast cell monolayers [ 1871 and by reduced hemolysis on blood-agar plates
[93], indicating a participation of phospholipase activity in hemolysis. One transposon
insertion was mapped in an ORF located adjacent to the hly gene on the chromosome of
L. monocytogenes [ 138,1871. The gene, plcA, was cloned 17,119,13I ] and it encoded a
protein of 34 kD which exhibits high homology to several gram-positive phospholipases
and contains a typical transport signal sequence. The enzyme called phosphatidylinositol-
specific phospholipase C (PI-PLC) was purified from culture supernatant liquids of an
overexpressing L. monocytogenes strain [ 681 and was highly specific for phosphatidyl-
inositol with no detectable activity on phosphatidylethanolamine, phosphatidylcholine, or
phosphatidylserine. It also does not cleave phosphatidy linositol-4-phosphate or phosphati-
dylinositol-4,5-bisphosphate,but it is active, albeit with low specific activity, on glyco-
sylated phosphatidylinositol-anchored proteins [59].
Besides the highly specific PI-PLC, L. monocytogenes produces a second phopholip-
ase C which hydrolyzes phosphatidylcholine (lecithin), and it is thus a phosphatidylcho-
Pafhogenesis of Listeria monocytogenes 107

line-specific phospholipase C (PC-PLC) or lecithinase [611, also called broad-spectrum

phospholipase C. A 32-kD protein was detected in the supernatant liquid of a L. monocyto-
genes culture which showed phospholipase activity on egg yolk overlays [93]. The protein
was purified to homogeneity [61,67] and was a zinc-dependent phospholipase C of 29
kD. The pH optimum of this enzyme is between pH 6 and 7, and its activity is stimulated
by 0.5 M NaCl and 0.05 mM ZnSO,. Besides phosphatidylcholine, it also hydrolyzes
phosphatidylethanolamine, phosphatidylserine, and with lower efficiency, sphingomyelin.
Phosphatidylinositol is not a suitable substrate. The purified protein exhibits weak hemo-
lytic activity but is not toxic to mice [61].
The gene plcB, encoding PC-PLC, is part of the lecithinase operon which consists
of mpl, actA, plcB, and the three small ORFs, X, Y, and Z. The gene was cloned and
sequenced [196], and the deduced amino acid sequence yielded a protein of 289 amino
acids with a 25-amino acid N-terminal transport signal and a putative propeptide of 26
amino acids. Maturation of the 32-kD precursor of PC-PLC occurs after secretion, since
both forms of the protein can be found in the supernatant liquid and is obviously accom-
plished by the metalloprotease of L. rnonocytogenes [ 153,1541.
Use of in-frame deletions in the plcA gene enabled clear demonstration that PI-PLC
is required for efficient escape of L. monocytogenes from the phagosome of mouse bone
marrow-derived macrophages. However, the mutation in plcA has only a slight effect on
virulence [ 181. It is assumed that PI-PLC acts in concert with listeriolysin inside the acidi-
fied phagosomal vacuole of the host cell to mediate lysis of the vacuolar membrane. The
broad pH optimum of PI-PLC, ranging from pH 5.5 to 7.0, is consistent with its postulated
function in the acidified phagocytic vacuole of infected cells. However, cooperative mem-
brane permeabilization by PI-PLC and LLO in in vitro assays is independent of phospho-
lipid hydrolysis, since composition of artificial membranes used as targets does not influ-
ence the permeabilization activity of PI-PLC when acting together with LLO [69]. To
further assess the role of PI-PLC, the plcA gene was expressed in L. innocua, which lacks
the prJA-dependent virulence gene cluster and is, therefore. unable to escape from the
host cell vacuole. The PI-PLC-expressing L. innocua strain cannot escape from the phago-
some of 5774 macrophages, but it exhibits limited intracellular growth inside vacuoles
which appear to be structurally intact [ 1691. These data suggest that PI-PLC alone is unable
to open the phagosomal membrane but affects the vacuole in a way which alters its func-
tion but not its structure.
The role of PC-PLC in escape from vacuoles is not clear and differs between cell
types. In bone marrow-derived macrophages, PC-PLC has no role in lysis of the vacuole
[ 1771. However, in the human Henle 407 epithelia-like cell line, where escape of L. mono-
cytogenes occurs at low efficiency independent from LLO [ 129,1521, PC-PLC is required
for lysis of the phagocytic vacuole together with the metalloprotease. PI-PLC is not re-
quired in this system, but the efficiency of escape was reduced in a hly, plcA double mutant
[129].
The way in which the metalloprotease contributes to pathogenicity and intracellular
replication of L. rnonocytogenes is still unclear. Transposon mutants in the rnpl gene are
less virulent but grow normally inside mammalian cell lines [ 1541. The reduced virulence
was attributed to the lack of proteolytic processing of the 32-kD PC-PLC proform
[153,183]. However, mutants within frame deletions in rnpl, also impaired in PC-PLC
maturation and ActA degradation, are as virulent as the isogenic wild-type strain when
injected intraperitoneally in the mouse (D. A. Portnoy, personal communication) [9]. The
108 Kuhn and Goebel

way in which Mpl contributes to lysis of the vacuole in Henle 407 cells is not known,
but the most favorable hypothesis suggests that Mpl is necessary to activate PC-PLC as
shown in broth culture [ 1541.
Located immediately downstream of the hly gene, the mpl gene [36,134] encoding
a zinc-dependent metalloprotease, is the first gene of the lecithinase operon [36,134,196].
The deduced amino acid sequence of this protease shows high homology to several met-
alloproteases from Bacillus species and yields 5 10 amino acids with a typical N-terminal
signal sequence and a putative internal cleavage site. Like other metalloproteases, the
enzyme is activated by proteolytic maturation resulting in a 35-kD mature protein [36,134].
A 60-kD protein is detected with an antiserum raised against Bacillus stearothermophilus
thermolysin which probably represents the proform of the metalloprotease. Only small
amounts of the postulated 35-kD mature form of the protein were detected in the superna-
tant liquid of a L. monocytogenes culture [36].
The role of the virulence gene cluster products LLO, PI-PLC, PC-PLC, and Mpl in
escape from the phagocytic vacuole and in intracellular growth has been analyzed in some
detail during the last decade, as just described. A ClpC ATPase of L. monocytogenes
was recently identified as a new type of virulence factor being involved in intracellular
multiplication. The gene encoding the ClpC ATPase, called clpC, was identified by Tn917
mutagenesis with selection of mutants dependent on iron [161]. The clpC mutants are
highly susceptible to stress from iron limitation, elevated temperatures, and high osmolar-
ity. Virulence of these mutants is severely impaired in the mouse with restricted capacity
to grow in bone marrow-derived macrophages [ 1621. Molecular mechanisms by which
the ClpC ATPase of L. rnonocytogenes protects against stress and promotes intracellular
multiplication are unknown, but obviously the PrfA-dependent virulence machinery
(e.g., the virulence gene cluster products) is not significantly affected in clpC mutants
[162].

INTRACELLULAR MOTILITY AND CELL-TO-CELL SPREAD

Intracellular movement of L. monocytogenes inside the host cell cytoplasm as well as
intercellular spread mediated by actin polymerization were initially described by Mounier
et al. [ 1431 and Tilney and Portnoy [ 1941. Their work was followed by a series of studies
describing the cell biology of the process. It was shown that L. monocytogenes moves
rapidly through the cytoplasm at a speed of up to 1.5 pm/s with help of formed actin
tails. The rate of actin assembly, which occurs at the barbed ends of actin filaments near
the bacterial surface, equals the rate of actin-based motility with actin polymerization
providing the propulsive force for intracellular movement. Such motility also takes place
in cytoplasmic extracts from Xenopus oocytes [28,164,19 1,192,1931. Mutants defective
in intracellular motility were obtained by transposon mutagenesis. These mutants have
either lost the ability to initiate actin polymerization [ 1871 because of an insertion into a
gene, called actA, or are unable to rearrange the polymerized actin filaments into actin
tails [ 1141. The gene actA located downstream from mpl in the lecithinase operon codes
for a proline-rich protein (ActA) of 639 amino acids (Fig. 4). Its apparent molecular weight
determined by SDS-PAGE is 92 kD [38,196]. ActA is a surface protein consisting of three
domains: the N-terminal domain with the transport signal sequence, the central proline-
rich repeat region, and the C-terminal part which includes a membrane anchor [38,99,196].
Mutations in the actA gene resulted in avirulence in mice [38], cessation of intracellular
Pathogenesis of Listeria monocytogenes 109

actin polymerization around bacteria, and lost intracellular motility [38,99]. Inside host
cells, the actA mutant forms microcolonies which are located near the nucleus [38].
To elucidate the role of ActA in actin filament assembly, actA was transfected into
mammalian cells [52,150,1511. Expression of the complete ActA protein (including the
membrane anchor) results in targeting of the protein to mitochondria, which subsequently
recruits actin to these organelles, suggesting that ActA alone is sufficient to polymerize
actin. However, the mitochondria did not move intracellularly [ 150,1511. Expression of
ActA lacking its signal sequence and its membrane anchor resulted in increased amounts
of F-actin in the transfected cells. ActA fused to a plasma membrane anchor which targeted
the fusion protein to the plasma membrane resulted in actin polymerization and formation
of aberrant protuberances on the cell surface [52]. From these assays, it appears that ActA
is sufficient to induce actin assembly.
To prove that ActA is also sufficient to promote intracellular movement, the nonmo-
tile species L. innocua was engineered to express ActA. The recombinant bacterium pro-
duced actin tails and moved in cytoplasmic extracts as did the wild-type L. monocytogenes
strain. In all parameters tested, the recombinant L. innocua strain expressing ActA was
indistinguishable from L. monocytogenes [ 10I].
The ActA protein is distributed asymmetrically on the surface of L. monocytogenes
but is not found within the actin tail [145]. After cell division, it is not present at the new
bacterial pole but is concentrated at the old pole [100,190). Using streptococci coated
asymmetrically with genetically engineered ActA protein, this asymmetrical distribution
of the ActA protein was shown to be required and sufficient to direct actin-based motility
[178]. In a cell-free system, these streptococci, but not uniformly coated ones, moved
efficiently in cytoplasmic extracts [ 1781.
The precise mechanisms by which ActA allows actin recruitment and intracellular
movement are still unknown. However, expression of mutated forms of ActA either in
mammalian cells [ 1513 or in L. rnonocytogenes [ 1 16,1791made it possible to define regions
of the ActA protein with specific functions in actin polymerization and movement. Dele-
tion of the N-terminal domain of ActA was followed in both systems by total abolishment
of actin polymerization and intracellular movement, thus showing the absolute necessity
of this domain in ActA function. In contrast, deletion of neither the proline-rich repeat
domain nor the C-terminal domain prevented actin assembly. However, the actin tails
produced by L. rnonocytogenes strains expressing ActA without proline-rich repeats were
appreciably shorter, and the number of repeats deleted corresponded with reduction in
speed, pointing to a stimulatory function for this region. Earlier work suggested a more
prominent function of the proline-rich repeats, since polyproline peptides, peptides repre-
senting one internal ActA repeat, or a naturally occuring polyproline peptide, blocked
actin assembly and motility after microinjection into L. nzonocytogenes-infected cells
[ 185,1861. It was speculated that the polyproline peptides would bind profilin [ 1861, which
in turn was suspected to be directly involved in actin-mediated motility of L. rnonocyto-
genes [ 1921, thereby inhibiting movement by inhibiting the association of profilin with
the repeat region.
Among actin binding proteins localized on actin tails--a-actinin, tropomyosin, vin-
culin, talin, fimbrin, villin, ezrinkadixin, profilin, the vasodilator-stimulated phosphopro-
tein (VASP), Mena, and Arp3 [20,28,34,63,98,190,192,200]-only profilin, Mena, and
VASP are associated with the surface of moving bacteria and colocalize with ActA. VASP,
a natural ligand of profilin [156], recently was shown to bind directly to the proline-rich
repeats of ActA [20] and can stimulate actin assembly by binding to ActA and enhancing
110 Kuhn and Goebel

the profilin concentration near the bacterium. Mena, which is closely related to VASP,
also binds ActA and profilin directly and might function in concert with VASP to recruit
profilin-actin complexes to the site of actin polymerization [63]. However, this model is
questioned by results of studies in which profilin was depleted from cytoplasmic extracts.
In such experiments, profilin-depleted extracts still supported actin assembly and bacterial
movement [ 1281. The recently described Arp2/3 complex consisting of eight host cell
proteins found in actin tails of moving bacteria may represent the host-cell actin polymer-
ization machinery [200]. The pure complex is sufficient to initiate ActA-dependent actin
polymerization on the surface of L. monocytogenes in a cell-free system and is thought
to interact at least transiently with ActA. Identification and purification of the Arp2/3
complex as a constituent of actin tails represents a great step forward toward the full in
vitro reconstitution of L. monocytogenes motility with purified components.
As recently shown, the 92-kD ActA surface protein is cleaved by the listerial me-
talloprotease (see above) resulting in a major 72-kD degradation product and, dependent
on the strains, additional smaller degradation products [66,145]. These products are either
found on the bacterial surface or in the supernatant fluid as 65- and 30-kD fragments
[ 1451. Whether degradation also occurs inside the cytoplasm of the infected host cell is
unknown. Additionally, the ActA protein is phosphorylated inside host cells, which yields
three distinct forms of this protein with slightly different sizes in SDS-PAGE [15]. How-
ever, a genetically engineered ActA variant which was fully functional but lacking the
C-terminal region is no longer phosphorylated inside host cells, suggesting that phosphory-
lation may not be necessary for movement [ 1 161.
As just mentioned, L. monocytogenes can spread from cell to cell without leaving
the cytoplasm by forming microvilli-like protrusions on the host cell surface which are
phagocytized by neighboring cells. The mechanism of microvilli formation and of induc-
tion of phagocytosis by neighboring cells are totally unknown. In cells infected with Shi-
gella flexneri, which uses a similar mechanism of cell-to-cell spread, proteins of the
cadherin family are critical for the spreading mechanism [ 1651. Whether this is also true
for L. monocytogenes remains to be clarified. Once inside the double membrane-bound
vacuole, bacteria again have to escape into the cytoplasm.
On monolayers of 3T3 fibroblasts, pZcB mutants form only small plaques, suggesting
that the cell-to-cell spread is impaired in these mutants. Electron micrographs of pZcB
mutants inside mammalian cells [ 1961 show numerous bacteria possessing actin tails
which are trapped in vacoules surrounded by a double membrane. This indicates that these
pZcB mutants cannot lyse the double membrane of the vacuole which is formed when
listeriae spread from cell to cell. Plaque formation capacity (which is thought to be a
strong indicator of intercellular spread) of different mutants revealed that in addition to
the broad-spectrum phospholipase PC-PLC, PI-PLC and the metalloprotease also contrib-
uted to plaque formation, most likely by supporting lysis of the double-membrane vacuole
[ 1771. The importance of LLO in this step has not yet been revealed.
L. ivanovii, a species pathogenic only to animals, is also invasive to most mammalian
cells tested, and the intracellular life cycle of this bacterium is similar to that of L. monocy-
togenes. Inside host cells, L. ivanovii polymerizes F-actin-like L. monocytogenes albeit
at a reduced rate, with actin tail formation and cell-to-cell spread also being observed
[90]. Recent cloning and sequencing of the actA-related gene from L. ivanovii [72,105]
showed surprisingly little sequence homology with the actA gene of L. monocytogenes.
On the protein level, some homology exists at the N- and C-termini and in the proline-
rich repeat sequences between the two proteins which are both active in actin polymeriza-
Pathogenesis of Listeria monocytogenes 111

tion [105]. The ActA-related protein of L. ivanovii is larger in size (1044 amino acids)
than ActA of L. monocytogenes (639 amino acids) because of two insertions which are
missing in ActA of L. monocytogenes and an increased number of proline-rich repeats
[72,105]. Despite the overall low-sequence similarity of the two ActA proteins, the mecha-
nism of actin polymerization seems to be similar, since host microfilament proteins that
bind to L. monocytogenes ActA also bind to L. ivanovii ActA [20,62]. Additionally, L.
ivanovii actA can replace L. monocytogenes actA in an L. rnonocytogenes actA mutant
[711.

PRFA AND REGULATION OF VIRULENCE GENE

EXPRESSION IN L. MONOCYTOGENES
Positive Regulatory Factor A
First indications for coordinate regulation of virulence genes in L. monocytogenes by a
trans-acting factor were obtained from analysis of spontaneously occurring nonhemo-
lytic mutants of L. monocytogenes which carried deletions in a region upstream from
the hly gene [70,121]. Cloning and sequencing of the locus affected by the deletion
led to identification of the prfA (positive regulatory factor A ) gene. Its product, PrfA,
a cytoplasmic protein of 27 k D [122,133] regulates all virulence genes of the virulence
gene cluster. The p$A deletion mutants can be complemented in trans by introduction of
the cloned p$A gene again to yield a wild-type phenotype [ 1221. Site-specific mutations
or transposon insertions in the prfA promoter or in the prfA coding region block transcrip-
tion of the entire gene cluster (i.e., plcA, hly, mpl, actA, and plcB [21,133]), indicating
that the p$A gene encodes a transcriptional activator required for expressing the L. mono-
cytogenes virulence gene cluster. Additional evidence for this presumptive role of PrfA
was provided by transcriptional activation of the cloned hly gene by PrfA in B. subtilis
~511.
A plrfA-like gene with high sequence similarity to pr3';4 from L. monocytogenes is
also present in the closely related species L. ivanovii, where it also controls a set of viru-
lence genes similar to those of L. monocytogenes [ 1151. The amino acid sequence of PrfA
suggests that it is a member of the Crp/Fnr family of transcriptional activators which have
been primarily identified in gram-negative bacteria. Like all members of this family, PrfA
contains a conserved helix-turn-helix motif in its C-terminal part. In addition, adjacent to
this motif PrfA carries a sequence containing a leucine zipper and a second helix-turn-
helix motif at its N-terminus [14,115,172] (Fig. 5).
A 14-bp palindromic sequence which was first identified in the promoter region
of the hly gene [ 1381 was present in promoters of all PrfA-dependent genes and located
about 40 bp upstream from the transcriptional start site. The 14-bp palindrome is, how-
ever, not perfectly conserved in all promoters, and these differences could contribute

PrfA Nm HTH

I I
HTH LZ

I I
C
7 30 169 194

FIGURE5 Schematic structure of PrfA from L. rnonocytogenes Sv 1/2a EGD. HTH,

he I ix-tu r n- he I ix m ot iv; LZ, I e u c i ne-zi p pe r.
7 72 Kuhn and Goebel

to differential regulation of the adjacent genes by PrfA [ 14,104,1731. Meanwhile, it

was shown that PrfA binds directly to these palindromic sequences [8,172], thereby
activating virulence genes. Purified PrfA alone is, however, not able to bind specifically
to the target sequence, but it does so after addition of PrfA-free extracts from various
Listeria species, indicating that it requires additional factor(s) for binding [8]. The putative
PrfA-activating factor (Paf) is most likely a protein which is negatively influenced in its
activity by iron [8]. This iron regulation of Paf might also be the key to explain observa-
tions of either iron-repressed LLO expression or iron-induced internalin expression
[23,26].

PrfA-Dependent Promoters and Transcripts-

The PrfA Regulon
Transcriptional organization of the virulence gene cluster is complex. The listeriolysin
gene, hly, is the only gene transcribed in a monocistronic mRNA from two PrfA-dependent
promoters, P1 and P2, located in the intragenic region between hly and plcA [138]. A
third hly promoter, P3, downstream from P1 and P2, recently identified and shown to be
PrfA-independent, results in low-level transcription of the hly gene [37]. Three transcripts
of the p$A gene were identified: a long (2.1 kb) transcript, which is cotranscribed with
the plcA gene and autoregulated by PrfA, and two shorter transcripts (0.8 and 0.9 kb)
transcribed from three distinct promoters located in front of the pr$A gene [50]. Transcrip-
tion of pr$A from one of the promoters seems to be negatively regulated by PrfA. Synthesis
of the bicistronic plcA-pr$A transcript depends on the activity of the plcA promoter [49],
and is necessary for full expression of PrfA-dependent genes [18]. Translation of the
monocistronic pr$A transcript(s) appears to be very inefficient, since PrfA-dependent viru-
lence genes are poorly expressed even in the presence of large amounts of this transcript
when the synthesis of the PrfA-regulated bicistronic transcript is blocked [65]. The three
genes of the lecithinase operon are transcribed from at least two PrfA-regulated promoters:
one, located in front of the mpl gene, yields a 5.4- to 5.7-kb transcript comprising mpl,
actA, and plcB and an additional mRNA of 1.8 kb comprising mpl alone [ 111. A second
promoter, located directly in front of the actA gene, leads to a 3.6-kb bicistronic transcript
comprising actA and plcB [ 1I].
Early in the infectious process, it is believed transcription of p$A via the p$A
promoters results in synthesis of a limited amount of PrfA sufficient to activate the
high-affinity PrfA-dependent hly and plcA promoters. This, in turn, would allow syn-
thesis of plcA-p$A transcripts which leads to enhanced PrfA synthesis. Higher cellular
levels of PrfA activate the mpl and actA promoters, which seem to have a lower affinity
to PrfA because of base mismatches in their palindromic PrfA-boxes [50]. A high
cellular level of PrfA also leads to downregulation of the monocistronic pr$A trans-
cripts [18,49,50,65]. Recent results [lO], however, suggest that both the amount of the
PrfA protein and the quality of the palindromic binding sites are critical parameters for
PrfA-mediated gene expression. The quality of the PrfA protein itself, which differs in
its C-terminal part between different L. monocytogenes strains, seems to influence PrfA
function.
The only known genes which do not belong to the virulence gene cluster but are
also regulated by PrfA are inlC [39,48] and inlAB [42]. Of the multiple promoters upstream
from inlA, which result in 2.9- and 5 .O-kb monocistronic and bicistronic transcripts, only
Pathogenesis of Listeria monocytogenes 113

one is PrfA dependent and harbors a rather incomplete palindromic PrfA-box [ 10,421.
The inZC gene is, however, transcribed from a single PrfA-dependent promoter which
contains a conserved PrfA-box at position -40 from the transcriptional start point. A second
possible PrfA binding site is located downstream from the transcriptional start site in a
position different from those of all other known PrfA-boxes in the promoter regions of
the PrfA-dependent genes [48]. The significance of this second PrfA-box for regulation
of inlC expression, however, is unknown.

Environmental Signals Affecting Virulence Gene

Expression
Pathogenic bacteria living in the free environment must sense and adapt to their surround-
ings by regulating expression of genes needed for living both inside or outside of their
host. Facultative intracellular pathogens also must be able to recognize whether they are
inside or outside their individual mammalian host cell.
An increasing number of signals have been shown to affect virulence gene expres-
sion in L. monocytogenes (reviewed in ref. 14). These signals can be classified into either
physicochemical signals (temperature, iron, glucose, cellobiose, salt, pH, activated char-
coal) or stress conditions (heat shock, oxidative stress, nutritional stress, growth inside
host cells). The mechanisms of altered gene expression are either unknown or only poorly
understood in some instances, but in all systems analyzed, PrfA plays a role in regulation
of environmentally modulated gene expression.
At temperatures below 30"C, the PrfA-dependent genes are not transcribed because
of a lack of prfA transcription. A shift in temperature to 37C results in the onset of pr$A
expression followed by transcription of virulence cluster genes [42,120]. Treating a culture
medium with activated charcoal probably depletes the medium of a signal molecule which
in turn would result in increased transcription of prfA and the PrfA-dependent genes [ 1571.
Carbohydrates modulate virulence gene expression in a complex and poorly understood
manner. Glucose directly influences prfA gene expression and thereby interferes with PrfA
regulation. Increasing the glucose concentration in the medium leads to acidification which
reduces LLO expression by unknown mechanisms [29,50]. The disaccharide cellobiose
inhibits hZy and pZcA expression without a reduction in prfA mRNA levels, probably by
reducing the amount of active PrfA by posttranscriptional mechanisms [97,146]. The
mechanisms of stress-mediated altered gene expression are even less understood. Heat-
shock conditions increase hZy, pZcA and actA expression. p60 expression is inhibited by
heat shock and also by oxidative stress (H202)[ 181,1821. Shift of L. munocytugenes from
a rich medium to a nutritionally stressful minimal essential medium (MEM) induces ex-
pression of virulence cluster genes as well as other surface-associated proteins [ 1571. The
pattern of induction of known PrfA-regulated transcripts in MEM indicates that the PrfA-
controlled genes are differentially regulated in the presence of apparently constant levels
of PrfA.
Phagocytosis and intracellular localization are two natural stress factors, and numer-
ous proteins are selectively induced during phagocytosis of L. monocytogenes by macro-
phages [79]. A genetic assay to isolate genes preferentially expressed inside mammalian
cells resulted in identification of genes involved in nucleotide biosynthesis, an arginine
transporter, and pZcA [97]. Experiments directly measuring bacterial mRNA levels inside
host cells revealed that the genes hZy, actA, and inZC are heavily expressed inside the
114 Kuhn and Goebel

mammalian cell, most likely by PrfA-dependent mechanisms [ 1 1,481. The complexity of

regulation of gene expression by environmental stimuli is, despite all progress, far from
being understood.

ROLE OF SUPEROXIDE DISMUTASE AND CATALASE

IN VIRULENCE
Possible roles of catalase and superoxide dismutase in virulence of L. monocytogenes
have been reviewed recently [78,11 I]. Both enzymes act in concert to detoxify potentially
harmful superoxide radicals. Superoxide radicals generated by the oxidative burst in a
phagocytic cell are converted into hydrogen peroxide by action of superoxide dismutase
(SOD), which is then cleaved by catalase into water and molecular oxygen. Bacterial
catalases and superoxide dismutases were long suspected of being important virulence
factors of intracellular bacteria, but no correlation of superoxide dismutase expression
with virulence was found in L. monocytogenes [ 1991. The gene for SOD from L. monocyto-
genes [ 121 was recently cloned in Escherichia coli. The nucleotide sequence of the gene,
called lmsod, revealed an ORF coding for a protein of 202 amino acids with high homology
to the manganese-containing SODS from other organisms. The gene, which is conserved
in all other Listeriu species, was mutagenized and virulence of the mutant was tested in
mice, but no difference in survival of the bacteria in the spleen and liver of infected
animals was observed between the lmsod mutant and the wild-type strain [ 131.
Catalase mutants obtained by transposon mutagenesis show wild-type virulence in
infected mice [ 1 171. Whereas catalase-negative L. monocytogenes mutants are killed by
mouse resident macrophages already at low serum concentrations, killing of the wild-type
bacteria requires high serum concentrations, suggesting that resistance to fully activated
macrophages is partially mediated by catalase activity [ 1951. Meanwhile, the catalase gene
(cut) of L. monocytogenes was cloned and sequenced [13]. Recent construction of frame
catalase and SOD mutants as well as a catalase/SOD double mutant has shown that cata-
lase and SOD alone obviously are dispensible for the bacterium. The double mutant, how-
ever, is drastically reduced in its ability to grow inside liver and spleen of infected mice
and is also unable to grow inside mouse bone marrow-derived macrophages, suggesting
a role for both enzymes in virulence and intracellular survival [13].

HOST CELL RESPONSES TO INFECTIONS BY

L. MONOCYTOGENES
Differential Regulation of Cytokine and Cytokine
Receptor Expression
In this chapter, we will not discuss the immunological aspects of cell-mediated immunity
against L. monocytogenes which were reviewed recently [95], but we will concentrate on
what is known about the response of mammalian host cells to L. monocytogenes infection
on the molecular and genetic levels. We will focus on host genes involved in signal trans-
duction and altered gene expression during infection by L. monocytogenes.
The initial studies on host response done with primary mammalian cells and estab-
lished cell lines infected with L. monocytogenes primarily determined cytokine activities
whose importance in nonspecific and T-cell-mediated immunity during experimental lis-
teriosis is well documented [ 1401. However, these studies could not discriminate between
Pathogenesis of Listeria monocytogenes 7 75

release of preformed proteins and de novo synthesized proteins. More recent studies deter-
mined expression of the affected host genes more specifically by semiquantitatively mea-
suring expression on the transcriptional level using the highly sensitive reverse tran-
scriptase-polymerase chain reaction (RT-PCR) method.
The early reports showed that primary mouse embryonic fibroblasts infected with
L. monocytogenes released interferon-a@ (IFN-a@) into the culture medium [83]. In-
terleukin-1 (IL- 1) production by mouse peritoneal macrophages was observed with viable,
virulent L. rnonocytogenes strains but not with killed or avirulent Listeria. Northern blot
analysis further showed that the increase in IL-1 secretion correlates with an increase
in IL- 1a-specific mRNA after infection of macrophages with L. monocytogenes [ 1411.
Appreciable amounts of tumor necrosis factor (TNF) and IL-6 are secreted in alveolar
macrophages after infection with viable L. monocytogenes [84]. IL-6 is induced in embry-
onic fibroblasts even by heat-killed L. monocytogenes albeit to a lesser extent than by
infection with viable bacteria. TNF is secreted only after treatment with killed but not
with viable L. monocytogenes [841. Differences between killed and viable Listeria in
induction of TNF-a also were observed after infection of mouse peritoneal cell prepara-
tions consisting mostly of macrophages [203). Release of the proinflammatory cytokines,
IL-lp, IL-6, and TNF-a, occurs in human polymorphonuclear granulocytes and in the
human epithelial cell line HEp-2 after L. monocytogenes infection [4]. The granulocytes
secrete all three cytokines in response to infection, whereas the HEp-2 epithelial cells
secrete IL-6 and small amounts of TNF-a but no IL-lp. Transcription of the respective
genes also is induced in these host cells. Using the human epithelial cell line Caco-2, we
could only detect IL-6-specific mRNA expression on L. monocytogenes infection which
was, however, already induced by adherent L. monocytogenes, since cytochalasin D treat-
ment did not inhibit IL-6 expression [ 1081. Mouse peritoneal macrophages also secrete
IL-6 after L. monocytogenes infection [ 1061. Hemolytic L. monocytogenes strains are less
efficient in IL-6 induction than are nonhemolytic mutants, probably because of cell damage
caused by the high level of secreted listeriolysin. Inhibited maturation of IL-lp by L.
monocytogcnes in mouse peritoneal macrophages was recently proposed as a novel mecha-
nism of how L. monocytogenes may escape the host cell response, since infection of the
macrophages by L. monocytogenes results in intracellular accumulation of unprocessed
IL- 1 p precursor [61.
In a recent study using the mouse macrophage-like cell line P388DI and different
well-defined mutants of L. monocytogenes to analyze cytokine induction after infection,
we showed [ I 101 that viable L. monocytogenes rapidly induced IL- 1 a, IL- 1p, IL-6, and
TNF-a mRNAs in these host cells, whereas killed L. monocytogenes only induced IL- 1 p
mRNA. Nonhemolytic mutants which cannot escape into the cytoplasm and which do not
multiply are unable to induce I L - l a , IL-6, and TNF-a but still induce IL-lp mRNA.
In most instances, the amount of cytokines in the culture supernatant liquid of infected
macrophages correlates well with levels of induced mRNAs. The exception is IL- 1a, of
which only low levels are found in the supernatant liquid despite an appreciable induction
of IL- l a l p mRNA. Mouse bone marrow-derived macrophages infected with L. monocyto-
genes also induce proinflammatory cytokines [ 1 101. However, in these cells, a nonhemo-
lytic L. monocytogenes mutant induces the same types and amount of cytokines as the
wild-type strain, indicating that intracellular growth is not necessary for transcriptional
induction of these cytokines in bone marrow-derived macrophages [33].IL-6 is produced
in the bone marrow-derived macrophages independently of IL-1 and TNF [33]. The im-
munomodulating cytokines, IL- 10, IL- 12, and the IL- 1 receptor antagonist, also are in-
116 Kuhn and Goebel

duced in bone marrow-derived macrophages after L. monocytogenes infection [33,112].

Large amounts of the chemokine, IL-8, are secreted by the polarized human colon epithe-
lial cell line TE4in response to L. monocytogenes infection [47]. Induction of IL-8 secretion
is caused by an increase of IL-8-specific mRNA synthesis and is not mediated by secreted
TNF-a and occurs preferentially at the basolateral side. IL-8 may be an initial signal for
the acute inflammatory response after bacterial invasion of mucosal surfaces.
The transcription of cytokine receptor mRNAs in mouse bone marrow-derived mac-
rophages infected with L. monocytogenes seems to be regulated inversely to the respective
cytokines. TNF receptor type I and IFN-y receptor are transcriptionally downregulated
shortly after infection of mouse bone marrow-derived macrophages by L. monocytogenes,
whereas IL-1 receptor type I1 mRNA is unaffected [33]. However, infection with the
closely related but nonpathogenic species L. innocua does not alter cytokine receptor ex-
pression in bone marrow-derived macrophages.
Consequently, these events might diminish the ability to activate L. monocytogenes-
infected macrophages by cytokines, such as TNF-a and IFN-y, at least in vitro. This mech-
anism probably allows L. monocytogenes to evade the killing mechanisms of infected
macrophages in vivo, since L. monocytogenes surmounts this barrier of defense and es-
capes to other cell types in the course of infection.

Expression of Stress Genes, MHC Genes, and Other Genes

Identified by Differential PCR
Invasion of a mammalian cell by bacteria growing rapidly inside the cytoplasm is a likely
stress factor for the host cell. Immediately after infection, the macrophage-like cell line
5774 responds with enhanced synthesis of heat-shock protein 70 (HSP70) mRNA and
HSP70 protein [168]. Phagocytosis of the bacteria is necessary for this induction, since
cytochalasin D treatment, which blocks invasion, also prevents induction of HSP70
mRNA. The amount of HSP60 and HSP90 mRNAs is only slightly enhanced during infec-
tion. Another stress response protein, MAP kinase phosphatase (MKP-1 or PTP), which
seems to participate in signal transduction pathways, is significantly induced 1 h postinfec-
tion and stays at an induced level for several hours in infected macrophages [107]. Tran-
scription of both HSP70 and MKP-1 mRNAs is much less induced when macrophages
are infected with nonhemolytic mutants of L. monocytogenes, suggesting that escape of
bacteria into the cytoplasm is required for induced transcription of the stress genes.
MKP-1 mRNA is transiently induced in L. monocytogenes-infected HeLa cells where it
might contribute to dephosphorylation of the MAP kinase [ 1981 (see below).
Since immunity to L. monocytogenes, which replicates in the cytoplasm of the in-
fected macrophage, is mainly dependent on major histocompatibility complex (MHC)
class I-restricted CD8 T lymphocytes [95], expression of the MHC class I molecules is
critical for macrophage antigen presentation. We analyzed the expression of H-2K and
I-AP mRNA in 5774 and P388Di macrophage-like cell lines infected with L. monocyto-
genes compared with noninfected cells [ 1671. Expression of both genes was repressed on
infection of P388D1 but not 5774 macrophages. Class I1 MHC gene transcription was
already repressed at early stages of infection when most bacteria were inside a phagosome.
In contrast, class I MHC transcription decreased 2 to 4 h postinfection when the bacteria
replicated inside the cytoplasm [ 1671. L. monocytogenes infection not only repressed MHC
class I and I1 expression in resting P388Di macrophages but also lowered responsiveness
of macrophages to IFN-y treatment [ 1671, which is known to induce MHC I and I1 expres-
Pathogenesis of Listeria monocytogenes 117

sion [ 1471. MHC I and I1 expression also was repressed by L. rnonocytogenes infection in
P388D1 macrophages previously activated by IFN-y treatment. This suppression of MHC
expression in activated macrophages was, however, only detectable on infection with wild-
type L. rnonocytogenes but not with a p$A mutant unable to escape efficiently from the
vacuole into the cytoplasm [ 1671. Suppression of MHC gene transcription may represent
an important mechanism allowing L. rnonocytogenes to reduce macrophage-mediated anti-
gen presentation followed by T-lymphocyte activation.
Using a modification of the previously described procedure of differential PCR
[ 123,1701, induction or repression of host genes after infection by L. rnonocytogenes was
determined in a more general way. This method allows isolation of cDNAs representing
fragments of genes which are expressed differently in infected and uninfected macro-
phages. By this procedure we obtained several cDNA clones derived from macrophage
genes that were either transcriptionally activated or downregulated after infection by L.
monocytogenes [ 123,1701. Some of the cloned cDNAs were sequenced and subsequent
homology searches revealed that some of the sequences did not show any significant ho-
mologies to known genes [ 1081. One of the cloned cDNA fragments showed more than
99% homology to murine mitogen-activated protein kinase phosphatase (MKP-1) [ 1701
which was earlier described as being upregulated in macrophages on L. rnonocytogenes
infection [ 1681.

Activation of Signal Transduction Pathways

Enhanced expression of MKP-1 in macrophages and HeLa cells is also an indication that
signal transduction pathways are modulated by a L. rnonocytogenes infection. Phosphory-
lation of MAP kinase is observed after infection of Caco-2 and HeLa cells with L. rnonocy-
togenes and is mediated by the pore-forming activity of listeriolysin 0 [ 188,189,1981.
MAP-kinase is a part of signal transduction pathways which link extracellular signals to
gene expression [158]. We have recently shown that the entire Raf-MEK-MAP kinase
cascade is transiently activated on L. rnonocytogenes infection of HeLa cells [ 1981. Further
studies are needed to elucidate the role of this activation and to clarify if enhanced cell
proliferation occurs, probably for the benefit of invading bacteria. In this respect, expres-
sion of listeriolysin 0 in mammalian cells also results in a dramatic change of cell mor-
phology and formation of foci consisting of tightly connected cells. The hly-transfected
mammalian cells also exhibit an appreciably enhanced proliferation rate [ 3 2 ] .Because of
the presence of its transport signal sequence, we assume that listeriolysin is transported
via the Golgi system to the cytoplasmic membrane of the host cell where it may trigger the
Raf-MEK-MAP kinase cascade which may ultimately lead to enhanced cell proliferation.
Besides LLO-mediated activation of the Raf-MEK-MAP kinase pathway, LLO in syner-
gism with PI-PLC also triggered synthesis of phosphatidylinositol phosphates (IP3 and
IP4] and diacylglycerol in endothelial cells [ 175,1761. The molecular mechanisms as well
as their significance during a L. monocytogenes infection remain to be unraveled.
Differential gene expression is mediated by transcription factors like nuclear factor
KB (NF-~3)[5]which are particularly involved in transcription of many immunologically
relevant genes, including cytokine genes. We studied NF-KB DNA binding activity in
response to L. rnonocytogenes infection in the macrophage-like cell line P388DI [82]. A
rapid invasion-independent enhancement of the NF-KB DNA binding activity was ob-
served in these host cells within 10-20 min after adding the Listeria. NF-KB is induced
in biphasic kinetics on L. rnonocytogenes infection: The first transient induction of NF-
I18 Kuhn and Goebel

KB requires only adhesion of L. monocytogenes to P38SDI cells. This induction also occurs
with avirulent mutants of L. monocytogenes and the avirulent L. innocua with similar
efficiency. This event likely involves lipoteichoic acid, the cell wall component of L.
monocytogenes, since purified lipoteichoic acid shows the same transient triggering of
NF-KB. A second, but permanent, induction of NF-KB occurs after release of listeriae into
the cytoplasm of the host cell. This event occurs exclusively with virulent L. monocyto-
genes strains and requires the bacterial phospholipases PI-PLC and PC-PLC that are pro-
duced in the infected host cells cytoplasm [Sl]. Activation of NF-KB also occurs in
Caco-2 cells after infection with L. monocytogenes but with slower kinetics than seen in
the macrophages [SO]. The DNA binding activity of two other transcription factors, AP-
1 (activator protein-]) and NF-IL6, is not changed after infecting the P388D1 cell line
with L. monocytogenes, indicating that the observed activation of NF-KB by L. monocyto-
genes is a specific event [82].

Apoptosis
Programmed cell death, or apoptosis, induced by pathogenic bacteria was first documented
for Shigella jexneri using the mouse macrophage-like cell line 5774 [204]. Induction of
apoptosis was later shown for several facultative intracellular bacteria, including Salmo-
nella typhimurium [ 1421, Bordetella pertussis [96], Legionella pneumophila [ 1441, and
L. monocytogenes [75,159]. L. monocytogenes, originally described as being unable to
induce apoptosis in 5774 macrophages [204], was recently shown to induce apoptosis in
hepatocytes [159] and in dendritic cells with listeriolysin 0 being thought to trigger
apoptosis [75]. L. monocytogenes-infected hepatocytes undergo apoptosis in vitro as well
as in infected mice, and it was suggested that events of hepatocyte apoptosis which are
linked to neutrophil recruitment eliminate infected cells rapidly and thereby inhibit L.
monocytogenes spread [ 1591. In most instances, the mechanisms of apoptosis induction
by bacteria are totally unknown. However, for S. Jexneri, IpaB invasin was reported to
bind directly to an interleukin-converting-enzyme (ICE) protease and thereby interfere
with the apoptosis-controlling network of the host cell [22].

CONCLUSIONS
The last years have seen an enormous increase in our understanding of the molecular basis
of infectious diseases. Our knowledge of the genes determining virulence of L. monocyto-
genes and the role which the virulence gene products play in the infectious process is
rapidly expanding. However, many problems concerning virulence of L. monocytogenes
still remain unsolved.
For instance, L. ivanovii, a species largely nonpathogenic for humans [ 1711, resem-
bles L. monocytogenes in its intracellular life cycle [90]. Genes homologous to most of
the known virulence genes of L. monocytogenes are also detectable in this species
[72,77,105], and the complete PrfA-regulated gene cluster identified in L. monocytogenes
apparently also is present in L. ivanovii 1721. However, L. ivanovii is only virulent for
animals and avirulent for humans with an experimental L. ivanovii infection in mice yield-
ing a different outcome than that by L. rnonocytogenes [86]. What is the molecular explana-
tion for this obvious difference in the pathogenic potential of these two Listeria species?
Is it the result of a different mechanism in regulation of known virulence genes inside
infected cells or differences in specific activity of known virulence gene products? Are
Pathogenesis of Listeria monocytogenes 7 79

there as yet unknown virulence factors in L. monocytogenes which are absent in L. ivanovii
or vice versa?
Expression of L. monocytogenes virulence genes inside infected mammalian host
cells and tissues is another important but unsolved problem. The expression pattern of
known L. monocytogenes virulence determinants is already complex under in vitro growth
conditions and regulated by PrfA-dependent and PrfA-independent mechanisms. Very lit-
tle is known about how PrfA and other putative regulatory factors control these genes
while the bacteria reside inside host cells and tissues. Preferential synthesis of listeriolysin
inside the phagosome and of ActA and InlC inside the cytoplasm has been described
[ 1 1,481. However, the precise timing in expression of virulence genes as well as cellular
signals and bacterial sensors which may control their intracellular expression are largely
unknown. Most analyses of listerial virulence factors were done with commonly used
laboratory strains such as serotype 1/2a strain EGD or serotype I /2c strain L028. How-
ever, many human infections and most foodborne outbreaks have been associated with
serotype 4b strains [ 1491. In the future, differences in structure, function, and especially
regulation of virulence factors of different L. monocytogenes serotypes [ 1841 and clinical
isolates will likely gain much more interest.
Analysis of host cell responses to a L. monocytogenes infection is now becoming
a topic of major interest, as it represents a suitable model system for studying the molecular
basis of pathogen-host cell interactions. Research now concentrates on identification of
new host genes which are differentially expressed during various steps of a L. monocyto-
genes infection. Characterization of such host genes may help us to understand better the
strategies which these two partners are using in their intimate and sometimes very severe
cross talks. The molecular mechanisms of this intimate cross talk which require signal
transduction from pathogens to their host cells and vice versa are now being analyzed.
Elucidation of the interaction of bacterial and host cell proteins also will shed new light
on the coevolution of bacteria and their hosts. These central questions of pathogenesis of
facultative intracellular bacteria pertain not only to L. monocytogenes but also to several
gram-negative bacteria, such as Shigella, Yersinia, and Salmonella, and may lead to excit-
ing answers in the near future.

ACKNOWLEDGMENTS
We thank A. Demuth for carefully and critically reading this manuscript, J. Kreft and F.
Engelbrecht for providing figures, and all members of our laboratory for allowing us to
quote their unpublished results. We apologize to all who contributed to our current knowl-
edge on the infection biology of L. monocytogenes but were not mentioned in this chapter.
Work from the group at the University of Wiirzburg was supported by the Deutsche
Forschungsgemeinschaft through the grant SFB 165-B4.

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198. Weiglein, I., W. Goebel, J. Troppmair, U.R. Rapp, A. Demuth, and M. Kuhn. 1997. Listeria
monocytogenes infection of HeLa cells results in LLO mediated transient activation of the
Raf-MEK-MAP kinase pathway. FEMS Microbiol. Lett. 148:189-195.
199. Welch, D.F. 1987. Role of catalase and superoxide dismutase in the virulence of Listeria
monocytogenes. Ann. Inst. Pasteur/Microbiol. 138:265-268.
200. Welch, M.D., A. Iwamatsu, and T.J. Mitchison. 1997. Actin polymerization is induced by
Arp2/3 protein complex at the surface of Listeria monocytogenes. Nature 385:265-269.
201. Wood, S., N. Maroushek, and C.J. Czuprynski. 1993. Multiplication of Listeria monocyto-
genes in a murine hepatocyte cell line. Infect. Immun. 61:3068-3072.
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Listeria monocytogenes is essential for cell viability and its gene product, p60, has bacterio-
lytic activity. J. Bacteriol. 175:3491-3501.
203. Zhan, Y., and C. Cheers. 1995. Differential induction of macrophage-derived cytokines by
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Characteristics of Listeria
monocytogenes Important to
Food Processors

YUQIANLou
Bil Mar Foods, Inc., Zeeland, Michigan

AHMEDE. YOUSEF
The Ohio State University, Columbus, Ohio

INTRODUCTION
Todays food manufacturer relies on a variety of processing and preservation techniques
to produce a safe and wholesome product with a suitable shelf life. Preservation methods
ensure the safety and stability of food by inactivating or inhibiting growth of foodborne
spoilage and pathogenic microorganisms. Methods currently used in food preservation
involve physical, chemical, and biological factors. Physical preservation includes heating,
cooling, freezing, and irradiation. Chemical treatments include addition of antimicrobial
agents such as benzoates, propionates, and sorbates, acidifying agents such as acetic, and
lactic acids or curing agents such as sodium chloride and sodium nitrite. Preservation
by biological means (biopreservation) includes fermentations which control spoilage and
pathogenic microorganisms through gradual lowering of pH. Combinations of these pres-
ervation factors also are applied simultaneously or sequentially in food processing. In
addition to these conventional preservation methods, novel nonthermal processing tech-
nologies are being investigated to meet increasing consumer demands for minimally
processed food with fresh-like taste and texture. Ultra-high pressure, pulsed light or
electric fields, and oscillating magnetic fields are examples of these novel techniques.

131
132 Lou and Yousef

New nonthermal technologies when combined with mild conventional preservation

methods can produce microbially safe minimally processed foods. Treatment combina-
tions are commonly used in food processing and such use is best expressed as the hurdle
concept.
Growth, inhibition, or inactivation of Listeria monocytogenes in response to food
processing and preservation techniques will be detailed in this chapter. Throughout this
chapter, some basic terms and concepts are used which must first be defined for clarity.
The expression log refers to the microbial count in Log&FU/ml or g. When a measurable
increase in count occurs, it is described as such unless multiplication of the microorganism
is reported; in this instance, the increase will be described as growth. Microbial growth
is enhanced when the lag period decreases, the generation time decreases, and/or the gain
in count attained after a given growth period increases. In contrast, growth inhibition, or
simply inhibition, results from a reversal in the aforementioned growth parameters.
Growth inhibitors may also be described as bacteriostatic, and for Listeria as listeriostatic
agents, with such agents usually not causing measurable inactivation. A decrease in count
will be described as inactivation and defined in decimal reduction time (D-value) or a
decrease in log count, when such information is available. An agent that causes microbial
inactivation will be called bactericidal, but it may also be reported as listericidal when
it is active against Listeria. The D-value is the time of exposure to a deleterious factor
(e.g., heat) required to inactivate 90% (i.e., 1 log) of the population of a given microorgan-
ism. Hence, the D-value is a measure of resistance of the microorganism to the deleterious
factor; the larger the D-value, the greater the resistance. When the count does not change
appreciably, the status of the microorganism is best described as survival. The word sur-
vival also refers to the ability of the microorganism to maintain its viability during treat-
ment.

TEMPERATURE
Temperatures to which food is exposed may have lethal, growth-conductive, or preserving
effects on microorganisms in the product. In general, temperatures greater than 50C are
lethal to L. monocytogenes. At -0 to 45OC, the pathogen grows to various degrees when
present in a suitable medium. Temperatures below 0C freeze the culture or food and
preserve or moderately inactivate the pathogen. These three ranges of temperatures will
be addressed separately.

Growth Temperatures
Microorganisms grown at optimum incubation conditions exhibit short initial lag periods,
short generation times during exponential growth, and high cell counts or densities at the
stationary phase. Incubation at temperatures different than the optimum extends the lag
period and/or the generation time and may decrease the maximum attainable population.
In this chapter, growth parameters of Listeria monocytogenes as a function of incubation
temperature will be described.
The temperature range within which L. monocytogenes grows is of particular interest
to food processors, since this pathogen has common features of both psychrotrophic and
mesophilic bacteria. Under laboratory conditions, L. monocytogenes was originally re-
ported to grow at temperatures between 3 and 45C [160], with optimal growth occuring
between 30 and 37C [300,340]. In 1972, Wilkins et al. [394] examined the temperature
range for growth of L. monocytogenes in a medium containing 1% tryptone, 1% yeast
Characteristics of Listeria monocytogenes 133

extract, 0.3% K2HP04, and 0.1% glucose. Extrapolation of data from an Arrhenius plot
of exponential growth rates collected at various temperatures indicated that the bacterium
had maximum, optimum, and minimum growth temperatures of 45-5OoC, 38"C, and 3"C,
respectively, which generally agree with the growth temperatures most frequently men-
tioned in earlier textbooks. However, Bergey 's Manual of Systematic Bacteriology [341]
gives the minimum growth temperature as 1C. In support of this change from 3 to lC,
Junttila et al. [ 1911 found that growth of 78 L. monocytogenes strains on tryptose soy agar
occurred at a mean minimum temperature of l.l(t0.3)"C, with 10 and 2 strains of L.
monocytogenes serotype 1/2 growing at 0.8 and OSOC, respectively. In contrast, L. innocua
(19 strains), L. murrayi (1 strain), and L. grayi (1 strain) failed to grow at temperatures
below 1.7 (tr0.4), 2.8, and 3.OoC, respectively. Although researchers in Florida also ob-
served slight growth of some L. monocytogenes strains in laboratory media at 1"C, Walker
et al. [382] confirmed the ability of this pathogen to multiply at even lower temperatures,
with three L. monocytogenes strains exhibiting generation times of 62- 131 h in chicken
broth and pasteurized milk, respectively, during extended incubation at -0.1 to -0.4"C.
Lowest growth temperature was reported by Hudson et al. [ 1781. L. rnonocytogenes and
two other ps ychrotrophic pathogens, Aeromonas hydrophila and Yersinia enterocolitica,
grew at - 1.5"C in vacuum-packaged sliced roast beef with calculated lag times of 174,
110, and 49 h and generation times of 100, 33, and 32 h, respectively.
Growth of L. monocytogenes in laboratory media at 1C is very slow. However,
when incubated at 3-6"C, the growth rate of the pathogen increases with final populations
of approximately 1OS CFU/mL attained after several weeks of incubation [ 1 151. In a study
by Bojsen-Moller [39] in 1972, flasks containing Tryptose Phosphate Broth (TPB) were
inoculated with one of several L. monocytogenes strains and incubated at different temper-
atures. L. monocytogenes exhibited average generation or doubling times of 12.0, 5.0, and
2.6 h during incubation at 4, 10, and 15"C, respectively. In a more recent study [27], the
average generation times for 39 L. monocytogenes strains growing in Tryptic Soy Broth
(TSB) supplemented with 0.6% yeast extract were 43, 6.6, and 1.1 h at 4, 10, and 37"C,
respectively, whereas the respective average lag times were 151, 48, and 7.3 h. In the
latter study, L. monocytogenes strains reached maximum OD600-valuesof 0.74, 0.92, and
0.97 after incubation at 4, 10, and 37C for 336, 113, and 16 h, respectively. Since many
other bacterial species fail to grow at refrigeration temperatures, extended cold storage
of clinical, environmental, and food samples previously diluted in a nonselective medium
such as Tryptose Broth (TB) often was successful for isolating L. monocytogenes. This
Listeria-detection procedure, which forms the basis for cold-enrichment, was widely used
until the mid 1980s.
Significant growth variation among 39 strains of L. monocytogenes, especially at
refrigeration temperature, also was observed by Barbosa et al. [27]. The lag phase for 39
strains varied from 69.8 to 270 h at 4C and from 36.5 to 68.9 h at 10C. Scott A, the
strain most extensively used in Listeria-related research, had the longest (209 h) and the
second longest (62.8 h) average lag periods at 4 and 10C, respectively. However, when
strains of I;. rnonocytogenes were grouped according to serotypes, few differences in
growth parameters among serotypes were noticed. Therefore, the choice of L. munocytu-
genes strains for use in challenge studies, particularly at refrigeration temperatures, may
affect the results and conclusions regarding food safety. Greater safety margins will be
obtained if the hardiest L. monocytogenes strains are used in such studies.
Because of the safety concerns, researchers attempted to find nonpathogenic indica-
tor microorganisms which can replace L. monocytogenes, particularly for studies done in
pilot plants or food processing facilities. L. innocua, which is nonpathogenic and has
134 Lou and Yousef

similar or higher growth rates and resistance to common food preservation methods than
L. monocytogenes, was considered a suitable substitute for L. monocytogenes
[97,143,299]. L. innocua PFEI, a strain with antibiotic resistance which aids its enumera-
tion in foods, is reported to be a good thermal-resistance indicator of L. monocytogenes
[143]. When incubated in Brain Heart Infusion (BHI) broth at 2-46"C, this L. innocua
strain grew faster below 42"C, slower above 42"C, and had a shorter lag phase below
8C than L. monocytogenes, and thus was generally considered a good indicator of the
growth behavior of L. monocytogenes [97]. Similar or faster growth of L. innocua in a
laboratory broth and a cheese sauce, compared with that of L. monocytogenes, was also
noted by Petran and Swanson [299].
Prior treatment of a microorganism affects its behavior during subsequent growth.
Gay et al. [149] found that the lag phase of L. monocytogenes (Scott A and V7) and L.
innocua increased by a low inoculum ( 10' vs 1O3 CFU/mL) cold storage and preincubation
at 30" rather than 14C. Listeria strains tested by these investigators exhibited a slower
first logarithmic growth phase and a faster second phase under most of the conditions
tested.
The food medium can also influence growth and calculated growth parameters of
L. monocytogenes. Rosenow and Marth [322] measured growth parameters of four L.
monocytogenes strains in autoclaved samples of skim, whole, and chocolate milk and
whipping cream that were stored at 4-35C. Listeria growth rates were generally similar
in all four products at a given temperature and increased with an increase in incubation
temperature. Generation times in hours for listeriae in all four products were 29.7-45.6
at 4OC, 8.7- 14.6 at 8OC, 4.5-6.9 at 13OC, 1.7-1.9 at 2 1 OC, and a uniform 0.68 at 35C.
L. monocytogenes reached maximum populations of 1 07- 1O9 CFU/mL in all products that
were incubated 30-45 days at 4"C, 11-14 days at 8"C, 5.0-5.8 days at 13"C, 2.1 days
at 21 "C, and 1 day at 35C. In addition, numbers of listeriae failed to decrease substantially
in the four products during extended storage. Donnelly and Briggs [92] also reported
rapid growth of five L. monocytogenes strains in inoculated samples of whole, skim, and
reconstituted nonfat dry milk (1 1% total solids) during incubation at 4, 10, 22, and 37C.
Growth of L. monocytogenes at low temperatures is also stimulated by presence of
certain solutes in growth media. Such compounds include glycine betaine or carnitine
[209,352], which are known as osmoprotectants, osmolytes, or compatible solutes. Similar
osmolytes may exist in foods at measurable levels. Such osmolytes usually accumulate
in microbial cells during periods of osmotic stress. Compatible osmolytes may stabilize
the otherwise unstable physiological functions of cytoplasmic proteins or other structures
under osmotic stress [398]. KO et al. [209] found that osmolytes accumulated in cells of
L. monocytogenes at low temperatures and during chill stress. When L. monocytogenes
was surface-plated on a defined medium containing 130 pM glycine betaine, colonies
were observed after 32 days at 7"C, whereas no colonies were visible without this osmo-
protectant. When L. monocytogenes was grown in the defined liquid medium at 4"C, addi-
tion of 130 pM glycine betaine nearly doubled the specific growth rate [209].
Virulence of Listeria is increased when the bacterium is grown at low rather than
high temperatures. Durst [99] reported that 7 of 36 weakly virulent L. monocytogenes
strains became markedly virulent to mice by intraperitoneal injection after the cultures
were maintained on agar slants for 6 months at 4C. Similarly, Wood and Woodbine [397]
found that one strain of L. monocytogenes was more virulent to chick embryos when
grown at 4 rather than 37C. Thus the possibility exists that cold storage may enhance
virulence of some L. monocytogenes strains isolated from refrigerated foods.
Characteristics of Listeria mon ocytogen es 735

Freezing
Numerous reports of Listeria-contaminated frozen foods in the United States and else-
where have prompted investigators to examine viability of L. monocytogenes during frozen
storage. Survival of L. monocytogenes in laboratory media, buffers, and milk during freez-
ing and frozen storage was assessed by Hof et al. [ 17 I], El-Kest and Marth [ 107,108,109]
and El-Kest et al. [ I 121.

Viability in Frozen Culture Media

Although L. monocytogenes does not grow below - 1.5"C, this pathogen can readily sur-
vive at much lower temperatures. Nontheless, freezing and frozen storage will cause a
limited reduction in the viable population of L. monocytogenes. Using TPB, Hof et al.
[ 17 1] found that viable populations of L. rnonocytogenes, L. ivunovii, L. innocua, L. seelig-
eri, and L. welshimeri decreased by 50, 90, 90, 40, and 50%, respectively, following 3
weeks of frozen storage at -20C. These findings suggest that L. monocytogenes can
remain viable as long as or longer than most other Listeria slip. during extended storage
at subzero temperatures.
Survival and injury during frozen storage depends on L. rnonocytogenes strains used,
freezing menstruum, and freezing rate [ 106- 108,I 121. Milk or TB provided better protec-
tion of L. monocytogenes against death and injury than did phosphate buffer. After 4
weeks of frozen (- 18C) storage, death and injury rates for three strains of L. monocyto-
genes were 9 1-99% and 52-78%, respectively, when the cells were suspended in phos-
phate buffer. 42-92% and 33-56% in TB, and 38-61 % and I I-67% in milk [ 1081. Addi-
tion of 2-4% glycerol or 2% milk components to phosphate buffer markedly decreased the
extent of cell death and injury. Simulated milk ultrafiltrate, when compared with phosphate
buffer, caused almost no change in death rate but decreased cell injury during the first
24 h of frozen storage at - 18C 11071. Slow freezing at -- 18C was more lethal and
injurious to this organism than rapid freezing at - 198C; however, freezing at - 198C
followed by storage at - 18C resulted in cell death and injury rates that were similar to
those caused by combined freezing and storage at - I 8C [ I 121. No evidence of cell injury
or death was observed when L. monocytogenes cells were suspended in phosphate buffer
and stored at - 198C for I month. When TB was used in place of phosphate buffer, the
extent of cell injury increased for suspensions stored at - 198C but decreased for similar
suspensions stored at - 18C [ I 121. El-Kest et al. [ 1 121 found that after 1 month of frozen
storage in TB, at - 18"C, 45% of the population was sublethally injured compared with
72430% of L. monocytogenes cells in a virtually identical study [ l58].
As expected, multiple freezekhaw cycles were more detrimental to survival of lister-
iae than was a single cycle. Such treatment was far more damaging to the pathogen when
done at - 18C (>99% lethality and no detectable injury) rather than - 198C (16-34%
lethality and 1 1-27% injury) with generally similar behavior observed using TB and phos-
phate buffer [ I 121.
Although freezing and frozen storage cause a limited decrease in viability of L.
munocytogmes, such treatments can produce injury and thus sensitize L. monocytogenes
cells to antimicrobial agents. Damage to several sites on L. monocytogenes cells caused by
freezing and frozen storage at - 18C was observed by El-Kest and Marth [ I 101. Freezing
increased sensitivity of L. monocytogenes to lysozyme and lipase, which are two enzymes
occurring naturally in some foods. Treatment with both enzymes resulted in a greater
effect than when each enzyme was used alone [ 1 10,I 1 I]. However, adaptation of L. mono-
136 Lou and Yousef

cytogenes to sublethal levels of certain environmental stresses, such as low pH, ethanol,
NaCl, heat shock, or starvation can increase survival of this pathogen during freezing,
frozen storage, and freezelthaw cycles [237].
Viability in Frozen Foods
Oscroft [282] found that frozen storage of three L. rnonocytogenes strains in carrot or
chicken homogenate at - 18C for 29-84 days did not appreciably reduce the viable cell
counts. Results from other studies [ 164,284,2881demonstrated that L. rnonocytogenes pop-
ulations decreased only <1-3 logs in inoculated samples of packaged fish and shrimp,
canned milk, 10%Karo corn syrup, ground beef, ground turkey, frankfurters, carined corn,
and ice cream mix during 2-3 months of frozen storage at - 18 to -20C. A somewhat
greater decrease in viability was observed in samples of frozen tomato soup, possibly
because of the lower pH of the product. Gianfranceschi and Aureli [155] investigated
survival of two L. rnonocytogenes strains in five foods (spinach, mozzarella cheese, cod
fish, chicken breast, and beef hamburger) during initial quick freezing at -50C for 57 min
and subsequent frozen storage at - 18C for 240-300 days. Quick freezing and subsequent
storage reduced viable Listeria populations by only 0.1- 1.6 and 0- 1.O log, respectively.
Injury among survivors ranged from a nondetectable level to 90%. In contrast, Palumbo
and Williams [284] did not recover injured listeriae from a variety of frozen foods tested
except for tomato soup.

Thermal Inactivation
Thermal processing is the most widely used method to preserve food and to destroy harm-
ful microorganisms, thus rendering food safe for human consumption. The established
association of L. monocytogenes with raw milk in the 1950s gave rise to several early
studies dealing with the possible resistance of this organism to pasteurization. In 1983,
interest in this topic was revived as a result of a listeriosis outbreak in Massachusetts
that was epidemiologically linked to consumption of pasteurized milk. The literature now
contains a wealth of information on thermal resistance of L. rnonocytogenes in a wide
variety of foods. Although data concerning heat resistance of L. monocytogenes in fluid
milk will be presented now, the discussion regarding thermal inactivation of listeriae in
other foods has been reserved for other chapters which deal with the incidence and behav-
ior of Listeria spp. in meat, poultry, seafood, and products of plant origin.
Early studies on the thermotolerance of L. rnonocytogenes gave controversial results
because of the methods used in measuring heat resistance. The 1983 outbreak suggested
that L. rnonocytogenes, at levels that may exist in milk, can survive minimal high-tempera-
ture short-time (HTST) pasteurization. Subsequent studies on freely suspended cells
showed that minimal HTST pasteurization is adequate; however, results from investiga-
tions on resistance of intracellular L. rnonocytogenes were in conflict. Further efforts by
the US Food and Drug Administration (FDA) [44,63,240], Centers for Disease Control
and Prevention (CDC) [ 13,14,18], and the World Health Organization (WHO) [393] sup-
port HTST pasteurization as a safe process. The following is a discussion of these aspects
of thermal inactivation of L. monocytogenes given in the sequence just outlined.
Conflicting Results in Early Literature
Numerous conflicting reports concerning the unusual heat resistance of L. rnonocytogenes
in milk can be found in the early literature. In 1951, Potel [307] demonstrated that L.
Characteristics of Listeria monocytogenes 137

monocytogenes died rapidly in milk held at 80C. However, the following year, Ozgen
[283] reported that L. monocytogenes survived 15 s at 100C. In 1955, Stenberg and
Hammainen [364] published results of a study which examined the heat resistance of five
L. monocytogenes strains in milk at different pasteurization temperatures. Using small-
diameter capillary tubes filled with inoculated milk, these researchers demonstrated that
L. monocytogenes was not completely inactivated until the milk was held at 65C for 5
min, 75C for 2 min, or 80C for 3-5 min. Thermal resistance of L. monocytogenes also
was studied by Stajner et al. [362]. When milk contained approximately 5 X 105L. mono-
cytogenes CFU/mL, the organism survived heat treatments of 71 and 74C for 42 s but
did not survive heating at 85 and 95C for 15 and 5 s, respectively. In 1957, Dedie and
Schulze [82] examined thermal resistance of 54 strains of L. nzonocytogenes in milk using
0.2- to 0.3-mm diameter capillary tubes. According to their results, L. rnonocytogenes
survived 30--40 s at 65"C, 10 s at 75OC, and -1 s at 85C. Ikonomov and Todorov [182]
used a pilot plant-sized tubular glass pasteurizer to examine heat resistance of Listeria
in milk obtained from ewes and cows. The milk was pasteurized (63-65"C/30 min), inocu-
-
lated to contain 107- 108L. monocytogenes CFU/ml, and then repasteurized at tempera-
tures between 63 and 74C. They found that the pathogen survived 20 min at 63"C, 10
rnin at 65OC1, 3 rnin at 68"C, 1 rnin at 7OoC, 20 s at 72"C, and <20 s at 74C. Thus
results of virtually all these early studies indicate that L. monocytogenes can survive HTST
pasteurization at 71.6"C for 15 s.
Variability in Results Caused by Thermal Inactivation
Methods
Several different approaches have been used to determine thermal resistance and have
given rise to conflicting results. Findings from the early pasteurization study of Bearns
and Girard [28], which included an "open-tube" heating procedure became highly suspect
during the 1980s. Their experimental approach involved inoculating 20 X 150-mm screw-
capped test tubes of sterile skim milk with approximately 5 X: 10' to 5 X 107L. monocyto-
genes CFU/ml. All tubes were placed in a water bath at 61.7"C so that the milk surface
was 3-4 cm below the water level in the water bath. Tubes were held in a wire test tube
rack attached to a mechanical shaker and were allowed to bounce in the rack to aid in
mixing. Results obtained from direct plating of milk on Tryptose Agar (TA) indicate that
L. monocytogenes survived 35 min at 61.7"C provided that the organism was present at
an initial level 2 5 X 104CFU/mL. From these data, the authors calculated a D61.70C value
(i.e., the time necessary to decrease the population 90% at 61.7"C) of 10.9 min, which
indicates that L. monocytogenes, if present at populations > 103CFU/mL, can survive vat
pasteurization at 61.7"C for 30 min.
Using the method of Beams and Girard [28], Donnelly et al. [94] demonstrated that
complete inactivation of L. monocytogenes in milk with an initial population of 106-1O7
Listeria CFU/mL cannot be accomplished within 30 rnin at 62, 72, 82, or even 92C.
Extensive tailing of survivor curves was observed after an initial 3- to 4-log decrease
during the first 5 rnin of heating. These investigators concluded that the open-tube method
of Bearns and Girard [28] is unreliable to determine thermal inactivation rates of microor-
ganisms, and they offered several explanations for their conclusion. One explanation is
that condensate and splashed cells accumulated on the test tube cap, which was above
the level of water in the water bath and, therefore, not exposed to thermal-inactivation
temperatures. Condensate containing listeriae would be expected to drip back into the
heating menstruum, thus eventually establishing a constant low population of survivors.
138 Lou and Yousef

A more likely explanation is that the test tube walls were coated with cells of Listeria
during initial mixing. The test tubes were not completely submerged in the water bath;
therefore, cells on the test tube wall would not be exposed to thermal-inactivation tempera-
tures. Since a constant surface area is presumably coated with listeriae, low levels of
survivors likely would be detected throughout the inactivation process. Concurrent studies
by Donnelly et al. 1941 using a sealed-tube method demonstrated that L. monocytogenes
was rapidly inactivated in milk at 62C. The sealed-tube method involved adding I .5 ml
-
of sterile whole milk inoculated to contain 107L. monocytogenes CFU/mL to a 2-ml
vial, sealing it, and then submerging the vial in a water bath at the desired temperature
for various times. In contrast to results of Bearns and Girard [28], thermal-inactivation
profiles obtained by the sealed-tube method were linear for three strains of L. monocyto-
genes during the entire inactivation period and gave rise to D620C values between 0.1 and
0.4 min depending on the strain of bacterium. From the aforementioned results, it is appar-
ent that the inactivation rate for L. monocytogenes at pasteurization temperature depends
on the method used to study heat resistance of the bacterium. In 1987, Beckers et al. [29]
compared thermal resistance of L. manmytogenes in TB using an open-tube and sealed-
bag method and obtained results similar to those of Donnelly et al. [94] just described.
Heating in sealed tubes may produce anaerobic conditions in the heating menstruum.
It is well known that anaerobic recovery of heat-injured cells of many pathogens, including
L. monocytogenes, results in higher D-values [208,234]. Knabel et al. [208] found that
after heating a Listeria-broth mixture in thermal death time (TDT) tubes, the broth at the
bottom of the tubes became anaerobic, as indicated by the color change of the redox
potential indicator, resazurin. Although no color change was found in milk in TDT tubes,
some degree of anaerobiosis may also exist.
The shortcomings of the open-tube procedure can be eliminated by using the capil-
lary tube method [ 122,238,356,357,3661. In this method, a small sample of culture or
inoculated liquid food (e.g., 40 1 L ) is introduced into a sterile capillary tube (e.g., 0.8-
1.10 X 100 mm) and both ends are carefully heat sealed. The sample is then heat treated
for a specified time after which the capillary tube is rapidly cooled, sanitized, and crushed
inside a large test tube. The released sample is then diluted appropriately and plated to
enumerate survivors. Compared with oiher methods, the capillary tube procedure allows
uniform heating of the sample and minimizes come-up and cooling-down times. When
compared with the capillary tube, other methods require a larger sample size and thus a
significant part of the heat treatment occurs during come-up and cooling-down times. The
amount of heat the sample receives during the coming-up and cooling-down times must
be calculated to avoid inaccurate D-values. The capillary tube method was used by several
investigators to determine the thermal resistance of L. monocytogenes in liquid media
and foods [ 122,2381. Overall, thermal inactivation rates for L. rnonocytogenes were linear
throughout the entire course of heating in the range of 50-75C.
Thermal Inactivation of Freely Suspended
L. monocytogenes
As a result of the 1983 listeriosis outbreak in Massachusetts that was epidemiologically
linked to consumption of pasteurized milk, Bradshaw et al. [42] investigated thermal resis-
tance of freely suspended L. monocytogenes in raw milk. A culture of L. rnonocytogenes
strain Scott A (serotype 4b, clinical isolate associated with the outbreak in Massachusetts)
-
was diluted in phosphate-buffered water and inoculated into raw milk to yield 105CFU/
mL. Portions of 1.5 mL were dispensed into 13 X 100-mm borosilicate glass tubes, which
Characteristics of Listeria monocyt ogenes 139

were sealed and immersed in a water bath at temperatures ranging between 52.2 and
71.7"C. Inoculated samples of raw milk also were heated in a slug flow heat exchanger
at 7 1.7 and 74.4"C. Thermal processing of inoculated milk samples at seven temperatures
between 52.2 and 74.4"C led to D-values ranging between 28. I min and 0.7 s, respectively,
including a I)717C of 0.9 s and a Dh33oc of 19.9 s. These investigators also noted that the
thermal resistance of strain Scott A remained unchanged over a 2-year period. Survivors
from some heating trials also were tested and were no more heat resistant than the parent
culture, which suggests that the extensive tailing observed by Bearns and Girard [28]
cannot be explained on the basis of heat-resistant spontaneous mutants.
Working in France in 1988, Lemaire et al. [227] used open vessels and sealed capil-
lary tubes to assess resistance of L. monocytogenes strains to vat and HTST pasteurization,
respectively. When samples of inoculated milk were held at 60C in open vessels, DhOoC
values for 38 different L. monocytogenes strains ranged from 1.3 to 6.5 s. In contrast,
D7yC values of 0.06-1.5 s were obtained when L. monocytogenes was heated in sealed

capillary tubes, with strains of serotype 1 being generally more heat resistant than those
of serotype 4. These findings along with those of Bradshaw et al. [42] indicate that current
minimum vat (61.7"C/30 min) and HTST (7 1.6"C/ 15 s) pasteurization requirements estab-
lished by the FDA are probably adequate to destroy expected levels of L. monocytogenes
in raw milk.
In 1986, Donnelly and Briggs [92) reported results of a study that examined the
influence of milk composition and incubation temperature on thermal resistance of L.
monocytogenes. The researchers inoculated five L. monocytogenes strains into sterile
whole milk, skim milk, and reconstituted nonfat milk containing 11% solids. Following
-
incubation, they heat-treated milk containing I Ox L. monocytogenes CFU/mL in sealed
glass vials at temperatures between 55 and 65C. Thermal resistance of L. monocytogenes
was not significantly affected by prior growth in skim, whole, or reconstituted nonfat
milk with I I % solids. Additionally, thermal inactivation experiments using the most heat-
resistant strain resulted in D550C and D6,0cvalues of 24.0 and 0.1 min, respectively, and a
z-value of 4.3"C. After extrapolating the linear thermal inactivation plot through 7 I .7"C,
the authors concluded that the most heat-resistant strain used in their study would be
unable to survive in whole milk during HTST pasteurization.
Going one step further, Bradshaw et al. [43], in 1987, examined the thermal resis-
tance of L. monocytogenes strain Scott A in raw, autoclaved, and commercially sterile
whole milk (-3.25% milk fat) and raw and autoclaved skim milk (<0.5% milk fat).
-
Products were inoculated at 105L. monocytogenes CFU/mL, and thermal resistance
was determined by the sealed-tube method. Listeria-inactivation studies done with raw,
autoclaved, and commercially sterile whole milk yielded I371 7 c cvalues of 0.9, 2.0, and
2.7 s, respectively, indicating significantly ( P 5 .05) greater survival in presterilized than
in other samples of whole milk. When heated in presterilized skim milk, the D7,70cvalue
for strain Scott A was 1.7 s. Although their data indicate that L. monocytogenes should
not survive in properly pasteurized raw whole milk, their other findings raise questions
concerning the adequacy of pasteurization to inactivate L. monocytogenes in reprocessed
products.
Working in Canada, Farber et al. [ I361 inoculated 1200 L of raw whole milk to
contain 105L. monocytogenes (a mixture of 10 strains including Scott A) CFU/mL. After
heating milk at 60-72C for 16.2 s in a pilot plant-sized regenerative plate pasteurizer,
L. monocytogenes was recovered from milk heated up to 67.5"C but was not recovered
from milk processed at 69 or 72C. Scientists from the FDA [240] also found that L.
140 Lou and Yousef

monocytogenes cells (2.6 X 105CFU/mL) freely suspended in raw milk were inactivated
by the minimum HTST pasteurization process.
Thermal inactivation of L. monocytogenes in reconstituted nonfat dry milk (NFDM)
was investigated by El-Shenawy et al. [122] in 1989. Suspensions of L. monocytogenes
cells in reconstituted NFDM (10% solids) were placed in capillary tubes which were
heated in a water bath at 50, 55, 60, 65, 70, and 75C for various times. Overall, thermal
inactivation rates for L. monocytogenes were linear throughout the entire course of heating
with estimated D62.80C- and D7,,70C-valuesof 20 and 0.94 s, thus reaffirming that pasteuriza-
tion as defined by the FDA should inactivate freely suspended cells of L. monocytogenes.
In 1991, Bradshaw et al. [44] reported that, in raw and sterile milk, other Listeria species
were no more heat resistant than L. monocytogenes. Therefore, properly pasteurized milk
should be free of all Listeria spp.
Although L. monocytogenes was more resistant in presterilized or repasteurized milk
than in raw milk, all investigations described thus far showed that HTST pasteurization
will inactivate L. monocytogenes when the pathogen is freely suspended in milk at levels
up to 105CFU/mL. In contrast, Fernandez Garayzabal et al. [141] reported, in 1987, that
the pathogen, when inoculated at high levels into raw whole milk, could survive minimum
pasteurization. The Spanish researchers inoculated milk to contain 3 X 106, 1 X 107,or
2 X 10 L. monocytogenes CFU/mL and pasteurized the milk at 72 or 73C for 15 s
(HTST method) in a pilot plant-sized pasteurizer. Using cold enrichment, Listeria was
detected in five of seven batches of milk treated at 72C for 15 s, with an estimated
D720c-valueof 1.8-2.1 s. Listeria, however, was not detected in the three pasteurization
trials at 73C for 15 s. This experiment was criticized by Lovett et al. [240] for possible
overloading of the pasteurizer. Large initial populations may have been a factor in promot-
ing Listeria survival during pasteurization in the study of Fernandez Garayzabal et al.
[ 1411. Additionally, since numbers of L. monocytogenes in naturally contaminated raw
milk are typically very low, these findings do not discount the adequacy of minimum
HTST pasteurization.
The heat resistance of L. monocytogenes Scott A in heavy cream (38% milk fat)
and pasteurized ice cream mix (-10.6% milk fat) was investigated by Bradshaw et al.
[43] using the sealed tube method. The organism had D6x,90c-values of 6.0 and 7.8 s in
raw and autoclaved cream, respectively. Thermal processing of pasteurized ice cream mix
at 68.3, 73.9, and 79.4C resulted in D-values of 231.0, 31.5, and 2.6 s, respectively.
Again their data indicate that L. monocytogenes should not survive in properly pasteurized
ice cream mix and that the pathogen had increased heat resistance in reprocessed products.
Holsinger et al. [173] investigated the effect of components of ice cream mix on thermal
resistance of L. monocytogenes. The D600C of L. monocytogenes in the mix correlated more
closely with the level of high-fructose corn syrup solids (HFCSS) or stabilizer (guar gum
and carrageenan) than with that of milk fat. Therefore, higher thermal resistance was
conferred by higher levels of HFCSS or stabilizer.
Although the aforementioned studies were all done with cows milk, MacDonald
and Sutherland [243] compared the thermal resistance of L. monocytogenes in sheeps
and cows milk using the sealed-tube method. The authors found that sheeps milk had
a protective effect on L. monocytogenes during heating at 65C when compared with cows
milk. When Listeria was initially present in milk at 106-107 CFU/mL, a count of s103
Listeria CFU/mL was observed after heating the sheeps milk ( 5 and 10% fat) for 45 min
at 65C; however, when cows milk and sheeps skim milk were treated similarly, no
survivors were detected by direct plating. Thus milk fat in sheeps milk protected Listeria
Characteristics of Listeria monocytogenes 74 7

during heating, whereas milk fat in cow's milk did not provide similar protection. When
whole sheep's milk was inoculated to contain 1O6 L. monocytogenes CFU/mL and pasteur-
ized at 68, 70, 72, and 74C for 15 s in an APV plate pasteurizer, the pathogen was only
detected in milk processed at 68OC, which indicates the adequacy of minimum HTST milk
pasteurization. However, caution should be exercised when interpreting these data, since
detection of Listeria in this study was done by direct plating of pasteurized milk on a
selective agar rather than using a preenrichment procedure for enhanced detection of suble-
thally injured cells.
Therma I Inactivat ion of Intrace1Iular L. rnonocytogenes
Studies discussed thus far have dealt with thermal inactivation of freely suspended cells
of L. rnonocytogenes in milk and other fluid dairy products. However, in cases of naturally
acquired listerial mastitis, the pathogen is normally not freely suspended in milk but rather
exists as a facultative intracellular bacterium within phagocytic leukocytes (neutrophils
and macrophages) typically present in milk. The facultative intracellular nature of L. rnono-
cytogenes has led some investigators to speculate that cells of the pathogen inside leuko-
cytes may be partially protected from thermal inactivation and thus are more able to sur-
vive pasteurization than are freely suspended cells of the bacterium in milk.
Intracellular L. rnonocytogenes cells induced by in vitro methods [45,93] were used
in early studies [61,64]. In 1986, Bunning et al. [61] determined thermal resistance of
L. rnonocytogenes in parallel experiments using freely suspended bacteria in raw milk as
well as L. rnonocytogenes cells that were inside of mouse peritonea1 phagocytes. Phago-
cytes were elicited in mice by injecting 107heat-killed L. monocytogenes (strain Scott A)
cells into the peritoneum and then were harvested by peritonea1 lavage. Differential stain-
ing indicated that the cell preparation was made up of 70% macrophages, 25% neutrophils,
and 5% lymphocytes. Opsonized cells of L. monocytogenes (i.e., incubated in normal
mouse serum at 37C for 30 min) were incubated in the phagocytic suspension for 60
min to allow phagocytosis. Phagocytes containing listeriae (average of 2.7-19.1
organisms/cell) were washed thrice by centrifugation and suspended in raw milk to obtain
- l O5 intracellular ListerialmL. Thermal resistance determinations were done using the
sealed-tube method of Bradshaw et al. [42,43] described earlier in this chapter. Mean
D-values for suspensions of intracellular L. rnonocytogenes in raw milk held at 52.2, 57.8,
63.3, and 68.9"C were 3170.0, 490.0, 33.3, and 7.0 s, respectively, as compared with
D-values of 2290.0, 445.0, 33.4, and 7.2 s when freely suspended cells were heat treated
at the same temperatures. Extrapolation of the data led to 1 ~ 7 1 . 7 0 C -of~ a1.9 e ~ 1.6 s
l ~and
for phagocytized and freely suspended listeriae, respectively. Under these experimental
conditions, the intracellular position did not appreciably protect L. rnonocytogenes from
thermal inactivation during pasteurization.
Subsequently, several methods were developed to obtain bovine phagocytes con-
taining internalized cells of L. monocytogenes, and such phagocytes have proven useful
in evaluating thermal resistance of intracellular listeriae. Briggs et al. [45] enhanced pro-
duction of bovine phagocytes (93% neutrophils, 5% macrophages, and 2% lymphocytes)
by infusing Escherichia coli endotoxin into the mammary gland. This procedure produced
an average of 2.4 X 106phagocytes/mL of milk, of which 89% were viable. Although
only 39% of the endotoxin-induced phagocytes ingested 1,. monocytogenes (average of
27 listeriadphagocyte) as compared with 64% of normal bovine phagocytes, no difference
in bactericidal activity was observed between endotoxin-induced and normal phagocytes.
In another study, Donnelly et al. [93] developed an in vitro assay to analyze uptake of
142 Lou and Yousef

L. monocytogenes cells by bovine phagocytes. Somatic cells harvested from fresh mastitic
milk were composed of 6 1% neutrophils, 20% macrophages, and 19% lymphocytes. Al-
though 75% of the neutrophils ingested opsonized L. monocytogenes cells as compared
with only 41% of the macrophages, both cell types contained an average of 19 listeriae
per phagocyte. Maximum Listeria uptake by phagocytes occurred within 30 min of incuba-
tion at 37C. Following ingestion, listeriae were resistant to the bactericidal activity of
phagocytes.
In 1988, Bunning et al. [64] reported results of a study comparing thermal resistance
of freely suspended and phagocytized cells of L. monocytogenes, the latter having been
prepared as previously described using endotoxin-induced bovine phagocytes [45]. Sterile
-
whole milk was inoculated to contain 1 Oh intracellular (average of 26 bacterialphago-
cyte) or freely suspended (obtained by sonicating phagocytes) L. monocytogenes cells/
mL and heated at 57.8, 62.8, 66.1, and 68.9"C using the sealed-tube method or at 66.1,
68.9, 7 1.7, and 74.4"C using a slug flow heat exchanger. Using the sealed-tube method,
the predicted D,,,80c-valuefor intracellular L. monocytogenes was 53.8 s, indicating a safe
33.4-D margin of inactivation for vat pasteurization (62.8"C/30 min). Using the slug flow
heat exchanger, D 7 1 , 7 0 C - ~ apredicted
lue~ from linear regression analysis were 4.1 s for intra-
cellular and 2.7 s for freely suspended listeriae. Hence, the intracellular position of
L. monocytogenes did not significantly (statistically) increase heat resistance under the
defined parameters of this study. More important, these results indicate potentially unsafe
3.7- and 5.6-D margins of inactivation for intracellular and freely suspended listeriae,
respectively, using the present minimum HTST pasteurization requirements (7 1.7"C/ I5 s).
The aforementioned data on heat resistance of intracellular L. monocytogenes were
obtained using phagocytes that were artificially induced to engulf listeriae. Heat resistance
of intracellular L. monncytogenes cells in milk from naturally or artificially infected cows
was investigated by several groups of researchers [9 1,96,136,240].A study that examined
heat resistance of L. rnonocytogenes in milk from a naturally infected cow was reported
in 1962 by Donker-Voet [91]. Milk from this cow contained 2 X 103-2X 104extracellular
listeriae and >I O6 leukocytes/mL but otherwise appeared completely normal. Although
no attempt was made to examine bovine phagocytes for intracellular listeriae, the organism
was presumably present in some of the leukocytes. After pooling the milk for a week and
holding it at 4"C, milk was heated in a plate-pasteurizer at 54-76.5"C for 15 s and then
examined for surviving Listeria cells. Unfortunately, by the time enough milk was ob-
tained for a pasteurization trial, the milk was heavily contaminated with other microorgan-
isms, making isolation of listeriae from milk extremely difficult. Furthermore, leukocytes
may have disintegrated, and the bacterial cells they may have contained were liberated
and became freely suspended cells in the milk. I n this study, L. monocytogenes survived
a heat treatment of 59.0"C for 15 s but did not survive in milk heated at 262.3"C for
15 s. This experiment was repeated using naturally contaminated milk from the same cow
that was held for only 2 days at 4C. Pasteurized milk was added to the contaminated milk
to increase the volume of milk available for pasteurization. Although the initial Listeria
population was not determined in the diluted milk before heating, L. monocytogenes was
detected in milk processed at 63.7"C for 15 s. However, L. monocytogenes was not found
in milk heated at 66.3, 68.0, 70.0, or 72.8"C for 15 s.
Pasteurization studies using L. monocytogenes-contaminated milk obtained from
cows artificially infected with the bacterium were conducted in 1987 by Doyle et al. 1961.
A laboratory culture of L. monocytogenes strain Scott A was inoculated into the udder of
each of four Holstein cows. Once listerial mastitis had developed, milk from these animals
Characteristics of Listeria monocytoge nes 743

was pooled and held 2 days (and in one instance 4 days) at 4C until sufficient quantities
were available to process in a pilot-scale plate pasteurizer (Cherry Burrell, model 217SB-
1) at 71.7-73.9"C for 16.4 s (nine trials) or 76.4-77.8"C for 15.4 s (three trials). Before
pasteurization, milk contained < 102- 1.9 X 10'' free Listeria cells and 4.5 X 105-2.4 X
1O6 somatic cells/mL. In addition, the milk generally contained 103- 1O4 L. monocytogenes
cells within polymorphonuclear leukocytes (PMNLs) per milliliter (average of 1.5-9.2
listeriae/PMNL). During pasteurization, 1 00-mL samples of milk were taken after 2, 4,
and 6 min of operation and analyzed for L. monocytogenes using two direct-plating and
three enrichrnent procedures. L. monocytogenes was isolated from milk in six of nine trials
in which the milk was heated to 71.7-73.9"C for 16.4 s. In contrast, L. morzocvtogenes
was not detected in milk from the remaining three trials in which the milk was processed
at 76.4-773C for 15.4 s. Additional studies on the fate of L. monocytogenes within
PMNLs indicated that the organism was no longer detectable in PMNLs after 3 days of
storage at 4C. Disappearance of listeriae after 3 days was accompanied by partial degrada-
tion of PMNLs, with complete breakdown occurring after 4 days. These findings suggest
that holding raw milk at 4C for 4 or more days would elirninate any thermoprotective
effect for listeriae that might result from their engulfment by PMNLs.
In the study just described, Doyle et al. [96] contended that phagocytized L. monocy-
togenes (<U. 1% infectivity with 1.5-9.2 listeriae/PMNL) cells were protected during pas-
teurization of milk at 71.7-73.9"C for 16.4 s. In contrast, using an in vitro method of
phagocytosis, Bunning et al. [64] reported that the intracellular position of L. monocyto-
genes (42% infectivity with 26 Listerialphagocyte) did not augment heat resistance of the
organism despite much larger numbers of engulfed listeriae in this study than in that of
Doyle et al. [96]. Lack of agreement between these two studies might be the result of the
bacterium being in different physiological states. It is well known that bacteria are gener-
ally more heat resistant during the stationary than the logarithmic phase of growth. Thus
nongrowing listeriae within bovine phagocytes may be more heat resistant than actively
growing cells that are engulfed by phagocytes or are freely suspended in milk.
In 1988, Farber et al. [ 1361, in Canada, tested raw milk containing 103-104L. mono-
cytogenes CFU/mL (- 105 somatic cells/mL with 10-50% of macrophages containing
- 1-20 listeriae/macrophage) which was obtained from a naturally infected cow, pooled
for 2.0-2.5 days, held at 4C and then heated in a pasteurizer. According to these authors,
L. monocytogenes survived heat treatments of 64 and 66C for 16.2 s but failed to survive
processing at 2 67C for 16.2 s.
Knabel et al. [208] realized that if L. monocytogenes is shed from infected cows
which have developed fever, the pathogen may have grown at elevated temperatures. They
also believed that Listeria cells inside macrophages are exposed to anaerobic conditions.
Therefore, a study was initiated to investigate the heat resistance of L. rntinocytogenes in
relation to its growth at elevated temperatures before heat treatment and anaerobic recov-
ery of heat-treated cells. The investigators heat treated L. monocytogenes (previously
grown at 37 or 43C) in sterile, whole, and homogenized milk and compared thermal
resistance data obtained when the heat-treated cells were recovered by incubation under
aerobic and anaerobic conditions. When Listeria was grown at 43C before heating and
recovered by anaerobic incubation after the treatment, the D62suc-value was 243 s as com-
pared with 36 s when the pathogen was grown at 37C and plated aerobically after the
heat inactivation. The researchers concluded that if L. monocytogenes is present at high
levels in milk, it could survive the minimum low-temperature, long-time milk pasteuriza-
tion process. Their results about the effect of anaerobic recovery also suggested that a few
144 Lou and Yousef

heat-injured cells remaining after minimum HTST milk pasteurization may grow under
anaerobic conditions that may exist in phagocytes. The investigators suggested that previ-
ous studies included (a) sample preparation practices such as sonication or mechanical
agitation in the presence of glass beads that may have disrupted phagocytes in milk, and
(b) aerobic plating may not have permitted recovery of heat-injured cells, thus D-values
for such studies were underestimated. Bunning et al. [63] argued that the homogenization
of raw milk at all milk processing plants will disrupt the phagocytes and that anaerobic
conditions did not exist in heat-treated milk as suggested by Knabel et al. [208].
In response to the previous study [208], Farber et al. [ 1331 investigated the impact
of growth temperature (30, 39, and 43C) and anaerobic incubation on recovery of L.
monocytogenes (a mixture of 10 strains) during milk pasteurization in a regenerative plate
pasteurizer at 63, 66, 69, and 72C for a minimum holding time of 16.3 s. The milk was
preheated at 85C for 1 h and cooled before inoculation and then held at 4C overnight
to simulate commercial holding practice. Four detection procedures, direct plating, a three-
tube most probable number (MPN) method, cold enrichment, and a warm enrichment
procedure were used and combined with both aerobic and anaerobic incubation. The milk
was inoculated to contain 5.0 X 104L. rnonocytogenes CFU/mL, which possibly represents
a worst-case situation. Consistent with the study of Knabel et al. [208], Listeria grown
at higher temperatures were more heat resistant. When the milk was pasteurized at 72"C,
L. monocytogenes was detected in four of four, two of five, one of four, and zero of four
trials when the cells were grown at 43, 39 (with 3 days of holding at 4"C), and 3OoC,
respectively. Therefore, L. monocytogenes cells grown at higher temperatures can survive
the minimum HTST milk pasteurization process. Although anaerobic incubation did not
appreciably enhance recovery of Listeria by direct plating, survivors in five of seven trails
at 72C were only detected under anaerobic conditions. An approximate D720c-valueof
8.1 s was calculated for Listeria grown at 43C. Increasing the holding time at 4C from
overnight to 3 days decreased the heat resistance of this pathogen.
Lovett et al. [240] investigated inactivation of both freely suspended and intracellu-
lar L. monocytogenes Scott A during the minimum HTST pasteurization (71.7"C, 15 s)
in a two-phase slug flow heat exchanger. Freely suspended listeriae were obtained by
inoculating raw milk to contain 2.6 X 105L. rnonocytogenes CFU/mL. Raw milk was
also inoculated to contain 5 X 104Listeria CFU/mL, of which 3-91% (average of 54%)
were intracellular, obtained through an in vitro internalization process. Raw milk for heat
treatment was also obtained from experimentally infected cows; the milk contained 3.4 X
103L. monocytogenes CFU/mL, with 53% being internalized. Three different enrichment
procedures were followed for detection of L. monocytogenes in pasteurized milks. The
researchers did not detect L. monocytogenes in any of the 23 minimum HTST milk pasteur-
ization trials.
Recognizing the impact of heat shock on thermotolerance, Bunning and coworkers
[63] studied the effect of heat shock on inactivation of L. monocytogenes during minimum
HTST pasteurization of whole milk. Heat shocking (48"C, 15 min) of Listeria in milk
increased the D71.70C from 3.0_+ 1.0 to 4.6 2 0.5 s; the latter value is comparable to that
for intracellular L. monocytogenes as measured in a previous study [64]. The authors
considered D71.70c-values after heat shock and those obtained with intracellular Listeria as
representing the upper limit of heat resistance in Listeria. However, after assessing the
data through risk analysis, Bunning et al. [63] believed that this increase in D71.7"C is
not a convincing reason to raise the minimum HTST milk pasteurization temperature
(71.7"C, 15 s).
Characteristics of Listeria monocytogenes 145

Pasteurization Efficacy: Concluding Remarks

In the past, effectiveness of pasteurization was measured by the ability of this treatment
to rid milk of Mycobacterium bovis and Coxiella burnetti, the most heat-resistant non-
spore-forming human pathogens known at that time. After L. rnonocytogenes was con-
firmed as a serious food borne pathogen, adequacy and safety of heat treatments commonly
used in food processing, particularly the pasteurization process, were questioned. At least
three research groups [96,133,141] found that L. monocytogenes survived the minimum
HTST milk pasteurization process if the pathogen was present in sufficiently high num-
bers. However, numerous studies with freely suspended, intercellular, or heat-shocked L.
monocytogenes cells showed that the minimum HTST pasteurization is a safe process.
Alarmed by a few reports on unusually high heat resistance of L. monocytogenes, some
food processing authorities gage effectiveness of pasteurization by ability of the treatment
to inactivate L. monocytogenes. These authorities also consider pasteurization-equivalent
treatments adequate when such treatments eliminate at least 6 logs of L. monocytogenes.
These are the arguments that one must consider when making a conclusion about
adequacy of pasteurization: (a) the safety margin of minimum pasteurization (under condi-
tions, however, not commonly encountered in commercial processing) is not as great as
many scientists originally believed, (b) contamination levels of L. rnonocytogenes in com-
mingled commercial raw milk are much lower than those used in most thermal-inactivation
studies, (c) recovery of a few injured Listeria cells in pasteurized milk, if they exist, is
doubtful, (d) homogenization of milk destroys macrophages, thus protection of Listeria
against heat by cellular internalization is unlikely, and (d) the thermoduric microflora in
pasteurized milk is likely to compete with any surviving L. monocytogenes. Therefore,
we along with Ryser and Marth [333], the CDC [13,14,18], FDA scientists [44,63,240],
and the WHO [393] conclude that pasteurization is a safe process which reduces the
number of I,. monocytogenes occurring in raw milk to levels that do not pose an apprecia-
ble risk to human health. Although the minimum HTST milk pasteurization is considered
a safe process, most raw milk processing facilities have wisely adopted pasteurization
temperatures well above the minimum legal limit.

Thermotolerance Induced by Stress Adaptation

In nature, L. monocytogenes may be subjected to various environmental stresses, such
as high and low temperature, acidic and oxidative conditions, and starvation [ 145,2611.
Environmental stresses can induce stress-adaptative or stress-protective responses. For
example, incubating a microorganism at a high but sublethal temperature will induce the
so-called heat-shock response. Stress adaptation occurs in all bacteria, including L. mono-
cytogenes. Resistance of L. rnonocytogenes to heat or other lethal factors can be greatly
increased by heat shock or adaptation to other stresses. In this section, we will discuss
heat resistance of L. monocytogenes in relation to stress adaptation and implications of
the adaptive response in food processing.
Bacteria respond to heat shocking by synthesizing new proteins, termed heat-shock
proteins (HSPs) [3,76]. Induction of the heat-shock response or HSPs usually increases
the thermotolerance of microorganisms. As opposed to the intrinsic or basic thermotoler-
ance of microorganisms, heat-shock-induced thermotolerance is transient and nonherita-
ble and thus is called acquired or adaptive thermotolerance [388]. Temperatures at which
microorganisms are heat shocked affect the magnitude of the acquired (i.e., induced) ther-
146 Lou and Yousef

motolerance. Optimal heat-shock temperatures for maximal thermotolerance in mesophilic

organisms with a wide range of growth temperatures are usually between 45 to 50C; that
is, 10- 15C above the microbe's optimal growth temperature [232]. L. monocytogenes
has optimal heat-shock temperatures in this range [ 13 11. The magnitude of heat-shock-
related thermotolerance is also affected by the length of exposure to heat shocking, heating
menstruum, heating rates, physiological state of Listeria cells, and the method used to
recover injured cells.
Heat shocking L. monocytogenes in laboratory broth under optimal or near optimal
heat-shock conditions increased the heat resistance, expressed as D-values, by severalfold
[63,139,189,233]. Bunning et al. [63] found that heat shocking L. monocytogenes at 48C
for 15 rnin before heating in sterile, whole bovine milk increased the D,, 7uc-valuefrom
3.0 k 1.0 s (control) to 4.6 t 0.5 s. Feido and Jackson I1391 reported a heat-shock-
induced increase at D60oC from 3.9 min (control) to 17. I min. Linton et al. [233] observed
that DssoC-valuesof logarithmic-phase L. monocytogenes, after heat shock at 48C for 20
min, increased 2.3-fold; however, heat shocking as such failed to change the z-value, the
temperature change required to cause a 90% change in the D-value.
Quintavalla and Campanini [309] found that thermotolerance of L. monocytogenes
in a pork emulsion was two to three times more than it was in broth. Heat resistance of
microorganisms also can be greatly increased by the presence of NaCl and sucrose in the
heating menstruum. Curing salts increased heat resistance of the pathogen in beef samples
[2451.
The physiological state of the organism affects the magnitude of heat-shock-induced
thermotolerance. Heat shocking log-phase L. monocytogenes at 48C for 10-20 rnin in-
creased the DSsoc-value by more than twofold [233,234]. In contrast, heat shocking station-
ary-phase cells of L. monocytogenes in a sausage mix at 48C for 30 or 60 rnin did not
significantly increase thermotolerance, although heat shocking for 120 rnin increased the
D640C-value by 2.4-fold [ 1311.
Growth temperature affects the thermotolerance of L. monocytogenes. Heat resis-
tance of Listeria cultures usually increases as the growth temperature increases; however,
growing cells in the range between refrigeration and their optimum growth temperature
had no or only a slight effect on heat resistance of the bacterium [92,189,290,353,354].
Growth at temperatures above 37C mimics heat-shock conditions. When heated at 52C
for 1 h, L. monocytogenes cultures that were grown at 37 and 42C showed 3-4 logs
more survivors than did the cultures incubated at lower temperatures (5, 10, 19, and 28C)
[354]. Other studies proved that growing L. monocytogenes at an elevated temperature
(43C) significantly increased the resistance of the pathogen to heat [208,133]. Patchett
et al. [290] found that heat resistance (Dssoc-values)of L. monocytogenes growing at 10
or 30C in continuous cultures was not significantly different.
Recovery of heat-injured cells of L. monocytogenes or E. coli 0 157:H7 by anaerobic
incubation or adding exogenous oxygen scavengers, catalase, or superoxide dismutase
(SOD) to the plating medium increased measured D-values [203,207,208,234,27 1,2911.
Heating can completely inactivate catalase and SOD [79], and thus makes the heated
organism an obligate anaerobe. Anaerobic incubation or adding catalase or SOD probably
prevents formation of oxygen-derived compounds (such as superoxide and H202)which
are toxic to injured cells. Knabel et al. [208] found that growth at 43C before heat inacti-
vation in combination with anaerobic recovery of the injured cells resulted in heat resis-
tance (D6280C) that was at least sixfold greater than when the pathogen was grown at 37C
and enumerated under aerobic conditions. Heat-shocked (42"C, 10 min) logarithmic-phase
Characteristics of Listeria monocytogenes 147

L. monocytogenes cells had a D550c-valueof 18.7 min and 26.4 min when the enumeration
plates were incubated aerobically and anaerobically, respectively [234]. Incubation at 25C
improved recovery of injured L. monocytogenes cells [64].
Conditions similar to heat shock exist in food processing. Slow heating or cooking,
preheating, hot water washing, mild thermal processes, and holding food in warm trays
(as occurs in food service establishments) are examples of heat shock that may happen
during food processing and handling. Heat shock may result, as suggested by Farber and
Brown [ 131 1, when foods are minimally processed or when the food is too bulky to allow
rapid heating. Heat shock may occur during vat pasteurization of dairy products or produc-
tion of "sous-vide" processed refrigerated foods, both of which involve a long-time tem-
perature coming-up and low-temperature heating/cooking [ 2331. The thermotolerance of
L. monocytogenes, Salmonella typhimurium, and Enterococcus faecium was increased by
low heating rates [203,244,309,3 10,3651. Quintavalla and Campanini [309] found that L.
monocytogenes became more heat resistant during slow (OS"C/min) rather than fast heat-
ing. A nearly twofold increase in the D-value for L. monocytogenes was noted by Kim
et al. [203] when the pathogen was heated in ground pork at I.3"C/min compared with
8.OoC/min. Stephens et al. [365] investigated heat inactivation of a 17-h-old culture of L.
monocytogenes (Scott A) in Tryptic Phosphate Broth at 50--64"C by both instantaneous
heating (adding a small portion of concentrated cells into a large volume of preheated
medium) and slow heating (0.7- 1 1 "C/min). Compared with instantaneous heating, slow
heating at a rate between 0.7 and 5"C/min significantly increased the heat resistance of
L. monocytogenes. This increase was maximal at a heating rate of I0.7"Clmin with a
population 1.7 X 105-foldhigher than that after instantaneous heating.
The heat-shock-induced thermotolerance of L. monocytogenes persists for a variable
time. Acquired thermotolerance of stationary-phase L. monocytogenes lasted at least 24
h at 4C in a sausage mix [131], < 1 h at 35"C, and 2 4 h at the heat-shock temperature
(42C) [62].
Besides heat shock, adaptation to other environmental stresses may also increase
the thermotolerance of pathogens. Farber and Pagotto [ 1341 found that exposing a station-
ary-phase culture of L. monocytogenes to a laboratory broth at pH 4.0 for 1 h rather than
2 or 4 h increased the DSROc-value in sterile whole milk from 2.75 to 3.90 min. A gradual
decrease of pH to 4 during 4 or 24 h also significantly increased heat resistance. Replacing
HC1 with acetic acid failed to increase heat resistance [ 1341. Lou and Yousef [238] found
that starvation and adaptation of L. monocytogenes to sublethal levels of HCl, ethanol,
and hydrogen peroxide significantly increased the thermotolerance. Maximum thermotol-
erance was observed in cells exposed to 4-8% (v/v) ethanol, pH 4.5, and 500 ppm hydro-
gen peroxide; the corresponding averages of Ds60Cin a phosphate buffer (pH 7.0) were
4.1, 8.8, and 2.9 min, whereas nonadapted L. monocytogenes cells had a D560C of 1.O min.
In phosphate buffer, starvation at 30C for up to 163 h increased the D-value of the re-
maining viable cells to 13.6 min (2381. Sudden osmotic shock (holding cells in a Tryptic
Phosphate Broth with 3.0-9.0% [w/v] NaC1) and osmotic adaptation (growth at high NaCl
concentrations) significantly increased the thermotolerance of L. monocytogenes Scott A
[ 1901. Thermotolerance of L. monocytogenes at 60C increased during osmotic up-shift
until the cells became almost as heat resistant as the culture grown for 48 h at the same
high osmotic conditions. Increased thermotolerance was rapidly lost (<5 min) during an
osmotic down-shock [ 1901.
Heat-shock and other environmental stresses also affect the virulence of L. monocy-
togenes. Environmental stresses are sensed by pathogens as signals for expression of viru-
148 Lou and Yousef

lence factors to enhance survival [20,235,257,28 I]. Heat shocking may increase the viru-
lence of L. rnonocytogenes. When L. rnonocytogenes was heat shocked at 48C for 2 h,
listeriolysin 0 was almost totally lost; however, subsequent growth of the heat-shocked
cells at 37C resulted in a 40-fold increase in production of the listeriolysin, whereas
unshocked cells exhibited only a 2-fold increase [202].

ACIDITY
Growth a t Low pH
According to Bergey 's Manual of Systematic Bacteriology [34 11, L. monocytogenes can
only grow at pH values from 5.6 to 9.6, with optimal growth occurring at neutral to slightly
alkaline pH values; the latter was verified by Petran and Zottola [300]. The minimum pH
value for growth is based on the work of Seeliger [340], who, in 1961, reported that L.
monocytogenes failed to grow in dextrose (glucose) broth at pH <5.6 after 2-3 days of
incubation at 37C. In addition, subcultures from the medium were no longer routinely
successful.
Listeriosis outbreaks linked to consumption of fermented dairy products have re-
opened the issue of a minimum pH requirement for growth which has now been revised
downward. During 1987, Lang et al. [219] examined growth at 13C of L. monocytogenes
(strain Ohio, isolated from recalled Liederkranz cheese) in TB adjusted to pH 5.0 and 5.6.
Following lag periods of 2.0 days at pH 5.0 and 0.5 day at pH 5.6, the pathogen grew
and reached maximum populations of 1.5 X 108and 4 X 10' CFU/mL in TB adjusted
to pH 5.0 and 5.6, respectively. During logarithmic growth, the organism exhibited genera-
tion times of 13.1 and 4.4 h in media adjusted to pH 5.0 and 5.6, respectively. Thus,
although Listeria failed to grow in TB at pH 5.0 during the initial 2 days of incubation,
further incubation led to growth of the organism with maximum populations being reached
after approximately 21 days at 13C.
Subsequent investigations have shown that L. rnonocytogenes can proliferate in labo-
ratory media adjusted to even lower pH values. When inoculated into Trypticase Soy
Broth acidified with hydrochloric acid, according to George et al. [153], all 16 L. monocy-
togenes strains tested initiated growth at pH values as low as 4.39-4.63 during extended
incubation at 20 or 30C. Although results from other independent studies
[40,15 1,288,3591confirm the ability of L. monocytogenes to multiply in similar laboratory
media adjusted to pH 4.4-4.6 with hydrochloric, citric, or malic acid, Farber et al. [135]
observed growth of L. monocytogenes at 30C in double-strength BHI broth acidified with
hydrochloric acid to a pH value as low as 4.3. Furthermore, L. innocua, L. seeligeri, and
L. ivanovii also were reported to grow in BHI broth acidified with hydrochloric acid to
pH values as low as 4.53, 4.88, and 5.16, respectively [151]. Thus the minimum pH at
which L. monocytogenes and most other Listeria spp. can grow is well below pH 5 pro-
vided that these organisms are incubated at near-optimum temperatures and allowed suffi-
cient time to overcome an extended lag phase.
As might be expected, growth of Listeria at low pH values is markedly influenced
by incubation temperature and the type of acid added to the medium; the latter will be
discussed in some detail later in this chapter. In one study [397], TB previously adjusted
to pH 5.0 and 5.6 was inoculated to contain -1 X 103L. monocytogenes CFU/mL and
then incubated at 4 and 13C. Listeriae not only failed to grow when incubated in TB
Characteristics of Listeria monocytogenes 149

(pH 5) at 4"C, but populations of the bacterium decreased clO-fold during 67 days of
storage. In contrast, increasing the incubation temperature to 13C led to growth at pH
5, with the organism attaining a final population of -1 X 10' CFU/mL. George et al.
[ 1531 found that minimum pH values for growth of 16 L. monocytogenes strains in Trypti-
case Soy Broth increased in the range of 4.39-5.45 as the temperature of incubation de-
creased from 30 to 4C. Sorrells et al. [359] reported that four different L. monocytogenes
strains grew in TB acidified to pH values as low as 4.40 following 7-28 days of incubation
at 10C, whereas growth of this organism at pH 4.4 was previously only observed at
220C. Hence, some L. monocytogenes strains may be able to grow, albeit slowly, in
laboratory media adjusted to pH 4.4 and incubated at near-refrigeration temperatures. Bu-
chanan and Klawitter [54] also reported a similar effect of incubation temperature on
growth of Listeria in TPB acidified to pH 4.5 with HCl. At 37"C, L. monocytogenes Scott
A was completely inactivated after 50 h of incubation, with populations, remaining stable
at 10 and 5C. However, at 28 and 19"C, the organism grew to -107 and -10' CFU/mL
in -100 and -500 h, respectively.
Growth of L. monocytogenes in acid or acidified foods confirms the findings in
laboratory media. In a study prompted by the listeriosis outbreak in Canada linked to
consumption of contaminated coleslaw [337], Conner et al. [74] demonstrated that L.
monocytogenes can tolerate and, in some instances, grow in cabbage juice at pH values
<5.6. Juice expressed from fresh cabbage was adjusted with lactic acid to pH values of
3.8-5.6, inoculated with L. rnonocytogenes at 104CFU/mL, and incubated at either 5 or
30C. After 3 days at 3OoC, Listeria reached maximum populations of -109 CFU/mL in
cabbage juice which had an initial pH 2 5.2. Rapid growth of listeriae during this period
was followed by equally rapid destruction, with the organism being no longer detectable
after -15 days at 30C. In cabbage juice adjusted to pH 5 and incubated at 3OoC, Listeria
exhibited a 3-day lag period and then grew to maximum populations 2 10' CFU/mL after
7 days of incubation before numbers decreased. At pH 5 4.8, L. monocytogenes was
inactivated in samples incubated at 30C. Although incubation at 5C prevented growth
of L. monocytogenes in cabbage juice adjusted to pH 5 5.6, listeriae populations remained
constant in samples at pH 2 5.2 during 22 days of storage. Interesting findings were
reported by Parrish and Higgins [288] on potential growth of' L. monocytogenes in orange
juice with modified pH. Initial Listeria populations of 106 CFU/mL increased approxi-
mately 1 and 2 logs in orange serum adjusted to pH values of 4.8 and 5.0, respectively,
during the first 2 days of incubation at 30C before decreasing to nondetectable levels 6
days later.
In 1988, Ryser and Marth [331J examined growth of L. monocytogenes at different
pH values in whey collected during manufacture of Camembert cheese. Samples of whey
were adjusted to pH values between 5.0 and 6.8, filter sterilized, inoculated to contain
5 X 10'-1 X 103L. monocytogens (four strains) CFU/mL, and incubated at 6C. Although
no growth occurred in whey at pH 5 5.4, small numbers of the organism survived during
the entire 35-day storage period. In contrast to the study involving cabbage juice [74], all
four strains grew in whey at pH 5.6 after 3 days of incubation at 6C. Under these condi-
tions, the four Listeria strains had generation times ranging between 25.3 and 3 1.6 h and
-
attained maximum populations of 1 X 107CFU/mL after 24 days at 6C. As expected,
L. monocytogenes had significantly (P < .OS) shorter generation times in whey samples
at pH 6.2 (14.8-21.1 h) and pH 6.8 (14.0-19.4 h) than at pH 5.6. The organism also
attained higher final populations in whey at pH 6.2 and 6.8 than at pH 5.6.
150 Lou and Yousef

Survival at Low pH
Although growth of L. monocytogenes at pH < 4.3 has not yet been documented, this
organism appears to be fairly acid tolerant. According to Reimer et al. [314], L. monocyto-
genes was recovered from inoculated samples of citrate/phosphate buffer that were acidi-
fied to pH 3.3 and held 4 h at 37C. However, the pathogen survived < I h in a similar
buffer adjusted to pH 1.4. Resistance of L. monocytogenes to acid was measured in D-
values by Ahamad and Marth (41. The bacterium exhibited average D-values of 13.3 and
11.3 days when held at 7C in TB previously adjusted with citric acid to pH values of
4.0-4.1 and 3.6-3.7, respectively. Since these authors obtained average D-values of only
2.2 and 1.4 days for corresponding cultures incubated at 35C L. monocytogenes can
clearly tolerate exposure to acid far better at near-refrigeration than at ambient tempera-
tures. Such behavior raises concerns about the safety of certain refrigerated acid and low-
acid foods that are often subjected to postprocessing contamination. Growth temperature
and growth rate before acid challenge also affect acid resistance of L. rnonocytogenes.
Patchett et al. [290] measured the acid tolerance of continuous cultures of L. monocyto-
genes that were grown at different growth rates or temperatures (10 and 30C). At the
same growth rate, L. monocytogenes grown at the higher temperature was more acid resis-
tant, whereas at the same temperature (30"C), cultures grown at a slower growth rate were
more acid tolerant.
Survival of Listeria in acid foods also varies with pH and temperature of storage,
as previously observed with laboratory media. Conner et al. [74) demonstrated that inacti-
vation rates for L. monocytogenes in acidified cabbage juice were inversely related to pH
with the organism surviving 49 days at pH 5.0-4.8 as compared with <2 I days in samples
of cabbage juice adjusted to pH 4.6 and 4.4. Parrish and Higgins [288] investigated behav-
ior of L. monocytogenes at 4C in inoculated (- 10' CFU/mL) samples of orange serum
adjusted to pH values of 3.6-5.0. Survival of the pathogen ranged between 21 days at
pH 3.6 and >90 days at pH 4.8 and 5.0 with slight growth of listeriae during storage
limited to orange serum adjusted to pH 5.0. Listeria was inactivated faster at higher rather
than lower incubation temperatures, with the pathogen being eliminated after 5 and 8 days
from orange serum at 30C and adjusted to pH values of 3.6-4.0 and 4.2-5.0, respectively.
Although acidic fruit juices appear to be unlikely sources of L. monocytogenes, the fact
that this pathogen survived well beyond the normal shelf life of nonsterile orange juice
(orange serum) suggests that such products should not automatically be eliminated as
possible vehicles of infection in future epidemiological investigations of human listeriosis.
Proper acid development is critical to the safety and quality of fermented foods.
Behavior of L. monocytogenes in these foods depends on numerous extrinsic and intrinsic
factors, including the pH. Camembert [329] (a mold-ripened cheese), Brick cheese [332],
and white pickled cheese [ 1) supported growth of L. monocytogenes, with the pH of these
cheeses being 5.9-7.2, 6.9-7.3, and >6.0, respectively. In contrast, the bacterium was
inactivated rapidly in Parmesan [403], mozzarella [50], and water-buffalo mozzarella
cheese [378], with final pH values of these cheeses being 5.0-5.1 , 5.2-5.3, and 4.0, respec-
tively. In most other cheeses investigated, L. monocytogenes survived to various degrees.
The bacterium persisted at least 28 days in creamed and uncreamed cottage cheese at pH
5.02-5.68 [327], 70 to 2434 days in Cheddar cheese at pH 5.0-5.15 13281, > 1 15 days
in Colby cheese at pH 5.0-5.18 [401], 270 days in semihard Manchego-type cheese at
pH 5.10-5.80 [90], 290 days in Trappist cheese at pH 4.70-5.42 [214] and feta cheese
at pH 4.6 [287], <66-80 days in Swiss cheese [49], >50 days in blue cheese [286], and
Characteristics of Listeria monocytogenes 75 7

2180 days in cold-pack cheese food without preservatives at pH 5.21-5.45 [330]. Viable
counts of L. monocytogenes decreased in cottage cheese stored at 4- 12C [ 170,3021.Simi-
lar studies concerned with behavior of L. monocytogenes in fermented meats have shown
that this bacterium can survive in hard salami at pH 4.3-4.5 during refrigerated storage
[ 1881. When cows milk was inoculated to contain 103and 107Listeria CFU/mL, made
into yogurt, and stored at 4OC, the pathogen survived for 2 and 7 days, respectively, at
pH 4.2-5.0 [25 I]. Other investigators, however, reported that L. monocytogenes remained
viable 13-27 days in yogurt stored at 4C [70]. Survival of the pathogen in yogurt was
reduced when milk was fermented at 42C with thermophilic starters compared with fer-
mentations that were done at 37C with mesophilic starters [335].Although these and
other studies will be discussed in greater detail in later chapters of this book dealing with
behavior of Listeria in dairy and meat products, it may be concluded that L. monocytogenes
is unlikely to initiate growth in food products which have a pH 5 5.2.

Acid Adaptation and Acidoduric Properties

Acid adaptation can enhance survival of many microorganisms, including L. monocyto-
genes, when exposed to lethal acidic conditions. Extensive investigations on acid adapta-
tion have been done with Salmonella typhimuriurn and Escherichiu coli 1145,3251, but
fewer reports have dealt with L. rnonocytogenes [ 147,216,239,2751. Kroll and Patchett
[216] investigated the effect of acid shocking on growth and survival of L. monocytogenes
in Yeast Dextrose Broth at 37C. Acid shocking at pH 3.0 or 3.5 for 20 min or preincuba-
tion at pH 5.0 did not affect the growth rate of L. monocytogenes at pH 7.0, but the lag-
phase was prolonged by acid shocking at pH 3.0. Prior incubation at pH 5 rather than pH
7, increased survival of L. monocytogenes by 3 logs during acid shock at pH 3 for 40
min. Adaptation of exponentially growing L. monocytogenes for 1 h at 35C to three acidic
conditions, (a) pH 5.0, (b) pH 4.5, or (c) pH 5.0, followed by additional incubation at pH
4.5 significantly (P < .OS) increased survival at pH 3.5 in a citrate/phosphate buffer [239].
Acid resistance of the pathogen was significantly greater after adaptation to the mild acidic
conditions (a) or after stepwise increase to the high acid-condition (b) than to the high-
acid conditions (c) alone. The authors suggested that food fermentations, which involve
a gradual lowering of pH, could lead to acid adaptation of L. monocytogenes. ODriscoll
et al. [275]obtained acid-adapted L. monocytogenes by incubating exponentially growing
cells for 1 11 at 37C in Tryptic Soy Yeast Extract Broth (TSYEB) acidified to pH 5.5 with
lactic acid. This treatment markedly decreased inactivation of L. monocytogenes when the
bacterium was inoculated into the same medium at pH 3.5. Exposure to pH 3.5 for 1 h
reduced the population of unadapted cells by 3 logs, whereas numbers of acid-adapted
cells decreased <1 log. The authors found that lactic and acetic acid were more effective
than hydrochloric acid in inducing acid-adaptive responses in L. monocytogenes. De novo
synthesis of acid stress proteins is presumably required for induction of the acid-toler-
ance response [275].
Acid adaption also cross protects L. monocytogenes against a variety of deleterious
factors such as lethal doses of hydrogen peroxide, heat, NaC1, ethanol, and certain surface
active hydrophobic compounds [ 134,238,239,2751. Since acid adaptation increases the
general resistance, including acid tolerance, it is not surprising that acid-adapted cells of
L. monocytogenes, like those of S.typhimurium [228] and E. coli 0157:H7 [229], survive
better in both acidic and fermented foods than do unadapted cultures [ 1471. Compared
with unadapted cultures, acid-adapted (at pH 5.5 with lactic acid) L. monocytogenes cul-
152 Lou and Yousef

tures and unadapted cultures of an acid-tolerant mutant showed enhanced survival during
storage of cottage cheese (pH 4.71) for 15 days at 4OC, ripening of Cheddar cheese (pH
5.16-5.25) for 70 days at 8OC, storage of yogurt (pH 3.9) for 48 h at 4OC, active milk
fermentation (pH <4.8 or <5.5), and storage in acidic foods such as salad dressing (pH
3.0) and orange juice (pH 3.76) for 7 h at 4C. However, no significant differences were
seen in survival of both types of cells in mozzarella cheese (pH 5.6). The acid-adapted
L. monocytogenes culture and its acid-tolerant mutant ( 105CFU/mL) were partially inacti-
vated in yogurt during 48 h of storage at 4C with -3 and -5 log reductions for these
two types of cells, respectively, whereas the unadapted control was inactivated within
24 h. When salad dressing (pH 3, attained by adding acetic acid) was inoculated to contain
106L. monocytogenes CFU/ml and stored at 4OC, the unadapted cells were completely
inactivated in 15 min, whereas both types of acid-adapted cells survived up to 90 min.
L. monocytogenes (105 CFU/mL) also was added to milk being actively fermented with
S. thermophilus at 37C when the pH decreased to 4.8 or 5.5. During an additional 7 h
of fermentation (pH was reduced to 4.15), populations of unadapted L. monocytogenes
cells decreased >3 logs, whereas numbers of adapted and mutant cells decreased 5 1 log
[147].
O'Driscoll et al. [275] found that long-time acid challenge selected for acid-tolerant
mutants of L. monocytogenes. This is contrary to findings of Buchanan et al. [53]that no
subpopulation of acid-tolerant L. monocytogenes developed after treatment with a combi-
nation of 1.0% lactic acid, 6.3% NaCl, and 100 pg/mL NaN02at 19OC, a condition known
to cause tailing of inactivation curves. This discrepancy may have resulted from differ-
ences in methods used in these two studies to select mutants.
According to O'Driscoll et al. [275], acid-tolerant mutants have increased virulence
compared with that of the parental cells. Intraperitoneal injection of 105CFU mutant cells/
mL into mice resulted in death of three of four infected mice, whereas none of the mice
infected with parental cells showed any signs of infection. Additionally, higher counts of
mutant rather than parental L. monocytogenes were found in the spleen after injection
[275]. Virulent and avirulent Listeria strains also respond differently to stress [268]. Aviru-
lent strains of L. monocytogenes did not multiply [78] or were completely inactivated
[ 1691 inside macrophages, whereas virulent strains survived and multiplied inside the mac-
rophage [78,169]. Therefore, considering that the increased acid tolerance of acid-adapted
or acid-tolerant mutants of L. monocytogenes may help the pathogen to survive inside
macrophages, and that L. monocytogenes can produce hemolysin over a wide range of
pH values (55-29) [200], it would not be surprising to observe the increased virulence
of acid-adapted or constitutively acid-tolerant cells as demonstrated by O'Driscoll et al.
[275].
Environmental stresses presumably are used by L. monocytogenes and other patho-
gens as signals for expressing virulence factors and enhancing survival [20,211,212,235].
Although weak organic acids and their salts inhibit growth of L. monocytogenes, these
compounds may enhance the virulence of this pathogen, and so should not be over-
looked in assessing the safety of preserved foods. Kouassi and Shelef [211] tested the
effect of salts of five weak acids on growth of L. monocytogenes and associated secretion
of listeriolysin 0, the exotoxin important for the spread of the pathogen, in TSB (pH
7.2-7.4) at 35 and 20C. Citrate, acetate, lactate, and propionate increased secretion of
listeriolysin 0, with only sorbate inhibiting secretion of this toxin. The inhibitory effect
of sorbate was later confirmed by the same authors [212]. McKellar [255] found that
Characteristics of Listeria monocytogenes 153

listeriolysin 0 is stable at >pH 5.3 (lactic acid), and has maximum activity at pH
4.0-5.0.

WATER ACTIVITY
The moisture requirement for microbial growth can best be expressed in terms of water
activity (a,), which is defined as the ratio of the water vapor pressure of a food substrate
to the vapor pressure of pure water at the same temperature. Like most bacterial species,
L. monocytogenes grows optimally at a, -0.97 [300]. However, when compared with
most foodborne pathogens, this bacterium has a rather unique ability to multiply at a,
values as low as 0.90.
L. monocytogenes can grow in complex laboratory media containing up to 10%
NaCl [341]. Skovgaard [351] estimated the a, of such a medium at -0.93 and therefore
predicted that L. monocytogenes would not grow at a, < 0.93. The minimum a, for growth
of L. monocytogenes estimated by Skovgaard was confirmed by Sperber [360]. Using
liquid laboratory media adjusted with NaCl to various a, values, growth of L. monocyto-
genes at 35C was observed at an a, of 0.943 but not at 0.935. Similarly, adjustment of
a, using sucrose and glycerol allowed growth of L. monocytogenes at minimum a, values
of 0.941 and 0.932, respectively. More recently, growth of L. monocytogenes at lower a,
was observed by Petran and Zottola [300], Sorrells and Enigl [358], and Miller [260].
The bacterium grew in TSB containing 39.4% sucrose (a, = 0.92) when incubated at
30C for 24 h [300] or in BHI broth containing 12% NaCl (a, -0.92) during incubation
at 10 and 25C [358]. Tapia de Daza et al. [370] found that when the a, of TSB was
adjusted with glycerol, sucrose, or NaCl, two strains of L. monocytogenes grew minimally
(determined as detectable turbidity of the culture in 20 days) at a, values of 0.90, 0.92,
and 0.93 at 3OoC, and 0.92, 0.93, and 0.94 at 4"C, respectively. Nolan et al. [274] found
the minimum growth (at least 1 log increase in 22 days at 2 1 "C) a, of L. monocytogenes
in TSBYE to be 0.90, 0.92, and 0.92 when water activity was adjusted with glycerol,
NaC1, and sucrose, respectively. When L. monocytogenes Scott A was grown at 28C in
BHI broth adjusted to different a, values (0.99-0.80) with glycerol, NaC1, or propylene
glycol; the minimum a, values for growth at 28C were 0.90, 0.92, and 0.97, respectively
[260].
Although L. monocytogenes does not appear to grow at a, < 0.90, the bacterium
can survive for extended periods at lower a, values. Shaharnat et al. [343] reported that
the bacterium survived at least 132 days at 4C in Trypticase Soy Broth containing 25.5%
NaCl, which would be expected to have an a, of -0.83. Survival of L. monocytogenes
under reduced moisture depends on both the a, and the dominant solute in the medium.
In BHI broth adjusted to the same a, values, the pathogen survived longest with glycerol
and shortest with propylene glycol and NaCl yielded intermediate survival [260]. Nolan
et al. [274] also reported generally shorter survival of L. monocytogenes in NaC1-adjusted
than in sucrose-or glycerol-adjusted TSYEB.
Survival of L. monocytogenes during processing and storage of food may depend
on a, of the medium. When sucrose/phosphate buffer solutions were inoculated to contain
- 104-105 L. monocytogenes CFU/mL and held at 140"C, Sumner et al. [369] found that
the pathogen was about four times more heat resistant in buffer having an a, value of
0.90 as compared with 0.98. Thus, given the inverse relationship between a, and thermal
resistance along with the ability of L. monocytogenes to grow at an a, value of 0.92 and
754 Lou and Yousef

ferment concentrated sucrose solutions, this organism also may be important to companies
that manufacture foods containing high levels of sugar, as has already been demonstrated
for Karo corn syrup stored at refrigeration temperatures 12841.
Of more practical importance to food processors, Johnson et al. [ 1881 found that L.
monocytogenes survived at least 84 days at 4C in fermented hard salami which had an
a, between 0.79 and 0.86. Extended survival of listeriae in sausage occurred despite the
presence of 5.0-7.8% NaCI, 156 ppm sodium nitrite, and a pH of 4.3-4.5. The authors
suggested that L. monocytogenes might survive longer at an a, of 0.9 1, which is occasion-
ally found in commercial hard salami. However, they also predicted that growth of the
bacterium in such sausage would be unlikely given the combination of salt, sodium nitrite,
low pH, and low storage temperature.
Additional information concerning the relationship between a, and growth/survival
of L. monocytogenes can be obtained from several dairy-related studies. Using pasteurized
whole milk inoculated to contain -500 L. monocytogenes CFU/mL, Ryser and Marth
[328] manufactured Cheddar cheese which, according to Marcos and Esteban [250], had
a, values between 0.972 and 0.979. The organism survived as long as 224 and >434
days in Cheddar cheese (pH 5.0-5.1) ripened at 13 and 6OC, respectively. Since Listeria
reportedly grows well within this a, range, the combined effects of low pH and low-
ripening temperature probably played a dominant role in preventing growth of listeriae.
Camembert cheese, also prepared by Ryser and Marth [329], had a, values between 0.959
and 0.984 [250], which should have allowed growth of L. monocytogenes. However, lister-
iae populations remained constant or decreased in cheese at pH 4.6 to -5.5 during the
first 20-30 days of ripening. Initiation of rapid Listeria growth in cheese at a pH between
-5.5 and 6.0 illustrates that pH rather than a, is primarily responsible for determining
growth characteristics of listeriae in Camembert cheese. Parmesan cheese was made of
pasteurized milk inoculated with 104- 10sListeria CFU/mL [4031. Listera was inactivated
rapidly in this cheese and was not detectable after 2- 16 weeks of ripening. A combination
of low moisture (30.1-3 1.4%), low pH (5.0-5. I), and heat treatment during curd cooking
(51C for -45 min) likely contributed to the rapid demise of Listera in this cheese.

ANTIMICROBIAL COMPONENTS IN FOOD

Some food components that are either naturally present or added during formulation and
processing have antimicrobial activity and thus contribute to food preservation. Of these
antimicrobial components, some are applied mainly to control foodborne microflora,
whereas others have dual or multiple functions. Control of L. monosytogenes by these
components has been heavily investigated during the past decade. The following is an
account of selected food components with antimicrobial activity in relation to control of
L. monocytogenes in food.

Salt (i.e., sodium chloride or NaCl), an important ingredient in defining the water activity
(a,) of many foods, affects microbial growth and survival in such foods. Salt, however,
also exerts antimicrobial effects that can not be explained by its ability to lower a food's
a,. Therefore, resistance of L. monocytogenes to salt and interaction of this important
ingredient with the pathogen are described here in some detail.
Characteristics of Listeria monocytogenes 755

Salt Tolerance
According to Bergey 's Manual of Systematic Bacteriology [34 11, L. monocytogenes can
grow in Nutrient Broth (NB) supplemented with up to 10% !w/v) NaCl. Although this
viewpoint concerning tolerance of Listeriu to NaCl is apparently based on results from
Larsen [224], preliminary data from one investigative team [lSl], reported in 1988, indi-
cate that one strain of L. monocytogenes grew at 8-30C during extended incubation in
-
BHI broth (pH 5.0) that contained up to 12% NaCl. Under identical conditions, single
strains of L. ivanovii, L. seeligeri, and L. innocua were only slightly less halotolerant,
with growth ceasing in the presence of >10% NaCl. In agreement with these findings,
Sorrells and Enigl [358] found that two strains of L. monocytogenes grew in TSB con-
taining 10% NaCl at 35C or 12% NaCl at 10 and 25C. Listeria may grow to high
numbers in the presence of moderate amounts of salt. Hudson [ 1771 inoculated BHI broth,
which contained 6.5% NaCl, with 106 CFU L. monocytogeizeslml. The bacterium in-
creased by at least 3 logs after 15 and 26 days at 10 and 0-4"C, respectively.
Lang et al. [219] found that growth of L. monocytogenes in TB containing 6% NaCl
was markedly influenced by pH. In their study, TB containing either 0 or 6% NaCl (w/
v) was adjusted to pH 5.0, 5.6, 6.2, and 6.8 with HCI, inoculated to contain -5 X 102
L. monocytogenes CFU/mL, and incubated at 13C. When grown in salt-free media at
pH 5.0, 5.6, 6.2, and 6.8, this organism had generation times of 13.1, 4.4, 3.5, and 2.9 h,
as compared with 77.8, 7.2, 5.0, and 6.3 h in the same medium containing 6% NaCl,
respectively. Thus the combination of pH 5 and 6% NaCl was most effective in inhibiting
growth of Listeria. Borovian 1401 also reported that L. monocytogenes grew at 10C in
culture media adjusted to pH 4.5 and 6.0 and containing 5 4 and 5 7 % NaCl, respectively.
These findings agree with those of Lang et al. [219].
Extended survival of listeriae occurs at a wide range of salt concentrations. Studies
at ambient temperatures demonstrated that L. monocytogenes can persist at least 150 days
in pure salt 13481 and 545 days in 0.85% NaCl 13051. In 1955, Stenberg and Hammainen
[364] reported that 10 L. monocytogenes strains survived > 1 year at 20-24C in NB
containing I % glucose and 10% NaCl. Listeriae also survived 34-68 days and 24 days
in the same medium containing 12 and 24% NaCI, respectively. When Stenberg and Ham-
mainen [364] stored organs (liver, heart, kidney) from Listeria-infected mice in salt solu-
tions at 4"C, L. monocytogenes remained viable for 238-246, 88-1 12, and 27 days in
solutions containing 3, 6, and 12% NaCI, respectively.
Survival of Listeria in the presence of salt varies with the storage temperature. In
experiments by Shahamat et al. [343], L. monocytogenes was inactivated in Trypticase
Soy Broth containing 10.5, 13.0, and 25.5% NaCl after 14, 9, and 4 days of incubation
at 37"C, respectively. Survival times in media containing 25.5% NaCl increased from 3
days at 37C to 24 days at 22C and to > 132 days at 4C. Sorrells and Enigl [358] found
that -106 CFU/mL of L. monocytogenes (two strains) in TSB, which contained 12 and
14% NaCI, decreased to a nondetectable level in 14-21, and 36 days at 35 and 25"C,
respectively, whereas reductions of only -2 logs occurred when listeriae were kept in
14% NaCl for 36 days at 10C. In a study by Hudson [ 1771, L.monocytogenes populations
(106 CFU/mL) in BHI broth containing 26.5% NaCI, decreased 4, 2, and 0 logs at 10,
0-4, and -- 18"C, respectively, after 33 days of storage with D-values of 6 and 19 days
at 10 and 0-4"C, respectively. Presence of 16.5% NaCl in the same medium did not affect
the Listeriii count after 33 days of storage at all three temperatures. These data indicate
156 Lou and Yousef

that survival by Listeria in concentrated salt solutions can be increased dramatically by

lowering the incubation temperature.
Several studies have examined the fate of L. rnonocytogenes in salted foods and
food-related products. Conner et al. [74] determined growth patterns of L. rnonocytogenes
in cabbage juice supplemented with 1 5 % NaCI. Two Listeria strains grew at 30C and
pH 6.1 in cabbage juice containing 1% NaCl but failed to grow in the presence of 11.5%
NaC1. In another study, Kukharkova et al. [218] demonstrated that L. rnonocytogenes
survived >60 days in meat stored at 4C in a 30% NaCl brine solution which also con-
tained nitrate. According to Sielaff [348], L. rnonocytogenes was detected in infected beef
that was immersed in a solution of 22% NaCl and stored 100 days at 15-20C. Results
just described indicate that this pathogen is likely to survive for long periods in salted
foods, particularly meat. The high osmotic tolerance of L. rnonocytogenes indicates that
immersing products such as cheese and salmon in brine solution (6-26% NaCl) is not a
reliable preservation method to control L. rnonocytogenes.
Physiological Response t o Salt
Studies by Brzin [47,48] during the mid 1970s demonstrated that L. rnonocytogenes under-
goes various morphological changes when grown in media containing high levels of NaCl.
Listeria cells were elongated (maximum length 55 pm) and filamentous when incubated
at 37 or 30C for 24 h on 5% human serum agar containing 8-9% NaCl and 0.4-0.6%
agar. Under these conditions, cell multiplication was inhibited without simultaneous inhi-
bition of cell growth (elongation). Attempts to grow listeriae on the same medium con-
taining 9% NaCl led to complete inhibition of cell division and either partial or total
cessation of cell growth, which ultimately led to fewer elongated and deformed cells.
Microscopic changes in Listeria cells grown on salt agar also were associated with
changes in colonial morphology. When incubated at 30 or 37C in the presence of 8-9%
NaC1, L. monocytogenes produced star-like colonies characterized by a rough surface,
irregular border, and longer than usual straight or coiled protrusions. Such colonies con-
tained large numbers of elongated filamentous cells. In addition to long twisted filamentous
forms, occasional fusiform and spheroplast-like forms also were observed, particularly
for cells from small colonies. In contrast, when grown on the same medium and incubated
at 22 or 10C, colonies became progressively smoother and tended to develop regular
borders, Cells from these colonies were less elongated and filamentous with microscopic
changes being most pronounced in cells from small rather than large colonies. Altered
morphological forms of L. rnonocytogenes persisted only as long as the bacterium was
grown on a medium containing 8-9% NaCl. Listeriae reverted back to their typical nonfil-
amentous, nonelongated form after 24 h of incubation on salt-free media. These results
were recently verified by Isom et al. [ 1831, who found that when L. rnonocytogenes was
grown in TSB, filament formation started above 1000 mM NaCl and peaked at 1200- 1500
mM. Interestingly, elongated cells also developed when L. rnonocytogenes was grown in
media that were (a) adjusted to pH 5-6 with citric acid, (b) adjusted to pH > 9, or (c)
supplemented to contain 11.75 mM H2O2.
Osmoprotectants or osmolytes, which are involved in osmotic shock response, are
required for growth and survival of L. rnonocytogenes in high-osmotic feeds. L. rnonocyto-
genes primarily utilizes glycine betaine (trimethylglycine), carnitine (P-hydroxy-y-N-tri-
methyl aminobutyrate), proline, and K+ as osrnoprotectants [289] with glycine being the
most effective and preferred [35]. Addition of 1 mM glycine betaine, 1 mM carnitine, or
10 mM proline significantly stimulated growth of L. rnonocytogenes at 10 and 37C in a
Characteristics of Listeria monocytogenes 157

minimal medium containing 3% NaCl, with final numbers reaching 109CFU/mL. A popu-
lation of only 10' CFU/mL was reached in the absence of osmoprotectants [35]. In accor-
dance with these findings, KO et al. [209] and Smith [352] reported that exogenous addition
of betaine stimulated growth of L. monocytogenes in a defined medium with a high content
of NaC1. Foods usually contain enough osmoprotectants to support microbial growth. Plant
foods are rich in betaine, whereas foods of animal origin are high in choline (the precursor
of betaine) and carnitine [35]. Processed meats (bologna, frankfurters, wieners, ham, brat-
wurst, salami) contain betaine at 0.34-0.48 nmol/mg and carnitine at 0.23-0.95 nmol/
mg [352]. Processed and ready-to-eat meats, which are high in salt and low in a,, can
contain L. monocytogenes. The capacity of L. rnonocytogenes to grow on the surface of
processed meats is reportedly related to the organism's ability to accumulate high levels
of betaine and carnitine (200- 1000 nmol/mg cell protein), with salami supporting neither
good growth nor the accumulation of betaine or carnitine [352].

Organic Acids and Their Salts

Growth and inactivation rates for L. monocytogenes vary markedly in the presence of
different acids. Most organic acids permitted in food are applied as acidulants (e.g., acetic
and lactic acids), whereas others, particularly their salt forms, are used as preservatives
(e.g., potassium sorbate and sodium benzoate). Effectiveness of these weak organic acids
as antimicrobial agents is related to the amount of the undissociated form present. Concen-
tration of the undissociated form of a weak organic acid which is related to pH of the
medium and the pK, of the acid can be calculated using the Henderson-Hasselbalch equa-
tion. For example, at pH 5,35.5% of acetic (pK, 4.74) and 5.8% of L-lactic acid (pK, 3.79)
will be undissociated. Undissociated organic acids can pass through the cell membrane,
dissociate inside the cytoplasm, and interfere with metabolic processes of the microbial
cell. The antimicrobial action of these acids is attributed to cytoplasm acidification, as
well as the specific antimicrobial effect of the particular anionic species.
A selection of organic acids and their salts will be addressed in relation to control
of L. monocytogenes in food. A more comprehensive account of these acids and their role
in food preservation can be found elsewhere [95]. The mechanism of microbial inhibition
just discussed appears applicable to some of the acids that will be discussed in this chapter.
However, it is not clear how some of the organic acid derivatives (e.g., parabens) inactivate
microorganisms or inhibit growth.
Acidifying Agents
Behavior of L. monocytogenes in media containing organic acids is affected by both pH
and the incubation temperature. Experiments to determine the effect of lactic acid on
growth of L. rnonocytogenes at different pH values and incubation temperatures were
conducted by Bojsen-Mgller [39]. Polymyxin-Tryptose Phosphate Broth containing 0,
0.003, 0.03, and 0.3 M lactic acid was adjusted to pH 5, 6, and 7 using HCl or NaOH,
-
inoculated to contain l O3 L. monocytogenes CFU/mL, and incubated at 35 or 4C. When
incubated at 35C and pH 5, Listeria populations decreased in broth that contained 0.3
and 0.03 M lactic acid, whereas rapid growth occurred with 0 and 0.003 M lactic acid
after a prolonged lag period. At pH 5 and 4"C, Listeria was eliminated after 27-42 days
from broth containing 0.3 M lactic acid; populations of the pathogen remained unchanged
in the same medium containing the three lower concentrations of lactic acid. At pH 6 and
in the presence of 0.3 M lactic acid, the listeriae population increased 2 logs at 35C and
158 Lou and Yousef

did not grow at 4C. However, at pH 6 and 4C Listeria growth occurred at the three
lower concentrations (0,0.03, and 0.003 M) of lactic acid. At pH 7, listeriae grew at 35"C,
regardless of lactic acid concentration; however, at 4C and the same pH, the pathogen
only grew in media containing 10.03 M lactic acid with a 7-day lag time.
Using an experimental design similar to that of Bojsen-MQIler [39], Ahamad and
Marth [4] examined the ability of acetic, citric, and lactic acid to prevent growth of L.
monocytogenes in TB during extended incubation at 7-35C. As expected, the pathogen
was markedly affected by type and concentration of acid as well as incubation temperature.
The presence of as little as 0.05% acetic acid (pH 5.8-5.9) caused noticeable inhibition
of Listeria, with deleterious effects of acetic as well as citric and lactic acid being more
evident at low rather than high incubation temperatures. Increasing the concentration of
acetic, citric, or lactic acid to 0.2% (pH 4.4-4.6) completely suppressed growth of the
organism at all incubation temperatures, with death of the pathogen occurring in the pres-
ence of 10.3% (pH < 4.2-4.3) acetic, citric, or lactic acid. In contrast, citric acid was
less inhibitory than acetic acid, with growth of listeriae occurring in all samples with 0.1%
citric acid regardless of temperature. The relationship between incubation temperature
and inhibition of L. monocytogenes was most pronounced with lactic acid; the pathogen
proliferated in the presence of 0.1% lactic acid at all temperatures except 7C. Results
from a follow-up study [ 5 ] showed that during extended incubation at both 13 and 35"C,
the presence of 0.3 and 0.5% citric acid in TB was most injurious to L. monocytogenes
followed in order by similar concentrations of lactic and acetic acid. Acid-injured listeriae
survived approximately nine times longer at 13" than 35C.
In 1989, Sorrells et al. [359] published results of a study that examined the effect
of pH, acidulant, time, and temperature on growth and survival of L. monocytogenes in
TSB acidified to pH values of 4.4-5.2 with hydrochloric, acetic, lactic, malic, or citric
acid. Based on average minimum pH values permitting growth of four L. monocytogenes
strains at 10, 20, and 35"C, acetic acid was again most inhibitory (pH 5.04) followed by
lactic (pH 4.73), citric (pH 4.53), malic (pH 4.46), and hydrochloric acids (pH 4.46).
These findings generally agree with those of Ahamad and Marth [4] and several other
investigators [33,116,135]. As in the previous study by Ahamad and Marth [4], longest
survival of listeriae occurred at lower rather than higher incubation temperatures. How-
ever, since the inhibitory activity of the various acids tested was markedly different when
based on equal molar concentrations of acid rather than pH, these data again indicate that
differences in antilisterial activity of acidulants depend on both type and concentration of
acid rather than on pH alone.
According to results just described and those from subsequent studies [4,184,213,
359,4001, acetic acid is most listericidal and listeriostatic followed by citric acid when
used on an equal weight (% w/w) or molar concentration basis at the same pH. However,
based on equal molar concentrations of undissociated organic acid at the same pH, the
order was reversed (i.e., citric >lactic ?acetic acid) for growth inhibition at 2 5 3 7 C
[51,400]. Differences in listericidal activity among the three acids were much greater at
low rather than at high concentrations, and diminished as the concentration of undissoci-
ated acid increased to 3 mM [5I].
Citric and lactic acids have different effects on survival of listeriae. Although high
levels of both acids inactivated listeriae in a similar pattern, low levels (0.1-0.5 M) of
citric acid, especially at pH 5-6, protected listeriae from death [51,521. Low concentrations
(50 mmol/mL) of citric acid were also noted by Young and Foegeding [400] to enhance
growth of L. monocytogenes at pH 4.7-5.0.
Characteristics of Listeria monocytogenes 159

Besides cytoplasm acidification caused by undissociated organic acids, specific in-

hibitory effects of these undissociated species on metabolic processes of L. monocytogenes
were also noted [ 184,4001. Ita and Hutkins [ 1841 grew L. monocytogenes in TSB (with
0.6% yeast extract), adjusted the pH of the culture to 6.5-3.5 using acetic, lactic, or citric
acid and held the culture for 4-6 h at 37C. Although at low external pH (pH,) values,
citric and lactic acids were more effective than acetic acid in lowering the intracellular
pH (pH,), acetic acid was the most bactericidal. After 24 h of incubation at pH 3.5, acetic
acid produced a pH, near S and decreased the population by -4 logs, whereas citric acid
decreased the pH, to <4 and only caused < I log reduction. Young and Foegeding [400]
also demonstrated that at an equal pH,, growth of Listeria was in this order: acetic >lactic
>citric acid. In a more recent study, Kouassi and Shelef [213] investigated the influence
of different salts of weak acids (0-5% sodium propionate, acetate, lactate, and citrate) on
metabolism of L. monocytogenes in a defined medium at pH 6.7-6.8 during incubation
at 35C. Cell growth was inhibited by 2 1 % propionate, 2 3 % acetate, and 2 5 % lactate,
with the relative inhibitory activity in this order: propionate > acetate > lactate > citrate.
Citrate at 5% only slightly inhibited growth. Of these salts, only lactate (1 -5%) supported
growth [213]
Lactates
Antimicrobial effects of sodium and potassium lactates were reviewed by Shelef [345].
These organic salts are used at 1-4% as additives in baked goods and meat and poultry
products. The mechanism of antimicrobial activity of lactates is not well understood; how-
ever, cytoplasmic acidification, specific anionic effect, a,-lowering and chelating action
may all contribute to the inhibitory properties. Generally, 2-4% of sodium or potassium
lactate is listeriostatic [345]. Combinations of lactate with NaC1, nitrate, or low tempera-
ture potentiated the overall antibacterial effect against L. monocytogenes [68,298,389].
Lactates, which do not change the pH of foods, were more effective in inhibiting microor-
ganisms including L. rnonocytogenes ( I O3 CFU/g) in meats than in laboratory broths 13451.
Shelef and Yang [347] found that >5% lactate was required to inhibit L. monocytogenes
in TSB whereas 2.6% lactate in meat inhibited growth at refrigeration temperatures and
4% lactate did so at all temperatures tested. These authors also noted that lactate was
more effective in comminuted beef than in comminuted chicken. In a subsequent study,
Chen and Shelef [68] examined the antimicrobial activity of three lactates (sodium, potas-
sium, and calcium) in cooked strained beef, which contained different moisture contents
and was stored at 20C. They found that all three lactates were equally effective in inhib-
iting growth of L. rnonocytogenes. In cooked strained beef prepared without lactates,
125% moisture (a, = 0.932) was required for complete growth inhibition, whereas at 55%
moisture (a, = 0.963), growth of L. monocytogenes (Scott A) was completely inhibited by
2 4 % sodium lactate or a combination of 2-3% lactate and 2% NaCI. When 103--104L.
monocytogenes CFU/g were inoculated into pork liver sausage containing 55% moisture
and 2% NaC1, incubation at 5C but not 20C increased the inhibitory effect of the three
lactate salts [389]. Although L. rnonocytogenes populations increased, 5 and 4.5 logs in
pork liver sausage at 20 and 5C after 10 and 50 days, respectively, the count of the
organism in the presence of 4% lactate changed by - 1.33 to 1.4 logs at 20C and by
- 1.49 to 0.88 log at 5OC, with calcium lactate being the most antilisterial. Results of
Pelroy et al. [298] are in accord with these findings. The investigators found that L. mono-
cytogenes (- 10 CFU/g) was completely inhibited in vacuum-packaged, cold-processed,
and smoked comminuted salmon containing 2% sodium lactate and 3% water-phase NaCl
160 Lou and Yousef

and stored for 40-50 days at 5C. Similar inhibition for up to 35 days at 10C was observed
in the presence of a combination of 3% lactate and 3% water-phase NaCl or of 2% lactate,
3% NaC1, and 125 ppm NaN02. L. monocytogenes grew appreciably in control samples
that contained 3% water-phase NaCl or 3% NaCl plus 125 ppm NaNO, [298]. The antilis-
terial activity of lactate in broth and bologna-type sausages was modeled recently by Hout-
sma et al. [175]; the model can be applied to predict Listeria growth in the presence of
lactates in this product.
In 1995, Buncic et al. [60] reported on the antilisterial activity of sodium lactate
and other antimicrobial agents in a buffered BHI broth (pH 5.5) held at 4C. When used
alone or in combination with sodium nitrite ( I 25 ppm) and/or polyphosphate (0.5%) so-
dium lactate (4%) prevented growth of L. monocytogenes (initial population 107CFUI
mL) during 7 weeks of incubation at 4C; however, no bactericidal effect was observed.
The antilisterial activity of sodium lactate was improved by addition of nisin (400 IU/
mL) but not 0.3% sorbate. Lactate and nisin had a synergistic effect against L. monocyto-
genes which was further enhanced by addition of polyphosphate (0.5%). Combinations
of lactatehisin and lactate/nisin/polyphosphatedecreased Listeria population by 2.2-2.4
and 4.2 logs after 28 and 20 days, respectively. In contrast, nisin alone only resulted in
an initial 1.1 -log decrease, but listeriae grew during prolonged refrigerated storage.
Sodium Diacetate
Sodium diacetate (CH,COOH CH,COONa), which contains acetic acid (about 40%) and
sodium acetate, is considered a generally recognized as safe (GRAS) additive by the
FDA. It is used as an acidulant, flavoring agent, and antimicrobial agent in foods [67,346].
Shelef and Addala [346] investigated the antilisterial activity of sodium diacetate in BHI
broth at 35, 20, and 5C. After adding sodium diacetate (18-35 mM) to BHI broth, the
resulting mixture of pH 6.3-5.25 was inoculated to contain about 103L. monocytogens
CFU/mL. Inhibition of L. monocytogenes increased with increasing diacetate levels and
decreasing incubation temperatures. The minimum inhibitory concentrations (MICs) of
diacetate in BHI broth were 35, 32, and 28 mM at 35, 20, and 5OC, respectively. Based
on equal levels of undissociated acetic acid at different pH values, sodium diacetate was
more effective and had lower MICs at 35C than did acetic acid; the MICs were 5 , 20,
30, 40, and > 100 mM for sodium diacetate, and 5 , 20, >50, >100, and > 150 mM for
acetic acid, at pH 4.7, 5.0, 5.5, 6.0, and 6.5, respectively.
The same study also assessed the antimicrobial activity of diacetate in meat [346].
Sodium diacetate added to ground beef (pH 5.6) or beef slurry (pH 5.6) greatly inhibited
growth of aerobic microflora during storage at 5C. Addition of 21 and 28 mM sodium
diacetate decreased the pH of ground beef from 5.6 to 5.17 and 5.10, respectively. Growth
of aerobes was measured with and without adjusting the sodium diacetate-containing
ground beef to pH 5.6. Populations of aerobes, after storage at 5C for 8 days, reached
7.12 and 6.21 logs and 5.98 and 5.1 1 logs in pH-adjusted and pH-unadjusted ground beef
containing 2 1 and 28 mM sodium diacetate, respectively. However, these organism, also
increased from 3.38 to 9.72 logs in the sodium acetate-free control. Similar trends were
seen in beef slurry. When 15 different aerobic bacteria were tested, sodium diacetate gener-
ally was more inhibitory to gram-negative than to gram-positive bacteria, although some
exceptions were noted [3461.
Schlyter et al. [338] investigated the antibacterial activity of sodium diacetate alone
or in combination with the commercial shelf-life extender, ATLA 2341 (a fermentation
product from lactic acid bacteria, Quest International Bioproducts, Sarasota, FL) in turkey
Characteristics of Listeria monocytogenes 161

slurry (pH 6.2) at 25C. During 7 days of incubation, the L. monocytogenes population,
initially present at 3.7 logloCFU/g, increased to 8.6 logs in the control and 6.8 logs in
the slurry, which was treated with 0.3% (21 mM) sodium diacetate. Additionally, sodium
diacetate at 05% (35 mM) inhibited growth and caused a slight decrease in the viable
Listeria population. The presence of ATLA 2341 at 0.25-0.75% did not appreciably affect
growth of listeriae; however, a synergistic inhibitory effect against Listeria was observed
when the additive, at the levels just indicated, was used in combination with 0.3 and 0.5%
diacetate. In a subsequent study by the same group [339], adding 0.3 and 0.5% sodium
diacetate to turkey slurry made the product listericidal when held at 4 and 2SC, respec-
tively. Antilisterial activity of sodium diacetate was synergistically enhanced by 2.5%
sodium lactate or 5000 units pediocin/mL but not by 30 ppm of sodium nitrite. The count
of Listeria in turkey slurry, which contained pediocin only, decreased by 0.9 log and then
increased and reached a final population (8.0 logs) similar to that of the control. Combined
use of pediocin and 0.3% diacetate at 4C or pediocin and 0.5% diacetate at 25C gave
counts of Listeria in the product that were -7 logs lower than those in the additive-free
controls.
Sodium Propionate
In 1987, Lang et al. [219] found that L. monocytogenes grew at 13C in TB at pH 5
supplemented with 0 and 6% NaC1; however, growth was prevented by addition of 5000
ppm propionic acid at both salt concentrations as well as by the combination of 6% NaCl
and 0.1% propionic acid. Using TB at pH 5.6 and containing 0, 1000, or 5000 ppm propi-
onic acid, L. monocytogenes grew to final populations of 107--10*and 104- 105CFU/mL
in the presence of 0 and 6% NaC1, respectively. Generation times calculated for listeriae
in the salt-free medium at pH 5.6 and containing 0, 1000, and 5000 ppm propionic acid
were 4.4, 10.3, and 16.1 h, respectively, rather than 7.2, 18.1, and 42.1 h, respectively,
in the same medium containing 6% NaCl. Similar behavior by L. monocytogenes was
subsequently noted during extended incubation at 4-30C in BHI broth (pH 5.9) con-
taining 4.0% NaCl and 0.15% potassium sorbate [151].
El-Shenawy and Marth [ 1 171 demonstrated that >2000 ppm sodium propionate can
inhibit growth of L. monocytogenes in TB at pH 5. Generation times for L. monocytogenes
in TB at pH 5.6 and without sodium propionate decreased from 68 to 49 min as the
incubation temperature increased from 4 to 35C. In TB at pH 5.6 and containing 3000
ppm sodium propionate, generation times decreased from 3.0 days to 4.5 h as the incuba-
tion temperature increased from 4 to 35C. Using TB at pH 5 and containing 3000 ppm
sodium propionate, Listeria populations decreased 1 log during 67 days of incubation at
4C. When the same medium was incubated at 35"C, numbers of L. monocytogenes de-
creased -3 logs, with the organism no longer being detected after 78 days,
In a follow-up study, El-Shenawy and Marth [ 1201 investigated the antilisterial activ-
ity at 13 and 35C of sodium propionate in combination with common organic acids. TB
was prepared to contain 0, 500, 1500, or 3000 ppm sodium propionate and the pH of the
medium was adjusted to 5.0 or 5.6 with HC1 or one of four common organic acids (acetic,
tartaric, lactic, and citric). Decreasing the pH from 5.6 to 5.0 enhanced the antilisterial
activity of propionate. Organic acids, when compared with HCl, greatly enhanced the
antilisterial activity of propionate, with acetic acid being the most effective followed by
tartaric, lactic, and citric acids. Lowering the incubation temperature from 35 to 13C not
only diminished the growth rate of L. rnonocytogenes, but also decreased the maximum
population of the bacterium for all combinations of propionate and organic acids.
162 Lou and Yousef

When used at 3000 ppm, Ryser and Marth [330] found that sodium propionate was
less effective than sorbic acid in eliminating four strains of L. monocytogenes from cold-
pack cheese food at pH 5.20-5.45. Cheese food was inoculated to contain -5 X 102L.
monocytogenes CFU/g and stored at 4C; the pathogen survived an average of 142 and
130 days in product that contained sodium propionate and sorbic acid, respectively. In
contrast, the pathogen was present in cheese food made without preservatives at levels
-
of I X 102CFU/g after 6 months of refrigerated storage.
Potassium Sorbate
Since receiving GRAS status in the United States during the 1950s, potassium sorbate
and sorbic acid have been widely used to extend the shelf life of many foods, including
butter, cheese, meat, cereals, and bakery items. Although most effective against yeasts
and molds, these antimicrobial agents also inhibit a wide range of bacteria, particularly
aerobic catalase-positive organisms. Consequently, the ability of potassium sorbate and
sorbic acid to inhibit L. monocytogenes has been assessed in laboratory media and several
foods. Moir and Eyles [265]measured the minimum inhibitory concentrations (MICs) of
sorbate against L. monocytogenes Scott A in buffered BHI broth. These authors reported
MICs of 400-600 and >5000 mg/L at pH 5 and 6, respectively, when the culture was
incubated at 35C and 1500 mg/L in broth of pH 6 which was refrigerated at 5C.
According to data collected by El-Shenawy and Marth [ 1 1.51, the ability of potassium
sorbate to prevent growth of L. monocytogenes is related to temperature and pH. In the
absence of potassium sorbate, generation times for L. monocytogenes in TB at pH 5.6
decreased from I . 13 days to 49 min as the incubation temperature increased from 4 to
35C. Addition of 2500 ppm potassium sorbate prevented growth of Listeria at 4C and
led to complete demise of the organism after 66 days, whereas listeriae grew with a genera-
tion time of 9 h in the same sorbate-containing medium incubated at 35C. The lower the
storage temperature and pH of the medium, the greater was the effectiveness of sorbates
against L. monocytogenes. In a subsequent study, El-Shenawy and Marth [ 1 191 investi-
gated the antibacterial activity of sorbate in the presence of other organic acids. TB was
prepared to contain 500, 1500, and 3000 ppm potassium sorbate and pH of the medium
was adjusted to 5.0 or 5.6 using HCI or organic acids (acetic, tartaric, lactic, or citric),
inoculated with L. monocytogenes, and incubated at 13 or 35C. When compared with
HCl as an acidulant, the antilisterial activity of sorbate was enhanced more by organic
acids, with acetic and tartaric acids being more effective than lactic and citric acids.
Working with food, Ryser and Marth [330]found that four strains of L. monocyto-
genes were eliminated faster from cold-pack cheese food at pH 5.45 that contained 3000
ppm sorbic acid (4100 ppm potassium sorbate) rather than from the same product at pH
5.2 manufactured without preservative. After inoculating cheese food containing sorbic
acid with one of four L. monocytogenes strains at a level of -5 X 1O2 CFU/g, the pathogen
survived an average of 142 days at 4C. Although L. monocytogenes failed to grow in
cheese food with a pH of 5.21 prepared without sorbic acid, the pathogen survived during
-
the normal 6-month shelf life of the product at potentially hazardous levels of 1 X 1 O2
CFU/g. Since potassium sorbate works best at pH < 6.0 and is generally ineffective at
pH > 6.5, it is not surprising that Dje et al. [SS] found potassium sorbate to be of little
use in inactivating L. monocytogenes in samples of reconstituted nonfat dry milk.
More recently, the antilisterial activity of potassium sorbate and other antimicrobial
agents was investigated by Buncic et al. [60] in buffered BHI broth (pH 5.5) at 4C.
Potassium sorbate (0.3%) prevented growth of L. monocytogenes (initially 1O7 CFU/mL)
Characteristics of Listeria monocyt ogenes 163

during 6 weeks of incubation; however, no bactericidal effect was observed. Strong listeri-
cidal effects were observed when sorbate was used in combination with sodium nitrite
(125 ppm), polyphosphate (0.5%),or nisin (400 IU/mL). Nitrite alone was only inhibitory
to L. monocytogenes, whereas nisin alone only caused an initial 1.1 -log decrease, with
Listeria survivors subsequently growing during prolonged incubation. Combined use of
sorbate and nitrite caused a 6.7-log reduction in 6 weeks. Adding polyphosphate to this
sorbatehitrite combination resulted in 6.4-log reduction in about 4 weeks. The combina-
tion of sorbate and nisin caused a 4.5-log reduction in 5 weeks, and this synergistic effect
was further increased by addition of nitrite (125 ppm). No antilisterial interaction was
observed between sorbate and 4% sodium lactate. A combination of lactate, sorbate, and
nisin prevented growth of Listeria but did not cause significant inactivation. Adding nitrite
to the three agent combination reduced populations by 3.7 logs after 37 days, with the
same reduction being achieved in I2 days by further incorporation of 0.5% polyphosphate.
Sodium Benzoate
El-Shenawy and Marth [ 1 141 reported that sodium benzoate is more inhibitory to L. mono-
cytogenes than is either potassium sorbate or sodium propionate. In this study, TB was
supplemented with 0-3000 ppm sodium benzoate in increments of 500 ppm, adjusted to
-
pH 5.0 and 5.6 with hydrochloric acid, inoculated to contain 103L. monocytogenes CFU/
mL, and incubated at 4, 13, 21, or 35C. At pH 5.6, L. monocytogenes was inactivated
in the presence of 22000 ppm sodium benzoate after 60 days of incubation at 4C. At
pH 5 , the organism was completely nonviable in TB containing 2 1500 ppm sodium benzo-
ate after 24-30 days of incubation at 4"C, whereas lower concentrations of sodium benzo-
ate led to gradual decreases in numbers of listeriae during 66 days at 4C. Inhibition of
Listeria by benzoate decreased at incubation temperatures above 4C and at pH values
higher than 5.
Inhibition and inactivation of L. monocytogenes in the presence of sodium benzoate
is affected by (a) temperature (i.e., more rapid at higher than lower incubation tempera-
tures), (b) concentration of benzoic acid (i.e., more rapid at higher than lower concentra-
tions), and (c) pH (i.e., more rapid at lower than higher pH values) as well as the type
of acid used to adjust the growth medium. When TB was acidified to pH values of 5.0
and 5.6 with acetic, tartaric, lactic, or citric acid rather than hydrochloric acid, El-Shenawy
and Marth [ I 161 found that the antilisterial activity of sodium benzoate was greatly en-
hanced. For example, 1500 ppm sodium benzoate led to complete inactivation of L. mono-
cytogenes after 96 h at 35C when acetic or tartaric acid was used to adjust the pH of the
medium to 5 ; under the same conditions, the pathogen remained viable at least 78 h longer
when the pH of the medium was adjusted with hydrochloric acid. The authors concluded
that acetic and tartaric acid were most effective in enhancing the antilisterial effects of
sodium benzoate followed by lactic and citric acid.
Using a minimal glucose-citrate medium (lacking a nitrogen source) adjusted to
pH 5.5 with sodium hydroxide, Yousef et al. [404] demonstrated that death rates for L.
monocytogenes were affected far more by incubation temperature than by the presence
of 1000-3000 ppm benzoic acid in the medium with D-values decreasing - 100-fold as
the incubation temperature was increased from 4 to 35C. Injured listeriae also were de-
tected after plating samples on restrictive and nonrestrictive media. The extent of cell
injury was somewhat greater at lower than higher incubation temperatures. These data
along with the isolation of an apparent sodium benzoate-resistant strain of L. monocyto-
genes from an animal-based dairy ingredient [ 1021 suggest that use of benzoic acid alone
164 Lou and Yousef

to control Listeria in food is questionable. However, Emme et al. [ 1251 found that repeated
exposure of L. monocytogenes to 1000 ppm benzoic acid did not increase resistance of
the organism to this widely used food additive.
Parabens and Other Benzoic Acid Derivatives
Parabens are esters of p-hydroxybenzoic acids. Of these esters, methyl, propyl, and heptyl
parabens are approved in several countries for direct addition to food. Since the pK, of
these derivatives is higher than that of benzoic acid, the molecules remain undissociated
at pH values up to 8.5. Although benzoic acid is effective as an antimicrobial agent only
in acidic foods, parabens retain activity over a wide range of pH values [81].
Payne et al. [294] examined the ability of methyl and propyl paraben to inhibit
growth of L. monocytogenes on Tryptose Phosphate Agar plates during 18 h of incubation
at 35C. Using eight strains of the pathogen, propyl and methyl paraben yielded minimum
inhibitory concentrations of 5 12 and >5 12 ppm, respectively. Moir and Eyles [265] treated
a culture of L. monocytogenes at 35 and 5C with methyl paraben in buffered BHI broth
and measured the MICs. Overall, L monocytogenes was more resistant to this paraben
than were other psychrotrophic bacteria, namely, Pseudomonas putida, Yersinia enterocol-
itica, and Aeromonas hydrophila. When Listeria in broth of pH 6 was incubated at 5C
in the presence of 1000 ppm methyl paraben, the count decreased only -2 logs in 4
months, with 86-99.9% of the viable cells being injured after 2 months. The MIC, defined
as the lowest concentration preventing visible growth in buffered BHI broth after 10 days
of incubation at 30C or 3 months at 5"C, was 300-700 ppm at pH 5 and 3OoC, 1300-
1600 ppm at pH 6 and 3OoC,and 600 ppm, at pH 6 and 5C. Therefore, the MIC decreased
as incubation temperature or pH decreased.
Parabens also exhibited antilisterial activity against Listeria when the additive was
tested in food. Using reconstituted nonfat dry milk (10% solids) inoculated to contain 10'
or 103L. monocytogenes CFU/mL, Payne et al. [294] found that populations of listeriae
were approximately three to four orders of magnitude lower in samples containing 1000
rather than 0 ppm propyl paraben following 24 h of incubation at 35C. When these
experiments were repeated at refrigeration temperatures [881, Listeria counts in milk sam-
ples containing 1000 ppm propyl paraben remained constant during 10 days at 4C.
Dje et al. [88] also investigated behavior of L. monocytogenes in 10% (w/v) aqueous
suspensions of raw chicken meat and frankfurters to which 1000 ppm propyl paraben was
-
added. Although L. monocytogenes attained maximum populations of 1O8 CFU/mL in
propyl paraben-free chicken suspensions following 24 h at 35"C, numbers of listeriae
increased only approximately 10-fold to a maximum of 105CFU/mL in similar suspen-
sions containing 1000 ppm propyl paraben. However, addition of 1000 ppm propyl para-
ben to frankfurter suspensions failed to prevent growth of L. monocytogenes, with similar
-
growth rates and maximum populations of 108CFU/mL appearing in samples prepared
both with and without propyl paraben after 24 h of incubation at 35C. Since L. monocyto-
genes and propyl paraben are primarily present in the water and lipid phases of these
suspensions, respectively, the higher percentage of fat in frankfurter than in chicken sus-
pensions likely accounts for increased growth of listeriae in the former.
Antilisterial activity of p-aminobenzoic acid (pK, of 4.8) relative to common or-
ganic acids (formic, propionic, acetic, lactic, and citric) was investigated by Richards et
al. [3 181. The authors reported that p-aminobenzoic acid had greater inhibitory activity
against L. monocytogenes, S. enteritidis, and E. coli than did other organic acids. The
MIC of p-aminobenzoic acid, measured against L. monocytogenes in BHI broth after 24
Characteristics uf Lister i a mon ocytogenes 165

h of incubation at 37OC, was 9-12 mmol/L at pH 6.7-6.8 compared with MICs of 18-
20 mmol/L for formic acid at pH 6.1-6.2, 18-25 mmol/L for propionic acid at pH 6.0-
6.3, 18-30 mmol/L for acetic acid at pH 5.5-6.2, 35-40 mmol/L for lactic acid at pH
5.2-5.4, and 12-14 mmol/L for citric acid at pH 5.2-5.4. When BHI broth was adjusted
to pH 6.5 with the acids included in the study, their antilisterial potency was as follows:
p-aminobenzoic >propionic >formic >acetic >citric >lactic acid. Antilisterial activity
of p-aminobenzoic acid was pH dependent, with higher activity being observed at lower
pH values; however, this acid showed some inhibitory activity even at near neutral pH
values near neutral.

Fatty Acids and Related Compounds

Interest in the potential food applications for fatty acids and related compounds that pos-
sess antimicrobial properties also continues to increase with potential applications in food.
Antilisterial activity of free fatty acids, particularly those of medium chain length, has
been demonstrated. In addition to their primary function as food emulsifiers, some fatty
acid esters, particularly monoacylglycerols (monoglycerides) and esters of sucrose, inhibit
a wide spectrum of microorganisms, including L. monocytogenes.
Free Fatty Acids
Pfeiffer et al. [301] investigated the individual effects of 100 ppm butyric, caprylic, and
caproic acids on growth of four L. monocytogenes strains in TB at pH 5.6 during incubation
at 13C. Butyric and caproic acid failed to inhibit growth of L. nzonocytogenes. In contrast,
the average generation time for L. monocytogenes was about twice as long (9.40 h) in
TB containing caprylic rather than caproic or butyric acid or in the control without fatty
acids. Along with the slower growth rate, slightly lower Listeria populations were ob-
served after 14 rather than 7 days for butyric and caproic acid.
Wang and Johnson [385] tested the antilisterial activity of fatty acids which are
naturally present in milk and suggested that some of these compounds could be effectively
used as antilisterial agents in dairy products. According to results of this study, L. monocy-
togenes Scott A, grown in BHI broth, at 35OC, was inactivated by 10, 20, 100, 200, and
200 pg/mL of glycerol monolaurate, lauric (C 12:0), linolenic (C 18:3), linoleic (C18:2),
and potassium salts of conjugated isomers of linoleic acids (K-CLA), respectively, with
corresponding concentrations of 10, 10, 20, 50, and 100 pg/mL inactivating the pathogen
at pH 6. In contrast, 200 pg/mL of myristic (C14:0), palmitic (C16:0), or stearic (C18:
0) acid were not inhibitory. In skim and whole milk, K-CLA showed strong listeriostatic
activity, which was enhanced by the presence of 2000 pg/niL citrate, 100-200 pg/mL
butylated hy droxyanisol (BHA), ascorbate, or a-tocopherol.
Kinderlerer and Lund [205] examined the MICs of hexanoic and octanoic acids,
both of which have a pK, of 4.85, against 10 L. monocytogenes strains and 2 L. innocua
strains growing in TSB supplemented with yeast extract and glucose (TSYGB) and ad-
justed to pH 5.5 and 5.0. Octanoic acid exhibited lower MICs against listeriae than hexa-
noic acid, and MICs varied among different strains of L. monocytogenes. After 162 h of
incubation, MICs for octanoic acid were <1.41-3.49 and 1.41-3.49 mmol/L at pH 5.0
and c3.18--7.86 and 3.18-7.86 mmol/L at pH 5.5 for L. munocytogenes and L. innocua
strains, respectively. Hexaonic acid MICs were 6.89-8.61 mmol/L after 138 h of incuba-
tion at pH 5.0 and > 10.33 mmol/L after 89 h of incubation at pH 5.5 for L. monocytogenes
and L. innocua strains except for one L. rnonocytogenes strain with MICs of <6.89 and
166 Lou and Yousef

<0.861 mmol/L at pH 5.0 and 5.5, respectively. Therefore, some strains of L. innocua
appear to be more resistant to hexanoic acid than L. rnonocytogenes. Free fatty acids are
present in certain cheeses, with blue cheese being reported to contain 4.6 and 1.9 mmol/
kg of hexanoic and octanoic acids, respectively [246]. Therefore, concentrations of these
two fatty acids comparable to those found in cheese may have marked antilisterial activity
in acidic foods. However, the antilisterial activity of these two fatty acids would be ex-
pected to decrease dramatically in blue cheese during ripening as the pH increases from
about 4.6 to 6.2 [286].
Fatty Acid Monoesters
Some fatty acid monoesters of glycerol (i.e., monoacylglycerols, which are commonly
known as monoglycerides) and sucrose are potentially useful as antimicrobial food addi-
tives. Monolaurin, the lauryl glycerol monoester, and sucrose laureate were studied exten-
sively and will be discussed in some detail below.
The antilisterial activity of monolaurin was first reported in 1992 by Oh and Marshall
[276] and Wang and Johnson [385]. Oh and Marshall [276] noted that monolaurin was
more antilisterial than other common antimicrobial agents (sorbate, propyl paraben, ter-
tiary butyl hydroxyquione [TBHQ], propyl gallate, and butylated hydroxyanisol [BHA])
and inhibited four L. rnonocytogenes strains (- 10' CFU/mL) in a laboratory broth at 35C
with MICs of 3-4, 7, 9, and 10 pg/mL at pH 5.0, 5.5, 6.0, and 7.0, respectively. Thus
different L. rnonocytogenes strains had similar sensitivity to monolaurin. Wang and John-
son [385] found that L. rnonocytogenes was inhibited by 10 pg/mL monolaurin in BHI
broth at pH 5 and 6. When L. monocytogenes was cultured on BHI agar plates, the MICs
for monolaurin were 96, 14, 7, and 5 pg/mL at pH 7.0, 6.0, 5.5, and 5.0, respectively
[3 121. A monolaurin MIC of 16 pg/mL for L. monocytogenes on Tryptic Soy Agar (TSA)
also was reported by Bal'a and Marshall [25].
Inhibition of microorganisms, including L. rnonocytogenes, is more pronounced us-
ing monolaurin than other fatty acid monoesters. The minimum bactericidal concentrations
at which 103-10" L. rnonocytogenes CFU/mL was completely inactivated in BHI broth
(pH 6) after 24 h at 37C were 25,50, and 75 yg/mL for monolaurin (MC,,), monocaprin
(MC,,), and monomyristin (MC,,), respectively, whereas a concentration of 300 pg/mL
monocaprylin (MC,) was only inhibitory [387]. According to these authors, L. rnonocyto-
genes was not inhibited by 300 pg/mL of monopalmitin (MC,,), monostearin (MC,,),
monoolein (MC18:,),or monolinolein (MCIX:?).
Gram-positive bacteria are more sensitive to monolaurin than are gram-negative
organisms. L. monocytogenes was the most resistant among the gram-positive bacteria
that were tested by Razavi-Rohani and Griffiths [3 121. These investigators used a spiral
gradient method and found that growth of L. monocytogenes on BHI agar was completely
inhibited by a minimum of 96 pg/mL monolaurin, whereas complete inhibition of six
other gram-positive bacteria (Bacillus, Stuphylococcus, and Lactococcus) required a mini-
mum of 8-24 lg/mL monolaurin. In contrast, concentrations as high as 3170 pg/mL
failed to inhibit nine gram-negative bacteria [3 121.
Monolaurin in combination with other treatments or antimicrobial agents, such as
low temperature, low pH, organic acids, chelating agents, and antioxidants, exhibited en-
hanced antimicrobial activity. Gram-negative bacteria were inhibited by some of these
combinations. In the presence of 5 4 % NaCl, growth of gram-negative bacteria on BHI
agar was inhibited by <2 pg/mL monolaurin [312]. As described previously, the MICs
of monolaurin decreased as the pH decreased [276]. Oh and Marshall [277] investigated
Characteristics of Listeria monocyt ogen es 167

the antilisterial activity of monolaurin (5-9 pg/mL) at different temperatures (7, 15, and
35C) and pH values (5.0,5.5, and 7.0) in TSBYE. Although monolaurin was listeriostatic
at most temperature/pH combinations, listericidal activity was detected using 8-9 pg/mL
of monolaurin at pH 5.0 with L. monocytogenes strain Scott A (initially inoculated at
- 1O3 CFU/mL) reportedly undetectable at this concentration after 20.0-22.0, 6.0, and 0.5
days at 7, 15, and 35"C, respectively. The study just described provides evidence that
inhibition of L, monocytogenes by monolaurin is highly temperature-dependent. Although
Wang and Johnson [385] found that monolaurin at 200 pg/mL was listericidal in skim
milk at 4"C, no antilisterial activity was observed at 30C.
Oh and Marshall [278] reported a greater listeriostatic effect in TSBYE at 35C
when 18 pM monolaurin was used in combination with sublethal concentrations of organic
acids (acetic, benzoic, lactic, or citric) than when monolaurin or the acids were used sepa-
rately. When tested in crawfish tail meat at 4OC, 336 mM lactic acid decreased L. rnonocy-
togenes populations from about 103CFU/g to nondetectable levels in 10 days; however,
similar inactivation could be achieved using a combination of 0.72 mM monolaurin and
224 mM lactic acid. When used alone, 224 mM lactic acid resulted in complete inhibition
12781. Bal'a and Marshall [25] investigated the combined effect of NaCl (2.5-7.8%), pH
(5.4-7.8), temperature ( 5 , 15, 25, and 35"C), and sublethal levels of monolaurin (2-8 pg/
L) on growth of L. monocytogenes on double (salt-pH) gradient plates and found signifi-
cant interactions between these factors. More recently, Razavi-Rohani and Griffiths
[3 I2,3 131 detected enhanced antimicrobial activity of monolaurin when this compound
was used in combination with ethylenediaminetetraacetic acid (EDTA), BHA, low pH,
or NaC1; however, no marked enhancement was observed in the presence of lysozyme.
The presence of EDTA not only reduced the MICs for all gram-positive bacteria (70 pg/
mL for L. rnonocytogenes), but also sensitized gram-negative bacteria to monolaurin
(MICs of 90-1500 pg/mL). However, no decrease in monolaurin MICs was caused by
the presence of EDTA at pH 1 6 . Sodium citrate and monoglyceride citrate, two other
chelating agents tested, decreased the antimicrobial effect of monolaurin [3 121.
Antilisterial activity of monolaurin increased in the presence of organic acids and
antioxidants [386]. According to Wang and Johnson, inhibitory activity of monolaurin ( 5
pg/g) against L. monocytogenes in BHI broth was enhanced by other antilisterial agents
such as BHA (100 pg/g), TBHQ (30 pg/g), propyl gallate (200 p/g), acidulants (0.1%
acetic or lactic acid), and potassium sorbate (0.1%) [386]. Propyl gallate (200 p/g) and
lactic acid ( 0.2%) enhanced the bacteriostatic activity of monolaurin and monocaprin
against L. rnonocytogenes in seafood (imitation crab meat and cooked shrimp) and Cam-
embert cheese, respectively, which were stored at 4C. However, when tested in foods,
0.1% glycine, sodium citrate, propylene glycol, or Tween 20,O. 1-0.2% potassium sorbate,
200 pg/mL lysozyrne, or 200 pg/mL BHA did not enhance activity of monoacylglycerols
[386]. Oh and Marshall [280] reported that combined use of 200 pg/g rnonolaurin and
0.5% lactic acid significantly inhibited growth of Listeriu in crawfish meat homogenate
stored at 4"C, whereas these additives had little or no effect on growth of Listeria when
used separately.
Antilisterial interactions occur not only between monolaurin and other factors but
also among different monoacylglycerols as well. Wang et al. [387] reported an additive
effect between monolaurin ( 100 pg/mL) and monocaprin ( 100 pg/mL) and a synergistic
effect between monomyristin (200 pg/mL) and monocaprin (200 pg/mL) when these com-
pounds were tested in skim milk at 4C. The authors also noted strong antilisterial activities
by monoacylglycerols synthesized from coconut oil. These monoacylglycerols had a MIC
168 Lou and Yousef

(10 pg/mL), which was lower than that of monolaurin (25 pg/mL) when both agents
were tested in BHI broth (pH 6) with incubation at 37C. Compared to milk fat-derived
monoacylglycerols, which were not inhibitory to L. monocytogenes at 300 pg/mL, coconut
monoacylglycerols are rich in lauric (C12), myristic (C14), and capric (C10) acids, and
thus may contain higher levels of monolaurin, monomyristin, and monocaprin. Demon-
strated antilisterial activity of these three dominant monoacylglycerols and the synergistic
interaction between them may account for the high antilisterial activity of coconut mono-
acylglycerols. When tested in refrigerated skim milk, 2% milk, and whole milk, monoca-
prin, monolaurin, and coconut oil-derived monoacylglycerols showed strong antilisterial
activity. At >200 pg/mL, monocaprin was more effective than monolaurin in inactivating
L. monocytogenes in all three refrigerated milks, possibly because of the higher water
solubility of monocaprin.
High temperatures of incubation and high fat content of the growth medium de-
creased the effectiveness of monoacylglycerols. In skim milk containing 250 pg/mL of
the coconut-derived monoacylglycerols, L. monocytogenes (- 1O3 CFU/mL) was inacti-
vated after storage at 4C for 7 days but grew to 107- 1 Ox CFU/mL at I 3 and 23C. Concen-
trations of 250-400,500-750, and 750- 1000 pg/mL of coconut-derived monoacylglycer-
01s were required to inactivate L. monocytogenes in refrigerated skim milk, 2% milk, and
whole milk, respectively [387]. Compared with 1000 pg/g monolaurin or monocaprin,
coconut monoacylglycerols ( I000 pg/g) and a combination of monolaurin (500 pg/g) plus
monocaprin (500 pg/g) exhibited greater listericidal activity in beef frankfurter slurries
(pH 5.0 and 5.5) and seafood salad (pH 4.9) stored at 4C. However, at 12OC, Listeria
grew rapidly in beef frankfurter slurries (pH 5.5) with or without such levels of these
monoacylglycerols. When present in turkey frankfurters (pH 5.6 and 6.1) stored at 4"C,
the monoacylglycerols ( 1000 pg/g) were bacteriostatic, with half this concentration being
listeriostatic in cooked shrimp (pH 7.1), imitation crab meat (pH 6.5), and Camembert
cheese (pH 6.2) [386]. Although 10-96 pg of monolaurin/mL can inhibit Listeria growth
in culture media [25,276,277,312,385,3871, much higher concentrations are required to
elicit similar levels of inhibition in foods. Monoacylglycerols may interact with food com-
ponents such as lipids and protein, and thus become less available for contact with microor-
ganisms and so less capable of exerting an antimicrobial effect. Wang and Johnson [385]
found that antilisterial activity of monolaurin decreased dramatically as the fat content of
milk increased. Although 100 pg/mL and 2200 pg/mL monolaurin was listeriostatic and
listericidal, respectively, in skim milk at 4OC, 200 pg/mL monolaurin showed no such
activity in whole milk.
Levels of monolaurin required to inhibit L. monocytogenes vary with the type of
food. Oh and Marshall [280] found that 200 pg/g monolaurin did not significantly inhibit
growth of L. monocytogenes in refrigerated crawfish tail meat homogenate packaged in
air or a modified atmosphere. However, this level of monolaurin significantly decreased
the growth of Listeria in vacuum-packaged meat samples. Earlier these authors [278]
found that 0.72 and 1.44 pM monolaurin extended the generation times of L. monocyto-
genes in crawfish tail meat stored at 4C from 16.7 (for untreated control) to 28 and 48
h, respectively. In 1997, Wang and Johnson [386] reported that antilisterial activity of
monoacylglycerols was much greater in beef frankfurter slurries and seafood salad than
in turkey frankfurter slurries, summer sausage, cooked shrimp, imitation crabmeat, yogurt,
and cottage and Camembert cheeses. Monolaurin had greater antilisterial activity in 2%
chocolate milk than in 2% milk, possibly because of the enhancing effect of cocoa.
Since monolaurin is poorly soluble in water at high concentrations and has a soapy
Characteristics of Listeria monocytogenes 169

flavor, the antimicrobial activity of more water-soluble monoacylglycerols and water-solu-

ble derivatives of monolaurin were investigated. Although less inhibitory than monolaurin
in broth cultures, the more water-soluble monocaprin at >200 pg/mL showed higher
antilisterial activity than similar concentrations of monolaurin in refrigerated milk [387].
However, monocaprylin (MC,) was only inhibitory at 300 pg/mL when tested in broth
cultures [387]. When the water-soluble triglycerol 1,2-laureate was used alone, it did not
inhibit L. monocytogenes; however, in the presence of 380 pg/mL EDTA, the pathogen was
inhibited by this monolaurin derivative at a MIC of 380 pg/mL on agar plates [312,313].
Beside monoacylglycerols, fatty acid esters of sucrose, which are commonly used
as food emulsifiers, exhibit antimicrobial activity against a wide range of spoilage and
pathogenic bacteria. Monk et al. [267] found that 400 pg/mL of sucrose monolaurate was
lethal to L. monocytogenes (Scott A) in TPB at 3OoC, whereas lower concentrations (100-
200 pg/mL) exhibited a strong listeriostatic effect. L. monocytogenes was generally more
sensitive than S. aureus to sucrose monolaurate alone or in combination with various
enhancers, such as EDTA and organic acids. As with monolaurin, EDTA (50-200 pg/
mL) synergistically enhanced the antilisterial effect of sucrose monolaurate; this enhanced
inhibition was more pronounced as the incubation temperature decreased from 30 to 15
or 5C. A similar effect of EDTA on sucrose monolaurate activity was previously reported
by Sikes and Whitfield [349]. However, addition of 0.1%acetic or lactic acid to the growth
medium decreased inhibition during the initial 32 h of incubation but resulted in signifi-
cantly lower final populations during subsequent incubation. Sucrose monolaurate alone
or in combination with EDTA synergistically increased the thermal inactivation of L.
monocytogenes 12671. Monolaurin was reported by Oh and Marshal1 [279] to enhance
destruction of L. monocytogenes in biofilms on stainless steel.
Similar to the findings for monolaurin, higher concentrations of fatty acid esters of
sucrose were required to inhibit L. monocytogenes in foods, particularly those with high
fat contents, than in laboratory media. Sikes and Whitfield [349] reported decreased antilis-
terial activity of sucrose monolaurate, present alone or in Combination with EDTA and
BHA, when the fat content of their model food system increased. Monk and Beuchat [266]
found that when added to uncooked ground beef, 0.30% sucrose laureate or 0.32% sucrose
palmitate sigriificantly inhibited growth of L. monocytogenes (Scott A) at 5"C, whereas
0.32% sucrose stearate or 0.15% sucrose oleate showed no antilisterial activity. Such levels
of these esters had little or no effect on S. aureus and psychrotrophic bacteria in ground
beef.

Sodium Nitrite
Studies undertaken by Shahamat et al. [342] during the 1970s examined effects of various
concentrations of sodium nitrite and sodium chloride on growth of L. monocytogenes in
TSB at different temperatures and pH values. When incubated at 37, 22, and 4C in broth
at pH 7.4, L. rnonocytogenes grew at nitrite concentrations as high as 25,000, 30,000, and
10,000ppm, respectively. Inhibitory effects of nitrite were enhanced at pH 6.5, particularly
at lower incubation temperatures, with complete inhibition being caused by 1500 ppm
nitrite at 4C. MICs of nitrite were further reduced at all three incubation temperatures
and at pH 5.5, with 600 ppm nitrite being sufficient to inhibit growth at 4C. The bacterio-
static activity of nitrite was greatest at pH 5 and at 22 and 3 7 T , with no growth reported
at nitrite concentrations >800 and 400 ppm, respectively. Addition of 3% sodium chloride
to TSB failed to increase the bacteriostatic action of sodium nitrite. Although MICs for
170 Lou and Yousef

nitrite were only slightly lower with 5.5 or 8.0% sodium chloride at pH 7.4 or 6.5, the
combination of 5.5 or 8.0% sodium chloride and pH values of 5.0 or 5.5 led to MICs for
nitrite that were generally 8- to 20-fold lower than controls prepared without sodium chlo-
ride. Inhibitory effects of nitrite were again most pronounced at 4C when the chemical
was combined with sodium chloride.
The study by Shahamat et al. [342] was criticized by McClure et al. [254], who
found that autoclaving caused nitrite to decompose. After autoclaving nitrite-containing
TSB at pH 5, no nitrite was detected. Since only 10-72% nitrite was detected after auto-
claving nitrite-containing TSB at pH 5.3-6.7 [254], McClure et al. [254], together with
Buchanan et al. [%I, believed that the antimicrobial activity of nitrite was seriously under-
estimated by Shahamat et al. [342]. Using filter-sterilized rather than autoclaved sodium
nitrite, McClure et al. [254] reported much greater antilisterial activity of nitrite in TSB.
Filter-sterilized nitrite, at 50 pg/mL and pH 5 , prevented visible growth for 48 h; however,
200 pg/mL autoclaved nitrite under the same conditions did not retard Listeria growth.
As mentioned earlier, behavior of L. monocytogenes in food and culture media de-
pends on the interactive effects of temperature, pH, type of acidulant, salt content, a,, and
types and concentrations of food additives that may be present in the system. The effective-
ness of sodium nitrite as an antilisterial agent also is strongly influenced by these same
factors. Buchanan et al. (581 used a factorial design to determine the effect of sodium
nitrite (0- 1000 ppm) in combination with incubation temperature (5-37"C), initial pH
(4.5-7.5), sodium chloride (0.5-4.5%), and atmosphere (aerobic vs anaerobic) on growth
of L. monocytogenes in TPB. Although lag periods, generation times, and maximum popu-
lations were all affected by these five interacting variables, sodium nitrite was most
listeriostatic when used in conjunction with low pH, increased sodium chloride, refrigera-
tion temperatures, and anaerobic conditions that simulated vacuum packaging. McClure
et al. [254] reported that antilisterial activity of nitrite strongly depended on pH. At pH
2 6, nitrite, even at 400 pg/mL, had little antilisterial activity. However, below pH 6,
nitrite, even at 50 pg/mL, exhibited antilisterial activity. Visible growth (defined as 0.03-
unit increase in ODhO0 in 21 days at 5-30C) of this pathogen in TSB at 20C was prevented
by 50 pg/mL sodium nitrite and pH 5 5.3. Autoclaving nitrite dramatically decreased its
antilisterial activity.
Buchanan et al. [53] found that inactivation of L. monocytogenes by NaNO, was
affected by several factors, with lactic acid being the most influential. At high levels of
lactic acid (low pH), the listericidal action of nitrite increased, whereas low levels of lactic
acid had little effect on nitrite activity. The antilisterial activity of sodium nitrite and other
antimicrobial agents also was studied by Buncic et al. [60]. Growth of L. monocytogenes
(initially 107CFU/mL) in buffered BHI broth (pH 5.5) incubated at 4C for up to 7 weeks
was prevented by addition of 125 ppm sodium nitrite or a combination of 125 ppm sodium
nitrite and 0.5% polyphosphate. Polyphosphate (0.5%) alone did not prevent growth of
Listeria. This is consistent with results from several research groups [58,254,342], who
noted that nitrite inhibited L. monocytogenes at low pH values. Nitrite and nisin also had
a synergistic effect against L. monocytogenes. When used alone, nisin (400 IU/mL), re-
sulted in an initial 1.1 -log reduction, with Listeria survivors eventually growing during
extended incubation. However, a combination of nisin and nitrite (125 ppm) not only
prevented Listeria regrowth but caused a further (1.4 log) reduction when compared with
nisin alone.
Working with meat products, Johnson et al. [ 1881 found that growth of L. monocyto-
genes was suppressed at 4C in hard salami (pH 4.3-4.5) that contained 5.0-7.8% NaCl
Characteristics of Listeria monocytogenes 171

plus 156 ppm sodium nitrite. Although their findings agree with those of Shahamat et al.
[342], the combination of low water activity (a, 0.79-0.86) and low pH were probably
more important in preventing growth of listeriae than was the addition of 156 ppm sodium
nitrite. Findings of Glass and Doyle [ 1561 also indicate that 3.5% sodium chloride plus
156 ppm sodium nitrite in sausage batter at pH 6.2 controlled growth of L. monocytogenes
in the product during fermentation at 90F. Under these conditions, additional acid devel-
opment through fermentation is essential to prevent growth of listeriae.

Antioxidants
Antioxidants such as BHA, butylated hydroxytoluene (BHT), TBHQ, and propyl gallate
comprise an important category of food additives. Although primarily used to prevent
oxidation of fat, some of these antioxidants also possess antimicrobial activity.
Limited trials on destruction of L. monocytogenes by BHA were initiated by Al-
Issa et al. 161 during the early 1980s. Tryptone Soy Broth containing 50 ppm BHA was
-
inoculated to contain 105L. monocytogenes CFU/mL and examined for numbers of the
bacterium. The Listeria population decreased -3 logs during the first 12 h at 37C and
remained at a level of -102 CFU/mL after 24 h of incubation. The same authors also
found that successive subculturing of L. monocytogenes in a medium containing glycerol
followed by inoculation into Tryptone Soy Broth containing 50 ppm BHA led to rapid
Listeria growth with populations reaching 10'- 1O9 CFU/mL after 24 h at 37C. Develop-
ment of BHA resistance correlated with a high lipid content in the cell wall and membrane
from prior growth in a medium containing glycerol. Payne et al. [294] investigated the
potential of BHA, BHT, TBHQ, and propyl gallate to inhibit growth of L. monocytogenes
on Tryptose Phosphate Agar during 18 h of incubation at 35C. Using an agar dilution
method, TBHQ was the most effective antioxidant tested with a MIC of 64 ppm followed
by BHA, propyl gallate, and BHT with MICs of 128, 256, and 5 12 ppm, respectively.
Although these findings may at first appear promising for the food industry, L. mono-
cytogenes is far more likely to encounter sublethal levels rather than MICs of antioxidants
in food. Consequently, Yousef et al. [405] examined the growth kinetics of L. monocyto-
genes strain Scott A in TB containing BHA (100-300 ppm), BHT (300-700 ppm), and
TBHQ (10-30 ppm) during 54 h of incubation at 35C. Overall, these findings agreed
with those of Payne et al. [294] in that TBHQ again was most inhibitory to L. monocyto-
genes followed by BHA and BHT. According to the authors, L. monocytogenes exhibited
increasingly longer lag periods and generation times as well as lower maximum popula-
tions in the presence of BHA at 100-200 ppm, with concentrations 2300 ppm proving
to be lethal. Since all three growth parameters were increasingly affected as the sublethal
concentrations of BHA increased, the organism was probably unable to detoxify this anti-
oxidant metabolically. Therefore, addition of up to 200 ppm BHA to food, as permitted
by the FDA, will likely contribute to overall keeping quality and safety of some products.
Unlike BHA, L. monocytogenes was unaffected by (-300 ppm BHT; however, poor solu-
bility of BHT in TB prevented critical analysis of this antioxidant at concentrations >300
ppm. Interestingly, increasing the concentration of TBHQ from 10 to 30 ppm led to an
exponentially longer lag period for L. monocytogenes, but it did not appreciably affect
generation times or maximum populations. Hence, unlike BHA, these observations suggest
that L. monocytogenes can metabolically detoxify sublethal concentrations of TBHQ to
safe levels and initiate growth thereafter. From this, it appears that BHA may be of greater
benefit than TBHQ for inhibiting growth of listeriae in food.
172 Lou and Yousef

Payne et al. [294] examined the antilisterial activity of TBHQ in reconstituted

NFDM (1 0% solids) that was inoculated to contain 10 L. monocytogenes CFU/mL; addi-
tion of 150 ppm TBHQ prevented growth of the pathogen and variably inactivated it
during 24 h of incubation at 35C. Although numbers of listeriae increased nearly 100-
fold in similar samples inoculated to contain - 10' L. monocytogenes CFU/mL, maximum
populations were still approximately three orders of magnitude lower than those observed
in TBHQ-free control samples. Results obtained by repeating these experiments at refriger-
ation temperatures were more encouraging [88], with original Listeria inocula of 10' and
1O3 CFU/mL being reduced to nondetectable levels or remaining constant, respectively,
during 10 days at 4C. Although these preliminary findings suggest that addition of TBHQ
to foods at FDA-permissible levels of 1200 ppm may be of some benefit in inhibiting
and/or inactivating L. monocytogenes, present FDA regulations [ 151 stipulate that TBHQ
and all other such additives can only be used as antioxidants and cannot be added indis-
criminately to foods for other purposes.
Chung et al. [7 I ] investigated the antimicrobial activity of propyl gallate, another
antioxidant food additive. The authors used a well diffusion method and measured the
zone of inhibition after 48 h of incubation at 32C. Propyl gallate prevented growth of
two strains of L. monocytogenes, but ellagic acid, the hydrolytic product of propyl gallate,
failed to inhibit the bacterium. The authors further examined the effect of this antioxidant
against L. monocytogenes growing in cabbage juice at room temperature. Results were
consistent with that of the well diffusion method; propyl gallate but not ellagic acid exhib-
ited antilisterial activity. Propyl gallate at 500 pg/mL prevented growth of L. monocyto-
genes (initially 6.3 X 10' CFU/mL) in cabbage juice and 250 pg/mL allowed -2-log
increase after 4 days; however, a final population of 108 CFU/mL was attained in the
control.

Liquid Smoke
Many commercially available liquid smoke products used in processed meats and sausages
can inactivate common foodborne organisms, including E. coli, S. aureus, and Lactobacil-
lus viridescens. These artificial liquid smoke flavorants owe their activity to the presence
of phenolic compounds and acetic acid, both of which are bactericidal at relatively low
concentrations.
After L. monocytogenes was recognized as a possible health hazard in ready-to-eat
meat and sausage products, several investigators examined the potential of various liquid
smoke compounds to inactivate L. monocytogenes in phosphate buffer and culture media
commonly used to isolate this pathogen from meat products. Using sterile phosphate buffer
at pH 5.64 and inoculated to contain 1 X 105L. monocytogenes CFU/mL [259], three of
five liquid smoke compounds (Charsol- 10, Aro-Smoke P-50, and CharDex Hickory, Red
Arrow Products, Manitowoc, WI) tested at a concentration of 0.5% reduced numbers of
listeriae to nondetectable levels after 4 h at ambient temperature. When the concentration
of liquid smoke products was decreased to 0.25%, numbers of listeriae were still reduced
to nondetectable levels within 4 h using either CharSol- I0 or Aro-Smoke P-50. However,
CharDex Hickory was far less effective at the lower concentration with 24 h of incubation
required to inactivate the pathogen completely. Listericidal activity of these liquid smoke
compounds also appeared to be at least partially related to levels of acetic acid present
in the various preparations.
Subsequently, Wendorff 13921 found that the same liquid smoke compounds were
Characteristics of Lister ia monocyt ogenes 173

far less listericidal when added to USDA-recommended Listeria Enrichment Broth rather
than phosphate buffer. Interactions between liquid smoke constituents and protein in the
enrichment broth were probably at least partially responsible for the observed decrease
in listericidal activity. Although three of five liquid smoke compounds were effective
against L. monocytogenes in buffer and to a lesser extent in culture media, later work
by Wendorff [392] showed that concentrations of liquid sinoke needed to inactivate L.
monocytogenes in processed meats were well above organoleptically acceptable limits.
In 1992, Faith et al. [128] reported that adding 0.2 and 0.6% (v/v) of liquid smoke
(CharSol Supreme, Red Arrow Products, Manitowoc, WI) to wiener exudate inactivated
L. monocytogenes with D-values of 36 and 4.5 h, respectively, whereas Listeria in the
-
smoke-free exudate grew from initial levels of 10sto 108CFU/mL after 3 days at 25C.
The authors further investigated the antilisterial activity of selected smoke components
in TB at pM 7. When the culture was incubated at 37"C, they found that among 11 phenol
compounds tested, only isoeugenol retarded growth of Listeria. Although final maximum
populations were similar, lag-phase duration increased linearly from -3 to 21 h as isoeu-
genol levels increased from 0 to 200 ppm. It was estimated that an isoeugenol concentra-
tion of 236 ppm was required to increase the lag phase by one order of magnitude. L.
monocytogenes in the presence of 100 ppm isoeugenol L. monocytogenes also was inhib-
ited to a greater extent when the pH of TB was adjusted to 5.8 compared with 7.0.

Spices, Herbs, and Plant Extracts

Although primarily used as flavoring and seasoning agents, many spices contain specific
chemicals and/or essential oils that can inactivate or inhibit various pathogenic and spoil-
age organisms. Consequently, surveys were made to identify spices that might be useful
in inhibiting growth of L. monocytogenes in food.
Ting and Deibel [375] reported results from one such survey in which a concentra-
tion gradient plate method was used to study the effect of 13 different spices on three L.
monocytogenes strains. Although the pathogen remained completely viable in the presence
of 3% (w/v) black pepper, chili pepper, cinnamon, garlic, mustard, paprika, parsley, and
red pepper during extended storage at 4 and 24"C, Listeria was sensitive at 24C to cloves,
oregano, sage, rosemary, and nutmeg, with calculated MICs of 0.60-0.70, 0.50-0.70,
0.70-0.90, 0.90-1 .O, and 1.1- I .4%, respectively. The latter five spices also were inhibi-
tory to L. monocytogenes at 4C. When added to TSB, growth of the pathogen at both
incubation temperatures was prevented by as little as O S % cloves, oregano, or sage. List-
ericidal effects were observed with 10.5% clove at 4 and 24C and 10.5% sage at 4C.
When exposed to 0.5% clove, the Listeria population decreased >2 logs after 24 days at
4C or after 24 h at 24C. A reduction in L. monocytogenes population of >5 logs was
observed with 1% cloves in 7 days at 4C or 3 h at 24C. Sage levels 0.5 and 1.0%
resulted in :.3- and >5-log decreases in Listeria populations, respectively, after 14 days
of incubation at 4C. Unfortunately, further experiments showed that cloves and oregano
were both no longer active against listeriae when present in a meat slurry at a level of
1%.
The ability of commercially available spices to prevent growth of L. monocytogenes
in TB also was investigated by Bahk et al. [24]. With the exception of an increased lag
phase, behavior of listeriae during extended incubation at 35 and 4C was relatively unaf-
fected by 0.5% ginger, onion, garlic, or mustard as well as ginseng, saponin, or mulberry
extract at concentrations 50.3%. However, unlike the previous study, addition of 0.5%
174 Lou and Yousef

cinnamon was somewhat inhibitory to L. monocytogenes, with the pathogen attaining max-
imum populations that were 1.5-2.6 orders of magnitude lower than in controls. Growth
of listeriae at 35C also was partly suppressed by 0.5%cloves, with the pathogen attaining
a maximum population 1.6 logs lower than that observed in controls. However, Listeria
populations decreased steadily in TB containing 0.5% cloves during extended incubation
at 4C.
Working in Thailand, Stonsaovapak and Chareonthamawat [367a] used a similar
experimental design to test nine dried native Thai spices, namely, cinnamon, black pepper,
white pepper, cloves, cardamom, coriander, star anise, nutmeg, and cumin seed at concen-
trations of 1,3, and 5% for activity against L. rnonocytogenes in TSB containing 10' CFU/
mL. As in the previous two studies [24,375], cloves exhibited strong listeriocidal activity
with concentrations 2 1%, reducing Listeria populations >8 logs and >6 logs after 7 days
of incubation at 35 and 4OC, respectively. When exposed to concentrations 2 1 % for 7
days, nutmeg and star anise also reduced numbers of listeriae 5-8 logs at 35C and
2-6 logs at 4C. Using 2 1 % white pepper, black pepper, or coriander, Listeria population
decreased 2-5 logs and 1-2 logs at 35 and 4OC, respectively, with these and the other
spices being most effective at concentrations of 5%. However, little if any inhibition was
observed using cardamom regardless of concentration or incubation temperature.
Antilisterial activity of 32 plant essential oils was investigated by Aureli et al. [22]
in Italy. The essential oils were dissolved in ethanol at a concentration of 1 5 (v/v) with
antilisterial activity assessed on TSA plates using the disc diffusion method. Five essential
oils showed activity against four strains of L. rnonocytogenes. Essential oils of origanum
and thyme were most active against Listeria, followed by oils of cinnamon, clove, and
pimento. Further tests with thyme, origanum, and cloves showed that the three oils main-
tained antilisterial activity at a 150 (v/v) dilution. Although nutmeg, rosemary, and sage
were found to the antimicrobially active by Ting and Deibel [375], the essential oils of
the three spices failed to inhibit listeriae. Essential oils that did not inhibit listeriae were
those of basil, camomile, celery, coriander, cumin, estragon, fennel, garlic, ginger, laurel,
lemon, mandarin, marjoram, neroly, onion, orange, parsley, pepper, peppermint, petti-
grain, saffron, and vanilla. Survival of L. rnonocytogenes ( IO5-1O6 CFU/mL) in a saline
solution containing 0.1% (v/v) of essential oils of origanum, thyme, cinnamon, cloves,
or pimento was similar among five strains of the pathogen. Oil of pimento had the greatest
activity and that of cinnamon the least. Essential oil of pimento decreased the population
of Listeria from > 103CFU/mL to an undetectable level in 1 h. All five oils reduced the
viable count to undetectable levels after 4 h of incubation at room temperature. In minced
pork stored for 8 days at 4 and 8OC, adding 100 pL of the diluted (15, vol/vol) essential
oil of thyme to 25 g of product, decreased the population of L. rnonocytogenes by -1
-
log, whereas the organism grew from 1 X 107to 6 X 107and 2 X 108CFU/g in untreated
controls at 4 and 8OC, respectively.
In 1993, Hefnawy et al. [ 1661 reported on the sensitivity of L. rnonocytogenes strains
Scott A and V7 to 10 spices in TSB at 4C and found the latter strain to be generally
more resistant. L. monocytogenes Scott A decreased from an initial population of 105to
<1 CFU/mL by 1% sage in 1 day; I % allspice in 4 days; 1% red pepper, paprika, garlic
powder, or cumin in 7 days; 5% black pepper in 4 days; or 5% mace in 7 days, whereas
5% nutmeg partially inactivated the organism and 1-5% white pepper enhanced growth.
For strain V7, only 1, 3, and 5% sage reduced the initial population (-107 CFU/mL) to
< I CFU/mL in 7, 4, and 4 days, respectively, whereas 3-5% allspice, mace, or nutmeg
showed partial inactivation.
Characteristics of Listeria monocytogenes 175

Pandit and Shelef [285] investigated the antilisterial activity of 18 spices and herbs
by measuring growth of L. monocytogenes on BHI agar with (0.1%) and without spices/
herbs. The authors found that only rosemary and cloves showed antilisterial activity, and
contrary to previous studies [22,375], cinnamon, nutmeg, oregano, and sage had no activ-
ity. Other noninhibitory spices and herbs were ajowan, allspice, asafoetida, cardamom,
cumin, fenugreek, ginger, marjoram, black mustard, red hot pepper, and turmeric. Growth
of Listeria on agar plates was completely inhibited by 0.5% rosemary or 1.0% cloves.
An aqueous extract of rosemary only prevented growth of Listeria in BHI broth at 35C
for the first 24 h, whereas the same levels of rosemary or its ethanol extract had significant
listericidal activity. The authors further investigated the antilisterial activity of rosemary
oil and its four major components (cineole, borneole, camphor, and pinene), rosemary
oleoresin, rosemary oil encapsulated in modified starch, and an antioxidant fraction ex-
tracted from rosemary by liquid carbon dioxide. Rosemary oil ( 10 pL/ 100 mL) was listeri-
ostatic during 48 h of incubation, whereas oleoresin (up to 100 mg/ 100 mL) did not inhibit
listeriae. Encapsulated oil was listeriostatic at 1 pg/lOO mL, and listericidal at 5 pL/lOO
mL. The antioxidant extract at 0.02 g/lOO mL was listericidal. Of the four rosemary oil
components, only pinene at 0.1 pL/ 100 mL suppressed growth of Listeria, whereas the
other three components had no activity at concentrations up to 1 pL/lOO mL. When added
to refrigerated (5C) pork liver sausage, 1% rosemary, 0.5% rosemary oil, 0.3% antioxi-
dant extract, or 5% encapsulated oil suppressed growth of Listeria, with maximal popula-
tions of -log, -108, -105, and -105 CFU/mL being attained after 25, 25, 50, and 50
-
days, respectively, whereas 1O3 CFU/mL of Listeria in the control reached 109CFU/
mL after 25 days.
Tassou et al. [372], working in Greece, investigated the antimicrobial effect of the
essential oil of mint on L. monocytogenes and S. enteritidis in a broth culture and in three
foods; tzatzilu (cucumber and yogurt salad, pH 4.3), taramosalata (fish roe salad, pH 4.9),
and piit6 (pH 6.8). Concentrations of mint oil tested in this study were 0.5, 1.0, 1.5, and
2.0%, v/w, and incubation temperatures were 4 and 10C. Although gram-positive bacteria
generally are more sensitive to essential oils than gram-negative microbes, the authors
found that L. monocytogenes was more resistant to mint essential oil than S. enteritidis.
Antibacterial activity varied between foods and pH values. The presence of 1-2% essential
oil in tzatziki decreased salmonellae numbers to undetectable levels after 3-6 h, whereas
>2 logs of the organism remained viable after 6 days in the untreated control that initially
contained l O7 L. monocytogenes CFU/g. However, in the same treated food, populations
of L. rnonocytogenes remained unchanged for the first 4 days and decreased gradually
during subsequent storage, with >4 logs being detected after 8 days at both temperatures.
Compared with the control, the presence of mint oil decreased the rate of death of L.
monocytogvnes in the product. Populations of L. rnonocytogenes and S. enteritidis de-
creased slightly in taramosalata made with and without mint essential oil during 9 days
of incubation at 4 and 10C. The essential oil was without antimicrobial activity in pit&,
which had an almost neutral pH. In all pgt6 samples, salmonellae populations decreased
at 4C or decreased and then increased at 10C, whereas listeriae increased > 1 and >3
logs at 4 and IOOC, respectively, after 6 days of incubation. Greater antimicrobial activity
of mint oil was observed in broth than in food, with both organisms being inhibited by
0.5-2.0% mint oil in broth at pH 6.6.
Because of the potential link between consumption of chocolate milk and listeriosis,
Pearson and Marth [297] studied the effect of caffeine and theobromine, two methylxan-
thine compounds in cocoa, against L. monocytogenes strain V7 (initially 103CFU/rnL)
176 Lou and Yousef

in a modified TPB and skim milk at 30C. L. monocytogenes grew similarly in both media.
Although limited antilisterial activity was observed with 2.5% theobromine, the authors
found that 0.5% caffeine had antilisterial activity in both substrates and increased the lag
phase from <3 (control) to 6-9 h, increased generation times from 1.2 to 2.17 h, and
decreased the final maximum population from 8.6 to 7.2 logs. A combination of 2.5%
theobromine and 0.5% caffeine had slightly more antilisterial activity than did 0.5% caf-
feine alone.
In conclusion, essential oils are more effective in broth than in foods. Because of
their hydrophobic nature, antilisterial activity of essential oils is adversely affected by
high fat and protein contents in food. Antimicrobial activity of essential oils is enhanced
by low pH, high salt content, and low storage temperatures.

Lysozyme
Lysozyme is an important natural enzyme which prevents bacterial growth (particularly
gram-positive organisms) in foods of animal origin, including hens eggs and milk. Lyso-
zyme is particularly attractive as a food preservative, since the enzyme is active between
4 and 95OC, stable over a wide range of pH values, specific for bacterial cell walls, and
is not harmful to humans. Although not yet approved as a food additive in the United
States, lysozyme has been used successfully in Europe to prevent blowing caused by
Clostridium spp. in Gouda, Edam, and other brine-salted cheeses.
In 1987, Hughey and Johnson [ 1791evaluated four L. monocytogenes strains isolated
during foodborne listeriosis outbreaks for their susceptibility to lysozyme. After nongrow-
ing cells of L. monocytogenes in the stationary growth phase were suspended in phosphate
buffer containing 10 mg of lysozyme/L, 70-80% of the cells were lysed after 6 h, as
determined by optical density measurement. In 1994, Johansen et al. [ 1871 tested the ability
of egg white lysozyme to lyse five L. monocytogenes strains, which were suspended in
phosphate buffer of pH 7. At 200 U/mL, lysozyme caused lysis of the five strains, and
the rate of lysis was maximum at a lysozyme concentration of 1000 U/mL. Sensitivity
of Listeria to lysozyme varied with the temperature at which the bacterium was grown
before the treatment. Cells grown at 5C were the most and those grown at 37C the least
sensitive, with cells grown at 25C showing intermediate sensitivity. A similar effect of
Listeria growth temperature on lysozyme sensitivity was noted by Smith et al. [355]; the
authors found that L. rnonocytogenes grown at 37C was 1.8-2.5 times more resistant to
lysis by lysozyme than were cells grown at 5, 12, and 19C.
In contrast to the listericidal effect of lysozyme in nutrient-poor buffers, Listeria
can grow in nutrient-rich media containing lysozyme. Growth inhibition detected in such
media depends on the concentration of lysozyme, pH of the medium, incubation tempera-
ture, and the presence of various growth modifiers. According to Hughey and Johnson
[179], four strains of Listeria grew in BHI broth containing 20-200 mg of lysozyme/L.
Similarly, cells of L. monocytogenes in the logarithmic growth phase were not lysed after
12 h of exposure to 100 mg of lysozyme/L. In a more recent investigation [187], the
presence of 10,000 U/mL lysozyme in TSB at 5C extended the lag phase from 0 to 8
days at pH 7.0, and from 9 to 60-70 days at pH 5.5. A similar pattern was seen at 25C
with a lag phase at pH 5.5 of 4 h without lysozyme and 37 h with 50,000 U/mL lysozyme.
The authors attributed the pH-dependent increase in bacteriostatic activity of lysozyme
to the growth-retarding effect of low pH, which allowed cell lysis to proceed faster than
Characteristics of Listeria monocytogenes 177

cell multiplication. The lytic action of lysozyme was not affected as the pH was lowered
from 7.0 to 5.5.
Various chemical treatments also have been examined for their ability to potentiate
lysis of growing cells of L. monocytogenes in the presence of lysozyme [ 1791. Addition
of 0.85% lactic acid stopped growth of L. monocytogenes and led to gradual lysis of
cells by lysozyme. EDTA was the most effective potentiator of cell lysis, whereas 0.05%
potassium sorbate, 0.25% glycine, 5 mM sodium acetate, 0.95% ethanol, 0.01% sodium
dodecyl sulfate, 5 mM thioglycolate, 5 mM dithiothreitol, and 10 mM ascorbic acid were
relatively ineffective. The inhibitory action of EDTA and lysozyme toward Listeria also
was tested using BHI agar. Although 100 mg of lysozyme/L or 1 mM EDTA failed to in-
hibit growth of two of four L. monocytogenes strains, the combination of EDTA and lyso-
zyme led to substantial growth inhibition as compared with the additive-free control [ 1791.
The antimicrobial activity of lysozyme against L. monocytogenes suspended in water
was enhanced by prior treatment with lipase, an enzyme naturally existing in milk, or fro-
zen storage for up to 6 weeks [ I 10,1111. Enhancement of lysozyme activity against L. mono-
cytogenes suspended in buffer or broth by lipase also was observed by Liberti et al. [23 11.
When compared with results using laboratory media, the antilisterial activity of lyso-
zyme is variably reduced in foods. Hughey et al. [I801 found that lysozyme was more
effective in controlling L. monocytogenes in vegetables than in meat products. A combina-
tion of lysozyme and EDTA inactivated L. monocytogenes (at 10" CFU/g) in fresh green
beans, fresh corn, shredded cabbage, shredded lettuce, and carrots during storage at 5OC,
whereas Listeria in control samples grew to 106-107CFU/g. In fresh pork sausage (brat-
wurst), lysozyme was only listeriostatic for 2-3 weeks and did not prevent growth during
extended storage of the food. During ripening of Camembert cheese, lysozyme alone or
in combination with EDTA decreased listeriae by about 1 log during the first 3-4 weeks,
and then the Listeria count increased slowly and reached 106- 10' CFU/g after 55 days
of ripening. Carminati and Carini [66] found that lysozyme at 25 and 1000 ppm only
caused 22 and 57% reductions in count, respectively, for two of four L. monocytogenes
strains inoculated into sterile skim milk. Addition of lysozyme to heat-treated skim milk
that contained Listeria did not inhibit outgrowth of survivors.
Kihm et al. [201] found that minerals in milk protected L. monocytogenes against
lysozyme. Hen's egg white lysozyme (100 mg/L) had no antilisterial activity in whole
milk. However, removal of minerals from milk by cation exchange slightly enhanced
activity of lysozyme at 4C. Prior heating (62.5"C for 15 s) of L. monocytogenes in a
phosphate buffer sensitized the pathogen to lysozyme in MES (2-[N-morpholino] ethane-
sulfonic acid) buffer or demineralized milk. Furthermore, heating the pathogen at 55C
in the presence of lysozyme greatly increased inactivation of the pathogen in demineralized
rather than undemineralized milk. Although lysozyme alone did not inhibit Listeria growth
in milk [20 I ] , Payne et al. [295] found that lysozyme plus EDTA had an interactive antimi-
crobial effect against L. monocytogenes growing in ultrahigh-temperature (UHT) milk.
Although lysozyme activity is affected by many food components, adding lysozyme
to foods is potentially beneficial to control this pathogen. As discussed previously, lyso-
zyme alone or in combination with EDTA inactivated Listeria in vegetables and retarded
growth of the pathogen in fresh pork sausage and Camembert cheese [ 1SO]. Lysozyme
(500 ppm), when added to acidified sterile skim milk (pH 5.3) that was stored at 4C for
6 weeks (i.e., simulation of cheese making), enhanced inhibition of Listeria [66]. Egg
white lysozyme contributed to the high antilisterial activity of some mayonnaise products
178 Lou and Yousef

[126]. Consistent with this finding, Wang and Shelef [383] found that raw egg albumin
at levels >15% was bactericidal to L. monocytogenes in TSB at 35"C, with this activity
being primarily attributed to lysozyme.
Wang and Shelef [384] later found that L. monocytogenes growth in raw cod fish
fillets could be retarded by lysozyme alone or in combination with EDTA. The fish fillets
were dipped for 10 min at 20C in solutions of lysozyme (3 mg/mL), EDTA (5-25 mM),
or a combination of lysozyme (3 mg/mL) and EDTA (25 mM), inoculated with about 103
CFU/g L. monocytogenes, and then monitored for Listeria growth during storage at 20C
for 3 days or at 5C for I7 days. The authors found that lysozyme plus EDTA had substan-
tial antilisterial activity at both storage temperatures. Listeria populations in the control
and in samples pretreated with 5-10 mM EDTA increased to about 108CFU/g after stor-
age at 2OoC, whereas the pathogen only increased <2 logs CFU/g in samples pretreated
with 15 and 25 mM EDTA. In lysozyme-treated samples stored at 2OoC, final Listeria
populations were about 10-fold lower than in the control. Lysozyme and EDTA (25 mM)
interacted synergistically and resulted in > 1-log decrease of listeriae in the first 18 h; the
pathogen never grew to a level exceeding the initial population during the entire storage
period. When control and treated samples were stored at 5C for 17 days, Listeria popula-
tions increased 1 log in the control, remained almost unchanged in EDTA-treated samples,
and decreased up to 1 log in samples treated with lysozyme or with the combination of
lysozyme and EDTA.

Hydrogen Peroxide
Although hydrogen peroxide is used as a preservative, particularly for raw milk in some
parts of the world, use of this antimicrobial agent in the United States is very limited.
The FDA permits adding up to 0.05% (w/w) hydrogen peroxide to raw milk intended to
be made into certain kinds of cheese. It has also been approved by the FDA for sterilizing
multilayer packaging materials used in aseptic processing systems.
The antilisterial effect of hydrogen peroxide at levels permitted by the FDA was
investigated in milk by Dominguez et al. [89]. These investigators found that L. monocyto-
genes was eliminated from autoclaved milk that had been inoculated to contain 9.5 X 107
L. monocytogenes CFU/mL, treated with 0.0495% hydrogen peroxide, and held for 24 h
at 15C. However, when raw milk was treated with 0.0495% hydrogen peroxide, inocu-
lated to contain -2 X 105L. monocytogenes CFU/mL, and incubated at 4OC, numbers
of listeriae increased slightly as compared with the natural microflora. In another experi-
ment by the same researchers, samples of autoclaved milk containing 50.0495% hydrogen
peroxide were inoculated to contain a mixed culture of L. monocytogenes, S. aureus, and
Enterococcus faecalis (each organism at --I X 107CFU/mL) and incubated for 48 h at
4, 15, and 22C. Although L. monocytogenes populations decreased approximately 10-,
16-, and 40-fold during the first 24 h of incubation at 4, 15, and 22OC, respectively, the
organism grew during the second 24 h and reached populations 2 1 X 107CFU/mL. In
a subsequent study, Kamau et al. [ 1951 observed that 0.6 mM (i.e., 0.002%) H202slightly
inhibited growth of L. monocytogenes in bovine milk that was mildly heated (57"C, 20
min), cooled, and stored at 10C compared with the control without H202.Thus hydrogen
peroxide was relatively ineffective in decreasing numbers of listeriae in raw milk or milk
containing equal numbers of S. aureus and E. faecalis.
The impact of hydrogen peroxide on destruction of L. monocytogenes by heat was
investigated by two research groups. Kamau et al. [ 1961 reported that the presence of 0.6
Characteristics of Listeria monocytogenes 179

mM H202in milk did not enhance inactivation of L. monocytogenes by heat. Lou and
Yousef [238] found that adaptation of L. monocytogenes to H202increased the resistance
of this pathogen to heat. The authors added 500 ppm hydrogen peroxide into a culture of
L. monocytogenes in the exponential growth phase, incubated the culture for an addi-
tional 1-2 h at 35"'-,, and then determined heat resistance of treated Listeria cells in a
H20,-free phosptidte buffer. Compared with the unadapted culture, H202adaptation in-
creased DS(,oC-values by 2.9-fold. The same authors 12391 also reported an increase in
resistance of L. monocytogenes to a lethal level (i.e., 0.1%, w/w) of H202after the bacte-
rium was adapted to pH 4.5-5.0, 500 ppm H202,5% ethanol, 7% NaCI, or heat shocked
at 45C for 1 h.

Lactoperoxidase System
The lactoperoxidase (LP) system, a naturally occurring antimicrobial system in milk, has
been proposed as a means for extending the shelf life of raw milk when extended refriger-
ated storage is not possible, as in certain developing countries. Proper functioning of this
system depends on adequate levels of lactoperoxidase, thiocyanate, and hydrogen perox-
ide. Lactoperoxidase in milk represents 1 % of whey proteins [3 151, which is an adequate
amount for functioning of the lactoperoxidase system. Thiocyanate, however, is present
in bovine milk at only 1-7 pprn [36,37] and H202needs to be added exogenously or
generated by exogenous enzymes, such as glucose oxidase. In the LP system, lactoperoxi-
dase catalyzes the oxidation of thiocyanate (SCN-) by hydrogen peroxide to hypothiocya-
nous acid (HOSCN) and hypothiocyanate (OSCN-); these endproducts are responsible for
inactivating the microflora common to milk, including S. aureus, Salmonella typhimurium,
psychrotrophic pseudomonads, and some lactic acid bacteria.
Siragusa and Johnson [350] reported results of a study which examined inhibition
of L. monocytogenes by the LP system. Their model LP system contained equimolar con-
centrations (0.3 mM) of potassium thiocyanate and hydrogen peroxide in TSB fortified
with 0.5% yeast extract. After addition of 0.37 U lactoperoxidase/mL, flasks were inocu-
lated with 1,. monocytogenes in the late logarithmic growth phase. L. monocytogenes had
lag periods of 147.3-159.6, 46.6-55.5, 16.4-17.1, and 7.1 h in the presence of the LP
system and 61.4-77.4,23.5-32.5,7.5-10.3, and 4.3-5.7 h in the control (with or without
0.3 mM H202)when the pathogen was incubated at 5, 10, 20, and 3OoC, respectively.
Although the LP system appreciably extended the lag phase, maximum specific growth
rates were not affected. When the LP system was tested in sterile reconstituted skim milk
at 2OoC, the lag phase of L. monocytogenes was extended from 9 h (control) to 12-36 h.
Maximum Listeria populations also were lower with the LP system than in controls. Thus,
in this particular study, the LP system was bacteriostatic rather than bactericidal to L.
monocytogenes, and it was more effective at low than at high incubation temperatures.
In a subsequent study, Kamau et al. [ 1951 activated the lactoperoxidase system by
adding 2.4 mM SCN- and 0.6 mM H202to preheated (57"C, 20 min) bovine milk, which
contained adequate residual lactoperoxidase (9.2 mg/mL). Concentrations of SCN- and
H202used in this study did not have measurable antimicrobial activity against L. monocyto-
genes. When the LP system was activated at 35"C, L. monocytogenes (initially 104CFU/
mL,) decreased slightly in the first 2 h and began to grow after 8 h. At 10C, the LP
system inhibited Listeria for 96 h before appreciable growth was observed. The times
required to achieve half of the maximum growth were 16.9, 11.7, and 10.6 h at 35C and
436, 170, arid 137 h at 10C in milk (a) with activated LP systems, (b) with 0.6 mM H202,
180 Lou and Yousef

and (c) without additives, respectively. At 35OC, Listeria grew in all milk samples at a
similar maximum specific growth rate (0.162-0.221 h-I), whereas at IOOC, the pathogen
had a lower specific growth rate (0.0047 h-I) in the LP system-activated milk than in
that of the control (0.0103-0.0123 h-I).
Although Kamau et al. [195] and Siragusa and Johnson [350] reported that the LP
system was mainly bacteriostatic toward L. monocytogenes in a laboratory medium and
preheated milk, several other research groups noted appreciable bactericidal activity of
the system. El-Shenawy et al. [I211 found that initial L. monocytogenes populations of
30-50 CFU/mL decreased to nondetectable levels following 2 h of exposure to the LP
system at 35C. Using selective and nonselective plating media, these researchers also
demonstrated that the pathogen was not sublethally injured during exposure to the LP
system. Denis and Ramet [86] reported that the LP system completely eliminated L. mono-
cytogenes (initial populations - 10'-10' CFU/mL) from TSB with 0.65% yeast extract
following 5 1 , 2-6, and 4-10 days of incubation at 30, 15, and 4OC, respectively, de-
pending on the initial inoculum. However, unlike the previously described model broth
systems, these authors added glucose oxidase to their LP system. Since this enzyme oxi-
dizes glucose to gluconic acid, the resulting lowering of pH likely increased the bacteri-
cidal effect of the LP system beyond what would have been observed in similar model
systems having pH values near neutrality. Furthermore, L. monocytogenes is also inhibited
and/or inactivated in TB and milk containing 20.75% gluconic acid during extended
incubation at 13 and 35C [ 1181. Hence, these findings likely reflect the combined effects
of the LP system, pH, and gluconic acid rather than that of the LP system alone.
Additional investigations dealing with antilisterial activity of the LP system in UHT
milk rather than in culture media appeared in the scientific literature. Using two different
UHT milk-based LP systems containing lactoperoxidase (30 mg/L), potassium thiocya-
nate (84 mg/L), glucose (10 g/L), and glucose oxidase (2 mg/L) both with and without
urea peroxide (376 mg/L) as a hydrogen peroxide-generating mechanism, Earnshaw and
-
Banks [ 1011 found that initial L. monocytogenes populations of 104CFU/mL decreased
to 102CFU/mL in both LP systems during 6 days of incubation at 10C. Denis and Ramet
[86] also found that L. monocytogenes populations decreased in a similar UHT milk-
based LP system containing lactoperoxidase, potassium thiocyanate, and glucose oxidase.
However, unlike the previous study, their LP system completely eliminated the pathogen
(initial populations of 101-104CFU/mL) from UHT milk following 6-21 and 7-30 days
of incubation at 15 and 4OC, respectively, with estimated D-values of approximately 5
and 8 days at these same temperatures. Thus, as expected, the LP system was more detri-
mental to listeriae at higher rather than lower temperatures. In contrast, without the LP
-
system, the pathogen attained populations of 1OS and 1O4 CFU/mL following 7 days of
incubation at 15 and 4OC, respectively.
Several groups investigated antilisterial activity of the LP system in raw milk con-
taining naturally occurring levels of lactoperoxidase. El-Shenawy et al. [121] used an LP
system in raw milk containing naturally occurring levels of lactoperoxidase along with
0.25 mM thiocyanate anion and 0.25 mM hydrogen peroxide, and they found that L.
monocytogenes was often only slightly inhibited. In samples inoculated to contain 104-
and 107L. monocytogenes CFU/mL, the pathogen attained maximum populations of 2 108
CFU/mL after overcoming an extended lag phase. However, this LP system was far more
effective in raw milk inoculated to contain Listeria populations (i.e., 1O2 CFU/mL) similar
to those that have been observed in cases of naturally occurring listerial mastitis. Under
these conditions, the pathogen was completely inactivated after 2-4 and 12-24 h of incu-
Characteristics of Listeria monocytogenes 181

bation at 35 and 4C. Thus, as was true for microbiological media and UHT milk, the LP
system was again more effective in raw milk stored at higher rather than lower tempera-
tures. In a subsequent study, Gaya et al. [ 1501 investigated antilisterial activity of the LP
system activated by adding equal concentrations (0.25 mM) of sodium thiocyanate and
H 2 0 2to raw bovine milk stored at 4 and 8C. The authors reported D-values of 4.1- 1 1.2
days at 4C and 4.4-9.7 days at 8C for four L. monocytogenes strains added to the LP
system-activated milk. Lactoperoxidase activity decreased during incubation, with the
loss being more rapid at 8C than at 4C. In a more recent study, Zapico et al. [407]
reported that the activated LP system in goat's milk remained bactericidal against three
L. monocytogenes strains for 3-9 days at 4C and 1-7 days at 8C. Bacteriostatic activity
against Listeria was observed at 20C.
The LP system can be used in conjunction with thermal processing to increase de-
struction of listeriae in raw milk. Kamau et al. [ 1961 reported that the LP system (0.24
mM SCN- and 0.6 mM H202)enhanced thermal inactivation of L. monocytogenes in
preheated (57"C, 20 min) bovine milk containing 9.2 pg/mL of lactoperoxidase. Biphasic
heat inactivation curves were observed when the LP system was activated, with most of
the population being heat sensitive and inactivated rapidly during heating. The D-values
(based on the heat-resistant fraction of the population if biphasic inactivation curves oc-
curred) in milk (a) with the activated LP system, (b) with 0.6 mM H202,and (c) without
any additives (control) were 10.7, 29.4, and 30.2 min at 52.2"C, 1.6, 11.1, and 8.2 min
at 55.2"C, and 0.5,2.6, and 2.3 min at 57.8"C, respectively. When the LP system-activated
milk was held at 35C for different periods before heating, thermotolerance of L. monocy-
togenes decreased as the holding time increased, with the D 55.2"C being only 6.8 min after
16 h of holding time.
In summary, L. monocytogenes is susceptible to the LP system, especially at low
incubation temperatures. The LP system also can be used in combination with other treat-
ments, such as heat to increase inactivation of listeriae. This system will likely prove to
be useful for decreasing numbers of naturally occurring listeriae in raw milk before milk
processing facilities receive the product.

Lactoferr in
The presence of iron in culture media stimulates growth of some microorganisms. Lacto-
ferrin, a glycoprotein found in mammalian milk, exerts its antimicrobial activity through
binding of iron. Thus the antimicrobial activity of lactoferrin is affected by its degree of
iron saturation and iron availability in the medium. The degree of saturation of lactoferrin
with iron can be reduced by dialysis, and the resulting product is known as apo-lactoferrin.
Both lactoferrin and apo-lactoferrin exhibit antilisterial activity. Recently, lactoferrin was
found to inhibit invasion of L. monocytogenes into cultured intestinal cells [ 191.
Payne et al. [296] studied the effect of bovine lactoferrin and apo-lactoferrin, with
52% and 18% iron saturation, respectively, on growth of L. inonocytogenes in UHT milk
with 2% fat. After 18 h of incubation at 35"C, two strains of L. monocytogenes grew in
the presence or absence of lactoferrin (46 mg/mL), but the count of Listeria was 1.6- 1.8
logs lower in treated milk than in the control. Compared with lactoferrin, apo-lactoferrin
had greater antilisterial activity. When added to milk incubated at 35"C, apo-lactoferrin
was strongly listeriostatic at 15 mg/mL and listericidal at 30 mg/mL. Addition of 0.125
M ferric ammonium citrate eliminated the inhibitory effect of 30 mg/mL apo-lactoferrin
against Listeriu.
182 Lou and Yousef

These researchers [295] subsequently investigated the antimicrobial activity of apo-

lactoferrin, EDTA, lysozyme, or their combinations in UHT milk. When applied separately
or in combinations, these substances did not inhibit P. Jluorescens and S. typhimurium.
However, inhibition of L. monocytogenes and E. coli 0 157 :H7 was observed using these
compounds, with a combination of 15 mg/mL apo-Iactoferrin and 150 mg/mL lysozyme
retarding growth of L. monocytogenes.
Lactoferricin, a small antimicrobial peptide (25 amino acid residues) resulting from
hydrolysis of bovine lactoferrin by gastric pepsin, has strong antilisterial activity in culture
media [381]. The MICs of lactoferricin for four L. monocytogenes strains ( 106CFU/mL)
at 37C were 0.3-0.6 pg/mL in 1% peptone and 1-3 pg/mL in Peptone-Yeast Extract-
Glucose (PYG) broth. The presence of up to 10 mg/mL of various sugars or starch did
not affect the antilisterial activity of lactoferricin. Addition of gelatin or bovine serum at
10 mg/mL slightly increased the MICs of lactoferricin. However, up to 100 mM NaC1,
KC1, or NH4C1 and up to 5 mM of Mg,C12 or CaC1, increased the MICs to 6-9 pg/mL
for one of the most resistant Listeria strains. Lactoferricin maintained its antilisterial activ-
ity over a pH range of 5.5-7.5. This peptide was bactericidal to L. monocytogenes growing
in PYG broth at 37C; treatment of 104-106 CFU/mL of L. monocytogenes with 31 pg
lactoferricin/mL for 60 min reduced the viable population to below a detectable number
(i.e., <100 CFU/mL) for three strains and to 300 CFU/mL for the fourth strain.
In a subsequent study [30], in addition to L. monocytogenes, lactoferricin inhibited
a wide range of pathogenic and spoilage bacteria: E. coli, Salmonella enteritidis, Yersinia
enterocolitica, Campylobacter jejuni, S. aureus, Corynebacterium diphtheriae, Clostrid-
ium perfringens, Klebsiella pneumoniae, Proteus vulgaris, Streptococcus mutans, and
Pseudomonas aeruginosa. Levels of 0.3- 120 pg/mL of lactoferricin were required to
inhibit these organisms. As with L. monocytogenes, this peptide inhibited E. coli 0 1 11
over a pH range of 5.5-7.5, with the greatest activity occurring at slightly alkaline pH
values.

BIOPRESERVATION
The terms biological preservation, biopreservation, and biocontrol all refer to the use of
microorganisms or their metabolic products to inhibit or inactivate undesired microorgan-
isms in foods. Biopreservation, as a means of naturally controlling pathogens and spoilage
microorganisms, especially in minimally processed foods, has been extensively studied
and excellent reviews are available [2,162,174,256,272,31 1,3361. Lactic acid bacteria
(LAB) and their metabolic products are commonly used in biopreservation, since these
bacteria are used in many traditional foods and are GRAS. Biopreservation by LAB occurs
because these bacteria compete with other microorganisms for nutrients and/or because
they produce antimicrobial compounds, such as weak acids, hydrogen peroxide, diacetyl,
and bacteriocins. The discussion in this section will focus on biopreservation with bacterio-
cins or bacteriocin-producing LAB which target L. monocytogenes in food.
Bacteriocins are antimicrobial substances that have a peptide or protein component
essential for their activity. Although most bacteriocins have a narrow spectrum of inhibi-
tion and only inhibit closely related species, some bacteriocins, such as nisin and pediocin,
have a relatively broad spectrum and can inhibit some less closely related organisms. A
large number of LAB bacteriocins are active against L. monocytogenes. Although their
modes of action vary, bacteriocins usually destabilize the cytoplasmic membrane of sensi-
tive cells, increase membrane permeability, and dissipate the proton motive force by form-
Characteristics of Listeria monocytogenes 183

ing water-filled transmembrane pores or channels [ 1851. Bacteriocins produced by LAB

are grouped into four distinct classes [206]. Bacteriocins of class I are lantibiotics, lanthio-
nine-containing peptides, such as nisin. Class I1 includes small (<10 kD), non-lanthio-
nine-containing, relatively heat-stable bacteriocins, such as pediocin PA- 1, P02, or AcH.
Class 111bacteriocins form large (>30 KD) heat-labile molecules. Class IV includes bacte-
riocins with nonpeptide moieties.
Nisin-producing strains of Lactococcus lactis subsp. lactis have been used legally
in the United States and elsewhere to manufacture certain cheeses and other dairy products
that require a mesophilic fermentation. Although bacteriocin-producing LAB may be
added as a starter culture or fermented ingredient to various foods, legal issues arise when
foods are supplemented with purified bacteriocins, as discussed by Fields [ 1421 and Post
[306]. Approval from US regulatory agencies, mainly the FDA and the USDA, is required
for the application of purified bacteriocins [ 1421. According to the US Code of Federal
Regulations [ 1381, a company can self-affirm whether a bacteriocin of interest is GRAS;
however, the company is required to justify application of the bacteriocin if requested by
the FDA [ 142,2721. Of purified bacteriocins, only nisin has been approved by the FDA
for use in pasteurized cheese spreads [ 1 1,1371. Approvals for use of nisin in other food
products were subsequently granted by the FDA.
Drawbacks of biopreservation are limiting large-scale application of this technology
in the food industry. When bacteriocins are added to foods, they usually show only a
modest antimicrobial effect, with the targeted organism often becoming bacteriocin-resis-
tant by one or more mechanisms [237,262,263,272]. Additionally, common LAB bacterio-
cins are not active against gram-negative bacteria. Destabilization of the outer membrane
of gram-negative bacteria by other factors or treatments is required for LAB bacteriocins
to be active against these bacteria [367]. Therefore, a bacteriocin is normally used in
food processing as a hurdle in combination with other treatments. Several research groups
reported data indicating the value of bacteriocins in combined treatments. In these studies,
bacteriocins were tested in combination with sublethal heat, acid, and freezing-thawing
[ 193,249,2721, lysozyme and chelating agents [367], other bacteriocins [ 1611, high hydro-
static pressure [ 165,1941, and pulse electric field [ 1941.
Biocontrol of L. monocytogenes in food can be achieved by (a) adding bacteriocin-
producing microorganisms to foods, (b) fermenting foods with bacteriocin-producing
LAB, (c) adding bacteriocin-containing fermentates, (d) adding bacteriocin crude extracts
or purified bacteriocins, or (e) incorporating bacteriocin-containing food ingredients [272].
It should be cautioned, however, that in the United States, application of purified bacterio-
cins to food is subject to the legal restrictions discussed earlier. Most studies on biopreser-
vation have dealt with nisin, pediocin, and the LAB producing these bacteriocins. There-
fore, the following discussion will deal mainly with these two bacteriocins.

Nisin
Nisin is a bacteriocin produced by certain strains of L. lactis subsp. lactis and has proven
to be extremely useful in preventing outgrowth of Clostridium spp., including Clostridium
botulinum, in fermented dairy and meat products. In 1980, Mohamed et al. [264] reported
results from a series of experiments that were designed to determine the effectiveness of
nisin against Listeria. When NB at pH 7.4 contained 4-16 International Units (IU) of
nisin per milliliter, populations of L. monocytogenes decreased >5 logs during 28 h at
37C. After this initial decrease, Listeria grew rapidly and attained final populations of
184 Lou and Yousef

- 108CFU/mL. Decreasing the pH of the medium from 7.4 to 5.5 led to a 16-fold decrease
in the level of nisin required to inhibit the bacterium.
Strains of L. monocytogenes may vary in resistance to nisin. Using Trypticase Soy
Agar, Benkerroum and Sandine [311 found that six L. monocytogenes strains were variably
resistant to nisin with MICs ranging from 1.4 X 102to 1.18 X 105IU/mL. Several addi-
tional studies also have demonstrated various degrees of nisin resistance for L. monocyto-
genes. Although Tatini [373] found that 512-1024 ppm nisin was required to inhibit
growth of 12 L. rnonocytogenes strains in laboratory media, S. typhimuriurn and E. coli
remained viable in the presence of up to 10,000ppm nisin. Although these findings suggest
that L. monocytogenes may be less resistant to nisin than some other potentially hazardous
microorganisms, one must keep in mind that some unusually resistant strains of L. monocy-
togenes do exist [ 163,262,263,2371.In 1989, Harris et al. [ 1631 examined sensitivity and
resistance of L. monocytogenes to nisin. According to these authors, populations of lister-
iae decreased 6-7 logs when nisin levels in BHI agar were increased from 0 to 10 pg/
mL. However, a relatively stable population of nisin-resistant mutants (- 100- 1000 CFU/
mL) developed on agar plates containing 1-50 pg nisin/mL with nisin-resistant mutants
occurring at a frequency of 10-6-10-x in media containing 50 pg nisin/mL. Although all
nisin-resistant mutants selected from agar plates were more resistant than their parent
strains, further testing revealed that nisin resistance was related to ability of nisin-resistant
strains to bind nisin rather than to specific genes coding for nisin resistance in plasmid
DNA. Similar nisin-resistant mutants also were obtained by Ming and Daeschel [262,
2631. Besides nisin resistance observed in spontaneous mutants, Lou [237] found that
acid adaptation or starvation increased resistance of L. monocytogenes to nisin and
pediocin.
As indicated by the earlier findings of Mohamed et al. [264], antilisterial activity
of nisin is strongly influenced by various environmental factors, including pH. Benker-
roum and Sandine [3I] determined the sensitivity of one L. monocytogenes strain to nisin
in Tryptose Soy Broth adjusted to pH values of 3.5-7.0. Populations increased -1 log
in broth cultures at pH 7.0 and 6.48 during the first 12 h of incubation, but no increase
in count was observed in similar samples adjusted to pH 5 5.94. Enhanced activity of
nisin against Listeria at lower pH values also has been observed by Harris et al. [ 1631.
Furthermore, data from Tatini [3731 indicate that average minimum nisin concentrations
of 5 12, 1365,2560, and 2496 ppm were required to inhibit growth of several L. monocyto-
genes strains on Trypticase Soy-Yeast Extract Agar adjusted to pH values of 5.0, 5.5,
6.0 and 6.5, respectively. Thus increased susceptibility of L. monocytogenes to nisin at
pH values <6 appears to be fairly well established.
The antilisterial action of nisin is further complicated by incubation temperature and
the presence of sodium chloride. According to Tatini [373], minimum concentrations of
nisin necessary to inhibit growth of L. monocytogenes were typically two to four times
greater at 35 than at 4C. In addition, when L. monocytogenes was incubated at 4C in
broth containing 1400 pprn nisin, lag periods for the various strains tested increased from
16 to 69 days as the nisin concentration increased from 0 to 400 ppm. In 1989, Harris et al.
[ 1631 also reported that addition of 2% sodium chloride enhanced the listericidal activity of
nisin in laboratory media, particularly at levels of <10 pg/mL.
Although many European countries have allowed direct addition of nisin to food
for some time, this practice was not permitted in the United States until 1989, when FDA
officials amended the food standard for pasteurized process cheese to allow addition of
not more than 250 pprn nisin to the finished product [ 1 1,16,17]. However, since allowable
Characteristics of Lister ia monocyt ogenes 185

levels of nisin may not completely inhibit L. monocytogenes in pasteurized process cheese
spreads that have been subjected to postpasteurization contamination, addition of nisin to
such products should not preclude use of proper sanitary practices.
Nisin-producing starter cultures provide some protection against L. monocytogenes
during cheese manufacture. Mainsnier-Patin et al. [248] inoculated L. monocytogenes into
milk which was used to make Camembert cheese. Nisin-producing or nonproducing starter
cultures were used in making the cheese. Counts of L. monocytogenes in the final product
were 2.4 log lower in cheese made with the nisin-producer than that made with the nonpro-
ducing strain. In another study, Zottola et al. [409] made Cheddar cheese with a nisin-
producing L. lactis starter and then prepared pasteurized processed cheese using this Ched-
dar cheese as an ingredient. Over 56 days of storage, populations of L. rnonocytogenes
decreased more rapidly in processed cheese made with rather than without the nisin-pro-
ducing culture.
The psychrotrophic and facultative nature of L. monocytogenes makes this patho-
gen a potential safety hazard for many minimally processed and vacuum/modified
atmosphere-packaged foods which require refrigeration. Incorporating bacteriocin or
bacteriocin-producing LAB into these products could be an effective way of minimizing
L. monocytogrnes growth and survival. However, sensory changes caused by addition of
biopreservatives must also be considered to ensure consumer acceptance [256].
Adding 10,000IU/mL nisin to cooked pork tenderloin that was packaged in air and
refrigerated prevented growth of inoculated L. monocytogenes but not Pseudomonas fragi.
However, use of nisin (1000 and 10,000IU/mL) in combination with modified atmosphere
packaging ( 100% CO2 or 80% CO2 + 20% air) inhibited growth of both bacteria, and
this inhibition was more pronounced at refrigeration than at room temperature [ 1291. Lac-
tic acid bacteria, such as Leuconostoc spp., often spoil minimally heat-processed vacuum-
packaged meat products. Some of the spoilage LAB are sensitive to nisin and nisin-produc-
ing Lactococcus spp. Thus Yang and Ray [399] suggested that biocontrol of these spoilage
LAB with nisin or nisin-producing strains could be an effective solution.

Pediocin
Pediocin, a wide-spectrum bacteriocin produced by certain strains of Pedicoccus acidilac-
tici, is a potent inhibitor of L. monocytogenes. Several research groups have reported
that P. acidilactici strains H, PAC1.O, and PO2 produced pediocin AcH, PA-1, and P02,
respectively. Luchansky et al. [24 I ] later reported that the restriction enzyme fragments
generated from plasmids encoding for these three pediocins were identical, with the three
producer strains also yielding identical genomic DNA fingerprints. These findings, in com-
bination with the DNA sequences for pediocin AcH and PA-1 (250a, 270a) indicate that
the three bacteriocins are similar in structure.
Control of L. monocytogenes by pediocin in several foods was explored. In a series
of studies on meat and meat products, Luchansky and his coworkers investigated the
antilisterial effect of pediocin AcH in wiener exudate, wiener packages, and turkey sum-
mer sausage [8524 1,4061. In refrigerated exudate from beef wieners, pediocin AcH de-
creased L. nzonocytogenes numbers by 0.74 log within 2 h. When the exudate was inocu-
lated with L,. nzonocytogenes and a pediocin-producing strain of P. acidilactici and kept
at 25OC, the count of L. monocytogenes increased initially and then markedly decreased
during extended incubation compared to the control. Pediocin activity in wiener exudate
was detected during the late stages of P. acidilactici growth [406]. Degnan et al. [85]
186 Lou and Yousef

later investigated survival of L. monocytogenes in vacuum-packaged beef wieners which

contained pediocin- or non-pediocin-producing P. acidilactici. Pediocin was produced
during early to late logarithmic phase. When packages were temperature abused at 25C
for 8 days, listeriae populations increased by 3.2 log CFU/g in the absence of any Pedio-
coccus strain, remained unchanged in the presence of the non-pediocin-producing strain,
and decreased by 2.7 logs in the presence of the pediocin producer. Results from several
other research groups are consistent with these findings. Berry et al. [32] found that the
bacteriocin-producing strain P. acidilactici JD 1-23 provided more protection against L.
monocytogenes during storage of vacuum-packaged frankfurters at 4C compared with a
plasmid-cured derivative of JDl-23. When either of these strains was used at a level of
107CFU/g, growth of Listeria was inhibited for up to 60 days. However, some antilisterial
activity also was observed at low levels ( 103-104CFU/g) of the pediococcus strains. When
pediocin was tested against L. monocytogenes attached to fresh beef muscle [241], pretreat-
ment of the muscle with pediocin slightly decreased L. monocytogenes attachment [ 1131.
According to Goff et al. [157], pediocin that was bound to heat-killed producer cells
remained strongly active in irradiated raw chicken breast meat which was stored at 5C.
Bacteriocin activity is usually adversely affected by certain food components. Degnan et
al. [83] improved pediocin activity in food slurries by using pediocin encapsulated in
phosphatidylcholine-based liposomes or by combining pediocin with 0.1% Tween 80.
Control of L. monocytogenes by pediocin-producing starter cultures was observed
during the manufacture of dry or semidry sausage. Berry et al. [34] noted -2-log reduction
in numbers of L. monocytogenes using a bacteriocin-producing strain of Pediococcus and
< 1-log reduction with a nonbacteriocinogenic starter during fermentation of semidry
sausage. Foegeding et al. [ 1441 compared the antilisterial activity of two starters, a pedio-
cin-producer, P. acidilactici PAC 1.O, and its isogenic pediocin-negative derivative, during
dry sausage production. The investigators observed that pediocin produced in situ was
partially responsible for listeriae inactivation during the sausage fermentation and drying.
Antilisterial activity from a pediocin-producing strain also was demonstrated during manu-
facture of turkey summer sausage [241]. During the 12-h fermentation, the presence of
pediocin and non-pediocin-producing strains decreased L. monocytogenes populations by
3.4 and 0.9 log CFU/g, respectively. Pediocin (-5000 AU/g) was recovered from sausage
even after 60 days of refrigerated storage. Besides use of bacteriocins and their producing
bacteria, LAB fermentates can also be used to improve control of L. monocytogenes in
food [84,338].
Several criteria for selection of suitable biocontrol microorganisms for use in meat
or meat products were proposed by McMullen and Stiles [256]. Biopreservation microor-
ganisms should be psychrotrophic, produce bacteriocins early in the growth cycle, and
exhibit little negative effect on product quality. Bacteriocins produced by these bacteria
should be active (bactericidal) and stable in the food environment. The authors concluded
that nisin-producing lactococci are poor biocontrol organisms, since they do not grow well
at chill temperatures or in meat products. Pediocin-producing pediococci are also poor
meat biopreservatives, because antilisterial activity occurs only at abuse temperatures
[85,241,406]. McMullen and Stiles [256] recognized the merit of using alternative bac-
teriocin producers in meat. Carnobacterium piscicola LV 17 and Leuconostoc gelidum
UAL 187 grow well, produce broad-spectrum bacteriocins at refrigeration temperatures,
and proved to be good biocontrol organisms in meats; their application in meats was
reviewed by these authors.
Potential benefits of pediocin to the dairy industry were explored by several research
Characteristics of Listeria monocytogenes 187

groups. Pucci et al. [308] inoculated commercial samples of cottage cheese (pH 5.1),
cheese sauce (pH 6.0), and half-and-half (pH 6.6) to contain 102-104L. monocytogenes
CFU/g and then added a crude extract of pediocin PA-1 to these products. According to
these authors, viable numbers of L. monocytogenes decreased rapidly in all foods during
the first day of refrigerated storage. Although the pathogen attained populations of 103- 105
CFU/mL or CFU/g in cheese sauce and half-and-half following 7- 14 days of refrigerated
storage, these levels were still approximately 2-5 logs lower than those observed in corre-
sponding samples prepared without PA-1 powder. Motlagh et al. [270] studied the effec-
tiveness of pediocin AcH (up to 1350 AU/mL), produced by P. acidilactici H, in control-
ling L. monocytogenes in reconstituted dry milk, ice cream, and cottage cheese at 4 and
10C. After 1 h of storage at 4"C, this treatment decreased numbers of L. monocytogenes
- - -
by 1 log. When milk was inoculated to contain 102or 104L. monocytogenes CFU/
mL and incubated at 4C for 28 days and at 10C for 12 days, 1350 AU/mL pediocin
reduced Listeria populations about 2- to 4-logs during the first day of storage but did not
inhibit growth of Listeria survivors. Liao et al. 12301 prepared a pediocin P02-containing
powder through fermentation of whey permeate with P. acidilactici P02. When the pow-
der was added to Listeria-contaminated whole milk, the antilisterial activity of this prepa-
ration was clearly demonstrated. Addition of pediocin 5, produced by P. acidilactici UL5,
to 1% fat milk reduced the viable L. monocytogenes by -3 logs after one day of storage
at 4C [176].
In addition to meat and dairy products, other foods may benefit from biopreservation
by pediocin and pediocin-producing strains. Choi and Beuchat [69J added a crude bacterio-
cin extract from P. acidilactici M to kimchi during fermentation. This treatment immedi-
ately reduced numbers of L. monocytogenes in the inoculated product and inhibited growth
by the organism during 16 days of fermentation. Adding pediocin PA- 1, curvaticin FS47,
or lacticin FS56 to liquid whole egg dramatically reduced the heating time required to
inactivate L. monocytogenes in this product [272].

Other Bacteriocins
In addition to nisin and pediocin, several other bacteriocins also are effective against
L. monocytogenes. Lactobacillus bavaricus MN, a meat isolate, inhibited growth of L.
monocytogenes in a model beef gravy at 10C, with this inhibition being attributed to a
bacteriocin [395]. In a subsequent study, Winkowski et al. [396] found that L. bavaricus
MN, when coinoculated with L. monocytogenes into three model beef systems, beef cubes
and beef cubes with gravy andlor 0.5% glucose, significantly inhibited Listeria growth
at 4 and 10C The inhibition was greater at 4C than at 1O"C, and increased with addition
of glucose to gravy or with use of a higher inoculum of this Lactobacillus strain. Other
bacteriocin-producing strains of L. bavaricus were reported [222,223]. Some of the many
other bacteria that produce antilisterial bacteriocins are Lactobacillus salivarius M7 [46],
Lactobacillus curvatus FS47 and LTH 1174 [148,374,379], L. sake [3 191, L. sake Lb674
and LTH673 [ 172,3741, Lactobacillus plantarum MCS [65], Leuconostoc carnosum LA54
[ 1981, Leuconostoc mesenteroides [771, Propionibacterium thoenii P 127 [ 2421, Carno-
bacterium piscicola [55,56,252], and Enterococcus spp. [2 1,1971.

MODIFIED ATMOSPHERE
Buchanan and Klawitter [54] investigated aerobic versus anaerobic incubation in relation
to growth and survival of Listeria in TPB at pH 4.5. Under aerobic conditions, L. monocy-
188 Lou and Yousef

togenes Scott A was undetectable after -50 h at 37"C, survived without change in numbers
- - -
at 10 and 5"C, and grew to 107and 10' CFU/mL at 28 and 19C in 100 and -500
h, respectively. When the experiments were repeated under anaerobic conditions, a similar
trend in Listeria growth and survival as related to incubation temperature was observed.
However, anaerobic incubation was more conducive to Listeria growth or survival than
aerobic incubation. Anaerobic incubation at 19C decreased the length of the lag phase
from 80.6 (aerobic) to 27.3 h and the generation time from 19.1 (aerobic) to 6.8 h. Al-
though anaerobic incubation at 37C initially decreased the count by -2 logs, the popula-
tion gradually increased to a level close to that of the initial inoculum. The authors sug-
gested that anaerobiosis improved recovery of acid-injured cells at 37C and led to
subsequent long-term survival of repaired cells. Using similar experimental conditions,
George and Lund [ 1521 examined the effect of anaerobiosis on growth of two L. monocyto-
genes strains when incubated at 20C in TPB and Tryptone Soya Broth which was supple-
mented with yeast extract (3 g/L) and glucose (10 g/L) (TSYGB) and adjusted to pH 4.5
with HCI. Contrary to findings of Buchanan and Klawitter [54], the authors noted that
the anaerobic condition (generated through flushing with nitrogen), when compared with
aerobic incubation, inhibited growth in both media. When the investigators changed incu-
bation conditions for TPB from aerobic to anaerobic, the generation time of Listeria in-
creased from 4.22-4.98 to 6.12-6.7 1 h, increased the lag-phase duration from 45.8-47.5
to 73.8-80.1 h, and decreased the maximal population from 9.55-10.09 to 8.74-8.86 logs.
Although these results are conflicting, it is generally believed that Listeria grows
well under both aerobic and anaerobic conditions [54,58]. The capacity for anaerobic
growth at refrigeration temperatures makes L. monocytogenes a potential threat to the
safety of foods packaged under vacuum or modified atmosphere. Sous vide-processed
foods also fall into this category. Such foods are vacuum packaged, then cooked, chilled,
and finally stored refrigerated. Two gases, N2 and CO2, are commonly used in modified
atmosphere packaging. For packaging of fresh meats, vegetables, and fruits, limited levels
of O2 or air may be incorporated to maintain food quality. Aerobic microflora are greatly
inhibited by modified atmosphere packaging; however, some psychrotrophic or anaerobic
pathogens, such as L. monocytogenes, A. hydrophila, Y. enterocolitica, and Clostridium
botulinum are potentially capable of growing under these conditions.
L. monocytogenes can grow in food which has been packaged under vacuum or N2
gas. However, incorporation of CO2 improves the antilisterial activity of the packaging
atmosphere [23,130,140,178,2151. Growth of L. innocua was not seen in cottage cheese
packaged under 100% CO2during 28 days of storage at 5C; however, the organism grew
after 7 days in containers packaged under air and 100% N2 [140]. Fang and Lin [130]
reported that growth of L. monocytogenes was inhibited in raw pork tenderloin packaged
under 100% CO2 when stored for 10 days at 20C or 20 days at 4C. Avery et al. [23]
also found that when L. monocytogenes was inoculated into packaged fresh beef striploin
steaks (pH 5.3-5.5) counts of the pathogen decreased slightly under saturated CO2 atmo-
sphere during storage at 5 to 10C but increased by 3 logs in vacuum-packaged steaks
[23]. Kraemer and Baumgart [215] investigated growth of L. monocytogenes at 4, 7, or
10C in sliced frankfurter-type sausage that was packaged under 0, 20, 30, 50, or 80%
CO2,with the remainder being under N2packaging Growth of L. monocytogenes decreased
as levels of CO2 increased, and complete inhibition occurred under 80% CO2. Packaging
under 50% CO2 resulted in only partial inhibition. Concurrent with this work, Farber and
Daley [132] observed that L. monocytogenes was inhibited by 270% CO2 in modified
Characteristics of Listeria monocytoge nes 189

atmosphere--packaged turkey roll slices stored at 4 and 8C; however, the pathogen grew
in packages containing 30 and 50% CO2.
Because of the threat from some foodborne pathogens, especially C. botulinum,
modified atmosphere-packaged foods rely heavily on refrigerated storage to prevent out-
growth of C. botulinum and toxin production. However, refrigeration alone can not guaran-
tee food safety [317]. Growth of L. monocytogenes, A. hydrophila, and Y. enterocolitica
was observed in vacuum-packaged sliced roast beef stored at - 1.5"C [ 1781. Adding addi-
tional microbiological hurdles should increase the safety of modified atmosphere-pack-
aged foods. Wederquist et al. [390] found that inhibition of L. rnonocytogenes increased
when 0.5% oodium acetate, 2% sodium lactate, or 0.26% potassium sorbate were added
to vacuum-packaged bologna stored at 4C. Safety of raw pork tenderloin packaged under
a modified atmosphere was improved by incorporation of nisin [ 129,1301. Furthermore,
Degnan et al. [ 851 observed that inoculation of pediocin-producing P. acidilactici into
vacuum-packaged beef wieners decreased numbers of the coinoculated L. monocytogenes
during storage at abusive temperatures.

NONTHERMAL PROCESSING TECHNOLOGIES

Processing of food by nonthermal means is not new. For centuries, relatively shelf-stable
foods were produced by adding salt and curing agents. Food fermentation, which has been
known and practiced for a long time, also may be considered a nonthermal process to
extend the shelf life of perishable foods like milk, meat, and fruit juice. Increased consumer
demand for foods with a fresh-like taste and texture have led to development of several
novel-technologies. Some newly developed physical treatments which inactivate microor-
ganisms without significantly increasing the temperature of the food can yield products
with fresh-like qualities. Such nonthermal physical treatments with potential applications
in nonthermal processing of food include irradiation, high hydrostatic pressure, high-inten-
sity pulsed light, high-intensity pulsed electric fields, and oscillating magnetic fields. These
modern nonthermal technologies are gradually gaining acceptance from regulatory agen-
cies and consumers, with irradiation now approved in the United States for use on selected
foods such as strawberries.
The ability of these nonthermal processes to control L. monocytogenes will be dis-
cussed in this section. Although these technologies are perceived as nonthermal, some
heat may be generated during their application. However, provisions are always made to
dissipate this heat, and thus control of pathogens is accomplished mainly by nonthermal
means.

Irradiation
The entire electromagnetic spectrum consists of at least six distinct forms of radiation that
differ in wavelength, frequency, and penetrating power, of these forms, microwaves and
ultraviolet and gamma radiation are of primary interest to food manufacturers. In food
processing, microwave radiation is mainly used for its heating properties; thus discussion
of effects of this form of radiant energy on L. monocytogenes are not covered in this
section. Ultraviolet (UV) radiation, which is nonionizing, ranges in wavelength from 136
A
to 4000 and has some application in food processing. The poor penetrating power of
UV radiation restricts its use to a few specialized beverage applications, eradication of
190 Lou and Yousef

airborne contaminants and treatment of food contact and ?on-food contact surfaces.
Gamma radiation, which has a shorter wavelength (0.1- 1.4 A) than UV, is better suited
for external and internal decontamination of foods. Information concerning the ability of
gamma and ultraviolet radiation to inactivate L. monocytogenes in laboratory media will
be reviewed now. Findings from similar food-related studies are discussed elsewhere in
this book.
Gamma Irradiation
Literature on sensitivity of L. monocytogenes to gamma irradiation is less controversial
than that addressing thermal inactivation. Results from gamma irradiation studies con-
ducted in the United States [72,123,181] and Hungary [371] are strikingly similar, with
reported D-values ranging from 0.28 to 0.6 1 kGy for 12 different L. monocytogenes strains.
-
In addition, exposure to a gamma radiation dose of 1.7-4.0 kGy was generally sufficient
to reduce numbers of L. monocytogenes, L. ivanovii, and L. seeligeri by six to seven orders
of magnitude. Overall, these findings suggest that Listeria spp. are likely to be at least
equally, if not slightly more, resistant to gamma radiation in culture media than are other
commonly encountered non-spore-forming foodborne pathogens such as S. typhimurium
(D-value = 0.28 kGy) [377], S. aureus (D-value = 0.24 kGy) [377], and Y. enterocolitica
(D-value = 0.1 1 kGy) [ 1241. Although differences between L. monocytogenes strains
likely account for most of the observed variation in D-values, radiation sensitivity of L.
monocytogenes is also affected by age of the culture, irradiation menstruum, and the type
of medium used to enumerate the pathogen after irradiation. According to Huhtanen et
al. [ 1811, 1.5- and 2.5-h-old cultures of L. monocytogenes were somewhat more resistant
to gamma radiation than those incubated 5 and 18 h before exposure. Furthermore, surviv-
ing cells previously exposed to high radiation doses were no more resistant than the parent
culture. Consequently, observed differences between sensitivity of young and old cultures
probably resulted from innate differences between strains rather than from development
of radiation-resistant mutants. These authors also reported that 12-h-old centrifuged cul-
tures of L. monocytogenes were most resistant to 1.0 kGy gamma radiation when resus-
pended in fresh culture media or the original culture supernatant liquid followed in order
by phosphate buffer and distilled water. Inability of distilled water effectively to scavenge
cell-damaging free radicals produced during irradiation is likely responsible for decreased
resistance of the pathogen in water than in culture media that contain high concentrations
of free radical-quenching organic compounds. It is not surprising that L. monocytogenes
is more resistant to gamma radiation when present in foods than in culture media. Two
independent investigations [ 123,2921 have shown that D-values for radiation resistance
are markedly affected by the type of plating media used to enumerate the pathogen after
irradiation. In both studies, a significantly higher ( P < .OS) D-value resulted from in-
creased recovery of the pathogen with nonselective or semiselective rather than highly
selective plating media. These findings indicate that substantial numbers of listeriae were
sublethally injured during exposure to gamma irradiation. Since repair and subsequent
growth of injured cells is frequently inhibited by some of the selective agents used in
highly selective media, D-values for organisms exposed to irradiation or any other poten-
tially injurious treatment always should be determined using a plating medium with low
selectivity.
Andrews et al. [9] found that sensitivity of L. monocytogenes in TSB to gamma
radiation was affected by broth temperature (-80, 4, or 20C) during treatment and the
initial count (103,106,and 109CFU/mL). Under this wide range of conditions, the organ-
Characteristics of Listeria monocytogenes 191

ism exhibited D-values of 0.4 1-0.62 kGy. L. monocytogenes was significantly more resis-
tant to irradiation at room temperature (20C) than at refrigeration (4C) or freezing
(-80C) temperatures. D-values obtained with an initial count of 109CFU/mL were sig-
nificantly lower than those with 106CFU/mL.
Andrews and Grodner [8] later reported that gamma radiation was more effective
in inactivating L. monocytogenes when split into two equal doses than when the same
dose was applied as a single treatment. At 2OoC, the split dose of gamma radiation with
1-2 h between treatment decreased D-values from 0.50-0.58 kGy (single irradiation) to
0.41-0.42 kGy. However, a similar trend was not observed at refrigeration (4C) or sub-
freezing (- 80C) temperatures.
Ultraviolet Radiation
In 1971, Collins [73] determined the susceptibility of L. monocytogenes to UV radiation
emitted from a 14-W cold cathode mercury vapor lamp. Tryptone Soy Agar plates con-
-
taining 109 L. monocytogenes were exposed to a radiation output of 40 W/cm2 at 40
cm from the source for 30, 60, 90, and 120 s and then incubated for 3 days at 37C.
Populations of L. monocytogenes decreased 10-fold during the first 60 s of irradiation (D-
value of 60 s) after which the rate of inactivation increased sharply with a D-value of
- 15 s. L. monocytogenes was much more resistant to radiation than E. coli or Serratia
marcescens, which are commonly used to test the effectiveness of UV lamps.
Yousef and Marth [402] also reported that L. monocytogenes was inactivated by
exposing the bacterium to UV energy. Following 4 min of exposure to short-wave (254
nm) ultraviolet energy (100 pW/cm2), numbers of L. monocytogenes (strain Scott A) de-
creased approximately 7 logs on Tryptose Agar plates that were previously spread with
a 24- or 48-h-old culture of the test organism. In contrast, L. monocytogenes numbers
remained constant after 10 min of exposure to long-wave (364 nm) UV energy. Increasing
the intensity of short-wave UV radiation to 550 pW/cm2 nearly doubled the rate at which
L. monocytogenes was inactivated. These investigators also found that dry rather than
moist Listeria cells were more resistant to radiation. Exposing a dried film of L. monocyto-
genes cells in a Petri plate to short-wave UV energy (100 pW/cm2) decreased the popula-
tion by 2 rather than 7 logs for moist cells on Tryptose Agar. Fortunately, when present
in food processing environments, numbers of listeriae appear to be relatively low. Hence,
results from the aforementioned study suggest that UV energy may be of some practical
importance in reducing airborne contaminants, including listeriae, in food production and
storage areas.
High-Intensity Pulsed Light
Pulsed light, which has wavelengths ranging from -200 nm (UV) to 1 mm (near-infrared)
with peak emissions at 400-500 nm, inactivates microorganisms with flashes of intense
sunlight-like radiation. Currently employed pulsed light has intensities about 20,000 times
that of sunlight. Literature currently available on the effect of pulsed light on L. monocyto-
genes is limited to one 1995 report by Dunn et al. [98]. They found that L. monocytogenes
present on surfaces was inactivated to a greater extent by pulsed light than by UV energy.
-
Treatment with a single flash of pulsed light at 0.5-1.0 J/cm2 inactivated 10s CFU/cm2
of various microorganisms, including L. monocytogenes, on an agar surface, and several
-
such flashes inactivated up to 10' CFU/cm2. Although pulsed light is much more effec-
tive in inactivating organisms than UV light, both types of radiation have limited penetrat-
ing power and thus are only useful for inactivating microorganisms on surfaces of foods
192 Lou and Yousef

and packaging materials or in transparent food ingredients, such as water and some bever-
ages. When these authors used pulsed light to treat wieners that were previously surface
inoculated with L. innocua, populations decreased about 100-fold, with similar pulsed
light treatments being shown to be effective in extending the shelf life of baked foods,
seafood, and meat.

High Hydrostatic Pressure

Although most microorganisms except certain ocean-dwelling species grow best under
normal atmospheric pressure, exposure to hydrostatic pressure >600 atm often induces
cellular changes that can be lethal to non-spore-forming organisms. Hence, exposure to
high hydrostatic pressures has been suggested as another means of inactivating certain
spoilage and pathogenic organisms in raw and pasteurized milk as well as in meat, poultry,
seafood, fruits, and vegetables.
Recognizing the proven ability of high hydrostatic pressure to inactivate Salmonella
spp. in laboratory media, Styles et al. [368] examined the behavior of L. monocytogenes
strains Scott A and CA in phosphate-buffered saline solution during exposure to pressures
of 35,000-50,000 psi (-240-345 MPa) at ambient temperatures. Strains Scott A and CA
were fairly barotolerant, exhibiting D-values of 56.4 and 31.6 min, respectively, in the
presence of 35,000 psi. At 50,000 psi, the D-values for Scott A and CA were 2.9 and 6.7
min, respectively. When these experiments were repeated using raw milk and UHT pro-
cessed milk, 60 and 80 min at 50,000 psi were required, respectively, to inactivate an L.
monocytogenes population of 1 X 106 CFU/mL. Thus both Listeria strains were more
resistant to high hydrostatic pressure when suspended in milk than in buffer.
Working with a higher range of pressures, Patterson et al. [293] found that exposure
to 375 MPa (54,400 psi) for 15 rnin at ambient temperatures was sufficient to inactivate
> 105L. monocytogenes CFU/mL in a phosphate buffer, whereas similar reductions in Y.
enterocolitica, S. typhimurium, S. enteritidis, E. coli 0 157:H7, and S. aureus required
275, 350, 450, 700, and 700 MPa, respectively. Sensitivity to high pressure varies among
Listeria strains, with the type of media also influencing the degree of protection against
inactivation. L. monocytogenes was more resistant to inactivation by pressure when present
in UHT milk than in buffer or poultry meat.
Bacterial inactivation during high-pressure treatment of foods is also temperature
dependent. Resistance of L. innocua to inactivation by high hydrostatic pressures at differ-
ent temperatures was studied by Gervilla et al. 11541 in ewe's milk. Applying 200 MPa
(29,000 psi) of pressure at different temperatures (2-50C) for up to 15 rnin resulted in
51-log decrease in population, whereas treatment at 500 MPa (72,500 psi) for 5 min
decreased the count of L. innocua from 107-108CFU/mL to < I CFU/mL regardless of
temperature. High-pressure treatment was least effective at 20-30C; this temperature
dependence was most obvious at 350 MPa. Results of kinetic studies yielded D-values
of 3.12 and 4 rnin at 2 and 25"C, respectively, when L. innocua was treated in ewe's milk
at 400 MPa.
Effectiveness of high hydrostatic pressure also depends on the growth history and
physiological state of treated bacteria. Lanciotti et al. [220] grew L. monocytogenes in
BHI broth at different temperatures (3-37"C), pH (5.0-6.5), and a, (0.94-0.99) before
treatment with high pressure. Cultures of L. monocytogenes grown or preconditioned at
lower temperatures (3-2OoC), pH 6 or high a, value (20.96) were most tolerant of high
hydrostatic pressures.
Characteristics of Listeria monocytogenes 193

Lanciotti et al. [22 1] investigated the effectiveness of continuous homogenization

at pressures of 15 to 200 MPa (2 175-29,000 psi) on microbial inactivation in milk and two
biphasic (oil and aqueous) model food systems. The authors found that homogenization
markedly reduced the initial load of microorganisms, including L. monocytogenes, and
changed the microstructure of treated foods in a way to minimize growth of survivors.
L. monocytogerzes populations decreased linearly at a rate of 0.0025 log CFU/g per bar
as the pressure increased. Homogenization in both model systems at 40-90 MPa resulted
in a 100-fold decrease in numbers of L. monocytogenes, with the remaining population
decreasing an additional 10-fold after 10 days of storage at 3-4C. Treatment at 190 MPa
-
reduced the initial population of 107CFU/g to < 1 CFU/g. The authors suggested that
decreasing space availability, as evidenced by the small water droplet sizes, may account
for the stability of processed foods.

COMBINED TREATMENTS
Since total reliance on any single preservation method (e.g., heat, acidity, salt) usually
causes quality deterioration, many food processors use several treatments in combination
to process and preserve food. The well-known hurdle concept emphasizes the combined
use of antimicrobial factors to inhibit growth or eliminate microorganisms from food.
When preservation factors (hurdles) are combined, an additive antimicrobial effect often is
observed. However, combined hurdles sometimes act synergistically to enhance microbial
inhibition and inactivation beyond the additive effect. In other circumstances, however,
one hurdle may negate the antimicrobial effect of another hurdle. Further complications
may arise when hurdles are applied in sequence, with time gaps, rather than simulta-
neously. When used intermittently, a mild hurdle may stress an organism and elicit an
adaptive response which will in turn protect the microorganism against subsequent expo-
sure to more severe hurdles. This phenomenon of adaptation and protection is receiving
great attention in relation to efficacy of preservation by multiple hurdles and microbial
safety of the resulting food. In this section, examples illustrating the interaction between
hurdles will be presented in relation to control of L. monocytogenes in food. It should be
cautioned, however, that the outcome of interaction between hurdles depends heavily on
the conditions under which these hurdles are applied.
A two-hurdle interaction was demonstrated by Johansen et al. [ 1871, who found that
antilisterial activity of lysozyme was synergistically enhanced by low pH values. Another
example of a two-hurdle interaction was presented by Mainsnier-Patin et al. 12491, who
found that adding nisin to skim milk dramatically reduced the heating time required to
inactivate L. monocytogenes. Results of a study by Conner et al. [75]illustrates the nega-
tive interaction between two hurdles, refrigeration and high acidity. The investigators
observed that at maximum growth-limiting pH values, L. rnonocytogenes populations
decreased from -104 to < 10 CFU/mL in 1-3 weeks at 35C; whereas at 10C, listeriae
survived for 6- 12 weeks.
Interaction between multiple hurdles was presented by Bala and Marshal1 [25] who
investigated the combined effect of NaCl (2.5-7.8%), pH (5.4-7.8), temperature (5, 15,
25, and 35C), and sublethal levels of monolaurin (2-8 pg/L) against L. monocytogenes
grown on double (salt-pH) gradient plates. Addition of monolaurin to the gradient plates
reduced salt and pH tolerance of the pathogen. Complicated interactions between preserva-
tion factors (hurdles) were evident in a recent study by Lou [2371, who noted that antiliste-
rial activity of nisin was affected by pH and the presence of NaCl. Addition of NaCl
194 Lou and Yousef

(3.5-7.5%), to Trypticase Soy Broth decreased the bactericidal action of nisin against L.
monocytogenes. However, the presence of 3.5-5.5% NaCl interacted synergistically with
nisin to inhibit outgrowth of the pathogen on Trypticase Soy Agar plates.
Inhibition of L. monocytogenes by multiple hurdles was studied by Buchanan and
coworkers [58]. A factorial design was used to determine the combined effect of incuba-
tion temperature (5-37C), initial pH (6.0-7.5), sodium chloride (0.5 vs 4.5%), sodium
nitrite (0-1000 ppm), and atmosphere (aerobic vs anaerobic) on growth of L. monocyto-
genes in TPB. Although lag periods, generation times, and maximum populations were
all affected by these five interacting variables, sodium nitrite was most listeriostatic when
used in conjunction with low pH, increased sodium chloride, refrigeration temperatures,
and anaerobic conditions that simulated vacuum packaging.
Research using predictive microbiological modeling is likely to be valuable in as-
sessing the safety of foods preserved by multiple hurdles. Additive, nullifying, or syner-
gistic antimicrobial effects of multiple hurdles can be estimated by predictive models.
Consistent with these objectives, Buchanan et al. [5 1,52,57] attempted to predict behavior
of L. monocytogenes in response to an array of extrinsic factors. Buchanan et al. [57] used
a factoriallsupplemental central composite design to assess quantitatively the effects of
temperature (5, 10, 19,28, 37C), pH (4.50, .5.25, 6.00, 6.75, 7.50), sodium chloride (0.5,
1.5, 2.5, 3.5, 4.5%), sodium nitrite (0, 50, 100, 150, 200, 1000 ppm), and atmosphere
(aerobic vs anaerobic) on the growth kinetics of L. monocytogenes strain Scott A in TPB.
After growth curves were constructed from each experiment using regression analysis to
obtain best fit Gompertz equation curves, results were analyzed by response surface
analysis to generate a polynomial model that could mathematically predict lag periods,
exponential growth rates, generation times, and maximum populations for L. monocyto-
genes in association with any of the five variables examined. Overall, changes in response
of the organism to the five environmental factors were most evident as altered specific
growth rates and lag periods. L. monocytogenes also achieved similar maximum popula-
tions in all instances except those that involved growth of the pathogen under environmen-
tal extremes in the presence of high concentrations of sodium nitrite.
As a result of these and other studies, Buchanans group developed useful mathemat-
ical models to quantify behavior of L, monocytogenes in response to multiple environmen-
tal factors or hurdles [53]. These models were incorporated into a computer program called
the Pathogen Modeling Program. As of 1997, the program is available as version 5.0 for
Microsoft Windows and can be downloaded from a USDA site on the internet or requested
from the developers. The L. monocytogenes module of this program can be used to predict
lag time, growth rate, maximum population, and time required to attain a given count of
Listeria under a wide range of environmental conditions.
Interaction between hurdles becomes even more complicated when the history of
Listeria cells to be inactivated by the multiple hurdles is considered. Adaptation of L.
monocytogenes during sublethal exposure to various preservation techniques (or stress)
may protect the pathogen against subsequent exposure to the the same, different, or any
combination of stresses at normally lethal levels. Kroll and Patchett [216] reported that
adaptation to pH 5 greatly increased survival of L. monocytogenes at pH 3 as compared
with the unadapted cultures. According to Lou and Yousef [238,239], adaptation of L.
monocytogenes to sublethal levels of acid, ethanol, and hydrogen peroxide and starvation
increased resistance of L. monocytogenes to lethal levels of these factors and heat. This
stress adaptation, or hardening, complements the hurdle concept, since such hurdles
in foods can be applied simultaneously or sequentially. When applied sequentially, hurdles
Characteristics of Listeria monocytogenes 795

may not deliver the desired effect. Stress adaptation to the first encountered hurdle, which
' 'hardens' ' pathogens and increases their resistance to subsequent preservation factors,
may counteract hurdle build-up.

SURVIVAL, ATTACHMENT, AND BlOFlLM FORMATION ON

SURFACES
L. monocytogenes is a very hardy organism, being able to survive up to 2 1 years in refriger-
ated laboratory media [SO] as well as 10 days in tap water incubated at 22C and 6, 3,
and 1 day in distilled water stored at 22, 30, and 40"C, respectively [87]. Moreover, this
pathogen is also relatively resistant to drying.
These observations have led to questions concerning the ability of L. monocytogenes
to survive on various types of materials common to food processing facilities. In an early
study, Durst and Sawinsky [ 1001 moistened various inert materials with a 24-h-old NB
-
culture containing 1O9 L. monocytogenes CFU/mL and stored the materials in sterile
Petri plates at ambient temperature. L. monocytogenes survived <24 h on glass, iron, and
aluminum, <48 h on paper and plastic, 7 but not 42 days on porcelain, 6 but not 12
months on wood, and at least 1 year on gauze. L. monocytogenes also survived more than
1 day on stainless steel [324], 165 days on contaminated wool stored at 8-22C [26], 105
days on dried threads stored at room temperature [376], 20-30 days on tiles [369], and
at least 20 days on 250-pm diameter glass beads [391].
Of particular interest to the dairy industry is a 1987 study by Stanfield et al. [363]
which examined survival of three L. monocytogenes strains on exterior surfaces of waxed
cardboard and plastic milk containers. Both container types were contaminated by swab-
bing their surfaces with a heavy suspension of an 18- to 24-h-old unstressed or stressed
(heated at 56C for 30 min) L. monocytogenes culture, and then containers were stored
at -0.8-6.6"C for 14 days. Unstressed cells of L. monocytogenes were recovered after
14 days of storage from at least one site on the surface of plastic and waxed cardboard
containers.
L. monocytogenes, like several other foodborne pathogens, can attach to surfaces
and form biofilms. When biofilms are formed, they are hard to remove by normal cleaning.
Biofilms also protect microorganisms, including L. monocytogenes, from antimicrobial or
sanitizing agents and often serve as a source of recontamination for processed foods.
Herald and Zottola [ 1681 examined the ability of a culture of L. monocytogenes
grown at 10, 21, and 35C in Trypticase Soy Broth at pH 5, 7, and 8 to attach to stainless
steel. A small stainless steel chip was placed inside a culture vial containing L. monocyto-
genes and then incubated at 21 or 35C for 18-24 h or at 10C for 36-48 h. Following
incubation, analysis of the chips using scanning electron microscopy (SEM) revealed that
the pathogen adhered to stainless steel at all pH values and temperatures studied; however,
cells with fibrils were observed only at 2 1 and 10C. Amounts of exopolymeric attachment
material were greater when the organism was incubated at 10 rather than 35C and in-
creased with the length of incubation. The ability of L. monocytogenes to attach to various
surfaces on food processing facilities was subsequently reported by other researchers.
Mafu et al. 12471found that L. monocytogenes attached to stainless steel, glass, polypropyl-
ene, and rubber, which are common materials in food-contact surfaces, after short contact
times (20-60 min) at both ambient and refrigeration temperatures. Formation of extracel-
lular material around the attached cells was revealed by SEM after 60 min of incubation
196 Lou and Yousef

at both temperatures. According to Krysinski et al. [217], L. monocytogenes adhered to

stainless steel and polyester or polyester-polyurethane conveyor belts at levels of -2 X
104CFU/cm2 after incubating these materials in a culture medium at 35C for 24 h. Black-
man and Frank [38] observed variable adherence of L. monocytogenes to surfaces of stain-
less steel, Teflon, nylon, and polyester floor sealant after 7 days of incubation in TSB at
21C with markedly less attachment occurring at 10C. Kim and Frank [204] found that
L. monocytogenes grown in a minimal medium exhibited an attachment rate 50-fold higher
than cells grown in TSB. L. rnonocytogenes attachment to Teflon and buna-n rubber and
secretion of biofilm matrix materials were also reported by Mosteller and Bishop [269].
Since floor drains frequently harbor L. monocytogenes, Spurlock and Zottola [361]
investigated growth of the pathogen in a model floor drain and its attachment to free-
standing cast iron drains at ambient temperature. L. monocytogenes grew and remained
on these drain surfaces at 106-108 CFU/cm2 regardless of drastic changes in pH from
alkaline (pH 9.0) to acidic (pH 4.5). Attachment of L. monocytogenes to cast iron was
clearly seen in SEM images taken after several hours of contact, with attachment material
also being observed on drains after 9 and 29 h of incubation in TSBYE and 0. I % reconsti-
tuted nonfat dried milk, respectively.
Environmental flora in food processing facilities may interfere with or enhance at-
tachment of L. monocytogenes to surfaces or its growth in biofilms. Sasahara and Zottola
[334] found that in a flowing system, Pseudomonas fragi, a bacterium which strongly
attaches to and produces exopolysaccharide materials on surfaces, enhanced surface at-
tachment of L. monocytogenes, but P. fragi itself failed to attach to these surfaces in this
flowing system. In contrast, Jeong and Frank [I861 reported that when environmental
isolates from meat or dairy plants were statically incubated in 0.2 and I .O% TSB at 10C,
these bacteria either inhibited or minimized attachment of L. rnonocytogenes to stainless
steel, although considerable L. monocytogenes attachment and biofilm growth occurred
in all instances. In pure culture biofilms, L. monocytogenes populations began to increase
after 9 and 13 days and reached 106CFU/cm2 after 17 and 25 days, respectively [ 1861.
L. monocytogenes on surfaces can be protected by the presence of food components,
and this protection increases as the layer of food thickens, possibly from the slower loss
of moisture from the thick food layers. When compared with phosphate-buffered saline
(PBS) solution, various milk residues (e.g., raw milk and pasteurized whole milk) en-
hanced survival of L. monocytogenes on stainless steel and buna-n rubber, and sometimes
promoted growth of Listeria [ 1671. However, cottage cheese whey, as a residue, did not
increase survival of L. monocytogenes. With PBS as a residue, L. monocytogenes popula-
tions (initially, -104 CFU/cm2) decreased -1 log at 6C and 75.5% relative humidity
(RH) after 10 days or became undetectable at 25C and 32.5% RH after 3-5 days. How-
ever, numbers of L. monocytogenes inside a single layer of pasteurized whole milk in-
creased >2 logs at 25"C/75.5% RH after 3 days and remained unchanged at 6OU32.5 or
75.5% RH and 25"C/32.5% RH. Of particular interest to dairy processors are the findings
of Al-Makhlafi et al. [7], that attachment of L. rnonocytogenes to silica surfaces preab-
sorbed with milk proteins was highest with P-lactoglobulin and lowest with bovine serum
albumin. Preabsorbed a-lactoglobulin and p-casein had an intermediate effect on attach-
ment of Listeria.
Although L. monocytogenes attaches to all types of surfaces, the ability of this bacte-
rium to form a biofilm on buna-n rubber, a gasket material, is relatively low. Buna-n
rubber is bacteriostatic to L. rnonocytogenes under certain conditions, with this activity
remaining after 20 cycles of a simulated clean-in-place process [ 167,3201. According to
Characteristics of Listeria monocytogenes 197

Ronner and Wong [320], buna-n rubber was strongly bacteriostatic to L. monocytogenes
growing in Peptone Glucose Phosphate (PGP) broth (a low nutrient medium) and slightly
bacteriostatic in TSB. Four of seven strains of L. monocytogenes growing in PGP formed
less dense biofilm populations on buna-n rubber than on stainless steel.
Temperature and moisture also affect survival of L. monocytogenes on surfaces.
Palumbo and Williams (284a) suspended a mixture of seven L. monocytogenes strains in
seven menstrua (distilled water, tryptone broth, nonfat dry milk, canned milk, glycerol,
light Karo syrup, and beef extract), and then dried these cell suspensions on glass plates
which were stored at 5 or 25C and I-75% RH. Enhanced survival was observed at 5C
rather than at 25C and at lower rather than higher RH. In a subsequent study, Helke and
Wong [ 1671 investigated survival of L. monocytogenes on surfaces under 32.5 and 75.5%
RH and temperatures of 6 and 25C. The authors found that survival of Listeria was higher
at 6C than at 25C and, in contrast to the previous study, survival was greater under
humid (75.5% RH) rather than dry (32.5% RH) conditions.
Microorganisms embedded in biofilms are more resistant to heat, sanitizers, and
other antimicrobial agents than are freely suspended (planktonic) cells. Frank and Koffi
[ 1461 prepared L. monocytogenes in three states: (a) planktonic cells, obtained by growing
the bacterium in TSB for 38 h at 21"C, (b) adherent single cells, prepared by immersing
glass slides in a planktonic cell culture for 4 h at 2 I "C; the attached Listeria was mostly
single cells, and (c) adherent microcolony cells, made by incubating the glass slides with
attached L. monocytogenes cells for 14 days at 2loC, during which the slides were periodi-
cally washed with a saline solution and incubated in fresh media; the attached cells in
this instance were mostly in microcolonies. The investigators found that Listeria was more
sensitive to banzalkonium chloride (n-alkyl dimethyl dichlorobenzyl ammonium chloride,
a quaternary ammonia sanitizer) and dodecyl benzene sulfonic acid, an anionic acid sani-
tizer (DBSA) when present in the planktonic rather than in the adherent single cell or
microcolony state. Contact with either sanitizer ( 100-800 ppm) at ambient temperature
immediately reduced populations of the planktonic cells from 1Oh CFU/ml to undetectable
levels. In contrast, adherent single cells (initially 105- 106CFU/cm2) and adherent micro-
colony cells (initially 106- 107CFU/cm2) decreased 3-5 and 2-3 logs, respectively. The
few remaining adherent single cells became undetectable after 16 min of sanitizer expo-
sure, with adherent microcolonies surviving a maximum of 20 min. Survival of these
adherent cells was not caused by depletion of sanitizers, since the remaining sanitizers
produced similar inactivation when new microcolony slides were treated. When planktonic
cells were heat-treated ( 5 min at 55 or 70C) in the presence of 400 ppm benzalkonium
chloride or 200 ppm DBSA, the combined treatment, regardless of temperature, decreased
populations -5 logs from an initial - 106CFU/mL, whereas similar treatments decreased
counts of microcolonies only -2 and >5 logs, respectively.
In a subsequent study, Lee and Frank [226] investigated the sensitivity of stainless
-
steel-adhering single and microcolony cells (initially 1O5 L. monocytogenes CFU/cm2)
on stainless steel to hypochlorite and heat. They found that adherent single cells on stain-
less steel were more sensitive to hypochlorite and heat inactivation than adherent microcol-
ony cells. Exposure to a hypochlorite solution, which contained 200 ppm residual chlorine,
for 30 s decreased the population of adherent single and microcolony cells by 4.8 and 2.6
log units, respectively, with microcolony cells surviving up to 5 min of exposure to this
agent. Although heating at 65C for 30 s resulted in a 3.8-log reduction of both types of
adherent cells, only microcolony cells were detectable after 3 min of heating. Adherent
single and microcolony cells became undetectable after 30 and 60 s of heating at 72"C,
198 Lou and Yousef

respectively. Combined exposure to 65C for 30 s and 200 ppm chlorine decreased the
number of microcolony cells to undetectable levels.
Krysinski et al. [217] obtained adherent L. monocytogenes cells by growing the
organism in a culture medium for 24 h at 25C that contained pieces of stainless steel,
polyester belt, or polyester-polyurethane conveyer belt and then tested the effectiveness
of 10 sanitizers and 6 cleaners in inactivating or removing adhering L. monocytogenes
cells after 10 min of exposure. Although L. monocytogenes attached to these surfaces at
similar levels (-2 X 104CFU/cm2), protection provided by these surfaces against sani-
tizers or cleaning agents varied with the substrate, with polyester belt being most protective
followed by polyester-polyurethane belt and stainless steel. Although most of the sanitizers
and cleaners that were tested inactivated Listeria attached to stainless steel, none of these
agents could effectively eliminate cells attached to polyester-polyurethane belt. However,
detergent cleaning followed by sanitizing, a practice commonly followed in industry, was
more effective in controlling adherent L. monocytogenes cells than when either cleaning
or sanitizing was used alone.
Besides sanitizers, effectiveness of listeriaphages in inactivating surface-adherent
L. monocytogenes was also investigated [326]. Adherent cells were obtained by immersing
chips of stainless steel or polypropylene in a L. monocytogenes culture for 1 h at 26C.
Treatment of adherent L. monocytogenes cells with a phage suspension (3.5 X 108PFU/
mL) reduced populations of the pathogen by -3.4 logs, with mixtures of three different
phages proving to be most effective. Although use of QUATAL (containing 10.5% N-
alkyldimethyl-benzylammonium HCl and 5.5% glutaraldehyde as active ingredients,
Ecochimie LtLe, Quebec, Canada) at 50 pprn destroyed the adherent Listeria flora, a com-
bination of 108PFU/mL phage and 30 pprn QUATAL resulted in similar destruction.

SANITIZERS
Sanitizers have been widely used in the food industry to decrease populations of patho-
genic and spoilage organisms in food production and processing facilities. Most of these
sanitizing agents belong to one of four categories: (a) chlorine-containing compounds, (b)
iodophors, (c) quaternary ammonium compounds frequently called quats, or (d) acid
sanitizers. Additionally, ozone has been used for decades in some European counties, and
application of this sanitizer in the US food industry is likely to increase.
When in aqueous solutions, chlorine-containing compounds release hypochlorous
acid which accounts for their bactericidal action. Iodophors, water-soluble complexes of
elemental iodine, and nonionic surface-active agents owe their bactericidal activity to re-
lease of free elemental iodine and hypoiodous acid, which is enhanced under acidic condi-
tions. In contrast, quaternary ammonium compounds are best classified as noncorrosive
germicidal cationic detergents that remain active at relatively high pH values. Finally,
acid sanitizers such as phosphoric and citric acid-containing compounds are frequently
used in conjunction with rinsing agents in automated cleaning systems better known as
clean-in-place (CIP) systems. Unlike iodophors, acid sanitizers are nonvolatile and retain
their bactericidal activity at temperatures below 100C. Sanitizing agents must reduce
populations of a given test organism at least 5 logs during 30 s of exposure at ambient
temperatures before the particular agent is deemed to be effective.

Chlorine Compounds
In the absence of organic debris, chlorine rapidly inactivates most non-spore-forming
bacteria even when used at the very low concentrations found in chlorinated drinking
Characteristics of Listeria monocyto genes 199

water. Although the actual mechanism of disinfection is not fully understood, germicidal
activity of chlorine has generally been attributed to hypochlorous acid (HOCI), which
is generated in aqueous solutions of sodium hypochlorite and other chlorine-containing
compounds. Although HOCl can in turn dissociate to form the hypochlorite ion (OC1-)
and hydrogen ion (H'), depending on the pH of the solution, the neutral electric charge
of the former suggests that HOCl can more easily penetrate the bacterial cell membrane
than OCI-. Thus it is not surprising that the germicidal activity of HOCl is 80 times that
of OC1-. After diffusing into the cell, HOCl is thought to inactivate the organism by
inducing formation of toxic oxygen species or combining with proteins, which may in
turn inhibit key enzymatic reactions and alter cell membrane permeability.
Numerous studies have dealt with the lethal effects of various chlorinated sanitizing
agents on L. monocytogenes. Beginning in 1969, Baranenkov [26] found that hypochlorite
could effectively control L. monocytogenes on the surface of hen's eggs. Chloramine also
was later shown to be listericidal when used under acidic conditions at concentrations of
0.1-0.2% [ 2581. Subsequently, Lopes [236] reported that two solutions of chlorine-based
sanitizers (one containing 8.5% sodium hypochlorite with 8%)active chlorine and the other
containing 25.8% sodium dichloro-s-triazinetrione) containing I00 pprn active chlorine
both reduced L. monocytogenes populations by more than 5 logs after 30 s of exposure.
These findings were subsequently confirmed by Rossmoore and Drenzek [324]. Further
tests by Lopes [236] revealed that the organic chlorine-based sanitizer was slightly more
effective against L. monocytogenes than the sodium hypochlorite-based sanitizer; the for-
mer had a lower pH which would in turn lead to higher concentrations of HOCI, the most
bactericidal form of chlorine. A chlorine dioxide-based sanitizing agent also has been
approved by the FDA for use in the food industry. According to its manufacturer [ 121, the
unusual effectiveness of this formula against L. monocytogenes and other microorganisms
results from a special activator which converts large quantities of stabilized chlorine diox-
ide to the free form.
Following the published report by Lopes [236], Brackett [41] determined the germi-
cidal effect of reagent-grade sodium hypochlorite and household bleach on two L. monocy-
togenes strains (Scott A and LCDC 8 1-861 ) previously associated with outbreaks of food-
borne listeriosis. After 20 s of exposure to 2 5 0 ppm available chlorine, both compounds
led to substantial reductions in numbers of viable L. monocytogenes in phosphate buffer.
However, Listeria populations remained relatively stable for an additional 4.6 min, and
in several instances listeriae survived 1 5 min with free residual chlorine levels that ap-
proached 40 ppm. Since 10 ppm available chlorine was ineffective, results of this study
indicate that the minimum chlorine concentration needed to kill L. monocytogenes lies
between 10 and 40 ppm.
Effectiveness of chlorine against L. monocytogenes also was examined in depth and
later reviewed by El-Kest and Marth [103-1051. Cells of L. monocytogenes strain Scott
A were harvested from 24- and 48-h-old slants or broth cultures, washed by centrifugation
in 20 mM phosphate buffer solution or 0.3 12 mM phosphate buffer dilution water, and
then exposed at 25C to sodium hypochlorite solutions at pH 7 (25C) that contained 0.5-
10.0 ppm available chlorine. Using a solution containing 5 ppm available chlorine, num-
bers of survivors decreased -6 logs after only 30 s, with the organism no longer being
detectable by direct plating on TA after 1 h. (Results from Rosales et al. [32 I ] also showed
that populations of L. monocytogenes, L. ivanovii, and L. seeligeri decreased >5 logs
-
following 30 s of exposure to distilled water (pH 7) containing 2 2 5 pprn hypochlorite
[i.e., 223.8 ppm available chlorine]). Exposing L. monocytogenes to 0.5, 1.0, 2.0, 5.0,
and 10.0 ppm available chlorine resulted in corresponding D-values of 61.7, I 1.3, 6.7,
200 Lou and Yousef

4.9, and 4.7 s. Although disinfecting activity clearly increased with increasing concentra-
tions of available chlorine, the effectiveness of sodium hypochlorite also was affected by
several additional factors. Increased resistance of L. monocytogenes to chlorine was ob-
served using (a) 24- rather than 48-h-old cultures, (b) cells harvested from broth rather
than agar slants, and (c) cultures exposed to solutions containing 20 mM rather than 0.3 12
mM phosphate. Five and 10 ppm of available chlorine was partially neutralized in the
presence of 0.05 and 0.1% peptone (nitrogenous compound) [103]. Given the findings
indicating that hypochlorite concentrations of up to 400 ppm were of little use against
L. monocytogenes, L. ivanovii, or L. seeligeri when these organisms were suspended in
reconstituted NFDM (10% solids) [321], it is clear that antimicrobial activity of chlorine
can only be maintained if organic material is effectively removed before exposure.
In addition to the factors just discussed, Lee and Frank [225] found that resistance
of L. monocytogenes cells in late exponential phase to hypochlorite solution (1-5 ppm
available chlorine) was greater when the organism was grown at 35C than at 6 or 21C.
Exposure to 1 ppm available chlorine for 5 min at ambient temperature decreased popula-
tions of the organism previously grown at 6, 21, and 35C by 3.4, 3.1, and 2.1 logs,
respectively. Furthermore, L. monocytogenes grown in a nutrient-poor medium ( 15-fold
diluted TSB) was 10-times more resistant to chlorine than when grown in regular TSB
[225].
Additional work by El-Kest and Marth [105] demonstrated that populations of L.
monocytogenes decreased most rapidly in sodium hypochlorite solutions at 5C followed
by 35 and 25C. Marked variation in chlorine sensitivity also was observed among the
three L. monocytogenes strains tested. However, since dissociation of HOCl to OC1- and
Hi increases with increasing pH, resistance and/or survival of L. monocytogenes in the
presence of chlorine compounds ultimately depends on the pH of the suspending medium.
For example, exposing the pathogen to 1 ppm available chlorine for 30 s led to population
decreases of -4.0, 3.0, and 0.7 logs at pH 5, 7, and 9, respectively. Hence, for chlorine
to be effective against listeriae and other microorganisms, it is imperative that such solu-
tions have pH values <7.
Although the work of El-Kest and Marth [103-1051 clearly indicates that the mini-
mum listericidal concentration of free chlorine lies between 1 and 5 ppm (similar to that
observed for many other non-spore-forming bacteria) depending on pH, temperature, the
presence of organic material, and bacterial strain, earlier studies [41,10I] conducted under
less controlled conditions showed minimum listericidal concentrations of free chlorine
that were markedly higher. Similar problems also were probably encountered by Mustapha
and Liewen [273], who found that a minimum of -100 ppm sodium hypochlorite was
required to reduce L. monocytogenes populations >4 logs in sterile distilled water during
2-5 min of exposure.
Chlorine is used extensively in fresh vegetable processing. Therefore, Zhang and
Farber [408] investigated the efficacy of several chlorine-based compounds against a cock-
tail of five L. monocytogenes strains on the surface of freshly cut lettuce and cabbage at
refrigeration and ambient temperatures. Sanitizers tested by these investigators included
chlorine from a hypochlorite-containing bleach, chlorine dioxide and a sodium chlorite-
based oxy-halogen compound. Immersing Listeria-contaminated vegetables in solutions
containing 200 ppm chlorine, 5 ppm chlorine dioxide, or 200 ppm Salmide for 10 min
resulted in maximum reductions of 1.3- 1.7,0.8-1.1, and 0.6 logs, respectively, for lettuce,
and 0.9-1.2, 0.4-0.8, and 1.8 logs for cabbage. The presence of surfactants reduced the
effect of chlorine. The authors also tested trisodium phosphate and lactic acid on lettuce
Characteristics of Listeria monocytogenes 201

and cabbage. Trisodium phosphate (0.1 and 0.2%) failed to inactivate listeriae, whereas
0.1% lactic or acetic acid reduced populations by only 0.5 and 0.2 log, respectively.
Many researchers have investigated the ability of commonly used sanitizers to inacti-
vate L. rnonoc.ytogenes on various types of food contact surfaces. Mustapha and Liewen
[273] found that destruction of L. rnonocytogenes was greater on smooth rather than pitted
stainless steel surfaces. However, cells incubated on either surface for 1 h were more
resistant to the lethal action of sodium hypochlorite than those remaining on such surfaces
24 h before exposure. Lower moisture levels on stainless steel surfaces incubated 24 rather
than 1 h may have enhanced the listericidal effect of sodium hypochlorite. In contrast to
these findings, Rossmoore and Drenzek [324] reported that L. rnonocytogenes populations
decreased 5 logs on relatively moist surfaces of glazed and unglazed ceramic tile as well
as stainless steel chips following exposure to 100 ppm sodium hypochlorite as directed
by the manufacturer. Furthermore, in no instance was L. monocytogenes more resistant
than single cultures of Pseudornonas or Serratia. However, when the same three surfaces
were treated with 1 and 10% solutions of milk and blood, Listeria populations decreased
1-4 logs in the presence of 100 ppm sodium hypochlorite.
As mentioned earlier, commonly used sanitizers are generally less effective against
L. rnonocytogenes in biofilms than when the cells are freely suspended. Lee and Frank
[226] reported that microcolonies of L. rnonocytogenes adhering to stainless steel
(-105CFU/cm2) decreased 2.6 logs after 30 s of exposure to 200 ppm chlorine (from a
hypochlorite solution), with some cells surviving a 5-min treatment. Mosteller and Bishop
[269] found that 200 ppm chlorine was sufficient to inactivate more than 5 logs of freely
suspended L. rnonocytogenes cells. However, a similar treatment failed to inactivate 3
logs of L. rnonocytogenes when a milk biofilm (initially 1O4-1O5CFU/cm2)was formed
on surfaces of Teflon and buna-n rubber. Resistance to sanitizers, including chlorine, in-
creased when L. rnonocytogenes biofilms were prepared on surfaces of polyester or polyes-
ter-polyurethane instead of stainless steel [2 171. Therefore, although freely suspended L.
rnonocytogenes can be controlled by 100 ppm chlorine, a higher level of chlorine is re-
quired to eliminate L. rnonocytogenes from biofilms.

Ozone
Ozone, a powerful sanitizing gas, is a better alternative to chlorine in many food processing
applications. Although used in European countries for decades, ozone is only approved
in the United States for treatment of bottled drinking water. Recently, a panel of experts
representing academia, food processors, and utility companies self-affirmed the GRAS
status of ozone, thus permitting its use in food processing applications [ 1591.
Ozone can be applied as a sanitizer in its gaseous form or as ozonated water. Ozo-
nated water is bactericidal to various microorganisms, with vegetative cells being more
sensitive to ozone than molds or bacterial spores. Use of ozone, in the form of ozonated
water, in food preservation and for decreasing microbial loads of meat and poultry and
of food plant effluents has been investigated [ 127,192,3441. Several factors affect the
bactericidal activity of ozonated water; organic matter such as food components quickly
react with ozone and reduce its effectivity.
Restaino et al. [316] investigated the lethality of ozonated water with and without
20 ppm organic matter, soluble starch (SS), or bovine serum albumin (BSA), against four
gram-positive (including L. rnonocytogenes) and four gram-negative (E. coli, S. typhirnu-
riurn, Y. enterocolitica, and P. aeruginosa) bacteria, two yeasts (Candida albicans and
202 Lou and Yousef

Zygosaccharomyces bailii), and mold spores (Aspergillus niger). Initial ozone concentra-
tions of 0.15-0.20 ppm produced by the ozone generator were higher in deionized water
with or without 20 ppm SS than in BSA-containing deionized water. Biphasic inactivation
curves were observed for bacteria and yeasts. Vegetative cells were inactivated instantly
(decreased >4 logs) after contact with ozone, with a much slower decrease in microbial
counts occurring during extended incubation. Gram-positive bacteria were generally more
resistant to ozone than gram-negative organisms, with the four gram-negative species hav-
ing similar sensitivity to ozone; however, L. monocytogenes was an exception. Contact
with ozone instantly inactivated >5 logs of L. monocytogenes but only -3 logs of the
other gram-positive species. The presence of organic matter during ozonation decreased
the lethality of ozone; however, the type of organic matter was more important than the
concentration. Incorporating 20 ppm SS had little effect on lethality of ozone toward
Listeria, whereas 20 ppm BSA significantly decreased the inactivation rate.

Antiseptic Soaps
Cross contamination of foods by food handlers or raw products in food service facilities
is a potential threat to public safety. Kerr et al. [ 1991 found that 12 and 7% of food workers
carried Listeria spp. and L. monocytogenes on their hands, respectively. Therefore, elimi-
nating L. monocytogenes from hands should decrease the incidence of Listeria in many
foods and enhance overall food safety. According to one report [32 I], full-strength solu-
tions of three commercially available antiseptic soaps, namely Mikro-x, Isoderm, and Zer-
obac were strongly listericidal, with populations of L. monocytogenes, L. ivanovii, and L.
seeligeri decreasing 7 logs following 30 s of exposure. Isoderm (a chlorine/quaternary
ammonium compound-based soap) remained almost equally effective when diluted 1 :4,
whereas Zerobac (an iodophor-based soap) retained strong listericidal activity at a dilution
of 1:8.
In another study [2 101, fingers of human volunteers were inoculated to contain 105
or 109L. monocytogenes CFU/finger to test the effectiveness of moist soap and a commer-
cially produced finger wipe containing isopropyl alcohol and citric acid as active ingredi-
ents. Overall, numbers of listeriae on fingers were generally reduced no more than 2-4
logs after 5 s of rubbing in phosphate buffer and moist soap, respectively. Therefore, a
population decrease of approximately 2 logs can be attributed to physical removal of the
pathogen during rubbing. In contrast, L. monocytogenes populations consistently de-
creased 2 4 logs after rubbing fingers with finger wipes for 5 s. Thus, strong listericidal
activity of these particular finger wipes and the ease with which they can be used should
make such products beneficial for food handlers in the food service industry.
In 1996, McCarthy 12531 checked inactivation of L. monocytogenes on latex gloves
by five commercially available hand-washing sanitizers. The latex gloves were artificially
contaminated by dipping them for 30 s into a PBS solution or crab cooking water that
contained - 1 O5 Listeria CFU/mL and then were treated with various hand-washing sani-
tizers. Dipping contaminated gloves into PBS containing a commercial chlorine bleach
solution (50 and 100 pprn chlorine), Zepamine A ( I95 ppm active quaternaries) or Ultra-
Kleen (a peroxide-based powder at 56 g/3.8 L) decreased counts of L. monocytogenes on
surfaces of gloves to undetectable levels, whereas treatment with Zep-i-dine (25 ppm
titratable iodine) and Zep Instant Hand Sanitizer that contains 60% ethanol reduced popu-
lations only 2 logs. Using nutrient-rich crab cooking water instead of PBS dramatically
Characteristics of Lister ia mon ocyt ogen es 203

decreased the effectiveness of both 50 ppm chlorine and Zep-i-dine and slightly decreased
the effectiveness of 100 ppm chlorine and Zepamine A. According to this study, only
Ultra-Kleen maintained the same effectiveness in cooking water.

Other Sanitizing Agents

Interest in the listericidal activity of non-chlorine-based sanitizers dates back to at least
1969 when Raranenkov [26] reported that iodine monochloride could effectively eliminate
L. monocytogenes from the surface of hens eggs. Shortly thereafter, creosote [59], phenol
[258,303], formaldehyde [303], and sodium hydroxide [258] were added to the list of
listericidal agents along with mercuric [258] and quaternary ammonium compounds (e.g.,
cetylpyridinium bromide) [258]. Sodium hydroxide at concentrations of 1 -2% solubilizes
the cytoplasmic membrane of Listeria [304], whereas bactericidal concentrations of ethyl
alcohol, phenol, and formaldehyde inhibit certain key enzymes, including succinic and
xanthine dehydrogenase [ 3031.
During the 1970s, several sanitizing agents were evaluated for treating soil samples
inoculated with L. mnnocytogenes. According to Vranchen et al. [380], 5 days of exposure
to 5 L of 3% formaldehyde solution was required before L. monocytogenes was eliminated
from 1 m2of soil. In a later study [ 101, 1 m2 of soil was Listeria-free 3 h after treatment with
0.5 L of an aqueous solution containing 3% quaternary ammonium compound. Although
sanitization of soil has little direct bearing on the food industry, such practices may be
useful in decreasing Listeria populations on farms that have experienced cases of listeriosis
in domestic livestock.
Following reports of foodborne listeriosis in the mid 1980s, a series of studies were
done to determine the listericidal activity of non-chlorine-based sanitizers that are rou-
tinely used by the food industry. According to experimental evidence presented in 1986 by
Lopes [236), acid anionic, iodophor, and quaternary ammonium compounds are effective
against L. mnnncytogenes when used at concentrations recommended by manufacturers.
Numbers of L. monocytogenes were reduced more than 5 logs after 30 s of exposure to
two different acid anionic sanitizers that contained 200 ppni of active ingredients (5%
dodecyl benzene sulfonic acid and 30% orthophosphoric acid or 2.6% sulfonated oleic
acid and 15% orthophosphoric acid). Similar reductions in Listeria populations were ob-
tained with a quaternary ammonium compound diluted to contain 200 pprn of the active
ingredient n-alkyl dimethyl benzyl ammonium chloride ( 12- 16 carbon atoms in the alkyl
group). An iodophor sanitizer diluted to contain 12.5 ppm titratable iodine was equally
effective against this pathogen. Thus all sanitizers tested showed effective antilisterial
activity when used at concentrations recommended by the manufacturer.
Two years later, Rosales et al. [321] found that populations of L. monocytogenes,
L. ivanovii, and L. seeligeri decreased >5 logs following exposure to aqueous solutions
containing 12.5- 100 ppm iodophor (pH 2.7-5.0), 12.5- 100 ppm quaternary ammonium
compound (pH 4.9-6.8), 100-400 ppm acid sanitizer (pH 2.4-3.l), 400 ppm phenolic
compound (pH 7.9), and 50- 100 ppm of a combined quaternary ammonium compound/
acid sanitizer preparation (pH 2.8-3.0). When these experiments were repeated using 10%
reconstituted nonfat dry milk (10% solids) rather than aqueous solutions of sanitizers,
reductions in Listeria populations of >5 logs only were observed for two of three iodo-
phors, one of three quaternary ammonium compounds, and one quaternary ammonium
compound/acid sanitizer preparation at concentrations of 200-400, 400, and 400 ppm,
204 Lou and Yousef

respectively. Although all aqueous solutions proved to be listericidal at concentrations

recommended by the manufacturer, only one iodophor, quaternary ammonium compound,
and phenolic sanitizer were listericidal at recommended concentrations when the test or-
ganism was suspended in milk.
Information from two additional investigations dealing with listericidal effects of
various non-chlorine-based sanitizers also became available in 1989. In the first study,
Mustapha and Liewen [273] reported that aqueous solutions containing 100-800 ppm of
one quaternary ammonium compound (i.e., n-alkyl dimethyl dichlorobenzyl ammonium
chloride) exhibited greater listericidal activity than similar solutions of sodium hypochlo-
rite when L. monocytogenes was exposed to these sanitizing agents in vitro. Moreover, a
50-ppm aqueous solution of the quaternary ammonium compound was equally effective
when the pathogen was present on smooth as well as pitted surfaces of stainless steel
chips, with populations decreasing >4 logs following short-term exposure.
In the second of these two investigations, Rossmoore and Drenzek [324] examined
the ability of four quaternary ammonium compounds as well as peroxyacetic acid, glutaral-
dehyde, MCI (S-chloro 2-methyl 4-isothiazolin 3-one and 2-methyl 4-isothiazolin 3-one),
dodecyl benzene sulfonic acid/orthophosphoric acid, and sulfonated oleic acid/orthophos-
phoric acid to inactivate L. monocytogenes, Pseudomonas, and Serratia on glazed/
unglazed ceramic tile and stainless steel. When used as recommended by the manufacturer,
peroxyacetic acid, glutaraldehyde, MCI, and one of four quaternary ammonium com-
pounds reduced numbers of listeriae >5 logs regardless of the type of surface tested, and
population decreases of 3-5 logs were noted for the three remaining quaternary ammo-
nium compounds. Since L. monocytogenes was consistently more sensitive to these sani-
tizers than the other two organisms tested, destruction of Pseudomonas spp (i.e., a fre-
quently used group of indicator organisms for general sanitation) also should guarantee
elimination of listeriae from properly treated surfaces.
To better simulate conditions that are likely to exist in dairy and meat processing
facilities, these researchers repeated the study just described [324] using surfaces that were
precoated with 1 and 10%solutions of milk or blood before exposure to the same sanitizing
agents. Not surprisingly, destruction of listeriae by most sanitizers was only 1-4 logs in
the presence of increasing concentrations of milk and blood, with the latter being most
detrimental to germicidal activity. However, since peroxyacetic acid and glutaraldehyde
maintained peak listericidal activity in the presence of up to 10%milk and blood, these two
sanitizers appear to be best suited for controlling listeriae within milk and meat processing
facilities.
Since water-based chain conveyor lubricants also may serve as a potential source
for spoilage and pathogenic microorganisms, including L. monocytogenes, incorporation
of sanitizing agents into lubricants has been suggested as one means of minimizing the
spread of microbial contaminants in food processing facilities. Although L. monocytogenes
populations in inoculated samples of sanitizer-free lubricant (pH 9.5) decreased only 2
logs during 14 days of storage at ambient temperatures [324], numbers of listeriae de-
creased more than 5 logs following 30 min of exposure to lubricant containing as little
as 25 ppm glutaraldehyde [323]. Furthermore, data collected during a field investigation
[324] showed that addition of 85 ppm glutaraldehyde to a conveyor chain lubricant reduced
general bacterial contamination along a dairy floor conveyor belt by an average of 4.4
logs/60 cm2. Although L. monocytogenes was not directly used in the latter study, this
pathogen still appears to be equally, if not more, sensitive to glutaraldehyde than Pseudom-
Characteristics of Lister ia monocytogenes 205

onus and other microbial contaminants. Hence, if L. rnonocytogenes was present initially,
this bacterium was likely to be eliminated during exposure to glutaraldehyde.
As is true for steel and tile surfaces, conveyor lubricants also come in contact with
various organic materials in food processing facilities. Hence, Rossmoore and Drenzek
[324] examined behavior of L. rnonocytogenes in lubricants containing 25-50 ppm glutar-
aldehyde, 5- 10 ppm MCI, and 500- 1000 ppm parachlorometaxylenol in combination
with 1% added milk and blood. In the presence of 1% milk, 50 ppm glutaraldehyde was
most effective, with numbers of listeriae decreasing >5 logs following 3 h of exposure.
However, in samples containing 1% blood rather than 1% milk, only 1000 ppm parachloro-
metaxylenol retained sufficient bactericidal activity to reduce Listeriu populations >5 logs
within 24 h. Although parachlorometaxylenol exhibited similar activity in the presence
of milk, addition of 5-10 ppm MCI was of little value in decreasing numbers of listeriae
in lubricant containing 1% milk or blood.
In addition to lubricants, several water-based cooling system fluids used in the dairy
and meat industry also are subject to sporadic contamination with pathogenic microorgan-
isms, including L. rnonocytogenes. Consequently, Rossmoore and Drenzek [324] also ex-
amined the potential benefit of adding low concentrations of glutaraldehyde, parachloro-
metaxylenol, and MCI to sweet water (i.e., potable refrigerated water containing a
corrosion inhibitor) and an aqueous solution of 35% propylene glycol, both of which are
commonly used in the cooling section of pasteurizers and other types of heat exchangers.
According to this report, L. rnonocytogenes populations in inoculated samples of sweet
-
water (pH 9.3) and 35% propylene glycol (pH 8.8) decreased only 1 and 3 logs, respec-
tively following 14 days of storage at 3.5"C. In sharp contrast, addition of 25 ppm glutaral-
dehyde to sweet water and propylene glycol containing 1% milk completely inactivated
L. rnonocytogenes populations of 105 CFU/mL in less than 1 h, as did addition of 100
ppm parachlorometaxylenol to propylene glycol. Inclusion of 100 ppm parachlorometaxy-
lenol in sweet water and 10 ppm MCI in both coolants was at best only marginally effec-
tive, with the pathogen surviving at least 48 h in several instances. Thus, although a low
concentration of glutaraldehyde will inactivate L. rnonocytogenes in sweet water and pro-
pylene glycol, use of parachlorometaxylenol for such a purpose should be limited to solu-
tions of propylene glycol.

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Conventional Methods
to Detect and Isolate Listeria
monocytogenes

W. DONNELLV
CATHERINE
University of Vermont, Burlington, Vermont

Die Methode ist alles

German proverb (Raiovich [ 1221)

INTRODUCTION
Listeria monocytogenes is a nonfastidious organism that can be subcultured on most com-
mon bacteriological media (i.e., Tryptose Agar, Nutrient Agar, and Blood Agar); however,
attempted isolation or reisolation of Listeria from inoculated or naturally contaminated
food and clinical specimens by use of nonselective media is often unsuccessful. Difficulties
encountered in isolating L. monocytogenes date back to initial characterization of this
pathogen in 1926 when Murray and his coworkers [ 1061 stated, The isolation of the
infecting organism is not easy and we found this to remain true even after we had estab-
lished the cause of the disease. Although efforts to isolate L. monocytogenes from blood
and cerebrospinal fluid of infected patients have met with considerable success mainly
because of the presence of Listeria in pure culture, obvious difficulties arise when food
and clinical specimens (tissue biopsies and autopsy specimens) contain small populations
of L. monocytogenes in combination with large numbers of other organisms.
Direct plating, cold enrichment, selective enrichment, and several rapid methods all

225
226 Donnelly

can be used in various combinations to detect L. monocytogenes in food, clinical and

environmental samples. Early attempts to isolate small numbers of Listeria from samples
containing large populations of indigenous microflora relied on direct plating and often
ended in failure. In 1948, Gray et al. [64] introduced the cold enrichment procedure as
an alternative method to isolate L. monocytogenes from highly contaminated samples.
Although this method has contributed much to our present-day knowledge concerning the
epidemiology of listeriosis, the prolonged incubation period necessary to obtain positive
results is a serious disadvantage. Major improvements in selective enrichment and plating
media have since decreased analysis times from several months to less than 1 week. Out-
breaks of foodborne listeriosis coupled with the high mortality rates associated with spo-
radic cases of illness and the advent of mandatory Hazard Analysis Critical Control Point
(HACCP) programs have underscored the need for faster and more efficient methods to
detect small numbers of Listeria in a wide range of foods.
The purpose of this chapter is to review and update the development of various
enrichment broths, as well as plating media and methods, used to isolate Listeria spp.,
including L. monocytogenes, from clinical, environmental, and food samples. Numerous
enrichment broth and plating media formulations have been used during the past 50 years
for selective cultivation of Listeria, the most important of which are detailed in Appendix
I. Detection and isolation of Listeria remains complicated by the inability of researchers
to identify a single procedure that is sufficiently sensitive to detect I;. monocytogenes in
all types of foods within a reasonable time. Furthermore, many selective enrichment broths
and plating media fail to allow repair and/or growth of sublethally injured Listeria fre-
quently present in processed foods [26] or food processing environments. Despite these
inherent shortcomings, research efforts in response to foodborne listeriosis outbreaks have
led to development of numerous regulatory procedures, including the U.S. Food and Drug
Administration (FDA) and the U.S. Department of Agriculture-Food Safety and Inspec-
tion Service (USDA-FSIS) procedures [74,76] which have been adopted in the United
States as standard methods to isolate L. monocytogenes from a wide variety of foods
and food processing environments. However, in an effort to detect more rapidly and reli-
ably both healthy and sublethally injured Listeria in the wide range of foods currently
being examined, these methods and others that are less widely accepted will undoubtedly
undergo further modifications as selective enrichment broths and plating media used in
these procedures continue to be improved.

COLD ENRICHMENT
Difficulties in isolating L. monocytogenes typically arise when small numbers of Listeria
are present in environmental and clinical food samples containing large numbers of indige-
nous microorganisms. Hence, numbers of Listeria must be increased, relative to that of
the background flora, before the bacterium can be detected. Thirteen years after the first
description of L. monocytogenes by Murray et al. [106], Biester and Schwarte 1131 ob-
served that Listerella (Listeriu) could be frequently isolated from naturally infected sheep
organs that were held refrigerated in 50% glycerol for several months. Although the organ-
ism was only rarely isolated after initial plating of diluted specimens, these authors failed
to comment on the significance of cold storage. Following similar chance observations,
a young graduate student, M. L. Gray, recognized the benefits of low-temperature incuba-
tion for recovering L. monocytogenes from clinical specimens. In 1948, Gray et al. [64]
reported that in three of five bovine listeriosis cases, L. monocytogenes was only isolated
Methods to Detect and lsolate L. monocytogenes 227

after brain tissue diluted in Tryptose Broth, was stored for 5-13 weeks at 4C and then
plated on Tryptose Agar. Although a few Listeria colonies were observed after directly
plating the remaining two brain tissue samples on Tryptose Agar, the bacterium was more
readily isolated following cold enrichment. These results clearly showed the ability of L.
monocytogerzesto multiply to detectable levels in the presence of other microbial contami-
nants during extended storage at 4C.
Gray\ cold enrichment method, in which samples hornogenized in Tryptose Broth
were incubated at 4C and plated weekly or biweekly on Tryptose Agar during 3 months of
storage, was soon adopted as the standard procedure for recovering L. monocytogenes.
Normally only a few weeks of cold enrichment are required before Listeria can be de-
tected; however, in one instance 1621, 6 months of refrigerated storage was necessary
before L. monocytogenes could be isolated from calf brains. Although the cold enrichment
procedure is clearly slow and laborious, this method greatly enhances the likelihood of
isolating Listeria from a variety of specimens, including food.
In 13 studies summarized by Bojsen-Mgller 1171, Listeria was identified in 995
tissue and organ specimens from naturally and experimentally infected domestic animals.
Using both direct plating and cold enrichment procedures, Lipteria was isolated from 684
of 995 (68.7%) specimens, whereas 307 of 995 (30.8%) specimens required cold enrich-
ment before the bacterium could be detected. Furthermore, cold enrichment failed to detect
Listeria in only 4 of 684 (0.6%) samples that were previously positive by direct plating.
A study by Ryser et al. [ I3 I ] stressed the importance of colcl enrichment for recovery of
L. monocytoqenes from cottage cheese manufactured from milk inoculated with this patho-
gen. Using direct plating, L. monocytogenes was recovered from 43 of 1 12 (38.4%) cottage
cheese samples stored at 3C for up to 28 days, whereas cold enrichment of the same
samples in Tryptose Broth for up to 8 weeks yielded Listeriu in 59 of 1 12 (52.7%) samples.
Thus, cold enrichment was necessary to detect this pathogen in 16 of 1 12 ( 14.3%) cheese
samples. Ryser and Marth also found cold enrichment to be of great value in detecting
low levels of L. monocytogenes in Cheddar [ 1321, Camembert [ 1331, and brick cheese
[ 1351 manufactured from pasteurized milk inoculated with the bacterium.
Despite the proven success of cold enrichment, the mechanism by which numbers
of L. monocytogenes are enhanced during prolonged incubation at 4C is not fully under-
stood. Although cold enrichment exploits the psychrotrophic nature of L. monocytogenes
and simultaneously suppresses growth of indigenous nonpsyc hrotrophic organisms, Gray
and Killinger [62] indicated that, at times, growth of Listerig was too rapid to attribute
enhanced growth of this pathogen to mere multiplication. When this procedure was first
described in 1948, Gray et al. [64] suggested possible involvement of an inhibitory factor
in bovine brain tissue that suppressed growth of competing organisms. However, this
theory has been dispelled by subsequent studies which demonstrated enhanced growth of
Listeria during cold enrichment of such diverse samples as mouse liver [ 1441, oat silage
[61], feces [ 1171, sewage [46], cabbage [66], raw milk 11441, and cheese 1131-1351. A
more plausible explanation is that in many clinical specimens, Listeria may exist within
monocytes, rnacrophages, or other phagocytic cells, with colcl storage facilitating release
of the intracellular organism. More recent research on the role of cold-shock proteins,
cold-acclimating proteins, and other mechanisms which enable psychrotrophic growth of
L. monocytogenes may help further explain the preferential growth of Listeria during cold
enrichment [K,8I]. For instance, anteiso-C15 fatty acid reportedly plays a critical role in
adaptation of L. monocytogenes to cold temperatures [4], with mutants deficient in this
fatty acid being shown to be cold sensitive.
228 Donnelly

As previously reviewed by Ryser and Marth [ 1361, over 20 media formulations have
been successfully used to cold enrich a diverse group of samples that were either naturally
or artificially contaminated with L. monocytogenes. Since incubation at 4C is in itself
partially selective for growth of L. monocytogenes, nonselective broths such as Tryptose
Broth and Oxoid Nutrient Broth No. 2 (ONB2) rapidly emerged as media of choice, with
Tryptose Broth generally recognized as being superior. In earlier studies, cold enrichment
was used as the sole enrichment procedure and was followed by plating a portion of
the enriched sample on Tryptose Agar at intervals during 2-12 months [139]. Following
incubation, plates were examined under oblique lighting for typical bluish green, Listeria-
like colonies.
Although growth of L. rnonocytogenes is favored at 4OC, other organisms, including
Proteus, Hafiia, Pseudornonas, enterococci, and certain lactic acid bacteria, also can mul-
tiply in nonselective media at refrigeration temperatures [2], thus making detection of
Listeria more difficult. To prevent overgrowth by non-Listeria organisms, investigators
began adding inhibitory agents to various nonselective cold enrichment broths. In 1972,
Bojsen-MQller [ 171 recognized that supplementing Tryptose Phosphate Broth with poly-
myxin B substantially reduced populations of gram-negative rods (i.e., Escherichia coli,
Pseudomonas aeruginosa, and Proteus spp.) and enterococci while at the same time
allowing rapid growth of L. monocytogenes. Unfortunately, certain species of lactic acid
bacteria resistant to polymyxin B can ferment lactose to lactic acid and reduce the pH to
the point where L. rnonocytogenes fails to grow at 4C. Attempts at maintaining a pH of
7.2 by adding 0.1 M MOPS (3-N-morpholino propane sulfonic acid) to cold-enriched raw
milk samples were unsuccessful [68].
Recovery of L. monocytogenes also is enhanced when cold enrichment is used as
a secondary enrichment preceded by a selective primary enrichment at 30-37C. Ban-
nerman and Bille [7] subjected numerous cheese and cheese factory environmental sam-
ples to secondary cold enrichment in FDA Enrichment Broth (Listeria Enrichment Broth
[LEB]) that were previously incubated at 30C for 48 h (primary warm enrichment). After
plating enrichments on two selective agars, 34 and 62 of 96 isolates were obtained using
warm and cold enrichment, respectively. Thus cold enrichment for 28 days resulted in a
29.2% (28 of 96) increase in recovery of L. monocytogenes from cheese and cheese factory
samples. However, with the advent of improved selective media and methods, most inves-
tigators have concluded that cold enrichment offers no advantages over selective enrich-
ment [70]. In addition, the lengthy incubation period necessary for cold enrichment makes
this procedure impractical for routine regulatory analysis of foods.

SELECTIVE ENRICHMENT AND PLATING AT 30-37C

The principle of enrichment at elevated temperatures (30-37C) is based on selective
inhibition of indigenous microflora through addition of inhibitory agents while at the same
time allowing unhindered growth of Listeria. Given the many months required for cold
enrichment, the scientific community soon became aware of the need for a shorter incuba-
tion period. In 1950, Gray et al. [63] isolated L. monocytogenes from contaminated mate-
rial that was inoculated into Nutrient Broth containing 0.05% potassium tellurite and incu-
bated at 37C for 6-8 h before being plated on Tryptose Agar with or without 0.05%
potassium tellurite. Even though subsequent studies showed both potassium tellurite-
containing media to be partially inhibitory to Listeria [80,89,111,122], Gray and his col-
leagues can still be credited with introducing both the first cold-enrichment procedure and
Methods to Detect and Isolate L. monocytogenes 229

the first warm enrichment media for selective isolation of L. rnonocytogenes. Since 1950,
various combinations of selective agents have been added to basal media (i.e., Tryptose
Broth, ONB2, and Tryptose Phosphate Broth) to obtain media suitable for selective enrich-
ment of Listeria at 30-37C. Mavrothalassitis [99] reported an optimum incubation tem-
perature of 30C for enrichment of L. rnonocytogenes from heavily contaminated samples.
Results from at least two additional studies [33,109] also showed that laboratory cultures
of L. rnonocytogenes, L. seeligeri, and/or L. ivanovii were more susceptible to commonly
used Listeria selective agents (i.e., ceftazidime, cefotetan, laxamoxef, and fosfomycin)
when incubated at 37 rather than 30C. Hence, most Listeria enrichments are done at
30C. Ryser and Marth [ 1361 previously reviewed the wide range of media formulations
that have been developed for selective enrichment of L. rnonocytogenes from environmen-
tal and clinical food specimens.

Selective Agents
Modest, nonspecific nutritional requirements of L. rnonocytogenes have led to difficulties
in formulating media that enhance growth of this pathogen. Consequently, efforts have
primarily focused on inhibition of the indigenous bacterial fIora by taking advantage of
the resistance of L. monocytogenes to various selective agents and antibiotics. The major
advances that have contributed to our present-day ability to isolate Listeria from heavily
contaminated environments are shown in Table 1. Although tnany inhibitory agents have
proven to be at least somewhat useful for selective isolation of L. rnonocytogenes from

TABLE
1 Recognition of Selective Agents Useful in Isolation of Listeria
Year Compound Role in selective media References
1950 Potassium tellurite Selective/differential for Liste- 20,63,80,83,89,100,111,122,145
ria, which reduces tellurite to
tellurium, producing black
colonies
1960 Lithium chloride/ Amplification of Listeria in the 38,49,65,66,68,87,92,100,132,
phen ylethanol presence of gram-negative 133,144
bacteria
1966 Nalidixic acid Inhibitory to gram-negative bac- 1,16,42,45,60,77,80,112,113,
teria through interference 124,141
with DNA gyrase
1971 Acriflavin(e)/ Inhibitory to gram-positive 3,15,36,41,42,47,65,75,78,79,
trypaflavin(e) cocci 1 12,113,122,123,125,126,
127
1971 Polyniyxin B Prevents growth of gram- 17,35,38,92,112,127,142
negative rods and strepto-
cocci
1986 Moxalactam Broad spectrum; inhibitory to 74,87,103,112
many gram-positive and
gram-negative contaminants,
including Staphy Lococcus,
Proteus, and Pseudomonas
1988 Ceftazidime Broad-spectrum cephalosporin 7,03,96,109
antibiotic
230 Donnelly

naturally and artificially contaminated biological specimens, others have demonstrated

very little value when added to basal media, as previously reviewed by Ryser and Marth
[ 1361. Throughout the following discussion of selective agents, one must keep in mind
that formulating media selective for L. monocytogenes is not a straightforward process,
as many selective agents can partially inhibit growth of this pathogen, particularly when
the organism is sublethally injured.
Potassium Tel Iu rite
Many selective media, including the early formulation by Gray et al. [63], contain inhibi-
tory substances that are now of questionable value. As previously described, in 1950, Gray
et al. [63] examined the potential usefulness of potassium tellurite and sodium azide in
Listeria-selective media. Sodium azide prevented growth of L. monocytogenes in Tryptose
Broth whereas potassium tellurite was quite selective for the pathogen. However, shortly
after these findings were published, Olson et al. [ 1 1 I] observed that potassium tellurite
prevented growth of numerous L. monocytogenes strains. Other investigators [80,83,
89,100,1221 have substantiated these findings and have discouraged the use of potassium
tellurite as a selective agent. The advantage of adding potassium tellurite to selective
media is that the resulting L. monocytogenes colonies appear black from reduction of
potassium tellurite to tellurium. Unlike the typical black-yellowish and gray colonies pro-
duced by gram-positive cocci, the marginal zone of Listeria colonies appears green when
the organism is grown on media containing potassium tellurite and viewed with oblique
illumination [ 1391. A modification of Vogel Johnson agar (MVJA) was evaluated by Bu-
chanan et al. [20] for isolating Listeria from foods. Selective agents, including moxalac-
tam, nalidixic acid, bacitracin, and potassium tellurite, permitted growth of Listeria while
suppressing background contaminants. Furthermore, the ability to distinguish colonies
readily was not predicated on the need for obliquely transmitted light. Buchanan et al.
[23] also found that Lithium chloride-Phenylethanol-Moxalactam Agar (LPM) and MVJA
generally gave comparable recovery of Listeria from naturally contaminated samples of
fresh meat, cured meat, poultry, fish and shellfish. Adding both tellurite and mannitol to
MVJA greatly aided in differentiating Listeria colonies from those formed by naturally
occurring contaminants, including various species of enterococci and staphylococci. How-
ever, Smith and Archer [ 1451 reported that potassium tellurite prevented repair of heat-
injured L. monocytogenes.
Lit hiu m C hIo ride/P he ny Iet ha no I
Using the combination of phenylethanol and lithium chloride, McBride and Girard [ 1001
succeeded in amplifying numbers of L. monocytogenes in the presence of gram-negative
bacteria. The usefulness of phenylethanol and lithium chloride as Listeria-selective agents
has since been confirmed by other investigators, resulting in the earlier widespread use and
acceptance of McBride Listeria Agar (MLA) as a plating medium for L. monocytogenes
[38,49,65,66,68,87,92,100,132,133,144].A modification of MLA (omission of sheep
blood and addition of cycloheximide as an antifungal agent) was once recommended by
the FDA for analyzing food samples suspected of harboring Listeria [92,93]. Ryser and
Marth [ 134,1351 and Yousef and Marth [ 1521 reported that increasing the lithium chloride
concentration to 0.5% (0.05% lithium chloride in the original formulation [loo]) increased
selectivity of the medium without appreciably decreasing recovery of healthy Listeria
[ 134,135,1521.
Methods to Detect and Isolate L. monocytogenes 231

Nalidixic Acid
Beerens and Tahon-Caste1 [ 111 were first to report the usefulness of nalidixic acid in
isolating L. rnonocytogenes from heavily contaminated pathological specimens. Increased
isolation of Listeria using media containing nalidixic acid primarily resulted from inhibi-
tion of indigenous gram-negative bacteria [60]. The benefits of adding nalidixic acid to
otherwise noninhibitory media were soon confirmed in many laboratories [ 16,42,77,80,
1 12,113,1411. After discovering the benefits of adding nalidixic acid to enrichment broth
[ 1 11, Ralovich et al. [ 1241 effectively used serum agar containing nalidixic acid to isolate
L. monocytogenes from feces, organs, and other clinical specimens. Although the micro-
bial background flora was largely inhibited on this medium, streptococci and other nali-
dixic acid-resistant organisms occasionally persisted. Nalidixic acid was eventually recog-
nized as one of the most important selective agents, and it is now used alone or more
commonly in combination with other selective agents for isolating L. monocytogenes from
food and clinical specimens. Farber et al. [45] developed an improved Listeria-selective
plating medium by combining the positive attributes of McBride Listeria Agar and LPM
Agar. In their formula for Farber Listeria Agar, oxolinic acid was substituted for nali-
dixic acid. Both agents function by interfering with the activity of DNA gyrase, an enzyme
needed to maintain proper DNA structure and resealing of chromosomal nicks [60].

Trypaf lavine/Acriflavi ne
Despite successful use of nalidixic acid, Ralovich et al. [125,126] found that growth of
certain gram-positive cocci and gram-negative rods in the presence of this selective agent
complicated the isolation of Listeria. Such difficulties led to inclusion of trypaflavine, a
known inhi bitor of gram-positive cocci, in media containing nalidixic acid. This medium
soon became known as Trypaflavine Nalidixic Acid Serum ,4gar (TNSA). The end result
was the selective inhibition of virtually all other bacteria, whereas growth of L. monocyto-
genes was only slightly decreased [ 15,1111. Following successful use of this medium in
many European studies [ 15,78,112,113,125], Ralovich et al. [ 1221 endorsed TNSA as the
plating medium of choice for isolating L. monocytogenes from contaminated materials.
Additional work revealed that contaminating organisms, predominantly streptococci, grew
infrequently on clear media containing both antibiotics and were generally discernible
from L. rnonocytogenes with the naked eye. In 1972, Seeliger [140] reported that the
combined use of acriflavine and nalidixic acid greatly suppressed gram-negative organisms
and fecal streptococci without apparently affecting recovery of L. rnonocytogenes. These
findings were subsequently confirmed by Bockemuhl et al. [ 161, who reported easy recov-
ery of L. monocytogenes from enriched fecal samples using an agar medium that contained
nalidixic acid and acridine dye. Confirmation of these findings in other European labora-
tories [3,42,47,65,79] led to widespread use of trypaflavinehalidixic acid as Listeria-
selective agents. In 1974, Hofer [75] proposed using a medium prepared from Tryptose
Agar containing nalidixic acid, trypaflavine, and thallous acetate. Trypaflavine can be
replaced by other acridine dyes, including xanthacridine, acriflavine, or proflavinehemi-
sulfate [123]. According to Gregario et al. [65], use of nalidixic acid together with either
acriflavine or trypaflavine gave rise to media that were equally inhibitory to background
microflora, suggesting that similar results can be obtained by substituting acriflavine for
trypaflavine. Based on results from European laboratories [36,41,78,123], a Serum Agar-
or Blood Agar-based medium containing trypaflavine, acriflavine, and nalidixic acid ap-
232 Donnelly

peared to be satisfactory for selective isolation of L. monocytogenes from samples con-

taining a mixed microbial flora. In 1984, Rodriguez et al. [ 1271 developed a blood agar
medium containing acriflavine and nalidixic acid (Rodriguez Isolation Medium [RIM])
that was far superior to the earlier formulations of Ralovich et al. [ 124,1261. During the
last decade, numerous media containing acriflavine and nalidixic acid with or without
other antibiotics have been developed for selective isolation/enrichment of Listeria from
food and environmental samples, including Merck Listeria Agar [ 18,671, which is com-
mercially available in Europe.
Potassium Th iocyanate
In 1961, Fuzi and Pillis [55] proposed a medium containing 0.35% potassium thiocyanate
for selective enrichment of L. monocytogenes. Although reported useful by some research-
ers [42,88,141], others found that potassium thiocyanate inhibited L. monocytogenes
[83,89,125]. Despite these reports, several studies demonstrated that an enrichment broth
containing this selective agent in combination with nalidixic acid was useful in isolating
L. monocytogenes from cabbage [66] and milk [68,144] and other dairy products [85]. In
1972, Ralovich et al. [125] endorsed Levinthals Broth and Holmans Medium, both of
which contain nalidixic acid and trypaflavine, for selective enrichment of Listeria. Results
obtained by Slade and Collins-Thompson [ 1441 demonstrated that growth of L. monocyto-
genes in ONB2 containing both nalidixic acid and potassium thiocyanate can be improved
by adding acriflavine.
Thallous Acetate
During the early 195Os, thallous acetate was employed as a selective agent for lactic acid
bacteria; however, it was not until 1969 that Kramer and Jones [83] recommended the
combined use of thallous acetate and nalidixic acid in Listeriu-selective media. Three
years later, Khan et al. [80] found that, unlike potassium tellurite, thallous acetate used
alone or together with nalidixic acid did not adversely affect recovery of L. monocytogenes
from biological specimens and silage samples. In 1979, Leighton [89] demonstrated that
the combined use of thallous acetate and nalidixic acid completely suppressed growth of
E. coli strains that were previously resistant to nalidixic acid. Greater inhibition of gram-
positive bacteria also occurred when both selective agents were used together rather than
separately. Although Leighton [891 recommended a medium composed of Tryptose Phos-
phate Broth, thallous acetate, and nalidixic acid for recovery of L. monocytogenes from
mixed bacterial populations, thallous acetate (as well as potassium thiocyanate, potassium
tellurite, and lithium chloride) altered the colonial morphology of L. monocytogenes from
the smooth to the rough form. In view of this experience, most of the currently used
formulations of Listeria-selective media omit thallous acetate.
Polymyxin B
In 1971, Despierres [35] reported that the combination of polymyxin B and nalidixic acid
was useful for recovering L. monocytogenes from feces, with these antibiotics preventing
growth of many background organisms, including Enterococcus faecalis. That same year,
Ortel [ 1 121 proposed another medium containing polymyxin B and bacitracin to isolate
L. monocytogenes from stool samples. According to Bojsen-MQller [ 171, gram-negative
rods and enterococci failed to grow in Tryptose Phosphate Broth containing polymyxin
B, whereas growth of L. monocytogenes was relatively unaffected. After examining six
different enrichment and isolation media, Rodriguez et al. [ 1271 concluded that little if
Methods to Detect and Isolate L. monocytogenes 233

any benefit was gained by adding polymyxin B to media already containing nalidixic acid
and acriflavine. Doyle and Schoeni [38] successfully isolated L. monocytogenes from milk
and clinical and fecal samples after enrichment in a selective broth containing polymyxin
B, acriflavine, and nalidixic acid that resembled Isolation Medium I1 developed by Rodri-
guez et al. [ 1271. Although the selective enrichment broth developed by Doyle and Schoeni
gained some attention [92], the necessity for polymyxin B in this medium remains some-
what questionable. Siragusa and Johnson [ 1421 successfully isolated L. monocytogenes
from yogurt using a medium containing polymyxin B, nalidkic acid, and acriflavine. Their
medium reportedly prevented growth of Streptococcus thermophilus and Lactobacillus
delbrueckii subsp. bulgaricus, and thus making it particularly suitable for isolating L.
monocytogenes from certain fermented dairy products.
M o x a lacta m
Results from antibiotic susceptibility tests [ 1 121 led Lee and McClain [87] to add moxalac-
tam (a broad-spectrum antibiotic which is inhibitory to many gram-positive and gram-
negative bacteria, including Stuphylococcus, Proteus, and Pseudomonas) to MLA con-
taining 0.25% phenylethanol and O S % lithium chloride. The result was a highly selective
medium for recovery of L. monocytogenes from raw beef and many other foods. This
medium, Lithium chloride-Phenylethanol-Moxalactam(LPM) Agar, is recommended by
the USDA-FSIS for isolating L. monocytogenes from raw meat and poultry [ 1031 and also
has been incorporated into the current FDA procedure as a second selective plating me-
dium [74].

Ceftazidi m e
Bannerman and Bille [7] used Columbia Agar Base in combination with acriflavine and
ceftazidime (AC Agar), a broad-spectrum cephalosporin antibiotic, to isolate L. monocyto-
genes from cheese samples. AC Agar was found to be superior to FDA-Modified McBride
Listeria Agar (MMLA) [93,96], recovering approximately 50% more L. monocytogenes
isolates frorn soft cheese and cheese manufacturing environments, than did FDA-MMLA.
Except for ii few enterococci, the combination of acriflavine and ceftazidime inhibited all
other non-Listeria organisms, including yeasts and molds. However, van Netten et al.
[109] reported that PALCAM Agar, which contains polymyxin B and lithium chloride
along with half or less the concentration of acriflavine and ceftazidime found in AC Agar,
was superior to the latter medium. After comparing 13 different plating media, these au-
thors also concluded that media containing both ceftazidime and 1.5% lithium chloride
afforded more selectivity than did phenylethanol alone. However, increased selectivity
results in decreased recovery of stressed or sublethally injured cells that are frequently
present in foods.

SELECTIVE MEDIA FOR ISOLATION AND ENRICHMENT

OF LISTERIA
Isolation Media
McBride Listeria Agar
MLA was the first widely used plating medium for selective isolation of L. monocytogenes.
This medium, introduced by McBride and Girard [ 1001 in 1960, is prepared from Pheny-
234 Donnelly

lethanol Agar to which lithium chloride, glycine, and sheep blood are added. At least
seven subsequent changes in the original formulation of MLA have led to considerable
confusion as to the exact composition of this medium. Ironically, the first reported modifi-
cation of MLA by Bearns and Girard [9] dates back to 1959, nearly I year before the
original formulation appeared in the literature [ 1001. This medium, named Modified
McBride Medium (MLA2) by the authors and known today as one of several Modified
MLAs, is similar to the original formulation except that sheep blood is omitted and glycine
anhydride is substituted for glycine, with the anhydride form reportedly being less inhibi-
tory to L. rnonocytogenes than glycine [87]. In most instances, MLA2 was more Listeria-
selective than Nalidixic Acid Agar [49,112], Acriflavine Nalidixic Acid Agar [ 1441, or
Acridine Nalidixic Acid Agar [49]. The selectivity of MLA2 can be further improved,
without affecting recovery of Listeria, by increasing the lithium chloride content to 0.5%.
With the addition of sheep blood, this medium became partially differential and inhibitory
to background microflora, and hence it was better suited than MLA2 for recovering L.
monocytogenes from brick [ 1351, feta [ 1 151, and blue cheese [ 1 161, as well as cold-pack
cheese food [ 1341.
An earlier report in which glycine was found partially to inhibit L. monocytogenes
[87] prompted many individuals to prepare the aforementioned forms of MLA with glycine
anhydride, which is far less inhibitory to Listeria. Nevertheless, two widely used formula-
tions of the original MLA containing glycine have been commercially available since
1985 from Difco Laboratories, Detroit, MI and Bethesda Biological Laboratories (BBL)
Cockeysville, MD. Although addition of blood provides one means of identifying possible
L. monocytogenes colonies (virtually all are at least somewhat P-hemolytic) and enhances
growth of the pathogen in certain B vitamin- and/or amino acid-deficient media, many
individuals prefer to omit blood from the various formulations of MLA and examine the
plates under oblique illumination for blue to bluish green Listeria-like colonies. In 1987,
Lovett et al. [96] added cycloheximide to blood-free MLA2 and named this particularly
useful medium FDA-Modified McBride Listeria Agar (FDA-MMLA). Although one ear-
lier study claimed that TNSA was superior to MLA2, subsequent data indicated that FDA-
MMLA [93,94,96] and MLA2 [58,66,68,96,126,144], which contain glycine anhydride,
were the MLA formulations of choice for isolating Listeria spp. from foods, particularly
dairy, vegetable, and seafood products, with the FDA formulation serving for many years as
one of two plating media (the other being LPM agar) in the widely used FDA procedure [95].
LPM Agar
In 1986, Lee and McClain [87] added 4.5 g of lithium chloride and 20 mg of moxalactam to
MLA2 and named their new medium Lithium chloride-Phenylethanol-MoxalactamAgar.
Although this selective medium (commercially available in the United States from BBL
and Difco Laboratories) is particularly well suited for isolating Listeria from raw meat
and poultry, as evidenced by its inclusion as the medium of choice in an earlier version
of the USDA procedure, LPM Agar has since been replaced by Modified Oxford Agar
[27], which produces black L. monocytogenes colonies, each with a black halo following
24 h of incubation.
Oxford/MOX Agar
In 1989, Curtis et al. [34] developed an agar medium that eliminated the need for oblique
illumination. Their medium, Oxford Agar, was prepared from Columbia Agar base to
which a number of selective agents, including colistin sulfate (20 mg/L), fosfomycin (10
Methods to Detect and lsolate L. monocytogenes 235

mg/L), cefotetan (2 mg/L), cycloheximide (400 mg/L), lithium chloride (15 g/L), and
acriflavine (5 mg/L), were added. Esculin and ferric ammonium citrate also were added
as differential agents to produce black Listeria colonies from esculin hydrolysis. This
medium was slightly modified by McClain and Lee by incorporating moxalactam, with
this new medium being designated Modified Oxford Agar (MOX) [27]. In May of 1989,
the USDA-FSIS procedure was changed to incorporate MOX as the recommended plating
medium. Late in 1990, the FDA modified its procedure by replacing FDA-MMLA with
Oxford Agar (OXA). In the present version of the FDA method [74], two selective media
must now be used, either both PALCAM and OXA or OXA and LPM (or LPM plus
esculin and Fe3+).These changes have decreased reliance on the sometimes tedious Henry
illumination technique and have brought U.S. regulatory procedures into closer compli-
ance with international regulatory protocols.
PALCAM Agar
In 1988, van Netten et al. [ 1081 reported that RAPAMY Agar, a modification of TNSA
developed by Ralovich et al. [ 1261 that includes acriflavine, phenylethanol, esculin, manni-
tol, and egg yolk emulsion, was suitable for enumerating Listeria spp. Virtually identical
populations were observed when overnight broth cultures of L. monocytogenes, L. seeli-
geri, and L. ivanovii were surface-plated on RAPAMY and nonselective agar, with growth
of all non- Listeria organisms tested, except Enterococcus fizecalis and Enterococcus fae-
cium, being completely inhibited on the selective medium. Like OXA [34], RAPAMY
Agar also produced distinctive black Listeria colonies that were surrounded by a dense
black halo from esculin hydrolysis. Although such characteristic colonies were present
against a deep red background (inability to utilize mannitol) on RAPAMY Agar, E. fae-
calis and E. faecium generally produced colonies with blue-green halos. Although attempts
to eliminate growth of these two species of enterococci by adding cefoxitin (moxalactam)
to this medium failed, results suggested that RAPAMY Agar could be used to quantify
Listeria spp. in thermally processed and dried foods having total aerobic plate counts of
5 1O6CFU/gand enterococcus counts of 5 1 02CFU/g. However, as might be expected,
high populations of enterococci severely hampered detectioin of Listeria spp. in chicken,
minced meat, and mold-ripened cheese.
Further attempts by van Netten et al. [107] to eliminate growth of enterococci by
adding fosfomycin (20 mg/L) to RAPAMY Agar met with only limited success. Addition
of lithium chloride (1.5%) to RAPAMY Agar inhibited many Listeria spp.; however, an
improved selective and differential medium was obtained by adding lithium chloride to
RAPAMY Agar and omitting nalidixic acid. The resultant medium was named ALPAMY
Agar, because it contains acriflavine, lithium chloride, phenylethanol, esculin, mannitol,
and egg yolk emulsion agar. In a study with pure cultures, ALPAMY Agar allowed unin-
hibited growth of all 10 L. monocytogenes strains tested but completely prevented growth
of single strains of L. seeligeri and L. ivanovii. Selectivity tests showed that ALPAMY
Agar supported growth of only 2 of 41 non-Listeria organisms-one strain each of Staph-
~ZOCOCCUS aureus and Micrococcus spp., both of which were readily differentiated from
Listeria colonies. Subsequent studies indicate that ALPAMY Agar is far superior to
RAPAMY Agar for detecting Listeria in raw milk and soft cheeses manufactured from raw
milk, as well as in raw vegetables and chicken. This medium is the forerunner to PALCAM
agar [ 1091, which contains polymyxin B and lithium chloride along with half or less the
concentration of acriflavine and ceftazidime found in AC A.gar. It is recommended that
PALCAM Agar plates be incubated for 48 h at 30C under microaerobic conditions (5%
236 Donnelly

oxygen, 7.5% carbon dioxide, 7.5% hydrogen, and 80% nitrogen). This medium, along
with L-PALCAMY enrichment broth, is the basis for the Netherlands Government Food
Inspection Service (NGFIS) method for Listeria isolation.
Other Selective Plating Media
Interest in foodborne listeriosis during the 1980s led to development of many additional
Listeria-selective media for examining milk and dairy products. In 1984, Martin et al.
[98] developed Gum Base Nalidixic Acid Medium (GBNA)-a synthetic agar-free solid
medium superior to the MMLA of Bearns and Girard [9] for isolating L. monocytogenes
from raw milk [68]. Bailey et al. [5] also found that a modified version of this medium
containing lithium chloride and moxalactam was suitable for isolating L. monocytogenes
from raw chicken. A selective agar medium [66] based on the enrichment broth of Doyle
and Schoeni [38], from which acriflavine was omitted and Fe'' was added, compared
favorably with the original formulation of MLA [ 1001. Supplementation of selective [66]
and nonselective [30] media with Fe3+enhances growth of L. monocytogenes and may
be beneficial for isolating sublethally injured cells from food samples containing a mixed
microbial flora.
As indicated previously, attempts to isolate L. monocytogenes from food products
have focused on enhancing the selectivity of currently available blood-free plating media
which are normally viewed under oblique illumination, as well as development of alterna-
tive media that incorporate differential agents other than blood to aid microbiologists in
identifying Listeria colonies in mixed cultures. In 1987, Buchanan et al. [20] found the
combination of moxalactam, nalidixic acid, and bacitracin to be effective in allowing
growth of Listeria spp. while preventing growth of most other foodborne organisms, in-
cluding micrococci and streptococci. These selective agents were used to formulate MVJ
on which L. monocytogenes colonies appear entirely black (reduction of tellurite) on a
red background (inability to use mannitol). Thus suspect Listeria colonies could be readily
identified on MVJ without using oblique illumination. Adding the same three selective
agents to the MMLA of Bearns and Girard [9] resulted in Agricultural Research Service
Modified McBride Listeria Agar (ARS-MMLA) which could be used in conjunction with
oblique lighting to quantitate Listeria in a wide range of dairy and meat products. In a
subsequent study, Buchanan et al. [22] found that MVJ was slightly superior to ARS-
MMLA for recovery of L. monocytogenes from inoculated samples of milk, dairy prod-
ucts, meat, and coleslaw. Although ARS-MMLA was more selective than MVJ, the black
Listeria-like colonies that appeared on MVJ were more readily discernible. Initial compari-
sons of ARS-MMLA and MVJ with LPM Agar indicated that both new media functioned
well. In a follow-up study, Buchanan et al. [21] assessed the ability of MVJ and LPM
Agar to detect Listeria in retail samples of raw meat, fish, and shellfish. Listeria popula-
tions were generally too low to be detected by direct plating on either medium. However,
using USDA LEB I in a three-tube/24-h most probable number (MPN) method, compara-
ble isolation rates were obtained for both MVJ and LPM Agar. The differential capability
of MVJ was again extremely useful in selecting presumptive Listeria colonies.
Oblique Illumination
Except for plating media that contain esculin, xylose, mannitol, or other differential agents,
most formulations of Listeria-selective plating media can be classified into one of two
categories based on presence or absence of blood. Recognition of Listeria-like colonies
on blood-free media such as MMLA, TNSA, and GBNA is greatly facilitated when colo-
Methods to Detect and Isolate L. monocytogenes 237

Mirror

FIGURE1 Oblique illumination technique developed by Henry [73].Angles of re-

flected light (p) and transillumination (a)equal 45" and 135", respectively.

nies are observed under oblique illumination with a binocular scanning microscope. Using
the Henry technique [73] in which plates are examined under obliquely transmitted white
light at an angle of 45" (Fig. I), Listeria colonies are small, round, finely textured, bluish
green to bluish gray with an entire margin. In 1984, Martin et al. [98] compared the
appearance of L. rnonocytogenes on Nalidixic Acid Agar and Tryptone Soya Gum Base
Nalidixic Acid Medium and found that the uniformly transparent nature of the gum-base
medium greatly enhanced the bluish green color of Listeria colonies when observed under
oblique illumination, as described by Henry [73]. Noting that the angle of transmission
in the Henry method is 135O, Lachica [84] found that the bluish green hue of Listeria
colonies was more easily observed if plates were viewed from the backside at an angle
of 45" with a 5 X magnification hand lens while colonies were directly illuminated with
a high-intensity beam of light that traveled perpendicular to the bench surface (Fig. 2). This

View

W!Jso
5x Hand Lens

FIGURE2 Modified Henry technique developed by Lachica [841. Angle of transillumi-

nation (a)equals 135".
238 Donnelly

latter method has eliminated many of the problems (i.e., reproducibility and convenience)
associated with the classical technique developed by Henry [73] nearly 60 years ago.
Given enough experience, either of these two lighting techniques can be used easily to
differentiate probable Listeria colonies from background organisms, even on heavily con-
taminated plates. However, these procedures are time consuming and are not readily adapt-
able for routine use in large testing laboratories.
p-Hemo Iysis
Addition of blood to solid media also can be used to differentiate Listeria, including L.
rnonocytogenes, from other microorganisms. When grown on media containing blood,
such as MLA, L. rnonocytogenes colonies are typically surrounded by a narrow zone of
P-hemolysis. In some instances, P-hemolytic activity is so weak that the clearing zone
cannot be observed until the colony is gently removed from the agar surface. In 1989,
Blanco [ 141 proposed overlaying previously inoculated plates of blood-free Listeria selec-
tive agar with a thin layer of blood agar so that the P-hemolytic activity associated with
pathogenic Listeria could be directly observed after reincubation. According to these au-
thors, hemolysis was more readily observed using this procedure than when blood was
incorporated into plating media before incubation. However, further work using highly
contaminated samples such as raw milk showed that the success of this procedure primarily
depended on selectivity of the initial plating medium, with highly selective media yielding
the best results.
Comparative Evaluation of Direct Plating Media for
Recovery of Listeria from Foods
The need for reliable media in routine food analysis precipitated several studies to identify
the most suitable direct plating media. Golden et al. [57], Hao et al. [66], and Cassiday
et al. [28] collectively compared 20 selective plating media for their ability to recover
uninjured cells of L. rnonocytogenes from samples of pasteurized milk, Brie cheese, ice
cream mix, raw cabbage, dry cured/country-cured ham, and/or raw oysters inoculated to
contain approximately 102,104,and 106L. monocytogenes CFU/g or mL. Gum Base Nali-
dixic Acid Tryptose Soya Medium (GBNTSM), MLA2, FDA-MMLA, and Modified Des-
pierres Agar (MDA) were consistently superior to nine other media used by Golden et
al. [57] for enumerating all three inoculum levels of Listeria in samples of pasteurized
milk and ice cream mix. Ability to recover low levels of Listeria from both products was
facilitated by the lack of significant levels of non-Listeria contaminants. Five of 14 plating
media used in this study failed to recover L. rnonocytogenes from inoculated samples of
pasteurized milk as well as Brie cheese and were therefore omitted for analysis of ice
cream and raw cabbage. Examination of Brie cheese containing approximately 102and
104 L. rnonocytogenes CFU/g indicated that none of the nine remaining direct plating
media was sufficiently selective to prevent overgrowth of Listeria by molds, yeasts, and
gram-positive cocci. Despite these inherent difficulties in detecting small numbers of Liste-
ria, Modified Rodriguez Isolation Medium 111 (MRIM III), MLA2, FDA-MMLA, and
MDA were judged to be satisfactory when Brie cheese contained 1 1 0 6Listeria CFU/g.
However, subsequent results from the same laboratory [29] indicate that LPM Agar was
superior to these four media for isolating Listeria rnonocytogenes from Brie cheese. With
raw cabbage, enumeration of Listeria was a problem only at the lowest inoculum level
where large populations of microbial contaminants (i.e., gram-positive and gram-negative
rods as well as gram-positive cocci) typically interfered with recovery. At the two higher
Methods to Detect and Isolate L. monocytogenes 239

inoculum levels, L. rnonocytogenes was readily quantitated by direct plating on MDA,

GBNTSM, and MLA2. However, this same investigative team [29] later obtained even
better results using LPM Agar.
One year earlier, Hao et al. [66] successfully recovered L. rnonocytogenes from
inoculated samples of cabbage using GBNA, Doyle and Schoeni Selective Enrichment
Agar (DSSEA), DSSEA + ferric citrate, DSSEA + acriflavine + ferric citrate, Thiocya-
nate Nalidixic Acid Agar (TNAA) + glucose + ferric citra.te, and MLA2, but concluded
that DSSEA + acriflavine + ferric citrate and MLA2 outperformed the other media tested.
When results from the previous three studies are combined, LPM Agar, GBNTSM, MLA2,
FDA-MMLA, and MDA generally emerged as the plating media of choice for detecting
uninjured Listeria in dairy and vegetable products. Overall, these findings agree with those
of at least four other studies [56,72,85,90] in which LPM Agar outperformed other popular
plating media, including FDA-MMLA, RIM 111, and/or MVJ for recovery of L. rnonocyto-
genes from raw milk, ice cream, yogurt, soft cheese, and/or vegetables inoculated with
the pathogen. In addition, Rodriguez et al. [ 1281 found that. Rodriguez Isolation Medium
(RIM) 111 containing 6 rather than 12 g of acriflavine hydrochloride was superior to the
original formulation of MLA for isolating L. rnonocytogenes from artificially contaminated
raw milk and hard cheese. Although the best media for recovering Listeria from dairy
products and vegetables remain to be defined, OXA, MOX, LPM, and PALCAM Agar
appear to be the present plating media of choice in the United States for selective isolation
of Listeria from such products as evidenced by their inclusion in the FDA and USDA
procedures [7 1,74,76,94].
Given the inherent differences that exist between the natural microflora found in
various foods, one can easily surmise that Listeria-selective plating media best suited for
dairy products and vegetables might be somewhat less than ideal for analysis of meat,
poultry, and seafood. Consequently, Cassiday et al. [28] evaluated 10 selective plating
media for their ability to enumerate L. rnonocytogenes in artificially contaminated dry-
and country-cured ham as well as raw oysters. According to their results, MDA, FDA-
MMLA, and LPM Agar recovered approximately equal numbers of uninjured Listeria
from dry-cured ham. However, ease in differentiating L. rnonocytogenes colonies from
those formed by background contaminants led these authors to recommend LPM Agar
for analysis of dry-cured ham. Not surprisingly, LPM Agar also was equal or superior to
three other plating media [i.e., MRIM 111, MVJ, and University of Vermont Agar (UVM)]
that were deemed acceptable for isolating Listeria from country-cured ham. Unlike both
types of ham, high populations of indigenous microflora in rilw oysters greatly complicated
detection of Listeria on virtually all 10 plating media. Although MRIM I11 and MVJ
supported less growth of Listeria than other marginally acceptable plating media, including
MLA2, FIIA-MMLA, and GBNTSM, MRIM I11 and MVJ were somewhat more reliable
for differentiating L. rnonocytogenes from background contaminants. Therefore, these au-
thors hesitantly recommended MRIM I11 and MVJ for examination of raw oysters.
Several less extensive studies also have dealt with the ability of various plating
media to recover Listeria from meat, poultry, and seafood. According to a 1988 report
by Loessner et al. [90], recognition of L. rnonocytogenes in inoculated samples of raw
ground beef and scallops was only possible using LPM Agar. Among the three other
plating media tested, RIM I11 and the original formulation of MLA proved to be insuffi-
ciently selective, whereas MVJ was inhibitory to the L. rnonocytogenes strain tested. Un-
like these findings, Garayzabel and Genigeorgis [56] indicated that LPM Agar and RIM
111 were acceptable for detecting Listeria in raw meat with both media superior to FDA-
240 Donnelly

MMLA. Bailey et al. [5] found that LPM Agar and GBNA fortified with lithium chloride
and moxalactam were both superior to unfortified GBNA and MLA for recovering L.
monocytogenes as well as other Listeria spp. from naturally contaminated raw poultry.
Incubation Conditions
Most plating media used to isolate Listeria are normally incubated aerobically at 30-37C.
Plates containing popular selective media such as LPM Agar or MOX Agar normally are
incubated for 48 h, whereas plates containing pure or near-pure cultures of Listeria on
nonselective media can generally be examined after 24 h. Since growth of L. monocyto-
genes is reportedly enhanced under conditions of reduced oxygen [ 1391, inoculated plates
[38,107,108,131-133,1351 as well as selective enrichment broths [38] have been incubated
under microaerobic conditions (5% 02:10% CO2:85% N2). These latter conditions are
recommended when using PALCAM Agar.

Enrichment Media
Several food-related listeriosis outbreaks during the 1980s emphasized the need for more
sensitive Listeria detection methods. The logical approach was to use some of the previ-
ously described enrichment broths containing selective agents and to incubate samples at
an elevated temperature, generally 30C. In response to numerous requests from the food
industry, several enrichment schemes have been developed that include one or two selec-
tive enrichments.
An outbreak of listeriosis which was epidemiologically linked to consumption of
pasteurized milk [53] led Hayes et al. [68] to develop a two-stage enrichment procedure
for isolating L. rnonocytogenes from raw milk. Primary cold enrichment in ONB2 followed
by secondary enrichment at 35C in ONB2 containing potassium thiocyanate (KSCN) and
nalidixic acid, and plating on GBNA yielded the highest number of positive milk samples.
No statistically significant difference in recovery of Listeria was observed using either
Stuart Transport Medium or selective enrichment broth containing potassium thiocyanate
and nalidixic acid. Although 15 milk samples were positive when plated on GBNA me-
dium as compared with 11 on MLA2 without blood, the difference was not statistically
significant. The authors concluded that primary cold enrichment in ONB2 followed by
secondary selective enrichment at 35C and plating on GBNA medium were most useful
for identifying positive raw milk samples.
Slade and Collins-Thompson [ 1441 developed a somewhat shorter two-stage enrich-
ment procedure to isolate Listeria from foods. Their method was tested using raw milk
inoculated to contain approximately 100 L. monocytogenes CFU/mL. Results showed that
Tryptose Broth was superior to ONB2 as a primary cold enrichment medium. In addition,
diluting milk samples 1 : 10, rather than 1 :5 , increased the number of Listeria isolations
on selective media. The more dilute samples probably maintained a higher pH ( 1 6 ) during
cold enrichment as a result of fewer lactic acid bacteria and less lactose being present,
which in turn led to faster growth and increased detection of Listeria on solid media.
Original MLA without blood was the only medium tested that proved to be useful for
plating primary cold enrichments, since Tryptose Agar and Trypaflavine Nalidixic Acid
Agar were typically overgrown by competing microflora. Favorable results were, however,
obtained using Tryptose Agar after secondary enrichment at 37C. Addition of acriflavine
to Thiocyanate Nalidixic Acid Broth proved beneficial for recovery of L. monocytogenes.
Thus, following 7-14 days of cold enrichment in Tryptose Broth, L. monocytogenes was
Methods to Detect and Isolate L. monocytogenes 24 1

most frequently isolated after plating samples enriched in Thiocyanate Nalidixic Acid
Broth on either MLA+blood or Tryptose Agar.
A shortened enrichment procedure and a two-stage cold/selective enrichment
procedure were developed in Canada by Farber et al. [44] for isolating Listeria spp. from
raw milk. In the shortened enrichment procedure, milk samples underwent primary and
secondary enrichment at 30C as well as primary cold enrichment in two selective media
(FDA Enrichment Broth and University of Vermont Medium [UVM]). Although no single
step within the procedure was completely satisfactory for isolating Listeria from raw milk,
the two steps that were most helpful involved surface plating the primary FDA Enrichment
Broth culture on MLA2+blood after 1 day of incubation ai: 30C and surface plating the
30-day-old cold enriched FDA Enrichment Broth culture (initially incubated 7 days at
30C) on MLA2+blood. Collectively, these steps detected Listeria spp. in 31 of 51
(60.8%) positive raw milk samples. Although I I isolation:; were made after 1 but not 7
days of primary selective enrichment at 30C, 6 isolations were only possible after 7 days
of primary selective enrichment. Thus, incubating the primary selective enrichment at
30C for 7 days before plating on MLA2+blood markedly enhanced recovery of Listeria
from raw milk.
The two-stage cold/warm enrichment method, which was the second of two proce-
dures developed by Farber et al. [44], also detected Listeria spp. in raw milk samples.
Using this procedure, Listeria spp. were isolated from 12 samples that were negative using
the shortened enrichment procedure. Similarly, 10 samples that were positive for Listeria
spp. using the shortened enrichment procedure were negative with the two-stage cold/
warm enrichment method. Thus, when used alone, neither procedure detected Listeria in
all positive samples. Following cold enrichment, similar numbers of samples were positive
for Listeria spp. after enrichment in FDA Enrichment Broth and UVM. However, eight
raw milk samples were only positive after 2 weeks of cold enrichment as compared with
three samples in which Listeria was only detected after 4 weeks of cold enrichment. These
results are similar to those of Doyle and Schoeni [38], who also observed that Listeria
spp. could be more readily isolated from raw milk and soft, surface-ripened cheese [39]
during the first 2 weeks of cold enrichment.
Food-associated outbreaks of listeriosis along with the discovery of L. monocyto-
genes in many European varieties of soft- and smear-ripened cheese prompted two Swiss
investigators, Bannerman and Bille [7], to develop a two-stage selective/cold enrichment
procedure to recover Listeria spp. from cheese and dairy plant surfaces. Their isolation
method is similar to the shortened enrichment procedure just described [44] with the ex-
ception that the secondary selective enrichment step has been eliminated and AC Agar
has been included as an additional selective plating medium. Using this method, Listeria
spp. were isolated from 157 of 1099 (14.3%) cheese and environmental samples. A total
of 99 samples were positive for Listeria using both plating media. Following selective
enrichment, 56 of 99 (57%) and 35 of 99 (35%) samples were positive after surface-plating
enrichment cultures on AC Agar and FDA-MMLA, respectively. Increased selectivity of
AC Agar was presumably responsible for detection of approximately 50% more Listeria
isolates as compared with FDA-MMLA.
Important information concerning presence of Listeria spp. in food and environmen-
tal samples can be gained using the three procedures just described as well as procedures
developed by Hayes et al. [68] and Slade and Collins-Thompson [ 1441; however, the need
for cold enrichment in these procedures increased the length of analysis to 30-40 days.
Hence, although cold enrichment will likely remain an important research tool, the time
242 Donnelly

constraints of this method negate its use in any isolation procedure that is to be adopted
by the food industry as a standard method.
Rodriguez et al. [129] developed a complicated scheme to isolate Listeria from raw
milk which more importantly paved the way for subsequent development of several widely
used enrichment media, including UVM Enrichment Broth [27,37]. Their protocol in-
cluded three noninhibitory collection (primary enrichment) media, three selective (second-
ary) enrichment media, and one selective plating medium, RIM 111, all of which were
previously described by Rodriguez et al. [ 1271. The three selective enrichment media used
in this protocol contained nalidixic acid and trypan blue with or without polymyxin B,
whereas nalidixic acid and acriflavine were used as selective agents in the plating medium.
Milk was added to all three collection media, with Collection Medium B streaked onto
RIM I11 after 7 and 15 days of storage at 4C. Collection Medium A was incubated at
4C for 24 h, subcultured in all three secondary enrichment media, which were incubated
at 22C until a color change occurred, and then samples were streaked onto plates of RIM
11. A portion of Collection Medium A also was diluted in Collection Medium C, which
was streaked on to RIM I11 following 7 and 15 days at 4C. According to these authors,
11 L. monocytogenes isolates were obtained after primary cold enrichment, with Collection
Medium C accounting for 9 of 11 isolations. Although results for Collection Medium C
appear impressive, the increased number of isolations using this medium may have re-
sulted from a more dilute sample, approximately 1 :40 as compared with approximately
1:8 in Collection Media A and B. Under these conditions, Collection Medium C should
have maintained a higher pH during cold enrichment, since fewer lactic acid bacteria
and less lactose were likely present, thereby enhancing the growth environment for L.
monocytogenes. In contrast to cold enrichment, 49 L. monocytogenes isolates were ob-
tained following secondary enrichment at 22C with 16, 32, and 1 colony originating from
Rodriguez Enrichment Media 1, 2, and 3, respectively. Recovery of only one Listeria
isolate using Rodriguez Enrichment Medium 3 is not surprising considering that Collection
Medium A was diluted approximately 1 :68 in Collection Medium C after only 24 h of
enrichment at 4C. Since transfer of the culture after 24 h of cold enrichment provides
little opportunity for appreciable growth of L. monocytogenes, the organism was likely
diluted out of the sample. Overall, primary cold enrichment of milk samples diluted ap-
proximately 1 :8 followed by secondary enrichment in Rodriguez Enrichment Media 1
and 2 at 22C and plating on an isolation medium containing nalidixic acid and acriflavine
provided the best opportunity for detecting L. monocytogenes in raw milk.
UVM Broth
Selective media originally recommended by the FDA [93,96] and USDA [102,103] for
enrichment of food samples containing L. monocytogenes were modifications of media
proposed by Ralovich et al. [126] and Rodriguez et al. [127] as modified by Donnelly
and Baigent (University of Vermont Medium) [37], respectively. Donnelly and Baigent
explored the use of several selective enrichment media to inhibit growth of raw milk
contaminants and select for L. monocytogenes. The most successful medium for this appli-
cation was a modification of Rodriguez Enrichment Medium III [127]. This medium,
designated LEB, by Donnelly and Baigent [37], consisted of proteose peptone (5.0 g/L),
tryptone (5.0 g/L), Lab-Lemco powder (5.0 g/L), yeast extract (5.0 g/L), sodium chloride
(20.0 g/L), disodium phosphate-2-hydrate ( 12.0 g/L), potassium phosphate monobasic
(1.35 g/L), esculin (1.O g/L), nalidixic acid (40 mg/L), and acriflavine HCl (12 mg/L).
McClain and Lee [ 1021 modified this formula to contain 20 mg/L nalidixic acid, and this
Methods to Detect and lsolate L. monocytogenes 243

formulation was known as USDA LEB I. These authors further modified LEB I to contain
25 mg/L acriflavine and used this medium, LEB 11, for secondary enrichment of meat
and poultry samples. USDA-FSIS currently recommends use of UVM Broth (LEB I) for
primary enrichment of meat and poultry samples [27,76].
Fraser Broth
Fraser Broth [54] is a modification of USDA LEB I1 which contains lithium chloride (3.0
g/L) and ferric ammonium citrate (0.5 g/L). This medium reportedly was advantageous
for detecting Listeria spp. in enriched food samples. Since Listeria will turn Fraser Broth
black from esculin hydrolysis within 48 h of incubation [ 191, this broth has now replaced
USDA LE,B I1 in the USDA protocol as the preferred secondary enrichment medium for
meat and poultry samples [76].
In 1986, Doyle and Schoeni [38] used the microaeraphilic nature of L. monocyto-
genes in developing a shortened one-step enrichment procedure to isolate this organism
from milk as well as fecal and biological specimens. In their protocol, the sample was
placed inside an Erlenmeyer flask equipped with a side arm and then diluted 1 :5 in Doyle
and Schoeni Selective Enrichment Broth (DSSEB). Following 24 h of incubation at 37C
in an atmosphere of 5% 0,:10% CO,: 85% N,, a portion of the sample was streaked onto
plates of MLA (original formulation with blood), which were similarly incubated under
microaerobic conditions. Using DSSEB, L. monocytogenes was consistently isolated from
raw milk samples inoculated to contain 10 L. monocytogenes CFU/mL. In addition, about
two and five times as many L. monocytogenes isolates were recovered from fecal and
biological specimens using DSSEB rather than cold enrichment and direct plating, respec-
tively.
Another enrichment procedure, which is partially based on microaerobic incubation,
was developed by Skovgaard and Morgen [143] to isolate Listeria spp. from heavily con-
taminated samples, including feces, silage, minced meat, and poultry. In this two-step
enrichment procedure, microaerobic incubation (24 h/30C/95% air: 5% CO,) of the sam-
ple in USIIA LEB I is followed by aerobic secondary selective enrichment in USDA LEB
11, after which untreated and KOH-treated samples are surface plated on LPM Agar. Using
this isolation scheme, which, with the exception of microaerobic incubation, closely re-
sembles the original USDA procedure, numerous fecal, silage, minced beef, and poultry
samples were positive for Listeria spp., including L. monocytogenes. Based on these re-
sults, the authors concluded that their method was suitable for detecting Listeria in heavily
contaminated materials, including samples of raw ground beef and poultry. Although both
procedures just described decrease the Listeria detection time to approximately 3 days,
incubating enrichment cultures under microaerobic conditions is particularly awkward and
not feasible for large-scale testing programs.
A large listeriosis outbreak in which coleslaw was implicated as the vehicle of infec-
tion prompted Hao et al. [66] to compare various media and methods to detect L. monocy-
togenes in cabbage. Preliminary results clearly demonstrated a need for some type of
enrichment procedure before L. monocytogenes could be isolated from inoculated samples.
After comparing results from various plating and enrichment media, these investigators
proposed a two-step enrichment procedure for isolating L. ,monocytogenes from cabbage.
A cold enrichment period of 14 or 30 days at 5C in ONB2 or Brain Heart Infusion Broth
(BHI) led to increased recovery of Listeria from cabbage following secondary enrichment
(30C/48 h) in FDA Enrichment Broth or ONB2 containing potassium thiocyanate and
nalidixic acid. A comparison of nine selective plating media, both with and without an
244 Donnelly

additional 5 mg of Fe3+/L,led to the recommendation of Modified Doyle/Schoeni Selec-

tive Agar I1 and MLA with glycine anhydride rather than glycine (MLA2) for isolating
L. monocytogenes from cabbage. Both media contained 5% sheep blood, which was bene-
ficial for picking Listeria-like colonies. As was true for the cold enrichment broths, several
popular plating media, including FDA-MMLA and LPM Agar, were not examined in this
study. Although these two plating media have gained widespread acceptance, their efficacy
in isolating L. monocytogenes from cabbage and other vegetables should be determined
before recommending this procedure for use in routine analysis of such products.
Despite repeated efforts toward developing an effective enrichment medium for re-
covery of L. monocytogenes, no one single selective enrichment broth has proven to be
totally reliable for analysis of food products containing Listeria. Nevertheless, several
enrichment broths have moved to the forefront, including the FDA Enrichment Broth [74],
UVM Broth [76], and Fraser Broth [76], all of which are commercially available from
BBL or Difco Laboratories. Truscott and McNab [ 1471 developed a selective enrichment
medium called Listeria Test Broth (LTB) as an alternative to UVM Broth for detecting
L. monocytogenes in meat products. After primary and/or secondary enrichment of 50
frozen ground beef samples in both enrichment broths, L. monocytogenes was detected
in 19 of 50 (38%) and 16 of 50 (32%) samples using UVM and LTB, respectively. Al-
though Listeria recovery rates for these two broths are not appreciably different, neither
medium alone was able to detect the pathogen in all 29 samples that were positive. In
addition, L-PALCAMY Broth, which was developed by van Netten et al. [ 1091, has shown
superior results to USDA LEBs I and I1 as well as the Tryptose Broth-based antibiotic
medium of Beckers et al. [ 101 for detecting L. monocytogenes in naturally contaminated
cheese, minced meat, fermented sausage, raw chicken, and mushrooms. However, given
wide variations in both the type and number of naturally occurring microbial contaminants
in our food supply, development of a single enrichment broth for truly optimal recovery
of Listeria from all types of food appears unlikely.

OFFICIAL METHODS FOR ISOLATING

L. MONOCYTOGENES FROM FOOD
Heightened worldwide interest in foodborne listeriosis coupled with the advent of manda-
tory HACCP programs for meat and seafood products in the United States has led to
development of more reliable commercial screening methods for Listeria. Two protocols
developed in the United States by the FDA and USDA-FSIS have emerged as standard
methods to isolate L. monocytogenes from dairy foods, seafoods, vegetables and meat
and poultry products, respectively. Despite widespread use of these methods in the United
States, Canada, and Western Europe, both procedures are still plagued with difficulties
that include the inability to isolate Listeria from all positive samples as well as difficulties
in recovering injured cells. In response to these concerns, the USDA-FSIS and FDA proto-
cols have been modified to enhance recovery of injured Listeria. Working in cooperation
with the International Dairy Federation (IDF), other official European agencies have devel-
oped somewhat similar protocols which are partially based on current FDA methodology.
In this section, positive and negative aspects of the most widely used Listeria testing
protocols will be discussed, along with identification of some of the most critical steps
involved in isolating L. monocytogenes from different foods.
Methods to Detect and Isolate L. monocytogenes 245

FDA Method
The FDA method, originally developed by Lovett et al. 33,961, is the most frequently
used procedure in the United States for detecting L. monocytogenes in milk, milk products
(particularly ice cream and cheese), seafood, vegetables, and food processing environ-
ments. The original protocol [93] has been modified as shown in Figure 3 [74]. The enrich-
ment medium (LEB M52 [74]) consists of TSBYE supplemented with monopotassium
phosphate (anhydrous) 1.35 g/L; disodium phosphate (anhydrous) 9.6 g/L; and pyruvic
acid (sodium salt 10% w/v aqueous solution) 11.1 ml/L. A 25-mL liquid or 25-g solid
sample is added to 225 mL of LEB without selective agents, mixed, and incubated at
30C for 4 h. Following addition of selective agents (acriflavine HCl 10 mg/L; nalidixic
acid sodium salt 40 mg/L; cycloheximide 50 mg/L), the sample is incubated an additional
44 h at 30C for a total incubation period of 48 h. LEB was modified by increasing its
buffering capacity, thereby positioning this medium to be used in conjunction with DNA
probe and other rapid methods which are less sensitive than conventional cultural methods.
In a further modification for nondairy foods, the acriflavine concentration was reduced
from 15 to 10 mg/L so as to conform to that used for milk; and dairy products. After 24
and 48 h, LEB cultures are streaked onto OXA [34] and LPM [87] Agar prepared either
with or without esculin/Fe3+.PALCAM [ 1091 agar may be used in place of LPM agar. This

I 1
Add 25g or 25 ml sample
to 225 ml LEB
I 1

Stomach or blend
6 Incubate 4h at 30C

Add selective agents

acriflavine, nalidixic acid and cycloheximide
A

II 20h U 44h
30C 30C

Streak to Streak to

F a n d LPM I
I I

35C 1 U 30C II 35C

24,48h 24,4811 24,48h

L
Examine for Listeria-like
colonies

FIGURE3 FDA procedure for isolating I_. monocytogenesfrom foods. (From Ref. 74.)
246 Donnelly

substitution brings the FDA method in closer alliance with other protocols used outside the
United States and decreases reliance on Henrys oblique illumination technique. OXA and
PALCAM plates are incubated (with optional use of a C02-air atmosphere) at 35C for
24-48 h, with LPM plates being incubated at 30C for 24-48 h. LPM plates can be viewed
using Henry illumination, or alternatively esculin and ferric iron salt may be added to
LPM to obtain Listeria colonies with black halos as also appear on OXA and PALCAM.
It is recommended that five or more typical colonies be picked from OXA and
PALCAM or LPM and transferred to TSAYE for confirmation. The selection of five colo-
nies increases the likelihood that multiple species of Listeria, if present, will be identified.
TSAYE plates are incubated at 30C for 24-48 h or 35C if colonies are not being used
for wet mount motility confirmation. Purified isolates are subjected to a series of standard
biochemical tests, with a total of 10-1 1 days being required to isolate and confirm the
presence of Listeria in food samples via the FDA procedure.
Present versions of the FDA procedure have greatly shortened and simplified the
isolation of Listeria spp. from many foods as compared with earlier methods that were
developed to detect the pathogen in clinical specimens, with revised procedures affording
many improvements over the original FDA protocol. In 1987, Doyle and Schoeni [39]
compared the original FDA classic cold enrichment and shortened enrichment procedures
for their ability to recover L. rnonocytogenes from 90 samples of commercially produced,
soft, surface-ripened cheese that was previously identified as likely to contain L. rnonocyto-
genes. Although L. rnonocytogenes was isolated from 41 of 90 (46%) cheeses, no single
procedure detected the pathogen in all positive samples. A total of 21 samples were posi-
tive after cold enrichment as compared with only 16 and 13 samples that were positive
using the FDA and shortened enrichment procedures, respectively. Thus, the latter two
protocols failed to recover L. rnonocytogenes from 5 of 21 (23.8%) and 8 of 21 (38.1%)
samples that were positive following cold enrichment. Furthermore, since Listeria was
never isolated from the same positive sample by all three protocols, it appears that the
original FDA method was inferior to cold enrichment. Similar results were obtained by
Doyle et al. [40] when these same three enrichment procedures were used to isolate L.
rnonocytogenes from raw milk samples after HTST pasteurization. Researchers in Canada
[45] and England [ 1181 found negligible differences between numbers of Listeria recov-
ered from naturally contaminated samples of raw milk and soft cheeses analyzed by the
FDA and cold enrichment procedures, although both methods again failed to detect Liste-
ria in all positive samples. These variable findings for the original FDA and cold enrich-
ment procedures have been attributed to nonuniform distribution of Listeria within sam-
ples. However, Doyle and Schoeni [39,40] found cold enrichment superior to the FDA
method for analysis of soft, surface-ripened cheese, where nonuniform distribution of
Listeria is expected, as well as in pasteurized milk. Hence, variations in the ability of the
FDA and cold enrichment procedures to detect Listeria in dairy products probably result
from inherent differences between the two methods (media, incubation conditions) and/
or the presence of microbial competitors rather than nonuniform distribution of Listeria
in the product. Although these results indicate that cold enrichment was generally superior
to the original FDA protocol, the time-consuming nature of cold enrichment makes this
procedure unacceptable as a commercial screening method for L. rnonocytogenes.
International Dairy Federation Method
Using the original FDA method as a starting point, the IDF initiated development of a
reference method in 1988 [146] to recover L. rnonocytogenes from dairy products.
Methods to Detect and lsolate L. monocytogenes 24 7

Development of the IDF method essentially followed that of the FDA protocol as previ-
ously reviewed by Ryser and Marth [ 1361 with the eventual elimination of both preenrich-
ment for detecting sublethally injured Lister-ier and the KOH treatment of the enrichment
broths before plating on Listc.r-icr-selective media. The present IDF method [4a] received
AOAC approval in I993 based on results from an AOAC collaborative study [ 1474 which
assessed the ability of this method to recover L. nzorzoc~toSeizL.sfrom inoculated samples
of raw milk. ice cream, Camembert cheese. Limburger cheese, and skim milk powder.
The AOAC-approved IDF method (Fig. 4) closely resembles the FDA protocol (see
Fig. 3) with the sample enriched i n IDF enrichment broth which contains the same concen-
trations of selective agents found in LEB. Following 48 h of incubation at 30C enrich-
ments are plated on Oxford Agar as opposed to the FDA procedure which calls for Oxford
Agar and either LPM without esculin/Fe or PALCAM. This method which requires a
minimum of 4 days to obtain presumptive results continues to be popular among Europe-
ans for detecting Lister-icr in dairy products.
USDA-FSIS Method
The USDA-FSIS devised a method for detecting L. i,zonoc~itoserie.sin meat and poultry
products (Fig. 5 ) 1761. The original USDA protocol developed in 1986 by Lee and McClain
[87,103] differs from both the original and revised FDA procedures in that both primary
and secondary enrichment steps are included for detecting Listrr-ici. The original USDA
procedure enabled Lister-iir detection within 3 days compared with 9- 1 1 or 5-6 days using
the original and revised FDA methods, respectively. The original USDA procedure was
revised in May of 1989 [27] and differs from the original method in that (a) LEB I1 has
been replaced by Fraser Broth [ 541 as the secondary enrichment medium; (b) LPM Agar
has been replaced by MOX; and ( c ) the regulatory sample size has been increased to 25
g. Fraser Broth and Modified Oxford Agar will both blacken during incubation, because
Lister-io spp. and other contaminants can hydrolyze esculin, with colonies of Listerici ex-
hibiting black halos on Modified Oxford Agar following 24-48 h of incubation. However.

FIGURE4 IDF procedure for isolating L. rnonocytogenes from milk and dairy prod-
ucts. (From Ref. 4a.)
248 Donnelly

Add 25g Meat sample

to 225 ml UVM Broth
stomach 2 min.

U Incubate at 30C
for 20, 2411

0 1 ml + I0 ml Fraser Broth 1
U 26 f 2h
35C

I Streak to MOX

U 35C
24, 4811 U 48h
35C

Examine for -+ ifnegative +

black colonies
Streak to MOX

U 35C 24, 48h

Examine for
black colonies

FIGURE5 USDA procedure for isolating L. monocytogenes from meat and poultry
products. (From Ref. 76.)

MOX is more selective than LPM or Oxford Agar [34], with staphylococci and strepto-
cocci both generally unable to grow on MOX.
Reported inadequacies in the prior [27] USDA procedure were related to the use of
Fraser broth for secondary enrichment. False-negative results caused by reliance on Fraser
broth darkening and a 24-h secondary enrichment have been reported by several labora-
tories [6,82]. Kornacki et al. 1821 compared recovery of L. monocytogenes from Fraser
broth incubated for 26 versus 48 h. L. monocytogenes was isolated from 60 of 1088 meat
product and environmental swab samples from meat and dairy plants. False-negative rates
as high as 6.7% were attributed to the inability of L. monocytogenes to be detected in
Fraser broth at 26 h but not at 48 hours, and to the failure of Fraser broth to blacken.
Furthermore, investigators failed to detect L. monocytogenes in eight Fraser broth enrich-
ments that were positive by primary enrichment. These findings clearly stress the impor-
tance of incubating Fraser broth enrichments for 48 h.
The USDA-FSIS has therefore recommended several modifications. All Fraser broth
enrichment cultures should be streaked following 24-26 h of incubation regardless of
color. Once cultures have been streaked to MOX, Fraser broth cultures should be reincu-
bated at 35C for an additional 24 h. MOX plates streaked from 24- to 26-h Fraser broth
enrichment cultures should be examined for the presence of Listeria-like colonies. If pres-
ent, isolation should proceed. If absent, a second MOX plate should be streaked from the
48-hr Fraser broth enrichment culture. Ferron and Michard [48] compared the FDA and
Methods to Detect and Isolate L. monocytogenes 249

USDA enrichment procedures using 300 pastry samples supplied by 100 different suppli-
ers in western France. The USDA procedure was deemed superior, detecting 69% of all
positive samples compared with the FDA procedure which detected only 34%.
Netherlands Government Food Inspection Service
Using the Netherlands Government Food Inspection Service (NGFIS) protocol, food sam-
ples are enriched in L-PALCAMY enrichment broth for 48 h at 30C. After 24 and 48
h, 0.1 mL of L-PALCAMY enrichment broth is plated onto PALCAM Agar. Plates are
incubated at 30C for 48 h under microaerophilic conditions (5% oxygen, 7.5% carbon
dioxide, 7.5% hydrogen, and 80% nitrogen) [ 1091, after which presumptive Listeria colo-
nies are black and surrounded by a dense black hole from txulin hydrolysis.
Lund et al. [97] examined 300 raw milk samples for the presence of Listeria using
three primary enrichment media. A total of 84 positive sarnples were identified by one
or more of these media. PALCAMY was the most effective medium, identifying 50 of
84 positive samples, followed by UVM and LEB, which identified 46 and 42 Listeria-
positive samples, respectively. Given that the best of these primary enrichment broths
identified only 50 of 84 (59.5%) Listeria-positive samples, the use of two or more primary
enrichment broths identified an additional 34 samples and increased the overall incidence
of Listeria by almost 41%. These results once again highlight the inadequacy of relying
on a single primary enrichment broth for Listeria detection.
Noah et al. [ 1 101 evaluated the impact of more than one test procedure on recovery
of Listeria species from naturally contaminated seafood and seafood products. A total of
21 1 samples were evaluated using five different protocols. The FDA procedure [95] was
used as a control against which the efficacy of the other procedures was evaluated. A total
of 60 samples were identified as Listeria-positive by at least one of the procedures. Of
these samples, the FDA procedure missed seven samples which were subsequently found
to harbor Lzsteria via other procedures. The overall incidence of Listeria increased I 1.7%
using more than one testing procedure.
Hayes et al. [69] assessed the USDA-FSIS and cold enrichment procedures for re-
covery of 1,. monocytogenes from suspect food samples. Both procedures identified L.
monocytogenes in 28 of 51 positive samples. The USDA-FSIS procedure identified 21
samples missed by cold enrichment, whereas the cold enrichment procedure identified an
additional 2 samples that the USDA-FSIS procedure missed. Three enrichment methods
were also compared by Hayes et al. [70] during an examination of foods obtained from
the refrigerators of patients with active clinical cases of listeriosis. A total of 2229 food
samples were examined in this study, of which 11% were positive for L. monocytogenes.
Overall, the USDA-FSIS [27], FDA [95], and NGFIS [ 1091 methods were not statistically
different in their ability to isolate Listeria from 899 samples included in the comparative
evaluation. The FDA procedure [95] identified 65% of all L. monocytogenes-positive
foods, whereas the USDA-FSIS and NGFIS procedures detected L. monocytogenes in
74% of foods shown to be positive. Although none of these widely used Listeria detection
methods proved to be highly sensitive when used independently, use of any two methods
improved detectability from 65 to 74% (for individual protoco'ls)to 87-9 1% for combined
protocols.

CONSIDERATIONS FOR RECOVERY OF INJURED L/STR/A

Most conventional and rapid detection procedures for Listeria use highly selective enrich-
ment media to facilitate growth over competitive background flora. However, these highly
250 Donnelly

selective enrichment procedures will not generally recover sublethally injured Listeria
which could exist in various heated, frozen, or acidified foods; or within heated, frozen,
and sanitized areas of food processing facilities. Sublethal injury of Listeria as a result
of heating, freezing, drying, irradiation, or exposure to chemicals (i.e., sanitizers, preserva-
tives, acids) is well documented [ 1,12,24,25,26,28,31,33,56,58,59,101,130,132,138].Un-
der ideal conditions, such injury is reversible with Listeria being capable of repairing
sublethal damage in foods. Repair of heat-injured L. monocytogenes has been reported in
whole and 2% milk stored at 4C [105].
Several investigators have attempted to improve the sensitivity of current detection
systems by focusing on recovery of injured Listeria that may be present in food products
and food processing environments. All current detection procedures, with the exception
of cold enrichment, involve selective enrichment and/or selective plating. Cold enrichment
is not feasible for routine testing, since several months of incubation may be necessary
to obtain positive results. By failing to consider recovery of injured Listeria, current meth-
odologies underestimate the true incidence of this organism. Several previous studies have
reported on the ability of commonly used plating media to recover injured Listeria. Among
the most commonly used selective agents examined, phenylethanol, acriflavine, poly-
myxin, and sodium chloride were found to inhibit recovery of both thermally stressed and
nonstressed Listeria [31,86,145,148,149,151].Furthermore, when examined for ability to
recover quantitatively thermally stressed Listeria, LEB agar, modified McBride's Agar
(MMA), LPM Agar [87], and FDA enrichment broth agar showed significantly impaired
recovery [221.
Warburton et al. [ 1501 examined the ability of the modified FDA and USDA meth-
ods to recover stressed cells and low levels of L. monocytogenes in food and environmental
samples. Although the modified FDA and USDA methods were comparable in their abili-
ties to isolate stressed and low level populations of L. monocytogenes, these authors failed
to assess the extent of injury within bacterial populations following exposure to sublethal
stress. The percentage of injury existing within a population of bacterial cells can pro-
foundly affect comparative results of media performance. Thus, it is difficult to determine
whether valid conclusions can be drawn from such studies.
Busch and Donnelly [26] developed an enrichment medium capable of resuscitating
heat-injured Listeria. This medium, Listeria Repair Broth (LRB), permits complete repair
of injured Listeria within 5 h at 37C after which various selective agents can be added
to inhibit the growth of competing microflora upon continued incubation. In studies com-
paring the efficacy of LRB in promoting repair/enrichment of heat-injured Listeria with
that of existing selective enrichment media, repair was not observed in FDA enrichment
broth [95],phosphate-buffered Listeria Enrichment Broth (PEB; Gene-Trak Systems, Fra-
mingham, MA), or UVM Enrichment Broth [ 1031. Final Listeria populations in selective
enrichment media after 24 h of incubation at 30C were 1.7 X 108to 9.1 X 10' CFU/
mL compared with populations in LRB which consistently averaged 2.5 X loll to 8.2 X
10'' CFU/mL [26].
Studies with LRB were extended to examine the potential for repair of freeze-injured
and sanitizer-injured L. monocytogenes [50,138]. Although variation in susceptibility of L.
monocytogenes to freeze injury was recorded, in general, L. monocytogenes is not severely
injured by freezing [43,58,114]. Percentage of injury ranged from only 40 to 60% after
Listeria populations were frozen at -9" to - 11"C for 24 h [50].As storage time increased,
an increase in percentage of injury increased to a maximum of only 70-80%. To examine
reversibility of freeze injury, low-level populations of freeze-injured L. monocytogenes
Methods to Detect and lsolafe L. monocytogenes 251

cells were added to UVM Enrichment Broth, FDA Enrichment Broth, and LRB. Repair
of freeze-injured populations occurred quickly, probably because of the low initial degree
of injury, with the pathogen again attaining high populations in LRB.
Sallam and Donnelly [ 1381 examined the ability of four commonly used dairy plant
sanitizers tc) induce injury in L. monocytogenes when exposed to sublethal concentrations.
UVM broth failed to support growth of sanitizer-injured cells, whereas LRB permitted
their recovery. Flanders et al. [52] examined the efficacy of using a repair step to increase
recovery of injured Listeria from environmental sponge samples obtained from dairy pro-
cessing plant environments. The USDA-FSIS Listeria isolation protocol using UVM mod-
ified Listeria Enrichment Broth was compared with a modified USDA-FSIS format which
utilized LRB as the primary enrichment medium. UVM and LRB broths also were used in
conjunction with a rapid DNA hybridization (Gene-Trak) and ELISA (Organon Teknika,
Durham, NC) assay. Of 80 sites positive by any method, UVM and LRB showed similar
recovery rates (87.5 and 88.8%, respectively). However, combining the cultural methods
with either rapid method for each broth increased detection to 97.5-98.8% [52]. Flanders
et al. [51] also evaluate the abilities of LRB, LRB containing ceftazidime (LRBC), and
UVM to enhance recovery of Listeria from dairy plant environmental samples. Although
no single broth could detect all Listeria-positive sites, LRBC identified 67 of 89 positive
sites (75.3%), and LRB and UVM each detected 60 of 90 positive sites (66.7%). Combin-
ing results from any two broths increased recovery from 66.7 to 75.3% to 82.2-94.4%.
The combination of LRBC and UVM detected 94.4% of positive samples, whereas LRBS
and LRBC identified 91.1% of positive samples. Pritchard et al. [121] also compared the
ability of UVM, LRB, and LRBC to isolate Listeria from dairy plant environments. Of
80 positive samples identified, 54 samples came from UVM medium, 56 were from LRB,
and 57 came from LRBC. A total of 26 samples (32.5% of positive samples) were identi-
fied by either LRB or LRBC but not by UVM media. Combining UVM with either LRB
or LRBC again substantially increased the number of positive samples identified. When
results from UVM and LRB are combined, 65 to 80 (8 1.3%) positive samples were identi-
fied. Using both UVM and LRBC, 74 of 80 (92.5%) positive samples were identified.
Despite the improved recoveries obtained by combining medja, these results illustrate the
severe limitations associated with the current regulatory procedures used to assure absence
of Listeria in foods and food processing environments.
Ryser et al. [ 1371 evaluated the ability of UVM and LRB to recover different strain-
specific ribotypes of L. monocytugenes from meat and poultry products. Forty-five paired
25-g retail samples of ground beef, pork sausage, ground turkey, and chicken underwent
primary enrichment in UVM and LRB (30C/24 h) followed by secondary enrichment in
Fraser Broth (35"C/24 h) and plating on modified Oxford Agar. A 3-h nonselective enrich-
ment period at 30C was used with LRB to allow repair of injured Listeria before adding
selective agents. Listeria spp. were detected in 73.8% and 69.4% of the 180 meat and
poultry samples tested using LRB and UVM, respectively. Although these differences
were not statistically significant, combining UVM and LRB results increased overall Liste-
ria recovery rates to 83.3%. Thus, enrichment in LRB for repair of injured cells in conjunc-
tion with the USDA-FSIS method has potential to improve recovery of Listeria from meat
and poultry products.
In the above study, following 24 h of incubation at 35"C, Listeria colonies were
biochemically confirmed and selected isolates were ribotyped using the automated Ribo-
printer Microbial Characterization System, (E.I. du Pont de Nemours and Co., Inc., Wil-
mington, DE). A total of 36 different Listeria strains comprising 16 L. monocytogenes
252 Donnelly

(including 4 known clinical ribotypes), 12 L. innocua, and 8 L. welshirneri ribotypes were

identified from selected positive samples (15 samples of each product type, 2 UVM and
2 LRB isolates/sample). Twenty-six of 36 (13 L. monocytogenes) ribotypes were detected
using both UVM and LRB; whereas 3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L.
monocytogenes) Listeria ribotypes were observed using only UVM or LRB, respectively.
Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocytogenes),
21 (12 L. monocytogenes),20 (9 L. monocytogenes), and 19 (1 1 L. monocytogenes) differ-
ent Listeria ribotypes, respectively, with some Listeria ribotypes being confined to a partic-
ular product. More importantly, striking differences in both the number and distribution
of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes
of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples
of each product were examined. When a third set of six samples per product type was
examined from which two Listeria isolates were obtained using only one of the two pri-
mary enrichment media, UVM and LRB failed to detect L. monocytogenes (both clinical
and nonclinical ribotypes) in two and four samples, respectively (Table 2). These findings
stress the complex microbial ecology of Listeria in foods and the limitations of existing
detection procedures fully to characterize the total Listeria population. Furthermore, two
of the L. monocytogenes ribotypes missed using UVM were known clinical ribotypes
which were linked to sporadic and epidemic cases of human listeriosis in England and
Scotland [ 1041. Continuing work [ 1201 on enrichment of dairy environmental samples in
UVM and LRB has shown that combining these two primary enrichment media into a
single tube of Fraser broth for secondary enrichment yields a significantly higher ( P <
.OS) percentage of Listeria-positive samples than when either LRB or UVM are used

TABLE
2 Ribotypes of Listeria spp. Recovered from 10 Samples of Raw Chicken
Following Primary Enrichment in UVM or LRB and Secondary Enrichment
in Fraser Broth

No. of isolates

Ribotype Listeria spp. UVM LRB

1-909-3 L. innocua 0 1
5-418-3 L. monocytogenes 2 0
5-415-4 L. innocua 4 0
5-4 13-2 L. monocytogenes 2 0
2-864-3 L. welshimeri 2 0
1-916-la L. monocytogenes 3 3
5-408-1 L. rnonocytogenes 2 0
1-909-4 L. innocua 5 5
1-910-7 L. innocua 0 1
5-426- 1 L. innocua 0 1
1-923-1a L. monocytogenes 0 3
5-408-4 L. monocytogenes 0 2
1-907- 1a L. monocytogenes 0 1
1-919-2 L. monocytogenes 0 1
1-864-7 L. monocytogenes 0 1
1-915-7 L. monocytogeries 0 I
Recognized clinical ribotypes associated with known epidemic or sporadic cases of human listeriosis.
Source: Adapted from Ref. 137.
Methods to Detect and lsolate L. monocytogenes 253

alone. These findings, combined with reports of L. innocm being able to outgrow L.
monocytogenes in UVM (and Fraser Broth) [32,119] suggest that different ribotypes of
L. monocytogenes may vary somewhat in nutritional requirements or their ability to com-
pete with other ribotypes of L. monocytogenes and/or other Listeria spp. Refinement of
existing Listeria recovery methods should consider the nutritional needs associated with
those specific genetic types widely distributed in foods.
Roth and Donnelly [ 1301 assessed survival of acid-injured L. rnonocytogenes in four
different acidic foods and also examined the efficacy of LRB and UVM to recover acid-
injured Lisreria from such foods. L. monocytogenes was injured in lactic (pH 3.0) and
acetic (pH 3.5) acids. Two levels of injury were produced anld monitored; one population
with 99.999b injury and the second with approximately 95% injury. The four acidic food
systems studied at 4 and 30C included fresh apple cider (pH 3.3), plain non-fat yogurt
(pH 4.2), fresh coleslaw (pH 4.4), and fresh salsa (pH 3.9). Acid-injured Listeria was
added to each acidic food and monitored by selective and nonselective plating. Simulta-
neously, sainples were enriched in both LRB and UVM followed by standard isolation/
identification procedures with survival of healthy L. monncytogenes also monitored. Al-
though acid-injured cells failed to repair in the acidic foods tested, the pathogen did survive
for more than a week. Storage temperatures did affect the survival rate of acid-injured cells
in that 4C storage was bacteriostatic and 30C was bacteriocidal. Parameters involved in
survival of acid-injured Listeria include the degree to which the bacterial population is
injured (percentage of injury), storage temperature, and the pH of the food. At time points
where differences were detected, LRB proved to be superior (22 of 54) in its ability to
detect injured Listeriu compared with UVM (3/54). Hence, use of LRB is recommended
when examining acidic foods for L. monocytogenes.

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148. Warburton, D.W., J.M. Farber, A. Armstrong, R. Caldeira, T. Hunt, S. Messier, R. Plante,
N.P. Tiwari, and J. Vinet. 1991. A comparative study of the FDA and USDA methods
for the detection of Listeria monocytogenes in foods. Int. J. Food Microbiol. 13:lOS-118.
149. Warburton, D.W., J.M. Farber, A. Armstrong, R. Caldeira, N.P. Tiwari, T. Babiuk, P. Lacasse
and S. Read. 1991. A Canadian comparative study of modified versions of the FDA and
USDA methods for the detection of Listeria monoc-ytogenes. J. Food Prot. 54:669-676.
150. Warburton, D.W., J.M. Farber, C. Powell, N.P. Tiwari, S. Read, R. Plante, T. Babiuk, P.
Laffey, T. Kauri, P. Mayers, M.-J. Champagne, T. Hunt, P. LaCasse, K. Viet, R. Smando,
and F. Coates. 1992. Comparison of methods for optimum detection of stressed and low
levels of Listeria monocytogenes. Food Microbiol. 9: 127- 145.
151. Werner, B.S., and D.V. Lim. 1990. Growth of Listeria monocytogenes in different media.
Abstr. Ann. Mtg, Amer. Soc. Microbiol., Anaheim, CA. May 13-19, Abstr. P-41.
152. Yousef, A.E., and E.H. Marth. 1988. Behavior of Listeria monocytogenes during manufacture
and storage of Colby cheese. J. Food Prot. 5 1 :12- 15.
Rapid Methods for Detection of
Listeria

CARLA.BATT
Cornell University, Ithaca, N e w York

INTRODUCTION
Presence of Listeria monocytogenes in food products is a safety problem that warrants
attention and improvements in detection and tracking. Although normal, healthy adults
are primarily unaffected by this pathogen, infants and immunocompromised persons are
at far greater risk [38]. The traditional techniques developed :sincethe 1980s for detecting
and enumerating L. monocytogenes are not sufficiently rapid to assure the safety of perish-
able food products before consumption. Regulations limiting contamination of ready-to-
eat foods to a zero-tolerance have been the driving force behind development of rapid
tests, prompting an intense effort in both commercial and academic laboratories. These
techniques are only useful as a survey tool and as a method to track an alleged foodborne
outbreak. The need to develop quicker and more precise methods for detecting Listeria
is also a function of the similarity between L. monocytogenes and other members of the
Listeria genus. Distribution of a ready-to-eat food containing L. monocytogenes typically
leads to a class I recall. This chapter is an attempt to review objectively most of the
literature on the subject published to date with emphasis on experiences from my labora-
tory. My group has explored many different formats to detect L. monocytogenes, and over
the years, several different rapid methods have been assessed. We have focused on L.
monocytogenes because of its significance to humans (1 400 cases occurred per year during
the late 1980s [38]) and its usefulness in models for development of rapid methods.

261
262 Batt

Microbiology-Based Methods
Classic microbiology-based methods for detecting and enumerating Listeria involve en-
richment in selective media, which may include incubation at refrigeration temperatures
[31,49]. Selective enrichment media which allow only Listeria to grow have also been
developed. The wide range of Listeria-selective plating media currently available is daunt-
ing. Even though several comparative studies have been reported, no single detection
scheme appears to be so vastly superior as to be adopted universally [ 141. Recovery of
injured Listeria cells has emerged as another important issue. Sublethal thermal processing
in addition to other intrinsic and/or extrinsic factors can injure Listeria. Although injury
is not a new phenomenon, the potential significance of injured Listeria in foods deserves
greater consideration in the formulation of enrichment/recovery media.

RAPID METHODS FOR DETECTION OF

L. MONOCYTOGENES
Antibody- (monoclonal or polyclonal) and nucleic acid probe-based systems, the latter
alone or in conjunction with amplification, have been developed to detect both L. monocy-
togenes and Listeria spp. Determining whether to use nucleic acid probes or antibodies
to detect pathogenic microorganisms is partly a matter of personal preference, with factors
such as simplicity, cost, speed, and sensitivity also being of importance. Amplification-
based methods (most notably polymerase chain reaction, PCR) have superior sensitivity
as compared with standard nucleic acid probes or immunoassays. However, PCR is some-
what more complicated in terms of setup and operation (Fig. 1). Only recently have reagent
additions in PCR been simplified and the process made more amenable to routine use as
seen by the introduction of the BAX system by Qualicon (Wilmington, DE). When coupled
to more direct measures of PCR product accumulation [3], these advances will likely result
in a system suitable for routine testing.

Nucleic Acid-Based Probes

Since 1987, nucleic acid probes have become a viable tool to detect viruses, bacteria, and
other microorganisms in food, clinical, and environmental samples. Target sequences that
can be used include (a) ribosomal RNA, (b) mitochondrial DNA, (c) plasmid DNA, and
(d) chromosomal DNA.
The key criterion for selection of any target nucleic acid is that its presence defines
the organism in question with little or no probability of existing in another microorganism
that might be found in the same ecological niche. There is no way of ensuring that a
targeted nucleic acid sequence will be found only in the microorganism for which the
detection system is being developed. This is especially true where the sequence is cryptic
and chosen simply because of its uniqueness within a selected test population. In cases
where a specific toxin gene sequence is selected, there is an assumption that it will not
be widely distributed in nature.
The use of 16s rRNA as a distinct signature for a bacterium has become a universal
method when no other obvious nucleic acid sequence uniquely defines the desired target
[80]. Databases of 16s rRNA sequences covering a wide diversity of microorganisms can
be searched to identify regions that are characteristic of the targeted microorganism. A
DNA probe based on the sequence for 16s rRNA which can detect all Listeria spp. [49,50]
Rapid Methods for Detection of Listeria 263

Step 1. Annealing
I), Primer
Template

-
Step 2. Extension

Step 3. Cycle
0-
__t_

7 -0

FIGURE1 Polymerase chain reaction.

has been developed by Gene Trak Inc. (Framingham, MA). Although the exact sequence
is proprietary, it is clearly derived from one of the variable regions of the 16s rRNA. A
novel solution hybridization assay has been formatted where final quantification is accom-
plished using an enzymatic marker [48]. Briefly, 16s rRNA is released by alkaline lysis
from cells grown in an enrichment broth. Then a capture tag consisting of the complemen-
tary sequence to a unique region of 16s rRNA and a poly-A (polyadenylic acid) tail is
allowed to hybridize to the target 16s rRNA. This hybrid is then removed from solution
through the poly A tail using a poly-T (polythymidylic acid) s,equencethat has been immo-
bilized on a polystyrene solid support (Fig. 2). Detection is accomplished using an anti-
body coupled to horseradish peroxidase and directed against a fluorescein marker cova-
lently linked to the detector probe. The detector probe recognizes sequences in 16s rRNA
as spatially distinct from the region recognized by the capturleprobe. Therefore, oxidation
of a substrate (tetramethyl benzidine) in the presence of hydrogen-peroxide by horseradish
peroxidase indicates the presence of Listeria. A more recent refinement of this approach
uses a 16s rRNA probe that is specific for L. monocytogems [58a]. Unique 16s rRNA
sequences that define L. monocytogenes have been reported [2 11, but achieving specificity
in the assay requires precise temperature control.
Virulence genes are frequent targets for nucleic acid--based probe methods, since
these genes are essential for pathogenicity and are typically conserved among a given
species. A probe derived from a putative delayed-type hypersensitivity (DTH) factor iso-
lated from L. monocytogenes 1/2a hybridized to all L. monocytogenes serogroups and L.
ivanovii but not to any other Listeria spp. tested [64]. The exact nature of the DTH gene
-
264 Batt

AAAAAAAAAA

i
FIGURE2 Sandwich hybridization capture assay.

has not yet been reported, and therefore its role in L. rnonocytogenes pathogenicity cannot
be determined. It does, however, appear to be an effective tool for detecting Listeria,
although its species specificity is not absolute for L. rnonocytogenes. For example, the
DTH gene appears to be absent from L. rnonocytogenes serogroup 4a yet present in L.
ivanovii. Thus far, it has only been used as a nucleic acid probe in colony hybridization
assays and the entire 1.1-kb DTH, which contains a fragment labeled with 32P,served as
the probe.
A sequence from what was first believed to be a putative L. rnonocytogenes
a-hemolysin gene [33] was reportedly specific for L. rnonocytogenes [22,24]. However,
subsequent analysis showed that this gene encoded for a major secreted protein (msp)
rather than a hemolysin [34]. Despite its nebulous quality, this sequence has proven to
be useful in developing nucleic acid-based detection systems for Listeria. Initially, a
colony hybridization protocol was used where suspect colonies were transferred to nitro-
cellulose filters and probed with this 32P-labeledfragment. Good specificity was shown
toward Listeria spp. which were P-hemolytic (CAMP-positive). Subsequent refinements
of this approach have included the evaluation of four synthetic 20-bp oligonucleotide
probes in lieu of the entire 500-bp fragment. Two probes which were tested against a
range of Listeria spp. hybridized to all L. monocytogenes isolates and one weakly hemo-
lytic isolate of L. seeligeri [24]. The origin of this probe has been clarified by the reported
cloning and sequencing of an invasion-associated protein (iap) [52].
Pathogenicity of L. rnonocytogenes depends on a number of factors, including the
production of one or more hemolysins. Transposon mutagenesis (Tn916) disrupts the cod-
ing sequence for listeriolysin 0 and renders L. monocytogenes avirulent to mice. The gene
coding for listeriolysin 0 has been cloned [23,54,61] and sequenced [61]. Interestingly,
Rapid Methods for Detection of Listeria 265

when the listeriolysin 0 gene is introduced into Bacillus subtilis, the organism gains the
ability to grow in macrophage-like cells in culture [8].
The listeriolysin 0 gene is presumably unique to L. monocytogenes and therefore
is an obvious target for developing a detection system. It does, however, share some amino
acid homology with other hemolysins, including streptolysin 0 and pneumolysin. The
listeriolysin 0 gene has been used as a probe in Southern hybridization analysis of DNA
purified from several Listeria spp. [ 191. A 610-bp fragment internal to the region coding
for listeriolysin 0 appears to hybridize only with hemolytic strains of L. monocytogenes.
However, under nonstringent conditions, a probe derived from sequences on the 3 of the
listeriolysin 0 gene hybridized with hemolytic strains of L,. ivanovii and L. seeligeri. Al-
though some nucleotide sequence conservation between the hemolysin genes in Listeria
apparently exists, a detailed sequence analysis will be required to determine the exact
extent of homology. Datta et al. [23] used two synthetic oligonucleotide listeriolysin 0
probes in a colony hybridization assay to detect L. monocytogenes and obtained good
specificity [24]. Such listeriolysin probes can likely be adopted to several assay formats
for analyzing food samples.

Nucleic Acid Amplification-Based Methods

The sensitivity of a nucleic acid-based detection system is a function of several parame-
ters, including the number of copies of the target within a single cell. The use of 16s
rRNA has the obvious advantage in that each cell contains over 100 copies which in turn
makes such an assay far more sensitive than an assay based on a single copy target.
As an alternative to using high-copy number target sequences, nucleic acid-based
amplification methods employing the ligase chain reaction (LCR) [78,79], PCR [4,9,15,
32,35,36,39,63,66,72], and most recently nucleic acid sequence-based amplification
(NASBA) [ 12,741 have been reported.
Ligase Chain Reaction
LCR is an amplification method that uses target DNA as a template for ligation of oligonu-
cleotides designed to abut one another. By using a pair of diametrically opposed comple-
mentary oligonucleotides, each ligated pair can serve as a template for subsequent rounds
of amplification (Fig. 3). LCR can be used to discriminate between two target sequences
that differ only in a single nucleotide because of the extreme sensitivity of DNA ligases
to mismatches on the 5 end of the substrate. The strength of LCR therefore lies in its
specificity as compared with PCR, which is more sensitive.
Temperature cycling allows products from one round to dissociate from their target
and then anneal and serve as a template in a subsequent round. Key to the process is the use
of a thermostable DNA ligase which retains activity after being exposed to temperatures
sufficient to dissociate the products [2].
The 16s rRNA which is sufficiently diverse for phylogenetic determinations also
can serve as a target for LCR-based assays. In studies documenting the utility of LCR as
a means to detect L. monocytogenes, 32P-labeledoligonucleotides were used and the ligated
products were detected by autoradiography after electrophoretic separation of the sub-
strates and products [79]. Subsequent improvements included use of nonradioactive labels
and a capture step which obviated the need for electrophoresis [78]. Enhanced sensitivity
was achieved by introducing a preliminary PCR amplification which utilized a common
set of 16s-rRNA primers. This approach is a generic model for developing a PCR-LCR
assay for virtually any microorganism.
266 Batt

FIGURE3 Ligase chain reaction.

Polymerase Chain Reaction

PCR involves the enzymatic amplification of a targeted nucleic acid sequence using a
thermostable DNA polymerase and flanking oligonucleotide primers that uniquely define
the target. The most commonly used DNA polymerase is from Thermus aquaticus and is
termed Taq polymerase. Since amplification is exponential, the target can be amplified
over one million times with respect to other sequences within the cell through cycles of
denaturing, annealing, and extending. The power of PCR prompted its obvious application
for detection of L. monocytogenes.
A wide variety of PCR-based assays have been developed for L. monocytogenes
which target several different genetic sequences. These sequences are largely derived from
virulence genes which are unique to L. monocytogenes and essential for the organisms
pathogenicity. These virulence factors, all of which were previously described in Chap. 5 ,
include (a) listeriolysin (&A), a gene encoding a thiol-dependent hemolysin which is
involved in escape from intracellular vacuoles [ 1,3,13,15,36,46], (b) invasion-associated
protein (icy)[ 16,44,56], (c) phospholipase B @lcB) [20], and (d) DTH [76].
In addition to virulence genes, any other nucleic acid sequences which are unique
to L. monocytogenes can serve as targets for PCR-based assays. Several genes sequences
sufficiently divergent to differentiate species including the ribosomal RNA operon and its
Rapid Methods for Detection of Listeria 267

intergenic regions are likely to be highly conserved among all L. monocytogenes but PCR-
based assays have been employing 16s rRNA 14 I ,44,75,77], the intergenic spacer region
that lies between the I6 and 23 rRNA. (In some instances, the assay was diagnostic only in
the size and restriction pattern of the PCR products providing not definitive identification
[ 26,401.)
Finally, cryptic sequences have been discovered which are unique to L. morzocyto-
genes and use repetitive element sequence-based PCR (rep-PCR) [4S] and subtractive
hybridization [ S I ] . Distribution and conservation of these cryptic sequences within all L.
rnono~yt0geize.sstrains cannot, however, be intuitively deduced and must be proven by
large-scale screening studies.
Formats for PCR-based assays are varied and differ in their complexity as well
as utility. The most comtnon read-out for PCR-based assays is gel electrophoresis
accompanied by ethidium bromide staining, with the presence of a particular PCR product
being diagnostic. The disadvantages of gel electrophoresis are the lack of quantification
and the difficulty in automating post-PCR processing. Alternative means of detecting PCR
products posthybridization include (a) reverse dot-blots. (A labeled PCR product is cap-
tured by an oligonucleotide primer immobilized on a membrane [ IS].), (b) microtiter plate
capture (the labeled PCR product is captured specifically or nonspecifically in the well
of a microtiter plate [ 1 S]), (c) macroporous hydrophobic cloth [ 1 I 1, (d) immunodetection
of RNA:DNA hybrid [ 101, (e) fiberoptic biosensors [73].
A 5 nuclease PCR detection assay was first developed and perfected using L. mono-
cytogenes a\ the target organism (Fig. 4). As a nucleic acid target, listerolysin 0 (hlyA)
was chosen iis the nucleic acid target, because this sequence is unique to L. nzorzocytogenes.
We have previously used this gene as a target for a reverse dot-blot PCR assay [IS].
Although this assay was extremely sensitive, the post-PCR handling steps, including prod-
uct capture and secondary enzyme-conjugate addition, introduced potential problems in
assay throughput and contamination. The latter is of particular concern, since PCR product
contamination through aerosols frequently leads to false-positive results.
Initial work in my laboratory has reliably demonstrated the ability of the 5 nuclease
PCR detection assay to quantify L. monocytogenes in pure culture [3]. The specificity of
the PCR primers and reactions and the parameters that were used in this assay have been
documented and were supported by our data [ IS). Among all Listerici spp., significant
ARQs and amplification products, the latter observed on ethidium bromide-stained agar-
ose gels, were only obtained for L. monocytogerzes. Furthermore, addition of competing
organisms did not affect the assay until the ratio of competing to target organisms exceeded
10.
The 5 nuclease PCR detection assay using the hlyA fluorogenic probe was linear
over a range of 5 X 10 to S X 10 L. monocyfogerzes CFU with SO CFU [3] easily
detected. The yes or no assignment is an accurate scoring method which can be
used for positive and negatijre samples. Non-L. monocytogrnes strains can give a weak
positive signal only when >S X 105copies of the template are present. Even then, the
signal generated is >30 times weaker than the signal obtained from an equivalent number
templates. This assay is now being used as a format to develop
of L. r?zorzoc:~togene.s
methods for detecting L. r,zonocytogsnL.s in dairy, feed, and clinical samples.
Nucleic Acid Sequence-Based Amplification
Nucleic acid sequence-based amplification (NASBA or 3SR [29]) is a system where nu-
cleic acid targets are amplified using a series of enzymes, including a RNA polymerase
268 Batt

I Polymerization I n
foyard
5 prlmer probeU3,
v -
3 5
3
5
+ 5

Q
reyerse
primer

-
Strand displacement
5
(QI
T 3
c
t

3 5
5 3
5
~~

5 3
3 5
5 3

5
3
- w El b
5

5
5 3
5

FIGURE
4 Schematic of 5 nuclease PCR detection. Polymerization is initiated by Ta9
DNA polymerasefrom both the forward and reverse primer extending along the target
strand. Strand displacement occurs when Taq DNA polymerase encounters the
fluorogenic probe and begins to displace it from the target. The probe is labeled with
a reporter (R) and quencher (Q) dye. Cleavage of the fluorogenic probe by Ta9 DNA
polymerase releases the reporter dye. Polymerization is completed when each exten-
sion strand reaches the end of the target.

and a reverse transcriptase (Fig. 5). A target RNA molecule is first reverse transcribed to
cDNA using reverse transcriptase. RNAase H is added to digest the template RNA which
occurs only after hybridization with cDNA. The newly formed cDNA is then used as a
template for a second round of synthesis again using reverse transcriptase. The primer for
this second round carries a T7 promoter as a tail on its 5 end and therefore introduces
this promoter sequence into the second round of synthesized cDNA. At this stage, the T7
promoter containing cDNA is a substrate for RNA synthesis by T7 RNA polymerase.
Copious amounts of T7 RNA polymerase-synthesized RNA are then produced. This in-
crease in RNA can be detected easily by gel electrophoresis or sandwich hybridization,
since amplification is typically on the order of 106fold,
Since NASBA uses RNA templates, it is amenable to detection of L. monocytogenes
with 16s rRNA. Probes specific for L. monocytogenes have been developed [74]. NASBA
assays have used hZyA mRNA as the target with sensitivities as low as 10 CFU/g being
reported [12]. In this latter study, enrichment was used to induce hZyA, a problem in
mRNA based methods where the initial level per cell cannot be predicted.
Problems in Amplification Methods
Two major problems with PCR-based assays (and in general all methods that employ
enzymatic amplification) are false negatives caused by PCR inhibition and false positives
resulting from detection of nonviable cells. The former has been addressed by development
of several template purification methods which range in complexity and utility.
 -
Rapid Methods for Detection of Listeria 269

ss RNA target

Primer Annealing
F
RNA-DNA
1I
Reverse Transcription (RT)
intermediate

............................ RNAse H digestion

Primer Annealing

ds DNA

ss RNA
+I
ds DNA synthesis (RT)

RNA synthesis
(T7 RNA poiymerase)

FIGURE5 Nucleic acid sequence-based amplification.

Sample preparation is a subject of intense interest but few determined efforts. Sev-
eral different approaches to sample preparation have been proposed including:
Target cell capture. In general, the most noted example of cell capture involves use
of immunomagnetic beads to which target-specific antibodies are attached [70].
The beads are used to capture cells from solution and then the recovered cells
are subjected to DNA extraction or culture enrichrnent [35]. L. rnonocytogenes
also has been recovered after centrifugation and washing to remove inhibitory
compounds in milk [20] and other foods [63].
Detergent or solvent extraction. Phenol, chloroform, and ether are examples of sol-
vents that can remove compounds that inhibit PCR [43]. Sodium iodide will gen-
erally solubilize food components and make the isolation of amplifiable DNA
possible [%I. Detergents including Tween 20 also can enhance the sensitivity
of PCR by solubilizing inhibitory compounds [68]. A two-phase solvent extrac-
tion using polyethylene glycol and dextran is reportedly effective for soft cheeses
[%I.
Filtrution. For liquid foods, most notably milk, filtration is a simple means of con-
centrating cells [20,72]. Certain filters are amenable to solvent solubilization
which aids in DNA release.
DNA capture. In addition to cell capture, target DN,4 can be captured after cell
lysis. DNA can be absorbed onto several matrices in a nonspecific manner; that
is, silica [43].
PCR cocktail. Few of the PCR inhibitors are known in specific terms. For L. monocy-
togenes and its detection in milk, calcium is thought to be a PCR inhibitor. Conse-
270 Batt

quently, increasing the amount magnesium in PCR is useful [7]. Addition of bo-
vine serum albumin or proteinase inhibitors might help spare the DNA polymerase
during amplification [65].

Viability has been a frequently cited but still unresolved problem. Amplification of archi-
val DNA from various sources documents the ability of DNA to survive well beyond
the life of the organism [57].Therefore, false positives often arise from samples whose
processing history ensures that all L. monocytogenes are nonviable. Two approaches ad-
dressing viability of target cells in PCR-based assays have been proposed. The first is to
have a mandatory culture enrichment period, where a positive result would require growth
of the target organism. The second approach involves targeting of mRNA rather than
DNA, since mRNA is less stable than DNA and should degrade in a manner that parallels
cell death. Efforts to use mRNA as a template for detecting viable L. monocytogenes have
been reported [42]. The utility of this approach may be limited because of strain differences
in target gene expression which will alter the number of mRNA molecules per cell. Second
is the difference in the history of the contaminating L. monocytogenes strain in the food
before and after processing. Since mRNA destruction is a kinetic process, thermal pro-
cessing and the time between processing and assay will be critical. Our efforts to pursue
mRNA as a target in a single-step PCR 5 nuclease assay have used the hlyA gene as a
target [ 3 2 ] . The thermostable DNA polymerase Tth has both reverse transcriptase and
DNA polymerase activity. It can be used in a single buffer reaction that contains a tempera-
ture-sensitive chelator which controls the availability of manganese. Manganese is critical
for Tth switching from reverse transcriptase to DNA polymerase activity [62]. A correla-
tion between viability (as determined by plate counts or staining with a fluorogenic esterase
substrate) and the ARQ of the assay was observed. Selection of PCR primers that hybrid-
ized to the most distal portions of the hlyA gene gave a more accurate result in monitoring
viability as compared with PCR primers that amplified an internal region.
A second means to ensure that only viable L. monocytogenes cells will be amplified
is to have a requisite enrichment period before the PCR assay. Although this might seem
to be the antithesis of rapid methodologies, the enrichment need only be a few hours
and total assay times of less than 8 h are still reasonable. We have used membrane filtration
to concentrate cells from liquid foods, including raw milk [30]. Hot detergent facilitates
filtration after which the collected cells, still on the membrane filter, are placed onto a
nutrient-soaked absorbent pad. The cells are enriched for less than 4 h, processed using
a chelating reagent, and then boiled. The total assay time is less than 8 h and sensitivities
of <I0 viable L. monocytogenes CFU are routinely obtained.

Antibody-Based Detection Systems

Use of immunological assays for detecting bacteria is by no means new. For example,
the classic methodology for Salmonella involves a series of enrichment and selective plat-
ing media followed by a fluorescent polyclonal antibody assay for final confirmation of
the organism.
The limitations of polyclonal antisera are obvious, with bacterial cross reactions
hindering their use for primary identification of a genus. They have been used, however,
for serological analysis of isolates, thus proving their effectiveness in establishing the
epidemiological relationship between suspected outbreaks of foodborne illnesses. One of
the earliest demonstrations of immunological detection of Listeria was reported by Eve-
land [28], who detected L. monocytogenes in spinal fluid from a patient with meningitis
Rapid Methods for Detection of Listeria 27 7

using rabbit polyclonal antibodies raised against heat-treated L. rnonocytogenes (serotypes

1, 2, 3, 3b, 4a, and 4b) conjugated directly to fluorescein isothiocyanate.
The future for the use of immunological reagents as a rapid method for detecting
microorganisms improved dramatically with the advent of hybridoma technology [5 11.
Monoclonal antibodies are produced by hybridomas, which are the result of fusing an
antibody-secreting splenocyte to a plasmacytoma cell. These hybridomas can then be
grown in culture and the antibody subsequently harvested from the medium. Alternatively,
hybridoma cells can be injected into an appropriately primed mouse and allowed to estab-
lish a tumor. This ascites tumor is a highly productive source of monoclonal antibodies
which are secreted into the interstitial fluid.
One of the first monoclonal antibodies against Listeria antibodies was raised by
immunization with semipurified flagella extracts of Listeria spp. [30]. This antibody re-
acted with all Listeria spp. except L. gruyi, L. rnurrayi, and L. denitrijkans and did not
react with other gram-positive bacteria, including Staphylococcus aureus and Streptococ-
cus faecalis. An assay using these antibodies has been formatted where the bacteria are
spotted onto a nitrocellulose filter and then detected with the monoclonal antibody in
conjunctiori with a secondary peroxidase-coupled antibody.
A monoclonal antibody has been characterized which reacts with a heat-stable anti-
gen from Listeria [ 17,581. The antibody was raised by immunizing mice with a heat-
treated L. rnonocytogenes lysate and subsequently fusing the splenocytes to a mouse
myeloma fusion partner. Hybridomas were screened by a direct binding assay using a
heat-treated lysate from an L. rnonocytogenes culture. Although the exact nature of the
antigen is unknown, it has been commercialized by Organon-Teknika (Durham, NC) and
is available. It requires (as do all rapid methods developed to date) a preenrichment step,
after which the culture is collected and heated to produce an extract. Detection of this
Listeria antigen is accomplished using an ELISA format with two different monoclonal
antibodies which first capture and then detect the trapped antigen. The two monoclonal
antibodies recognize different epitopes on the antigen, thereby avoiding competition. The
monoclonal antibody used for detection is directly conjugated to horseradish peroxidase,
and tetramethylbenzidine is used as the chromogenic substrate. The total time involved
in the actual assay is approximately 2 h. The heat stability of this antigen (which varies
in size frorn 30 to 38 kD) is potentially problematic, because samples that are heavily
contaminated with Listeria are then thermally processed. In this situation, a false-positive
reaction is possible, although given the current need for enrichment, only viable Listeria
will be detected. Some efforts to document the utility of this assay have been reported [ 5 ] .
Hybridomas which produced monoclonal antibodies ag,ainst L. rnonocytogenes were
isolated by immunizing mice with both live and heat-killed L. rnonocytogenes [82]. Only
a limited number of Listeria strains were tested using a radioimmunoassay and shown
not to cross react with some of the hybridomas isolated. McLauchlin et al. [59] character-
ized two monoclonal antibodies, CL 17, which recognized L. rnonocytogenes serotypes 1/ 2 ,
and 3; and CL2, which reacted with serotypes 4b, 4 (not 4b), and L. innocua serotype 6a.
These monoclonal antibodies could be used to detect L. monocytogenes in soft cheese by
direct fluorescence microscopy, although some samples which were known to contain L.
rnonocytogenes (based on standard microbiological tests) were negative by this analysis
[601.
As mentioned previously, selective detection of L. rnonocytogenes is of great impor-
tance, and for this reason, we have isolated a monoclonal antibody which will specifically
recognize I,. rnonocytogenes [ 371. We have characterized several murein monoclonals
272 Batt

produced by fusing spleen cells isolated from BALB/c mice immunized with live L. mono-
cytogenes to NS-1 plasmacytoma cells. The immunogen consisted of live L. monocyto-
genes Scott A cells that were injected directly into the spleen of the mice [71]. Live cells
(as opposed to heat- or formalin-killed cells) and direct injection were chosen to provide
the most direct presentation of an unaltered (or minimally processed) antigen to the spleen.
Hybridomas were screened by direct ELISA assay, and of the 150 hybridomas tested,
three reacted most strongly with L. monocytogenes Scott A [27]. Although monoclonal
antibodies Mab 20-10-2, Mab 36-6-12, and Mab 59-9-16 reacted to some extent with L.
innocua and L. ivanovii in the direct binding assay, greater specificity for L. monocyto-
genes was seen in an indirect ELISA assay (Fig. 6). These antibodies were used to trap
L. monocytogenes, which was then detected by a rabbit anti-L. monocytogenes polyclonal
antiserum.
Siragusa and Johnson [69] also attempted to isolate a monoclonal antibody specific
for L. monocytogenes. These antibodies resulted from immunizations with heat-treated L.
monocytogenes as previously reported [ 171. Both immuno-dot-blot of heat-treated whole
cells and Western analysis of sodium dodecylsulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) separated cell extracts were used to demonstrate reactivity. Unfortunately,
their monoclonal reacted not only with L. monocytogenes but also with L. welshimeri, L.
innocua, and possibly others. Later efforts by this group targeted another antigen that
produced monoclonal antibodies which preferentially reacted with L. monocytogenes [6].
Further specificity in terms of serotype-specific monoclonal antibodies which detect L.
monocytogenes 4b has also been reported [47].

0.9

0.8

0.7

0.6
U)

g
0
0.5
0
0.4

0.3

0.2

0.I

0
L rnonocytogenes L. ivanovii L innouca L seeligeri S. faecalis
Organism

FIGURE6 Indirect ELISA using MAB20-10-2 to trap antigen and rabbit polyclonal
anti-L. rnonocytogenes serum for detection. Bound antibodies were detected using
goat antirabbit alkaline phosphatase conjugate and p-nitrophenol phosphate. 2 x
106cells/mL; 5 x 105cells/mL. (From Ref. 37.)
Rapid Methods for Detection of Listeria 273

Immu noassay Formats

Unlike nucleic acid probe-based assays, most immunoassays utilize a relatively standard
ELISA format. Cells are either directly absorbed or immunocaptured onto wells of a micro-
titer plate. The captured cells are then detected using an antibody which carries either a
hapten (i.e., biotin) or an enzyme reporter, with the latter being chromogenic, fluorescent,
or chemiluminescent either directly or through the use of substrates. Beyond ELISA for-
mats, other assays employ flow cytometry-which can detect irnmunocaptured cells in solu-
tion rather than having to immobilize them on a solid support [25].Antibodies conjugated
to magnetic beads also have been used not directly for detection but for capture, followed
by standard microbiological plating [70].

Commercial Test Systems

Many companies have entered the market with rapid methods to detect either Listeria or
more specifically L. monocytogenes. Most of these assays are extensions of formats used
to detect other microorganisms (or their toxins), with their components altered specifically
to detect Listeria. Assays based on nucleic acid hybridization or antibody-antigen interac-
tions are available as well as one that employs nucleic acid amplification. All of these
assays require some prior enrichment to increase selectively the target population to detect-
able levels. At best, these assays can be completed within 24 h, although this is dependent
on the food source and intended level of sensitivity. Barring approval of the test method
by an appropriate regulatory agency, positive samples must be confirmed using standard
microbiological culture methods. Therefore, these rapid methods are most often used as
quick screening tools to examine large numbers of samples.
One ELISA for Listeria has been developed by Organon-Teknika. This ELISA is
formatted for a 96-well microtiter plate and the readout is colormetric. To obviate the
liquid handling normally associated with microtiter plates, Tecra has developed and incor-
porated an antibody-coated dipstick into its unique system. After initial cell capture, an
interim culture replication step helps increase cell numbers. The readout is colormetric.
BioMerieux (St. Louis, MO) also has developed a immuno test strip for the Vidas instru-
ment that employs a solid phase receptacle and a fluorescent readout.
In general, most immunological assays for Listeria have not been successfully al-
tered to specifically detect L. monocytogenes. Despite apparent successes in developing
antibodies specific for L. monocytogenes (this authors work. inclusive), incorporation of
these antibodies into commercial assay formats has not yet followed. One exception is
the development of an immunomagnetic bead-colony immunoassay by Vicam (Water-
town, MA) which is rather complex and is claimed by the manufacturer to be specific for
L. monocytogenes. Cells from the sample are collected on immunomagnetic beads which
are subsequently cultured on an agar plate. Suspect colonies are than screened using a
filter immunoassay.
A nucleic acid hybridization assay which uses a sandwich capture format is available
from Gene Trak. Two assays have been developed, one which detects all species of the
genus Listeria and the other which is specific for L. monocytogenes. Both assays target
16s rRNA and have a colorimetric readout. In each assay, a capture probe binds to a
region of the 16s rRNA while a second probe binds to a spatially separated region of the
16s-rRNA molecule. The complex is then removed from solution using a poly-A tail which
hybridizes to a poly-T-coated solid support.
Finally, a PCR-based amplification method for detecting L. monocytogenes has been
274 Batt

developed by Qualicon (Wilmington, DE; a subsidiary of DuPont). Although the nature

of the amplicon is proprietary, it is reportedly specific for L. monocytogenes. All assay
components are contained in a single tablet which is added to the sample after processing.
This assay requires gel electrophoresis to confirm the presence of the appropriate amplifi-
cation product.

FUTURE DEVELOPMENTS
The long-term goals in development of any rapid method dictate that the test be fast,
simple, sensitive, accurate, and, for commercial purposes, inexpensive. However, at least
some of these desired performance attributes are mutually exclusive: for example, as an
assay is made more sensitive, the accuracy, as it is defined by the number of false positives,
increases. Most attention concerning monoclonal antibodies or nucleic acid probes for
Listeria identification (or in fact other microorganisms) is focused on the reporter mole-
cules and associated detection instrumentation. Advances in chemiluminescent-based
reporters which have sensitivities in excess of 100-fold greater than existing enzymatic-
based systems will be applicable to Listeria detection.
All of the rapid assays developed to date (July 1997) require prior enrichment, with
this step taking up to 48 h. Therefore, any claims that an assay can be completed within,
for example, 4 h, are not entirely truthful. Continued efforts to further improve media
formulations for recovery of Listeria from foods should prove beneficial as a prelude to
any rapid detection method. Another area of concern is the significance of injured Listeria
cells in a given food product and their potential for recovery either during enrichment or
in the food during long-term storage. As mentioned previously, antibodies or nucleic acid
probes can, in theory, detect both injured and dead cells. If future rapid assays are devel-
oped to detect microorganisms in food without any prior enrichment, the significance of
injured populations will need to be addressed.
At issue is whether detection systems specific for L. monocytogenes are advanta-
geous over genus detection of all Listeria. The most obvious argument for an L. monocyto-
genes-specific test is based on the fact that virtually all cases of human listeriosis are
caused by L. monocytogenes. In an ideal world, the goal would be to create a rapid test
which detects Listeria spp., which are pathogenic in humans. Until we have elucidated
the factors mediating pathogenicity of L. monocytogenes, such a goal is not feasible.

ACKNOWLEDGMENTS
The support of the Northeast Dairy Foods Research Center is greatly appreciated. The
author thanks Mary Lou Tortorello and Jerrie Gavalchin for their assistance. The author
also thanks Liz Borod for her help in the preparation of this manuscript.

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Subtyping Listeria
monocytogenes

LEWISM. GRAVES,
BALASWAMINATHAN,
AND SUSANB. HUNTER
Centers for Disease Control and Prevention, Atlanta, Georgia

Most bacterial species have sufficient phenotypic and genotypic diversity to allow for
identification of different subtypes. Therefore, phenotyping and genotyping systems used
singly or in combination often provide useful subtyping schemes for pathogenic bacteria.
The various subtyping systems reviewed in this chapter provide different degrees of dis-
crimination among Listeria monocytogenes isolates. By using these systems in epidemio-
logical studies to distinguish individual strains or groups of strains, it has been possible
to obtain information on relationships between isolates, identify disease outbreaks, identify
the source of infections in outbreaks and sporadic disease settings, and determine modes
of transmission for the organism.
We present a broad overview of the typing methods that have been applied to L.
monocytogenes and, where appropriate, discuss briefly the strengths and weaknesses of
each. In this review of the usefulness of the most commonly used subtyping methods for
L. monocytogenes, we have separated them into two major categories: conventional meth-
ods (i.e., serotyping, phage typing) and molecular methods (i.e., multilocus enzyme elec-

Use of trade names is for identification only and does not imply endorsement by the Public Health Service or
by the U.S. Department of Health and Human Services.

279
280 Graves et al.

trophoresis, DNA restriction analysis). At present, each of these methods has some utility;
however, with the extraordinary developments in nucleic acid technology and the wide
availability of these technologies, some conventional methods may cease to be used in
the future.
L. monocytogenes is among the first pathogenic bacteria for which a concerted and
internationally coordinated attempt has been made to evaluate critically various available
subtyping methods and to standardize the more useful methods. In 1996, Bille and Rocourt
organized the World Health Organization (WHO) Multicentre Listeria monocytogenes
Subtyping Study. The result from Phase I of this study were published in a special issue
of the International Journal of Food Microbiology [25] and will be referenced throughout
this chapter.

CONVENTIONAL METHODS
Serotyping
Serotyping has been a classic tool for epidemiological and sporadic case studies of L.
monocytogenes [27,38]. Strains of L. monocytogenes differ in the antigenic determinants
expressed on the cell surface. Such antigenic variations are produced by many different
surface structures, including lipoteichoic acids, membrane proteins, and extracellular or-
ganelles (e.g., flagella and fimbriae). These differences can be identified by serological
typing (serotyping). Strains of Listeria species are divided into serotypes based on somatic
(0)and flagellar (H) antigens [71]. Flagellar antigens as well as 0 antigens must be identi-
fied to type strains of serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, and 3c. The remaining serotypes
all have the same flagellar antigens (A, B, and C). Serotypes 1/2a, 1/2b, 1/2c, 3a, 3b,
and 3c can be identified with two 0 antisera (one with antibodies to factor I and the other
with antibodies to both factors I and 11) and three H antisera (one with antibodies to factors
A and B, one reacting with C, and one reacting with D). With antisera for 0 factors V
and VI, VII and IX, VIII, X, XI, and XV, strains of serotypes 4a, 4b, 4c, 4d, 5 , 6a, and
6b can be typed [80]. Serotype 4bX is a variant of serotype 4b and was implicated in an
outbreak in the United Kingdom that was traced to contaminated piit6 [47].
Most (>95%) human infections are caused by strains of L. monocytogenes belong-
ing to serotypes 1/2a, 1/2b, and 4b. Therefore, serotyping alone is of limited value in
epidemiological investigations. In the WHO Multicentre L. monocytogenes Subtyping
Study, Schonberg et al. [69] found that all 80 strains tested by serotyping were typeable.
However, for only 49 (61.3%) strains was there complete agreement between the six par-
ticipating laboratories on the serotype (21 of serotype 1/2a and 28 of serotype 4b). Intra-
laboratory reproducibility, assessed on 11 duplicate strains, ranged from 82 to loo%, with
a median value of 91%. Interlaboratory reproducibility varied from 64 to 95%; no labora-
tory correctly identified the two serotype 4bX strains in the set. Schonberg et al. [69]
concluded that a critical need exists for good-quality antisera prepared from standardized
strains. Also, they emphasized the need to absorb these antisera completely and efficiently
to produce good-quality factor sera.
Serotyping has poor discriminating power when compared with other subtyping
methods. Isolates from foods and the environment are frequently nontypeable with stan-
dard typing antisera. Nevertheless, serotyping provides valuable information for rapid
Subtyping L. monocytogenes 281

screening of groups of strains isolated during suspected outbreaks. Serotype information

allows elimination of isolates that are not part of an outbreak and facilitates efficient
application of other more sensitive but time-consuming subtyping methods.

Bacteriophage Typing (Phage Typing)

Numerous lytic bacteriophages have been identified for Listeria spp. [42], L. monocyto-
genes isolates can be characterized by their patterns of resistance or susceptibility to a
standard set of phages, as demonstrated by Rocourt et al. [63]. Until the recent advent
of molecular subtyping, phage typing was often used in conjunction with serotyping for
epidemiological investigations because of its high discriminating power [4,45,63,68]. The
WHO Multicentre L. monocytogenes Subtyping Study on phage typing was done using
an international phage set in five laboratories and unique phage sets in two laboratories
[44]. With the international phage set, 20-5 1% of the isolates were nontypeable. Non-
typeability with the international phage set was a greater problem among strains of sero-
groups 1/2 and 3 (%-to 72%) than for serogroup 4 (11-22%). The two laboratories
that used unique phage sets had fewer problems with nontypeable strains. One of these
laboratories was able to type all strains of serogroup 4 and 81% of strains of serogroups
1/2 and 3. The reproducibility of phage typing among the participating laboratories was
79% using criteria for interpretation previously proposed for the international phage set [46].
Based on the aforementioned findings, McLauchlin et al. [44] recommended that
the phages in the international set be reviewed and additions be considered to increase
typeability of strains. Lemaitre et al. [41] proposed a method that facilitates detection of
induced phages; this procedure may be useful for identifying additional typing phages.
McLauchliri et al. [44] suggested that better interlaboratory reproducibility may be
achieved by standardization of phage suspensions, propagation strains, and methodology.
Use of centrally propagated phages, as is done by the Central Public Health Laboratory,
London, for phage typing of Salmonella serotypes may be helpful.
Despite its high discriminating power and easy applicability to large numbers of
strains, phage typing is available only at selected national and international reference labo-
ratories because of the need to maintain stocks of biologically active phages and control
strains. Although the procedure is technically not very demanding, it suffers from consider-
able experimental as well as biological variability. The percentage of nontypeable strains
may vary with the standard phage set used. Nevertheless, phage typing remains the only
practical method that can be rapidly applied to type strains in massive outbreaks. Rocourt
et al. [64] phage-typed more than 16,000 isolates in 1 year while investigating an outbreak
in France in 1993 in which pork tongue in jelly was implicated as the vehicle of infec-
tion.

Bacteriocin Typing
Bacteriocins (monocins) were first isolated from L. rnonocytogenes in 1961 and character-
ized by Sword and Pickett [78] and Hamon and P6ron [35]. Monocins are resistant to
trypsin, sensitive to heating at 56C for 30 min, and stable a.t 4C. In monocin typing, an
isolate is assessed for susceptibility to a set of bactericidal peptides produced by selected
strains [54,85]. Curtis and Mitchell [18] studied monocin interactions of 97 strains of L.
monocytogenes using an improved production method involving standardization of the
monocins against the type strain of Listeria ivanovii. Only serotype 4 strains acted as
282 Graves et al.

indicators. A typing system using 8 producer and I 1 indicator strains showed poor discrim-
ination. Bacteriocin typing has limitations similar to those described for phage typing.
Bannerman et al. [2] typed 100 strains of L. monocytogenes from sporadic cases
and epidemic outbreaks by a combination of monocin typing and phage receptorheverse
phage receptor methods. The combination monocin-phage receptor subtyping method had
a discrimination index of 0.99 for 87 epidemiologically unrelated strains, which was the
highest of seven subtyping methods evaluated. The authors suggested that the monocin-
phage reversal method was simple enough to be done in a nonspecialized laboratory and
was highly discriminatory and reproducible. However, they cautioned that the method and
the indicator test strains must be rigorously standardized.

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing is of limited use for typing Listeria species, since L.
monocytogenes susceptibility patterns have remained relatively constant for many years.
However, in recent years, plasmids conferring resistance to chloramphenicol, macrolides,
and tetracyclines have been found in L. monocytogenes [34,581.

MOLECULAR METHODS
Multilocus Enzyme Electrophoresis
Characterization of prokaryotes and eukaryotes by multilocus enzyme electrophoresis
(MEE) is based on differences in electrophoretic mobility of their metabolic enzymes.
These differences in electrophoretic mobility are a result of charge differences resulting
from amino acid substitutions in the polypeptide sequence; these charge differences, in
turn, reflect changes in the nucleotide sequence of the DNA encoding the polypeptide
[72]. In MEE, cell extracts containing the soluble metabolic enzymes are electrophoresed
in nondenaturing starch gels. After electrophoresis is completed, the gel is sliced, and
each slice is treated with a specific chromogenic substrate for a specific enzyme (e.g.,
aldolase) to render the enzyme band visible. Mobility variants of each enzyme are consid-
ered to be different electromorphs and are subjectively designated by different numbers.
Combinations of a set of electromorphs (usually 10-20) constitute an electrophoretic type
(ET), with each ET representing a multilocus genotype. Some isolates may present null
results (absence of activity for specific enzymes); this complicates analysis of MEE data.
In the early 1990s, MEE was used in the United States and Europe for epidemiologi-
cal investigations of listeriosis outbreaks [3,31,521 and to determine the extent to which
contaminated foods are involved in sporadic listeriosis [57]. Also, MEE has been useful
for taxonomic and genetic characterization studies of L. monocytogenes [7,8,56]. Boerlin
et al. [8] used MEE to estimate the genetic relatedness between various Listeria species.
The MEE data not only allowed identification of different genotypes within a population,
but also provided an estimation of the genetic relatedness between strains.
Although MEE is a very powerful tool for population genetic, taxonomic, and evolu-
tionary studies, it is only moderately discriminatory for use as a subtyping tool in epidemi-
ological investigations.
Caugant et al. [ 171 coordinated evaluation of MEE for the WHO Multicentre L.
monocytogenes Subtyping Study. Seven laboratories participated in the study, assaying a
Subtyping L. (ionocytogenes
I 283

total of 24 enzymes. Reproducibility and the discriminating power of the method varied
greatly between the laboratories. Null alleles were reported by five laboratories; in some
instances, these could be attributed to less than optimal activity of the enzyme in the
cytoplasmic extracts applied to gels, whereas in others, it was clearly related to characteris-
tics of the strains. Caugant et al. [ 171 concluded that to asceirtain immediate epidemiologi-
cal relationships of L. monocytogenes strains, one will need, in some instances, to supple-
ment MEE with other methods providing further discrimination. Similar conclusions were
reached by Norrung and Gerner-Smidt [5 11, who reported an overall discrimination index
(DI) of 0.83 for MEE. When results of MEE were combined with those of restriction
endonuclease analysis, the DI increased to 0.92. Further, MEE is a labor-intensive method
that requires techniques and equipment available in relatively few laboratories. For these
reasons, this method presently has relatively limited application in epidemiological studies.

Chromosomal DNA Restriction Endonuclease Analysis

Chromosomal DNA restriction endonuclease analysis (REA) using frequently cutting re-
striction endonucleases has proven useful for typing L. monocytogenes [24,29,50,83].The
number and size of restriction fragments generated by digesting a given piece of DNA
are influenced by the recognition sequence of the enzyme <andcomposition of the DNA.
Thus, enzymes with a four-base recognition sequence are expected to yield more and
smaller restriction fragments than enzymes with six-base recognition sequences. However,
an enzyme whose recognition sequence is composed of only guanine (G) and cytosine
(C) will cut DNA with a low G+C content less frequently and consequently will generate
fewer and larger restriction fragments than an enzyme recognizing sequences of adenine
and thy mine. Selection of an appropriate restriction endonuclease is based on the recogni-
tion sequence of a restriction endonuclease and the G + C content of the test organism
[28,55]. Because of the high specificity of restriction endonucleases, complete digestion
of a given DNA with a specific enzyme provides a reproducible array of fragments. Within
a size range from 30 to 1 kb, the fragment can be separated by agarose gel electrophoresis
to obtain DNA fingerprint patterns.
For L. monocytogenes, the advantages of REA are that it is universally applicable,
sensitive because the entire genome is evaluated, cost effective, and relatively easy to
do. Gerner-Smidt et al. [29] evaluated REA for the WHO Multicentre L. monocytogenes
Subtyping Study. Of the restriction endonucleases tested, HaeIII, HhaI, and CjoI were
most useful for REA typing of L. monocytogenes. The set of 80 strains was divided into
27 REA types with HaeIII, 24 with HhaI, and 25 with CfoI. DNA fragment patterns
obtained with HhaI and CjoI were nearly identical and were the easiest to interpret by
visual examination; EcnRI divided the strain set into only 14 REA types, and the patterns
were difficult to compare.
The major limitation of REA is the difficulty of cornparing the complex profiles,
which consist of hundreds of bands that may be unresolved and overlapping. Patterns
generated by some restriction enzymes may not be stable. Differentiation criteria (the
minimum number of band differences that are indicative of differences between the iso-
lates) for each enzyme need to be clearly established before discrimination between strains
can be reliably evaluated [29]. Consequently, REA is not the method of choice for large-
scale comparisons or for building a dynamic database of DNA patterns for comparative
evaluation of new strains.
284 Graves et al.

Restriction Fragment Length Polymorphism Analysis:

Ribotyping and Other Methods
Ribotyping is one form of Southern hybridization analysis in which strains are character-
ized for restriction fragment length polymorphisms (RFLPs) associated with ribosomal
operon(s). Southern blot analyses only detect the particular restriction fragments associ-
ated with specific chromosomal loci, thereby significantly reducing the number of DNA
fragments to be analyzed [75]. Ribotyping involves transferring chromosomal DNA re-
striction fragments that have been electrophoretically separated on a gel matrix onto a
nitrocellulose or nylon membrane. After immobilization, DNA fragments are hybridized
with an appropriately labeled 16+23S ribosomal RNA (rRNA) or rDNA probe [33,76].
Because the genes coding for rRNA are highly conserved, Escherichia coli rRNA [76]
or a cloned ribosomal operon (rrnB) of E. coli [ 121 can be used to probe L. monocytogenes.
In general, ribotype patterns appear to be stable and reproducible after in vitro and in
vivo passage of strains [12]. Therefore, this method may be best suited for long-term
epidemiological or phylogenetic studies. Figure 1 shows a gel containing genomic DNA
restriction fragments from L. monocytogenes. Figure 2 shows a typical nylon membrane
containing ribotype patterns obtained after Southern blotting and probing with a cloned
E. coli rrnB operon (plasmid pKK3535) labeled with digoxigenin.
Ribotyping has been widely used for subtyping L. monocytogenes [ 1,13,19,31,32,39,
49,5 11, with most investigators using EcoRI as the restriction endonuclease. Baloga and
Harlander [I] compared HaeIII and Hind111 to EcoRI and concluded that EcoRI was the
most discriminating enzyme for subtyping L. monocytogenes. Nocera et al. [49] reported
that 69 of 96 serotype 4b isolates clustered in two closely related EcoRI ribotypes; they
found bacteriophage typing to be more discriminating than ribotyping. Norrung and
Gerner-Smidt [5 11 found ribotyping to be less discriminating than bacteriophage typing,
REA, or MEE for subtyping 99 clinical, food, and slaughterhouse isolates of L. monocyto-
genes.
Swaminathan et al. [77] evaluated ribotyping and another probe derived from repeat
sequences of L. monocytogenes DNA for the WHO Multicentre L. monocytogenes Subtyp-
ing Study. Six laboratories did ribotyping using EcoRI enzyme to restrict the L. monocyto-
genes DNA and rRNA or DNA as the probe for Southern hybridization. A seventh labora-
tory used NciI to restrict the DNA, and two probes, one randomly cloned and the other
containing repeat sequences cloned from L. monocytogenes DNA. The overall Simpsons
index of diversity (DI) for five laboratories that ribotyped most or all study strains ranged
from 0.83 to 0.88. A DI value of 0.91 was obtained for the combination of two probes
used by one laboratory. Also, DI values for strains of serotypes 1/2a or 1/2c were greater
than DI values for strains of serotypes 1/2b, 3b, 4b, or 4bX for ribotyping as well as the
randomly cloned probes used by one laboratory. The investigators concluded that although
ribotyping satisfies two requirements for a good subtyping method, namely, typeability
and reproducibility, its discriminating ability for serotype 4b strains may not be adequate
for epidemiological investigations. They recommended that ribotyping should be supple-
mented by other methods such as pulsed-field gel electrophoresis for molecular epidemiol-
ogy of L. monocytogenes strains of serotype 4b.
Probes other than rRNA and rDNA have been used to type L. monocytogenes. Saun-
ders et al. [66] selected cloned DNA fragments of L. monocytogenes from a bacteriophage
lambda gene library to type 64 isolates of serogroup 1/2 using restriction enzyme NciI
and found good discrimination between epidemiologically unrelated isolates. Ridley [62]
Subtyping L. monocytogenes 285

FIGURE1 Genomic fingerprints of EcoRI-digested DNA o f Listeria monocytogenes

strains isolated f r o m humans and food. Lanes 2 and 14, human isolates (serotype I /
2a); lanes 3-13 and 15-20, food isolates (serotype 1/2a). Lane 21, human isolate (sero-
type 4b); lanes 22 and 23, food isolates (serotype 4b). Lanes 1 and 24 molecular size
marker.

evaluated the same probe to type 862 isolates representing serogroups 1/2, 3, and 4. Al-
though useful for subtyping serogroup 1/2 isolates, the method did not adequately dis-
criminate between serogroup 4 isolates. The cloned probe evaluated by Ridley [62] and
another probe derived from repeat sequences of L. monocytogenes DNA were assessed
in the WHO Multicentre L. monocytogenes Subtyping Study [77]. Like ribotyping, the
two cloned probes did not adequately discriminate between epidemiologically unrelated
serotype 4b isolates. Also, these probes did not discriminate between a serotype 4d
isolate and other serotype 4b isolates associated with a listeriosis outbreak. However, the
repeat sequence probe discriminated between serotype 4b isolates and serotype 4bX iso-
lates [77].
The RiboPrinter (Qualicon, Wilmington, DE) is an automated ribotyping system
that generates, analyzes, and stores riboprint patterns of bacteria. The first version of the
286 Graves et al.

FIGURE2 Ribotype profiles obtained by Southern blot analysis of genomic finger-

prints of EcoRI-digested DNA of Listeria monocytogenes from Figure ? .

RiboPrintcr was contigured t o generate ribotype patterns using only EcoRI and was used
to generate a database of patterns for 1346 isolates of L. r i i o r i o c ~ ! ~ t o g c r I c . v[ 13,371. The
RiboPrintcr was used by Ryser et al. 1651 to demonstrate that different ribotypcs of L.
were fiivorcd b j, d i ffe re n t selec t i ve c nri c h me n t pro tocol s. W i edma n n t:t
r i i o i I o( ~j~togc~iic.s
al. I841 characterized 133 isolates of L. rilorloc:\.togcrlc..v using the RiboPrintcr and tested
for pol y morph i s nis i n vi rii 1e nce-;is soc i ii t ed ge lies. They conc 1uded that the i sol ;it e s coii 1d
be separated into three distinct phylogenetic lineages: hiiman isolates were found i n lin-
eages 1 and 2. but lineage 3 nfiis coniposeed exclusi\,ely of animal isolates.
Pol y merase chain react io ti - ri bot y ping ( PC R-ri boty pi ng ) is ;i met hod that cx ploi ts
iisc of oligonucleotide primers designed to be complementary t o conser\.ed regions of the
5s. 16s. and 23s regions of the rRNA genes. These primers are amplified with purified
or criidc preparations of template DNA bj, PCR. The resulting PCR products may bc
digested with ;i restriction cndonuclease of choice or added t o an agarose gel, elcctropho-
rcsed. and \,i sii;I I i zed by et h i d i ii m bromide staining . The potential of PC R- ri bo t y p i ng for
discriminating between and within \.arious species of Listc~rier.;is well ;is strains of L.
r i ~ o r ~ o c ~ ~ ~ thas i ~ ~ ~explored
o g ebeen .~. by Sontakke and Farber 1741. who analyzed 49 strains
of L. riioiioc!togeiie.s and 12 isolates of other Listc~r-iuspecies. Gcnomic DNA isolated
from bacteria wiis sub.jcctcd to PCR amplification using the region of DNA encoding 16s
and 5s rRNA. They found that PCR-I-ibotyping distinguished beti\wn L. riiorioc:\toScric,s
serotypes 1 /2a and 1 /2b w;ith no o\-erlap i n composite ~vfiles.The sensitii~itj~ of this
metliod for differentiating serotypc 1 /2a and 1 /2b isolates appears to be ;is good ;is that
Subtyping L. monocytogenes 287

of other molecular methods. However, the PCR-ribotyping method was less discriminatory
for serotype 4b strains [26]. Sontakke and Farber [74] concluded that PCR-ribotyping
could be considered as an alternate molecular subtyping technique. However, they recom-
mended combining PCR-ribotyping with another highly discriminatory molecular subtyp-
ing method for confirming associations between isolates of serotype 4b.

DNA Macrorestriction Analysis by Pulsed-Field Gel

Electrophoresis
DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) has revolution-
ized precise separation of DNA fragments greater than 40 kb. Schwartz and Cantor [70]
developed PFGE, a variation of agarose gel electrophoresis in which the orientation of
the electric field across the gel is changed periodically (pulsed) rather than being kept
constant as in conventional agarose gel electrophoresis used for REA. This technology
separates large fragments of unsheared microbial chromosomal DNA obtained by embed-
ding intact bacteria in agarose gel plugs, enzymatically lysing the cell wall, and digesting
the cellular proteins. Intact DNA is digested using one or more restriction endonucleases
that cut infrequently, thus producing large fragments. Subsequent RFLP analysis allows
differentiation of clonal isolates from unrelated ones. PFGE analysis has been used for
epidemiological subtyping of L. tnorzocytogenes by several investigators [ 10, 1 1,14,16.36].
Figure 3 shows patterns obtained when genomic DNA from L. monocytogenes was di-
gested with restriction endonucleases ApaI and AscI.

FIGURE3 PFGE separation of Apal (lanes 2-7) and Ascl (lanes 9-14) macrorestriction
fragments of Listeria rnonocytogenes genomic DNA f r o m sporadic case isolates.
Lanes 1, 8, and 15, Xbal-digest o f Escherichia col; 0 1 5 7 : H7 strain.
288 Graves et al.

Brosch et al. [ 101 first demonstrated the usefulness of PFGE for subtyping L. mono-
cytogenes by applying the method to type serotype 4b strains. Using ApaI, SmaI, and
NotI, they showed PFGE can distinguish between closely related strains indistinguishable
by other typing methods. The applicability of PFGE to subtype L. monocytogenes sero-
types 1/2 and 3 was subsequently demonstrated [ 151. The applicability of PFGE for out-
break investigations was shown by Buchrieser et al. [ 141, who typed 75 L. monocytogenes
strains isolated during six major and eight smaller listeriosis outbreaks. PFGE divided
these strains into 20 subtypes. Strains within each major epidemic (Switzerland [ 1983-
19871, California [ 19851, and Denmark [ 1985-871) demonstrated indistinguishable pat-
terns, whereas strains responsible for other outbreaks were characterized by specific com-
binations of patterns. Variations within PFGE patterns occurred more frequently within
epidemiologically unrelated isolates. PFGE was used to demonstrate the link between
contaminated chocolate milk and febrile gastroenteritis among a group of people who
attended a cow show in Illinois [20,59]. Destro et al. [21] also found that RAPD and
PFGE were the most useful methods for tracing dissemination of L. monocytogenes in a
shrimp processing plant.
Brosch et al. [9] evaluated PFGE in the WHO Multicentre L. monocytogenes Subtyp-
ing Study. Four participating laboratories evaluated PFGE by analyzing 80 coded strains
of L. monocytogenes. Two restriction endonucleases (ApaI and SmaI) were used by all
laboratories; one laboratory used an additional restriction endonuclease (AscI).Agreement
among the four laboratories ranged from 79 to 90%. Sixty-nine percent of the strains
were placed in exactly the same genomic group by all four laboratories, with most of the
epidemiologically related strains being correctly identified. This study validated previous
claims that PFGE is a highly discriminating and reproducible method for subtyping L.
monocytogenes and is particularly useful for subtyping serotype 4b isolates, which are
not subtyped satisfactorily by most other subtyping methods [9].
The major disadvantages of PFGE are the time required to complete the procedure
(2-3 days), the requirement for large quantities of expensive restriction endonucleases,
and the need for relatively expensive, specialized equipment for electrophoresis.

Random Amplification of Polymorphic DNA

Arbitrarily primed polymerase chain reaction (AP-PCR) and random amplified polymor-
phic DNA (RAPD) analysis are PCR-based methods in which a single arbitrarily selected
primer is allowed to anneal to nearly complementary sequences of target DNA by anneal-
ing at very low temperatures (37C). Typically, the primer anneals to several locations
on the target and amplifies an array of DNA fragments of different sizes, yielding a DNA
pattern suitable for typing. RAPD uses primers of 10-bp length, whereas AP-PCR was
developed with longer primers [8 1,861. RAPD was first applied to subtyping of L. monocy-
togenes by Mazurier et al. [43]. They used a 10-mer primer (HLWL 74) to analyze 104
L. monocytogenes isolates, which included representative strains from six outbreaks. All
but one of the outbreak-associated isolates were classified by RAPD in complete agree-
ment with phage typing. Mazurier et al. [43] suggested that RAPD offers an attractive
alternative to phage typing. Lawrence et al. [40] used a different 10-mer primer to type
91 isolates from raw milk, food, and veterinary, environmental, and clinical sources and
obtained 33 different patterns. Farber and Addison [26] applied RAPD to type 52 L. mono-
cytogenes isolates representing 11 serotypes and concluded that the method offered much
promise as a subtyping method for L. monocytogenes. Niederhauser et al. [48] used a 19-
Subtyping L. monocytogenes 289

mer primer to subtype 57 L. monocytogenes isolates and reported that the method allowed
the tracing of L. monocytogenes contamination in several food outlets to be traced back
to a food processing plant. Boerlin et al. [6] did an extensive evaluation of RAPD by
typing 100 L. monocytogenes isolates which had been characterized by serotyping, phage
typing, MEE analysis, REA, and ribotyping. They found R.APD to be highly discriminat-
ing for subtyping. ODonoghue et al. [53] found RAPD to be useful for subtyping
serogroup I /2; also, they found that the method distinguished serotype 4bX strains in-
volved in a pit&-associatedoutbreak from other serotype 4b isolates.
Wernars et al. [82] evaluated RAPD in the WHO Multicentre L. monocytogenes
Subtyping Study. Using three different 10-mer primers, the median reproducibility of
RAPD results obtained by the six participating laboratories was 86.5% (range 0-100%).
Failure in reproducibility was caused primarily by results obtained with one particular
primer. The authors concluded that RAPD is a rapid and relatively simple technique for
epidemiological subtyping of L. monocytogenes isolates that can produce reproducible
and useful results.
Despite the simplicity and high discriminating ability of RAPD, much more work
is still needed before RAPD typing becomes a widely used standard technique. Its primary
drawback is the inconsistent reproducibility of patterns. E%ecauseRAPD conditions are
less stringent to facilitate initiation of the polymerization reaction at sites having one or
more sequence mismatches, polymerization is initiated withi various efficiencies. The final
quantities of DNA produced may vary widely among the different fragments amplified
from a given isolate. Such variation is inherent in RAPD analysis and introduces two
specific problems. First, comparison and interpretation of patterns with differences in in-
tensity become quite difficult. Second, because some of the products may represent rela-
tively inefficient reactions, the actual fragments obtained from a single isolate may vary
in different amplification reactions. Consequently, a well-standardized RAPD protocol
must be followed and used consistently to obtain reliable results.

Repetitive Element-Based Subtyping

Repetitive element-based (REB) subtyping is a PCR method incorporating use of primers
based on short extragenic repetitive sequences [79] or generic rRNA intergenic spacer
oligonucleotide [30]. Sequences are typically present at many sites around the bacterial
chromosome such that when two sequences are sufficient1:y near to each other, the DNA
fragment between those sites is effectively amplified. Because the number and location of
the repetitive sequences are quite variable, the number and size of the interrepeat fragments
generated can similarly vary from strain to stain. Ericsson et al. [23] subtyped 133 strains
of L. monocytogenes serotype 4b using REB. A segment of 2916 bp from L. monocyto-
genes ancl containing parts of the two genes inlA and inlB was amplified by the PCR.
The PCR product obtained was digested with the restriction enzyme A M . The resulting
fragments yielded two distinct groups, one containing 37 types and the other 96 types.
These results indicate that REB may be useful for subtyping L. monocytogenes serotype
4b strains.

DNA Sequence-Based Subtyping

We anticipate that within the next 5- 10 years, definitive identification of L. monocytogenes
and epidemiological subtyping of strains will be based on :DNA sequencing. At this time,
DNA sequence-based subtyping is a not a useful alternative, because the sequencing
290 Graves et al.

TABLE
1 Characteristics of Phenotypic and Molecular Subtyping Methods Used in the WHO Multicentre L. rnonocytogenes
Subtyping Study

% Intralaboratory Interlaboratory Discriminatory

Method Typeability reproducibility (96) reproducibility (%) power (DI) Reference Recommendations
Serotyping 100 82- 100 83 0.68 69 For standardization, reagents should be pro-
duced and distributed by one laboratory. Sero-
typing is not very discriminatory, but is a use-
ful prerequisite, especially in outbreak
investigations.
Phage typing 49-80a NA 79 ND 44 Centrally propagate phages and standardize
phage suspensions, propagation strains and
methodology.
MEE 100 27-9 1 ND 0.83-0.93 17 Not sufficiently discriminating to be used alone
for epidemiological investigations.
REA 100 88-97 98 0.93-0.98 29 Develop standardized nomenclature for types.
Ribotyping I00 80- 100 ND 0.83-0.88 77 Not sufficiently discriminating to be used alone
for epidemiological investigations.
PFGE 100 ND 84 0.95-0.96 9 Highly discriminating and useful for epidemio-
logical investigations; standardize method.
RAPD I00 0-100 for 3 0- 100, median 0.75-0.95 for 82 Highly discriminating and useful for epidemio-
primers 86.5 three primers logical investigations. One primer gave seri-
ous reproducibility problems. Standardize
method and address problems with reproduc-
ibili ty.
DI, Simpsons index of diversity: ND, not done; NA, not available.
The remaining isolates did not give a strong reaction with any of the phages in the international phage set.
Subtyping L. monocytogenes 291

methods are still too complex and appropriate targets on the genome of L. monocytogenes
have not been identified. Furthermore, a database of DNA sequences of suitable targets
of epidemiologically related and unrelated strains to facilitate interpretation of DNA se-
quencing dala is also not yet available. However, L. monocytogenes is an attractive candi-
date for implementing DNA sequence-based subtyping for the following reasons. There
is an excellent set of well-characterized, epidemiologically related and unrelated strains
of L. monocytogenes that have been subtyped by all available phenotypic, protein-based,
and DNA-RFLP-based subtyping methods. These strains will be invaluable for devel-
oping a database of DNA sequences for L. rnonocytogenes. Recent characterization of
numerous virulence-associated genes of L. monocytogenes also has provided critical infor-
mation concerning sequence heterogeneities in these genes [ 22,60,6 1,67,73,87].
The virulence-associated genes of L. monocytogenes that have been sequenced as
of November, 1997, include iup (encodes an invasion-associated protein), inlA (a family
of genes involved in internalization of the organism into the host cell), hlyA (encodes a
P-hemolysin), plcA (encodes a phosphatidyl inositol-specific phospholipase C which is
involved in lysis of the membrane during cell-to-cell spread of L. monocytogenes), mpl
(encodes a metalloprotease), actA (encodes factors involved in actin polymerization), and
the lmu operon (induces delayed-type hypersensitivity reactions in L. monocytogenes-
immune mice). In addition, theJEaA gene that encodes flagellin protein, thejuR gene that
appears to modulate DNA topology, and genes encoding 16s and 23s ribosomal RNA
in L. monocytogenes have been sequenced. Some virulence-associated genes such as
hlyA are highly conserved and may not be suitable targets for strain identification. Others
such as the inlA operon and genes encoding cell surface structures such as the cell mem-
brane and flagella, may be more polymorphic and hence more useful for discrimination
of strains.
Rasmussen et al. [61] sequenced internal fragments of theJuA, iup, hly, and 23s
rDNA genes from different L. monocytogenes serotypes of clinical, food, and environmen-
tal origin. A 150-bp region of the hly gene was sequenced in 75 strains, with 27 strains
sequenced for the other genes. Although the DNA sequence data for hly, iupJEaA were
useful for identifying three lineages, the genetic diversity within the sequencing targets
was insufficient for adequate subtyping. As methods for DNA sequencing are simplified
and methods for direct sequencing of 1- to 2-kb fragments are developed, DNA sequencing
may become viable for subtyping L. monocytogenes.

COMPARISON OF METHODS USED TO SUBTYPE

L. MONOCYTOGENES
Besides the WHO Multicentre L. monocytogenes Subtyping Study, several additional stud-
ies have compared various subtyping methods using different sets of L. monocytogenes
isolates. Baloga and Harlander [ 1 3 used genomic DNA fingerprinting, ribotyping, serotyp-
ing, and MEE to study 28 strains of L. monocytogenes. In theiir study, DNA fingerprinting
was more discriminating than ribotyping. Norrung and Gerner-Smidt [5 I ] compared MEE,
ribotyping, REA, and phage typing. Overall, phage typing was the most discriminatory,
with a DT of 0.88 followed by REA, MEE, and ribotying with DIs of 0.87, 0.83, and 0.79,
respectively. Differences in discrimination were observed depending on 0 serotype.
For serotype 1, REA gave the best discrimination, whereas phage typing gave the best
results for serotype 4. Nocera et al. [49] also characterized 134 strains of L. monocytogenes
292 Graves et al.

(96 of serotype 4b) from an outbreak using ribotyping and four other typing methods
(serotyping, phage typing, MEE, and REA). For serotype 4b isolates, phage typing gave
the highest DI, followed by REA, MEE, and ribotyping, with various combinations of
these methods yielding higher DIs. Graves et al. [3 11 compared ribotyping and MEE using
305 isolates of L. monocytogenes. Although MEE was more discriminating than ribotyping
for this set of isolates, neither of these methods provided adequate discrimination for
serotypes 1/2b and 4b.
At the beginning of this chapter, we referred to the World Health Organization
(WHO) Multicentre Listeria monocytogenes Subtyping Study. Phase I of this study used
a set of 80 coded L. monocytogenes strains that included 11 sets of duplicates. An overview
of the study was reported by Bille and Rocourt [ 5 ] . Because several different methods
were compared using a well-defined set of isolates, these studies are very useful in compar-
ing the utility of the various subtyping methods. Table 1 shows a comparison of the meth-
ods used in the study. On the basis of these results, serotyping, phage typing, REA, PFGE,
and RAPD were selected for standardization in Phase 11. This effort should provide a
selection of standardized subtyping methods that can be employed in a variety of epidemi-
ological investigations to make multicenter comparisons of data possible.

ACKNOWLEDGMENTS
We thank Thomas Donkar and Eric Renner for their assistance with the literature review
for this chapter.

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10
Foodborne Listeriosis

ELLIOTT. RYSER
Michigan State University, East Lansing, Michigan

HISTORICAL OVERVIEW
Discovery of several listeriosis outbreaks during the 1980s that were positively linked to
consumption of cheese and raw vegetables has led to inclusion of Listeria rnonocytogenes
in the current list of bonafide foodborne pathogens. However, in retrospect, the concept
of listeriosis as a foodborne illness actually can be traced back to when L. rnonocytogenes
was first isolated. Nine years before Murray et al. [167] described L. rnonocytogenes in
1926, Atkinson [42] reported an outbreak of meningitis among five 2- to 9-year-old Austra-
lian children, caused by a small gram-positive, diphtheroid-type bacillus that was probably
L. rnonocytogenes. Similarly, two additional listeriosis outbreaks [67,21I ] accounted for
7 of 36 listeriosis cases recorded in the literature between 19 17 and 1943 [ 1361. Hence,
to explain these three small outbreaks, along with at least 35 other listeriosis outbreaks
that have included 2195 documented cases since 1949 (Table I), one might reasonably
postulate that food-to-human transmission of L. rnonocytogenes occurred in at least some
instances.
Early animal feeding studies also support the notion that listeriosis can be acquired
through consumption of contaminated food. The first accurate description of L. rnonocyto-
genes in 1926 by Murray et al. [ 1671 included trials in which three of six 32-day-old
rabbits were successfully infected via the oral route. Results from subsequent postmortem
examinations agreed with the previously observed pathological findings for naturally oc-
curring listeriosis in rabbits. Fourteen years later, Julianelle [ 1321 reported that white mice
died of generalized listerial infections after consuming drinking water inoculated with

299
300 Ryser

TABLE
1 Apparent and Confirmed Common-Source Outbreaks of Listeriosis
Involving 10 or More Cases
Number Possible vehicle of
Location Year of cases infection Reference
Halle, East Germany 1949- 1957 - 100 Raw milk, sour milk, 44,l 13,185,186,
cream, cottage cheese 192,215
Jena, East Germany 1954 26 Unknown 227
Soviet Union 1956 19 Pork, mouse 116
Bremen, West Germany 1960- 1961 81 Unknown 98
Halle, East Germany 1966 279 Unknown 177
Auckland, New Zealand 1969 13 Unknown 49
Anjou, France 1975- 1976 162 Unknown 71
Johannesburg, South Africa 1977- 1978 14 Unknown I25
Western Australia 1978- 1979 12 Raw vegetables 220
Massachusetts, USA I979 20 Raw vegetables, milk 122
Auckland, New Zealand 1979- 1980 10 Unknown 190
Auckland, New Zealand 1980 22 Shellfish, raw fish 144
East Cambria, England 1981 11 Cream 104,156
Slovakia 1981 49 Unknown 190
Maritime Provinces, Canada 1981 41 Coleslaw 210
Christchurch, New Zealand 1981-1982 18 Unknown 93
Houston, Texas, USA I983 10 Unknown 69
Saxony, West Germany 1983 25 Unknown I69
Massachusetts, USA 1983 49 Pasteurized milk 99
Vaud. Switzerland 1983- 1987 122 Vacherin Mont dOr 47.66
cheese
Los Angeles, CA, USA I985 142h Mexican-style cheesea 145
Denmark 1985- 1987 35 Unknown 200,208
Linz, Austria 1986 20 Raw milk, vegetables 3,226
Los Angeles, CA, USA 1986- 1987 33 Raw eggs 214
Los Angeles, CA, USA 1987 11 Butter 154
England 1987 23 Unknown 157
England, Wales, Northern 1987- 1989 366 Pritt2 159,195
Ire 1and
New York, NY, USA 1989 10 Shrimp I94
Denmark 1989- I990 26 Blue-mold or hard 131
cheese
Western Australia 1990 11 Processed meats or p h i 139, 234
France 1992 279 Jellied pork tonguea 106,126,207
France 1993 39 Pork piit6 rilletes 126
Italy 1993 18 Rice salad 206
Illinois, Michigan, Wiscon- I994 66 Chocolate milk 75
sin, USA
France I995 33 Brie de Meaux cheesea 126
France 1997 14 Pont IEvCque cheese -c
Italy 1997 1594 Sweet corn -c

a Vehicle of infection positively identified.

Estimated number of cases as high as 300.
Goulet. 1998. Personal communication.
Foodborne Listeriosis 301

L. monocytogenes. From these observations, both authors concluded that ingestion of L.

monocytogenes followed by penetration of the gastrointestinal tract by the bacterium is
one means by which listeriosis can be acquired.
As you will recall from the previous discussion of animal listeriosis, lactating cows
can shed L. monocytogenes in milk for long periods as a consequence of listeric mastitis
or abortion. Of greater importance are reports that clinically normal cows can shed listeriae
in their milk for at least 12 months [ 123,2171. Although L. monocytogenes was not isolated
from both the udder and milk of mastitic dairy cattle until 1944 [237], Burn [67] postulated
as early as 1936 that milk could serve as a vehicle of infection in humans. Two years
later, Schniidt and Nyfeldt [2 I I ] also suspected a causal relationship between listeriosis
in humans and dairy cows during a small listeriosis outbreak in Denmark but were unable
to confirm milk as the vehicle of infection.
Given the preceding information, it is not surprising that consumption of raw milk
was suspected as the most likely cause of the first documented foodborne outbreak of
listeriosis. This outbreak, which occurred in Halle, Germany, between 1949 and 1957
(Table l), was accompanied by additional outbreaks in Jena, Germany, and Prague,
Czechoslovakia, with all three outbreaks prompting numerous investigations dealing with
various epidemiological aspects of the disease. In 1955, the scientific literature on Listeria
that had accumulated over nearly 50 years was admirably reviewed by the late H. P. R.
Seeliger in his monograph entitled Listeriosis (in German). Interest in this disease was
so great that over 150 publications appeared in the literature between 1955 and 1957.
Consequently, Seeliger extensively revised and updated his monograph in 1957 (in Ger-
man) and again in 1961 (in English) [215] to the point where his book has been, until
recently, a leading source of information on the subject.
During this period of keen interest, Seeliger [2 151 and other European researchers
[57,124,138,23I] emphasized the likely importance of food in disseminating listeriosis,
which, in furn, resulted in some of the first studies dealing with the organisms suggested
ability to survive during pasteurization of milk, as described in Chapter 6. Although a
heightened awareness of L. monocytogenes led to documentation of at least nine listeriosis
outbreaks (613 cases) in the 20-year period from I960 to 1980 (Table 1 ), a lack of any
clear link to food along with continued difficulties in isolating this organism from food and
environmental sources are two likely reasons for a leveling off of interest in foodborne
listeriosis during this period. Although L. monocytogenes was not yet included in the list
of recognized foodborne pathogens published by the World Health Organization (WHO)
in 1976 [ 5 ] ,3 years later, this pathogen was placed under the heading of Bacteria Not
Conclusively Proved to Be Foodborne in the second edition of the well-known book
Foodbornc. Infections and Intoxications, edited by Riemann and Bryan [ 1931.
Ascribing a source to listerial infections has proven difficult because of a highly
variable incubation period ( 1 -2 I days) before clinical symptoms appear and the unavail-
ability of food samples for analysis at the time of onset. However, in 1981, the status of
L. monocytogenes began to change when Schlech et al. [210] reported that 17 of 41
(41.5%) people died of listeriosis after consuming coleslaw from which L. monocytogenes
was later isolated. This outbreak provided the first conclusive evidence that humans can
contract li steriosis by consuming contaminated food and also demonstrated that foods
other than dairy products can become contaminated with L. monocytogenes and thus con-
stitute a health risk to certain segments of the population. Two years later, three additional
listeriosis epidemics were documented (Table l), including one widely publicized out-
break in which consumption of pasteurized milk was epiderniologically linked to 49 cases
302 Ryser

of listeriosis (including 14 deaths) in Massachusetts. However, it must be emphasized that

L. monocytogenes was never isolated from the incriminated milk.
Any remaining doubt concerning the ability of L. monocytogenes to produce food-
borne illness completely vanished in June of 1985 when consumption of Jalisco brand
Mexican-style cheese was directly linked to at least 142 cases of listeriosis, including 48
deaths, in Los Angeles [ 1451. Thus the two aforementioned listeriosis outbreaks involving
consumption of contaminated coleslaw and Mexican-style cheese, along with another ma-
jor outbreak in Switzerland that resulted from consumption of contaminated Vacherin
Mont dOr soft-ripened cheese, have generated worldwide concern over the presence of
Listeria in dairy products and many other foods, including meat, poultry, eggs, seafood,
and vegetables. In response to this heightened awareness, eleven additional outbreaks
(eight European, two United States, one Australian) have been identified since 1987, in-
cluding two confirmed outbreaks involving pi%&(366 cases) and jellied pork tongue (279
cases) and the largest outbreak to date (1594 cases of gastroenteritis) possibly linked to
Italian sweet corn (Table 1). Consequently, L. monocytogenes has now moved to the ranks
of a bonafide foodborne pathogen in virtually all food microbiology textbooks [2,46,73,82,
83a, 130,162,166,180,191].
Since concern about foodborne listeriosis originally centered around dairy products,
particularly soft cheeses [212], it is only fitting to begin this chapter by reviewing the
known cases of listeriosis in which nonfermented and fermented dairy products were sus-
pected and/or proven as vehicles of infection. Special attention will be given to the three
cheese-related listeriosis epidemics documented in California, Switzerland, and France as
well as the 1983 outbreak in Massachusetts that was supposedly linked to consumption
of pasteurized milk and a more recent epidemic traced to pasteurized milk in Illinois [75].
Similar evidence for involvement/possible involvement of other foods, including red meat,
poultry, eggs, seafood, fish, vegetables, and fruits in cases of human listeriosis will be
presented in the remaining pages of this chapter.

RAW MILK
Sporadic cases of bovine mastitis and abortion in which L. monocytogenes was intermit-
tently shed in milk over several lactation periods have been recorded in the literature for
more than 50 years. Dairy cows that appear healthy also can serve as reservoirs for L.
monocytogenes and secrete the organism in milk. Once obtained from the cow, milk may
be further contaminated through inadvertent contact with feces and silage, both of which
often contain Listeria and are normally present in the dairy farm environment. Considering
the present estimate that 3-4% of the milk supply contains detectable levels of L. monocy-
togenes, it is easy to understand why raw milk was suspected as one of the most likely
sources of infection in several large European outbreaks of listeriosis.
The first evidence for foodborne transmission of L. monocytogenes can be found in
a series of anecdotal reports from Germany [ 1 13,2 151. During the reconstruction period
that followed the end of World War 11, a sharp increase in the number of stillborn infants
was observed at an obstetrical clinic in Halle, with approximately 100 cases recorded up
to 1952. Working in an antiquated laboratory, Potel [ 1841 concluded that these stillbirths
resulted from infection with Corynebacterium infantiseptica. However, in 1952, Seeliger
suggested and later confirmed that this rash of stillbirths was caused by L. monocytogenes
[ 1131. Unpasteurized milk as well as sour milk, cream, and cottage cheese were suspected
by Seeliger [215] as possible vehicles of infection in several cases observed in Halle.
Foodborne Listeriosis 303

Subsequently, Potel [ 1861 isolated identical serotypes of L. monocytogenes from a mastitic

cow and from stillborn twins delivered by a woman who had consumed this milk before
parturition. On this basis, Potel can be credited with the first description of foodborne
listeriosis in humans. During this outbreak, raw milk for pregnant women was generally
available only through the black market, which in turn suggests that consumption of raw
milk was likely responsible for additional cases of listeriosis [ 1131. After this listeriosis
epidemic ended in 1957, two subsequent outbreaks involving 180 and 160 cases were
identified in Halle during 1960 and 1961 [98] and in 1966 [177], respectively. Fischer
[98] also described a cluster of listeriosis cases that occurred in Bremen, Germany, be-
tween 1960 and 1961. Although neither the mode of transmission nor the primary reservoir
for L. monocytogenes were identified in these outbreaks, involvement of milk appears
unlikely, since most of these cases occurred during a time in which production and sale
of milk were both rigidly controlled.
Since the postwar listeriosis outbreak in Halle in which raw milk was linked to at
least one stillbirth, only two additional case studies have been published in which raw
bovine milk has been mentioned as a possible cause of listerial infection. In the first of
these cases, reported in 1973 [61], a 28-year-old Canadian woman went into premature
labor and delivered an infant who died of listeriosis 33 h after delivery. Shortly before
giving birth, the mother recalled purchasing raw milk and cream; however, these products
were no longer available for testing. The second case involved a 43-year-old male acquired
immunodeficiency syndrome (AIDS) patient in California who contracted listerial menin-
gitis in 1987. Following rigorous antibiotic therapy and complete recovery, the patient
admitted being a regular consumer of commercially available raw milk. Although the
investigating team made no attempt to confirm raw milk as the vehicle of infection, the
possible role of raw milk in this case suggests that current (1997) efforts by the U.S. Food
and Drug Administration (FDA) and the U.S. Centers for Disease Control and Prevention
(CDC) to inform AIDS patients about the potential threat of listeriosis, salmonellosis, and
other foodborne illnesses associated with consumption of certain high-risk foods (e.g.,
raw milk, surface-ripened cheese, undercooked poultry) should continue. Boiling has been
often suggested as one means of eliminating microbial pathogens from raw milk and en-
hancing the safety of this product. However, given one additional report [ 1741 of a 60-
year-old, j mmunocompromised Indian man who developed listerial septicemia and menin-
gitis after ingesting boiled raw milk, individuals at risk of developing listeriosis would be
well advised to consume only commerciallyproduced milk that has been properly pasteurized.
The threat of contracting milkborne listeriosis generally appears to be confined to
susceptible individuals who routinely consume raw bovine milk, with no listerial infections
yet linked to consumption of raw milk from other animals, including ewes and goats.
Thus, even when given the ability of L. monocytogenes to infect humans and produce
mastitis in animals, it is still surprising to learn that researchers in Yugoslavia [230] were
able to link one case of neonatal listeriosis to consumption of contaminated human breast
milk. According to their 1988 report, a 24-day-old infant girl contracted listeriosis after
receiving breast milk from her mother. Thirteen days after onset of symptoms, L. monocy-
togenes serotype 4b was isolated from the infants cerebrospinal fluid and blood as well
as the mothers milk. The infant recovered fully 3 days after cessation of breast-feeding.
Interestingly, excess breast milk from the mother was given to a newborn litter of three
Doberman puppies, and all three dogs became ill with vomiting, diarrhea, and bloody
stools. One of the animals died, and the same serotype of L. monocytogenes as found in
breast milk was detected in a stool specimen from one of the two survivors. Although
304 Ryser

this is currently the only report linking listeriosis to consumption of human breast milk, the
medical profession should be aware of the possibility for such transmission, particularly in
apparent nosocomial cases of neonatal listeriosis.

PASTEURIZED MILK
Until 1985, the only proven foodborne listeriosis outbreaks associated with dairy products
involved consumption of raw milk. However, this changed when Fleming et al. 1991 epide-
miologically linked consumption of a specific brand of whole and 2% pasteurized milk
to 49 cases of listeriosis in Massachusetts between June and August of 1983. As seen in
Table 2,42 (86%)of the cases occurred in adults and seven (14%) in mother-infant pairs.
Fourteen of 49 individuals died giving a mortality rate of 29%. Two years later, Todd
[225] calculated the total cost of this outbreak at $1.89 million ($1.37 million-deaths,
$387,000-hospitalization, $70,00O--investigation, $6 I ,000-financial/legal costs) or
$38,6 14 per case excluding legal settlements.
Although all adults had underlying conditions that resulted in immunosuppression,
symptoms expressed during the course of illness varied depending on the persons age
and degree of immunosuppression. Forty of 49 isolates were available for serotyping, with
32 (80%) being identified as serotype 4b, which was later defined as the epidemic strain.
Two case-control studies, one matched for neighborhood of residence and the other
for the patients underlying condition, indicated that development of listeriosis was
strongly associated with drinking a specific brand of pasteurized whole or 2% milk. Further
epidemiological investigations showed (a) a correlation between increased consumption
of the specific brand of whole or 2% milk and contracting listeriosis, (b) a lower incidence

TABLE
2 Characteristics of Adult and Perinatal
Listeriosis Cases Identified in Massachusetts Between
June 30 and August 30, 1983.
Number of cases (%)

Case profile Adult Perinatal

~ ~~

Total cases 42 (86)

Total fatalities 12 (29)
Sex: M/F 271 15
Clinical syndrome
Meningitis 13 (31)
Septicemia 29 (62)
Death in utero
Underlying condition
Cancer
Cirrhosis/alcoholism
Diabetes
Corticosteroid therapy
Renal transplant
Myelofibrosis
Chronic hepatitis
Intravenous drug abuse

Source: Adapted from Ref. 99.

Foodborne Listeriosis 305

of disease among individuals who drank skim or 1% milk produced by the same dairy,
(c) an association between several listeriosis cases in Connecticut and consumption of the
same brand of whole or 2% milk, and (d) an association with a specific phage type of L.
monocytogenes (phage type 2425A), which was isolated from all 19 listeriosis victims
who reportedly drank the specific brand of whole or 2% milk.
Although these epidemiological studies strongly suggest that this outbreak resulted
from consuming whole or 2% milk, the microbiological findings were far less convincing
in that L. rnonocytogenes was never isolated from the incrirninated pasteurized milk. The
milk implicated in this listeriosis outbreak was processed at a single dairy factory and
pasteurized at 77.2C (1 7 1 O F ) / 18 s [ 1881, which is well in excess of the minimum require-
ment (7 1.7C [ 161F]/15 s) specified in the Pasteurized Milk Ordinance. Furthermore, no
defect was identified that could have led to improper pasteurization and no source of
postpasteurization contamination was ever found within the dairy factory. Shortly after
the outbreak ended, a survey conducted by the CDC [99,112,119] indicated that 15 of
124 (12%) raw milk samples collected from the factory milk supply, individual farms,
and a milk cooperative that supplied the factory contained L. monocytogenes. Several
different serotypes were identified, including 1a, 3b, 4a/b, and 4b, the epidemic serotype.
Using DNA macrorestriction analysis [ 1261, the epidemic strain was later reported to be
genetically similar to isolates responsible for two subsequent outbreaks traced to p2te in
the United Kingdom [ 1591and France [ 1261 (Table 10) but distinctly different from strains
implicated in two North American outbreaks involving coleslaw and Mexican-style cheese
[235].However, neither the phage type nor the restriction enzyme type that was epidemio-
logically linked to this presumed outbreaks of milkborne listeriosis was ever recovered
from raw milk or the incriminated pasteurized milk.
Although the epidemiological evidence gathered by Fleming et al. [99] suggests this
outbreak resulted from drinking a particular brand of pasteurized whole or 2% milk, the
means by which L. monocytogenes may have found its way into the milk remains unclear.
Postpasteurization contamination of the milk cannot be excluded; however, it seems un-
likely, since inspections failed to recover L. rnonocytogenes from the dairy factory environ-
ment. Since whole and skim milk were processed each day using the same equipment, it
is also difficult to postulate a means by which only whole milk would have been subjected
to postpasteurization contamination.
To further support the involvement of pasteurized milk in this outbreak, the authors
concluded that intrinsic contamination of the milk and siirvival of some organisms de-
spite adequate pasteurization is both consistent with the rr:sults of this investigation and
biologically plausible; the latter conclusion was based on the now faulty but at the time
frequently quoted pasteurization study by Bearns and Girard [48]. Consequently, the ap-
parent association of listeriosis with consumption of pasteurized milk raised immediate
product safety concerns, which in turn led to numerous studies examining L. monocyto-
genes heat resistance (see Chap. 6). Nearly 2 years after the outbreak was reported in the
New England Journal of Medicine, Donnelly et al. [81 I found that the open-tube
method used by Bearns and Girard [48] was flawed and concluded that freely suspended L.
monocytogenes cells were unlikely to survive normal high-temperature, short-time (HTST)
pasteurization at 71.7C (161F) for 15 s. Knowing that this pathogen can exist within
milk leukocytes, and that milk in the Massachusetts outbreak was not clarified but rather
passed through a milk sock (coarse filter) that did not remove leukocytes, many researchers
postulated that the presence of L. monocytogenes within these leukocytes enhanced the
organisms resistance to pasteurization. Subsequent studies addressing this issue have gen-
306 Ryser

erally shown insignificant differences in the degree of thermal resistance between freely
suspended and internalized L. monocytogenes cells [84,1731. Using milk containing rela-
tively large numbers of intracellular listeriae, several workers demonstrated that L. mono-
cytogenes can survive minimum requirements for pasteurization; however, commingling
of milk from many farms before pasteurization would result in much lower levels of
listeriae (i.e., 5 10 CFU/mL) [38,146,218] with many, if not most, organisms being present
only extracellularly as a result of leukocytic breakdown during the first 24-48 h of cold
storage. Hence, on this basis, both the scientific community and the WHO maintain that
L. monocytogenes will not survive minimal milk pasteurization at 16l0F/15 s [236]. In
support of this position, L. monocytogenes has not yet been demonstrated to have survived
pasteurization in a commercial dairy product that met minimum HTST pasteurization re-
quirements.
Although numerous FDA Class I recalls of pasteurized dairy products, including
2%, 1%, and skim milk as well as chocolate milk, ice milk mix, ice milk, ice cream
mix, ice cream, ice cream novelties, sherbet, butter, and various cheeses, have been well
publicized in the United States, in virtually all instances L. monocytogenes was present
in the immediate manufacturing environment, which strongly suggests postpasteurization
contamination. Given this information, the likelihood of L. rnonocytogenes having sur-
vived pasteurization at -77.2"C (17 1OF)/ 18 s in the Massachusetts outbreak appears re-
mote at best.
Since L. monocytogenes was never isolated from pasteurized milk implicated in
the Massachusetts outbreak or the dairy factory environment, some investigators have
questioned the role of milk [80]. In 1988, several design flaws were discovered in the
case-control studies [80] that included missing questions and data on questionnaires, as
well as a disproportionate number of follow-up interviews between cases and controls,
any of which might have given an unfair bias. Contrary to the authors [99], cases were
generally clustered around the Boston area in a manner that was not consistent with the
milk distribution pattern. Furthermore, inconsistencies in data were noted between expo-
sure to implicated milk and isolation of the L. monocytogenes strain supposedly responsi-
ble for the epidemic [go]. Discovery of these discrepancies in the various case-control
studies prompted a lawsuit against the CDC; however, as one might expect, a definitive
answer concerning involvement of pasteurized milk in this outbreak was never reached
through the judicial system.

Chocolate Milk
In July of 1994, Dalton et al. [75]reported that 54 of 60 (90%) previously healthy individu-
als who attended a summer picnic in Illinois developed listeriosis 9-32 h (median 20 h)
after consuming one or more 8-oz cartons of pasteurized chocolate milk with four, three,
and five related cases later reported in Illinois, Wisconsin [ 1891, and Michigan, respec-
tively. Unlike most foodborne listeriosis epidemics recorded to date, gastrointestinal symp-
toms predominated among picnickers, with victims most commonly experiencing diarrhea
(79%), fatigue (74%), fever (72%), chills (65%), headache (65%), myalgia (59%), abdomi-
nal cramps (55%), nausea (47%), and vomiting (26%) over several days. Only four indi-
viduals required short hospitalization, with one pregnant woman delivering a healthy baby
5 days after experiencing a 6-h bout of diarrhea.
In support of the aforementioned findings, the clinical strain of L. monocytogenes
identified as serotype 1 /2b was soon isolated from multiple unopened containers of choco-
Foodborne Listeriosis 307

late milk at levels of 8.8 X 108 to 1.2 X 109 CFU/mL (Le., mean infective dose of
2.9 X 10'' CFU/person), with the product's taste and quality reportedly being poor. During
a follow-up environmental survey of the implicated milk processing facility, investigators
recovered the epidemic serotype of L. monocytogenes from a floor drain beneath the choco-
late milk filler and from a valve connected to the chocolate: milk pasteurizer. Based on
multilocus enzyme electrophoresis, ribotyping and pulsed-field gel electrophoresis, the
clinical, chocolate milk, and factory environmental isolates of L. monocytogenes serotype
1/2b were indistinguishable from each other, thereby confi.rming chocolate milk as the
vehicle of infection.
Although properly pasteurized at >87"C/ 18 s after the addition of chocolate flavor-
ing and immediately cooled to <8"C, the product was held unrefrigerated for 2 h in a
malfunctioning and improperly sanitized tank before being pumped to the filling machine
over a 7-h period. In all likelihood, this outbreak occurred as a result of postpasteurization
contamination. However, given the ability for rapid growth of L. rnonocytogenes in tem-
perature-abused chocolate milk [ 1991 (see Chap. 1 l), inadequate and/or nonexistent
refrigeration during packaging (7 h), and transit (2.25 h) to the picnic are obvious contrib-
uting factors along with consumption of the product 3 days before the expiration date.

OTHER NONFERMENTED DAIRY PRODUCTS

A search of the early literature has uncovered only a few inconclusive reports suggesting
that listeriosis can be contracted by consuming nonfermented dairy products other than raw
and pasteurized chocolate milk. After the first massive listeriosis outbreak was reported in
Halle, Gerinany, between 1949 and 1957, Seeliger [215] indicated that in addition to raw
milk, sour milk and cream also were considered as possible sources of infection in several
cases. Although such products could certainly contain L. rnonocytogenes, the exact role
of raw milk and cream in these cases was never determined. During the latter half of 1981,
an apparent common-source listeriosis outbreak was recorded in East Cambria, England, in
which identical phage and serotypes of L. monocytogenes were isolated from 1 I patients
[ 1561. Even though epidemiological evidence indicated a possible association with con-
sumption of pasteurized cream [45,104], the exact mode of transmission was never veri-
fied. Similarly, Ralovich [ 1901 learned of a personal account in which L. monocytogenes
was isolated from a cream-based rice soup associated with illness; however, the etiological
role of Listeria was never proven.
Following the widely publicized 1985 listeriosis outbreak in California in which
numerous deaths were directly linked to consumption of contaminated Mexican-style
cheese, Los Angeles County officials instituted an active surveillance program for listeri-
osis and made the disease reportable [ 1521. These efforts wentually led to discovery of
a cluster of 1 I perinatal listeriosis cases among Hispanics during November and December
of 1987 [151]. Seven of 11 L. monocytogenes isolates obtained from the victims were of
serotype 1 /2a. Although a subsequent case-control study identified butter as a possible
vehicle of infection (odds ratio = 4), this first-time association between consumption of
butter and listeriosis was not culturally confirmed. Absence of Listeria spp. from butter
tested by the FDA during the Dairy Initiatives Program 1381 and evidence indicating that
large numbers (-92-97%) of L. monocytogenes are lost in buttermilk and washings when
butter is manufactured from inoculated cream [176] suggest that butter is not a major
vehicle for transmission of listeriae. Nonetheless, L. rnonocytogenes can occasionally be
found in hutter with at least five Class I recalls issued thus far without incident.
308 Ryser

Attempts to implicate frozen dairy products as vehicles of infection in listeriosis

cases are of relatively recent origin. In 1986, FDA officials investigated an incident in
which L. monocytogenes was isolated from amniotic fluid following the premature delivery
of an infected infant who died 5 days later [ 1 I 1. After learning that the 2 1-year-old mother
consumed ice cream sandwiches 3 days before delivery, all suspect product was withdrawn
from store shelves; however, follow-up studies failed to incriminate ice cream as the
source of infection.
Since 1985, enhanced surveillance of the dairy industry by the FDA has prompted
over 50 recalls of Listeria-contaminated frozen dairy products, including ice cream, ice
milk, sherbet, and ice cream novelties as well as ice milk, ice cream, and milk shake mix.
Consumption of such products could potentially constitute a public health risk. Hence,
preliminary epidemiological results from the CDC suggesting a possible link between
consumption of ice cream and a cluster of 31 listeriosis cases, including 14 deaths, in
Philadelphia, Pennsylvania, appeared to have some merit [ 171. However, inability to iso-
late L. monocytogenes from ice cream eliminated this product as the vehicle of infection
[ 18,2131. After repeated attempts to (a) identify a common epidemic strain and (b) isolate
Listeria from cheese and other dairy products as well as meats and vegetables ended in
failure, the CDC finally retracted their previous statement and concluded that a common-
source outbreak of foodborne listeriosis had not occurred in the Philadelphia area [ 16,2 131.
Although well over 4 million gallons of frozen dairy products thought to be contami-
nated with Listeria have thus far been recalled from the market at a cost in excess of $88
million [19], not one case of listeriosis has been directly linked to consumption of frozen
dairy products marketed in the United States. The only proven case comes from Belgium
[4], in which an immunocompromised 62-year-old man developed listerial meningitis after
consuming commercially prepared ice cream containing 1O4 L. monocytogenes serotype
4b CFU/g, with this pathogen coming from contaminated cream. Apparent low levels of L.
monocytogenes in frozen dairy products resulting from postpasteurization contamination
combined with the organisms obvious inability to grow during frozen storage [51] are
but two reasons why these products appear to constitute a very minimal risk to public
health.

FERMENTED DAIRY PRODUCTS

Certain species of lactic acid-producing bacteria can be used to ferment fluid milk into
a wide array of dairy products, including cultured buttermilk, sour cream, and yogurt as
well as hundreds of cheese varieties. Thus far, no listeriosis outbreaks have been positively
linked to consumption of contaminated yogurt, sour cream, or cultured buttermilk; how-
ever, the same cannot be said for cheese. As early as the 1950s, cheese was suspected of
playing a role in foodborne listeriosis. However, since June of 1985, three major listeriosis
outbreaks have been directly linked to contaminated soft cheese, thus confirming its role
in foodborne listeriosis. The first such outbreak involved consumption of Mexican-style
cheese in California and was followed by a second major outbreak in Switzerland, which
was traced to contaminated Vacherin Mont dOr soft-ripened cheese. Most recently, Brie
de Meaux and Pont 1Eveque cheese were responsible for two smaller listeriosis outbreaks
in France. These outbreaks will now be reviewed in some detail, after which several spo-
radic cases of cheeseborne listeriosis also will be discussed.
As just mentioned, the notion that humans can contract listeriosis by consuming
Listeria-contaminated cheese is not new. Along with raw milk, sour milk, and cream,
Foodborne Listeriosis 309

Seeliger [2 15) suggested a possible relationship between consumption of cottage cheese

and several cases of listeriosis that occurred in Halle, Germany, mentioned earlier. None-
theless, it must be stressed that cottage cheese was never confirmed as the vehicle of in-
fection. A search of the scientific literature revealed no additional reports suggesting involve-
ment of cheese in cases of listeriosis during the 30 years that followed the Halle incident.

Mexican-Style Cheese: California, 1985

In June of 1985, the ability of cheese to serve as a vehicle for foodborne listeriosis became
evident when consumption of Jalisco brand Mexican-style cheese was linked to a massive
listeriosis outbreak in southern California. This outbreak, which later proved to be among
the deadliest of all known outbreaks of foodborne disease recorded in the United States,
prompted much dairy-related research worldwide, most of which is reviewed elsewhere
in this book. Before dealing with the facts of this outbreak, it is appropriate to review
chronologically the detective work conducted by various. governmental agencies (Fig.
1) that linked this outbreak to Mexican-style cheese and also led to a nationwide recall
of the product on June 17, 1985.
According to information appearing in local newspapers, the first listeriosis case
associated with this outbreak was diagnosed in Los Angeles County during the first week
of January 1985, with one and three additional cases being documented during the second
and fourth weeks of January, respectively. Unknown to public health officials, the rate of
listerial infections continued to increase, with six and nine cases being reported in Los
Angeles County during February and March, respectively.
Several important chance happenings led to discovery of this outbreak in the weeks
that followed. The first hint of a possible problem was uncovered at the Los Angeles
County-University of Southern California (USC) Medical Center-a vast medical com-

Fi 1.
2.
3.
Coordinate e n t i r e i n v e s t i g a t i o n
I n i t i a t e case-control s t u d i e s
Serve a s a c e n c r n l l a b o r a t o r y f o r L i S t e r i a - t e S t i n g

Collect l o c a l Los Angeles County Dept. 1. I n v e s t i g a t e c a s e s of l i s t e r i o s i s

l i s t e r i o s i s data of Health S e r v i c e s - 2. Spread news of ourbreak t o
Lead I n v e s t i g a t o r Spanish communit y

r--l
Orange County
Health Care Agency He a 1t h S e r v i c e s

J a l i s c o Mexican L3ok f o r c a u s e o f
P r o d u c t s , Inc. 4 contamination

FIGURE1 Primary roles o f local, state, and federal agencies in investigating the 1985
listeriosis outbreak in California.
310 Ryser

plex at which about 17,000 infants are delivered each year [ 128,1291.In early April, Carol
Salminen, a nurse epidemiologist who monitored infection rates at this facility, uncovered
five additional listeriosis cases among Hispanic motherhnfant pairs during the previous
2 weeks. Under normal circumstances, only three to five listerial infections would be
observed annually at this hospital. After consulting with the medical director of the labor
and delivery service, who had developed a personal interest in listeriosis and maintained
a log of listerial infections over the previous 10 years, Salminen informed health officials
at the Los Angeles County Department of Health Services on May 6 that nine listeriosis
victims had been treated at the USC Medical Center since January of 1985. Once notified,
health officials at the Los Angeles County Department of Health Services and the Orange
County Health Care Agency began surveying area hospitals for additional cases. After
the reported number of listerial infections increased from 16 to 67 in just a few days, Los
Angeles County health officials contacted the California Department of Health Services
and the CDC on May 10 for investigative assistance. At the same time, 200 area hospitals
in the counties of Los Angeles and Orange were requested to report all listeriosis cases
to the health department.
Ten days later, health officials and epidemiologists from the CDC began the first
of two case-control studies in which listeriosis victims, mostly previously healthy Hispan-
ics, were interviewed about various environmental factors, behavioral patterns, and con-
sumption of over 60 food items, including fresh fruits and vegetables, water, milk, and
cheese. On May 29, an open package of Jalisco brand Mexican-style cheese was taken
from one of the victims refrigerators and sent to the CDC in Atlanta, Georgia, for analysis.
After CDC investigators provided preliminary confirmation of L. monocytogenes in this
opened package of Mexican-style cheese on June 8, 20 packages of Mexican-style cheese
of various brands, including two packages of Queso Fresco and Cotija Jalisco brand
cheese, were purchased at area markets near the victims place of residence and sent to
the CDC for analysis [145]. On June 10, results from the case-control study described
earlier clearly demonstrated that individuals who had consumed Mexican-style cheeses
were at increased risk of contracting listeriosis. Armed with this information, investigators
immediately began a second case-control study in which individuals were questioned
about the names and brands of Mexican-style cheese consumed. On June 12, statistical
analysis of these data revealed a definite link between consumption of Jalisco brand Mexi-
can-style cheese and development of listeriosis [ 1451. FDA officials were immediately
advised of the impending problem with Jalisco cheese. Confirmation of L. monocytogenes
serotype 4b in two unopened packages each of Jalisco brand Queso Fresco and Cotija
Mexican-style cheese-the last piece of evidence needed to initiate a Class I recall of the
product-was provided by the CDC on June 13. (Subsequent studies eventually identified
the epidemic L. monocytogenes strain in 82% of all Jalisco brand products purchased at
area supermarkets.) Armed with this information, the California Department of Food and
Agriculture immediately closed the Jalisco cheese factory and announced a statewide re-
call for these two varieties of Mexican-style cheese, -80% of which was sold through
retail outlets in Los Angeles and Orange Counties. On June 14, state officials expanded
this recall to include the firms entire line of 44 products (predominantly cheese), consump-
tion of which was already blamed for at least 28 deaths. Hence, in the weeks that followed,
health officials were faced with the enormous task of checking -28,000 Los Angeles-
area supermarkets, family-owned grocery stores, and restaurants to ascertain that all Jali-
sco brand products were removed from shelves. FDA officials also ordered a Class I recall
of all Jalisco brand products distributed in California and in 12 other primarily western
Foodborne Listeriosis 31 1

states [6]. Three days later, this recall was expanded to include all 26 states in which these
products were sold as well as the United States Protectorates of Guam, American Samoa,
and the Marshal1 Islands [8]. When this recall was completed on June 22, nearly 250 tons
of Jalisco brand Mexican-style cheese and other dairy products were ready for burial in
a landfill site overlooking the San Gabriel Valley.
Even though the number of individuals who actually contracted listeriosis after
eating the tainted cheese has been a debatable issue for some time, the exact figure will
never be known, since mild listerial infections in individuals who did not seek medical
attention obviously went unreported. Newspaper accounts have placed the total number
of listeriosis cases occurring in California between January 1 and August 15 at nearly
300, including 85 fatalities. Although about half of these cases were concentrated within
the Hispanic communities of Los Angeles and Orange County, a substantial number of
listeriosis victims also reportedly resided in the San Diego area, which made the collection
of reliable data more difficult. In addition, at least 16 cheese-related listeriosis cases were
uncovered outside California (Arizona, Colorado, Oregon, Texas, and Connecticut) with
three fatalities being reported in Texas.
Although the total number of listeriosis cases reported in Los Angeles County during
the 12-month period immediately following the outbreak decreased to 94, the calculated
-
annual crude incidence rate of 12 cases/million population is still approximately twice
the national average [ 1521. Numbers of reported listerial infections have continued to
decrease in Los Angeles County, with 1990 and 1994 rates of nonperinatal and perinatal
listeriosis decreasing from 6 to 3 cased1 million population and 17 to 6 cases/100,000
live births, respectively [72,223]. The fact that these rates were previously well above the
national average is not too surprising when one considers that this outbreak certainly made
area physicians, hospital personnel, and public health authorities keenly aware of this
disease. In all likelihood, these factors in combination with mandatory reporting of this
formerly obscure illness were largely responsible for the abnormally high incidence of
listeriosis in Los Angeles County.
In 1988, Linnan and 14 other members of the investigative team [145] published
their findings concerning 142 listeriosis cases that were linked to consumption of Jalisco
brand cheese in Los Angeles County between January 1 and August 15, 1985. Although
nearly 160 additional cases occurred elsewhere in California (Orange, San Diego, and
Fresno Counties) and in other states, logistical concerns limited their studies to Los
Angeles County. During the 7.5-month epidemic period, 93 reported listeriosis cases
(65.5%) involved pregnant women or their offspring with 49 (34.5%) affecting nonpreg-
nant adults (Fig. 2, Table 3). Forty-eight of the 142 listeriosis victims died, giving an
overall mortality rate of 33.8%. Thirty deaths occurred among the 87 early fetalheonatal
cases; however, no late fetal/neonatal or maternal deaths were reported. All but 1 of the
49 nonpregnant adults had a predisposing condition such as cancer (3 patients), steroid
dependency (12 patients), chronic illness (23 patients), age :>65 ( 5 patients), or AIDS (3
patients), which placed these individuals at greater risk (than the normal population) of
developing listerial infections [91,2121.
After identifying the epidemic L. monocytogenes strain as belonging to serotype 4b,
all 105 clinical isolates available for study were phage typed and compared with the strain
isolated from Jalisco brand Mexican-style cheese. Results showed that 86 of 105 (82%)
clinical isolates were serotype 4b, with the remaining 19 non--serotype 4b isolates originat-
ing from listeriosis victims whose illnesses were presumably not related to consumption
of contaminated cheese. Of the 86 isolates identified as L. monocytogenes serotype 4b,
372 Ryser

N onpre g n an I I n
...
...

Mate

~~~
4

2
0
&
...
...
...
...
..
.
....
..
...
...
...
...
.... ..
....
.*:.

...
...

Jan MU *Pr May Jun Jul

Week of Positive Culture

FIGURE2 Listeriosis cases classified according t o risk group in Los Angeles Country,
January 1 t o August 15, 1985. Arrow designates the time of recall 11451.

TABLE 3 Clinical and Demographic Data o n 142 Listeriosis Cases Occurring in Los
Angeles County, California, Between January 1 and August 15, 1985

Fetal or neonatal
Non pregnant
Variable Early Late Maternal adults
No. of patients 87 6 93 49
Mean age 32 weeks 38 weeks 26 yr 58 yr
gestation gestation
Race or ethnic group:
number (%)
Hispanic - - 81 (87) 14 (29)
White - - 10 (1 ) 26 (53)
Black - - 0 7 (14)
Asian - - 2 (2 2 (4)
Fatalities (%) 30 (34) 0 0 18 (37)
Epidemic phage type (%) - - 75 27
Mean birth weight (kg) 2.54 3.15 - -
Septicemia (%) 88 17 52 71
Meningitis (%) 2 67 0 14
Septicemia -t meningitis (5%) 6 17 0 14
Other positive culture (9%) 4 0 48 2
Source: Adapted from Ref. 145.
Foodborne Listeriosis 3 13

63 (73%) sfrains were of the same phage type as strains isolated from the contaminated
cheese. In several follow-up subtyping studies, clinical and cheese isolates were of the
same multilocus enzyme electrophoresis type [55,110], ribotype [ 1 101, and pulsed-field
gel electrophoresis type [65,163], and they also gave identical restriction enzyme [235]and
random amplified polymorphic DNA patterns [74], thereby confirming the involvement of
Jalisco brand cheese in this outbreak (Fig. 3). The 23 remaining clinical isolates belonging
to nonepidemic phage types presumably represented non-cheese-related background cases
of listeriosis that occurred throughout the year. Sporadic sale of tainted cheese in a few
family-owned grocery stores and restaurants, along with a likely listeriosis incubation
period of 3 days to 2 weeks, are both, at least partly responsible for those cases which
occurred beyond the middle of July with secondary infections being spread by fecal shed-
ding also a probable contributing factor [ 1531.
Although these statistics are fairly typical for human listeriosis cases, the most strik-
ing feature in Table 3 is that 81 of the 93 motherhfant pairs that contracted listeriosis
were of Hispanic origin. Furthermore, many of these economically disadvantaged Hispan-
ics sought treatment at the Los Angeles County-USC Medical Center. Clustering of cases
at a single medical facility was instrumental in uncovering, this outbreak of foodborne
listeriosis, since this epidemic would have likely gone unnoticed if the cases had been
distributed evenly among the nearly 200 major hospitals in metropolitan Los Angeles.
As previously mentioned, epidemiologists and health officials used data collected
from two case-control studies to trace this outbreak first to1 Mexican-style fresh cheese
and then to Jalisco brand Queso Fresco and Cotija cheese. The fact that these strong-

J an Feb MU APT May Jun Jul Aug

Week of Positive Culture

FIGURE3 Listeriosis cases classified according t o epidemic and nonepidemic phage

types in Los Angeles County, January 1 t o August 15, 1985. Arrow designates the
time of recall.
314 Ryser

flavored cheeses are a common part of the Hispanic diet and not widely consumed by
other individuals was another key in determining the exact source of this epidemic.
Thus recognition of this outbreak was largely possible because of the use of the
Los Angeles County-USC Medical Center by many economically disadvantaged listeri-
osis victims and the predominance of cases among Hispanics, which in turn precipitated
the involvement of Mexican-style cheeses (i.e., products which few other groups of indi-
viduals consume on a regular basis). Hence one can easily speculate that this clustering
of cases would not have been observed if a nonethnic food such as Cheddar cheese, milk,
and ordinary fruits or vegetables-all of which are consumed by most individuals-had
been contaminated with L. monocytogenes. Under such circumstances, an epidemic would
probably have gone undetected. Without the increased awareness that this outbreak
brought to the scientific community, additional cases of foodborne listeriosis, including
the 1987 outbreak linked to consumption of Vacherin Mont dOr soft-ripened cheese,
would likely have gone unnoticed, as the Swiss outbreak previously had for almost 10
years. Hence although Murray et al. [ 1671 can be credited with the first accurate description
of L. monocytogenes, those individuals who investigated the I985 listeriosis outbreak in
California (and to a lesser extent, outbreaks of listeriosis associated with coleslaw and
possibly pasteurized milk in Canada and Massachusetts, respectively) can be credited with
fostering the emergence of L. monocytogenes as a serious foodborne pathogen of world-
wide concern.
Once Jalisco brand cheese was positively identified as the vehicle of infection, local,
state, and federal investigators were confronted with the task of determining how the
cheese became contaminated (see Fig. 1). Additional testing of Jalisco brand cheese indi-
cated that L. monocytogenes was present in cheese manufactured from January to mid
June, thus indicating an ongoing problem at the cheese factory. Investigators then focused
their attention on three areas: (a) raw milk supply, (b) adequacy of pasteurization, and (c)
possible contamination of the cheese during manufacture, packaging, and/or ripening.
However, before interpreting the results from these investigations, it would be prudent to
deal with methods used to manufacture those varieties of Mexican-style cheese that were
directly linked to cases of listeriosis and also with behavior of L. monocytogenes in the
finished product.
Queso Fresco, or fresh cheese (also known as Ranchero, Estilo Casero, or Quesito),
is among the most popular and widely distributed Mexican-style cheeses. Unlike most
cheese varieties, Queso Fresco is traditionally prepared without a lactic acid bacteria starter
culture. Curd is formed by coagulating warm skim milk with rennet or a similar coagulant.
The resulting curd is drained in cheesecloth, salted, packed into hoops, and pressed under
weights for several days. The final product, which is consumed without additional aging,
has a slightly grainy texture and can be sliced or shredded for cooking.
Cotija, also known as Queso Sec0 (dry cheese) or Queso Anejo (aged cheese), is
another white cheese. After cutting, the curd is pressed in large round hoops and cured
at least 3 months to produce a dry, sharp-flavored, odorous cheese, which in some respects
resembles Italian Parmesan.
It is important to realize that these cheese-making procedures produce favorable
conditions for multiplication of L. monocytogenes in the final product. The relatively high
moisture content of these Mexican-style cheeses, and absence of a starter culture which
leads to pH values 1 5 . 6 in the finished product, both played crucial roles in allowing L.
monocytogenes to grow in the cheese during refrigerated storage [ 1211. According to Lee
Foodborne Listeriosis 3 15

[ 1421, surface and interior samples of frozen Jalisco brand cheese examined some months
after the recall contained 1.4 X 104and 5.0 X 104L. rnonocytogenes CFU/g, respectively,
which supports the hypothesis that the pathogen grew in cheese during refrigerated storage.
Numbers of listeriae increase approximately 10-fold during cheese making as a result of
entrapment within curd particles. If one assumes that the pathogen did not grow in cheese
during storage, then the milk from which the cheese was prepared would have had to
contain unreasonably high levels of listeriae (- 1000-5000 CFU/mL) to produce the popu-
lations observed by Lee in the finished product. It also is noteworthy that the epidemic
strain of L. rnonocytogenes that was designated as strain California (CA) by Ryser and
Marth [202] has since proven to be less hardy in Cheddar [202], Camembert [203], brick
[204], Colby (2401, feta [ 1791, and blue cheese [ 1781 than strains Scott A (clinical isolate
from the 1983 milkborne listeriosis outbreak in Massachusetts), V7 (raw milk isolate
from Massachusetts), and/or Ohio (OH) (isolated from Liederkmnz cheese manufactured
in Ohio).
Comprehensive sanitation inspections of the cheese factory were conducted immedi-
ately after the recall to assess the possibility that the cheese was contaminated during
manufacture, packaging, and/or storage. Although the factory received a satisfactory sani-
tation rating of 85 on a scale of 100, numerous problem areas were cited which included
suspended filth on electrical wires near cheese vats, peeling paint above a pasteurizer vat,
condensate dripping on cheese in a walk-in refrigerator, and a major ant infestation. Sev-
eral L. monocytogenes isolates from environmental samples (i.e., cooler condensate,
cheese curd, insects, pasteurizer) also yielded the same restriction enzyme profile as the
epidemic strain [235], thereby confirming several possible modes of transmission. Al-
though these environmental sources could have contributed to sporadic contamination of
the finished product, the fact that the epidemic strain was isolated from 22 of 85 lots of
cheese produced between January and mid June as well as from Cotija Fresco cheese
[235] indicates that an ongoing problem existed in the factory. Hence, contact between
cheese and the factory environment was likely not to be the major route of contamination
in this outbreak.
At the time of the recall, government officials considered faulty pasteurization as
one of the most likely means by which the cheese became contaminated. Initial factory
inspections uncovered various pasteurization problems related to record keeping and re-
cording charts; however, the time and temperature at which milk was pasteurized exceeded
minimum requirements (71.7C [16l0F]/15 s). Dye testing later revealed a number of
pin-sized holes in the pasteurization units heat-transfer plates which separate raw and
pasteurized milk. However, since further inspection demonstrated that the booster pumps
of the pasteurizer had maintained a higher pressure on the pasteurized rather than raw
milk side of the heat exchanger, raw milk would not have passed through the pinholes
found in the pasteurizer plates. Hence pasteurization failure was no longer suspected as
the source of contamination [7].
Final reports indicate that L. rnonocytogenes most likely entered the cheese during
manufacture through direct addition of raw milk. Toward the end of June 1985, investiga-
tors documented that the firm received nearly 700,000 pounds (- 10%) more raw milk
between April 1 and June 12, 1985, than could have been pasteurized given the capacity
of their pasteurizer. Additionally, on several days only 150,000 of 200,000 pounds of milk
received was pasteurized. These enormous discrepancies between raw milk received and
the quantity pasteurized suggest that unpasteurized milk was deliberately mixed with pas-
316 Ryser

teurized milk for cheese making [ 1451. This conclusion also is supported by the fact that
cheese supposedly prepared from pasteurized milk contained excessive levels of alkaline
phosphatase-a native, heat-labile enzyme normally destroyed during proper pasteuriza-
tion. However, some caution must be used in interpreting these results, since Pratt-Lowe
et al. [ 1871demonstrated that California Queso Fresco cheese occasionally contains micro-
organisms which produce a heat-labile alkaline phosphatase similar to that found in raw
milk. Under these conditions, cheese prepared from properly pasteurized milk may falsely
appear as having been manufactured from raw milk. Continued preparation of Mexican-
style cheese from a mixture of raw and pasteurized milk also is compatible with the pattern
of listeriosis cases that occurred over a period of 7 months. Toward the end of July 1985,
investigators visited 27 dairy farms that supplied raw milk to the cheese factory [ 145,1501.
Although no Listeria spp. were detected in raw milk or milk filters from dairy farms
supplying the Jalisco cheese factory, the same epidemic phage and restriction enzyme
type of L. monocytogenes was isolated from jocoque (a sour cream-like product), sodium
caseinate (a jocoque ingredient), and cottage cheese by-products produced by another
company that shared the same raw milk source with Jalisco cheese [235]. However, no
cases of listeriosis were epidemiologically linked to consumption of either of these prod-
ucts. According to Hird [121], the L. monocytogenes epidemic strain was uncommon in
California during the 1 1-year period preceding the outbreak, with only three to five L.
monocytogenes serotype 4b isolates belonging to the same epidemic phage type. Even
though the epidemic strain was never isolated from raw milk, evidence described in the
preceding paragraphs strongly suggests that raw milk was the probable source of L. mono-
cytogenes.
Major outbreaks of foodborne disease not only cause great human hardship but also
major financial difficulties from lost product and employee wages as well as medical bills
and lawsuits. Jalisco Mexican Products, Inc., was forced to close its doors and declare
bankruptcy shortly after its products were recalled, because the company could no longer
meet expenses. On March 27, 1986, Los Angeles County prosecutors filed 60 misde-
meanor charges against the president and vice-president of the company for alleged short-
cuts and inadequate safety precautions that routinely occurred in the factory during cheese
making [ 151. On May 20, 1986, the vice-president of the firm was sentenced to 60 days
in jail, 2 years of probation, and was fined $9300 in connection with manufacturing and
selling Listeria-contaminated cheese [ 141. In addition to investigative costs, which report-
edly totaled $617,204, the company also is believed to be facing up to $700 million in
lawsuits filed by some of the victims [225], making this one of the costliest and deadliest
outbreaks of foodborne illness in U.S. history.
After criticism for not recalling the contaminated Jalisco brand cheese sooner, state
and federal officials issued a Class I recall for Mexican-style cheese produced by a second
Los Angeles-area firm. Although this Listeria-contaminated cheese was supposedly pre-
pared from raw milk as shown by the presence of alkaline phosphatase [ 101, the recall
was subsequently downgraded to Class I1 (i.e., a situation in which use of the product
may cause temporary or medically reversible adverse health consequences) after labora-
tory results confirmed that these cheeses contained L. innacua-a nonpathogenic Listeria
species-rather than L. monocytogenes. Later investigators also showed that this cheese
was prepared from properly pasteurized milk. Hence, one must conclude that false-
positive results were obtained with the phosphatase test, as described earlier in this
chapter .
Foodborne Listeriosis 317

Vacherin Mont d'Or Soft-Ripened Cheese: Switzerland,

1983- 1987
Human listeriosis has been observed in Switzerland for many years [85], particularly in
the Canton of Vaud, which borders France to the west and Lake Geneva to the south.
The early scientific literature contains several reports of sporadic listerial infections that
occurred in and around Lausanne, the population center of Vaud. Over 20 years ago,
Piolino and de Kalbermatten [ 1831 reviewed five adult cases of listeriosis that were diag-
nosed at Vaudois University Hospital Medical Center in Lausanne (VUHC) between De-
cember 1964 and February 1967. Listeria monocytogenes serotype 4b was isolated from
four of five patients who exhibited symptoms of meningoencephalitis. Although no com-
mon source of infection was demonstrated among these individuals, the authors suggested
a possible role of domestic livestock in spreading listeriosis.
In 1981, Yersin et al. [239] reviewed 10 adult cases of listeriosis (ages 35-76 years)
that were diagnosed at VUHC between January 1974 and January 1980. (Ten cases of
neonatalhfant listeriosis also were treated at this hospital during the same period.) All
10 adult patients suffered from one or more underlying illnesses, which increased their
chance of developing listeriosis. During this 6-year period, two cases of septicemia, six
of meningitis-encephalitis, and two of encephalitis were recorded, including five deaths
(50% mortality rate). As in the previous study, none of the adult cases of listeriosis could
be traced to an exact source of infection.
Subsequently, Malinverni et al. [ 1481 noted that only 20 sporadic listeriosis cases
were diagnosed at VUHC between 1974 and 1982, with a mean of three cases per year.
Thus these figures reflect an endemic rate of approximately five listeriosis cases/ 1O6 popu-
lation in the Canton of Vaud during this 9-year period. In sharp contrast to these findings,
a cluster of 25 listeriosis cases (14 adults and 1 1 maternal/fetal) was observed at the same
medical facility between January 1983 and March 1984 [147,148], with 15 additional
cases being documented in surrounding hospitals (e.g., Geneva and Neuchatel) in western
Switzerland during the same 15-month period [ 13,1471.
This epidemic was somewhat atypical in that most adult listeriosis victims had been
in good health before the outbreak. In addition, an unusually high incidence of brain-stem
encephalitis was observed among patients. Eleven of 14 adults were treated at VUHC for
meningitis and/or encephalitis, 5 of whom eventually died, giving a mortality rate of 45%.
Septicemia was observed in the remaining three patients, two of whom were pregnant
women.
According to Bille [52] and Malinverni et al. [ 1471, 38 of 40 (95%) L. monocyto-
genes strains isolated from listeriosis victims during the epidemic period were of serotype
4b, whereas only 9 of 15 (60%) clinical isolates obtained during the previous 6-year epi-
demic period were serotype 4b. More important, 33 of 36 (92%) L. monocytogenes sero-
type 4b cultures were of two unique phage type configurations as compared with only 4
of 9 (44%) serotype 4b cultures obtained during the previous 6 years. This, in turn, sug-
gested that ii common-source listeriosis outbreak had occurred in western Switzerland
between January 1983 and March 1984.
Unlike endemic cases of listeriosis treated between 1974 and 1982, most listerial
infections recorded during the epidemic period were diagnosed during the winter months.
However, listeriosis cases were uniformly distributed throughout the general population
and were apparently unrelated to listeriosis in animals. Despite an in-depth investigation
318 Ryser

that included interviews with patients and a search for L. monocytogenes in several hun-
dred food items, neither the source nor the mode of Listeria transmission could be found.
Working under the assumption that a similar listeriosis outbreak was likely the fol-
lowing winter, public health officials initiated a case-control study using listeriosis cases
that were diagnosed in French-speaking Switzerland between November 1, 1984, and
April 30, 1985 [13,52]. Overall, 16 cases (7 adults and 9 motherhfant pairs) were identi-
fied and compared with 49 controls matched for age, sex, and underlying conditions.
Fifteen of 16 (94%) patients were infected with L. monocytogenes serotype 4b, with 5 of
16 (3 1%) isolates belonging to the same phage type. Although these five cases suggest a
possible epidemic focus, data obtained from questionnaires dealing with professional and
home exposure as well as types of food consumed (e.g., milk products and raw vegetables)
were inconclusive.
In response to the 1985 listeriosis outbreak in California linked to consumption of
Mexican-style cheese, Swiss officials initiated a series of surveys to determine the inci-
dence of Listeria spp. in different dairy products, the results of which are summarized in
Chapter 12. During one such survey of soft, semihard, and hard cheeses, Breer [62] iso-
lated L. monocytogenes from 5 of 25 surface samples of Vacherin Mont dOr, a soft,
smear-ripened cheese that is only manufactured from October to March and consumed
primarily in and around the Canton of Vaud. Subsequent test results indicated that all L.
monocytogenes isolates from Vacherin Mont dOr cheese belonged to serotype 4b and
also demonstrated that two L. monocytogenes phage types isolated from this cheese were
identical to most clinical strains isolated during the 1983- 1986 epidemic period. Investiga-
tors in Switzerland [52] then examined over 200 types of domestic and imported soft
cheeses, 8- 10% of which contained L. monocytogenes. However, based on serotyping
and phage typing, these strains as well as other food and dairy product isolates were
distinctly different from those found on the surface of Vacherin Mont dOr cheese.
A subsequent review of hospital records indicated that 122 listeriosis cases involving
57 adults (Table 4) and 65 motherhnfant pairs were diagnosed in the Canton of Vaud
between 1983 and 1987 [66] (epidemic rate of -50 cases/ 1O6 population/yr) as compared
with only 28 cases between 1974 and 1982 (endemic rate of -5 cases/106 population/
yr) [37]. Interestingly, 84% of the cases that occurred during the epidemic period were
identified between October and April. Thirty-four of 122 patients died, giving a mortality
rate of 28%, with 18 of 57 (32%) adult cases proving to be fatal.
Although the two previous case-control studies failed to uncover the source of this
epidemic, a third case-control study conducted in 1987 demonstrated that 31 of 37 (84%)
cases had consumed Vacherin Mont dOr cheese as compared with only 20 of 51 (39%)
controls [54]. In addition, investigators were able to isolate the epidemic strain of L. mono-
cytogenes from a piece of Vacherin Mont dOr cheese that had been partially consumed
by one of the victims. Armed with this information, Swiss authorities halted production
of Vacherin Mont dOr cheese on November 20, 1987, and recalled the product throughout
Switzerland [20,27,54].
Overall, 111 of 120 (93%) clinical isolates available from the epidemic period be-
longed to serotype 4b, with 98 of l l 1 (85%) serotype 4b strains matching the two epidemic
phage types that were isolated from Vacherin Mont dOr cheese. Several years later, clini-
cal and cheese isolates were found to be identical based on multilocus enzyme electropho-
resis [ 1711, pulsed-field gel electrophoresis [60,64], ribotyping [%I, restriction enzyme
analysis [64,235], randomly amplified polymorphic DNA patterns [74], and pyrolysis mass
Foodborne Listeriosis 319

TABLE
4 Description of 57 Adult Listeriosis Cases Diagnosed i n Western
Switzerland from 1983 to 1987
Manifestation
~ ~~~

Septicemia Meningitis Meningoencephalitis

Characteristic (n = 12) (n = 23) (n = 22)
Sex
Males 9 12 12
Females 3 11 10
Age (years)
Median 75 69 55
Range 44-85 3 1-96 37-79
>65 (%) 10 (83) 15 (65) 6 (27)
Underlying illness (%)a 12 (100) 18 (78) 10 (45)
S ymptoms
Median onset time 12 hours 2 days 3 days
Fever (%) 12 (100) 21 (91) 17 (77)
Vomiting/diarrhea (%) 4 (33) 11 (48) 11 (50)
Meningismus (%) 18 (78) 14 (64)
Altered mental state (%) 19 (83) 13 (59)
Outcome
Cured 9 15 6
Neurological sequelae - 1 8
Fatal (%) 3 (25) 7 (30) 8 (36)
Includes leukemia, cancer, alcoholism, immunosuppressive drug therapy, diabetes, and AIDS.
Source: Adapted from Ref. 66.

spectroscopy [ 1011, thus confirming Vacherin Mont dOr cheese as the infectious vehicle.
Interestingly, this epidemic strain also is of the same phage type [60,64,1811, enzyme type
[56], ribotype [56], and pulsed-field gel electrophoretic type [60,64] as strains isolated
during the 1985 listeriosis outbreak in California [181]. However, two distinct DNA re-
striction endonuclease profiles [ 1721 were eventually identified among the Swiss epidemic
strains corresponding to epidemic phage types I and 11, with Eioerlin et al. [60] also divid-
ing the epidemic electrophoretic enzyme type into two major subtypes using pulsed-field
gel electrophoresis.
The fact that the Swiss and California epidemic strains are closely related to each
other and are also phenotypically and genotypically similar to strains involved in major
outbreaks traced to coleslaw, cheese, and p2t6 in Canada, Denmark, and France, respec-
tively [65,110,126,163], raises some interesting questions as to why many of the most
serious outbreaks in North America and Europe have been confined to this one particular
strain. When Boerlin and Piffaretti [59] examined 181 Swiss L. rnonocytogenes isolates
from clinical, veterinary, food, and environmental sources, the Swiss epidemic enzyme
type comprised 26.5 and 24.2% of all strains collected during (1983-1987) and after
(1988- 1989) the Swiss outbreak, respectively (Table 5). Furthermore, the epidemic en-
zyme type was widely distributed, with this strain being recovered from humans (clinical
and fecal samples), animals (clinical and/or fecal samples from cows, sheep, and goats),
meat/meat products, cheese/milk, and the environment (soil, silage). Since this particu-
320 Ryser

TABLE
5 Swiss Epidemic and Nonepidemic L. monocytogenes Enzyme Types
Recovered from Various Sources within Switzerland During 1983-1989
Source of L. monocytogenes strain
Humans Animals Meat/Meat products Cheese/Milk Environment
Enzyme type (n = 43) (n = 49) (n = 40) (n = 19) (n = 30)
Epidemic (n = 1 ) 13 24 1 4 6
Nonepidemic (n = 49) 3O/2Oa 25/16 39/14 15/13 241 I3
aNumber of nonepidemic straindnumber of nonepidemic enzyme types.
Source: Adapted from Ref. 59.

larly virulent strain is dominant in ruminants and since a similar strain was responsible
for cheese-related outbreaks of listeriosis in California and Denmark, recovery of this L.
rnonocytogenes subtype from foods has taken on added public health significance.
However, the exact importance of this particular electrophoretic enzyme type as
compared with others in foodborne listeriosis cannot yet be adequately assessed until more
information is available regarding the distribution of different L. monocytogenes subtypes
in nature along with the ability of these various strains to grow and/or survive in dairy
products and initiate disease in both humans and laboratory animals.
Most of the tainted cheese was marketed in Switzerland; however, small quantities
were exported to other countries, including England and the United States. Hence, on
November 25, 1987, health officials in England warned the general public against consum-
ing Vacherin Mont dOr cheese, which was available at a few delicatessens and specialty
cheese shops in and around London [1,21]. Similarly, FDA officials in the United States
became concerned after a major newspaper reported that five specialty shops in New York
City and a chain of 37 stores in Connecticut had been distributing the cheese since Novem-
ber 1987 [20]. These recall efforts were largely successful, since all known listeriosis
cases linked to consumption of this cheese were confined to Switzerland.
Immediately after the recall, Swiss authorities began investigating possible routes
by which Vacherin Mont dOr cheese could have become contaminated. According to
Bille [53,54], the cheese implicated in this outbreak was produced at 40 different factories
located in western Switzerland. All contaminated cheese was reportedly prepared from
Listeria-free cows milk. Following coagulation of milk, the resulting curd was dipped
into wooden hoops and allowed to drain for 1-2 days. When thoroughly drained, the
hooped cheeses were transported to 1 of 12 cellars (i.e., caves) located throughout western
Switzerland and ripened for -3 weeks on wooden shelves during which time the cheeses
were turned daily and brushed with salt water. Once ripened, the cheeses were packaged
for sale and the wooden hoops were returned to the cheese factory.
From this description, it is apparent that ample opportunity existed for contamination
of Vacherin Mont dOr cheese, particularly during ripening. In fact, the epidemic strain
[5] was detected in 18.5% of surface (rind) samples from Vacherin Mont dOr cheese at
levels of 104-106 L. rnonocytogenes CFU/g and also on 6.8% of wooden shelves and
19.8% of brushes used in the ripening cellars.
In all likelihood, this outbreak began several years earlier when L. monocytogenes
entered one of the 40 cheese factories in raw milk from an infected dairy herd [59]. Al-
though this outbreak was first detected in 1983, the epidemic strain was initially isolated
from a listeriosis victim in 1977, which suggests that this outbreak may have been devel-
Foodborne Listeriosis 321

oping for at least 7 years. Investigations showed that nearly hall' of the 12 ripening cellars
were contaminated with one or both epidemic strains of L. monocytogenes, thus suggesting
cellar-to-cellar spread of the pathogen through production and distribution practices. This
theory is strongly supported by the fact that cheeses produced at all 40 factories were
normally transferred between different cellars for ripening and/or distribution. The prac-
tices of brushing cheeses with salt water, ripening cheese in wooden hoops, and returning
these hoops to the cheese factory also were important factors in disseminating L. manocy-
togenes to different ripening cellars.
Following the recall, all 40 factories in which Vacherin Mont d'Or cheese was manu-
factured were thoroughly cleaned and sanitized. More important, all wooden material (e.g.,
shelves, boxes, hoops) was removed from ripening cellars anti burned, The cellars were
then thoroughly cleaned, sanitized, and refitted with metal shelves and easily sanitized
equipment. Once this work was completed, experimental batches of Vacherin Mont d'Or
cheese were produced during a 2-month period and examined for the epidemic strain of
L. monocytogenes to assure government officials that the pathogen was eliminated from
all ripening cellars. These clean-up efforts proved to be highly successful, with only two
cases of listeriosis being reported in western Switzerland between January and September
of 1988 [53]. Although both of these cases resulted from nonepidemic strains, multilocus
enzyme electrophoresis later demonstrated that 45 of 145 (28%) human clinical and 44
of 116 (37%:) animal strains of L. monocytogenes isolated in Switzerland between 1988
and 1993 belonged to the previously identified epidemic enzyme type (601. After further
analysis by pulsed-field gel electrophoresis, 34 of these 26 I ( 1 3%) humadanimal isolates
matched the epidemic strain recovered from Vacherin Mont d"Or cheese, thus suggesting
a continued presence of this strain in the natural environment.
As a rr:sult of this outbreak which by one account cost an estimated $1.4 million
[232], several steps have been taken to control and limit the extent of listeriosis in Switzer-
land [52,53]. First, health authorities are systematically screening high-risk foods for L.
monocytogenes and have adopted a zero tolerance for the pathogen in 10-g samples. Sec-
ond, physicians and laboratories are now required to notify health officials of every new
case (i.e., clinical isolate) of listeriosis occurring throughout Switzerland. Finally, since
1990 the Swiss National Center for Listeriosis in Lausanne has been actively collecting
human, animal, food, and environmental isolates and further characterizing these Listeria
strains according to serotype, phage type, ribotype, enzyme type, DNA restriction pattern,
and pulsed-field gel electrophoresis profile. These efforts will serve to identify the exact
endemic rate of human listeriosis in the general population and lead to faster recognition
of possible future listeriosis outbreaks as well as the vehicleis involved.

Blue-Mold/Hard Cheese: Denmark, 1989-1990

Listeriosis has been a reportable disease in Denmark since 198 1, with two apparent com-
mon source outbreaks being identified, the first of which occurred during 1985- I987 and
included 35 cases of unknown origin [208]. Two years later, one specific phage type of
L. monocytogenes serotype 4b was identified as being responsible for 26 of 69 (38%)
listeriosis cases that were reported from March 1989 to December 1990 (Fig. 4) [131].
Although found throughout the country, epidemic cases were most frequently seen in
suburban Aarhus, Denmark's second largest city. Twenty-three cases occurred in adults,
13 of whom suffered from underlying illnesses (i.e., leukernia, cancer, AIDS, diabetes,
alcoholism), with three cases involving pregnant women. Prirnary manifestations included
322 Ryser

FIGURE4 Monthly distribution of epidemic and non-epidemic listeriosis cases in

Denmark during 1989 and 1990. (Adapted from Ref. 131.)

meningitis ( IS cases) and septicemia ( 8 cases). Six of 23 adults died while hospitalized,
giving a mortality rate of 26%.
Evaluation of 90 food history questionnaires given to epideriiic/nonepideinic cases
and matched controls showed a clear epidemiological link between consumption of Danish
blue-mold cheese and cases of listeriosis, with one brand of cheese being cited in particu-
lar. Unfortunately. investigators were unable microbiologically to confirm blue-mold
cheese as the vehicle of infection. However, one year earlier. the FDA issued a Class I
recall for L. rizorzoc.?.togc.rzr.s-contaminated Danish blue cheese that had been shipped to
the United States [ 23,251. Routine dairy inspections also indicated that the epidemic strain
was present in the dairy environment as evidenced by recovery of this strain from eight
different Danish hard cheeses. The fact that these extremely popular hard cheeses were
consumed by more than 90% of both patients and controls makes these cheeses another
plausible vehicle of infection. Inability to recover the epidemic strain from other packaged
foods lends additional support for involvement of Danish blue and hard cheeses. Follow-
up studies showed this L. rizorzoc:\~to,~crzes strain to be of international importance, since
the identical phage type also was responsible for two major foodborne outbreaks in Swit-
zerland (Vacherin Mont dOr cheese, 1983- 1987) and France (jellied pork tongue. 1992).

Brie de Meaux Cheese: France, 1995

A nationwide listeriosis surveillance program has been operating in France since 1987,
with the French National Reference Center (NRC) (Pasteur Institute. Paris) being responsi-
ble for collecting. serotyping. and phage typing L. 1izorzo~~togerie.s
isolates from human.
veterinary, and food sources [ 1261. Over 10,000 Listor-iir strains were received annually
during I993 and 1994, so routine characterization is limited to serotyping and phage typ-
Foodborne Listeriosis 323

ing. with molecular typing methods such as pulsed-field gel electrophoresis and ribotyping
being used only in epidemiological investigations. Using this surveillance system, three
minor outbreaks involving less than 16 cases were detected from 1987 to 1992, including
one in the Strasbourg area [ 143,196.198j. More important, three major foodborne listeri-
osis outbreaks were identified in 1992, 1993. and 1995. the most recent of which was
traced to Brie de Meaux cheese.
Between April 2 and 19 of 1995. the NRC received six L. rriorioc\tos:eiies human
isolatcs from hospitals in different regions of France which were soon identified as belong-
ing to an unusual phage type that had been previously responsible for only 33 cases of
listeriosis ( 1 [ < 1 % I to 8 [ 3 % ] cases annually) since 1987 [ 109,126). These findings
prompted the NRC to inform the Ministry of Health on April 28 about a possible outbreak.
A search of NRC records indicated that the same phage type had been recovered from 5
of 2200 food samples ( 1 delicatessen product, 4 Brie de Meaux cheeses) tested from
January to April. Further characterization of these strains by pulsed-field gel electrophore-
sis indicated that the six patient and four cheese isolates were identical.
Based on follow-up epidemiological investigations, all victims identified as of May
10 consumed Brie de Meaux cheese (a raw milk soft cheese) that had been cut and pur-
chased at either supermarkets or small local markets [ 1091. Additional investigative work
by the Ministry of Agriculture coupled with a case-control study soon implicated one
particular brand of Brie de Meaux cheese. with this product being recalled from the market
on May 18. Additional cheeses exported to Belgium were also recalled without incident
shortly thereafter [ 2241. Isolation of the epidemic phage type and pulsed-field gel electro-
phoresis type from eight Brie de Meaux cheeses and several other cheeses ripened at the
same facility confirmed the role of Brie de Meaux cheese in this outbreak [ 126,235). At
the time of the May 18 recall. 20 epidemic cases were documented (Fig. 5 ) , with the
victims residing in 8 of 22 French regions. Of these 20 cases, 1 I among pregnant women

FIGURE5 Distribution of epidemic and non-epidemic listeriosis cases in France from

January to July, 1995. (Adapted from Ref. 126.)
324 Ryser

led to two spontaneous abortions, four premature deliveries, and two stillbirths, with the
remaining nine cases involving immunocompromised adults (7 cases) and the elderly
(2 cases).
By the time this outbreak finally ended in July of 1995, 13 additional cases were
confirmed by phage typing and pulsed-field gel electrophoresis, bringing the total number
of cases to 33. Interestingly, this outbreak strain belonged to a new epidemic clone which
was clearly distinct from the two L. monocytogenes strains responsible for the other major
outbreaks in Canada (coleslaw 1981 ), California (Mexican-style cheese I985), Switzer-
land (Vacherin Mont dOr cheese 1983- 1987), United Kingdom (pGt6 1987- 1989), Den-
mark (blue-mold/hard cheese 1989- 1990), France (jellied pork tongue I992), and France
(pit6 1993) which either have been or will be discussed shortly.
In 1997, the NRC also identified 14 listeriosis cases over a 4-month period that were
directly traced to Pont IEvEque cheese produced in Normandy [241]. The implicated
cheese was manufactured from raw milk and contained L. monocytogenes serotype 4b at
a level of > 1000 CFU/g. Although some of this cheese was exported to Sweden, no
additional cases were reported.

Additional Reports of Cheeseborne Listeriosis

Other than listerial infections associated with the aforementioned outbreaks in California
and Switzerland, only two additional well-documented cases of nonfatal listeriosis have
been directly linked to consumption of contaminated cheese, both of which occurred in
England. The first case involved a healthy, nonpregnant 36-year-old woman who devel-
oped meningitis on January 9, 1986, 9 days after consuming a full-fat, soft French cheese
[ 12,1041. According to Bannister [45], identical phage types of L. monocytogenes serotype
4b were isolated from the womans cerebrospinal fluid and a partially consumed package
of cheese. However, the fact that six unopened packages of the same cheese failed to
yield viable listeriae suggests that the cheese may have become contaminated in the refrig-
erator rather than during manufacture.
In response to the 1987 outbreak of listeriosis in Switzerland and the discovery of
L. monocytogenes in an increasing variety of foods, health officials in England began
treating all reported listeriosis cases as possibly being foodborne. Follow-up questions
about different foods consumed by victims led to discovery of a second cheese-related
case of listeriosis in February of 1988 [ 1 171. According to official reports [30,44,158], a
previously healthy, nonpregnant 40-year-old woman was admitted to a London hospital
with meningitis following a 4-day bout with a flu-like illness. Identical phage types
of L. monocytogenes serotype 4b were eventually isolated from the womans cerebrospinal
fluid, stool, and an open package of Anari raw goats milk cheese (a Greek-style soft
cheese) from which the victim had consumed -85 g 24 h before onset of symptoms. Four
additional unopened packages of Anari cheese (from the same lot) purchased from a retail
store yielded the same L. monocytogenes strain at levels of 3-5 X 10 CFU/g. Conse-
quently, this cheese was withdrawn from the market in February of 1988, with production
not resuming until the summer of 1988.
Following news of this cheeseborne listeriosis case, McLauchlin et al. [ 1581 exam-
ined the extent to which other dairy products produced by this manufacturer were contami-
nated with listeriae and attempted to identify the exact source of contamination. The Anari
goats milk cheese responsible for the aforementioned case of listerial meningitis came
from a one-man, off-farm dairy factory at which Halloumi, Cheddar, feta, soft chive, and
Gjestost cheese as well as yogurt also were produced from goats milk. According to these
Foodborne Listeriosis 325

investigators, 16 of 25 (64%) retail cheeses and 12 of 24 (50%) cheeses obtained directly

from the factory over a period of 1 1 months yielded L. rnonocytogenes serotype 4b, with
all goats milk cheese varieties except feta testing positive for the pathogen. Although 22
of 24 (92%) positive cheeses contained < 10 L. rnonocytogenes CFU/g, the two remaining
cheeses that were purchased from a retailer 10 weeks before their sell date contained > 105
L. monocytogenes CFU/g, thus suggesting that the pathogen grew in the cheese during
retail storage. This hypothesis was subsequently confirmed using naturally contaminated
(<10 L. rnonocytogenes CFU/g) 2- to 3- day-old Anari and Ilalloumi cheeses that were
periodically analyzed for numbers of listeriae during 8 weeks of refrigerated storage. Al-
though no listeriae were detected in samples of raw goats milk. or yogurt obtained directly
from the factory, L. rnonocytogenes serotype 4b was recovered from shelving within the
factory, which in turn suggests that the cheese most likely became contaminated during
the final stages of manufacture or packaging. Most important, phage typing indicated that
66 of 68 (97%) L. rnonocytogenes isolates recovered from various cheeses and factory
shelving were identical to the strain isolated from the patients cerebrospinal fluid and
stool. With the aforementioned evidence, there appears to be little doubt that this case of
listerial meningitis resulted from consumption of Anari goats milk cheese in which L.
rnonocytogenes likely grew to high numbers during retail storage.
Although the only confirmed cases of cheeseborne listeriosis in the United States
have been those associated with consumption of Jalisco brand Mexican-style cheese in
1985, cheese has been suggested as a vehicle of infection in several additional cases.
These primarily unconfirmed reports include (a) isolation of Listeria from the blood of a
7-day-old California infant whose mother consumed a raw milk cheese 2 weeks before
delivery [9]; (b) three cases of listeriosis in Arizona in which the victims consumed non-
Jalisco brand soft Mexican-style cheese; (c) a possible association between listeriosis and
consumption of Italian cheese; (d) one case of listeriosis in California in which a woman
delivered an aborted fetus after eating Monterey Jack cheese prepared from raw milk; (e)
an alleged listerial abortion by a woman in New York who consumed contaminated feta
cheese; (9 one case in which L. rnonocytogenes serotype 4b was isolated from a 3-year-
old Washington state girl and cheese found in her familys refrigerator [34]; (g) isolation
of an identical L. monocytogenes strain (same phage type and electrophoretic enzyme
type) from ii listeriosis patient in Philadelphia and from cheese that the victim reportedly
consumed 1801; (h) one case involving a healthy woman from New Jersey who suppos-
edly contracted listeriosis after consuming Ricotta cheese containing 10- 1Oh L. monocyto-
genes CFU/g [ 1421; and (i) the report of a San Bernardino, California, woman who devel-
oped a fatal listerial infection after eating locally purchased soft Mexican-style
cheese [ 102 1.
Despite the recall of approximately 600 million pounds of French soft-ripened
cheese in 1986 (see Chap. 12), no cases of listeriosis were linked to consumption of this
cheese in the United States. However, several cases were documented in both Canada and
England [45]. In one of these Canadian cases, L. rnonocytogmes serotype Ib of the same
electrophoretic enzyme type was isolated from the blood of a 66-year-old man and opened
packages of imported soft cheese that he consumed [95]. The same strain was later identi-
fied in unopened packages of cheese produced by the same manufacturer, thus confirming
the role of cheese in this isolated case of listerial bacteremia. Additionally, ingestion of
homemade fresh cheese by a pregnant Italian woman was hlamed for the listerial death
of her infant three days after delivery [ 1681. Although the scientific literature contains
one additional report of an AIDS patient in England who contracted meningitis after con-
suming Staffordshire cheese, the two non-phage typeable strains of L. rnonocytogenes
326 Ryser

serotype 1/2a recovered from the patients cerebrospinal fluid and cheese were subse-
quently found to exhibit different DNA restriction enzyme patterns, thus negating cheese
as the vehicle of infection.

MEAT PRODUCTS
Foods of animal origin have long been recognized as potential vehicles of infection, with
meat-associated cases of salmonellosis and botulism being recorded in the scientific litera-
ture since the 1890s. Following confirmation of L. monocytogenes as a human and animal
pathogen during the 1920s, listeriosis was subsequently identified as a zoonosis, a disease
transmissible from animals to humans. Hence, when listerial infections in domestic live-
stock began to emerge with some regularity during the 1930s and 1940s, some individuals,
including Wramby [238), who in 1944 first identified Listeria in raw meat, began to specu-
late that consumption of meat products could play a role in the spread of human lister-
iosis.
Listeria-laden fecal material from asymptomatically infected livestock can readily
enter the slaughterhouse environment and contaminate retail raw meats. Meat processors,
veterinarians, and others who work closely with animal carcasses will also inevitably come
in contact with L. monocytogenes as evidenced by recovery of this pathogen from the
hands and gowns of Czechoslovakian line workers during the mid 1970s [86,87]. Although
evidence is somewhat conflicting, most of the earlier studies previously reviewed by Ryser
and Marth [205] also indicated that increased exposure to L. monocytogenes in the meat
industry can lead to higher fecal carriage rates among workers. According to Elischerova
and Stupalova [86,88], six clinically healthy Czechoslovakian meat workers who had an
opportunity to consume crude and semicrude product during work were asymptomatic
fecal shedders of L. monocytogenes. These same individuals also exhibited elevated 0
and H serum agglutination titers against L. monocytogenes serotype 1, which is compatible
with oral transmission of Listeria via meat products.
Since two of the three meatborne listeriosis outbreaks identified thus far have in-
volved consumption of pit&-a ready-to-eat meat, fish, or vegetable product that is com-
monly marketed in Belgium, France, Germany, the Netherlands, and the United Kingdom
and is consumed without reheating or further cooking, it is appropriate briefly to review
the manufacture and safety-related issues surrounding this product. Preparation of meat
pit& most often involves chopping pork liver with water, seasoning, salt, and sodium
nitrite. This raw product is then either (a) cooked in a mold, decorated, sliced, and sold
as loose or vacuum-packaged pit&;or (b) cooked in small hermetically sealed containers.
The high water activity and pH of typical pi& provide an ideal growth environment for
most bacteria, including L. monocytogenes, which has an estimated doubling time of 19
h in pit& stored at 7C [76]. Hence, thorough cooking of the raw product, addition of
preservatives, and proper packaging are all essential to preventing growth of listeriae to
dangerously high levels in this food during 3 or more weeks of refrigerated storage. With
this background information in mind, the role of pit6 in one of the largest listeriosis
outbreak thus far reported will now be assessed.
Numerous reports suggesting possible involvement of meat products in human lister-
iosis can be found in the scientific literature with over 60 primarily sporadic cases docu-
mented since 1955. However, the ability of meat products to serve as vehicles of listerial
infection was not fully realized until the late 1980s when consumption of pit&was deemed
responsible for over 350 cases of listeriosis in the United Kingdom. As a result of an
Foodborne Listeriosis 327

ongoing, nationwide listeriosis surveillance program in France, two additional meat-re-

lated outbreaks came to light in 1992 and 1993, with 279 and 39 cases being traced to
Listeria-contaminated jellied pork tongue and pork pSt6 rilletes, respectively. These
outbreaks will now be discussed in some detail followed by a review of the aforementioned
sporadic cases of suspected meatborne listeriosis reported since the 1950s.

Piite: United Kingdom, 1987-1989

In the United Kingdom, a national voluntary reporting program for human listeriosis has
been operating since 1967 under the direction of the Public Health Laboratory Service,
Communicable Disease Surveillance Center (England, Wales, Northern Ireland), and the
Communicable Disease Unit (Scotland), with the Listeria Reference Unit at the Central
Public Health Laboratory (Colindale) being responsible for confirming and characterizing
all strains received by serotype and phage type. As a result of these efforts, one cluster
of 23 listeriosis cases ( 1 0 maternofetal and 13 adult) documented from early June to No-
vember of 1987 involved an unusual serotype of L. monocytogenes designated 4b(x) that
was previously responsible for only 12 of 842 cases reported i n Britain between 1967 and
1986 [ 1571. Although all 23 clinical isolates were indistinguishable by phage typing and
monoclonal antibody typing, subsequent food consumption profiles obtained from 17 cases
failed to implicate a common food, brand, or supplier. From 1987 to mid 1989, a large
upsurge in human listeriosis cases was observed in the United Kingdom [ 1551 (Fig. 6).

300

250
I
$ 1
U)

200
%
0
b
-.I f \
a
E 150
\\\
3
z

O L_ _ ~ ~ - ~ _ _ _ _ J
198384 85 86 87 88 89 90 91 92 93 94
Year

FIGURE6 Reported cases of human listeriosis in England, Wales and Northern Ire-
land, 1983-1994. (Adapted from Ref. 155.)
328 Ryser

L. monocytogenes strains classified as serotype 4b phage type 6,7 and serotype 4b(x) were
responsible for 366 of 823 cases reported during this period [ 1951, with these two strains
being far less common before 1987 and after July 1989 (Fig. 7). Thereafter, the number
of human listeriosis cases decreased sharply to pre- 1987 levels.
During a routine food poisoning investigation in May of 1989, the Cardiff Public
Health Laboratory identified high levels of L. monocytogenes in pit6 taken from one vic-
tim's refrigerator. This chance finding prompted an immediate survey of primarily im-
ported pit6 sold from delicatessen display counters throughout southeast Wales between
May and August of 1989 [164,165]. Overall, L. monocytogenes was recovered from 75
of 216 (35%) pit& tested at levels ranging from <20 (42 samples) to >104 (10 samples)
CFU/g. More important, however, 32% of all positive samples harbored L. rnonocytogenes
serotype 4b(x), which was responsible for the aforementioned cluster of 23 cases identified
2 years earlier.
Results from this Welch survey prompted two actions in July of 1989: (a) a govern-
ment health warning to vulnerable individuals about eating pit6 [78] and (b) a far more
extensive survey [76] in which 1698 samples of pSt6 marketed in England and Wales
were examined for listeriae. Overall, I86 ( 10%) samples contained L. monocytogenes at
levels ranging from <200 (12 1 samples) to > 106(3 samples) CFU/g, with 37 of the I86
positive samples harboring > 103CFU/g. Investigators also noted that L. monocytogenes
levels were generally higher in (a) pSt6 prepared from fish rather than meat, (b) loose
slices rather than prepackaged pate, (c) samples marketed at >7"C, (d) pit6 tested at or

+ 4bX f 4b PT6,7 Other strains

1385 1986 1987 1988 1989 1990

Year

FIGURE7 Annual distribution of selected Listeria monocytogenes strains from hu-

man listeriosis cases reported in England, Wales, Scotland, Northern Ireland, and the
Republic of Ireland, 1985-1990. (Adapted from Ref. 159.)
Foodborne Listeriosis 329

beyond the sell-by date, and (e) samples having standard bacterial plate counts of >106
CFU/g, with these findings supporting the reported ability of this pathogen to grow in
p2t6 at near-refrigeration temperatures. More important, however, 5 1 of 107 (58%) piit6s
produced in Belgium by manufacturer Y contained L. monocytogenes, with 12 of these
samples yielding >103CFU/g. Follow-up investigations [ 1591 showed that 96% (48 of 50)
of all L. monocytogenes isolates from manufacturer Ys pGt6 belonged to either serotype 4b
phage type 6,7 or serotype 4b(x), with these two strains being responsible for 30-54%
of all human listeriosis cases reported during the epidemic period. (Fig. 7). In contrast,
only 19% (6 of 31) of pitis from other producers contained these two strains, with cross
contamination among pit& handled at delicatessen counters likely contributing to appear-
ance of these otherwise rare strains. A subsequent epidemiological investigation revealed
that 13 of 15 patients infected by these epidemic strains had consumed pit6 within 3
weeks of oriset compared with 6 of 17 patients infected with nonepidemic strains.
Illness was strongly associated with piit6 consumption; however, pit6 samples were
no longer available from the victims refrigerators to microbiologically confirm this food
as the vehicle of infection. Nevertheless, all available evidence, along with the fact that
the reported decline in listeriosis cases after mid 1989 coincided with both a government
warning concerning pit6 consumption and removal of manufacturer Ys pit6 from sale,
clearly points to this particular brand of imported pit6 as being responsible for the outbreak
observed from 1987 to mid 1989. A much lower incidence of L. monocytogenes in retail
p2t6 samples tested in 1990 as compared with 1989, along with the virtual absence of
both epidemic strains in pit6 and other foods examined after 1989 [ 1051, further support
involvement of manufacturer Ys pit6 in this outbreak.

Jellied Pork Tongue: France, 1992

As part of an ongoing Listeria surveillance program, the French National Reference Center
(NRC) noted in May 1992 that 29 clinical L. monocytogenes serotype 4b isolates received
during the previous 2 months belonged to an unusual phage type. Furthermore, this strain
was previously responsible for only 6 (1%) to 27 (7%) listeriosis cases annually, thus
suggesting a common source outbreak [ 126,1271. By the time this outbreak ended in De-
cember of 1992,279 phage typed cases (Fig. 8) involving 182 adults (53% with underlying
illnesses), 5 children and 92 pregnant women were documented, including 63 deaths and
22 abortions [106,149], making this outbreak one of the largest thus far reported. Among
the 73 live births, 7 newboms died, giving an infant mortality rate of 9.6%. Geographically,
cases were reported from every region of the country except the island of Corsica, with
as many as 10- 14 cases/million population being recorded in and around Limousin, Al-
sace, and the Rh6ne Valley.
Following a nationwide alert in May, the French Ministries of Health, Agriculture,
and Economy began investigating this outbreak. Initially, no correlations between develop-
ment of disease and consumption of various meats, cheeses, and pit& was observed from
144 cases and 288 matched controls [ 106,1081. However, in a subsequent case-control
study, 36 of 60 (60%) pregnant women who became ill recalled consuming jellied pork
tongue as compared with only 5 of 82 (6.1%) healthy controls. Thus, jellied pork tongue
was implicated as the vehicle of infection (odds ratio: 9.2) with product brand A (odds
ratio: 14.8) identified in a later case-control study.
Simultaneously, over 14,000L. monocytogenes isolates from food and related envi-
ronmental sources were serotyped and phage typed by the NRC [ 1271, with the epidemic
phage type eventually being identified in 135 delicatessen products, 40 cheeses, 40 meat/
330 Ryser

FIGURE8 Monthly distribution of epidemic and non-epidemic listeriosis cases in

France during 1992. (Adapted from Ref. 126.)

nieat products. 3 1 milk s~implcs.and 10 cnvironiiicntal samples. Among the 279 cliniciil
isolates. 249 strains exhibited the same pulsed-field gel clectrophoretic profile. with this
epidemic strain also subsequently being confirmed in I 12 saniples of jellied pork tongue
;is \veil ;is i n 19. 13, and 1 1 samples of other meat products (i.e.. ham. pit&. sausage).
cheeses. and miscellaneous foods. respectively. Furthermore. the epidemic strain was most
closely associated Lvith brand A jellied pork tongue. with high numbers being recovered
from st\re 11prc \io i i s 1y 11no pc ned con t ai ners and si x sam p 1c s s I iced at de 1i cate sse 11coii n tcrs .
Seve r;i 1 e n v i ron me 11t ;I 1 sam p 1es from brand A * s m ;in 11fac t u ri ng frici 1i ty e \re n tu a11y y i e I dcd
the epidemic strain [ 1271. with raw brine being identified iis the niost probable soiirce of
contamination during iii:iii~ifiictiire [ 207 I. These findings and results from the earlier c;ise-
control studies contirni brand A jellied pork tongue ;is the primary \.ehicle of infection.
w i t h ot he r cross con t am i 11;it ed foods at de 1i cat c sse 11 coil 11t er5 pre s i i 111;i b 1y ser\.i ng iis sec-
ondary \vehicles in approsiniately 19% of cases [ 1081. Support for the latter also comes
fro111 ii s 11b s c q 11e 11t c ;i se - c on t ro 1 s t LIct y i n which ;i st at i st i c a1 assoc i at i o11 LV ;is demonst r;i t c d
b e t ~ ~ eillnessii in patients who did not consume .jellied pork tongue and contact between
brand A jellied pork tongue and other foods at the delicatessen counter [ I08 I . Furthermore.
the epidemic strain was isolated from iitensils iised in slicing both brand A jellied pork
tongue and other delicatessen meats [ 107.197 1.
Based 011 phage typing. pulsed-tield gel electrophoresis. ribotyping. and multilocus
e11z y me e I ec t ro phore s i s. t h i s c pi de 111i c s trai 11 is phc 11ot y pi cal I y and ge 110tJ.1~ i c all y s i 111i 1;ir
to s t rai 11s re s po 11s i bl e for t he a f ore men t i oned chee se - re 1at ed 011 t b rcaks i 11 Cal i fo rn i ;i. Den-
111ark. id S\v i t ze 1.1 a11d. Th i s o bscr\.at i o n agai 17 con ti riii s that most 11i:i.j or 1is teri o s i s out-
breaks appear to be caused b!, ;i small group ot closely related strains. Gi\.cn the low
;it t ;ic k r:i tc and w i de ?cograph i c al d i s t ri bu t i 011 of 1i s te ri osi s cascs. con t i 11ued o ngo i rig sur-
i.eillance at the national Ic\.el is necessary for detection of future to,odbornc listeriosis
011t breaks.
Foodborne List eriosis 33 1

Pork Pate "Rillettes": France, 1993

A second markedly smaller meat-related outbreak was also documented i n western France
I year later. During late June and early July of 1993. 10 seemingly related cases of listcri-
osis were recorded at the Frcnch National Listeria Reference Center (NLRC) i n Paris. with
all I0 clinical isolates belonging to an unusual phage type of L. /,io/ioc:\'togciic'.~ serotype 4a
[ 1261. Since this epidemic strain was previously rcsponsible for only 2 ( < 1 % ) to 1 1 (3%)
1i ste ri osi s cases annual 1y si ncc 1 987. i n ve st i g at ors i m iii ed i ;I t e 1y 1au n c hed ;i c as e -c o 11 t ro 1
study in which ii pork pit& product (known locally ;is "rillettes" ) produced by onc manu-
facturer and sold through a single supermrirket chain was soon implicated as the \rehicle of
i n fect i on. One e n v i ron men t al i soI ate i de n t i tied from the i m pl i cat ed man 11 frict iirer 4 iii on t h s
earlier also matched the epidemic strain. These findings prompted a recall of the implicated
product and a series of public warnings through the mass media. By the time this outbreak
subsided in October. this single epidemic strain was responsible for 39 cases (i.e.. 15% -
of all human listeriosis cases reported from June to October) (Fig. 9). with approximately
80% of all listeriosis Lictims being identified :is iiiother/infiint pairs [ 1971.
Three weeks after the recall. investigators isolated the epidemic strain from 49
opened/unopcned containers of implicated pork pit6 that were eithcr recalled from the
s11perm ark e t . re t u 1-11ed by con s IIm e rs . o r retrieved fro171 v i c t i m ' s re fr i ge ra t or s. w i t h seven
environmental swab samples from the implicated factory also yielding the outbreak strain
[ 107.1261. These findings along with the fact that 38 of 39 clinical and SS of 56 pit&/
e n \iro n i n e n t a 1 i so1ates we re al so i dent i c ;i 1 based on pu 1sed-fi e 1d g e 1 e 1ec t ro ph ore si s con -
firmed pork pit6 as the infectious vehicle. According to follow-up DNA inacrorestriction
analyses. this epidemic strain was closely related to those responsible for the aforeinen-
tioned outbreaks that were epidemiologically linked to p i t i and pasteurized inilk i n En-
gland and Massachusetts. respectively [ 1261. However. only 0.2 and 0.1 c/r of - 17.400

FIGURE9 Monthly distribution of epidemic and non-epidemic listeriosis cases in

France during 1993. (Adapted f r o m Ref. 126.)
332 Ryser

food isolates received at the NLRC since October of 1993 have matched the epidemic
phage type and pulsed-field gel electrophoresis type, respectively, thus signaling the end
of this most recent outbreak of meatborne listeriosis.
Other than the three major outbreaks just discussed, the scientific literature primarily
contains only circumstantial evidence linking or, in some instances, only suggesting
involvement of meat products in cases of human listeriosis (Table 6). Beginning in 1955,
consumption of contaminated pork (probably undercooked) was suggested as the possible
cause of 27 listeriosis cases in the former Soviet Union [115,140]. The following year,
Gudkova et al. [ 1161 isolated L. monocytogenes from the viscera of pigs on a Russian farm
where several individuals contracted listerial infections, presumably after ingesting pork
from an infected group of pigs. In 1960, Olding and Philipson [ 1751 investigated one adult
and three perinatal cases of listeriosis that occurred within a three-block area of Uppsala,
Sweden, during the previous 2 years. Although repeated attempts to isolate L. monocyto-
genes from water, milk, vegetables, and meat ended in failure, the fact that meat was the
only food item obtained from the same source by all four individuals suggests the possible
involvement of unspecified meat products in this apparently common-source outbreak.
In the only other early recorded incident involving meat products from domesticated
animals, ground meat from a dead calf was suspected of transmitting L. monocytogenes
to the wife of a Dutch farmer in the early 1960s [ 1351. Although involvement of meat in
this case of listeriosis appears plausible, the remainder of the suspected meat was sterilized
during canning, thus eliminating any hope of confirming the causative agent.
During a review of listerial infections in Canada over a 21-year period, Bowmer et
al. [61] uncovered one case in which a pregnant woman in Newfoundland delivered an
infant who died 1 month later from listerial meningitis. Ten days before the infant became
ill, the mother recalled skinning, cooking, and eating two previously frozen hares that
were brought from New Brunswick, thus suggesting rabbit meat as a possible vehicle of
infection. Although less commonly consumed, it appears that rabbit meat also may serve

6 Human Listeriosis Cases in Which Consumption of Meat Products Was

TABLE
Suggested as a Possible Source of Infection
Number Possible vehicle of
Area Year of cases infection Reference
USSR I955 27 Pork 115, 140
USSR I956 19 Pork 116
Sweden 1958-59 4 Meat 175
The Netherlands early 1960s 1 Ground veal/beef I35
Newfoundland/Canada 1963 1 Rabbit 61
United States 1986-87 Unknown Uncooked hot dogs 214
Philadelphia, PA 1987 Unknown Salami 31
Italy 1988 I Cooked pork 70
United States 1988- 1990 1 Pork sausage 182
1988- I990 1 Ground beef 182
Spokane, WA 1989 1 Cooked ground beef 34
San Francisco, CA 1989 1 Cooked Cajun pork sausage 36
Victoria, Australia 1990- I995 1 Prepackaged sliced meat 222
1990- 1995 1 Sliced ham 222
1990- 1995 1 Sliced meats 222
Foodborne Listeriosis 333

as a potential source of L. monocytogenes, as evidenced by a long history of listerial

infections among rabbits [ 113,114,215,2331.In fact, the first type-strain of L. monocyto-
genes was isolated by Murray et al. [167] in 1924 from the blood of infected rabbits.
Several European scientists have expressed some concern about the incidence of L. mono-
cytogenes in rabbit meat, along with possible risks of consuming such potentially contami-
nated products.
Despite such circumstantial evidence suggesting that consumption of contaminated
meat products can lead to cases of human listeriosis, the possible involvement of meat
products in listerial infections received little if any further attention before 1981, primarily
because listeriosis had not yet been associated with any foods other than raw milk. How-
ever, this situation changed after three major listeriosis outbreaks were positively linked
to consumption of contaminated coleslaw, Mexican-style cheese, and Vacherin Mont dOr
soft-ripened cheese in 1981, 1985, and 1987, respectively. Several factors, namely, (a)
the long-time association of L. monocytogenes with domestic: livestock, (b) the ability of
L. monocytogenes to grow at refrigeration temperatures, and (c) questions from public
health authorities prompted numerous studies on the incidence and behavior of this patho-
gen in raw and processed meat products (see Chap, 13) and also led to increased surveil-
lance and reporting of listeriosis cases. After CDC officials in Atlanta began receiving
information about scattered cases of listeriosis occurring throughout the United States,
Schwartz et al. [2 141 initiated a retrospective epidemiological study to identify food prod-
ucts that might be associated with sporadic cases of listeriosis.
According to their 1988 report which appeared in the: British journal, Lancet, an
active L. monocytogenes surveillance program was established in Missouri, New Jersey,
Oklahoma, Tennessee, Washington, and Los Angeles County, California, in January of
1986. During the following 18 months (12 months in Los Angeles County), 154 listeriosis
cases were identified among 34 million people with approximately one third and two thirds
of the patients being classified as newborn infants and elderly or immunocompromised
adults, respectively. Overall, 82 of these 154 individuals agreed to participate in a retro-
spective case-control study in which patients responded to a series of questions concerning
demographic characteristics, underlying illnesses, medication, exposure to other sick indi-
viduals or animals, excavation work, and dietary history. The latter included questions
pertaining to consumption of raw fruits and vegetables, poultry, eggs, and dairy products
as well as raw, processed, and pickled meats. After comparing their answers with those
from 239 controls (individuals without listeriosis) that were matched to the cases in terms
of age and underlying illness, individuals who consumed uncooked frankfurters and un-
dercooked chicken were 6.1 and 3.2 times more likely to contract listeriosis, respectively,
than those who did not consume these products. Overall, epidemiological evidence from
this study suggested that consumption of these foods accounted for 30 of 154 (20%) listeri-
osis cases reported in the surveillance area with 1 in 1200-6000 and 1 in 1500-7500
individuals likely to contract listeriosis after consuming uncooked frankfurters and un-
dercooked chicken, respectively [32]. Given that about 1600 cases of listriosis occurred
annually in the United States during the late 1980s, these investigators speculated that 255
and 102 of these cases were attributable to eating uncooked frankfurters and undercooked
chicken, respectively.
Although this case-control study identified uncooked frankfurters and undercooked
chicken as risk factors in sporadic cases of listeriosis, it is important to stress that such
epidemiological investigations cannot establish causality. Furthermore, one must also re-
member that lack of an association with other foods does not necessarily mean that con-
334 Ryser

sumption of such products poses no risk of listeriosis. Several shortcomings of this retro-
spective case-control study were echoed by the scientific community [26], including, (a)
omission of questions concerning cooking methods and consumption of foods such as
seafood that until recently have seldom been associated with listeriosis, (b) limited ability
to identify risk factors when exposure was very common or very rare, and (c) difficulty
in obtaining accurate diet histories with the possibility of cases more clearly recalling
what they consumed before their illness than controls. Nonetheless, results from numerous
microbiological surveys (see Chaps. 13 and 14), along with a report by the American
Meat Institute indicating that 5- 10% of prepackaged frankfurters produced in the United
States were contaminated with L. monocytogenes [22], support the possibility of con-
tracting listeriosis from consuming uncooked frankfurters or undercooked chicken as was
suggested in the case-control study by Schwartz et al. [214] and a similar case-control
study [212] reported by the CDC several years later. During their work, these researchers
[31,2141 also identified another processed meat product consumed without further cook-
ing, namely salami, as a possible risk factor in a 1987 listeriosis outbreak in Philadelphia
that claimed 14 lives. However, CDC officials again lacked the bacteriological data to
positively link consumption of the salami to illness. In a subsequent case-control study
[ 1821, L. monocytogenes strains of the same electrophoretic enzyme type were recovered
from two patients and two unopened packages of pork sausage and ground beef that were
epidemiologically linked to illness. However, inability to recover and test these products
from patients refrigerators prevented CDC investigators from positively confirming the
vehicle of infection.
As mentioned earlier, piX, jellied pork tongue, and pork pgt6 rilletes were re-
sponsible for three major meat-related listeriosis epidemics in England and France, includ-
ing two of the largest outbreaks of foodborne listeriosis recorded worldwide (see Table
1). However, it must be stressed that as of July 1998, no American-produced raw, cooked,
or otherwise processed meat product has been conclusively proven as the vehicle of infec-
tion in any case of human listeriosis. Although it is important to remember that such a
causal relationship can only be shown conclusively by isolating the identical L. monocyto-
genes strain from the patient, product consumed, and unopened packages of the implicated
food, numerous North American and European surveys have uncovered low to moderate
levels of L. monocytogenes in a wide range of commercially available raw, processed, and
ready-to-eat meat products (see Chap. 13). Even before Schwartz et al. [31,2141 announced
preliminary results from their study, the meat industry [29] maintained that susceptible
individuals who consume Listeria-contaminated dry sausage, frankfurters, luncheon
meats, and other packaged pasteurized products are at low to moderate risk of contracting
listeriosis.
Since 1988, eight isolated cases and one small outbreak of listeriosis have occurred
worldwide where meat products were suspected as the most likely vehicle of infection
(see Tables 1 and 5 ) . In the first such case, a previously healthy Italian man contracted
nonfatal meningitis several days after consuming cooked homemade pork sausage that
was later shown to contain -3 X 106L. monocytogenes CFU/g [70]. According to investi-
gators, the clinical and sausage isolates were both identified as belonging to serotype 4,
the most common serotype encountered in clinical cases of listeriosis. Unfortunately, the
exact source of contamination was never determined; however, antiquated sausage-making
practices and storage of sausage at ambient rather than refrigeration temperature were
cited as major contributing factors in this isolated case of listerial meningitis. Nevertheless,
although numbers of listeriae present in this sausage were probably more than sufficient
Foodborne Listeriosis 335

to induce illness, some caution still must be used in evaluating the role of sausage in this
case, since both isolates were never characterized beyond serotype.
Four of these unconfirmed cases of possible meatborne listeriosis have been recorded
in the United States and include (a) a 76-year-old man from Spokane, Washington, who
died from an L. rnonocytogenes serotype 4b infection after consuming cooked ground
beef; however, only serotype l a was recovered from the ground beef thus making it an
unlikely source of infection [34], (b) a case-control study in which identical L. monocyto-
genes electrophoretic enzyme types were recovered from two patients as well as retail
packages of pork sausage and ground beef [212], and (c) an incident in which L. monocyto-
genes serotype 4b was isolated from cooked Cajun pork sausage that was consumed by
an elderly San Francisco man who developed a nonfatal case of listeriosis [36]. Approxi-
mately 1000 pounds of this sausage were subsequently recalled from the market after
investigators recovered L. rnonocytogenes serotype 4b from similar unopened packages.
Even though the patient and sausage isolates were not further classified, isolation of the
same L. monocytogenes serotype from unopened packages of sausage and the ability of
investigators presumably to trace the source of contamination to natural sausage casings
imported from China [83] provides reasonably convincing evidence that Cajun pork sau-
sage was directly responsible for this case of foodborne listeriosis. The remaining listeri-
osis cases (Table 6) as well as an outbreak which included six stillbirths or mid term
miscarriages among 11 pregnant women (Table 1 ) were identified in Australia during
routine surveillance programs with processed meats and piit6 cited as possible vehicles of
infection [ 139,162,2341 based on incomplete laboratory andlor epidemiological findings.
Continued surveillance of listeriosis cases by CDC officials uncovered a direct link
between consumption of contaminated turkey frankfurters and listerial meningitis in an
Oklahoma breast cancer patient [47] (to be discussed shortly) and also led to a nationwide
recall of the product [35] along with radical changes in the U.S. Department of Agricul-
ture-Food Safety and Inspection Service (USDA-FSIS) policy regarding the presence of
L. rnonocytogenes in cooked, ready-to-eat, or otherwise processed meat and poultry prod-
ucts. In the light of this information, some public health officials are now advising high-
risk individuals (i.e., pregnant women, immunocompromise:d adults, and the elderly) to
thoroughly reheat previously cooked and chilled meat and poultry products before con-
sumption. Hence, the proven ability of L. rnonocytogenes to grow and/or survive in many
refrigerated raw, processed, and ready-to-eat foods, including meat and poultry products,
together with extensive food histories now being obtained from many listeriosis victims
in the United States, make it highly probable that meat products, particularly frankfurters
and ready-to-eat meats, will be positively linked to cases of human listeriosis in the future.

POULTRY PRODUCTS
Shedding of L. rnonocytogenes in fecal material from both clinically and subclinically
infected domestic fowl 1791 appears to place poultry workers at a somewhat higher than
normal risk of contracting superficial listerial infections, particularly conjunctivitis. This
probable association between handling infected poultry and contracting conjunctivitis is
partially based on a 1951 report by Felsenfeld [96], who, 7 years earlier, identified listerial
conjunctivitis in two employees who dressed poultry in Illinois. On further investigation,
L. rnonocytngenes was isolated from the spleens of five birds that were not dressed in the
same shop but came from an area in Illinois in which avian listeriosis was previously
observed, thus suggesting poultry as the probable source of infection. Although reports
336 Ryser

of listerial conjunctivitis can be found in the early scientific literature, including several
cases in which patients had contact with birds suffering from undetermined illnesses [ l 111,
the 1951 report by Felsenfeld [96] remains one of the few instances where avian listeriosis
was linked to listerial conjunctivitis in humans.
A search of the early scientific literature has uncovered only two reports indicating
that contact with infected poultry may lead to the systemic infections for which L. rnonocy-
togenes is best known. In 1958, Gray [ 1111 cited numerous instances in which Central
European women gave birth to Listeria-infected infants following contact with sick or
dead birds; however, evidence for the link between listeriosis and contact with infected
poultry was only circumstantial.
Similarly, Embil et al. [89] identified a woman in Nova Scotia, Canada, who gave
birth to an infected infant who died of listeriosis 1 h after delivery. Although the mother
reportedly prepared poultry for sale in a family-owned store during the previous 8 months,
researchers again failed positively to link this listeriosis case to contact with raw poultry
by not isolating the pathogen from raw chickens sold at the store.
Given the preceding evidence, Kampelmacher [135] suggested as early as 1962 that
consumption of contaminated poultry might lead to cases of human listeriosis. Although
this view also was voiced 10 years later by Mir6 and Ralovich [159a], transmission of
L. monocytogenes from contaminated poultry was not documented until November 1988
[137]. As was true for meat products, failure positively to link consumption of contami-
nated poultry to human listeriosis was until recently primarily related to difficulties in
isolating L. monocytogenes from poultry and other foods containing a complex microflora
and to a generalized lack of concern about foodborne listeriosis.
Following the two major cheese-associated outbreaks in 1985 and 1987, public
health officials in the United States and England implemented active/semiactive surveil-
lance programs to obtain more accurate data on the incidence of listeriosis in the general
population. Attempts also were made to trace the source of reported infections to consump-
tion of dairy products and other foods such as poultry which at the time had not yet been
linked to listeriosis. As a result of these efforts, three cases of listeriosis were positively
linked to consumption of poultry products, which, in turn, has led to inclusion of poultry
in the list of foods that may pose a potential threat of listeriosis to susceptible individuals.
These three recently recognized cases will now be reviewed in some detail.
Worlung in England, Kerr et al. [33,137] identified the first case of listeriosis clearly
linked to consumption of contaminated poultry. According to their November 1988 report,
a 3 1-year-old pregnant woman with a 24-h history of flu-like symptoms was admitted to
a hospital and subsequently delivered an aborted 23-week-old fetus. On further investiga-
tion, the woman reportedly consumed a heated chicken dish prepared from cooked-and-
chilled chicken 5 days before onset of symptoms, with the remaining chicken being refrig-
erated and consumed 3 days later in a salad. Thus the woman had a maximum incubation
time of only 4 days before onset of symptoms as compared with the more typical 7-
30 days for listeriosis. Following bacteriological analysis, an identical phage type of L.
monocytogenes serotype 4 was found in samples of chicken and fetal liver. Other foods
in question were tested, with no evidence of Listeria contamination, thus confirming
chicken as the vehicle of infection.
Considerable research and regulatory activity, prompted by reports suggesting that
12-25% of cook-chill poultry products marketed in England may be contaminated with
L. rnonocytogenes, uncovered a second case of poultry-associated listeriosis early in 1989.
According to this report [134], L. rnonocytogenes serotype 1/2a was cultured from the
Foodborne Listeriosis 337

blood of a 52-year-old immunocompromised woman who was receiving steroids for sys-
temic lupus erythematosus. Three to 5 days before onset of vomiting and diarrhea, the
hospitalized woman and her 29-year-old son shared some ready-cooked chicken nuggets
which he had purchased at a fast-food restaurant. Detailed questioning later revealed that
he experienced a short-lived illness with diarrhea and vomiting on the same night that his
mother became sick. Subsequently, the sons stool sample yielded L. innocua as well as
L. rnonocytogenes serotypes 1/2a and 1/2c, with the DNA homology pattern of the sero-
type 1/2a isolate being identical to that of the L. rnonocytogenes strain originally isolated
from the womans blood. Although L. innocua and an L. rnonocytogenes strain of unre-
ported serotype and DNA homology pattern were recovered from uncooked chicken nug-
gets, these investigators failed to detect L. rnonocytogenes in a subsequent lot of cooked
chicken nuggets obtained from the same source. Nonetheless, infection in both the woman
and her son presumably was acquired from commercially cooked chicken nuggets of the
fast-food variety, which, although served hot, were most likely undercooked, thus allowing
L. monocytogenes to survive in sufficient numbers to cause illness.
Although this is only the second case of poultryborne listeriosis recorded in England,
similar cases have likely gone undetected because of inadequacies in reporting and diffi-
culties encountered in linking these illnesses to consumption of poultry or any other food.
These two cases of poultryborne listeriosis and a recent survey of listeriosis cases in Scot-
land which included identification of possible food-related risk factors associated with the
disease [68] prompted the Public Health Laboratory Service (PHLS) to conduct a national
case-control study in England and Wales which attempted to correlate consumption of
high-risk foods (i.e., poultry, pitt5, cheese, prepared salads, delicatessen items) with human
listerial infections [ 1181. A total of 124 cases diagnosed from July 1990 to January 1992
were identified from both the national voluntary reporting laboratory system and the PHLS
Listeria reference laboratory and matched to 459 controls according to age, sex, underlying
illness, and pregnancy status. After obtaining dietary histories, undercooked and ready-
cooked chicken consumed either hot or cold were statistically related to development of
listeriosis in both pregnant and nonpregnant individuals. Additional epidemiological stud-
ies are needed in England, the United States, and elsewhere to expand the number of
reported foodborne listeriosis cases and generate a more comprehensive list of foods that
pose a significant public health threat.
Despite the controversial nature of many epidemiological studies, such efforts have
already played an important role in identifying possible risk factors associated with food-
borne listeriosis. As you will recall from our aforementioned discussion of meatborne
listeriosis, undercooked chicken was identified as a high-risk vehicle of infection by CDC
officials during several case-control studies [ 182,212,2141 conducted in conjunction with
an active listeriosis surveillance program in Oklahoma and five other states. In two spo-
radic cases traced to turkey frankfurters and sliced turkey ham [ 1821, L. rnonocytogenes
isolates from unopened packages of the same product brand belonged to the same electro-
phoretic enzyme type as the patient isolate, thereby implicating turkey frankfurters and
sliced turkey ham as the source of infection.
During the first of these surveillance programs, CDC officials learned of a breast
cancer patient in Oklahoma who had been infected with L. rnonocytogenes and hospitalized
for listerial septicemia and meningitis in December 1988 [35,47]. In an attempt to identify
the vehicle of infection, investigators went to the womans home, obtained foods from her
refrigerator, and eventually isolated Listeria from various products, including an opened
package of turkey frankfurters that contained > 1.1 X 103L. rnonocytogenes CFU/g. A
338 Ryser

swab sample from the refrigerators interior also yielded the pathogen. Although CDC
investigators initially concluded that the woman had contaminated the food herself, public
health officials from Oklahoma began examining the same brands of retail products from
the womans refrigerator that were positive for L. monocytogenes. Interest soon focused
on turkey frankfurters after officials learned that the woman consumed one turkey frank-
furter daily after 45-60 s of heating in a microwave oven. Four months later, the same
strain and isoenzyme type of L. monocytogenes serotype 1/2a recovered from this patient
and the opened package of turkey frankfurters was also identified in five of seven unopened
packages of identical product purchased from nearby stores [243a], thereby confirming
turkey frankfurters as the vehicle of infection in the first poultryborne listeriosis outbreak
recorded in the United States.
Once the USDA-FSIS was notified of this case by the CDC on April 14, 1989,
government officials prompted the Texas manufacturer to issue an immediate recall for
approximately 600,000 pounds of turkey frankfurters that were marketed by retail and
institutional establishments in 23 states [35]. Joint investigations initiated by the CDC and
USDA-FSIS 1 day later eventually showed that six of seven retail lots of product produced
over a 37-day period contained the implicated L. monocytogenes strain at a most probable
number (MPN) level of <0.3 CFU/g. Furthermore, environmental testing of the produc-
tion facility showed that only 2 of 40 samples taken before sausage peeling harbored L.
monocytogenes as compared with 12 of 14 samples taken after peeling, with the implicated
strain also being recovered from a conveyor belt attached to the peeler. These findings
suggest that the factory was experiencing an ongoing contamination problem at a single
point during the sausage peeling process. Although large quantities of product contained
relatively few listeriae, the ability of L. monocytogenes to grow to hazardous levels in
such processed poultry products during refrigerated storage indicates that special precau-
tions must be taken to eliminate this pathogen from the food processing environment.
This incident has since prompted the USDA-FSIS to toughen its regulatory policy regard-
ing Listeria-contaminated poultry and meat products (see Chap. 13).

EGGS AND EGG PRODUCTS

Acquiring listeriosis through consumption of contaminated eggs and egg products has
been considered for nearly 40 years; however, unlike poultry products, no such cases have
been firmly documented. Geurden and Devos [ 1031, who in 1952 isolated L. monocyto-
genes from a necrotic lesion in the oviduct of an infected hen, were first to suggest that eggs
might serve as a possible vehicle of infection. Nonetheless, these authors also admitted that
a 35-year-old man who consumed raw eggs from this infected chicken flock showed no
signs of listeriosis. Three years later, Urbach and Schabinski [227] found that guinea pigs
who were fed artificially infected eggs in which L. monocytogenes had grown to very
high levels soon died of listerial septicemia, thus suggesting that humans also might de-
velop listeriosis by consuming contaminated eggs.
During the same year (1955), Dedii [77] reported an interesting case in which a
pregnant woman, who owned nine chickens, gave birth to a Listeria-infected infant. Al-
though 26 eggs from these chickens were negative for L. monocytogenes, the H and 0
agglutination titers of the chickens increased dramatically during the 4-week period in
which the eggs were collected, thus suggesting recent exposure to this pathogen. Subse-
quent attempts to isolate L. monocytogenes from 200 eggs laid by experimentally infected
hens ended in failure. However, the fact that L. monocytogenes was detected in feces and
Foodborne Listeriosis 339

nasal secretions from these birds suggests that externally contaminated eggs may constitute
a potential source of infection and a potential source for cross contamination of other
foods if the eggs are handled improperly.
As is true for meat and poultry workers, listerial infections among apparently healthy
individuals employed in the egg industry are extremely rare. In fact, a search of the litera-
ture uncovered only one documented case from 1965 in which a 39-year-old male egg
factory worker became infected with L. rnonocytogenes and subsequently died of meningi-
tis [ 135a1. Other than the fact that 29.1 and 10.6% of the egg factory workers carried L.
rnonocytogenes in their feces 4 and 16 months after the mans death, respectively, no
further evidence was reported to incriminate eggs in this fatal case of listerial meningitis.
Hence, at this time it appears that healthy egg factory workers need not take any special
precautions to guard against listeriosis.
Despite the inability to link listeriosis to consumption of eggs or egg products, the
recognized ability of L. monocytogenes to grow in egg products [ 1001 along with emer-
gence of this organism as a bonafide foodborne pathogen may change this picture in the
future. This prediction is supported by the fact that CDC officials [2 131 identified a possi-
ble cluster of listeriosis cases in Los Angeles County in which 6 of 33 cases and 4 of 101
matched controls consumed raw eggs (odds ratio = 6.4) over a 5-month period spanning
1986- 1987.
Although epidemiological investigations can never conclusively prove causality, the
possible associations discovered in such studies will likely prompt public health authorities
seriously to consider eggs and other foods (e.g., seafood, fruits) as potential vehicles of
infection, which, in turn, may lead to their implication in future cases of listeriosis.

SEAFOOD PRODUCTS
Despite the recent discovery of L. rnonocytogenes in a wide range of raw and processed
fish and seafoods, including finfish (i.e., smoked salmon, cod, trout), mussels, oysters,
shrimp, crabmeat, lobster tails, and surimi, most attempts directly to link consumption of
such products to cases of human listeriosis have proven unsuccessful. Nonetheless, a few
scattered reports attesting to possible involvement of seafoods in listerial infections along
with three reports of cases positively linked to consumption of fish, smoked mussels, and
artificial crabmeat have found their way into the scientific literature. Two early reports
from New Zealand include (a) a 1971 observation that two pregnant women delivered
Listeria-infected infants after presumably consuming raw fish sometime during their preg-
nancies [49], and (b) a cluster of 22 perinatal listeriosis cases between January and Novem-
ber of 1980 in which food histories suggested, at best, a weak association between con-
sumption of contaminated shellfishh-awfish and development of listeriosis [ 1441. In 1980,
Vilde et al. [228] published a report suggesting that a 48-year-old immunocompromised
French woman had contracted listeriosis after consuming contaminated oysters. More re-
cently, Arriold and Coble [39] identified two miscarriages among Australian women (one
case in Victoria and the other in New South Wales) in which smoked salmon was impli-
cated as the vehicle of infection, with Tan et al. [222] suggesting possible involvement
of smoked salmon and salmon cheese spread in two additional Australian cases of listeri-
osis. In one of the largest suspected seafood-related outbreaks thus far reported, 8 of 36
previously healthy adults attending a June 1989 party in New York City developed a
predominantly mild form of listeriosis characterized by fever, nausea, vomiting, diarrhea,
and musculoskeletal distress [ 1941. However, two cases of bacteremia caused by the epi-
340 Ryser

demic strain (an unusual enzyme type of L. monocytogenes serotype 4b) also occurred
among expectant mothers, with one pregnancy ending in a miscarriage. Although epidemi-
ological evidence most strongly implicated shrimp as the vehicle of infection, shrimp from
the party was unavailable for testing, and investigators also were unable to detect Listeria
in shrimp purchased 6 weeks later from the partys supplier. Thus, as already implied, a
causal link between consumption of seafood and listeriosis was never clearly proven in
any of these cases.
The first convincing evidence for direct involvement of fish or seafood in human
listeriosis is that of a 54-year-old Italian woman who in 1988 or 1989 contracted nonfatal
meningitis 3-4 days after consuming undercooked fish from which L. monocytogenes was
subsequently isolated [92]. The fact that L. monocytogenes isolates from the patients
cerebrospinal fluid and a leftover portion of the fish were of serotype 4 and were identical
based on phage typing and DNA restriction analysis confirmed fish as the vehicle of infec-
tion in this case of listerial meningitis. According to the investigators, survival and trans-
mission of the pathogen was most likely the result of undercooking, since the fish was
eaten and refrigerated after steaming. However, the mode by which this fish became con-
taminated could not be determined.
In August of 1991, the Tasmanian Health Department was notified about an other-
wise healthy 37-year-old woman and her 10-year-old son who developed malaise, chills,
fever, headache, vomiting, and diarrhea 3 days after eating 90 g of smoked mussels im-
ported from New Zealand [160,161]. On culturing, opened and unopened packages of
smoked mussels yielded L. monocytogenes at a level of 1.6 X 107CFU/g (i.e., oral in-
fective dose of 9 X log CFU), with contamination traced to three batches in which the
products shelf life was inadvertently overestimated by at least 3 months. Despite two
public warnings and withdrawal of the implicated samples from sale, one additional case
was reported a month later in an 83-year-old woman who developed similar symptoms
of gastroenteritis. Although results from strain-specific typing studies are not available,
the fact that all three victims became ill within 3 days of consuming smoked mussels
clearly points to this heavily contaminated product as the most probable source of
infection.
Working in New Zealand, Brett et al. [63] also identified two perinatal cases of
listeriosis in Aukland during November and December of 1992 in which the women gave
histories of consuming one particular brand of smoked mussels. During follow-up investi-
gations, an unopened package of smoked mussels obtained from one victims refrigerator
yielded a strain of L. monocytogenes serotype 112 that was indistinguishable from both
clinical isolates based on phage typing and pulsed-field gel electrophoresis. On further
searching, this unusual strain was identified in retail packages of smoked mussels marketed
in Aukland, New Zealand, and Brighton, England, as well as in environmental swab sam-
ples from the mussel processing factory, thereby confirming smoked mussels as the vehicle
of infection and the factory environment as the source of contamination. Although no
additional cases were confirmed in New Zealand, export of these mussels to England
combined with a reported association between mussel consumption and listeriosis in En-
gland [118] suggests that these mussels may have been responsible for additional cases
abroad. Given a 1995 report from Tasmania [219] in which L. monocytogenes was recov-
ered from 15.4% of premarket raw blue mussels and Pacific oysters collected in the wild
and from farms, public health concerns regarding shellfish safety need to be reexamined.
In 1997, Farber [94] also identified two cases of foodborne listeriosis among healthy
adults in Ontario, Canada. Samples of imitation crab meat, canned black olives, macaroni/
Foodborne Listeriosis 34 7

vegetable salad, spaghetti sauce with meatballs, and mayonnaise taken from the victims
refrigerator all yielded L. monocytogenes, with imitation crab meat containing 2.1 X 109
CFU/g. On further investigation, clinical strains and isolates from both opened and un-
opened packages of crab meat were identified as serotype 1/2b and were also indistinguish-
able by both randomly amplified polymorphic DNA analysis and pulsed-field gel electro-
phoresis, thereby confirming imitation crab meat as the vehicle of infection.
The aforementioned listerial infections along with other recently reported cases of
foodborne listeriosis that have been positively linked to consumption of dairy as well
as ready-to-eat meat and poultry products suggest it would be naive to assume that L.
monocytogvnes poses any less danger to public health when present in cooked and/or
ready-to-eat seafood than in other foods. Hence, FDA officials have maintained a policy
of zero tolerance for L. monocytogenes in all ready-to-eat foods and have (mid 1998)
issued numerous Class I recalls involving well over 45,000 pounds of contaminated
cooked/ready-to-eat seafood. These recalls and the fact that CDC officials now include
various shellfish and finfish in food history questionnaires given to listeriosis victims in
five states as well as Los Angeles County [24,28,212,214] make it likely that additional
listeriosis cases will be positively linked to fish and seafood in the future.

FOODS OF PLANT ORIGIN

Evidence for transmission of listeriosis through foods of plant origin probably dates back
to 1922 when investigators in Iceland described a listeriosis-like illness in silage-fed
animals [ 1 131. This apparent relationship between consumption of improperly fermented
silage and listeriosis in ruminants was clarified in 1960, and now numerous reports of
silage-related listeriosis outbreaks in sheep and cows can be found in the scientific litera-
ture. Although additional cases of animal listeriosis associated with consumption of other
types of animal feed have been primarily limited to scattered reports involving cattle that
grazed on Ponderosa pine needles in western Canada, one 1977 outbreak of listeriosis
in chinchillas was attributed to a batch of meal containing beet pulp [97]. However, L.
monocytogenes was never isolated from the incriminated feed.
Given this link between animal listeriosis and consuinption of contaminated plant
material, i1 is reasonable to suspect that raw vegetables (e.g., cabbage, lettuce) and fruits
are responsible for a certain percentage of listeriosis cases appearing in the human popula-
tion, as was first suggested by Blendon and Szatalowicz [58] in 1967. Evidence for
involvement of raw fruits in human listeriosis is currently limited to one 1984 unpublished
report frorn Connecticut suggesting a possible link between listeriosis and consumption
of unwashed strawberries, blueberries, and/or nectarines and one recent case of neonatal
listeriosis in Italy which was traced to olives and confirmed by DNA fingerprinting [71a].
In contrast, the ability of certain segments of the population to succumb to listeriosis after
consuming contaminated raw vegetables has been well documented over the past 15 years.
In 1986, Ho et al. [122] published results from an earlier epidemiological investiga-
tion in which raw vegetables were suggested as a possible vehicle of infection in an out-
break of listeriosis that occurred among patients in eight Boston-area hospitals during
September and October of 1979. However, it is noteworthy that 1 year before these find-
ings were published, Schlech et al. [210] published their landmark report describing the
first confirmed North American outbreak of foodborne listeriosis in which 41 Canadians
became ill in 1981 after consuming coleslaw contaminated with L. monocytogenes. Hence,
342 Ryser

it seems unlikely that the listeriosis outbreak in Boston would have been reported if
Schlech et al. [210] had not published their results first.
In the outbreak described by Ho et al. [ 1221, 23 patients admitted to Boston-area
hospitals acquired systemic listerial infections during September and October of 1979,
with L. monocytogenes isolates from 20 of 23 (87%) patients identified as serotype 4b
(Fig. 10). In contrast, only 19 listeriosis cases were identified at the same eight hospitals
during the 26-month period immediately preceding the outbreak. Unlike the previously
described foodborne outbreaks, which included pregnant women, neonates, and adults, all
20 outbreak-related cases of listeriosis from which L. monocytogenes serotype 4b was
isolated were adults ranging in age from 46 to 89 years, with half the victims being immu-
nocompromised as a result of cancer, chemotherapy, or steroid treatment, Overall, 18 of
20 (90%) and 8 of 20 (40%) patients suffered from listerial bacteremia and meningitis,
respectively. Although 5 of 20 (25%) patients died, two of these deaths were related to
underlying illnesses rather than listeriosis.
A series of epidemiological studies revealed that cases were more likely than con-
trols (i.e., listeriosis patients treated at the same eight hospitals during the 26-month period
preceding the outbreak) to have (a) become infected with L. monocytogenes serotype 4b
rather than another serotype, (b) acquired listeriosis during hospitalization, (c) exhibited
gastrointestinal symptoms, and (d) received antacids or cimetidine before onset of illness.
More important, results from food histories revealed that cases were more likely than
controls to have consumed tuna, chicken salad, and cottage cheese as well as hard cheese.
Although it was at first difficult to develop a scenario by which this apparent common-
source outbreak could have resulted from consumption of three seemingly unrelated foods
obtained from different distributors, hospital kitchen records revealed that all three foods

Type other than 4b

...
...
...
..,
..<
...
...
..<
...
...
. . I

...
....
....<
...
...
...
...
,,..
..
...
I . .

. . I

0
-R
2 3 4
5 6 7 8 9 10 11 I21
n
1977 1978 1979

FIGURE10 Number of listeriosis cases in eight Boston-area hospitals that resulted

f r o m infection with serotype 4b, non-serotype 4b, and untypeable strains of L. mono-
cytogenes between July 1977 and December 1979. (Adapted f r o m Ref. 122.)
Foodborne Listeriosis 343

were commonly used in salads containing lettuce, celery, and/or tomatoes. Thus, in light
of the 1981 Canadian listeriosis outbreak involving coleslaw., these researchers postulated
that the Boston outbreak was the result of victims having consumed raw lettuce, celery,
and/or tomatoes that were contaminated with listeriae. However, since no attempt was
made to isolate L. monocytogenes from these vegetables during the outbreak, the exact
source of infection will always remain in question.

Coleslaw: Canada, 1981

Two years after this presumed vegetableborne outbreak of listeriosis in Boston, Schlech
et al. [210] positively identified coleslaw as the vehicle of infection in a major outbreak
of listeriosis that occurred in the Maritime Provinces of Canada. According to their report,
34 perinatal and 7 adult cases of L. rnonocytogenes serotype 4b infection were diagnosed
between March 1 and September 1 of 1981 in Nova Scotia, New Brunswick, and Prince
Edward Island as compared with 22 cases in the 26-month period immediately preceding
the outbreak (Fig. 11). Perinatal cases were characterized by acute febrile illness in preg-
nant women followed by spontaneous abortion (5 cases), stillbirth (4 cases), live birth of
a seriously ill infant (23 cases), or live birth of a healthy infant (2 cases). Detailed clinical
and laboratory findings concerning 15 of these cases diagnosed at Grace Maternity Hospi-
tal in Halif'ax, Nova Scotia, were subsequently reported by Evans et al. [90]. Six cases
of meningitis and one case of pneumonia/septicemia were diagnosed in seven nonimmu-
nocompromised adults (six men and one nonpregnant woman) who were 2 1-8 1 years old.
Fifteen of 34 (44%) perinatal and 2 of 7 (29%) adult victims died, giving an overall
mortality rate of 41% [141].
Two case-control studies were subsequently initiated 1.0 assess possible risk factors
for acquisition of listeriosis. The first study examined medical, residential, occupational,
travel, and educational histories as well as exposure to animals and information concerning
gardening and outdoor activities, whereas the second case-control study involved a general

'' 7 Perinatai cases

14

12

I0

8 8
U
6

I 1979 I 1980 I 198 1 I

FIGURE11 Number of perinatal and adult cases of listeriosis recorded in the Mari-
time Provinces of Canada from January 1979 t o December 1981. (Adapted from Ref.
210.)
344 Ryser

history of foods consumed during the 3 months before onset of illness. Analysis of results
from the first case-control study failed to implicate a common environmental source for
this outbreak. Although initial data collected from food histories also failed to implicate
any particular food, results from a second questionnaire that included names of several
food items found in the refrigerator of a man who developed pneumonia and septicemia
indicated that cases were more likely than controls to have consumed both coleslaw and
radishes. Multivariate analysis later showed that ingestion of radishes was associated with
consumption of coleslaw rather than illness. However, repeated interviews with cases and
controls who had previously denied eating coleslaw revealed that 100% of the cases but
only 40% of the controls remembered consuming the product during the 3-month period
before the outbreak.
Armed with this information, investigators visited the home of one of the patients,
sampled foods from the refrigerator, and then isolated L. rnonocytogenes from coleslaw
but no other foods obtained from the patients refrigerator. The strain isolated from cole-
slaw was soon identified as serotype 4b, the same serotype isolated from all 34 victims.
To prove that the patient had not inadvertently contaminated the coleslaw, investigators
obtained two unopened packages from two different Halifax-area supermarkets and recov-
ered L. rnonocytogenes serotype 4b from each package. Audurier et al. [43] later reported
that 28 of 3 1 (90.3%) L. monocytogenes serotype 4b isolates obtained from blood, cerebro-
spinal fluid, and/or placental material between January and September of 1981 were of
the same phage type. Any remaining doubt concerning the role of coleslaw in this outbreak
was eliminated when clinical and coleslaw isolates were later shown to be of the same
electrophoretic enzyme type [40,411, pulsed-field gel electrophoretic type [65], ribotype
[ 1101, restriction enzyme pattern [235], randomly-amplified polymorphic DNA pattern
[74], and DNA macrorestriction pattern [ 1261 with this strain being closely related both
phenotypically and genotypically to those responsible for outbreaks in the United States
(Mexican-style cheese 1985) and Switzerland (Vacherin Mont dOr cheese 1983- 1987)
[126]. Thus Schlech et al. [209,210] can be credited with providing the first concrete
evidence that consumption of contaminated food, in this instance coleslaw, can cause
listeriosis in humans.
After coleslaw was confirmed as the infectious vehicle, Schlech et al. [210] at-
tempted to determine the route by which the coleslaw became contaminated. Investigators
soon traced the tainted coleslaw to a regional manufacturer whose product had been dis-
tributed exclusively in the Maritime Provinces of Canada. Although repeated microbiolog-
ical testing of environmental samples from the coleslaw factory failed to uncover the
contamination source, review of factory records along with recent data on animal listeriosis
revealed that the manufacturer had received at least 2250 kg of cabbage from a farmer
who also raised sheep. Furthermore, the farmer lost two sheep in his flock to listeriosis-
one in 1979 and one in 1981. A review of the farmers agronomic practices indicated that
cabbage was routinely fertilized with both raw and composted sheep manure obtained
from animals that were presumably fecal carriers of L. rnonocytogenes. Moreover, the
final October cabbage crop was held in a large cold-storage shed until early spring. Such
storage practices, which somewhat resemble cold enrichment, may have led to an increase
in numbers of L. rnonocytogenes in the cabbage. Although none of the implicated cabbage
crop (other than that which was previously manufactured into coleslaw and contaminated
with the epidemic strain of L. rnonocytogenes) was available for testing, and none of the
farm environmental samples, including raw sheep manure, yielded Listeria, the aforemen-
tioned circumstantial evidence still supports an indirect link between sporadic cases of
ovine listeriosis on a cabbage farm and cases of listeriosis in humans consuming a Listeria-
Foodborne Listeriosis 345

contaminated product. The fact that (a) crops grown in Canada, the United States, and
industrialized European nations are routinely fertilized with material other than raw ma-
nure, (b) 82% of all L. monocytogenes strains isolated during one large survey of raw
vegetables marketed in the United States were of serotype la [ 1201, and (c) most L. mono-
cytogenes strains isolated from animal sources, including feces, in North America are of
serotype 4 rather than 1 support transmission of L. monocytogenes from raw sheep manure
to cabbage. Hence, raw manure should not be used to fertilize vegetables that will be
consumed without cooking.

Additional Reports of Listeriosis Associated with Products

of Plant Origin
Despite the proven link between coleslaw and development of listeriosis in the Canadian
outbreak just discussed, consumption of raw vegetables, fruits, and other types of produce
has not generally been deemed to constitute a major public health threat 12121. This lack of
concern resulted because consumption of raw produce has until recently been infrequently
associated with most forms of foodborne disease. However, after Mexican-style and Vach-
erin Mont dOr soft-ripened cheese were directly linked to two separate listeriosis out-
breaks (see earlier discussion in this chapter), public health officials around the world
became increasingly interested in the possibility that nondairy foods such as meat, poultry,
seafood, and raw produce could transmit L. monocytogenes to humans.
As of May 1997, only one additional small outbreak and three sporadic cases have
been documented in which consumption of a product of plant (or microbial) origin was
directly linked to human listeriosis. According to a 1996 report by Simpson [2 161, a hospi-
tal laboratory on the Texas-Mexican border cultured L. monocytogenes serotype 4b from
five patients (three newborn infants, one pregnant woman, and one immunocompromised
adult) over a 5-week period, with two additional cases being identified over the following
2 months. Although 23 food samples initially collected from the victims houses were
negative for listeriae, frozen broccoli and cauliflower were implicated in a follow-up case-
control study. Investigators eventually recovered L. monocytogenes serotype 4b from
opened and later unopened packages of frozen vegetables. Using pulsed-field gel electro-
phoresis, the food and seven patient isolates proved to be identical, thereby confirming
commercially frozen broccoli and cauliflower as infectious vehicles in this unusual out-
break of listeriosis. In November 1988, Ken et al. [ 1371 reported that a 29-year-old preg-
nant woman in England miscarried after a 2-week history of pyrexia and rigors. An identi-
cal phage type of L. monocytogenes serotype 4b was subsequently isolated from placental
swabs and maternal/fetal blood samples as well as a 3-month-old bottle of vegetable rennet
(may have been microbial rennet) that was discovered in the womans refrigerator, thus
implicating vegetable rennet which was presumably used to produce a custard-like
product.
In another of these isolated cases, an 80-year-old previously healthy man in Finland
developed a nonfatal septicemic infection 1 day after consuming previously cooked but
not reheated homemade salted mushrooms that were later found to contain 3.8 X 106L.
monocytogenes CFU/g [133]. Listeria isolates from the patients blood and a portion of
the uneaten mushrooms belonged to serotype 4b and the same phage type, thus confirming
salted mushrooms as the vehicle of infection. According to the investigative team, the
implicated homegrown mushrooms were washed, cooked, salted to a level of 7.5% NaC1,
and then stored in a cold cellar for 5 months before consumption. Since this product,
which was presumably contaminated after cooking, had a pH of 5.9 with visible mold
346 Ryser

growth on the surface, the pathogen probably grew in these mushrooms during the 5
months of storage. Although the exact source of contamination was never determined, it
is noteworthy that the mans wife did not become ill after consuming thoroughly reheated
mushrooms from the same container. Hence, it appears that heavily contaminated vegeta-
bles also can be rendered safe by thorough cooking.
The final case of listeriosis directly linked to products of plant origin is markedly
different in that consumption of alfalfa tablets, a dry product in which L. rnonocytogenes
is presumably unable to grow, was directly responsible for a fatal case of listerial meningo-
encephalitis in a 55-year-old immunocompromised Canadian man [95]. As in the two
cases just discussed, L. rnonocytogenes strains of serotype 4b isolated from the victims
blood and cerebrospinal fluid as well as the remaining alfalfa tablets were all of the same
electrophoretic enzyme type, which in 1990 confirmed alfalfa tablets as the vehicle of
infection in the only listeriosis case thus far linked to ingestion of a nearly completely
dry product. However, since Czojka and Batt [74] recently found that these isolates yielded
slightly different randomly amplified polymorphic DNA patterns, the exact source of in-
fection now appears to be somewhat open to debate.
Although not directly linking consumption of raw vegetables to listerial infection,
considerable circumstantial evidence exists for involvement of vegetables in human listeri-
osis. In the first of these reports [43], a 74-year-old man contracted L. rnonocytogenes
serotype 1/2c septicemia and meningitis 1 1 days after repair of his perforated duodenal
ulcer in a London-area hospital. During the week before onset of illness, the patient con-
sumed hospital food supplemented with high protein puddings and sandwiches containing
various fillings, including cheese; however, hospital personnel failed to maintain an exact
record of foods eaten. In conjunction with a survey to determine the incidence of listeriae
in hospital-prepared foods, investigators isolated L. rnonocytogenes serotype 1/2a and L.
innocua from 1 of 15 and 2 of 15 samples of washed English round lettuce but failed to
detect Listeria spp. in 40 other food samples consisting primarily of dairy products and
other raw vegetables. Most evidence suggests that listeriosis has an incubation period of
1 to several weeks; however, three of four patients in England with confirmed cases of
foodborne listeriosis exhibited incubation periods of 5 1 week. Hence, although the incu-
bation period observed in this 74-year-old man is compatible with a hospital-acquired
listerial infection, isolation of different L. rnonocytogenes serotypes from the man and
washed English round lettuce appears to preclude direct involvement of lettuce in this
particular case of listeriosis. Nevertheless, since L. rnonocytogenes was recovered from
washed lettuce but from no other hospital-prepared food, consumption of lettuce still ap-
pears to be a possible risk factor in development of hospital-acquired listeriosis.
During the previously discussed 1986- 1987 case-control study in which consump-
tion of uncooked frankfurters and undercooked chicken was epidemiologically associated
with listeriosis, Schwartz et al. [214] isolated the same serotype and enzyme type of L.
rnonocytogenes from five Hispanic listeriosis patients who resided in Los Angeles County.
Of the four patients who voluntarily enrolled in the case-control study, all consumed let-
tuce as well as chicken and whole milk. Unfortunately, because matched controls con-
sumed these products at rates similar to those of cases, it was impossible to link consump-
tion of lettuce as well as chicken or whole milk to listeriosis. More recently, routine follow-
up investigations in Victoria, Australia, have provided some circumstantial evidence for
possible involvement of lettuce (one cluster of eight cases) [222], coleslaw (one case)
[222], raw vegetables/mayonnaise vegetable dip (one case) [201], and raw vegetables (one
case) [201] in sporadic cases of listeriosis. In the United States, active surveillance of a
Foodborne Listeriosis 347

large multistate population uncovered a small outbreak of listerial gastroenteritis during

1994 in which commercially prepared potato salad containing low levels of L. monocyto-
genes serotype 1/2a was the presumed vehicle of infection [221]. According to Salamina
et al. [206]. a similar listeriosis outbreak occurred 1 year earlier near Bologna, Italy, with
18 of 39 (46%) young (median age 28 years), previously healthy, nonpregnant adults
becoming ill after attending a party. Fourteen of 18 individuals developed listerial gastro-
enteritis characterized by diarrhea, fever, headache, vomiting, and abdominal pain (median
onset time 18 h), with the remaining four patients complaining of flu-like symptoms
(median onset time 43 h). Although four individuals with acute gastroenteritis were hospi-
talized, two of whom also developed septicemic infections, all patients were released 3-
14 days after antibiotic therapy. Analysis of the foods consumed showed that rice salad
(prepared from boiled rice, Swiss cheese, pickled vegetables, hard-boiled eggs and frozen
vegetables [i.e., peas, carrots]) was the most likely vehicle, since only those individuals
who ate the salad became ill. Unfortunately, none of the rice salad remained after initial
microbiological testing eliminated E. coli, Salmonella, B a c i h s cereus, and Stuphylococ-
cus aweus as possible causes. However, overwhelming evidence from the food-intake
survey, coupled with recovery of the epidemic strain of L. rnonocytogenes serotype 1/2b
(confirmed by both enzyme and phage typing) from several other foods consumed at the
party, reported temperature abuse of the rice salad as further evidenced by a total aerobic
plate count >109 CFU/g and probable cross contamination of other foods in the kitchen
points to rice salad as the most probable vehicle of infection. In 1997, Italy was the site
of another apparent foodborne outbreak of listerial gastroenteritis, this time the largest
to date involving 1594 cases, 123 of which required hospitalization (24 1). Preliminary
epidemiological findings suggest a possible link to consumption of sweet corn. Although
such epidemiological investigations alone can never prove causality, future studies similar
to those just described will continue to play a vital role in elucidating possible relationships
between listeriosis and consumption of vegetables as well as fruit, dairy, meat, poultry,
seafood, and other foods not yet thought to be associated with this disease.

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Incidence and Behavior of Listeria
monocytogenes in Unfermented
Dairy Products

ELLIOTT. RYSER
Michigan State University, East Lansing, Michigan

INTRODUCTION
Recognition of raw milk as a potential source of Listeria monocytogenes led to speculation
that consumption of such milk was at least partly responsible for the previously described
listeriosis outbreak in post-World War I1 Germany. After this listeriosis epidemic, only
scattered reports of individuals drinking raw milk, along with assurances that raw milk
was being properly pasteurized, virtually eliminated the threat of any further outbreaks
of milkborne listeriosis. Consequently, research in this area also subsided. However, in
1983, concerns about the possibility of milkborne listeriosis were rekindled when con-
sumption of pasteurized milk was epidemiologically linked to an outbreak of listeriosis
in Massachusetts. Two events, namely, publication of an article in the New England Jour-
nal of Medicine detailing this outbreak in Massachusetts and a report in June of 1985 that
as many as 300 people in California had acquired listeriosis after eating Mexican-style
cheese contaminated with L. monocytogenes, caused considerable concern in the United
States about the presence of Listeria in dairy products. This problem subsequently took
on international proportions with the 1987 report of another cheese-related outbreak in
which consumption of tainted Vacherin Mont dOr soft-ripened cheese was directly linked
to numerous cases of listeriosis in Switzerland. Despite considerable progress, such epi-
demics continue to plague the dairy industry, with pasteurized chocolate milk and Pont

359
360 Ryser

1EvSque cheese being responsible for the most recent dairy-related outbreaks of listeriosis
in the United States and France, respectively.
In response to questions raised by milk producers, dairy processors, health officials,
and the general public, a plethora of work has been conducted worldwide since 1983 to
determine the incidence and behavior of L. rnonocytogenes in unfermented (raw milk,
pasteurized milk, chocolate milk, cream, butter, ice cream, other frozen dairy desserts) as
well as fermented (cheese, yogurt, cultured milk) dairy products. The incidence and behav-
ior of L. rnonocytogenes in unfermented dairy products will be dealt with in this chapter;
similar information about fermented dairy products appears in Chapter 12.

INCIDENCE OF L/STR/A SPP. IN UNFERMENTED DAIRY

PRODUCTS
The dairy-related listeriosis outbreaks reported during the mid 1980s (see Chap. 10)
prompted scientists worldwide to determine the extent of Listeria contamination in raw
milk and in pasteurized dairy products such as milk, ice cream, ice cream novelties, frozen
desserts, nonfat dry milk, and casein. L. monocytogenes can readily enter dairy processing
facilities in the raw milk supply, which can in turn lead to contamination of the factory
environment. The occasional appearance of listeriae in pasteurized dairy products nearly
always has been associated with contamination of the product after pasteurization. Thus
it is fitting to begin this discussion by examining the incidence of Listeria spp. in raw
milk, which is a major source of this bacterium in dairy factory environments.

Raw Cows Milk

As you will recall from the discussion of animal listeriosis in Chapter 3, dairy cattle can
intermittently shed L. monocytogenes in their milk as a consequence of listerial mastitis,
encephalitis, or a Listeria-related abortion. Although milk from animals showing obvious
signs of listeriosis is unlikely to reach consumers, the scientific literature contains numer-
ous accounts in which mildly infected and apparently healthy dairy cattle have shed L.
monocytogenes intermittently in their milk for many months. Thus it appears that such
asymptomatic carriers of listeriae pose the greatest threat to public health.
The 1983 listeriosis outbreak in Massachusetts that was supposedly associated with
drinking a particular brand of pasteurized milk raised numerous questions about milk
safety. The well-publicized outbreak of 1985 in which consumption of contaminated Mexi-
can-style cheese was directly linked to at least 40 deaths in California prompted additional
concerns about the safety of dairy products manufactured in the United States. Since raw
milk is a potential source of L. monocytogenes, this fact together with recalls of Listeria-
contaminated pasteurized dairy products (i.e., milk, chocolate milk, ice cream) and im-
ported soft-ripened cheeses prompted nearly 70 surveys worldwide to determine the extent
of Listeria contamination in raw milk. Results of these surveys, which will now be de-
scribed in some detail, have been summarized in Tables l and 2.
The first large-scale survey of raw milk for Listeria spp. was prompted by the 1983
listeriosis outbreak in Massachusetts. During the 3-week period immediately following
the outbreak, Fleming et al. [ 1 101 and Hayes et al. [ 1291 examined 121 raw milk samples
collected from milk trucks (40 samples), milk cooperatives (72 samples), and bulk tanks
from four farms on which bovine listeriosis was diagnosed (9 samples), as well as 14 milk
socks (used to remove debris but not leukocytes from milk). All samples were analyzed for
L. monocytogenes in Unfermented Dairy Products 361

TABLE
1 Incidence of Listeria spp. in Raw Milk Produced in the United States and Canada
Number of positive samples (%)
Number of
Location samples L. inonocyogenes L. innocuu L. ri.eist'iirneri Others Ref.
USA
California 200 14 (7.0) 19 (9.5) 0 0 141
100 0 4 (4.0) 0 1 (1.0y 141
Massachusetts 121 15 (12.4) ND ND ND 129
Massachusetts, 939 15 (1.6) ND ND ND 87
Vermont
Minnesota 300 9 (3.0) 77 (25.7) 5 (1.7) 0 144
84 0 6 (7.1) 1 (1.2) 0 161
Nebraska 200 8 (4.0) 10 (5.0) 0 0 139
Ohio, Kentucky, 350 13 (3.7) 27 (7.7) 6 (1.5) 3 (0.9)a 141
and Indiana
Pennsylvania 251 1 79 (3.1) ND ND ND 88
Tennessee 292 12 (4.1) ND ND ND 177
Wisconsin 50 0 ND ND ND 89
55 0 0 0 0 20 1
Total 5 197 165 (3.2) 143 (11.1) 12 (0.9) 4 (0.3)
Canada
Alberta 426 8 (1.9) ND ND ND 106
252 4 (1.6) ND ND ND 200
Manitoba 256 4 (1.6) ND ND ND 80
Ontario 445 6 (1.3) 43 (9.7) 6 (1.3) 0 97
3 15 17 (5.4) 26 (8.2) 1 (0.3) 0 190
Total 1694 39 (2.3) 69 (9.1) 7 (0.9) 0 (0)
ND, not determined (omitted from total).
Two L. ivnnovii and one L. seeligeri
362 Ryser

TABLE
2 Incidence of Listeria spp. in Raw Milk Collected Outside of North America
Number of positive samples (%)
Number of
Location samples L. monocytogenes L. innocua L. welshimeri Others Ref.
Europe
Czechoslovakia 177 6 (3.4) ND ND ND 151
123 4 (3.3) ND ND ND 152
Denmark 1,227,053 278 (0.02) ND ND ND 167
Finland 256 13 (5.1) ND ND ND 167
59 1 (1.7) ND ND ND 167
France 1409 85 (6.0) ND ND ND 43
561 21 (3.8) ND ND ND 49
337 14 (4.2) 5 (1.5) 0 0 120,121
51 10 (19.6) 10 (19.6) 18 (35.3) 9 (17.6) 120,121
635 2 (0.3) ND ND ND 115, 197
80 3 (3.8) ND ND ND 174
50 2 (4.0) ND ND ND 174
Ireland 589 29 (4.9) 20 (3.4) ND ND 173
50 4 (8.0) ND ND ND 133
Italy 290 8 (2.8) 16 (5.6) 0 2 (0.7) 83
142 0 1 (0.6) 0 0 192
98 2 (2.0) ND ND ND 138
85 0 0 0 0 117
50 0 1 (2.0) 0 0 199
40 0 0 0 0 148
32 0 2 (6.3) ND ND 112
Netherlands 137 6 (4.4) ND ND ND 68
Poland 134 2 (1.5) ND ND ND 136
81 6 (7.4) ND ND ND 178
L. monocytogenes in Unfermented Dairy Products 363

Switzerland 4046 14 (0.4) ND ND ND 66

340 2 (0.6) ND ND ND 66
Turkey 77 14 (18.2) ND ND ND 187
United Kingdom
Great Britain 350 13 (3.6) ND ND ND 118
England/Wales 2009 102 (5.1) ND ND ND 157
361 13 (3.6) ND ND ND 126
Scotland 640 90 (14.1) ND ND ND 108
560 14 (2.5) 7 (1.3) 0 1 (0.2) 107
540 14 (2.6) ND ND ND 107
Northern Ireland 176 27 (15.3) 18 (10.2) 0 5 (2.8)a 127
113 6 (5.3) ND ND ND 119
Total 14,678 527 (3.6) 80 (3.4) 18 (0.8) 27 (1.2)
Elsewhere
Australia 169 1 (0.6) ND ND ND 95
150 0 4 (2.7) 0 0 130
Brazil 220 11 (4.8) 21 (9.5) 2 (0.9) 1 (0.4) 155
20 0 ND ND ND 73
Costa Rica 220 0 10 (4.5) 3 (1.4) 1 63
Egypt 236 7 (3.0) 20 (8.5) 0 0 93
Iran 190 4 (2.1) ND ND ND 172
Japan 120 0 ND ND ND 196
150 6 (4.0) 2 (1.3) 0 0 186
Mexico 100 0 7 (7.0) 2 (2.0) 0 142
Morocco 30 3 (10.0) ND ND ND 94
New Zealand 71 0 10 (14.1) 1 (1.4) 7 (9.9) 194
South Africa 982 67 (6.8) ND ND ND 204
Taiwan 80 5 (6.3) ND ND ND 74
Total 2738 104 (3.8) 74 (6.5) 8 (0.7) 9 (0.8)
ND, not determined (omitted from total).
aL. seeligeri.
L. grayi.
364 Ryser

L. rnonocytogenes using the multiple two-stage enrichment procedure. Although investiga-

tors at the U.S. Centers for Disease Control and Prevention (CDC) isolated the epidemic
serotype along with other serotypes of L. rnonocytogenes from 15 of 121 (12.4%) (includ-
ing 1 of 9 bulk tank samples) [ 1281 and 2 of 14 (14%) raw milk and milk sock samples,
respectively, the epidemic phage type was never detected.
Between October 1984 and August 1985, U.S. Food and Drug Administration (FDA)
officials surveyed 650 raw milk samples that were collected from bulk tanks in Massachu-
setts, Vermont, California, and the tristate area of Kentucky, Ohio, and Indiana. The sam-
ples were examined for Listeria spp. using the original FDA method [141]. Low levels
of various Listeria spp., including L. rnonocytogenes, were detected in raw milk samples
obtained from all states except California. Overall, 82 of 650 (12.6%) samples contained
Listeria spp., with L. rnonocytogenes being detected in 27 of 650 (4.2%) samples. Of the
27 L. rnonocytogenes strains isolated from raw milk, I6 were serotype 1, 10 were serotype
4, and 1 was nontypable. In addition, only 2 of the 27 L. rnonocytogenes strains proved
to be nonpathogenic to mice, and both were of serotype 4.
In 1988, Donnelly et al. [87] reported using an automated flow cytometric procedure
to analyze 939 samples of raw milk obtained from 54 farms in California. Unlike the FDA
study just described [ 1411, string samples (milk pooled from 25 to 40 cows), combination
samples (milk pooled from -200 cows), and samples of raw milk from bulk tanks were
tested for L. rnonocytogenes. Using this novel method of analysis, L. monocytogenes was
detected in 15 of 939 ( I .5%) samples of raw milk.
Researchers in Minnesota [ 1611 and Wisconsin [89,201] failed to detect L. rnonocy-
togenes in raw milk during three small surveys. However, this organism was found in 4.0
and 2.8% of raw milk samples obtained from bulk storage tanks and tank trucks in Ne-
braska [ I401 and Pennsylvania [88], respectively, with approximately equal numbers of
L. rnonocytogenes isolates being classified as serotype 1 , 4, or nontypable (non-serotype
1 or 4) in the latter study. More recent findings indicate a relatively stable incidence for
L. rnonocytogenes in bulk tank raw milk, with 3.0 and 4.1 % of such samples from Minne-
sota [ 1441 and Tennessee [177], respectively, being positive. In the Minnesota survey,
Listeria-positive samples also tended to have higher bacterial and somatic cell counts,
which are indicative of less stringent sanitation and mastitis control practices. The overall
findings from Table 1 indicate that L. rnonocytogenes was present in 0- 12.4% of the U.S.
raw milk samples examined. When the results are averaged, 3.2% of all raw milk processed
in the United States can be expected to contain low levels (i.e., <10 CFU/mL) of L.
rnonocytogenes at any given time with the incidence of L. innocua being appreciably
-
higher at 1 I . 1 %. Hence, it is imperative that all milk processors take special precautions
to prevent the spread of L. monocytogenes from raw milk to production, packaging, and
other sensitive areas within the factory.
Turning to the incidence of Listeria in Canadian raw milk (see Table l), Farber et
al. [97] and Slade et al. [ 1901, respectively, isolated L. monocytogenes from 6 of 445
(1.3%) and 17 of 315 (5.4%) raw milk samples obtained from bulk tanks located through-
out the province of Ontario. Davidson et al. [80] also reported that a similar percentage
of raw milk samples collected from four local dairies and 48 farms in Manitoba contained
L. rnonocytogenes. In the study by Slade et al. [ 1901, 14 of 17 (82%) L. rnonocytogenes
isolates belonged to serotype 1, with the remaining three strains being classified as serotypa
4. Although subsequent work [ 1091 demonstrated that none of these L. rnonocytogenes
strains harbored plasmid DNA, 8 of 22 (36%) L. innocua strains did carry plasmids ranging

in size from 10 to 44 MDa. Variable plasmid profiles among these isolates further suggests
L. monocytogenes in Unfermented Dairy Products 365

that contamination of raw milk on the farm is an ongoing process. Whereas these authors
did not quantitate L. monocytogenes in any of the samples examined, Slade and Collins-
Thompson [ 1891 reported that positive raw milks from bulk tanks in southwestern Ontario
always contained <5 L. monocytogenes CFU/mL when samples were analyzed by direct
plating and a most probable number (MPN) enrichment procedure. Thus the positive raw
milk samples encountered in three Canadian studies likely contained only low levels of
L. monocytogenes. In two subsequent surveys, L. monocylogenes was identified in 1.6
[200] and 1.9% [106] of all raw milk bulk tank samples examined in the province of
Alberta. However, both surveys also indicated substantially higher contamination rates
for commingled raw milk before processing with milk in 5 of 72 (6.9%) tank trucks [ 1061
and in 4 of 15 (26.6%) milk silos [200] testing positive for L. monocytogenes. As was
true for the three United States surveys [ 1411just discussed, L. innocua also was the most
common Listeria sp. isolated from Canadian raw milk, with 8.2 and 9.7% of the samples
being reported as positive. Overall, 2.3 and 9.1% of all Canadian raw milk samples con-
tained L. rnonocytogenes and L. innocua, respectively, as compared with 3.2 and 1 1.1 %
of raw milk samples examined in the United States. Thus the incidence of listeriae in raw
milk from both countries appears to be similar.
Public concern and economic hardships brought about by several recalls of French
Brie cheese imported into the United States prompted French scientists to begin surveying
raw milk for L. monocytogenes. Results from three such surveys [43,49,120] (see Table
2) conducted between 1986 and 1988 indicated that 85 of 1409 (6.0%), -21 of 561 (3.8%),
and 14 of 337 (4.2%) raw milk samples from French bulk tanks were positive for L.
monocytogrnes, with L. innocua being detected in about one third as many samples in
the latter study [120]. During January of 1986, 51 raw milk samples were submitted to
the French Central Laboratory of Food Hygiene. Both L. monocytogenes and L. innocua
were found in 19.6% of these samples. The unusually high incidence of listeriae ob-
served in this survey, as compared with other studies described thus far, may be the re-
sult of nonrandom sampling or seasonal variations, the latter of which will be discussed
shortly.
Frequent isolation of L. monocytogenes from dairy products prompted additional
surveys of the raw milk supply throughout Europe and later elsewhere, with primary focus
being given to countries with sizeable dairy industries (see Table 2). Results from two
comprehensive year-long surveys in the United Kingdom [ 1 181 and Scotland [ 1071 re-
vealed an incidence of L. monocytogenes in raw milk similar to that observed in the United
States and Canada, with actual Listeria populations in positive samples also being esti-
mated to be extremely low. As in North America, L. monocytogenes strains belonging to
serotype 1/2 appear to predominate in European raw milk [ 107,108,126,1781. Although
four subsequent surveys from the United Kingdom yielded similar findings [ 107,119,
126,1571, L. monocytogenes contamination rates of 14 to 15% have been reported from
both Scotland [ 1081 and Northern Ireland [ 1271, thus suggesting considerable local vari-
ability. Harvey and Gilmour [127] also found that 33% of raw milk samples collected
from processing centers harbored L. monocytogenes, which again emphasizes the impact
of commingling milk. However, levels of listeriae in such milk again appear to be quite
low, with actual numbers of L. monocytogenes typically being < 10 CFU/mL in positive
samples from Scotland [ 1081.
In 1987, Beckers et al. [68] reported culturing L. monocytogenes from 6 of 137
(4.4%) raw milk samples obtained from farms in the Utrecht region of The Netherlands.
As was true for raw milk tested in the United States and Canada, milk samples from The
366 Ryser

Netherlands again contained < 100 L. monocytogenes CFU/mL. Similar findings have
been reported from most other European surveys, with 3-5% of raw milk samples harbor-
ing low levels of L. monocytogenes. However, two nationwide surveys conducted in Swit-
zerland during and after the outbreak involving Vacherin Mont dOr cheese indicated a
far lower incidence, with only 0.4 and 0.6% of the raw milk samples being positive for
L. monocytogenes [66]. Several years earlier, Terplan [ 1971 detected L. monocytogenes
in only 2 of 635 (0.3%) raw milk samples obtained from farm bulk tanks in Wurtemburg,
Germany. Subsequent attempts to isolate this pathogen from 448 quarter-milk and 30
separator sludge samples failed, along with attempts to culture this organism from raw
milk samples obtained from tank trucks and storage tanks. More recently, findings from
an exhaustive survey in Denmark of over 1 million raw milk samples demonstrated an
L. monocytogenes contamination rate of only 0.2%, with several additional small-scale
Italian surveys yielding negative results. Thus, when the Danish results are excluded, 3.6%
of all European raw milk would be expected to contain low levels of L. monocytogenes
as compared with 3.0% of the raw milk produced in North America.
In response to the aforementioned North American and European surveys, investiga-
tors in Australia, New Zealand, the Middle East, South America, Africa, and the Orient
also have begun assessing the incidence of L. monocytogenes contamination in their own
raw milk supplies. In the first of these surveys, workers in New Zealand [ 1941 collected
and analyzed 71 raw milk bulk tank samples between August 1986 and March 1987 for
listeriae using both warm and cold enrichment. Although L. monocytogenes was appar-
ently absent from this milk as well as from milk examined in a later Australian survey,
isolation of other Listeria spp. from these samples strongly suggests that such milk is
unlikely to be completely free of L. monocytogenes. Although similar negative findings
have been obtained from surveys conducted in Brazil [73] and Costa Rica [63], Arias
et al. [63] also cited another survey in which 20% of hand-milked samples harbored L.
monocytogenes. Working in Japan, Takai et al. [196] reported the absence of Listeria spp.
from 120 raw milk samples, whereas L. monocytogenes was recovered from 6 of 150
(4.0%) samples in a subsequent Japanese survey [186]. Given these findings along
with additional reports of L. monocytogenes in raw milk from Egypt [93,1], Iran
[172], Turkey [124], Morocco [94], and South Africa [204], it is now clear that
milkborne listeriosis constitutes a worldwide threat. However, confirmation of dairy-
related listeriosis cases in such developing countries will likely remain very difficult
given their lack of resources and understandable preoccupation with far more immediate
health concerns.
Examination of data summarized in Tables 1 and 2 indicates that 3.2, 2.3, and 3.6%
of the raw milk produced in the United States, Canada, and Europe, respectively, can be
expected to contain low levels of L. monocytogenes, with similar contamination rates also
likely occurring in other parts of the world. Except for the European samples, L. innocua
was isolated from raw milk more frequently than L. monocytogenes, with the former found
in 11.1, 9.1, and 3.4% of the samples analyzed for all Listeria spp. in the United States,
Canada, and Europe, respectively. This observation also is supported by additional survey
findings from Australia, Costa Rica, Egypt, and New Zealand.
Although L. innocua is nonpathogenic, isolation of this organism and other listeriae
from dairy products and dairy processing facilities is taken very seriously in the United
States, since nonpathogenic listeriae and L. monocytogenes are assumed to occur in similar
environmental niches. Use in the United States of L. innocua as a potential indicator of
L. monocytogenes is supported by data from the raw milk surveys just described (see
L. monocytogenes in Unferrnented Dairy Products 367

Table 1) in that isolation of listeriae other than L. morzocytogenes from dairy products
and processing facilities suggests that the factory environment may be contaminated with
raw milk; that is, a product that can be expected to contain L. monocytogenes 3-4% of
the time. Although long theorized, the spread of identical L. nzonocytogenes strains from
the farm environment into the raw milk supply and ultimately to dairy processing facilities
now has been confirmed using various strain-specific typing methods, including restriction
fragment length polymorphism [ 1271 and automated ribotyping [64].
In addition to assessing the general incidence of listeriae in raw milk, several of the
surveys just described also dealt with seasonal variations in the incidence of L. monocyto-
genes and L. innocuci in raw milk produced in the United States [88,139,141] and Canada
(971. However, since all of the aforementioned studies differ in numbers and sizes of
samples analyzed as well as the Listericl isolation procedures employed, it is difficult to
make any definitive statement concerning the seasonal occurrence of Listerin in raw milk
[ 107,190]. Nonetheless, several distinct trends can be observed from selected data in Fig-
ure 1. First. the overall incidence of L. rnonocytogenes in raw milk was highest in spring
(5.8%) followed by winter (4.5%), fall (2.8%), and summer (2.5%). Second, a somewhat
similar seasonal variation also can be observed for the incidence of L. innocun in raw
milk, with the highest overall percentage of positive samples again occurring in winter
(1 1.7%) followed by spring (10.2%). summer (6.0%), and fall (4.2%). These findings
again suggest that L. innocirn can be used as a potential indicator for the presence of L.
inonocytogenes.
Although not fully understood, current herd management and feeding practices may
be at least partly responsible for seasonal differences observed in isolation rates for L.
inonocytogenes and possibly L. iiinocun. During cold winter months, silage comprises a
major component of the diet. While investigating a listeriosis outbreak, Donnelly [85]

FIGURE1 Seasonal variation in the incidence of L. monocytogenes and L. innocua

in raw milk from the United States [88,129,139,1411, Canada 1971, and England/Wales
[ 1571.
368 Ryser

observed that 8 of 44 Holstein cows fed Listeria-contaminated silage shed the organism
in their milk. Furthermore, milk from these animals was free of L. monocytogenes 1 month
after feeding of contaminated silage ceased. Ruminants that ingest contaminated silage
may either succumb to infection or carry L. monocytogenes asymptomatically ; however,
if the animal lives, the organism can be shed for many months in feces and in milk from
lactating animals. Extended survival of L. monocytogenes in fecal material, soil, and grass
can perpetuate the infectious cycle shown in Figure 2, particularly when animals are win-
tered in cramped quarters. Once dairy cattle resume grazing on pastures during late spring,
summer, and early fall, L. monocytogenes becomes dispersed over a wide area, which, in
turn, weakens the infectious cycle by decreasing the likelihood that animals will come in
contact with contaminated material.
Seasonal differences in the incidence of Listeria spp. in raw milk also may be related
to breeding practices. Dairy cattle typically bear their young in late winter or early spring.
During winter gestation, dairy cattle develop a weakened immune system as a direct result
of pregnancy, which, in turn, makes these animals more susceptible to listerial infections
and abortions. These events can then culminate in the shedding of L. monocytogenes in
milk and fecal material. Increased environmental stress and changes in habitat that occur
during winter, along with increased difficulties in providing proper herd hygiene, all can
serve to decrease the natural defense system in dairy cattle, which again increases the
likelihood for listerial infections. Once an asymptomatic animal begins shedding L. mono-
cytogenes in feces, the organism is likely to spread quickly to other animals that are housed
in close proximity to the shedder. In this way, confinement of dairy cattle may play an
important role in increasing the number of animals that shed L. monocytogenes in their
milk during late winter and early spring.

Survival and/or growth

Survival in
s o i l and grass / Ingestion
by ruminants

confinement
4
-
L. monocytogenes
excreted in feces

-
L. monocytogenes
secreted in milk

FIGURE2 Infective cycle for maintaining L. rnonocytogenes in ruminants.

L. monocytogenes in Unfermenfed Dairy Producfs 369

Raw Ewes and Goats Milk

Surveys in Europe, Australia, and the United States also have demonstrated that raw milk
from ewes and goats can occasionally contain L. monocytogenes (Table 3) with incidence
rates ranging from 0-2.2% (average of 1.6%) and 0-4.0% (average 2.4%), respectively.
These contamination levels are similar to those reported for cows milk. Working in Ver-
mont, Abou-Eleinin et al. recently recovered Listeria spp. from 35 of 445 (7.9%) bulk
tank samples of raw goats milk using three different enrichment methods. Seventeen
samples contained L. monocytogenes and 8 contained both L. monocytogenes and L. in-
nocua. Listeria contamination rates were higher in winter (14.3%) and spring (10.4%)
than in fall (5.3%) and summer (0.9%) with similar observations reported for cows milk
[ la]. Overall, 62.6% and 37.4% of those L. monocytogenes that were further characterized
belonged to serovars 1 and 4, respectively. Automated ribotyping of selected isolates also
indicated five distinct ribotypes, two clinically important ribotypes of which were eventu-
ally traced back to the farm environment.

Pasteurized Milk and Other Unfermented Dairy Products

The dairy industry has long been considered as the most regulated food industry in the
United States. The FDA was given responsibility under the Food, Drug and Cosmetic Act
and the Public Health Service Act to assure the public that this countrys milk supply is
both uniformly safe and wholesome. Dairy sanitation laws and regulations, including micro-
biological criteria for some dairy products, enforced by the FDA and state agencies are based
almost exclusively on the Public Health Service/FDA Grade .4Pasteurized Milk Ordinance.
In the United States, pasteurized milk and other unferniented dairy products prepared
from pasteurized milk, including ice cream, butter, and nonfat dry milk, generally have

TABLE
3 Incidence of L. rnonocytogenes in Raw Ewes and Goats Milk
Number of Number of samples
Type of milk Location samples positive (%) Ref.
Ewe England/ Wales 56 1 (1.8) 126
Italy 40 0 71
Italy 34 0 76
Spain 1052 23 (2.2)a 175
Turkey 302 0 78
Total 1484 24 (1.6)
Goat United States 450 17 (3.8) la
England/Wales 480 4 (0.8) 126
Italy 24 0 71
Portugal 25 1 (4.0) 79
Spain 1445 37 (2.6) 116
Australia 69 1 (1.4) 125
Total 2493 60 (2.4)

a Also 21 L. innocua, 4 L. welshimeri, 3 L. seeligeri, 3 L. grayi, and 2 I,. ivanovii

8 L. monoivtogenes and L. innocua, and 26 L. innocua.
370 Ryser

earned a reputation for being both safe and nutritious, with dairy products accounting for
<1.5% of all foodborne illnesses [67]. Pasteurized milk is responsible for 5 5 % of all
reported dairy-related illnesses [ 1501. Despite these impressive findings, public confidence
in the safety of pasteurized milk began to erode in July of 1982 following an outbreak
of yersiniosis in Tennessee, Arkansas, and Mississippi [ 1951 and again in 1983 when the
CDC claimed that consumption of pasteurized milk was responsible for 49 cases of listeri-
osis in Massachusetts. In 1985, the dairy industry was dealt another blow when at least
16,000 culture-confirmed cases of salmonellosis were associated with drinking a particular
brand of pasteurized milk produced in the Chicago area [183].
These three outbreaks, along with the previously discussed listeriosis outbreak in
California linked to consumption of contaminated Mexican-style cheese, prompted the
FDA to take corrective action in the form of a large-scale testing program commonly
referred to as the FDA Dairy Initiative Program [134] (Fig. 3). This program, begun in
April of 1986 in cooperation with individual state agencies and members of the National
Conference on Interstate Milk Shipments, was designed to examine every interstate milk
shipment (IMS) pasteurization facility in the United States for potential safety problems
related to pasteurization, postpasteurization contamination, cleaning and sanitizing regi-
mens, equipment maintenance, and educationaUtraining programs for dairy factory per-
sonnel. As part of the FDA Dairy Initiative Program, the agency also established the
Microbiological Surveillance Program, which was designed to detect L. monocytogenes,
Salmonella spp., Yersinia enterocolitica, Campylobacter jejuni, and C. coli (Campylo-
bacter omitted in 1987) as well as Staphylococcus aureus, which was added in 1987 for
dry and fluid milk [16]. Dairy products tested under this program included fluid milk,
nonfat dry milk, cream, butter, ice cream, ice milk, and other dairy commodities over

1 ! I

June to August, 1983: March to April, 1985: January to June, 1985:

Listeriosis outbreak in Salmonellosis outbreak Listeriosis outbreak in
Massachusetts associated in Chicago linked to California linked to
wich pasteurized milk pasteurized milk Mexican-style cheese

I I
April 1986: Begin dairy initiatives. Portion of IMS and non-PIS
inventory sampled f o r Listeria, Salmonella, Yersinia and
Campylobacter. Plant inspections in cooperation with
state agencies. 1174 samples analyzed during fiscal 1986.

1
~

October 1986: Continue sampling except omit Cam lobacter

1444 samples analyzed during f e

October 1987: Continue sampling remainder of inventory and the

following composite samples - Fluid milk: Listeria,

-I
Staphylococcus aureus and Yersinia; Dry milk: Listeria,
Salmonella, and Scaphylococcus aureus.

FIGURE3 FDA Dairy Initiatives Program. IMS, interstate milk shipment. (Adapted
from Ref. 134)
L. monocytogenes in Unfermented Dairy Products 371

which the FDA has jurisdiction. Analysis of cheeses (except cottage cheese) was covered
under a series of separate programs, which will be discussed in Chapter 12 concerning
fermented dairy products. Under provisions of this program, FDA inspectors collected 30
retail-sized containers of as many as five different products available from dairy factories
at the time of inspection. Duplicate 25-g or 25-mL samples obtained after combining 30
retail-sized samples per product were then analyzed for L. monocytogenes and other lister-
iae using the original and later versions of the FDA procedure.
Since raw milk containing L. monocytogenes will enter every dairy factory in the
United States from time-to-time, it is logical to assume that finished products also may
become contaminated with this pathogen. During the first 2 years of the FDA Dairy Initia-
tive Microbiological Surveillance Program (Table 4), L. monocytogenes was isolated from
2 of 350 samples of pasteurized whole milk and 5 of 415 samples of chocolate milk,
which suggests that approximately 0.67% of the pasteurized milk available in the United
States could contain this pathogen unless factories take corrective action to reduce this
value. In contrast, L. monocytogenes was isolated from 3 of 99, 23 of 659, and 30 of 351
samples of ice milk, ice cream, and novelty ice cream, respectively. Only two of the
positive ice cream samples were analyzed quantitatively for L. monocytogenes. One con-
tained an average of 15 L. monocytogenes CFU/g, whereas the other sample contained
between 1 and 5 CFU/g. Thus during the time of this survey, approximately 5% of frozen
dairy products manufactured in the United States presumably contained low levels of L.
monocytogenes.
Furthermore, L. innocua was isolated from three of four product categories (see
Table 4) which also contained samples positive for L. monocytogenes. Since both organ-

TABLE
4 Incidence of Listeria spp. in Unfermented Dairy Products Manufactured
in the United States during 1986 and 1987
Number of positive samples (%)
Number of -
Product samples tested L. rnonocytogenes L. innocua
Whole milk 350 2 (0.57) 0
Lowfat milk 182 0 2 (1.10)
Skim milk 98 0 0
Chocolate milk 415 5 (1.20) 1 (0.24)
Cream 52 0 0
Half and half 42 0 0
Ice milk 99 3 (3.03) 0
Ice cream 659 23 (3.03) 12a(1.82)
Novelty ice cream 35 1 30 (8.55) lob (2.85)
Butter 30 0 0
Nonfat dry milk 44 0 0
Casein/Milk protein hydrolysate 15 0 0
Other products 171 0 0
Total 2518 63 (2.50) 25 (0.99)
L. seeligeri also detected in 7 of 659 (1.06%) samples.
L. gruyi also detected in I of 351 (0.28%) samples.
Dairy blend whey, eggnog.
Source: Refs. 62 and 135.
372 Ryser

isms likely occupy similar niches in the natural environment and dairy processing facili-
ties, isolation of L. innocua from a dairy product should raise immediate concerns about
the possible presence of L. monocytogenes.
Greater success in isolating L. monocytogenes from chocolate rather than whole
milk is likely related to the organisms ability for enhanced growth in this product as
compared with other fluid dairy products. Reasons for increased growth of Listeria in
chocolate milk will be discussed shortly in conjunction with the behavior of listeriae in
autoclaved fluid dairy products.
The higher incidence of L. monocytogenes in frozen rather than fluid dairy products
coincides with the relatively complex handling of ice milk, ice cream, and particularly
ice cream novelties during manufacture and packaging. This, in turn, suggests that these
products are most likely contaminated after pasteurization through either direct or indirect
contact with listeriae within the dairy factory environment. This hypothesis is supported
by frequent isolations of L. monocytogenes from many areas within dairy factories, includ-
ing floors, ceilings, drains, and coolers. In addition, this organism also has been found in air
and condensate and on various pieces of equipment, including conveyor belts. A detailed
discussion of the incidence of L. monocytogenes in food processing facilities, including
dairy factories, can be found in Chapter 17.
Inability of the FDA to detect L. monocytogenes in skim and low-fat milk as well
as half and half, cream, and butter may have resulted from the separation processes used
for adjusting milk-fat content that these products undergo. Such centrifugal separation
processes tend to decrease levels of listeriae, particularly if leukocytes containing the
organism are still present after initial clarification of the milk.
Failure to isolate L. monocytogenes from nonfat dry milk and casein/ protein hydro-
lysates may be partly related to the heat treatments necessary to manufacture these prod-
ucts. This theory is supported by the work of Doyle et al. [90], who demonstrated that
populations of L. monocytogenes decreased 1 9 0 % during conversion of skim milk into
nonfat dry milk via spray drying. However, failure to isolate L. monocytogenes from dried
dairy products also may result from the generally recognized inability of the FDA method
to detect cells of L. monocytogenes that have been sublethally injured during thermal
processing. Thus the methodology employed to detect L. monocytogenes will predetermine
whether or not the organism can be isolated from a particular food.
According to the Food, Drug and Cosmetic Act of 1938, a food may be considered
adulterated and therefore unfit for human consumption if the product contains poisons or
other harmful substances (e.g., pathogenic microorganisms) at detrimental concentrations.
Although the oral infective dose for L. monocytogenes is presently unknown, evidence
from the California listeriosis outbreak involving Mexican-style cheese suggests that the
number of L. monocytogenes cells needed to induce this life-threatening illness may be
quite low-perhaps as few as several hundred to a few thousand total cells for certain
segments of the population. Although not directly applicable to the human population,
several independent studies involving immunocompromised mice have demonstrated LDSo
values (the dose of cells which is lethal to 50% of a given population) in the range of
approximately 10 [ 1231 and 4 to 480 [77,193] L. monocytogenes cells when the pathogen
was administered orally and intraperitoneally, respectively. Consequently, because of a
moral obligation to the public, the FDA has adopted and is continuing to uphold a policy
of zero tolerance regarding presence of L. monocytogenes in ready-to-eat foods.
In accordance with Title 21 of the United States Code of Federal Regulations, Sec-
tion 7.40 [ 1 1 I], the FDA can request that firms voluntarily recall any product that contains
L. monocytogenes in Unfermented Dairy Products 373

or is suspected of containing L. monocytogenes. These recalls can be classified into one

of three categories: Class I, Class 11, or Class 111. A Class I recall, which is the most
serious, is defined by the FDA as a situation in which there is a reasonable probability
that the use of or exposure to a violative product will cause rserious adverse health conse-
quences or death. Thus far, all recalls issued for products contaminated with L. monocyto-
genes have been categorized as Class I. In the unlikely event that a firm fails to comply
with the FDAs request to recall a product containing L. monocytogenes, FDA officials
can (a) initiate a seizure request in the U.S. District Court to have the product removed
from commerce (Title 21 Code of Federal Regulations, Section 334) or (b) obtain a legal
injunction to halt production and distribution of the contaminated product (Title 21 Code
of Federal Regulations, Section 332). In addition, the FDA also can take criminal action
against individuals of a company who are responsible for commercial distribution of a
contaminated product.
Adoption of the FDA Dairy Initiative Microbiological Surveillance Program in April
of 1986 fostered the beginning of a series of recalls issued for milk and unfermented dairy
products contaminated with L. monocytogenes (Table 5). Just 1 month after the surveil-
lance program began, a California firm voluntarily recalled an unknown quantity of ice
milk mix contaminated with L. monocytogenes. During the same month, approximately
I million gallons of fluid dairy products that comprised milk, chocolate milk, half-and-
half, whipping cream, ice milk mix, ice milk shake mix, and ice cream mix also were
recalled in Texas [ 1 1,121. However, other than this single incident, the remaining recalls
have been primarily confined to frozen dairy products such as ice cream, ice cream novel-
ties, ice milk, and sherbet, with only four additional recalls thus far being traced to nonfro-
Zen dairy products (i.e., butter).
In July of 1986, a large recall of Listeria-contaminated ice cream bars received
considerable media attention, which in turn did much to enhance the state of hysteria
concerning the presence of L. monocytogenes in dairy products IS]. The following month,
another nationwide recall was issued for frozen dairy products. As a result of this recall,
approximately 1 million gallons of products possibly contaminated with L. monocytogenes
and including ice cream (1 32 flavors), ice milk ( I 6 flavors), sherbet (9 flavors), and gelati-
da products (6 flavors) were reportedly buried at a Minnesota landfill site. One year later,
a similar recall was issued for contaminated ice cream, ice milk, and sherbet manufactured
in Iowa [26,30]. By the end of 1987, well over 500 Listeria-contaminated dairy products
were voluntarily recalled in the United States at a total cost to the dairy industry of well
over $70 million [29,149]. Although corrective measures instituted by the dairy industry
in response to governmental pressure reduced both the extent of Listeria contamination
in processing facilities and the number of Class I recalls issued for L. monocytogenes-
contaminated dairy products during 1988 and 1989 [49], recalls involving frozen desserts
and more recently butter continue to plague the industry. During the 6-year period from
1990 to 1995, 17 of 39 (44%) dairy-related L. monocytogenes recalls involved unfermented
dairy products, with Listeria being responsible for 3 I % of all dairy-related recalls issued
for any reason [ 1841. Unfortunately, product losses are also being substantially underre-
ported, since, under certain circumstances, manufacturers can retrieve their own product
without issuing a formal Class I recall. In one such instance, a manufacturer of Listeria-
contaminated ice cream was not required to issue a formal recall, because the product
had not yet reached the consumer [34]. Additional cases also likely occurred in which
contaminated products moved only as far as the companys warehouse and were recalled
internally by the manufacturer.
374 Ryser

5 Chronological List of Voluntary Class I Recalls Issued in the United States During 1986 and 1987 for Milk and Unfermented
TABLE
Dairy Products Contaminated with L. monocytogenes
Month/ year State of
Type of dairy product of recall manufacture Distribution Quantity Ref.
~~~~~~~~~~ ~ ~

Ice milk mix 5/86 California Arizona, Nevada, Unknown 9,ll

California
Milk (2 and 112% fat), 5/86 Texas Texas -1 million gal 11,12
chocolate milk, choco-
late milk (2% fat), half
and half, whipping
cream, vanilla and choc-
olate ice cream mix, ice
milk shake mix, and ice
cream mix
Ice cream bars 7/86 Virginia Eastern United States Large but unknown 5
Ice cream, sherbet, glacee 7/86 Wisconsin Minnesota, Wisconsin 8000 gal 8
Ice cream (1 32 favors), ice 8/86 Minnesota Illinois, Indiana, Iowa, - 1 million gal 10,13
milk (16 flavors), sher- Kentucky, Michigan,
bet (9 flavors), gelati-da Minnesota, Missouri,
products (6 flavors) North Dakota, Ohio,
South Dakota, Wis-
consin
Ice cream and ice cream 8/86 Iowa Illinois, Iowa, Minnesota, Unknown 6
novelties-bars, drum- Missouri, North Dakota,
sticks, slices, sundaes Wisconsin
Ice cream 9/86 Wisconsin Wisconsin Unknown 7
Ice cream 12/86 New York New York -835 gal 25,30
Ice cream 12/86 West Virginia West Virginia, Ohio 450 gal 15,26
Ice cream, ice milk, 1187 Iowa Arkansas, Delaware, Illi- -1 million gal 15,19,28,30
sherbet nois, Iowa, Kansas,
Maryland, Minnesota,
Missouri, Nebraska,
Oklahoma, Pennsylva-
nia, South Dakota, Wis-
consin
L. monocytogenes in Unfermented Dairy Products 375

Ice cream 4/87 New York New York, Pennsylvania -316 gal 27,30
Ice cream (48 flavors), ice 7/87 California California, Oregon -60,000 gal 20,40
milk (6 flavors), sher-
bert (5 flavors)
Ice cream nuggets 7/87 Maryland Connecticut, Florida, 20,400 boxes 21,28
Maryland, New Jersey,
New York, North Caro-
lina, Ohio, Pennsylva-
nia, Virginia
Ice cream, ice milk 7/87 Nebraska Colorado, Iowa, Kansas, -30,000 gal 17
Nebraska, North Da-
kota, South Dakota, Wy-
oming
Ice cream sundae cones 8/87 Florida Alabama, Arizona, British Unknown 22
West Indies, Florida,
Louisiana, Mississippi,
North Carolina, Ohio,
Puerto Rico, South Caro-
lina, Tennessee, Vir-
ginia, West Virginia
Chocolate ice cream 8/87 Kentucky Florida, Puerto Rico -956 gal 14
Ice cream nuggets 9/87 Maryland Nationwide Unknown 23
Ice cream novelties-sand- 9/87 Ohio Nationwide Unknown 24
wiches, bars, pieces,
slices, sundae cones
Ice cream bars 10187 Ohio Michigan, Ohio, Pennsyl- 5 1,780 bars 18
vania, West Virginia
Chocolate ice cream 2/88 Georgia Georgia, North Carolina Unknown 32,35
Ice cream, sherbet, ice 7/88 Ohio Ohio >1083 gal 31
milk
Ice cream 8/88 Connecticut Connecticut, New York, 5.6-8.4 gal 37
Massachusetts
Ice cream 8/88 Connecticut Connecticut 30 gal 36
Ice cream pies 9/88 Connecticut Connecticut, New York, 1700 pies 33
Massachusetts
376 Ryser

TABLE
5 Continued
Monthlyear State of
Type of dairy product of recall manufacture Distribution Quantity Ref.

Ice cream 9/88 Pennsylvania Pennsylvania 215 gal 38

Ice cream bars 12/88 New York New York - 128 gal 41
Ice cream 2/89 Connecticut Connecticut Unknown 42
Ice cream bars 11/89 Wisconsin Wisconsin -365 gal 44
Ice cream bars 2/90 New Mexico Alabama, Illinois, Pennsyl- - 1000 gal 46
vania, Texas
Sherbet, ice milk, ice 6/90 Ohio Indiana, Kentucky, Michi- Unknown 48
cream gan, Ohio
Ice cream 8/90 Ohio Indiana, Kentucky, Ohio -778 gal 47
Ice cream novelties, frozen 5/91 Tennessee Alabama, Georgia, Ken- Unknown 45
novelties tucky, Tennessee, Vir-
ginia, West Virginia
Ice cream, ice milk 619 1 Illinois Illinois, Indiana, Wis- >2275 gal 51
consin
Butter 619 1 North Carolina, Tennessee Pennsylvania -18,165 lb 50
Butter 619 1 North Carolina, Tennessee Pennsylvania -18,428 lb 50
Butter, butterine 3/92 Wisconsin Arizona, California, Flor- Unknown 52
ida, Illinois, Maryland,
Massachusetts, Michi-
gan, Minnesota, Missis-
sippi, Missouri, New Jer-
sey, Ohio, Wisconsin
Ice milk, ice cream 10192 Wisconsin Wisconsin -1 125 gal 53
Ice cream bars 6/93 Ohio Ohio, Kentucky, New En- 34,752 bars 54
gland
Chocolate milk 7/94 Wisconsin Michigan, Wisconsin Unknown 168
Whipping cream 8/94 Wisconsin Michigan, Wisconsin Unknown 168
Butter 8/94 California Maine, Michigan, New 36 Ib 55
Hampshire, Wisconsin
Butter 9/94 Wisconsin Pennsylvania, Wisconsin Unknown 168
Ice cream bars 9/94 Wisconsin Michigan, Wisconsin 167 gal 56
L. monocytogenes in Unfermented Dairy Products 377
Ice cream novelties 10195 Ohio Illinois, Indiana, Iowa, Large but unknown 57
Kentucky, Maryland,
Michigan, Ohio, North
Carolina, Pennsylvania
Ice cream, frozen yogurt, 10195 Ohio Georgia, Indiana, Ken- -420,000 gal 60
sherbert, sorbet, ice tucky Michigan, North
cream mix Carolina, Ohio, Pennsyl-
vania, South Carolina,
Tennessee, Virginia
Ice cream 12/95 California Arizona, Hawaii, Califor- >500 gal 61
nia, New Mexico,
Nevada
Ice cream 12/95 Michigan Michigan Unknown 61
Frozen yogurt 12/95 Ohio Connecticut, Delaware, -12,381 gal 58
Florida, Maine, Mary-
land, Massachusetts,
Michigan, New Hamp-
shire, New Jersey, New
York, Ohio, Pennsyl-
vania, Rhode Island,
Vermont, Virginia
Ice cream, sherbet 1/96 Ohio Ohio 2,864 gal 59
Chocolate ice cream 7/97 Michigan Michigan Unknown 205
Frozen strawberry yogurt 7/97 Pennsylvania Pennsylvania -42 gal 206
Ice cream bars 10197 Texas Arkansas, California, Flor- 29,814 cases 207
ida, Georgia, Illinois,
Kansas, Maryland, New
York, Ohio, Oregon,
Texas, Washington
State
Ice cream sandwiches 1/98 California California Unknown 208
Ice cream 3/98 North Carolina Delaware, Florida, Geor- 130,000 gal 209
gia, Kentucky, Mary-
land, North Carolina,
Pennsylvania, South Car-
olina, Tennessee, Vir-
ginia, West Virginia
378 Ryser

Despite millions of gallons of frozen dairy products that have been recalled both
formally and internally, it must be stressed that only one case of listeriosis has been posi-
tively linked to consumption of a contaminated frozen dairy product in Belgium [4] (see
Chapter 10). On this basis, the International Ice Cream Association and the Milk Industry
Foundation have contended that a Class I recall may be too harsh a response for a frozen
dairy product containing presumably very low levels of L. monocytogenes. However, until
the oral infectious dose and relative risk for susceptible individuals can be firmly estab-
lished, the FDA is likely to maintain its zero tolerance policy for L. monocytogenes and
continue requesting recalls of products containing this pathogen at any detectable level.
The U.S. government has developed one of the most stringent policies regarding
presence of L. monocytogenes in ready-to-eat foods, whereas most other countries have
adopted more relaxed policies (i.e. not >100 CFU/g or ml), particularly where consump-
tion of contaminated products that have not yet been firmly linked to cases of listeriosis
is concerned. As an example of the latter attitude, the Canadian government has decided
to confine all formal recalls to only those foods that have been linked to major outbreaks
of listeriosis, namely, coleslaw, soft cheese, and pasteurized milk, with the role of pasteur-
ized milk in foodborne listeriosis still being highly debated [ 1331. Hence, no recalls were
issued when researchers at the Health Protection Branch of Health and Welfare Canada
(analogous to the U.S. FDA) identified L. monocytogenes in 1 of 394 (0.25%) and 1 of
51 (2.0%) samples of ice cream and ice cream novelties, respectively [96], during their
own federal inspection program. Although subsequent investigations were presumably
conducted to identify (a) the source of contamination, (b) proper corrective measures, and
(c) possible links to human illness, Canadian officials maintained that recalling the two
contaminated lots would be inappropriate without proof that consumption of Listeria-
contaminated ice cream can lead to listeriosis. Many individuals and most manufacturers
will undoubtedly argue in favor of the more relaxed Canadian position. When one consid-
ers the numerous recalls of Listeria-contaminated ice cream in the United States, that
worldwide only one case of listeriosis has been positively linked to ice cream containing
unusually high numbers of listeriae, the inability of L. monocytogenes to grow in this
product during frozen storage and the normal exposure rate of the human population to
listeriae, it appears that the risk of contracting listeriosis from contaminated ice cream is
extremely low. Although current scientific data mandate the immediate removal of fluid
dairy products and cheeses that support growth of L. monocytogenes, it appears that a
scientifically valid argument can be made against recalling certain dairy products in which
listeriae will not proliferate such as ice cream and dried goods which, if contaminated,
typically contain very low numbers of listeriae as postpasteurization contaminants.
As a result of several large recalls of French Brie cheese and a listeriosis outbreak
in Switzerland that was traced to consumption of Vacherin Mont dOr soft-ripened cheese,
European scientists have logically focused their attention on the incidence of listeriae in
cheese. However, numerous recalls of unfermented dairy products in the United States
also have heightened public health concerns about the presence of listeriae in pasteurized
dairy products manufactured outside of North America.
In one of the first European surveys of finished products reported in 1988, research-
ers in Germany [ 1971 failed to isolate Listeria spp. from pasteurized milk (39 samples),
nonfat dry milk (1 1 samples), caseidcaseinate (30 samples) and various dried products,
including baby food (Table 6). During the same year, investigators in Hungary [ 1001 and
The Netherlands [69] also failed to recover L. monocytogenes from samples of pasteurized
milk, with similar negative findings being obtained from most other subsequent surveys
L. monocytogenes in Unfermented Dairy Products 379

of properly pasteurized milk and cream produced in Europe, Australia, the Middle East,
and North Africa (Table 6). However, L. monocytogenes was eventually demonstrated in
11 of 1039 (1.1%), 4 of 115 (3.5%), and 1 of 95 (1.1%) pasteurized milk samples examined
in the United Kingdom [ 119,126,1821 for a combined contarnination rate of 1.3%, with
these findings generally being similar to those observed in the United States. According
to Garayzabal et al. [113], 21.4, 89.2, 10.7, and 3.6% of pasteurized milk samples from
one particular milk processing facility in Madrid contained I,. monocytogenes, L. grayi,
L. innocua, and L. welshimeri, respectively. These same authors [ 114,1761 previously
reported similar Listeria contamination rates for raw milk entering the same processing
facility. Furthermore, after pasteurization these same samples had a total mesophilic aero-
bic plate count of 2.5 X 107CFU/mL, which is well above the maximum allowable limit
of 1 X 104CFU/mL for properly pasteurized milk in the United States. Hence, improper
pasteurization caused by leaking pasteurizer plates, as suggested by Northolt et al. [ 1561,
and/or postpasteurization contamination from the factory environment appear to be most
likely responsible for the unusually high incidence of listeriae in pasteurized milk sam-
ples from this particular dairy factory. Although results from these aforementioned surveys
of pasteurized milk, cream, and dried products are very encouraging, the isolation methods
used in these studies were generally unable to detect sublethally injured listeriae. Hence,
the true incidence of listeriae in pasteurized milk, cream, and dried products may well be
somewhat higher. To enhance recovery of injured cells, the International Dairy Federation
has recommended that such dairy products undergo preenrichment in a nonselective me-
dium (i.e., buffered peptone water) before primary enrichment in various selective broths
and plating on Listeria-selective media [37,198]. Further details concerning recovery of
sublethally injured listeriae can be found in Chapter 7.
Results from a 1989 International Dairy Federation survey [ 1331 indicated that pub-
lic health issues regarding the presence of listeriae in pasteurized milk were clearly spread-
ing beyond the continental boundaries of Europe and North America, with the many afore-
mentioned surveys from Table 6 attesting to these concerns. More recently, the safety of
several additional dairy products, including flavored milks, chocolate milk, ice cream, and
butter has attracted international attention with the FDA Initiatives Program, the many
Class I recalls of Listeria-contaminated dairy products, and fears of international trade
embargoes fueling these concerns. Following the 1987 discovery of L. monocytogenes in
Australian ricotta cheese, New Zealand and Australian officials instituted Listeria-moni-
toring programs for caseidcaseinate products as well as high-moisture cheese, pasteurized
milk, ice cream, and milk powders. Results from one 10-month survey begun in April
1988 [202] revealed the presence of L. monocytogenes in 1 of 206 (0.48%) samples of
pasteurized flavored/unflavored milk processed in and around Melbourne. Subsequent
identification of heat-labile alkaline phosphatase in the contaminated product (pasteurized
milk to which a pasteurized flavored syrup was added) suggested that improper pasteuriza-
tion was most likely responsible for the presence of L. monocytogenes in the final product.
However, unsatisfactory storage of the flavored syrup also may have contributed to con-
tamination. In keeping with Listeria policies developed in the United States and Canada,
Australian officials withdrew the affected product from the marketplace and prohibited
the sale of all subsequently produced product until 12 consecutive lots of Listeria-free
pasteurized flavored milk could be produced from the same product line.
As in the United States, recent foreign surveys also have shown a higher incidence
of L. monocytogenes in chocolate milk (1 1.6%), ice cream (2.0-13.9%), and butter (3.8-
6.7%) as compared with pasteurized milk and dried products which are seldom contami-
380 Ryser

TABLE
6 Incidence of Listeria spp. in Pasteurized Dairy Products Produced Outside the United States and Canada
Number of positive samples (%)
Country of Number of
Product origin samples L. monocytogenes L. innocua L. welshimeri Other Ref.
Milk Australia 77 0 0 0 0 125
33 0 ND ND ND 65
Brazil 220 0 2 (0.9) 0 0 155
20 0 0 0 0 73
Czechoslovakia 30 0 ND ND ND 151
15 0 ND ND ND 152
Germany 39 0 0 0 0 197
Hungary 100 0 ND ND ND 131
50 0 0 0 0 100
Italy 348 0 0 0 0 117
50 0 0 0 0 199
Morocco 20 0 ND ND ND 94
Netherlands 41 0 0 0 0 69
Poland 73 0 0 0 7a 178
Turkey 22 0 ND ND ND 187
United Arab Emirate 182 0 0 0 0 122
L. monocytogenes in Unfermented Dairy Products 381

United Kingdom
England/Wales 1039 11 (1.1) ND ND ND 126
Scotland 115 4 (3.5) ND ND ND 182
Northern Ireland 95 1 (1.1) ND ND ND 119
Chocolate milk Hungary 60 7 (11.6) 0 0 0 171
Flavored milk Australia 206 1 (0.5) ND ND ND 202
Ice cream Australia 166 23 (13.9) ND ND ND 65
Costa Rica 50 1 (2.0) ND ND ND 154
England/Wales 40 0 ND ND ND 118
Turkey 50 5 (10.0) 6 (12.0) 0 0 75
Cream Australia 12 0 0 0 0 125
England/Wales 40 0 ND ND ND 126
Hungary 15 0 ND ND ND 131
Morocco 20 0 ND ND ND 94
Butter Hungary 15 1 (6.7) 0 0 Ib 131
Italy 130 5 (3.8) ND ND ND 169
Nonfat dry milk Germany 11 0 0 0 0 197
Caseidcaseinate Germany 30 0 0 0 0 I97
Dry infant formula Germany 120 0 0 0 0 197
ND, not determined.
a Seven non-L. monocytogenes isolates.

One non-L. monocytogenes.

382 Ryser

nated (Table 6). The increased incidence of listeriae in ice cream and butter is clearly the
result of postpasteurization contamination during handling and packaging as evidenced
by the highest contamination rates in ice cream bars and novelties. The fact that Listeria
spp. are more commonly found in chocolate milk, as opposed to unflavored milk, is also
not surprising given that the added ingredients can serve as an additional source for
listeriae.

BEHAVIOR OF L. MONOCYTOGNS IN UNFERMENTED

DAIRY PRODUCTS
Although the psychrotrophic nature of L. monocytogenes and the ability of both normal
and diseased animals to shed this pathogen in their milk have been recognized for many
years, behavior of L. monocytogenes in raw milk and unfermented dairy products did not
receive serious attention until 1983 when an outbreak of milkborne listeriosis was
reported in Massachusetts. Research efforts prompted by this and two other dairy-related
outbreaks in the United States and Switzerland have given us an understanding of the
behavior of L. monocytogenes in raw and pasteurized milk as well as in chocolate milk,
cream, nonfat dry milk, and butter. The remainder of this chapter will describe results
from these studies along with information concerning behavior of this organism in ultra-
filtered milk and ice cream mix.

Raw Milk
Despite longtime recognition of L. monocytogenes as a raw milk contaminant, relatively
few studies assessing the behavior of this organism in raw milk can be found in the litera-
ture. In 1958, Dedie [82]found that L. monocytogenes survived 210 days in naturally
contaminated raw milk stored in an ice chest. Thirteen years later, Dijkstra [84]reported
results from a much longer storage study in which 36 samples of naturally contaminated
raw milk (obtained from cows that experienced Listeria-related abortions) were held at
5C and examined for viable L. monocytogenes over a period of 9 years. Although 4 of
36 (1 1%) samples were free of L. monocytogenes within 6 months, the pathogen was still
detected in 16 of 36 (44%) samples following 2 years of refrigerated storage. The number
of samples from which listeriae could be isolated continued to decrease, with 9 of 36
(25%) samples being positive after 4 years of storage. However, the pathogen was still
present in 4 of 36 (1 1%) raw milk samples after 8-9 years of storage. These early findings
emphasize the importance of establishing proper cleaning and sanitizing programs for all
phases of milk production. If routinely used, such programs will likely prevent this organ-
ism from finding an appropriate niche within the farm or dairy factory environment and
greatly reduce the threat of this pathogen surviving long term.
The studies just described adequately demonstrate that L. monocytogenes can persist
in raw milk for long periods; however, until several outbreaks of milkborne and cheese-
borne listeriosis were reported in the 198Os, little attention had been given to the potential
for growth of L. monocytogenes in raw milk.
In 1988, Northolt et al. [ 1561 examined the behavior of listeriae in samples of freshly
drawn raw milk that were inoculated to contain approximately 500 L. monocytogenes
CFU/mL and incubated at 4 and 7C. As shown in Figure 4, Listeria populations decreased
approximately 4- and 8.5-fold in raw milk during the first 2 days of incubation at 4 and
7C, respectively. These authors suggested that naturally occurring bacterial substances
L. monocytogenes in Unfermented Dairy Products 383

lo4 I

10 L
0 2
Raw Milk
L l L L
4 6
Days

FIGURE4 Growth of Listeria rnonocytogenes strains in raw milk incubated a t 4 and

7C (enumerated on Trypaflavine Nalidixic Acid Serum Agar). (Adapted from Ref. 156.)

in raw milk (i.e., lactoperoxidase and lysozyme) may have partially inhibited growth of
listeriae during the first 2 days of incubation. However, in a Canadian study which will
be discussed shortly [98], no such decrease was observed when incubated samples of
naturally contaminated raw milk were surface plated on FDA Modified McBride Listeria
Agar. Hence, a more likely explanation is that the plating medium Trypaflavine Nalidixic
Acid Serum Agar used by Northolt et al. [ 1561 was less than ideal for recovering listeriae,
as also was observed during concurrent work with pasteurized milk. Although L. monocy-
togenes failed to grow in raw milk samples incubated at 4C for up to 7 days, Listeria
populations increased approximately 10-fold during this period when the incubation tem-
perature was raised to 7C. Following 3 days of incubation at 4 and 7OC, Listeria popula-
tions began doubling every 3.5 and 1.0 day, respectively. Two years later, Wenzel and
Marth [203] reported that populations of L. monocytogenes strain V7 remained constant
in inoculated raw milk during 5 days of storage at 4 and 7"C, with numbers of listeriae
also being unaffected by the presence of a commercial raw milk lactic acid bacteria inocu-
lant designed to suppress the growth of primarily gram-negative psychrotrophic bacteria.
Since L. monocytogenes failed to grow during 3-5 days of incubation at 7"C, it
appears that the 3-day period during which raw milk is sometimes held in farm bulk tanks
is insufficient to allow growth of the organism. However, the temperature of raw milk in
farm bulk tanks will fluctuate every time freshly drawn raw milk at 37C is commingled
with bulk tank milk at -4C from previous milkings. In 1985, Oz and Farnsworth [I591
found that raw milk in farm bulk tanks attained temperatures of 30-3 1OC, 10- 14"C, I2"C,
and 9C when freshly drawn raw milk was added after the first, second, third, and fourth
milking periods, respectively. Moreover, 6 h were generally needed for the milk to cool
to 4C after each milking period. In view of these findings, it appears that temperatures
obtained after adding warm milk to farm bulk tanks may be sufficient to allow at least
limited growth of L. monocytogenes, particularly when raw milk from early millungs
384 Ryser

enters the bulk tank. Although the temperature of bulk tank milk will eventually decrease
to -4"C, exposure to temperatures as high as 9C when raw milk is trucked to processing
facilities during summer [99] also may lead to some multiplication of the pathogen.
Discovery of a naturally infected cow in Canada that shed freely suspended and
phagocytized cells of L. monocytogenes in milk (maximum of 104CFU/mL in milk from
one of four quarters of the mammary gland) continuously for nearly 3 years provided
Farber et al. [98] with a unique opportunity to study growth of L. monocytogenes in natu-
rally rather than artificially contaminated raw milk during extended storage. When raw
milk from this cow was analyzed for numbers of L. monocytogenes,no appreciable growth
of the pathogen was observed during the first 3 days and 1 day of incubation at 4 and
IO'C, respectively (Fig. 5). The delay in onset of growth was less than 1 day at 15C.
Immunological staining of milk smears indicated that some multiplication of L. monocyto-
genes had occurred within macrophages after I and 2 days of incubation at 15 and 10C,
respectively, with 1 0 4 0 % of the macrophages containing 1-20 intracellular listeriae.
Nonetheless, as previously noted by Doyle et al. [91], rapid deterioration of macrophages
shortly thereafter was followed by appearance of freely suspended listeriae in milk with
few intact macrophages remaining after 5 days regardless of incubation temperature. Fol-
lowing the lag phase, L. monocytogenes entered a period of logarithmic growth, with
generation or doubling times of 25.3, 10.8, and 7.4 h being calculated for raw milk samples
held at 4, 10, and 15OC, respectively. Although maximum L. monocytogenes populations
were approximately 2 X 107CFU/mL after 10, 7, and 3 days of incubation at 4, 10, and
15"C, respectively, the highest achievable population in raw milk was independent of

7.0 -

m- 4C
t- 10C
.- 15OC

0 2 4 6 8 10 12 14

FIGURE5 Growth of L. monocytogenes in naturally contaminated raw milk during

incubation at 4, 10, and 15C. (Adapted from Ref. 98.)
L. monocytogenes in Unfermented Dairy Products 385

incubation temperature (Fig. 6). As in the previous study by Northolt et al. [156], these
findings again stress the importance of maintaining raw milk at 1 4 C during storage and
transport to milk processing facilities.
Investigations dealing with behavior of listeriae in raw milk have not been limited
to cow's milk. Reports of ovine listeriosis in Europe prompted Ikonomov and Todorov
[132] to examine the behavior of L. monocytogenes in raw ewe's milk inoculated with
the pathogen. Their results show that L. monocytogenes remained viable for long periods
and persisted in the milk even after coagulation at 10 and 20C. In 1987, a pregnant
woman in the United States reportedly aborted after consuming feta cheese contaminated
with L. monocytogenes. Since feta and other cheeses such as Roquefort, Manchego, Gjeost,
and Chachcaval are traditionally manufactured from ewe's or goat's milk, interest in the
behavior of listeriae in these milks as well as in ethnic-type cheeses manufactured from
these milks has increased over the last several years.

Pasteurized and Intensively Pasteurized Milk

In addition to defining the growth pattern of L. monocytogenes in artificially contaminated
raw milk (Fig. 4), Northolt et al. [ 1561 also examined behavior of this organism in pasteur-
ized (72"C/I 5 sec) and intensively pasteurized whole milk (Fig. 6). Although L. monocyto-
genes failed to grow in raw milk incubated at 4C (Fig. 4), Listeria populations in pasteur-
ized milk increased nearly 10-fold during 7 days of incubation at the same temperature.

&ITS?'-Pasteurized Milk
I/ 4 "C

0 2 4 6 0 2 4 6

Days Days

FIGURE6 Growth of L. monocytogenes in high-temperature, short-time (HTST)-pas-

teurized and intensively pasteurized milk incubated at 4 and 7C. -: Enumerated
on Trypafiavine Nalidixic Acid Serum Agar,---: Enumerated on Nutrient Agar.
(Adapted from Ref. 156.)
386 Ryser

i-
91-

7 - -+- - - - - - - - -
6 -

-
-
a SkimMilk
5 -
---- Whole Milk

-A Chocolate Milk

Days

FIGURE7 Growth of L. rnonocytogenes strain California in fluid dairy products at

4C. (Adapted from Ref. 180.)

The organism also grew markedly faster in pasteurized than in raw milk when both prod-
ucts were incubated at 7C. In contrast to their data for raw and pasteurized milk, lag
times for L. rnonocytogenes were reduced considerably when the organism was grown in
intensively pasteurized milk incubated at 4 and 7C. Furthermore, numbers of listeriae in
intensively pasteurized milk increased approximately 100-fold following 3 and 6 days of
incubation at 7 and 4OC, respectively. When L. rnonocytogenes was later grown in ultra-
high temperature (UHT) sterilized milk, Rajkowski et al. [ 1711 reported generation times
of 4.7, 1.7, 1.0, and 0.9 h for samples incubated at 12, 19, 28, and 37"C, respectively.
Hence, these findings suggest that the growth rate for L. rnonocytogenes in milk is directly
related to the degree of heat applied to milk. Further work is needed to define more clearly
the effect of competing microorganisms on growth of listeriae in raw and pasteurized
milk as compared with intensively pasteurized and UHT-sterilized milk with biochemical
changes that occur in milk during thermal processing (i.e., protein denaturation, enzyme
inactivation, carmelization) also likely influencing listeriae growth in these products.

Autoclaved Milk, Cream, and Chocolate Milk

Except for the two studies just described [98,156] and an initial attempt by Pine et al.
[ 1661 to follow growth of L. rnonocytogenes in inoculated samples of pasteurized milk,
all remaining work dealing with behavior of Listeria in fluid dairy products has been
done using autoclaved samples. Although using such sterile products as growth media for
listeriae offers several major advantages, including the ability to accurately quantitate both
stressed and unstressed listeriae on nonselective plating media in the absence of other
L. monocytogenes in Unfermented Dairy Products 387

microbial competitors, readers should keep in mind that growth rates for L. monocytogenes
are likely to be somewhat faster in autoclaved than in pasteurized or especially in raw milk
products. Nevertheless, L. monocytogenes clearly can grow to dangerously high levels in
all three types of milk during extended refrigeration.
In 1987, Rosenow and Marth [180] published results from a definitive study in
which autoclaved (12 1"Cl15 min) samples of whole, skim, and chocolate milk as well as
whipping cream were each inoculated separately with four strains of L. monocytogenes
(Scott A, V7, V37CE, or California), incubated at 4, 8, 13, 21, or 35"C, and examined
for numbers of listeriae at suitable intervals by surface plating appropriate dilutions on
Tryptose Agar. Growth rates of L. monocytogenes were generally similar in all four prod-
ucts at a given temperature and increased with an increase in incubation temperature. At
4"C, listeriae began growing after an initial delay of approximately 5- 10 days depending
on the bacterial strain and type of product (see Fig. 7). All four strains generally attained
maximum populations of 2 1O7 CFU/mL after 30-40 days of incubation, with little change
in numbers occurring after 30-40 days of additional storage. Overall, chocolate milk sup-
ported development of the highest Listeria populations followed by skim milk, whole
milk, and whipping cream. Generation times for growth at 4C ranged between 28.16 and
45.55 h. Average generation times for L. monocytogenes in all four products are shown
in Table 7. Although these results clearly demonstrate the ability of L. monocytogenes to
reach potentially hazardous levels in fluid dairy products held at 4"C, more recent data
suggest that slow growth of this organism can even occur in milk held at 0C. Thus
the only way to avoid a public health problem with fluid dairy products is to prevent L.
monocytogenes from entering such products before, during, and after manufacture.
Increasing the incubation temperature from 4 to 8C decreased the lag period to
1.5-2 days (Fig. 8) and nearly tripled the growth rate for L. monocytogenes in all four
products (Table 7) [ 180,1811. After 10- 14 days of incubation, the growth curves at 4 and
8OC were similar, with highest Listeria populations again being found in chocolate milk.
Theoretical calculations based on these data indicate that Listeria populations could in-
crease from 10 to 4.2 X 106organismdqt (947 mL) of milk during 10 days of storage at
8C (46"F), a temperature that commonly occurs in some home and commercial refrigera-
tors. These findings, which since have been confirmed by Siswanto and Richard [188]
using skim milk, raise additional safety concerns about reclaiming and reprocessing re-
turned products that have likely undergone some degree of temperature abuse.
As is true for 8"C, 13C (55F) also represents a temperature that dairy products
occasionally encounter during transportation and storage. Following a 12-h lag period, all

TABLE7 Generation Times for L. rnonocytogenes in Autoclaved Samples of

Various Dairy Products
Generation time (h) at
Product 4C 8C 13C 2 1"Cb 350Cb
Whole milk 33.27 13.06 5.82 1.86 0.692
Skim milk 34.52 12.49 6.03 1.92 0.693
Chocolate milk 33.46 10.56 5.16 1.72 0.678
Whipping cream 36.30 11.93 5.56 1.80 0.683
aAverage generation times for four strains of L. monocytogenes.
Strain V7 only.
Source: Adapted from Ref. 180.
388 Ryser

9. -
8. -

7. -
-
6. -
-
5. -
-
- A-. Chocolare milk
4.

3. -

FIGURE8 Growth of L. monocytogenes strain California in fluid dairy products at

8C. (Adapted from Ref. 180.)

four Listeria strains grew nearly twice as fast at 13C as at 8C (see Table 7) and generally
attained levels of 2 106CFU/mL in all four products by the third day [ 1801. These genera-
tion times are somewhat longer than those observed by Farber et al. [98] when naturally
contaminated raw milk was incubated at 4 (25.3 h), 10 (10.8 h), and 15C (7.4 h). L.
monocytogenes also attained maximum populations that were approximately 1 0-fold lower
in raw than in sterile milk, which in turn suggests possible depletion of essential nutrients
by raw milk contaminants or production of substances inhibitory to growth of the patho-
-
gen. Maximum Listeria populations of 1 O9 CFU/mL were again observed in chocolate
milk, with numbers generally being 10-fold lower in skim milk, whole milk, and whipping
cream [ 1801. Increasing the incubation temperature to 2 1"C doubled the growth rate (see
Table 7) and led to maximum Listeria populations of I Ox- 10' CFU/mL within 48 hs. As
expected, L. monocytogenes grew most rapidly at 35"C, with populations of 1Ox- 10' CFU/
mL being observed after only 24 h of incubation.
In another study examining the influence of temperature and milk composition on
growth of listeriae, Donnelly and Briggs 1861 found that five L. monocytogenes strains
began growing in inoculated samples of autoclaved (121 "C/ 10 min) whole, skim, and
reconstituted nonfat dry milk (1 1 % total solids) after approximately 24-48, 2-24, 4-12,
and 0.5-4.0 h of incubation at 4, 10, 22, and 37OC, respectively. Although growth rates for
all Listeria strains were primarily determined by the incubation temperature, two strains
of L. monocytogenes serotype 4b grew considerably faster in whole rather than skim or
L. monocytogenes in Unfermented Dairy Products 389

reconstituted nonfat dry milk during incubation at 4 and 10C. These observations led
Donnelly and Briggs [86] to suggest a possible relationship between levels of milkfat and
the growth rate of L. monocytogenes in milk during refrigerated storage. Furthermore,
these authors suggested that enhanced psychrotrophic growth in whole milk may be related
to a listerial lipase produced by both P-hemolytic strains of L. monocytogenes serotype
4b. Unlike both of these strains, the three remaining L. monocytogenes strains of serotypes
1 and 3 failed to exhibit enhanced growth in whole milk at 10C and had little if any
hemolytic activity on McBride Listeria Agar containing sheep blood.
In contrast to what might be expected from the study just described, Rosenow and
Marth [ 1801 failed to observe any significant difference in growth rates among four strains
of L. monocytogenes (two serotype 4b, two serotype 1) when they were incubated in
autoclaved samples of whole and skim milk at 4, 8, 13, 21, and 35C. The pathogen also
attained lower maximum populations in whipping cream than in whole, skim, or chocolate
milk at all incubation temperatures. In support of these findings, Marshall and Schmidt
[ 1451 failed to observe enhanced growth of L. monocytogenes strain Scott A (serotype
4b) in whole rather than skim milk during 8 days of incubation at 10C. Finally, in a
study to be discussed in greater detail in Chapter I2 [ 1851, four strains of L. monocytogenes
(three serotype 4b and one serotype 1) frequently attained higher maximum populations
in whey samples that were defatted by centrifugation, filter sterilized, and incubated at
6C than would be expected to occur in autoclaved skim milk, whole milk, or whipping
cream after prolonged incubation at 8C. Thus, although some L. monocytogenes strains
are lipolytic as reported by Marshall and Schmidt [ 1461, one must presently conclude that
psychrotrophic growth of L. monocytogenes is not generally enhanced by the normal level
of milk fat found in fluid milk.
Recognizing the vital importance of carbohydrates in microbial metabolism, re-
searchers at the CDC [ 1661 attempted to define growth of Listeria spp. in terms of sugar
utilization. An initial experiment using aerobically incubated broth media indicated that
five strains of L. monocytogenes and one strain each of L. Jnnocua, L. seeligeri, and L.
ivanovii utilized only the glucose moiety of lactose, whereas single strains of L. grayi and
L. murruyi utilized both the glucose and galactose of lactose. Overall, maximum cell
populations, as determined by optical density, were directly proportional to the concentra-
tion of glucose ( S O . 125%) in the growth medium. However, marked differences were
observed in the ability of L. monocytogenes and L. innocua to utilize lactose, with three
strains of L. monocytogenes (isolated from Mexican-style cheese in connection with the
1985 listeriosis outbreak in California) being unable to grow in a medium containing
lactose as the only carbohydrate. Although these observations agree with several reports
[102,145,146] indicating that the pH of fluid milk is unaffected by L. monocytogenes
growth, Quinto et al. [I701 did report a sharp pH decrease in such milk after 16 and 24
days of incubation at 14 and 7"C, respectively, with these differences most likely being
related to strain variation.
Growth of L. monocytogenes in autoclaved samples of whole and skim milk was
generally similar to that previously observed by Rosenow and Marth [ 1801, with maximum
populations of 5 5 X IO* CFU/mL developing after extended incubation at 5 and 25C.
Except for L. seeligeri, the behavior of L. innocua and L. ivanovii did not differ markedly
from that of L. monocytogenes in these samples (Fig. 9). However, as noted by Northolt
et al. [ 1561, higher maximum populations and increased survival rates were again observed
when these organisms were grown in autoclaved rather than pasteurized whole milk. Ex-
amination of milk by gas-liquid chromatography indicated that lactic, acetic, isobutyric,
390 Ryser

L. rnonocvloaeneg + 0
Lseeliaeri m o
C ivanovil A A
\ innocua e o

9.0

8.0

7.0

6.0
0 4 8 12 16 20 24
Days

FIGURE9 Growth of Listeria spp. in pasteurized (open symbols) and autoclaved

whole milk (solid symbols) incubated at 5C. (Adapted from Ref. 166.)

isovaleric, and 2-hydroxy isocaproic acids were formed during incubation. Since this milk
initially contained -81 -85 mg of glucose/L, the aforementioned acids likely resulted, at
least in part, from fermentation of glucose. Considerably lower populations of L. monocy-
togenes as well as L. innocua, L. grayi, and L. murrayi also developed in glucose oxidase-
treated (an enzyme that degrades glucose) rather than untreated milk during both aerobic
and anaerobic incubation, and so it is evident that glucose is one of the major substrates
for growth of listeriae in milk. However, when incubated anaerobically in glucose oxi-
dase-treated milk, two lactose-negative L. monocytogenes isolates from Mexican-style
-
cheese still attained final populations of 10' CFU/mL; thus suggesting the involvement
of other as yet unidentified growth factors.
In the aforementioned study by Rosenow and Marth [ 1801, maximum populations
of L. monocytogenes were typically about 10-fold higher in chocolate milk than in other
fluid dairy products. To explain the enhanced growth .of L. monocytogenes in chocolate
milk, several investigators at the University of Wisconsin examined the effect of major
chocolate milk constituents (i.e., cocoa, sugar, and carrageenan) on growth of this organ-
ism in autoclaved skim milk and laboratory media. Rosenow and Marth [ 1791 found that
L. monocytogenes in Unfermented Dairy Products 391

growth of L. monocytogenes at 13C was only slightly enhanced in skim milk containing
5% cane sugar, and that the organism attained higher final populations when commercial
cocoa power (1.3%) and carrageenan stabilizer (0.5%) were used in place of cane sugar
(Fig. 10). Carrageenan also enhanced the growth rate of L. monocytogenes in the presence
of cocoa; however, the organism attained similar maximum populations regardless of the
presence or absence of carrageenan. These findings suggest that carrageenan may be more
important in increasing contact between cocoa particles and Listeria than as a source of
nutrients. Highest final populations and shortest generation times were observed when L.
monocytogerzes was grown in skim milk containing cocoa, sugar, and carrageenan. In
addition, maximum Listeria populations obtained in skim milk containing all three ingredi-
ents (see Fig. 10) were similar to populations observed in initial work with commercially
produced chocolate milk (see Figs. 7 and 8).
Subsequently, Pearson and Marth [ I621 examined growth of L. monocytogenes strain
V7 at 13C in skim milk containing various concentrations of cocoa, sugar, and carra-
geenan. Since some Listeria strains can utilize sucrose, it is not surprising that L. monocy-
togenes developed significantly higher final populations (see Fig. 1 1 ) and had shorter
generation times (5.05 vs 5.17 h) as the concentration of cane sugar (sucrose) in skim
milk was increased from 0 to 12%. (Peters and Liewen [ 1651 also reported that addition of
7% sucrose to ultrafiltered (concentrated) skim milk caused rnaximum L. monocytogenes
populations to increase rather than decrease.) A near-linear relationship between increas-
ing sugar concentration and maximum attainable populations of L. monocytogenes also
was observed for all but one combination of sugar, cocoa, and carrageenan tested; that is,

10

-- 2%milk(m)
rl
- ------ 2%m + sugar (s)

8b -- -A 2%m + cocoa (c) + can.

m - - - - - -+ 2 % m + c + s + c a r r .

r n20 ~ 40 ' 60 l 80
' l 100 ' 120 ' 140
' 1160 ' 180 I 200
' l
2 O

FIGURE10 Growth of L. rnonocytogenes strain V7 in 2% fat milk with added sugar,

cocoa, and carrageenan (carr.) at 13C. (Adapted from Ref. 179)
392 Ryser

8.90

8.80

8.70

8.60

8.50

8.40

0 3 6 9 12

Cane Sugar (O/., w/v)

FI URE 11 Maximum L. monocytogenes population in skim milk alone (m), skim

+
milk carrageenan (A), +
skim milk + cocoa (O), and skim milk cocoa + carrageenan
(+) with 0,6.5, and 12.0% cane sugar after 36 h of incubation at 13C. Any two points
differing by 20.07 loglo CFU/mL are significantly different (P < .05). (Adapted from
Ref. 162.)

12% sugar and 0.03% carrageenan (see Fig. 11). Although addition of 0.03% carrageenan
significantly lengthened generation times and decreased maximum populations compared
with those observed in skim milk without carrageenan, L. monocytogenes achieved highest
populations in skim milk containing 0.75% cocoa with or without carrageenan, which in
turn indicates that the apparent ability of cocoa to stimulate growth of this organism in
skim milk containing 0- 12% sugar is independent of carrageenan. Since cocoa contains
only trace amounts of fermentable carbohydrates, these authors theorized that cocoa en-
hanced growth of L. monocytogenes in skim milk by providing increased levels of peptides
and amino acids, particularly valine, leucine, and cysteine, which are reportedly essential
for growth. Additional work showed that agitation, combined with the presence of cocoa,
sugar, and/or carrageenan in skim milk, enhanced growth of the pathogen at 30C when
compared with growth in the same medium that was incubated quiescently. However,
growth of Listeria in skim milk alone was better without rather than with agitation. Thus
agitation most likely increased the availability of extractable nutrients from cocoa, which
in turn led to enhanced growth of the pathogen.
In 1968, anthocyanins in cocoa were reported to inhibit growth of salmonellae in
laboratory media; however, the inhibitory effect of cocoa could be neutralized with casein
[72]. These early findings prompted Pearson and Marth [164] to investigate the effect of
cocoa with and without casein on growth of L. monocytogenes strain V7. Using Modified
Tryptose Phosphate Broth containing 0.2% tryptose, addition of 0.75- 10% cocoa in-
creased the generation time for L. monocytogenes at 30C (1.02-1.12 h) as compared
with samples without cocoa (0.94 h). However, the pathogen generally attained higher
populations when grown in media with (1.1 - 1.5 X 109CFU/mL) rather than without (6.4
X 108CFU/mL) cocoa. Interestingly, when the same medium was inoculated to contain
L. monocytogenes in Unfermented Dairy Products 393

- 1 OS L. monocytogenes CFU/mL and agitated, the pathogen decreased to nondetectable

levels in samples containing 5-10% cocoa after 15-24 h of incubation at 30C. Nonethe-
less, the organism readily grew in the presence of 0.75% cocoa and attained higher maxi-
mum populations in media with ( I .9 X 109CFU/mL) rather than without (7.6 X 108CFU/
mL) cocoa during agitated incubation at 30C.
As previously reported for salmonellae, the presence of 1.5 or 3.0% casein neutral-
ized the inhibitory effect of cocoa toward L. monocytogenes, with the pathogen exhibiting
shorter lag phases and higher maximum populations in media containing both casein and
5.0% cocoa rather than cocoa alone and incubated quiescently at 30C. However, results
obtained during agitated incubation of cultures containing 5% cocoa were far more dra-
matic, with L. monocytogenes populations of 2.9 X 109rather than <10 CFU/mL devel-
oping in samples with rather than without 2.5% casein. Hence, these findings suggest that
the behavior of L. monocytogenes in laboratory media containing cocoa partially depends
on the concentration of one or more inhibitory substances than can be neutralized by
casein and that are more readily extracted during agitated rather than quiescent incubation.
Next, Pearson and Marth [I631 determined if theobromine and caffeine (i.e., two
methylxanthine compounds in cocoa that reportedly possess different degrees of antimi-
crobial activity) were responsible for the previously observed antilisterial activity of cocoa.
Overall, addition of 2.5% theobromine to both Modified Tryptose Phosphate Broth and
autoclaved skim milk with and without 0.5% caffeine did not markedly influence the
behavior of L. monocytogenes during incubation at 30C. This suggests that theobromine
is not responsible for suppressing or enhancing growth of listeriae in chocolate milk.
Unlike theobromine, addition of 0.5% caffeine to Modified Tryptose Phosphate Broth
doubled or tripled the length of the organism's lag phase, nearly doubled the organism's
generation time, and led to maximum Listeria populations approximately I 0-fold lower
than those obtained in caffeine-free media. Similar trends also were observed when auto-
claved skim milk instead of Modified Tryptose Phosphate Broth served as the growth
medium. Thus, although caffeine in cocoa may contribute to inhibition of L. monocyto-
genes in a broth medium, failure of casein in skim milk to neutralize the inhibitory effect
of cocoa indicates that caffeine also is not responsible for inhibition of listeriae as observed
in the previous study. Such efforts to identify Listeria-active components within cocoa
should be continued to better understand the behavior of this pathogen in chocolate milk.

Sweetened Condensed and Evaporated Milk

To our knowledge, Listeria spp. have not yet been isolated from commercially produced
sweetened condensed milk (i.e., a nonsterile concentrated fluid milk product containing
approximately 64% sucrose or glucose in the water phase, 8.5% milk fat, and 28% total
milk solids) or evaporated milk (an unsweetened commercially sterile concentrated fluid
milk product containing approximately 7.9% milk fat and 25.9% total milk solids). How-
ever, given the widespread incidence of Listeria in food processing facilities, it is conceiv-
able that listeriae could enter both of these products as postprocessing contaminants. Such
concerns prompted Farrag et al. [103] to examine the fate of three L. monocytogenes
strains in samples of commercially produced sweetened condensed and evaporated milk
that were inoculated to contain three different levels (- 1 03- 1O7 CFU/mL) of the pathogen.
Regardless of initial inoculum, Listeria populations in sweetened condensed milk
decreased 5 1.2 and 1.6-3.4 orders of magnitude following 42 days of storage at 7 and
2 1"C, respectively. This behavior was not surprising, since addition of sugar to this product
394 Ryser

during manufacture reduces its water activity (a,) to -0.83, which is well below the
minimum a, value of 0.90 reported for growth of L. monocytogenes. Unlike sweetened
condensed milk, the relatively high a, value for evaporated milk (-0.986) allowed profuse
growth of listeriae, with lowest inoculum levels increasing approximately 4 orders of
magnitude after 7 and 14 days of incubation at 21 and 7OC, respectively. In addition, no
decrease in numbers of listeriae was noted during continued incubation at either tempera-
ture. Thus, since L. monocytogenes can survive >42 days in sweetened condensed milk
and grow rapidly in evaporated milk, special precautions should be taken to prevent lister-
iae from entering these products during packaging, storage, and subsequent use.

Ultrafiltered Milk
Ultrafiltration, a mechanical process by which milk is filtered under pressure and concen-
trated, results in major compositional changes in the finished product when compared with
the starting material. During ultrafiltration, 94- 100% of the milk proteins and protein-
bound vitamins (i.e., vitamin BI2and folic acid) remain in the retentate along with milk
fat, whereas lactose is equally divided between the retentate and permeate. Increased use
of ultrafiltered milk in cheesemaking prompted El-Gazzar et al. [92] to investigate the
growth characteristics of L. monocytogenes in 2X and 4X retentate as well as the corre-
sponding permeate obtained from ultrafiltered pasteurized milk. When samples were inoc-
ulated to contain about 104CFU/mL of L. monocytogenes strain V7 or CA and incubated
at 4"C, growth of both organisms was enhanced 10- to 100-fold in retentate as compared
with unfiltered skim milk. Increasing the concentration of ultrafiltered (UF) skim milk
retentate from 2X to 5X also resulted in faster growth, with the pathogen attaining a
population of 106CFU/mL in 5X and 2X retentate after approximately 7 and 10-12 days
of refrigerated storage, respectively. L. monocytogenes also grew to dangerous levels in
permeate with maximum levels of 10' CFU/mL as compared with 10' CFU/mL in reten-
tate following 30 days of incubation. When identical tyndalized samples were incubated
at 32 and 4OoC, both Listeria strains grew similarly in skim milk and retentate, with
populations of 10' CFU/mL generally being reported after 24 h of incubation. However,
as was true for samples incubated at 4OC, maximum numbers of listeriae were again 10-
to 100-fold lower in permeate than in retentate and unfiltered skim milk. Hence, the same
care should be given to production of unfiltered milk to prevent contamination and subse-
quent growth of Listeria in the product during cold storage.

GROWTH OF L. MONOCYTOGENES IN MIXED CULTURES

Except for several early works assessing the behavior of L. rnonocytogenes in raw and
pasteurized milk, all studies described thus far have dealt with growth of L. monocytogenes
in the absence of competitive microorganisms. Although results from these studies have
been of great value to the dairy industry, one should remember that pasteurized dairy
products are not sterile. Psychrotrophic bacteria, belonging to the genera Pseudomonas
and Flavobacteriurn are typically present in raw milk and, like L. rnonocytogenes, can
grow in milk at refrigeration temperatures both before and after milk is pasteurized. Al-
though readily destroyed during pasteurization, these organisms universally appear in pas-
teurized dairy products as postpasteurization contaminants, often at levels > 100 CFU/mL.
Since L. monocytogenes is thought to enter dairy products primarily after pasteurization,
products that contain low levels of listeriae (probably <I0 CFU/mL) will likely contain
L. monocytogenes in Unfermented Dairy Products 395

higher populations of other psychrotrophs. Although the ability of psychrotrophic pseu-

domonads to stimulate growth of nonpathogenic as well as pathogenic bacteria in dairy
products has been recognized for more than 25 years; data concerning the behavior of L.
monocytogenes in mixed cultures is of far more recent origin.
After initial work [I011 with Tryptose Broth demonstrated that growth of L. monocy-
togenes was slightly inhibited by the presence of Pseudomonas jluorescens (see Chapter
6), Farrag and Marth [66] examined associative growth of L. monocytogenes (strains Scott
A, CA, and V7) with P. juorescens (strains P26 and B52) in autoclaved (1 2 I "C/ 15 min)
skim milk that was inoculated to contain equal populations (- I O5 CFU/mL) of both organ-
isms and incubated at 7 or 13C for 56 days. Growth of L. monocytogenes was generally
enhanced by the presence of P. jluorescens after 7 days of incubation, with the pathogen
-
attaining populations of 1O7 CFU/mL in mixed cultures. However, continued incubation
at 7C led to lower numbers of listeriae in mixed rather than pure cultures, with populations
of strain V7 being inhibited approximately eightfold by P. jluorescens B52 following 56
days of storage. Farrag and Marth [ 1051 later showed that inactivation of strain CA was
affected by initial levels of P. jluorescens P26, with highest populations being most detri-
mental to Listeria survival. However, populations of L. monocytogenes strains Scott A
and V7 remained unaltered in samples that were initially inoculated to contain P. jluo-
rescens P26 at levels of 103-106CFU/mL. When Farrag and Marth [102] increased the
incubation temperature to 13"C, growth of L. monocytogenes was neither enhanced nor
inhibited by either Pseudomonas strain during the first 7 days of incubation. However,
after 56 days of incubation, final populations of Listeria were as much as 20-fold lower
in mixed rather than pure culture. Although these findings and those of Quinto et al. [ 1701
indicate that P. jluorescens was more detrimental to survival of listeriae in skim milk
stored at 13 than 7"C, growth and survival of P. jluorescens was not appreciably affected
by the presence of L. monocytogenes, with pseudomonads consistently reaching popula-
tions of 108--1O9 CFU/mL. In a similar study, Marshall and Schmidt [ 1451 found that
growth of L. rnonocytogenes strain Scott A (used in the previous study) in autoclaved
skim and whole milk was not enhanced by the presence of Pseudomonas fragi during
8 days of incubation at 10C. Similarly, growth of P. fragi also was unaffected by L.
monocytogenes.
Some researchers have speculated that psychrotrophic pseudomonads may be able
to utilize some of the nutrients in milk faster than L. monocytogenes; thus suppressing
growth of listeriae in milk during refrigerated storage. Marshall and Schmidt [ 1451 investi-
gated this theory by inoculating samples of autoclaved whole milk, skim milk, and recon-
stituted nonfat dry milk (10% solids) with P. fragi or P. jluorescens (strain T25, P26, or
B52); incubating the samples for 3 days at 10C to obtain --106-107 P. fragi CFU/mL
or 104- 1O6 P. jluorescens CFU/mL; inoculating these Pseudomonas cultures with L. mono-
cytogenes; and then incubating the samples for an additional 8 days at 10C. Throughout
this study, addition of listeriae to all milks preincubated with P. jluorescens or P. fragi
did not significantly affect growth or survival of either pseudomonad. However, as shown
in Figure 12, L. monocytogenes grew faster and attained higher final populations in sam-
ples of whole milk that were preincubated with either of the two pseudomonads than in
whole milk that was not treated with pseudomonads. L. monocytogenes behaved similarly
in both whole and skim milk, with average generation times of approximately 7 and 8 h
in milks preincubated with P. jluorescens and P. fragi, respectively (Fig. 13). Although
accelerated growth of Listeria was observed in reconstituted nonfat dry milk preincubated
with either pseudomonad, generation times for listeriae in either of the two mixed cultures
396 Ryser

L. nionocyrogenes
--- +-
L. niotiocylogenes
+ P.fragi
------Q.---

L. nronocyrogenes
+ P . flicorescens

Days

FIGURE 12 Growth of Lisferia monocytogenes at 10C in whole milk preincubated

for 3 days with selected Pseudomonas spp. (Adapted from Ref. 145.)

generally did not differ significantly. As was true for whole and skim milk, L. monocyto-
genes attained populations of 1 X 107to 5 X 107CFU/mL in reconstituted nonfat dry
milk, with highest numbers occurring in milk preincubated with P. jluorescens rather than
P. fragi.
Flavobacterium is another genus of gram-negative psychrotrophic bacteria that is
frequently recovered from raw milk, pasteurized milk, and butter. Hence, Farrag and Marth
[104] also examined behavior of L. monocytogenes in the presence of flavobacteria in
skim milk at 7 and 13C. Growth of L. monocytogenes strains Scott A, CA, and V7 in
autoclaved skim milk was enhanced by the presence of F. lutescens during 14-42 days
of a 56-day incubation period at both 7 and 13"C, with these higher populations again
being attributed to proteolysis of milk proteins by F. lutescens. However, Flavobacterium
sp. ATCC 21429 failed to impact the growth of L. monocytogenes at 7C and proved to
be slightly inhibitory to the same three Listeria strains when samples were held at 13C.
One strain of Bacillus spp. [ 1431 also prevented Listeria growth in raw milk.
These results dispel the previous theory and indicate that L. monocytogenes can
readily compete with P. fragi, P. jluorescens, and certain Flavobacterium spp. for nutrients
in milk and at the same time can outgrow these organisms at refrigeration temperatures
L. monocytogenes in Unfermented Dairy Products 397

17 -
16 - L. monocytogenes
- L. monocytogenes
15 - P . fluorescens

L. monocytogenes
14 - P.fragi

13 -
12 -
11 -
10 -

6
Whole Skim Nonfat Milk Solids
Product

FIGURE13 Generation times of Listeria monocytogenes at 10C in various milks pre-

incubated for 3 days with P. fragi or P. fluorescens spp. (Adapted from Ref. 145.)

even if the ratio of L. monocytogenes to pseudomonads or flavobacteria is on the order

of 1 : 100,000. Enhanced growth of microorganisms, including L. monocytogenes, in the
presence of these psychrotrophs is now known to be related to increased levels of nutrients
that occur in milk as a result of proteolytic enzymes produced by these organisms
[104,146]. Since many of these enzymes are heat-stable and able to survive pasteurization,
raw milk must be handled properly and pasteurized within a reasonable time (i.e., 3-4
days) to prevent conditions that may favor growth of listeriae. As previously noted [156],
enhanced growth of listeriae in intensively pasteurized as compared to HTST-pasteurized
and raw milk also might be related to this phenomenon.

NONFLUID DAIRY PRODUCTS

Although the aforementioned studies demonstrate the ability of L. monocytogenes to grow
to potentially hazardous levels in fluid dairy products held at refrigeration temperatures,
concern about the behavior of this organism in dairy products extends well beyond fluid
milks and cream. As you will recall from Table 5 , nearly 50 recalls have been issued in
the United States for Listeria-contaminated ice cream. These recalls, along with FDA
reports suggesting that about 3.5% of the ice cream and 8.5% of the ice cream novelties
produced in the United States may be contaminated with presumably low levels of L.
monocytogenes, have prompted research on the fate of listeriae in frozen dairy products.
Additionally, behavior of Listeria during manufacture and storage of nonfat dry milk and
butter also has been investigated in the event that these products are inadvertently prepared
from skim milk and cream, respectively, that have been contaminated after pasteurization.
398 Ryser

Ice Cream
Frequently, pasteurized milk that has not been sold in retail stores is returned to dairy
factories and reprocessed into chocolate ice cream. Since large commercial refrigeration
units often fail to maintain a constant temperature of 4"C, virtually all reclaimed milk has
undergone some degree of temperature abuse during the period in which the product was
on sale. In addition to possible growth of L. monocytogenes during this 2-week period of
"cold enrichment,'' pseudomonads also can grow in milk and produce an environment
that is more favorable for growth of Listeria even after pasteurization.
The numerous Class I recalls issued since the late 1980s for Listeria-contaminated
ice cream prompted Berang et al. [70] to investigate the behavior of L. monocytogenes
in inoculated samples of chocolate ice cream (and chocolate milk as discussed earlier)
prepared from fresh skim milk and commercial skim milk that was held beyond the expira-
tion date. Although growth of listeriae was certainly not expected in ice cream held at
- 18 to -24"C, the pathogen survived equally well in both types of chocolate ice cream.
Hence, use of returned milk in chocolate ice cream did not appear to enhance Listeria
survival. Long-term survival of L. monocytogenes was also confirmed in a later study
[160] in which the pathogen persisted for 14 weeks in ice cream stored at - 18C with
no apparent cell death or injury. In 1996, Dean and Zottola [81] assessed the fate of L.
monocytogenes V7 in full-fat ( 10%) and reduced-fat (3%) soft-serve ice cream prepared
with and without 14 ppm nisin. Regardless of fat content, L. monocytogenes populations
remained constant in ice cream during freezing and 3 months of storage at - 18C. How-
ever, nisin effectively reduced Listeria survival in both full- and reduced-fat ice cream
during manufacture, with Listeria populations generally decreasing 2 and 3 orders of mag-
nitude in full- and low-fat ice cream, respectively, following 1 month of frozen storage
at - 18C. Although not currently approved in the United States as an ice cream ingredient,
incorporation of nisin into ice cream formulations appears to be an effective, albeit costly,
means of inactivating listeriae and reducing the number of Class I Listeria-related recalls
that continue to plague the dairy industry.
In 1989, Amelang and Doores [2,3] determined the generation times for L. monocy-
togenes in nine formulations of commercially produced ice cream mix that varied in type
and level of fat (cream, butter), sugar (cane sugar, corn sweetener), and milk solids (con-
densed milk, skim milk, whey powder). To simulate postprocessing contamination, all
-
samples were inoculated to contain 103L. monocytogenes strain Scott A or V7 CFU/
mL and incubated at 4, 2 1, and 35C. Overall, L. monocytogenes had average generation
times of 21.6, 1.08, and 0.79 h in ice cream mixes incubated at 4, 21, and 35"C, respec-
tively, with similar growth rates occurring in mixes containing 10, 14, and 15% fat and
held at the same temperature. It is noteworthy that these generation times are markedly
shorter than those calculated by Rosenow and Marth [ 1801 for growth of the same strains
in whole milk, skim milk, chocolate milk, and whipping cream (see Table 5). Although
L. monocytogenes generally behaved similarly in all ice cream mixes incubated at 4 and
2 1"C, differences in generation times were noted at 35C when the pathogen was cultured
in ice cream mixes made with alternative fat and milk solids. At 35"C, growth of listeriae
was somewhat enhanced in mixes containing butter rather than cream, skim milk powder,
or whey powder rather than condensed skim milk and egg yolk as additional sources of
solids. Although the pathogen grew most rapidly in ice cream mix containing a 50 :50
ratio of cream to butter, partial replacement of cane sugar (sucrose: glucose + fructose)
L. monocytogenes in Unfermented Dairy Products 399

with corn sweetener (glucose and maltose) or high fructose corn syrup failed to signifi-
cantly shorten generation times.

Butter
In 1988, Olsen et al. [158] examined the fate of L. monocytogenes during manufacture
and storage of butter in the event that the product is prepared from contaminated cream.
According to their report, pasteurized cream was inoculated to contain 104- 10' L. monocy-
togenes CFU/g and churned into butter. After removing the buttermilk, washed butter
grains were salted to a level of 1.2% and resultant butter was analyzed weekly for listeriae
during 10 weeks of storage at - 18, 4-6, and 13C. During manufacture -95% of the L.
monocytogenes population was lost in buttermilk, with the remaining 5% of the population
appearing in butter. The pathogen was present at levels of 1.7 X 104to 1.8 X 10' CFU/
g in cream as compared with 1.5 X 103to 1.6 X 104CFU/g in butter, indicating that like
Staphylococ-cus aureus [ 1531, L. monocytogenes also favors the water rather than lipid
phase during butter making. As shown in Figure 14, Listeria populations increased 1.9
and 2.7 orders of magnitude in butter stored at 4-6 and 13OC, with maximum numbers
being observed after 49 and 42 days of storage, respectively. These findings along with
similar results by Lanciotti et al. [ 1371 for commercially prepared light butter stored at 4
and 20C indicate that enough milk solids were trapped in the water phase (containing
-6% salt) to support growth of listeriae during storage. Numbers of listeriae then began
to decrease; however, the organism was still present at levels >104 CFU/g following 70
days of refrigerated storage. Although freezing the contaminated butter prevented growth

6.00
1
I-/ / \ / 4 to 6 O C

t
2.001'
0
'
20
* A '
40
' . a '
60
" ' J
80
Days

FIGURE14 Survival of L. monocytogenes in butter manufactured f r o m artificially

contaminated cream and stored at 12, 4-6, and -18C. Each line represents the aver-
age of 4 trials. (Adapted f r o m Ref. 158.)
400 Ryser

of L. monocytogenes, the organism was still present at levels of -103 CFU/g after 70
days of storage at -18"C, as was also reported by Slavchev et al. [191].
Thus far L. monocytogenes has not been isolated from pasteurized cream manufac-
tured in the United States; however, given the massive Listeria recall of Texas-produced
fluid dairy products, including half-and-half and whipping cream, in May of 1986 (see
Table 5 ) , one cannot assume that all pasteurized cream and butter manufactured in the
United States and elsewhere will be universally free of listeriae. As you will recall from
Chapter 10, one cluster of listeriosis cases in southern California was attributed to con-
sumption of contaminated butter [ 1471. Hence, since at least four Class I recalls have been
issued for L. monocytogenes-contaminated butter, and since growth of L. monocytogenes
has been demonstrated experimentally in both cream and butter during refrigerated storage,
it is necessary to ensure that cream is pasteurized and that recontamination of pasteurized
cream is prevented before and during its churning into butter.

Nonfat Dry Milk

Dried dairy products, including nonfat dry milk, whey, and casein, also may become con-
taminated with pathogenic microorganisms both before and after drying. Such concerns
have been raised recently in Australia and New Zealand [133]. Although all dry dairy
products examined thus far have been Listeria-free, methods used to detect listeriae in
these surveys were generally unable to recover cells that may have been injured during
the drying process.
Two factors, namely, the unusual thermal resistance of L. monocytogenes and the
report of a milkborne listeriosis outbreak in Massachusetts during 1983, prompted Doyle
et al. [90] to examine behavior of L. monocytogenes during manufacture and storage of
nonfat dry milk. Samples of concentrated (30% solids) and unconcentrated (10% solids)
-
skim milk were inoculated to contain 105- 106L. monocytogenes (strain Scott A or V7)
CFU/mL and dried to moisture contents of 3.6-6.4% in a gas-fired pilot plant-sized spray
dryer with inlet and outlet air temperatures of 165 t 2 and 67 t 2"C, respectively. All
samples of nonfat dry milk were stored at 25C for up to 16 weeks and periodically
analyzed for listeriae using both direct plating on McBride Listeria Agar (detects uninjured
cells) and cold enrichment in Tryptose Broth (detects injured and uninjured cells). Listeria
populations decreased approximately 1.O- 1.5 orders of magnitude during spray drying
regardless of whether or not nonfat dry milk was prepared from concentrated or unconcen-
trated skim milk. Strain V7 was generally hardier than strain Scott A during both spray
drying and storage of nonfat dry milk. Twelve to 16 weeks of storage at room temperature
were required to decrease populations of strain V7 > 1000-fold in nonfat dry milk, whereas
only 6 weeks of storage were necessary to obtain similar decreases in numbers of strain
Scott A. Overall, strains Scott A and V7 survived a maximum of 8 and 12 weeks in nonfat
dry milk, respectively. Although strain Scott A generally survived equally well in nonfat
dry milk prepared from concentrated and unconcentrated skim milk, strain V7 survived
2 weeks longer in nonfat dry milk manufactured from concentrated rather than unconcen-
trated skim milk. The higher moisture content of nonfat dry milk (i.e., 5.7 and 6.4%)
prepared from concentrated skim milk may have enhanced survival of listeriae in this
product during extended storage. Overall, populations of L. monocytogenes decreased
> 10,000-fold in nonfat dry milk during 16 weeks of storage at room temperature. Hence,
if commercially produced nonfat dry milk is ever found to contain L. monocytogenes,
L. monocytogenes in Unfermented Dairy Products 401

presumably at very low levels, it may be possible to eliminate this pathogen by holding
the product at room temperature for several months.

1. Abou-Donia, S.A., and A.K. Al-Medhagi. 1992. Detection and survival of Listeria monocyto-
genes in Egyptian dairy products. J. Dairy Sci. 75 (suppl. 1): 138.
1a. Abou-Eleinin, A.M., E.T. Ryser, and C.W. Donnelly. 1998. Unpublished data.
2. Amelang, J., and S. Doores. 1989. The effect of ingredients in ice cream formulations on the
growth of Listeria monocytogenes. Annual Meeting of the Institute of Food Technologists,
Chicago, June 25-29, Abstr. 468.
3. Amelang, J., and S. Doores. 1989. The effect of medium, growth phase and temperature on
the growth of Listeria monocytogenes in ice cream mix. Annual Meeting of the Institute for
Food Technologists, Chicago, June 25-29, Abstr. 469.
4. Andre, P., H. Roose, R. Van Noyen, L. Dejaegher, I. Vyttendaele, and K. De Schrijver. 1990.
Neuro-meningeal listeriosis associated with consumption of an ice cream. Med. Mal. Infect.
20:570-572.
5. Anonymous. 1986. Ice cream bars recalled. FDA Enforcement Report, July 16.
6. Anonymous. 1986. Ice cream recalled. FDA Enforcement Report, Oct. 22.
7. Anonymous. 1986. Ice cream recalled. FDA Enforcement Report, Oct. 29.
8. Anonymous. 1986. Ice cream, sherbet and glacee recalled. FDA Enforcement Report, Sept.
3.
9. Anonymous. 1986. Ice milk mix recalled. FDA Enforcement Report, June 25.
10. Anonymous. 1986. Large class I recall made of ice cream because of Listeria. Food Chem.
News 28(24):11-12.
11. Anonymous. 1986. Listeria causes class I recalls of ice milk mix, milk. Food Chem. News
28( 16):22.
12. Anonymous. 1986. Milk, chocolate milk, half and half, cultured buttermilk, whipping cream,
ice milk, ice milk mix and ice milk shake mix recalled. FDA Enforcement Report, June 25.
13. Anonymous. 1986. Sherbets, non-dairy products, ice milk products, gelati-da products and
ice cream recalled. FDA Enforcement Report, Aug. 27.
14. Anonymous. 1987. Chocolate ice cream recalled. FDA Enforcement Report, Sept. 16.
15. Anonymous. 1987. Class I recall made of cheese because of Listeria. Food Chem. News
28(50):52.
16. Anonymous. 1987. FDA launching two-year pathogen surveillance program. Food Chem.
News 29(31):10-12.
17. Anonymous. 1987. Ice cream and ice milk recalled. FDA Enforcement Report, Aug. 26.
18. Anonymous. 1987. Ice cream bars recalled. FDA Enforcement Report, Nov. 4.
19. Anonymous. 1987. Ice cream, ice milk and sherbet recalled. FDA Enforcement Report, Feb.
11.
20. Anonymous. 1987. Ice cream, ice milk and sherbet recalled. FDA Enforcement Report, Aug.
19.
21. Anonymous. 1987. Ice cream nuggets recalled. FDA Enforcement Report, Aug. 5.
22. Anonymous. 1987. Ice cream products recalled. FDA Enforcement Report, Sept. 2.
23. Anonymous. 1987. Ice cream products recalled. FDA Enforcement Report, Sept. 16.
24. Anonymous. 1987. Ice cream products recalled. FDA Enforcement Report, Sept. 23.
25. Anonymous. 1987. Ice cream recalled. FDA Enforcement Report, Jan. 28.
26. Anonymous. 1987. Ice cream recalled. FDA Enforcement Report, Feb. 11.
27. Anonymous. 1987. Ice cream recalled. FDA Enforcement Report, May 27.
28. Anonymous. 1987. Ice cream recalled because of Listeria, pottery because of lead. Food
Cheni. News 29(21):16-17.
402 Ryser

29. Anonymous. 1987. Milk industry has spent $66 million on recalls and related expenses, Witte
says. Food Chem. News 29( 17):29-30.
30. Anonymous. 1987. More ice cream recalled because of Listeria. Food Chem. News 28(48):
33.
31. Anonymous. 1988. Frozen dessert products recalled. FDA Enforcement Report, July 27.
32. Anonymous. 1988. Ice cream, cheese recalled because of Listeria. Food Chem. News 30(6):
27.
33. Anonymous. 1988. Ice cream pies recalled. FDA Enforcement Report, Dec. 28.
34. Anonymous. 1988. Ice cream products, cheese recalled because of Listeria. Food Chem.
News 30(9):47.
35. Anonymous. 1988. Ice cream recalled. FDA Enforcement Report, April 6.
36. Anonymous. 1988. Ice cream recalled. FDA Enforcement Report, Sept. 7.
37. Anonymous. 1988. Ice cream recalled. FDA Enforcement Report, Sept. 14.
38. Anonymous. 1988. Ice cream recalled. FDA Enforcement Report, Nov. 2 .
39. Anonymous. 1988. International Dairy Federation: Group E64-Detection of Listeria rnono-
cytogenes-sampling plans for Listeria rnonocytogenes in foods, Feb. 9. Brussels.
40. Anonymous. 1988. More cheese, ice cream linked to possible Listeria. Food Chem. News
29( 11):37-38.
41. Anonymous. 1989. Ice cream bars recalled. FDA Enforcement Report, Feb. 15.
42. Anonymous. 1989. Ice cream recalled. FDA Enforcement Report, April 19.
43. Anonymous. 1989. Le contr6le des rbsidus dans les produits laitiers. Bull. 1nf.-Minist. Agric.,
France I273:22-24.
44. Anonymous. 1990. Frozen yogurt recalled. FDA Enforcement Report, Feb. 7.
45. Anonymous. 1990. Ice cream and frozen yogurt novelties recalled. FDA Enforcement Report,
July 10.
46. Anonymous. 1990. Ice cream bars recalled. FDA Enforcement Report, April 25.
47. Anonymous. 1990. Ice cream recalled. FDA Enforcement Report, Nov. 7.
48. Anonymous. 1990. Sherbet, ice milk and ice cream recalled. FDA Enforcement Report, Dec.
5.
49. Anonymous. 1990. USDA, FDA officials report apparent decrease in Listeria isolations. Food
Chem. News 32( 1): 12- 15.
50. Anonymous. 1991. Butter recalled. FDA Enforcement Report, Aug. 7.
51. Anonymous. 1991. Ice cream and ice milk recalled. FDA Enforcement Report, June
19.
52. Anonymous. 1992. Butter and butterine recalled. FDA Enforcement Report, July 22.
53. Anonymous. 1992. Ice milk and ice cream recalled. FDA Enforcement Report, Dec. 30.
54. Anonymous. 1993. Ice cream bars recalled. FDA Enforcement Report, Sept. 29.
55. Anonymous. 1994. Butter products recalled. FDA Enforcement Report, Oct. 12.
56. Anonymous. 1994. Ice cream recalled. FDA Enforcement Report., Oct. 5.
57. Anonymous. 1995. Ice cream novelties recalled. FDA Enforcement Report, Dec. 13.
58. Anonymous. 1996. Frozen yogurt recalled. FDA Enforcement Report, Mar.6.
59. Anonymous. 1996. Ice cream and sherbet recalled. FDA Enforcement Report, April 10.
60. Anonymous. 1996. Ice cream, frozen yogurt, sherbet, sorbet and ice cream mix recalled.
FDA Enforcement Report, Jan. 3 1.
61. Anonymous. 1996. Ice cream recalled. FDA Enforcement Report, Jan. 3 I .
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12
Incidence and Behavior of Listeria
monocytogenes in Cheese and
Other Fermented Dairy Products

ELLIOTT. RYSER
Michigan State University, East Lansing, Michigan

INTRODUCTION
On June 14, 1985, L. monocytogenes emerged from relative obscurity to the front page
of many American newspapers because of a large listeriosis outbreak in California that
was directly linked to consumption of Mexican-style cheese manufactured in metropolitan
Los Angeles. By the time this outbreak subsided in August 1985, as many as 300 cases
of listeriosis were reported, including 85 deaths-at least 40 of which were traced to the
tainted cheese. In response to this foodborne outbreak of listeriosis, U.S. Food and Drug
Administration (FDA) officials added L. monocytogenes to their list of pathogenic organ-
isms that should be of concern to cheesemakers and began surveying various soft domestic
cheeses for listeriae.
Approximately 6 months later, isolation of L. monocytogenes from several imported
Brie cheeses purchased at a supermarket led to the eventual recall of approximately
300,000 tons of Brie cheese imported from France and to a real concern about the inci-
dence of this pathogen in other European cheeses. Recall of this cheese prompted two
corrective measures: (a) adoption of a cheese certification program by the United States
and France to prevent importation of Listeria-contaminated cheese and (b) initiation of
numerous large-scale surveys to determine the extent of Listeria contamination in virtually
all types of cheese manufactured in the United States, Canada, and Western Europe.

411
4 12 Ryser

Throughout 1986 and most of 1987, the impact of Listeria on European cheesemak-
ers was primarily in the form of economic losses from destruction of contaminated prod-
uct. However, L. rnonocytogenes struck again late in 1987 with the report of a large listeri-
osis outbreak in Switzerland (see Chap. 10) in which Vacherin Mont d'Or soft-ripened
cheese was incriminated as the vehicle of infection. Most recently, tainted Brie cheese
prepared from raw milk was responsible for a major listeriosis outbreak in France.
These cheeseborne listeriosis outbreaks have prompted worldwide efforts to deter-
mine the incidence of Listeria spp. in various cheeses and examine the behavior of L.
rnonocytogenes during manufacture and storage of numerous fermented dairy products.
The first portion of this chapter summarizes Listeria-related recalls of cheese in the United
States and results from surveys dealing with the incidence of listeriae in domestic and
imported fermented dairy products. The second half of this chapter addresses the fate of L.
rnonocytogenes during manufacture and storage of buttermilk, yogurt, and various cheeses
(including whey) and the potential for cheese ingredients, such as rennet, salt brine, and
coloring agents, to serve as vehicles of contamination during cheesemaking.

U.S. SURVEILLANCE PROGRAMS AND RECALLS FOR L.

MONOCYTOGNS IN DOMESTIC AND IMPORTED
CHEESE
Domestic Cheese
The concept of listeriosis as a foodborne illness is not new. As you will recall from Chapter
10, consumption of contaminated raw milk was believed to have caused several cases of
listeriosis in post-World War I1 Germany. In 1961, Seeliger [274] also suggested sour
milk, cream, and cottage cheese as possible vehicles of infection in this outbreak. Al-
though results from two Yugoslavian studies concerned with behavior of L. rnonocyto-
genes in various fermented dairy products (i.e., cultured cream, unsalted skim milk cheese,
Kachkaval cheese, and yogurt) were published in 1964 [195] and 1981 [281], no surveys
dealing with the incidence of listeriae in fermented dairy products were made before con-
taminated Mexican-style cheese was linked to the California listeriosis outbreak in June
of 1985.
Public health concerns about presence of L. rnonocytogenes in domestic and im-
ported fermented dairy products as well as other foods, such as meat, poultry, seafood,
fruits, and vegetables, can be traced either directly or indirectly to the 1985 listeriosis
outbreak in California. Less than 1 month after the first nationwide Class I Listeria-associ-
ated recall was issued for 22 varieties (-500,000 lbs.) of Mexican-style cheese contami-
nated with L. monocytogenes (Table l), the FDA developed a series of programs designed
to prevent the recurrence of such an outbreak [280] (Fig. 1).
The Domestic Soft Cheese Surveillance Program-the first of the dairy factory ini-
tiative programs-was instituted by the FDA in July of 1985 and involved on-site inspec-
tion of firms manufacturing soft cheese [ 141. Priority was given to manufacturers of Mexi-
can-style soft cheese, followed by firms producing other ethnic-type soft cheeses, such as
Edam, Gouda, Liederkranz, Limburger, Monterey Jack, Muenster, and Port du Salut, made
from raw, heat-treated (<71.7"C [16l0F]/15 s) or pasteurized (171.7"C [161"F]/15 s)
milk. In addition to determining the firm's compliance with good manufacturing practices
(i.e., use of proper pasteurization, cleaning, and sanitizing procedures), FDA inspectors
collected and analyzed cheese samples for L. rnonocytogenes using the original FDA pro-
L. monocytogenes in Fermented Dairy Products 4 13

cedure. Cheese samples also were tested for the presence of enteropathogenic strains of
E. coli and for phosphatase activity, which if present generally indicates improper pasteur-
ization of cheesemilk and/or subsequent contamination with raw milk. However, suitabil-
ity of the phosphatase test for cheese has since been questioned.
Less than 2 months into this program, FDA officials isolated a pathogenic strain of
L. monocytogenes from one sample of domestically produced Liederkranz cheese (see
Table 1). The manufacturer subsequently recalled the product nationwide. Following pre-
liminary FDA reports of further Listeria contamination, this recall was extended to include
all lots of Brie and Camembert cheese manufactured at the same facility [8,12]. However,
final laboratory reports indicated that both Brie and Camembert cheese were contaminated
with L. inrzocua, which is nonpathogenic, rather than L. monocytogenes. Although the
Domestic Soft Cheese Surveillance Program also was responsible for temporarily closing
two soft cheese factories in California that produced phosphatase-positive cheese [9], it
must be stressed that L. monocytogenes was never isolated from cheeses produced at either
facility.
In general, FDA inspections of other soft cheese factories uncovered problems simi-
lar to those encountered during inspections of Grade A fluid milk factories: (a) potential
bypasses of the pasteurizer, (b) postpasteurization blending of product, and (c) a general
lack of education and/or training of plant personnel [223]. Items of particular concern to
cheesemakers and that were not generally found during visits to Grade A milk factories
included defects in the pasteurization process, discrepancies in pasteurization/production
records, and a higher incidence (than in Grade A milk factories) of pathogenic microorgan-
isms (including L. monocytogenes) on environmental surfaces in production and storage
areas.
Inspections of domestic cheese factories continued throughout 1986, 1987, and 1988
under four separate programs (see Fig. l), with FDA officials reaching nearly half of the
400 soft cheese factories in the United States by April of 1986 and the remaining factories
(including follow-up inspections of problem factories) by late 1987 [46]. According to
FDA records [loll, L. monocytogenes was confirmed in 12 of 658 (1.82%) domestic
cheese samples analyzed during 1986. During these inspection programs, six Class I recalls
were issued for various ethnic-type soft and semisoft cheeses containing L. monocyto-
genes. In response to (a) a 1987 report of a woman who developed listeriosis in San
Bernadino, California, after consuming illegally produced Mexican-style cheese and (b)
the widespread availability of uninspected, unbranded Mexican-style cheese illegally pro-
duced from raw milk in metropolitan Los Angeles [71a], Genigeorgis et al. [175], in
conjunction with the California Department of Food and Agriculture, U.S. Department of
Agricultures Food Safety and Inspection Service (USDA-FSIS), the Immigration and
Naturalization Service, and the Los Angeles District Attorneys Office, surveyed 100 Cali-
fornia-produced soft Hispanic-style cheeses that were either seized or purchased under-
cover between June and November of 1988, Overall, two samples each were positive for
L. monocytogenes and L. innocua. These four Listeria-contaminated cheeses had a pH of
6.2-6.5 and were presumably prepared from raw milk as evidenced by a positive alkaline
phosphatase test. Given the ability of L. monocytogenes to grow in such cheeses during
refrigerated storage and marketing, Hispanic-style cheeses continue to constitute a signifi-
cant public health threat, with these varieties accounting for 7 of 21 recalls issued through
1996, including one large recall in June 1990 involving approximately 500,000 Ib of prod-
uct. As previously mentioned, although all products containing L. monocytogenes must
be retrieved from the marketplace, formal Class I recalls do not have to be issued for
4 14 Ryser

TABLE
1 Chronological List of Class I Recalls in the United States for Domestic Cheese Contaminated with L. monocytogenes
Date recall
Type of cheese initiated Origin Distribution Quantity (lb) Ref.
Jalisco brand soft Mexican-style: Cotija, 6/13/85 California Arizona, Arkansas, California, Colorado, -500,000 10, 11
Queso Fresco, and 20 other varieties Georgia, Guam, Hawaii, Idaho, Illinois,
Kansas, Louisiana, Marshal1 Islands, Mas-
sachusetts, Nevada, New Jersey, New Mex-
ico, New York, Oklahoma, Oregon, Rhode
Island, Samoa, Texas, Utah, Washington
state
Liederkranz (Brie,a Camembert) 8/ 14/85 Ohio Nationwide, Puerto Rico -10,000 8, 9, 12
Soft Mexican-style: Queso Fresco and 5 3/5/86 California Arizona, California, Oregon, Texas 127,607 20, 38
other varieties
Semisoft Salvador-style white 911 1/86 Virginia Virginia, Washington, DC 10,850 42, 54
Soft-ripened: Old Heidelberg 4117/87 Illinois Illinois, North Carolina, Ohio, Pennsylvania 1150 52
Soft-ripened: Bonbel and Gouda 5/6/87 Kentucky Nationwide -13,800 51
Raw milk sharp Cheddar 8/21187 Wisconsin California, Washington state -1400 53, 280
Soft Mexican-style: Cotija, Queso Fresco, 1/29/88 California Arizona, California, Florida, Texas, Washing- Unknown 64
+
and 8 other varieties Baby Jack and ton state
Monterey Jack
Mexican-style soft cheese 1 1/6/90 California Arizona, California, Idaho, Nevada, Oregon, 500,000 78
Washington state
Cheese spread 2/1/91 Florida Southeastern United States -1362 81
Mozzarella 2/14/91 Wisconsin Connecticut, Georgia, Illinois, Michigan, >89.000 80
New York, Ohio, Pennsylvania, Texas,
West Virginia, Wisconsin
Ricotta 711 1/91 New York Florida, New York 1109 82
Jack 10/28/91 Wisconsin Iowa, Minnesota, Wisconsin 12,500 79
Cold-pack cheese food 3110192 Wisconsin Arizona, California, Colorado, Florida, Geor- Unknown 83
gia, Illinois, Indiana, Maryland, Michigan,
Minnesota, New York, Ohio, Pennsylva-
nia, Tennessee, Texas, Vermont, Virginia,
Wisconsin
Queso fresco 10/14/92 Washington Oregon, Washington state Unknown 85
Limburger 1211 8/92 Wisconsin Wisconsin 1500 86
L. monocytogenes in Fermented Dairy Products 4 15

Cheese spread 3/4/93 Tennessee Alabama, Illinois, Indiana, Kentucky, Missis- 11,789 84
sippi, Tennessee
Cream cheese 10119/93 Wisconsin California, Florida, Georgia, Illinois, Indiana, 3075 88
Iowa, Minnesota, Nebraska, North Caro-
lina, Ohio, South Carolina, South Dakota,
Tennessee, Wisconsin
Queso prensado 4/15/94 Wisconsin Florida, New Jersey, Wisconsin 1429 94
Cream cheese and lox 511 1/94 Massachusetts Connecticut, Georgia, Massachusetts 20 89
Mexican-style soft white 5120194 Texas Texas Unknown 92
Mexican-style soft white 512 I 194 Texas Texas Unknown 91
Mexican-style soft white 5/23/94 Texas Texas Unknown 91
Queso blanco 5/24/94 Wisconsin New Jersey 1220 93
Goat milk cheese 6/15/94 California California, Colorado, Georgia, Illinois, Mas- -5,682 90
sachusetts, Michigan, New York, Oregon,
Texas
Torte loaf cheese 8111194 Missouri Illinois, Indiana, Kansas, Louisiana, Mis- 301 96
souri, Texas
Swiss cold-pack cheese food 811 1 194 Wisconsin Missouri, Ohio 510 95
Swiss I0/28/94 Ohio Pennsylvania 2270 97
Gorgonzola 2/2/96 Wisconsin California, Colorado, Florida, Georgia, Illi- 4500 98
nois, Minnesota, New Jersey, New York,
North Carolina, Pennsylvania, Tennessee,
Washington state, Wisconsin
Cream cheese with vegetables 10/30/97 Massachusetts Connecticut, Maine, Massachusetts, New 7,340 lOOa
Hampshire, New Jersey, New York,
Rhode Island, Pennsylvania, Vermont
Cream cheese 1 1114197 Massachusetts Connecticut, Maine, Massachusetts, New Unknown lOOb
Hampshire, New Jersey, New York,
Rhode Island, Vermont
Queso fresco 2/4/98 Wisconsin Nationwide 248,938 IOOe, lOOf
Queso fresco 3/23/98 Domestic Alabama, Florida, Georgia, North Carolina, Unknown lOOg
South Carolina, Tennessee, Virginia
Blue cheese 411 1/98 Wisconsin Nationwide Unknown lOOc
Blue cheese salad dressing 511198 Louisiana Nationwide Unknown lOOd

a Later found to contain only L. innocua.

 California listeriosis outbreak:
DOMESTIC

Domestic soft cheese surveillance

program - begun July 1985

1 l-
* 2

L. monocytogenes accidentally isolated

from French Erie cheese - January 1986 IMPORTED
' \
d
J./

r
Aged and ripened cheese survey -
begun January 1986

r
Continuation of s o f t cheese survey -
March 1987 cheese testing program - begun April 1986

1
Survey of cheese manufactured
I-
1
L. monocytogenes isolated from Italian
Romano cheese - June 1987
I 1
Italian cheese surveillance program
begun July 1987
I
-
I
-I

surveillance program -
Cheese under general pathogen
1988
I
I
status -
Survey of import cheese in domestic
begun December 1987 I
L

FIGURE1 Surveillance programs for Listeria spp. in domestic and imported cheese. (Adapted from Ref. 101.)
L. monocytogenes in Fermented Dairy Products 417

contaminated products that have not yet reached retail stores. Since such situations typi-
cally lead to nonpublished ''internal recalls" issued by the manufacturer, far more cheese
was likely destroyed during this 11-year period than has actually been reported. Several
such informal recalls involved a part-skim milk cheese manufactured in California [57]
as well as ricotta, Parmesan, and mozzarella cheese of uncertain origin [233].
Following a report by Ryser and Marth [259] that L. rnonocytogenes can survive
more than 1 year in Cheddar cheese (i.e., well beyond the mandatory 60-day aging period
for Cheddar cheese manufactured from raw milk), the FDA modified its Domestic Cheese
Program in August of 1987 to include cheese prepared from unpasteurized milk [46].
Between April and October of 1987, 181 samples of domestic aged (held a minimum of
60 days at 1:1.7"C [35"F]) natural cheese manufactured from raw milk, as well as similar
imported cheeses in domestic status, were collected from retail stores by FDA field person-
nel and analyzed for L. rnonocytogenes (Table 2). These efforts uncovered one positive
sample-a sharp Cheddar cheese manufactured in Wisconsin, which was subsequently
recalled from the market in July of 1987 (see Table 1).
Late in 1987, the FDA announced plans for a 2-year pathogen surveillance program
[43] which was designed to examine domestic and imported cheese as well as other high-
risk foods (i.e., milk, vegetables, and seafood) for the presence of L. rnonocytogenes and
other selected pathogens, including Vibrio cholerae, V. parahaernolyticus, Escherichia
coli, enteropathogenic E. coli, Staphylococcus aureus, Salmonella spp., Yersinia entero-
colitica, Clarnpylobacterjejuni, and C. coli. Under this program, samples of soft-ripened
and raw milk cheese as well as imported hard and artificial blended cheese were examined
for all of the aforementioned organisms except Vibrio spp. Domestic cheeses were col-
lected at the wholesale level, whereas samples of imported cheese were obtained from
retail stores. Although this program prompted only one Listeria -related recall of domestic
cheese during 1989 and 1990, five separate recalls of Anari and Halloumi cheese imported

TABLE
2 Incidence of L. monocytogenes in
"Domestic" Cheese Manufactured from Raw
Milk-FDA 1987a
Number of Number of
samples positive samples
Type of cheese analy zed (%>
Blue 18 0
Brick 5 0
Cheddar 71 l b (1.4)
Colby 8 0
Edam 4 0
Goat 6 0
Gouda 1 0
Monterey Jack 9 0
Swiss 42 0
Other 17 0
Total 181 1 (0.55)
~

aIncludes imported cheese in domestic status.

2 samples with L. innocua.
Source: Adapted from Ref. 101.
4 18 Ryser

from Cyprus were reported during this same 2-year period along with one additional recall
of Italian soft-ripened/semisoft cheese. However, since additional cheese-related recalls
after 1990 have been limited to three imported cheeses, the present FDA inspection pro-
gram in combination with increased vigilance on the part of cheesemakers appears to be
highly effective in limiting consumer exposure to both domestic and imported Listeria-
contaminated cheese.
Several other fermented dairy products also were examined for L. monocytogenes
in conjunction with the FDA Dairy Initiative Program [ l o l l (see Chap. 11). In 1986, 10
samples of cottage cheese were found to be free of listeriae. Other than cheese and cheese
food, 1% fat cultured buttermilk [33,35] and frozen yogurt [7 1 ] are the only other domesti-
cally produced, fermented dairy products known to have been contaminated with L. mono-
cytogenes. The first of these products was included in a 1986 Class I recall involving
approximately 1 million gallons of dairy products (fluid milk, chocolate milk, half-and-
half, whipping cream, ice milk, ice milk mix, ice milk shake mix, ice cream, and ice cream
mix), all of which presumably contained L. monocytogenes. Three years later, officials
from the Wisconsin Department of Agriculture, Trade and Consumer Protection issued a
statewide recall for one particular brand of frozen yogurt after routine testing revealed
the presence of L. monocytogenes in one sample of mandarin orange frozen yogurt [73].
Both of these products were retrieved from the marketplace without incident.

Imported Cheese
France
International concern over the potential health hazard of consuming Listeria-contaminated
cheese also is rooted in the California listeriosis outbreak of 1985. This outbreak and an
earlier link between consumption of French Brie and/or Camembert cheese and several
outbreaks of foodborne illness in the United States and Europe caused by enterotoxigenic
and/or enteropathogenic E. coli [2 17,234,2961prompted a meeting in September of 1985
between FDA officials and representatives of the French Embassy/French Delegation on
Food Safety and Food Distribution to discuss the FDAs plans for inspecting imported
soft cheese [ 131. Late in September, representatives from the Codex Committee on Food
Hygiene and the International Dairy Federation agreed with FDA officials that a Code
of Hygienic Practices should be developed for manufacturing fresh and soft cheese [7].
Use of raw milk (a known source of L. monocytogenes) to improve organoleptic properties
of certain cheeses was cited as a particular area of concern. Although a basic certification
program for soft cheese produced in France was in operation for some time, agreement
on a general Code of Hygienic Practices for manufacture of soft cheese was not reached
during the remainder of 1985.
In January of 1986, as part of a research effort to enhance recovery of listeriae from
cheese, FDA officials inadvertently isolated a pathogenic strain of L. monocytogenes from
two uninoculated control samples of French Brie cheese purchased at a local supermarket
[ 19,1011. Ironically, both cheeses were prepared from pasteurized milk in a cheese factory
certified by the French government under the existing soft-ripened cheese agreement. In
response to these findings, the first in a series of six nationwide recalls was issued in
February of 1986 for Listeria-contaminated French Brie cheese (Table 3). The following
week, the French firm that manufactured the tainted cheese agreed to cease all production
[19]. Shipment of additional cheese that was previously certified as Listeria free by an
L. monocytogenes in Fermented Dairy Products 4 19

independent French laboratory (certification by the French Ministry of Agriculture began

January 1, 1986) was also stopped pending identification of the contamination source. In
addition, all suspect lots of French Brie cheese en route to the United States were detained
on entry and tested for Listeria by the FDA before being released. Shortly thereafter,
sampling was extended to include virtually all lots of French Brie cheese produced by
this manufacturer.
The French cheese industry was dealt its most serious blow in March of 1986, when
approximately 660 million pounds of Brie cheese produced by five different manufacturers
were recalled in the United States (Table 3). This recall, which involved nearly 60% of
all Brie cheese marketed in the United States, immediately raised the possibility of block-
listing French firms that produced contaminated cheese [2 I ]. Consequently, FDA officials
immediately began testing all shipments of French soft-ripened cheese as well as 20% of
French soft/semisoft cheese and 20% of all other imported soft cheeses for Listeria, E.
coli, and phosphatase (see Fig. I ) [24].
Recognizing the danger of contracting listeriosis from consuming contaminated
French soft-ripened cheese, FDA officials drafted the following proposal to detain, test,
and certify all French soft-ripened cheeses exported to the LJnited States [72]:
FDA intends to detain any entry that is found to be Listeria-positive, regardless of the species
of ListcJriafound. French soft-ripened cheese without a certificate indicating negative results
for the Listeria analysis or with a positive analysis for Listeria .will be detained. . . . In addition,
all French soft-ripened cheeses are to be sampled and analyzed for the presence of Listeria.
[Under the previous agreement, cheeses were shipped with a certificate of analysis which
only indicated the level of E. coli and the absence of phosphatase.] The analysis may be
carried out by a private laboratory after the cheese has arrived in the United States, or alterna-
tively the analysis may be conducted so that the availability of the results will coincide with
the arrival of the shipment in the United States. The timing of the analysis is important to
ensure that the nature of the cheese tested, particularly the pH, is identical to that examined
by the FDA, if we decide to perform an audit on the entry. Thus, importers of French soft-
ripened cheese should provide FDA with certificates indicating the dates testing was initiated
and completed. Cheese shipments will not be released without a certificate indicating negative
results for Listeria analysis. . . .

In this proposal, FDA officials stressed that other methods used to detect Listeria
in cheese should conform to the 7-day FDA method described in Chapter 7 and also
suggested that 1 / 16-in-thick slices from the cheese surface (sample with highest pH) be
analyzed for listeriae rather than cross-sectional plugs of cheese.
Following FDA threats to halt importation of soft-ripened cheese, the French Minis-
try of Agriculture agreed to begin lot-by-lot testing in April of 1986, as outlined in the
March FDA proposal [25]. However, French authorities stressed that such a program
would not be practical on a long-term basis and hoped that the FDA would accept an
expansion ofthe existing factory/product certification program to include Listeria testing
in the near future. Beginning in May 1986, FDA officials announced that all shipments
of French soft-ripened cheese lacking certification of analysis for Listeria would be de-
tained [23]. Inspections during the next 2 months uncovered L. rnonncytogenes in two
French cheeses-a noncertified Brie and a 6-lb certified lot of Muenster [23]-both of
which were presumably recalled internally.
Continued problems with Listeriu-contaminated French soft-ripened cheeses
prompted FDA officials to revise the imported cheese surveillance program in August of
1986 [IS]. These changes allowed immediate detention of French cheeses that were: (a)
420 R yser

TABLE
3 Chronological List of Class I Recalls in the United States for Imported Cheese Contaminated with L. rnonocytogenes
Date recall Country of
Type of cheese initiated manufacture Distribution Quantity Ref.
Brie 2112/86 France Bermuda, Nationwide 57,000 2-6-lb wheels 17, 19, 26, 29
Brie 2114/86 France Georgia, New Jersey 40 cases 16
Brie 2/14/86 France Colorado, Connecticut, Florida, Louisi- 100 cases 16
ana, Maryland, Massachusetts, New
Jersey, New York, Ohio, Washing-
ton, DC
Brie 2/14/86 France Florida, New York, Washington, DC 10 cases 16
Brie 2/14/86 France Nationwide Unknown 16, 37
Brie 212 1186 France Oregon, Washington state Unknown 15, 37
Brie 2/24/86 France Illinois, Minnesota, New Jersey 909 cases 17, 39
Brie 3/14/86 France Nationwide -660 million lb 36, 40
Brie 411/86 France Colorado, Connecticut, Georgia, New -230 Ib 27, 36
Jersey, New York, North Carolina,
Texas, Washington, DC
Soft-ripened: Tourre de 1Aubier and 6/23/86 France New York, Ohio, Pennsylvania Unknown 28,31
Fromage des Burons
Soft-ripened: Tourre de 1Aubier 8/13/86 France California, Illinois, Maine, Massachu- 1056 Ib 32
setts, New Jersey, New York, Oregon
Semisoft: Morbier Rippoz 8118/86 France Illinois, Massachusetts, Michigan -1600 lb 28, 30, 34
Soft-unripened, full fat 4/16/87 France New Jersey, New York, Texas 15 wheels 47, 48
Semisoft 1/27/88 Italy Nationwide 410cartons 59
L. monocytogenes in Fermented Dairy Products 421

Semiseft: LP,mu!ette Dmish Esrem 2/ 11/88 EefiF-Ek CdifGrni2 Unknown 56, 57, 60
Semisoft: LAmulette Danish Esrom 21 12/88 Denmark East, Midwest, North, South -11,500 lb 56, 57, 61
Semisoft: LAmulette Danish Esrom 21 18/88 Denmark Florida, New Jersey, New York, Massa- -1,150 lb 56-58, 62
chusetts
Blue 4/6/88 Denmark California, Florida, Illinois, Massachu- -5,000 Ib 55, 56
setts, Michigan, Minnesota, New Jer-
sey, New York, North Carolina, Ore-
gon, Pennsylvania, Texas
Anari 5/26/89 Cyprus New York 50 cases 68
Anari 7/27/89 Cyprus Illinois, Texas 79 cases 69
Anari 8/9/89 Cyprus New York 80 cases 70
Halloumi 9/15/89 Cyprus Florida, New Jersey, New York 14,400 Ib 75
Italian soft ripened and semisoft 7/27/90 Italy California, Connecticut, New Jersey, Unknown 76
New York, Pennsylvania
Fontina 4/6/93 Sweden California, Connecticut, Maryland, Mas- 85,080 lb 87
sachusetts, Minnesota, New Hamp-
shire, New York, North Carolina,
Pennsylvania, Rhode Island, Washing-
ton state
Limburger 2/29/96 Germany Florida, Indiana, Maine, Massachusetts, 813 lb 100
New Jersey, New York, Ohio, Utah,
Virginia
jarisberg 6i7i96 Norway Alaska, Cdifoniia, Guam, Hawaii, 30,727 lb 99
Idaho, Montana, Nevada, Oregon,
Utah, Washington state
422 R yser

manufactured at a non-government-certified factory, (b) unaccompanied by a Listeria-

free government certificate, (c) positive for phosphatase, or (d) manufactured by one of
several firms that were block-listed for Listeria. Although these changes made importation
of French cheeses more difficult, sampling of cheeses that were manufactured at certified
factories and accompanied by Listeria-free certificates was decreased to the 20% level.
Between June and August of 1986, four additional Class I recalls were issued for
French semisoft/soft-ripened cheese contaminated with L. monocytogenes (see Table 3).
Two of three firms involved in these recalls were previously block-listed by the FDA [ 181.
An additional Class I recall issued for semisoft Morbier Rippoz cheese (see Table 3) was
accompanied by the following press release [34]:
Although Listeria is a rare cause of human illness, it can be life-threatening to pregnant
women and their fetuses, frail elderly persons or other persons with weakened immune sys-
tems. In healthy adults, it is a transient illness with such mild-to-moderate flu-like symptoms
as fever, headaches, and/or gastrointestinal tract distress.

The language used in such press releases also has received considerable attention.
These messages to the public must be firm enough to accomplish the goals of the recall
but not so alarming as to create an undue panic.
After considerable consultation, the governments of France and the United States
reached agreement on a French certification program for soft cheese [49,101]. Under this
program, which began February 15, 1987, cheeses were tested before shipping using meth-
ods that were mutually acceptable by both governments. French cheeses manufactured at
certified factories would be sampled at the 5% level, whereas other French cheeses (and
cheeses manufactured in other countries without certification programs) would be analyzed
at the 20% level. In the event of a Listeria-positive shipment, personnel at the French
cheese factory would be required to investigate the potential source of contamination and
analyze every lot of cheese for listeriae in at least the next 20 consecutive shipments
destined for the United States. Although a positive finding would not automatically result
in suspension of the certified status for a cheese factory under this program, FDA officials
reserved the right to initiate detentions and/or recalls if a product was found to contain
L. monocytogenes. After this certification program was accepted, only one additional recall
involving a French soft-ripened full-fat cheese has been reported (see Table 3).
Other Western European Countries
Despite the adverse publicity that the French cheese industry received throughout 1986
and 1987, it must be recognized that the problem of Listeria-contaminated cheese was
not limited to France. Between October and December of 1986, FDA inspectors isolated
Listeria spp. from 4 of 74 (5.4%) cheeses imported from Italy, two cheeses of which also
contained high levels of phosphatase [45]. After finding similar percentages of positive
samples during January, February, and March of 1987, FDA officials told representatives
of the Italian government either to submit a draft for a certification program (or recommend
an alternate solution) or face a ban on importation of potentially hazardous cheeses into
the United States. As of April 30, 1987, only 13 of all Italian cheese samples analyzed
complied with current FDA safety standards: free of Listeria, phosphatase and enteropath-
ogenic strains of E. coli. Additionally, 144 cheese samples examined as part of an import
alert were suspected of containing L. monocytogenes [44].
After isolating listeriae from Italian Pecorino Romano cheese prepared from goats
milk (see Fig. 1) in June of 1987, [280], the previous import alert was extended to include
L. monocytogenes in Fermented Dairy Products 423

both soft and hard varieties of Italian cheese [50]. (This was the first instance in which L.
monocytogenes was isolated from hard cheese.) Subsequently, the FDA ordered intensified
sampling of soft and hard cheese for the next 2 months [44].
Late in 1987, FDA officials also increased the number of cheeses sampled from
Austria, Denmark, Germany, Italy, and Switzerland as part of the agencys ongoing im-
ported cheese surveillance program (see Fig. 1) [57]. Although this action prompted the
recall of several Danish cheeses in early 1988 (see Table 3), no additional Class I recalls
were issued during the remainder of 1988 for imported cheese contaminated with L. mono-
cytogenes. Heightened concern over the presence of this pathogen in European cheeses
(which sterns from the 1987 cheeseborne outbreak of listeriosis in Switzerland) and subse-
quent initiation of corrective action are probably both responsible for the lack of Class I
recalls issued during the remainder of 1988 and early 1989. However, during the latter
half of 1989, FDA officials issued (a) four separate Class I recalls for Listeria-contami-
nated soft cheeses manufactured in Cyprus (see Table 3) and (b) an import alert for con-
taminated soft and hard cheeses produced by two Italian firm [ 1651. The overall situation
regarding presence of L. monocytogenes in imported cheese has greatly improved since
1986 [77] with only four additional recalls of imported cheese issued since 1990. However,
sporadic detection of listeriae in imported cheeses suggests that limited surveillance of
such products is still necessary to safeguard public health.

SURVEYS AND MONITORING PROGRAMS FOR LISTERIA

SPP. IN CHEESE PRODUCED OUTSIDE THE UNITED
STATES
In response to the 1985 cheeseborne listeriosis outbreak in California, scientists worldwide
began analyzing many types of cheese for Listeria spp. Given the possible ramifications
of selling Listeria-contaminated cheese and the fact that large quantities of European spe-
cialty cheeses are exported to the United States and Canada, high priority was given to
determining the incidence of listeriae in Western European cheeses.
Since 1986, over 50 surveys have dealt with the incidence of Listeria spp. in various
cheeses. However, since sampling designs (i.e., number and size of sample, site of sample
collection [cheese factory or retail store], age of sample, portion of cheese analyzed [sur-
face, interior, or both]) and methods for detecting and identifying listeriae vary widely
among these surveys, many of the Western European studies and those conducted else-
where need to be interpreted with some caution.

Canada
Reports from federal monitoring programs in both Canada and the United States [57,196]
indicate that the incidence of listeriae in Canadian cheese (and nonfermented dairy prod-
ucts as described in Chap. 11) is relatively low. In the only Canadian survey thus far
reported, Farber et al. [ 1661 examined 182 samples of soft and semisoft cheese for listeriae
using the original FDA method. The cheeses analyzed in this survey were produced at
61 different cheese factories, most of which were located in the provinces of Ontario and
Quebec. Although all cheeses examined were Listeria-free, 19 of 79 samples (24%) were
positive for phosphatase, which suggests that these cheeses may have been prepared, at
least in part, from raw milk. The only additional information concerning Canadian cheese
is the unconfirmed isolation of L. monocytogenes from Cheddar and Colby cheeses [207],
424 Ryser

both of which were manufactured from raw milk and held a minimum of 60 days at
2 1.7"C (235F) as required by the Canadian government.
Although the incidence of L. monocytogenes in Canadian-produced cheese appears
to be low, the pathogen has been detected in cheese exported to Canada from several
Western European countries, including Denmark, France, Switzerland, and Germany
[66,196]. In conjunction with the Canadian survey just discussed, Farber et al. [ 1661 also
examined 187 samples of Western European soft and semisoft cheese (98 different brands
from 12 different countries) that were regularly exported to Canada. Three soft and semi-
soft cheeses produced by the same manufacturer in France were positive for Listeria spp.
(Table 4). Two cheeses contained L. monocytogenes alone, whereas the third contained
both L. monocytogenes and L. innocua. In keeping with the previously described 1988
policy regarding foods that have been directly linked to major listeriosis outbreaks, Cana-
dian officials immediately recalled the contaminated cheese. Although some of the tainted
cheese was likely consumed before the recall, no cases of listeriosis linked to consumption
of this cheese were reported.
Despite being labeled as manufactured from pasteurized milk, the three French
cheeses from which listeriae were isolated yielded positive results with the phosphatase
test as did other cheeses imported from Denmark, Finland, and Switzerland. Such findings,
along with unpublished reports of phosphatase in pasteurized dairy products, have raised
serious questions as to the validity of the phosphatase test. Results from one study [244]
demonstrated that certain heat-labile, microbially produced alkaline phosphatases can
mimic the natural phosphatase found in milk and produce false-positive results in the
Scharer test. Hence, the ability of the phosphatase test to determine whether or not a
dairy product such as cheese was made from pasteurized milk (or from pasteurized milk
contaminated with raw milk) needs to be reexamined.

TABLE
4 Incidence of Listeria spp. in Soft/Semisoft European Cheeses Exported
to Canada Between October 1985 and March 1987

Number of positive samples (%)

Number of
samples Other
Country of origin anal yzed L. monocytogenes L. innocua Listeria spp.
Austria 2 0 0 0
Denmark 43 0 0 0
Finland 1 0 0 0
France 104 3 (2.9) 1 (1.0) 0
Germany 16 0 0 0
Greece 2 0 0 0
Italy 3 0 0 0
Netherlands 2 0 0 0
Norway 7 0 0 0
Portugal 2 0 0 0
Sweden 2 0 0 0
Switzerland 3 0 0 0
Total 187 3 (1.6) 1 (0.5) 0
Source: Adapted from Ref. 166.
L. monocytogenes in Fermented Dairy Products 425

France
Beginning in early 1986, sporadic Listeria-contamination problems have been associated
with French soft cheeses exported to the United States and Canada as well as England
[ 1961, Germany [74], the Netherlands [ 1 141, Norway [ 196,3081, Sweden [ 1961, and Aus-
tralia [ 1911. Hence, in an effort to bolster public confidence in the safety of cheeses pro-
duced in France, the French government, in cooperation with the Veterinary Service for
Food Hygience in France, conducted a series of systematic surveys to determine the inci-
dence of Listeria spp. in French cheeses destined for domestic and foreign markets (Table
5) [ 1 19,181 1. Overall, I .34% of predominantly soft, 30-day-old French cheeses examined
during 1986 and 1987 contained detectable levels of both L. monocytogenes and other
Listeria spp., with few differences observed between cheeses destined for domestic or
foreign consumption. It also is noteworthy that comparable levels of contamination were
seen in soft cheeses prepared from raw and pasteurized milk. These findings agree with
those of most other surveys which suggest that soft cheeses are most likely to become
contaminated with L. monocytogenes during the latter stages of manufacture and ripening.
Although L. monocytogenes also was recovered from 10.3% of soft/semisoft French
cheeses marketed in Sweden from 1989 to 1993 [210], most surveys have suggested con-
tamination rates of < 10% with I03 of 2275 (4.5%) French cheeses surveyed (Table 6)
reportedly harboring L. monocytogenes. However, contamination rates of 46.9 and 87.0%
have been reported for soft surface-ripened cheeses prepared from raw milk. In the latter
survey [279], L. monocytogenes populations of 106 CFU/g were detected on the cheese
surface, with serotype 1/2 predominating.
In another French survey [ 1SO], workers at the Veterinary Service for Food Hygiene
recovered I,. monocytogenes as well as L. innocua and other Listeria spp. from 0.3 to
3.5% of cottage, soft-ripened, and semihard cheeses examined (Table 6). However, unlike
the aforementioned survey, comparable contamination rates were observed for soft-rip-
ened cheese prepared from raw and pasteurized milk.
Additional efforts in France have focused on characterizing listeriae isolates from
cheese and other milk products. Listeria spp. recovered from French dairy products during
1986 included L. monocytogenes (370 strains), L. innocua (134 strains), L. seeligeri (17

TABLE
5 Incidence of Listeria spp. in French Cheese Destined for Domestic
(France) and Foreign Markets during 1986 and/or 1987
Number of positive samples (%)
Number of ~

samples Other
Market Type of cheese analyzed L. monocytogenes Listeria spp.
DomesticJ Soft 192 2 (1.0) 3 (1.6)
Other I35 0 I (0.7)
Foreignb Soft (pasteurized milk) 736 1 1 (1.5) 1 0 (1.4)
Soft (raw milk) 355 6 (1.7) 5 (1.4)
Total 1418 19 (1.34) 19 (1.34)

aResults from January to November 1987.

Results from January 1986 to September 1987.
Source: Adapted from Ref. 119.
426 Ryser

TABLE
6 Incidence of Listeria spp. in Cheeses Manufactured Outside the United States
Number of Other
samples L. monocy- Listeria
Country Type of cheese analyzed togenes L. innocua SPP. Ref.
Europe
Belgium Soft 886 62 (6.9) ND ND 210
Unspecified 929 214 (23) ND ND 151
Unspecified 262 35 (13.4) ND 68 (25.9) 300
Unspecified 37 0 0 0 119
Czechoslovakia Soft ripened 77 6 (7.8) ND ND 229
Sheep's milk 10 0 ND ND 229
Hard 33 0 ND ND 229
Unspecified 24 2 (8.3) ND ND 229
Denmark Soft/semisoft 46 0 ND ND 210
Unspecified 25 8 (32.0) ND 8 (32.0) 119
France Soft ripened (raw milk) 330 3 (0.9) 6 (1.8) 1 (0.3) 180
Soft, surface-ripened (raw milk 23 20 (87.0) ND ND 228
Soft (raw milk) 32 15 (46.9) 13 (40.6) 1 (3.1)" 164
Soft (heat-treated milk) 5 0 2 (40.0) 0 164
Soft ripened (pasteurized milk) 873 12 (1.4) 5 (0.6) 3 (0.3) 180
Soft (pasteurized milk) 32 3 (9.4) 5 (15.6) 0 164
Soft semisoft 174 18 (10.3) ND ND 210
Semihard 289 10 (3.5) 1 (0.3) 1 (0.3) 180
Blue 126 0 0 0 180
Cottage 149 2 (1.3) 0 0 180
Unspecified 242 20 (8.3) ND 61 (25.2) 257
Germany Soft 712 33 (4.6) 58 (S.1) 4 (0.6)" 287
Soft 248 3 (1.2) 16 (6.4) 0 272
Soft 166 7 (4.2) ND ND 290
Soft (raw milk) 22 2 (9.1) 2 (9.1) 0 164
Soft (unripened) 8 0 0 0 302
Soft (mold-ripened) 117 4 (3.4) 5 (4.3) 0 302
L. monocytogenes in Fermented Dairy Products 427

Soft (smear-ripened) 41 3 (7.3) 5 (12.2) 0 302

Soft/semisoft 256 23 (9.0) ND 7 (2.7) 119
Soft/semisoft 31 1 (3.2) ND ND 210
Semisoft 268 9 (3.4) 7 (2.6) 8 (?.O)a 287
Semisoft 45 0 1 (2.2) 0 272
Semisoft (unripened) 89 4 (4.5) 1 (1.1) 3 (3.4)a 302
Semisoft (mold-ripened) 12 0 0 0 302
Semisoft (smear-ripened) 7 2 (28.6) 0 0 302
Semihard 237 7 (3.0) 14 (5.9) 0 287
Semihard 108 6 (5.6) ND ND 290
Hard 42 2 (4.8)b 0 1 (2.4)a 289
Acid 61 2 (3.3) 22 (36.1) 1 (1.6)a 289
Acid 48 1 (2.1) ND ND 290
Fresh 149 0 0 0 289
Processed 21 0 0 0 289
Unspecified 89 8 (9.0) 17 (19.0) 0 273
Soft 25 0 ND ND 253
Soft 15 0 ND 4 (26.7)' 194
Semisoft 25 0 ND ND 253
Moulded 10 2 (20.0) ND ND 253
Mold-ripened 10 0 ND ND 253
Hard 1s 1 (6.7) ND 4 (26.7)' 194
Hard 10 0 ND ND 253
Curd 15 0 0 0 194
Processed 20 0 ND ND 253
Processed 15 0 0 0 194
Ireland Soft 40 1 (2.5) ND ND 196
Soft/semisoft 17 0 ND ND 141
Italy Soft 1284 65 (5.1) 140 (10.9) 218 (17.0)d 137
Soft 400 8 (2.0) ND ND 134
Soft 54 2 (3.7) 4 (7.4) 0 146
Soft 29 0 ND ND 174
Soft 21 2 (1.6) 2 (1.6) 0 222
428 Ryser

TABLE
6 Continued
Number of Other
samples L. monocy- Listeria
Country Type of cheese analyzed togenes L. innocua SPP- Ref.

Soft 30 0 1 (3.3) 0 293

Soft 12 1 (8.3) ND ND 243
Soft 8 0 1 (12.5) 0 256
Soft unripened 69 6 (8.7) ND ND 250
Soft unripened 18 0 ND ND 136
Soft ripened 136 9 (6.6) ND ND 250
Soft surface-ripened (mold) 16 0 ND ND 146
Soft surface-ripened 90 1 (1.1) 10 (11.1) 1 (1.1)C 293
Fresh 239 0 0 0 137
Fresh 38 2 (5.3) 2 (5.3) 0 146
Fresh 17 0 ND ND 174
Soft/semisoft 64 0 ND ND 136
Soft/semisoft 36 1 (2.8) ND ND 210
Semisoft 118 0 5 (4.2) 0 137
Ripened 50 1 (2.0) 0 0 256
Hard 99 0 0 0 137
Hard 40 0 0 0 146
Hard 10 0 ND ND 136
Goats milk 24 0 ND ND 124
Goats milk 21 1 (4.8) ND ND 136
Sheeps milk 40 0 ND ND 124
Unspecified 1846 24 (1.3) ND ND 109
Unspecified 373 22 (5.9) 50 (13.4) 70 (18.8)b 138
Unspecified 115 6 (5.2) 3 (2.6) 0 246
Unspecified 75 0 ND ND 236
Unspecified 62 3 (4.8) ND ND 127
Unspecified 52 4 (7.7) 0 0 119
Asiago 12 0 ND ND 124
L. monocytogenes in Fermented Dairy Products 429

Crescenza 212 0 ND ND 124

Gorgonzola 67 6 (9.0) 28 (41.8) 1 (l.l)a 246
Gorgonzola 58 3 (5.2) 21 (36.2) 0 242
Gorgonzola 44 4 (9.1) ND ND 243
Gorgonzola 40 2 (5.0) 4 (10.0) 0 146
Gorgonzola 25 2 (8.0) ND ND 174
Mozzarella 94 15 (16.0) 10 (10.6) 1 (l.l)a 246
Mozzarella 74 0 2 (2.7) 2 (2.7)e 146
Mozzarella 50 0 0 0 103
Mozzarella 30 0 0 0 164
Mozzarella 29 4 (13.8) 2 (6.9) 1 (3.4)' 242
Mozzarella 24 0 ND ND 124
Mozzarella 20 2 (10.0) ND ND 243
Mozzarella 14 0 ND ND 174
Pecorino 8 0 ND ND 124
Talleggio 45 0 7 (15.6) 2 (4.4) 242
Talleggio 38 0 ND ND 243
Taleggio 12 0 ND ND 124
Netherlands Soft (raw milk) 938 43 (4.6) ND ND 294
Soft (pasteurized milk) 484 10 (2.0) ND ND 294
Norway Soft (domestic) 850 0 ND NDg 254
Soft (imported) 90 10 (11.0) ND ND 254
Spain Fresh 23 ND ND 1 (4.3)h 220
Soft 14 ND ND 1 (4.3)g 220
Semihardlhard 20 0 0 0 220
Blue (raw milk) 11 0 ND ND 21 1
Unspecified -100 0 ND ND 196
Unspecified (raw milk) 49 1 (2.0) 0 1 (2.0)a 247
Unspecified (raw milk) 42 10 (23.8) ND ND 130
Unspecified (pasteurized milk) 21 0 ND ND 130
Queso fresco / cottage 91 7 (7.7) 4 (4.4) 2 (2.2)a 120
430 Ryser

TABLE
6 Continued
Number of Other
samples L. monocy- Listeria
Country Type of cheese analyzed togenes L. innocua SPP. Ref.

Sweden Soft/semisoft 27 0 ND ND 210

Switzerland soft 604 40 (6.6) 38 (6.3) 0 123
Soft (mold-ripened) 54 0 0 ND 41
Soft (smear-ripened) 18 4 (22.2) 3 (16.7) ND 41
Semisoft 205 4 (1.9) 0 0 123
Semisoft (mold-ripened) 26 1 7 (2.7) ND ND 123
Semisoft (smear-ripened) 343 33 (9.6) ND ND 123
Semisoft (smear-ripened) 69 6 (8.7) 13 (18.8) ND 41
Hard 88 0 0 0 123
Unspecified 17 0 ND 1 (5.9) 119
Turkey Kashor 30 0 ND ND 186
White 30 0 ND ND 186
Unspecified 224 4 (1.8) 7 (3.1) 0 190
Unspecified (raw milk) 40 2 (6.1) ND ND 186
Yugoslavia White-brined 170 9 (5.3) ND ND 151
United Kingdom
England Soft 25 1 1 (0.4) 9 (3.6) 0 213
Soft/semisoft 12 0 ND ND 210
England/ W ales Soft 1437 16 (1.1) ND ND 177
Soft 25 1 10 (4.0) ND ND 213
Soft 222 23 (10.4) ND ND 24 1
Soft 131 0 ND ND 118
Soft ripened 769 63 (8.2) ND ND 188
Soft unripened 366 4 (1.1) ND ND 188
Hard 66 1 (1.5) ND ND 188
Ewes milk 141 1 (0.7) ND ND 188
Goats milk 476 22 (4.6) ND ND 188
L. monocytogenes in Fermented Dairy Products 431

Northern Ireland Soft 33 0 0 1 (3.3)a 191

Scotland Soft 27 3 (11.1) ND ND 258
Unspecified 305 3 (1.O)C 0 1 (3.3)a 110
Elsewhere
Australia Soft 437 15 (3.4) ND 24 (5.5)' 102
Soft 28 1 (0.2) 0 0 102
Unspecified 338 6 (1.8) ND ND 298
Unspecified 126 1 (0.8) 1 (0.8) 0 187
Brazil Minas frescal 20 0 0 0 129
Costa Rica Soft 20 9 (45.0) ND ND 23 1
Egypt Damietta 50 1 (2.0) 1 (2.0) 0 167
Kareish 100 1 (1.0) 3 (3.0) 1 (l.O)h 167
Japan Domestic fresh 92 0 ND ND 232
Domestic soft/semisoft 94 0 ND ND 232
Domestic semi-hard/hard 105 0 ND ND 232
Imported fresh 94 0 ND ND 232
Imported soft/semisoft 418 16 (3.8) ND ND 232
Imported semi-hard/hard 403 0 ND ND 232
Imported other 55 0 ND ND 232
Jordan White brined 67 0 ND ND 161
Morocco Domestic fresh 20 0 ND ND 159
Imported mold-ripened 45 0 ND ND 159
United Arab Emirates Domestic 53 0 0 0 184
Imported 196 2 (1.0) 2 (1.0) 0 184

ND, not determined.

a L. seeligeri.

Isolated from sheep's milk cheese.

non-L. monocytogenes.
185 L. welshimeri, 18 L. ivanovii, 15 L. murrayi.
L. grayi.
63 L. welshimeri, 6 L. ivanovii, 1 L. murrayi.
g Lisreria sp.

L. welshimeri.
432 Ryser

strains), and L. ivanovii (1 strain), with 299 of 370 (80%) and 48 of 370 (13%) L. monocy-
togenes strains belonging to serovars I /2 and 4b, respectively. Additional surveys con-
ducted in France [ 1 19,1811 and Belgium [ 1031 from 1985 to 1990 indicated that a dispro-
portionately large number of L. monocytogenes strains isolated from cheese and other
dairy products were serovar 1/2. This situation appears to be reversed in the United States,
with isolates of serovar 4b typically outnumbering those of serovar 1/2.
Phage typing has become a useful means of characterizing particular L. monocyto-
genes strains isolated from dairy products and of trackmg the probable source of foodborne
listeriosis outbreaks. Although only 33 3 % of all L. monocytogenes strains isolated from
French dairy products during 1986 and 1987 were typeable using the available set of
phages, some phage types were unique to particular regions within France [ 1191. In some
instances, excellent correlations were observed between specific phage types and certain
cheese varieties, with some phage types even being specific to a particular dairy. Such
findings have lead to better control of the listeriosis problem within the dairy industry.
The inadvertent isolation of L. monocytogenes from French soft-ripened cheese by
FDA officials in January of 1986 prompted several additional surveys of French cheese
exported to other Western European countries. Working in The Netherlands, Beckers et
al. [ 114,1151 examined 69 samples of French soft cheese (i.e., Brie and Camembert) for
L. monocytogenes using both direct plating and cold enrichment. The pathogen was recov-
ered from 7 of 69 (10.1%) cheeses at levels ranging between 103and 106 CFU/g. Cold
enrichment uncovered three additional cheeses with L. monocytogenes for a total of 10
positive samples. Although all 10 Listeria-positive cheeses were prepared from raw milk,
comparable rates of contamination have frequently been reported for cheese manufactured
from raw and pasteurized milk [ 119,1801.

Germany
As mentioned in Chapter 10, Germany experienced a major outbreak of listeriosis shortly
after World War 11. This outbreak, which may have resulted from consuming contaminated
raw milk, led to an increased interest in listeriosis research, and this in turn prompted
Prof. H. P. R. Seeliger to publish his time-honored monograph Listeriosis in 1961. Dur-
ing the last 40 years, the late Prof. Seeliger emerged as one of the worlds leading au-
thorities on listeriosis. In addition, he operated a listeriosis research center at the In-
stitut fur Hygiene und Mikrobiologie der Universitat Wurzburg to which Listeria isolates
could be sent for biochemical and serological confirmation. Hence, it is not surprising
to learn that the incidence of listeriae in cheese has received considerable attention in
Germany.
Although spared from the heavy economic losses experienced by the United States
and France, Germany and most other European countries have not escaped the Listeria
problem completely unscathed. Despite rigorous testing, Listeria-laden German blue-
veined cheese was recalled from France [196], with a similar recall being issued for sour
milk cheese exported to Canada [66,196] and The Netherlands [196]. Consequently, a
series of Listeria-monitoring programs were introduced for German soft, semisoft, semi-
hard, and hard cheeses as well as cultures, cheese byproducts, and the general environment
within cheese factories. Since 1990, German officials have been enforcing a policy similar
to that adopted in Canada in which only contaminated foods previously associated with
foodborne listeriosis outbreaks are recalled from the marketplace.
Results from various surveys made since 1986 (see Table 6) indicate that 0-9.1 %
L. monocytogenes in Fermented Dairy Products 433

(average of 3.9%) and 0-28.6% (average of 3.6%) of the soft and semisoft cheeses mar-
keted in Germany contained L. monocytogenes, respectively, with the highest incidence
of listeriae generally occurring in smear-ripened varieties. With few exceptions, L. innocua
was isolated more frequently from soft and semisoft cheese than was L. monocytogenes.
Although somewhat similar average percentages were reported for the incidence of L.
monocytogenes in semihard (3.8%) and hard cheese (4.8%), the two hard cheeses that
contained L. monocytogenes were reportedly manufactured from ewes rather than cows
milk. Overall, it appears that the Listeria contamination rate for hard cheeses prepared
from cows milk may still be relatively low, as also was observed in Switzerland (see
Table 6). Hence, these results from Germany generally agree with those from other surveys
in that L. monocytogenes was found more frequently in high-rather than low-moisture
cheese.
Of the three remaining categories of German cheese shown in Table 6, only acid
curd cheese was positive for listeriae. The apparent absence of Listeria spp. from samples
of fresh (i.e., cottage) and processed cheese is not entirely unexpected, since procedures
used to manufacture these cheeses include relatively severe heat treatments. Even if a few
listeriae survived the manufacturing process, most, if not all, of the survivors would have
been sublethally injured during exposure to heat and/or acid and would therefore be unable
to grow in most selective enrichment broths that are commonly used for examining cheese.
In the only other study thus far reported, Weber et al. [302] examined various Ger-
man cheeses, including 11 types manufactured from ewes and goats milk, for listeriae
(Table 7). Although all cheeses prepared from ewes or goats milk were free of L. monocy-
togenes, L. innocua was detected in one sample of fresh goats milk cheese.
Despite the ability of lactating sheep and goats to shed L. monocytogenes in their
milk, as further evidenced by isolation of L. monocytogene:?from approximately 4 of 480
(0.8%) samples of raw goats milk in England [188], relatively few additional studies
have dealt with the incidence of listeriae in ewes and goats milk cheese. Nevertheless,
in addition to the aforementioned survey of German hard cheese produced from ewes
milk [287l, Tham [291] also reported isolating L. monocytogenes from one sample of 8-
week-old goat cheese marketed in Sweden.

TABLE
7 Incidence of Listeria spp. in Domestic and Imported Cheese
Analyzed in Germany Between October 1987 and June 1988
~

Number of Number of positive samples (%)

samples
Type of cheese/milka analyzed L. monocytogenes L. innocua
Fresh / Cow 21 2 (9.5) 0
Fresh/Goat 1 0 1 (100.0)
Soft/Cow 307 8 (2.6) 11 (3.6)
Soft/Goat 1 0 0
Soft/Ewe 3 0 0
Semisoft/Cow 144 19 (13.2) 8 (5.6)
Semisoft/Ewe 6 0 0
Semihard/Cow 22 0 11 (50.0)
Hard/Cow 4 0 0

aMilk from which cheese was manufactured.

Source: Adapted from Ref. 302.
434 Ryser

The incidence of L. monocytogenes in the remaining cheeses prepared from cows

milk was similar to those values obtained in other studies (see Table 6), with the pathogen
being detected in 2.6 and 13.2% of the soft and semisoft (presumably mold- and smear-
ripened varieties) cheeses examined, respectively. The unusually high incidence of L. mun-
ocytogenes in fresh curd cheese (9.5%) is probably the result of contamination during
later stages of manufacture and/or packaging as well as the small number of samples
examined. As was true for surveys of French soft-ripened cheese discussed earlier in this
chapter, most L. monocytogenes isolates were serovar 1/2 (22 strains) as also reported by
Schonberg et al. [273], with the remaining seven isolates being classified as 4ab or 4b.
However, since most clinical L. monocytogenes isolates from Germany are serovar 4b, it
appears that some questions remain concerning the ability of present isolation methods
to recover L. monocytogenes serovar 4b as compared with other serovars, from various
foods, including cheese.

Italy
Public health concerns raised in the United States following the isolation of L. monocyto-
genes from imported cheese prompted over 15 surveys of cheeses manufactured in Italy
(see Table 6). Overall, L. monocytogenes was recovered from 196 of 6382 (3.1%) Italian
cheeses surveyed, with this pathogen being most prevalent in Gorgonzola (7.3%), followed
by mozzarella (6.3%) and various soft cheeses (4.2%). In another study, Cantoni et al.
[128] reportedly isolated L. monocytogenes from 14 of 375 (3.7%), 14 of 216 (6.5%), and
5 of 95 (5.3%) samples of Gorgonzola (blue-veined), Tallegio (soft, surface-ripened), and
other Italian cheeses, respectively. Although a follow-up study demonstrated L. monucytu-
genes at levels of <100 to 12,000 CFU/g in Gorgonzola and Taleggio cheese, respectively,
the pathogen was never recovered from 1150 samples of soft, semisoft, semihard, or hard
cheese or from 72 samples of pasta filata-type cheese such as provolone and mozzarella.
When present, however, L. monocytogenes serovar 1 typically predominated [242] as has
been reported for other European cheeses.
Between January 1987 and September 1988, Massa et al. [222] also examined 54
soft rindless (i.e., Mascarpone, mozzarella, Crescenza) and 67 soft thin-rind (i.e., Italico,
Caciotta) cheeses produced by both large and small northern Italian factories for listeriae
and E. coli. Listeria rnonocytogenes was detected in only 2 of 47 (4.2%) thin-rind cheeses
manufactured by one small factory, with core samples from these two positive cheeses
being negative for the pathogen. Although all other thin-rind and rindless soft
cheeses were free of L. monocytogenes, two mozzarella cheeses contained detectable lev-
els of L. innucua. According to these investigators, E. coli populations in these cheeses
ranged from <10 to 8 X 105 CFU/g, with the two L. rnonocytogenes-positive cheeses
containing 2 104E. coli CFU/g. However, since 14 similar Listeria-free cheeses also con-
tained >103 E. coli CFU/g, E. coli is clearly a poor indicator organism for possible pres-
ence of listeriae.

Switzerland
Although several major Western European countries have experienced various degrees of
economic loss from Listeria-contaminated cheese, thus far only Switzerland, France, and
the United States have been forced to deal with major outbreaks of cheeseborne listeriosis.
Well before the 1987 listeriosis outbreak in Switzerland linked to consumption of Vacherin
Mont dOr soft-ripened cheese, Swiss officials began examining various cheeses for lister-
L. monocytogenes in Fermented Dairy Products 435

iae. Although these surveys apparently were prompted by the 1985 listeriosis outbreak in
California, an unusually high incidence of listeriosis in certain areas of Switzerland which
could not yet be explained may have provided added incentive to initiate these surveys.
A two-stage Listeria-monitoring program was later established for cheese and other dairy
products with random testing of 10-g samples obtained at both the factory and retail level
[ 1961. According to the Federal Bureau of Health, such samples must be completely free
of L. monocytogenes before the product is deemed acceptable.
Working in Switzerland, Breer [ 1231 examined 799 domestic and imported cheese
samples for listeriae during the winter of 1985/1986. Various Listeria spp. were detected
in 19.2% of the soft surface-ripened cheeses, all of which were traced to 10 Swiss and a
few foreign manufacturers. During follow-up investigations of these 10 cheese factories
in Switzerland, Listeria spp. were isolated from surfaces of various cheeses and also from
curing and smearing brines, waste-water sinks, and surfaces of wooden boards used in
cheese ripening. In addition, identical serovars of L. monocytogenes (1 /2b and/or 4b) and/
or L. innocua were isolated repeatedly from the same cheese factories. These findings
demonstrate that ample opportunity existed for cheese to become contaminated with lister-
iae during the later stages of manufacture and ripening.
Subsequently, Breer [ 1231 reported that 4.9 and 4.7% of all cheeses sold in Switzer-
land contained L. monocytogenes or L. innocua, respectively (see Table 6). Of equal im-
portance is the fact that both Listeria spp. were isolated more frequently from soft (6.6,
6.3%) than semisoft cheese (1.9,0%) and that neither organism was detected in 88 samples
of hard cheese.
During 1986, Breer [122] also found that 12.9 and 10.0% of soft surface-ripened
cheeses manufactured in Switzerland were contaminated with L. monocytogenes and L.
innocua, respectively (Table 8). The incidence of both Listeria spp. was generally twice
as high in smear- rather than mold-ripened cheese. As in previous studies, the rate of
Listeria contamination was typically independent of the type of milk (raw or pasteurized)
from which smear-ripened cheeses were manufactured.
These Swiss studies, along with several of the aforementioned German surveys,
indicate a greater likelihood of isolating L. monocytogenes and L. innocua from high rather
than low-moisture cheese, with special emphasis on mold- and smear-ripened varieties.

TABLE8 Incidence of Listeria spp. in Soft Mold- and Smear-Ripened

Cheeses Manufactured in Switzerland During 1986 from Pasteurized
and Raw Milk
Number of Number of positive samples (%)
samples
Type of cheese analyzed L. monocytogenes L. innocua
Mold-ripened
Raw milk 22 2 (9.1) 0
Pasteurized milk 9 0 1 (11.1)
Smear-ripened
Raw milk 17 3 (17.6) 4 (23.5)
Pasteurized milk 22 4 (18.2) 2 (9.1)
Total 70 9 (12.9) 7 (10.0)

Source: Adapted from Ref. 122.

436 Ryser

In support of this observation, Bannerman and Bille [ 11I] found that 110 of 449 (24.5%)
rinds from soft cheese produced in Switzerland were contaminated with Listeria spp.,
including a high percentage of samples with L. monocytogenes. During an additional sur-
vey made between October 1986 and September 1987 [41], L. monocytogenes was de-
tected in 4 of 18 (22.2%) and 6 of 67 (8.7%) smear-ripened soft (i.e., Limburger, Romadur,
Muenster, Reblochon) and semisoft (i.e., St. Paulin, Tilsiter, Mutschli, Raclette) cheeses,
respectively, with many cheeses also containing L. innocua. From the apparent widespread
distribution of Listeria within some cheese factory environments, it follows that cheeses
prepared from raw and pasteurized milk are equally likely to contain listeriae. Additional
information concerning the incidence and control of listeriae in dairy factories and other
food processing facilities is given in Chapter 17.

Other European Countries

As already implied, recent Listeria problems that have affected the European cheese indus-
try are not limited to France, Germany, Italy and Switzerland. Other European nations,
including Austria, Belgium, Denmark, England, Finland, Greece, Hungary, The Nether-
lands, Spain, and Sweden, also expressed concern in a March 1989 poll conducted by the
International Dairy Federation [ 1961. According to the survey, L. monocytogenes was
isolated from domestic cheese sold in Austria (soft cheese), Belgium (rind-type cheese),
Denmark (various soft cheeses), England (various cheeses), Ireland (soft farm-house
cheese), The Netherlands (young farm-house Gouda), and Sweden (goats milk cheese),
with this pathogen later being identified in cheeses from Czechoslovakia (soft-ripened)
[229], Greece (Feta) [ 1511, Ireland (soft) [196], Turkey (soft) 1186,1901 and Yugoslavia
(white brined) [ 15 11 (see Table 6). Denmark, England, and Sweden have also experienced
problems with soft or semisoft cheeses imported from Denmark, France, Germany and/
or Italy (Table 9). In contrast, Norway [ 1 10,3051 and Spain [ 1961 have been primarily
affected by imported soft and/or blue-veined cheeses. Thus, in addition to France, Italy,
Switzerland, and Germany, Austria, Belgium, Denmark [67], England, Finland, Greece,
Hungary, The Netherlands, and Sweden also have developed programs to actively monitor
the incidence of listeriae in domestic/imported cheese (especially soft surface-ripened
varieties) and/or manufacturing environments within cheese factories.
As of March 1989 [196], Austria, Denmark, France, Germany, Greece, Hungary,
Italy, Sweden and Switzerland had regulations regarding the sale of cheese and other foods
contaminated with L. monocytogenes, with most European countries now attempting to
prevent distribution and sale of cheese (and in some instances other ready-to-eat foods)
containing 2100 L. monocytogenes CFU/g. Since these observations along with the 1987
listeriosis outbreak in Switzerland involving Vacherin Mont dOr cheese both support the
widespread notion that L. monocytogenes poses a significant health threat to certain seg-
ments of the population, the European Economic Community Consumers Association pub-
lished a list of soft and semisoft cheeses manufactured in France and Switzerland that
should be avoided by susceptible individuals-pregnant women, immunocompromised
adults, and the elderly. This highly controversial list of cheeses (and brands when applica-
ble) included Brie (La Renommee), Muenster (Ermitage), Crkme de Bleu (Diapason), Lys
Bleu, Camembert (I Signy), Tilsit, Fourme de Bresse, Bleu de Bresse, Reblochon, Pont
LEveque, Gruykre, and Vacherin Mont dOr.
In February of 1988, a case of cheeseborne listeriosis was reported in England in
which a 40-year-old woman contracted meningitis shortly after consuming Anari-type soft
L. monocytogenes in Fermented Dairy Products 437

TABLE
9 Incidence of L. monocytogenes in Cheeses Marketed in Sweden from 1989 t o 1993
Type of cheese Type of cheese milk

Country of origin White mold Green/Blue mold Smear-ripened Other Heat-treated Raw
Austria __ 0/1" o/ 1 -

Denmark 0/19 0/27 0/46 -

England 0/12 - - 0/12 -

France 15/119 (12.6) 0123 3/18 (16.7) 0/14 5/144 (3.5) 13/30 (43.3)
Germany 0/8 1/19 (5.3) o/ 1 0/3 1/31 (3.2) -
Greece - __ - o/ 1 o/ 1 -
Italy 0/5 0/25 1/2 (50.0) 0/4 1/36 (2.8) -
Netherlands - 0/2 - 0/2 -

Norway o/ 1 - - o/ 1 -
Romania - - o/ 1 o/ 1 -
Spain - o/ 1 - - o/ 1 -
Sweden 0/3 0/13 0/3 0/8 0126 o/ 1
Total 15/154 (9.7) 1/ 122 (0.8) 4/26 (15.4) 0/3 1 7/302 13/31 (41.9)
~~~ ~~

aNumber of positive sampleshumber of samples analyzed (%).

Source: Adapted from Ref. 210.
438 Ryser

goats milk cheese that contained L. monocytogenes at levels >107CFU/g (see Chap. 10).
During follow-up investigations at the factory [224], the same L. monocytogenes strain
also was isolated from 8 of 11 and 4 of 8 factory and/or retail samples of Halloumi and
Cheddar cheese, respectively, as well as single samples of Gjestost and soft chive cheese.
In addition, L. innocua also was recovered from several samples of Halloumi and Cheddar
cheese. As in the previously described studies by Pini and Gilbert [241] and Massa et al.
[222], no clear relationship was observed between the presence of L. monocytogenes/L.
innocua and coliformslE. coli. According to several additional surveys, some Costa Rican
[2311 and Turkish cheeses [ 1481 contained 104L. monocytogenes and 2 102coliform CFUI
g. Hence coliforms appear to be relatively poor indicators of Listeria contamination.
The Public Health Laboratory Service in London coordinated a large-scale survey in
which various dairy products marketed in England and Wales were sent to 46 laboratories
throughout the country for Listeria testing. Results from this comprehensive survey (see
Table 6) indicated that 8.2, 1.1, 1.5 and 4.1% of the soft-ripened, soft-unripened, hard,
and goats milk cheese manufactured in England and Wales contained L. monocytogenes;
75, 42 and 7 isolates classified as serovar 1/2, 4b and 4, respectively. Among the soft
ripened varieties, 13 cheeses harbored > 103 L. monocytogenes CFU/g with 3 samples
exceeding 105 CFU/g. Of these 13 cheeses, 7 were prepared from raw milk, with only
one of the cheeses being manufactured in the United Kingdom. In contrast, only 2 of 33
cheeses prepared from ewes or goats milk contained >500 L. monocytogenes CFU/g.
Overall, the incidence of this pathogen was similar in imported (7.4%) and UK-produced
cheese. These incidence rates and serovar distribution patterns for L. monocytogenes in
soft-ripened and unripened cheese are generally similar to those observed in most other
European studies [209,2101.
As just suggested, numerous surveys for incidence of listeriae in cheese also have
been completed in many of these aforementioned countries which, with the exception of
Denmark and France, have not experienced major economic problems associated with
Listeria-contaminated cheese. Following the 1986 report of an English woman who con-
tracted listeriosis after consuming French soft cheese [ 1121, two English researchers [241]
examined 45 domestic soft cheeses as well as 177 soft cheeses imported from France,
Italy, Cyprus, Germany, Denmark, and Lebanon for Listeria spp. and E. coli. (Table 10).
Overall, L. monocytogenes was isolated from 2 of 45 (4.4%) English cheeses and 21 of
177 (11.9%) soft cheeses imported from France, Italy, and Cyprus. Populations of L.
monocytogenes in contaminated cheese ranged from <102 to 105 CFU/g, with 9 of 12
French cheeses containing 2 104CFU/g. Despite differences in media and methods used
in various surveys, the contamination rate of 14.1% for soft French cheeses calculated in
this study was close to the 14.5% previously observed for French soft cheese exported to
The Netherlands. As was true for previous surveys of French dairy products, all strains
of L. monocytogenes (except one nontypable strain) were of serovar 1/2 or 4b, with the
former predominating. Listeria innocua, the only other Listeria sp. detected during this
survey, was isolated from 9 of 85 (10.6%), 7 of 44 (15.9%), 2 of 45 (4.4%), and 1 of 6
(16.7%) soft cheeses produced in France, Italy, England, and Denmark, respectively, with
6 of 222 (2.7%) cheeses containing both Listeria spp. Although E. coli populations ex-
ceeded 10 CFU/g in 73 of 222 (32.9%) cheeses examined, no correlation was again ob-
served between the presence of L. monocytogenes or L. innocua and contamination with
E. coli. In fact, E. coli was detected at >10 CFU/g in only 10 of 23 (43.5%) cheeses that
contained the pathogen. In this study, 10 of 23 (43.5%) cheeses contaminated with L.
monocytogenes were prepared from pasteurized milk, whereas 2 and 11 of the remaining
positive cheeses were manufactured from raw milk and milk of undetermined processing,
L. monocytogenes in Fermented Dairy Products 439

TABLE
10 incidence of L. rnonocytogenes and E. coli in Soft Cheese Sampled in
England During 1987

L. monocytogenes
Number of Number of Number of samples
samples positive samples with >I0 E. coli
Country of origin analyzed Level/g CFU/g (%)
England 85 12 (14.1) <102-105 32 (37.6)
France 45 2 (4.4) <:102 14 (31.1)
Italy 44 7 (15.9) < 102-1 0 4 12 (27.3)
Cyprus 20 2 (10.0) <:102 3 (15.0)
West Germany 17 0 ND 9 (52.9)
Denmark 6 0 ND 2 (33.3)
Lebanon 5 0 ND 1 (20.0)
Total 222 23 (10.4) ND- 105 73 (32.9)
ND, not detected.
Source: Adapted from Ref. 241.

respectively. Thus, as in previous studies, the type of milk (i.e., raw or pasteurized) from
which cheese is made appears to be a poor indicator of possible Listeria contamination.
Problems regarding the occasional presence of listeriae in soft cheese also have
surfaced in the Scandinavian countries, with L. monocytogenes being recovered from 0.3%
of Norwegian cheeses [ 1511 and also identified in Danish Esrom and Blue Costello cheese
that was exported to Norway [196,308], Sweden [196], and the United States. Although
four Class I recalls were issued for Danish Esrom and Blue cheese in the United States
(see Table 3), both of these cheeses (-20% of which were contaminated) were on sale
for up to 2 months in Norway before being removed from the market, apparently without
incident [3O5]. Danish officials also took steps to prevent unsold cheese from reaching
consumers and have since developed a Listeria surveillance program [67] similar to that
instituted in the United States with routine testing of various cheeses as well as cheesemak-
ing facilities.
Additional concern over the microbiological safety of various cheeses also led to
isolation of L. monocytogenes serovar 1/2b from two presumably French soft-ripened
cheeses (one prepared from raw milk and one from pasteurized milk) that were exported
to Norway and Sweden [292]. Surface and interior samples from the raw milk cheese
contained 7.5 X 105and 1.0 X 102L. monocytogenes CFU/g, respectively, whereas corre-
sponding samples from the pasteurized milk cheese contained 4.0 X 106 and 1.O X 106
L. monocytogenes CFU/g. The reasons for nonuniform distribution of listeriae in soft-
ripened cheese will be explored in the second half of this chapter. Although cheese pre-
pared from raw milk contained 3 X 106to 7 X 106colifomr CFU/g, coliform tests indi-
cated that the remaining cheese manufactured from pasteurized milk was fit for consump-
tion. These findings reinforce the fact that coliform-free cheese may not necessarily be
free of L. monocytogenes.

Other Countries
Reports of Listeria-contaminated cheese in countries beyond North America and Europe
also are beginning to surface (see Table 6). In 1987, L. monocytogenes was recovered
440 Ryser

from ricotta cheese manufactured in Melbourne, Australia [298]. This event, along with
identification of L. monocytogenes in the same imported brands of Danish blue and French
brie cheese that were recalled in the United States [ 1911 prompted Venables [298] to
determine the incidence of listeriae in Camembert, blue vein, ricotta, cottage, pasta filata,
high-moisture, low-acid, and other cheese varieties manufactured in and around Mel-
bourne. Overall, L. monocytogenes was recovered from 6 of 338 (13%)cheeses produced
by five different manufacturers, with the pathogen identified as being present in pasta
filata (three samples), ricotta (two samples), and shredded (one sample) cheese. One cheese
also contained L. seeligeri. Simultaneous identification of L. monocytogenes in environ-
mental samples from all factories producing Listeria-positive cheese strongly suggests
that these cheeses were contaminated during manufacture and/or ripening. In keeping
with U.S. policies, attempts were made to remove tainted cheese from the marketplace.
Furthermore, after thoroughly cleaning and sanitizing the factory, government officials
required that Listeria-free cheese be produced for 12 consecutive days before being re-
leased to the public. Recent discovery of nonpathogenic listeriae in raw milk from neigh-
boring New Zealand also has prompted authorities in that country to institute a similar
environmental monitoring program for all cheesemakers who export their products.
Information regarding the presence of listeriae in dairy products produced elsewhere
is still reasonably scant, with results from a March 1989 IDF Survey [ 1961 indicating that
L. monocytogenes had not yet been isolated from any dairy products manufactured in
South Africa, Israel or the former Soviet Union. Most cheese-related surveys from other
countries have yielded negative results, with detection of L. monocytogenes being limited
to certain high-moisture domestic cheeses produced in Costa Rica [23 I], Egypt [ 1671, and
Venezuela [ 1471 along with imported (presumably European) soft cheeses marketed in
Japan [232] and the United Arab Emirates 11841.
Given the enormous volume of dairy products exported to other countries and the
fact that L. monocytogenes has been isolated from the natural environment of all seven
continents except Antarctica, it appears that developing countries are unlikely to remain
completely untouched by the problems associated with Listeria-contaminated foods. Con-
sequently, interest in the incidence of listeriae in dairy products and other ready-to-eat
foods will likely continue in the years ahead.

BEHAVIOR OF L. MONOCYTOGENES IN FERMENTED

MILKS
Before the well-known 1985 outbreak of cheeseborne listeriosis occurred in California,
very little information was available about the behavior of L. monocytogenes in fermented
milks and cheese. In fact, at the time of this outbreak, a search of the scientific literature
uncovered only four such studies, which were reported from Bulgaria [ 195,2951 and Yugo-
slavia [278,281] between 1965 and 1979. Hence, in addition to prompting numerous sur-
veys for Listeria spp. in cheese, discovery of cheese as an important vehicle in foodborne
listeriosis has led to well over 100 publications addressing the fate of L. monocytogenes
in fermented dairy products during manufacture and storage.
As described elsewhere in this book, cows, sheep, and goats can shed L. monocyto-
genes naturally in their milk during lactation. According to results from recent environ-
mental surveys of dairy processing facilities, ample opportunity exists for this pathogen to
enter pasteurized milk as a postpasteurization contaminant before the fermentation process
L. monocytogenes in Fermented Dairy Products 44 I

begins as well as afterward as a contaminant of the finished product. Thus far most studies
have dealt with behavior of L. monocytogenes in fermented dairy products inoculated with
the pathogen either before or after fermentation, with relatively few studies addressing
the fate of listeriae in fermented dairy products manufactured from naturally contaminated
raw milk.
Although the extent to which L. monocytogenes survives in cultured dairy products
is partly dictated by whether or not the pathogen enters the product before or after fermen-
tation, viability of Listeria in fermented dairy products, particularly cheese, depends on
the type of product in which the pathogen is found as well as the degree of acid tolerance
possessed by the contaminating strain [ 1721. Hence, to better understand the complex
interactions between the various factors that affect viability of listeriae in cheese (i.e.,
amount, activity and type of starter culture, a,, pH, salt content, temperature during manu-
facture and storage), it is appropriate to begin this section by first discussing the behavior
of L. monocytogenes in milk fermented with niesophilic arid thermophilic lactic starter
cultures. Commingled with this information will be data concerning the fate of this food-
borne pathogen during manufacture and storage of cultured buttermilk, cream, and yogurt.
The viability of L. monocytogenes in coagulants (e.g., calf rennet, microbial rennet, and
bovine-pepsin rennet extract), coloring agents (e.g., annatto), and starter distillates (e.g.,
natural flavor compounds derived from cultured milk) used in cheesemaking also will be
considered before our discussion of natural cheeses and cold-pack cheese food. Two addi-
tional areas of concern to cheesemakers, namely, the fate of L. monocytogenes in whey
and salt brine solutions, will be examined at the end of this chapter.

Starter Cultures, Cultured Milks, and Cream

Fermented or cultured buttermilk, cream, and yogurt were among the first dairy products
to be mass produced commercially using pure bacterial starter cultures. Today, two meso-
philic (optimal growth at 30C) lactic acid bacteria starter cultures, namely, Lactococcus
lactis ssp. luctis and Lactococcus lactis ssp. cremoris, (formerly Streptococcus lactis and
Streptococcus cremoris, respectively), are commonly used either alone or in combination
to manufacture cultured buttermilk and cream, whereas a mixture of two thermophilic
(optimal growth at or above 37C) lactic acid bacteria, namely, Streptococcus salivarius
ssp. thermophilus and Lactobacillus delbriickii ssp. bulgaricus (formerly Streptococcus
thermophilus and Lactobacillus bulgaricus, respectively), is used to produce yogurt. These
same mesophilic and thermophilic starter cultures also are used to produce over 400 variet-
ies of cheese that were recognized by the United States Department of Agriculture [297]
in 1978 with over 1200 cheese varieties now being in existance worldwide. The variety
of cheese to be produced depends, in part, on which of the lactic acid bacteria are used,
either alone or in combination, as the starter culture.
Mesophilic Starter CuItu res
Viability 01' L. monocytogenes in the presence of mesophilic lactic acid bacteria was first
examined by Schaack and Marth [269]. Samples of autoclaved skim milk were inoculated
to contain approximately 10' L. monocytogenes CFU/mL along with 0.1,0.5, 1.O, or 5.0%
of a S. cremoris or S. lactis milk culture and then fermented at 21 or 30C for 15 h to
simulate preparation of a conventional bulk starter culture for cheesemaking.
As shown in Figure 2, L. monocytogenes grew to some extent in all samples during
fermentation regardless of the species of lactic acid bacteriurn, inoculum level, or incuba-
442 Ryser

-1
mm] =0.10/0 = 1.0%
-E
2 1.50
c1
M

tl, 1.25
0
-g,
Y
.d

1 .o
0
a
'c 0.75

;
I
.- 0.50
M
c
cd
v 0.25

2 I "C 3ooc 21C 30C

crerrioris
S. _
- __ S. lactis
--

FIGURE2 Changes in populations of L. monocytogenes in skim milk following a 15-

hour fermentation at 21 and 30C with 0.1, 0.5, 1.0, or 5.0% (v/v) added S. cremoris
or S. lactis. (Adapted from Ref. 269.)

tion temperature. Maximum Listeria populations after 15 h of incubation at 21 "C were

-1.0-2.3 orders of magnitude lower in skim milk containing 0.1-5.0% of either starter
culture than in control samples without starter culture. Increasing the incubation tempera-
ture to 30C led to final Listeria populations that were -2.5-4.0 orders of magnitude
lower than in controls. As expected, growth of L. monocytogenes increased as the starter
culture inoculum level decreased from 5.0 to 0.1%. Although acid production by the starter
culture played a major role in limiting multiplication of Listeria, with the pathogen never
growing at pH (5.0, two situations were reported in which the organism was almost
completely inhibited at pH 5.6-6.0-well above the minimum pH value generally required
for growth.
Although still in use today, conventionally prepared bulk starter cultures have one
major disadvantage in that excessive acid development from an inherent lack of buffering
capacity frequently leads to some cell damage and partial loss of starter culture activity.
Consequently, in industrially prepared bulk starter cultures, the final pH is now normally
held constant at 5.1-5.2 by either adding (manual or automated) a neutralizer during the
fermentation process (Le., external pH control) or by using a specially prepared growth
medium containing chemical buffers which solubilize as the pH decreases (i.e., internal
pH control).
Given the popularity of internal pH-controlled media for starter culture preparation,
researchers at the University of Wisconsin investigated the ability of L. monocytogenes
strains Scott A, V7, and CA to compete with S. lactis [303] and S. cremoris [304] in one
commercially available starter culture medium having internal pH control. In both studies,
L. monocytogenes in Fermented Dairy Products 443

-
the starter culture medium was inoculated to contain 103 L. monocytogenes CFU/mL
together with either a 0.25 or 1.0% inoculum of S. lactis or S. cremoris and inoculated
at 21 or 30C for 30 h. Growth of the pathogen was only partially inhibited by S. lactis
and S. cremoris when compared with starter-free controls, with the greatest inhibition
occurring at the higher inoculum level and higher temperature. However, Listeria popula-
tions of 10'- 106and 104-10' CFU/mL developed in samples fermented with S. lactis and S.
cremoris, respectively, when these cultures were ready for use (pH 5.5) after 15 to 18 h of
incubation. Since neither conventional bulk starter technology nor internal pH-controlled
media will completely inhibit this pathogen, manufacturers of cheese and other fermented
dairy products should not discount the starter culture as a possible source for Listeria but
rather should adopt rigorous sanitation standards and Hazard Analysis Critical Control
Point (HACCP) programs to minimize Listeria contamination and potential product loss.
Ultrafiltered milk, a type of concentrated milk sometimes used for commercial man-
ufacture of certain cheeses including mozzarella, ricotta, cottage, and Cheddar, also pos-
sesses a higher buffering capacity than that of unfiltered milk because of higher concentra-
tions of proteins and insoluble salts. Consequently, listeriae may have greater opportunity
to grow in ultrafiltered as opposed to unfiltered milk during fermentation. El-Gazzar et
al. I1581 examined this question by inoculating samples of unfiltered skim milk as well
as retentate (concentrated twofold and fivefold by volume) and permeate from unfiltered
skim milk to contain 103-10' L. monocytogenes CFU/mL together with 107-108 L. lactis
subsp. cremoris CFU/mL. In contrast to the aforementioned studies involving skim milk
and internal pH-controlled bulk starter media, Listeria failed to grow in ultrafiltered milk
containing starter culture, with populations remaining constant in unfiltered slum milk and
decreasing up to 10- and 100-fold in 2-fold retentate and permeate, respectively, after 36
h of incubation at 30C. Increased inactivation of L. monoc-ytogenes in the permeate as
compared with retentate and unfiltered skim milk is again related to the lower buffering
capacity of the permeate which results from ultrafiltration. When these samples were re-
frigerated at 4"C, L. monocytogenes persisted 4-6 weeks in slum milk (pH 4.2), 3-5
weeks in retentate (pH 4.6), and 1 week in permeate (pH 4.1). Thus, fermentation of
ultrafiltered milk at 30C will not guarantee complete inactivation of Listeria even after
the finished product is moved to refrigerated storage.
Cultured Buttermilk
Cultured buttermilk is essentially pasteurized skim milk that has undergone a 12- to 15-
hour fermentation at 20C with S. cremoris or S. lactis (0.5% initial inoculum) and certain
flavor-enhancing lactic acid bacteria such as Leuconostoc cremoris or L. dextranicum.
After fermentation, the final product is packaged and refrigerated until consumed.
As part of a follow-up study [270], all 15-h-old fermented milk samples from the
aforementioned study by Schaack and Marth [269] were stored at 4C and examined for
L. monocytogenes. Survival of the pathogen ranged from an average of 5 weeks in skim
milk fermented at 30C with a 5.0% inoculum of S. cremoris to 12.5 weeks in skim milk
fermented at 21C with a 0.1% inoculum of S. cremoris (Table 11). Similarly, Listeria
viability averaged 2.5- 13.0 weeks in skim milk previously fermented at 30 and 2 1"C with
5.0 and 0.1% S. lactis, respectively. Slower inactivation of the organism in skim milks
fermented at 21 rather than 30C may be related to the rate of acid production during
fermentation, since pH values for fermented milks ranged between 4.3-6.0 and 4.2-4.6
immediately after 15 h of incubation at 2 1 and 3OoC,respectively. The fact that L. monocy-
togenes can survive 10.5 weeks in this refrigerated product (fermented 15 h at 21C with
444 Ryser

TABLE 11 Weeks of Survival of L. monocytogenes

in Skim Milks Fermented with S. cremoris or S.
lactis at 21 and 30C for 15 h and then Stored at 4C
S. cremo ris S. lactis
Inoculum (%) 21C 30C 21C 30C
0.1 12.5a 6.5 13.0 2.0
0.5 10.5 6.0 ND ND
1.o 8.0 4.5 8.5 3.0
5.0 9.0 5 .O 6.0 2.5
ND, Not determined.
a Average of two trials.

Source: Adapted from Ref. 270.

0.5% S. cremoris) emphasizes the importance of maintaining sanitary conditions during

manufacture of cultured buttermilk.
In addition to entering pasteurized skim milk as a postpasteurization contaminant,
L. monocytogenes also can be introduced into cultured buttermilk as a postfermentation
contaminant. Choi et al. [ 1331 studied the second of these scenarios. Samples of commer-
cially produced, cultured buttermilk (pH 4.2 1) were inoculated separately with each of
four strains to contain an average of 3.5 X 103L. monocytogenes CFU/mL and stored at
4C. Under these conditions, the pathogen survived an average of 22.8 days (Table 12),
with populations of three of four Listeria strains decreasing more than 100-fold during
the first 8- 12 days of refrigerated storage. It is important to realize that although L. mono-
cytogenes was inactivated faster when added directly to cultured buttermilk than when
skim milk was fermented into a buttermilk-like product in the previous study [270],
the pathogen still survived throughout the normal shelf life of the product. Furthermore,
results from an earlier study [ 1821 demonstrating that E. coli and Enterubacter aerogenes

12 Survival of L. monocytogenes in Inoculated Samples of

TABLE
Commercially Produced Buttermilk and Yogurt Stored at 4-5C
L. monocytogenes PH
inoculum Survival
Product (log,, CFU/mL) Initial Final (dayS >
Buttermilk 3.55 4.21 4.38 22.8
Plain yogurt
Brand Da 4.26 4.02 4.08 21.2
Brand Yb 4.36 4.03 4.09 24.7
Vanilla-flavored yogurt
Brand D 4.2 1 4.03 4.10 24.7
Brand Y 4.70 4.03 4.10 22.3
Plain low fat yogurt 2.10 4.10 4.10 3
Plain low fat yogurt 7.10 4.10 4.10 9
Custard-style.
Fluid-style.
Source: Adapted from Refs. 133, 279.
L. monocytogenes in Fermented Dairy Products 445

are inactivated faster than L. monocytogenes in cultured buttermilk imply that coliform-
free buttermilk may not necessarily be free of listeriae. These findings again emphasize
the importance of good sanitation in producing Listeria-free buttermilk.
Cultured Cream
Unlike cultured buttermilk, far less is known about the viability of L. monocytogenes in
cultured cream. In the only study reported thus far, Stajner et al. [281] manufactured
cultured cream from naturally contaminated raw cows milk containing approximately 5
X 1O5 L. monocytogenes CFU/mL. According to these Yugoslavian authors, viable lister-
iae were detected in the finished product throughout 7 days of storage at 3-5C.
Thermophilic Starter Cu Itures
Practically speaking, thermophilic fermentations used to produce yogurt and certain
cheeses (e.g., Swiss, Parmesan, mozzarella, and Romano) are not normally continued be-
yond 4-6 h. The only two exceptions are in production of Bulgarian buttermilk and acido-
philus milk, which require thermophilic fermentations of 10- 1 2 and 18-24 h, respectively.
Therefore, primary emphasis will be placed on behavior of Listeria during the first 6 h
of fermentation.
In addition to determining the fate of L. monocytogenes in the presence of mesophilic
starter cultures [269], Schaack and Marth [268] also investigated the ability of this organ-
ism to grow during fermentation of skim milk with thermophilic lactic acid bacteria. As
in the previous study, samples of autoclaved skim milk were inoculated to contain -103
L. monocytogenes CFU/mL. After adding 0.1, 1 .O, or 5.0% (Streptococcus thermophilus,
Lactobacillus bulgaricus, or a mixture of the two species, all samples were examined for
numbers of listeriae during 15 h of incubation at 37 and 42C.
Limited growth of L. monocytogenes occurred in all samples, with the organism
generally attaining maximum populations after 6 h of incubation at either temperature
(Fig. 3). At this point, Listeria populations were generally I .O-1.5 orders of magnitude
lower in fermented than in unfermented control samples, as was also reported by Luka-
sova, [212], indicating that growth of the pathogen was markedly suppressed by the ther-
mophilic starter culture, particularly when used at inoculuni levels of 5%. In addition,
greater inhibition of listeriae was consistently observed in rnilks fermented at 42 rather
than 37C.
Listeria monocytogenes behaved similarly in milks fermented with S. thermophilus,
L. bulgaricus, and a mixture of both starter cultures during the initial 6 h of incubation;
however, viability of the pathogen in milks fermented beyond 6 h varied with the species of
lactic acid bacterium used in the fermentation. Although populations of listeriae remained
relatively unchanged in all milks fermented 6-15 h with S. thermophilus (final pH of
4.55-4.90), the pathogen frequently survived only 9-15 h in milks fermented with L.
bulgaricus alone. Rapid inactivation of the pathogen coincided with pH values 1 4 . 0 which
developed in milks fermented 9-15 h with L. bulgaricus. The combination of L. bulgaricus
and S. thermophilus was more inhibitory to Listeria than was S. thermophilus alone but
less inhibitory than was L. bulgaricus alone. Although populations of listeriae failed to
decrease in milks fermented at 37C with the mixed starter culture, some inactivation was
noted in all corresponding samples incubated at 42C. As was true when L. bulgaricus
was used alone, inactivation of listeriae by the mixed starter culture again was most pro-
nounced in samples having pH values 54.0.
Following 15 h of incubation, all fermented milks in this study were stored at 4C
 446 Ryser

1.75
-E
h

5 1.50
LL
U
2
1.25
c!
c
.g
- 1.0

*I
1
a
0
a 0.75

3 0.50

B;:[
.
to
0)
2 0.25
...
6 ...
...
...
0
37oc 42C 37C 42C 37C 42OC
S, thermophilug -
L. bulrraricu$ -
L. bulearicus arid
S. IhermoohiluS

FIGURE3 Changes in populations of L. monocytogenes in skim milk following a 6-

hour fermentation at 37 and 42C with 0.1, 1.0, and 5.0% (v/v) added Lactobacillus
bulgaricus, Streptococcus thermopilus, or L. bulgaricus + S. thermopilus. (Adapted
from Ref. 268.)

and monitored for listeriae by Schaack and Marth [270]. Using S. thermophilus alone,
L. monocytogenes survived 21-32 and 5-15 weeks in milks fermented at 37 and 42OC,
respectively (Table 13). Failure of these milks to attain pH values 14.0 after fermentation
with S. thermophilus helps explain the unusually long survival of listeriae. As expected,
L. bulgaricus was most detrimental to listeriae with the pathogen surviving beyond 15 h
only in samples fermented with the lowest inoculum. Using a 0.1 % L. bulgaricus inoculum
the pathogen was eliminated from milks fermented at 37 and 42C following 7 and 3 days

TABLE
13 Weeks of Survivalaof L. monocytogenes
in Skim Milks Fermented with S. thermophilus or S.
thermophilus + L. bulgaricus (LEST) at 37 and 42C
for 15 h and then Stored at 4C

S. thermophilus LBST
Inoculum (%) 37C 42C 37C 42C
0.1 28.5 15.0 7.5 1.5
1.o 32.0 8.5 1.5 12 h
5 .O 21.0 5 .O 1.o 15 h
aAverage of two trials.
Source: Adapted from Ref. 268.
L. monocytogenes in Fermented Dairy Products 447

of refrigerated storage, respectively. Milks cultured with a combination of S. thermophilus

and L. bulgaricus yielded results that were between both extremes observed when the two
starter cultures were used separately (Table 13). Once again, L. monocytogenes survived
longer in refrigerated milks fermented at 37 than 42"C, with slower inactivation in the
former being attributed to slightly higher pH values. These findings are similar to those
of Zuniga-Estrada et al. [309], who found that Listeria persisted for only 8 h when milk
containing 1O3 L. monocytogenes and 1O6 S. salivarius subsp. thermophilusll. delbrueckii
subsp. bulgaricus CFU/mL was fermented at 42C to pH 4.4.
Yogurt
As previously mentioned, L. monocytogenes can enter yogurt either before the fermenta-
tion as a milk contaminant or afterward as a contaminant of the finished product. Schaack
and Marth [268,270] examined the behavior of Listeria under the first of these conditions
by inoculating yogurt mix to contain -103-104 L. monocytogenes (strains V7, OH, Scott
A, or CA) CFU/mL and 2% of a commercial starter culture containing S. thermophilus,
L. bulgaricus, and Lactobacillus acidophilus. Yogurt mix was fermented at 45C for 5 h
and then stored at 4C. Populations of all four Listeria strains increased an average of
2.5- to 10.0-fold in yogurt mix during the fermentation. After finished yogurt at pH 4.75
was refrigerated at 4"C, numbers of listeriae decreased, with strains V7, OH, Scott A, and
CA surviving 7-12, 7-12,4-12, and 1-5 days, respectively. The pH of yogurts in which
listeriae were last detected ranged from 3.88 to 4.1 1. However, several later studies have
shown that Listeria survival is more variable than previously observed, with L. monocyto-
genes persisting 2-7 days [221], 4 days [4], 3-5 days, 18-24 days [203], and 14-25 days
[251] in yogurts of pH 4.2, 4.0-4.3, 4.1-4.7, 3.8-3.9, and 4.5-5.0, respectively, during
refrigerated storage, with growth of the pathogen also general1y not being observed during
fermentation. Furthermore, in one French study [ 1401 involving highly acidic yogurt (pH
3.5) prepared from yogurt mix inoculated to contain 102-107 L. monocytogenes CFW/
mL, the pathogen was eliminated after only 1-2 days of storage at 4C. Although acid
development and differences in acid tolerance/injury during manufacture [ 1721 play major
roles in determining the fate of listeriae in yogurt, other factors including various starter
culture metabolites and antilisterial compounds (bacteriocins) produced by certain strains
of L. acidophilus [248] also may contribute to the rapid demise of Listeria in yogurt,
provided this lactic acid bacterium is in the product, with addition of 100 mgl-' lysozyme
to yogurt mix before fermentation also reportedly decreasing Listeria survival in the fin-
ished product [251].
Although nearly all yogurt in the United States is now prepared from cow's milk,
this product is sometimes manufactured from ewe's and/or goat's milk, particularly in
Europe and the Middle East. In a 1964 Bulgarian study [ 1951,L. monocytogenes viability
in yogurt prepared from naturally contaminated ewe's milk was markedly influenced by
the storage temperature, with the pathogen surviving <24-48 h and >6 days in yogurt
held at 18-22 and 10C, respectively. Faster demise of listeriae at the higher rather than
lower storage temperature was attributed to increased acid production by S. thermophilus
and L. bulgaricus. When Abdel-Gawad et al. [personal communication] recently prepared
yogurt from cow's, ewe's and goat's milk inoculated to contain 103-104L. monocytogenes
CFU/ml, 97-99.9% of the population was sublethally injured within 24 h of manufacture
with no cells repairing and/or surviving in any yogurt samples (pH 4.0N4.3) beyond 5
days of cold storage.
Many dairy industry personnel believe that contamination of yogurt is more likely
448 Ryser

to occur after rather than before fermentation. Choi et al. [ 1331 simulated postfermentation
contamination of yogurt by inoculating two commercial brands of plain and vanilla-fla-
-
vored custard-and fluid-style yogurt to contain 104- 1O5 L. monocytogenes CFU/mL.
The pathogen survived an average of 2 1.2-24.7 days in yogurt held at 4C (Table 12),
with most listeriae being inactivated during the first 8-12 days of refrigerated storage.
Khattab et al. [203] reported similar findings with listeriae persisting 18-24 days in experi-
-
mentally produced Egyptian yogurt (pH 3.8-3.9) inoculated to contain 106L. monocyto-
genes CFU/mL. After inoculating various formulations of experimentally produced yogurt
-
to contain 106L. monocytogenes CFU/mL, Griffith and Deibel [ I891 also detected the
pathogen in yogurt samples having a pH value of 4.3 following 28 days of storage at 4C.
Although the type of yogurt had no apparent effect on Listeria survival in these studies,
the point at which yogurt became contaminated greatly influenced survival of listeriae,
with Schaack and Marth [270] showing that the pathogen survived only 1- 12 days when
added to yogurt mix before fermentation.
Siragusa and Johnson [279] conducted a similar study in which three different brands
of commercial, unflavored, low-fat yogurt were inoculated to contain approximately 1O2
and 107L. monocytogenes CFU/g and then stored at 5C. Listeriae survived <3 days in
yogurt inoculated with low levels of the pathogen even though pH values of yogurt were
similar to those in the study by Choi et al. [133] (Table 12). Using the high inoculum,
viable listeriae were found for only 9 days, with populations decreasing approximately
100-fold each after 3 and 6 days of refrigerated storage. Ayre et al. [ 1061 reported similar
results using an L. monocytogenes inoculum level of 107CFU/g. In contrast, Ribeiro and
Carminati [251] examined the effect of yogurt pH on Listeria survival by inoculating
experimentally produced (pH 4.5), commercial fruit (pH 4.05) and plain yogurt (pH 3.76)
to contain -104 L. monocytogenes CFU/mL. Overall, the pathogen survived 2-3 days,
3-4 days, and 12-14 days in refrigerated yogurts having pH values of 3.76, 4.05 and 4.5,
respectively, thereby verifying the impact of pH on Listeria survival. However, Griffith
and Deibel [ 1891 reported that L. monocytogenes populations decreased approximately
four orders of magnitude in artificially acidified (pH 4.2) rather than fermented yogurt
during the first 6 days of storage at 4C. Hence, decreased tenacity of L. monocytogenes
in inoculated yogurt samples in these studies as compared with the work by Choi et al.
[ 1331 again demonstrates that factors other than pH also are contributing to the demise
of Listeria.
Many yogurts and cultured buttermilks marketed today have pH values of approxi-
mately 4.0 and 4.3, respectively. Since large populations of L. monocytogenes were inacti-
vated faster in yogurt than in buttermilk during refrigerated storage, one would expect a
lower incidence of listeriae in commercial yogurt. Evidence from FDA surveys discussed
earlier in this chapter supports this view, since thus far the pathogen has been detected
in commercial buttermilk but not yogurt, with 100 retail and farm-produced samples also
negative for L. monocytogenes in the United Kingdom [202]. However, as was true for
buttermilk, E. coli and Enterobacter aerogenes are also inactivated faster than L. monocy-
togenes in yogurt during refrigerated storage [ 1821. Hence, coliform-free yogurt may not
necessarily be free of Listeria. This is important to remember when results of coliform
tests on these products are interpreted. It is evident from this discussion that good sanita-
tion practices are of utmost importance in producing Listeria-free yogurt, buttermilk, and
other fermented milk products.
Since yogurt is occasionally used as an ingredient in other foods, Sikes [277] investi-
gated the fate of L. monocytogenes in low-moisture (1.9% water), medium-acid (pH 4.9)
L. monocytogenes in Fermented Dairy Products 449

yogurt-based dairy bars supplied to the U.S. military. These bars, which contained -34%
heavy cream, 27.5% yogurt, 27.5% cream cheese, and I I % of other ingredients (i.e., sugar,
sunflower oil, whey), were inoculated to contain approximately 1 X 10' L. monocytogenes
strain Scott A CFU/g and then periodically examined for numbers of listeriae during
extended storage at 25C. Results indicated that Listeria populations decreased only ap-
proximately 100-fold after 40 days of incubation, thus demonstrating the ability of this
organism to persist in low-moisture, medium-acid foods. As will soon be discussed, similar
behavior has been reported for L. monocytogenes in semihard cheeses, such as Cheddar,
Colby, and Gouda, which also have pH values near 5.0.

Traditional Fermented Milk Products

In certain African and Middle Eastern countries, traditionally fermented ethnic milk prod-
ucts such as ergo (Ethiopia) and labneh (United Arab Emirates) comprise a significant
portion of the daily diet. Although produced from commercially pasteurized milk under
generally adequate hygienic conditions, these products are also frequently prepared at
home (particularly in rural areas) from raw milk which is allowed to undergo a natural
souring (i.e., fermentation) at ambient temperature without the addition of starter cultures.
Hence, the likelihood for the presence of Listeria and other bacterial pathogens in raw
milk combined with an often questionable fermentation and a short product shelf life
makes the microbiological safety of such home-fermented milks particularly suspect.
Working in Zimbabwe, Dalu and Feresu [ 1421 examined the fate of L. monocyto-
genes in commercially fermented milk prepared from both raw and pasteurized milk. Com-
mercial fermented milk was prepared from pasteurized milk which was inoculated to con-
tain 104L. monocytogenes CFU/mL together with an active mesophilic starter culture and
then packaged and fermented 24 h at ambient temperature, whereas both traditionally
fermented milks were prepared by allowing raw milk to sour naturally in earthenware
pots during 24 h of incubation at ambient temperature. Overall, Listeria populations in-
creased 5 10-fold in all three milks during fermentation, with the final product attaining
a pH of 4.3--4.6. After 5 days of storage at 4 and 20C, numbers of listeriae generally
decreased about 10- and 100-fold, respectively. Consequently, survival of L. monocyto-
genes beyond the normal 3-7 day shelf life of these traditionally fermented milks poses
a legitimate public health concern.
In 1993, Ashenafi [ 1041 prepared ergo, a traditional Ethiopian fermented milk, from
boiled milk that was inoculated to contain 103L. monocytogenes CFU/mL along with
Lactobacillus and Streptococcus starter organisms from a previous batch of product. After
the first 12 h of a 24-h fermentation at 25OC, numbers of listeriae generally increased 10-
to 100-fold i n ergo (pH 4 . 5 - 5 3 , with the highest populations being observed in samples
fermented in unsmoked as compared with olive wood-smoked glass containers. During
the next 12 h, the pathogen was slowly inactivated as the pH of the product decreased to
1 4 . 0 . Continued ambient storage led to complete demise of Listeria in ergo fermented
in wood-smoked and unsmoked containers within 36-48 and 48-60 h, respectively. Con-
sequently, traditional preparation of ergo in rural areas using wood-smoked fermenting
vessels appears to be beneficial in minimizing Listeria survival in this product which is
typically prepared from raw milk and consumed immediately after the 24-h fermentation
period.
Labneh, a Middle Eastern yogurt-like product, is produced using a normal yogurt
starter culture of L. delbreuckii subsp. bulgaricuslS. salivarius subsp. thermophilus. Fol-
450 Ryser

lowing approximately 5 h of incubation at 42"C, the product is salted (1% NaCl), poured
into muslin bags, and hung in a cooler for 48 h. Gentle mixing to obtain a smooth consis-
tency follows, after which the finished product (pH 3.8) is packaged for sale. These manu-
facturing steps which afford many opportunities for contamination prompted Gohil et al.
[ 1851 to assess the fate of L. rnonocytogenes in labneh as a postfermentation contaminant.
When commercially produced labneh was inoculated to contain l O4 L. rnonocytogenes
CFU/g and stored at 4"C, the pathogen survived 2 and 7 days in product of pH 3.8 and
4.5, respectively, with the addition of 1% NaCl not appreciably altering Listeria survival.
Raising the holding temperature to 10, 20, or 30C led to more rapid demise of listeriae,
with samples generally being free of L. rnonocytogenes after 2-3 days of storage.

BEHAVIOR OF L. MONOCYTOGNS IN CHEESE

Considerable work has been done at the University of Wisconsin-Madison and elsewhere
to define the behavior of L. rnonocytogenes during manufacture and ripening of various
types of cheese. Most of these studies describe what might happen if cheese is prepared
from contaminated milk. Since North American and European surveys have indicated that
soft/semisoft cheeses ripened with mold or bacteria are most frequently contaminated with
L. rnonocytogenes, research dealing with such varieties will be discussed first, followed
by data on ripened cheeses of progressively lower moisture content, goat's milk cheese,
unripened cheese, whey cheese, and cold-pack cheese food. Whereas this chapter con-
cludes with a discussion of L. rnonocytogenes behavior in whey and brine solutions, we
will begin this section by first examining the viability of listeriae in coagulants, coloring
agents, and starter distillates, all three of which are commonly used in cheesemaking.

Coagulants
To produce cheese curd, milk must first be coagulated or clotted, which can be done either
by acidification or addition of a coagulating enzyme. In the first method, an active lactic
starter culture is used to lower the pH of the milk to 4.6-4.7 (isoelectric point of casein), at
which point the casein micelles in the milk precipitate and form a coagulum. Alternatively,
coagulation is occasionally accomplished by adding food-grade acids directly to milk.
Coagulation of milk by either means of acidification is primarily confined to the manufac-
ture of cottage cheese and a few ethnic varieties of fresh cheese.
The second method, in which a coagulating enzyme is added to destabilize the casein
micelles and clot milk at a near-neutral pH, is used to manufacture virtually all other
types of cheese. Coagulants presently used in cheesemaking include calf rennet extract,
chymosin, bovine pepsin-rennet extract, and microbial rennet. Traditionally, calf rennet
is extracted from the lining of the abomasum (fourth stomach) of suckling calves and
contains two enzymes-pepsin and chymosin (the latter is most important for coagulation
of milk). Shortage of calf rennet following World War I1 led to the use of bovine pepsin-
rennet, an extract obtained from the abomasum of somewhat older calves, that can be
substituted for calf rennet. Increased production costs of both calf rennet and bovine pep-
sin-rennet have in turn prompted development of several rennets of microbial origin. Thus
far enzyme preparations obtained from molds belonging to the genus Mucor (particularly
M. rniehei) have proven to be the most satisfactory substitutes for animal rennet.
Since two of four coagulants used in cheesemaking are of animal origin, and since
these animals sometimes carry L. rnonocytogenes, this pathogen might occasionally appear
L. monocytogenes in Fermented Dairy Products 451

in both crude enzyme preparations and finished coagulant at the time of shipping. Although
microbial rennet should be free of Listeria spp. when manufactured, the presence of lister-
iae within the rennet manufacturing facility or the cheese factory environment could con-
taminate any of these products if mishandled. Given the recovery of an apparent sodium
benzoate-resistant strain of L. monocytogenes from commercially produced calf rennet
extract [ 1571, the possible presence of L. monocytogenes in coagulants should be of con-
cern to cheesemakers, with the International Dairy Federation also contemplating the addi-
tion of rennet to its list of cheesemaking ingredients to be examined for Listeria
spp. [288].
During 1988 and 1989, El-Gazzar and Marth published results from three studies
examining the viability of listeriae in calf [ 1531, bovine pepsin [ 1541, and microbial rennet
[ 1561. In each of these studies, commercially produced, Listeria-free rennet was inoculated
to contain approximately l 03,l 04,l 05,or 1O6 L. monocytogenes CFU/mL and analyzed for
listeriae during 56-70 days of storage at 7C using both direct plating and cold enrichment.
All samples of calf and bovine pepsin-rennet inoculated with the two lowest levels
of Listeria were free of the pathogen after 14-28 days of storage at 7C (Table 14).
Even though 42-56 days of storage were required to eliminate the pathogen from samples
containing initial inocula of approximately 1O5 and 1O6 L. rnonocytogenes CFU/mL, the
reader is reminded that all four inoculum levels used in these studies were many times
greater than levels that might occur naturally in commercially produced coagulants. Hence,
barring contamination in the cheese factory, these findings suggest that calf and bovine
pepsin-rennet are normally held long enough in distribution channels to ensure cheesemak-
ers that both coagulants are Listeria-free. Inactivation of L. monocytogenes in calf rennet
and pepsin-rennet probably results from the combined effects of 5% propylene glycol,
2% sodium propionate, 0.1% (or more) sodium benzoate, 14-21 % salt, and a relatively
low pH of 5.6. Results from several studies assessing the viability of L. monocytogenes
in the presence of benzoic acid and sodium propionate are discussed in Chapter 6.
Unlike calf and bovine pepsin-rennet, more than 70 days of storage were required
to eliminate L. monocytogenes at even the lowest inoculuni level from microbial rennet
(see Table 14). Enhanced survival of listeriae in microbial rennet may be related to the
nature of the coagulant itself as well as the presence of fewer preservatives. Although L.
monocytogenes is unlikely to enter microbial rennet during manufacture, the relatively
high incidence of listeriae in cheese factories may lead to inadvertent contamination of
the coagulant during cheesemaking. Considering the tenacity of L. monocytogenes in mi-
crobial rennet and the long shelf life of this product, it may be prudent for cheesemakers
periodically to verify that the microbial rennet they are using is indeed Listeria-free.

Coloring Agents and Starter Distillates

Depending on local preference, various yellow-orange colorants such as annatto (an extract
from annatto seed [Bixia orellana]) and turmeric (an extract from the turmeric root [Cur-
cuma longa]) can be added to milk at the beginning of cheesemaking, with annatto most
commonly being used in the manufacture of Cheddar, Colby, Muenster, and brick cheese.
Although freshly prepared colorants are unlikely to contain microbial pathogens, inadver-
tent exposure of these coloring agents to L. monocytogenes in the cheese factory could
lead to production of a contaminated product. Thus, in addition to the aforementioned
coagulants, El-Gazzar and Marth [ 1551 also investigated the fate of listeriae in five com-
mercially available annatto/turmeric extracts that were inoculated to contain approxi-
452 Ryser

TABLE
14 Survival of L. monocytogenes Strain CA in Three Milk Coagulants Stored a t 7C
~~ ~ ~

Number/mL after days of storage

Product 0 7 14 28 42 56 70
Calf rennet extract 2.5 X 103 1.5 X 102( + ) a <10 ( - ) b <10 (-) <10 (-)
1.1 x 10' 3.5 x 102(+) <10 (-) <10 (-) <10 (-)
3.0 X 10' 6.0 X 104(+) 1.2 x 103 (+I 30 (+I <10 (-)
2.0 X 106 6.0 X 104(+) 4.5 x 107(+) 2.0 x 102(+) <10 (-) - -

Bovine pepsin-rennet extract 9.5 X 103 10 <10 (-) <10 (-) <10 (-) <10 (-) -

2.0 x 10' 30 <10 (-) <10 (-) <10 (-) <10 (-) -

7.5 x 105 1.0 x 103 1.0 x 10' <10 (+) <10 (+) <10 (-> -

1.0 x 106 2.0 x 104 1.0 x to2 <10 (+) <10 (+) <10 (-) -
Microbial rennet 6.0 x 103 1.5 x 103 7.6 X 10' 2.8 X 10' 3.3 x 102 1.2 x 10' 40
7.0 X 103 1.7 X 103 1.0 x 103 2.2 x 102 4.4 x 10' 1.4 X 102 90
2.0 X 10' 1.7 X 10' 2.3 X 10' 2.5 X 10' 2.3 X 10' 1.6 X 10' 8.5 X 103
1.0 x 106 1.5 x 105 4.0 X 10' 3.6 X 10' 9.3 x 10' 7.0 X 10' 9.0 X 10'

a (+) Positive result by cold enrichment.

( - ) Not detected after 6 weeks of cold enrichment.
Source: Adapted from Refs. 153, 154, 156.
L. monocytogenes in Fermented Dairy Products 453

mately 103--1O7 L. monocytogenes strain CA/mL and stored at 22C. Regardless of the
initial inoculum level, populations of listeriae immediately decreased 2 4 orders of magni-
tude in all colorants, with the pathogen being completely inactivated immediately after
addition to three of five extracts. The almost instantaneous death of Listeria in these three
colorants was attributed to the presence of propylene glycol and a pH of 13.3 (one extract).
Although Listeria populations of 5800 CFU/mL were observed in the two remaining
colorants immediately after inoculation, with the highest level of listeriae, these samples
were free of the pathogen following 7 days of ambient storage. Overall, these findings
indicate that the length of time that these colorants spend at ambient temperatures during
distribution and before use at the cheese factory is more than adequate to inactivate small
numbers of listeriae that might enter as chance contaminants.
Small levels of starter distillates, that is, mixtures of natural flavor compounds such
as diacetyl obtained by distilling specially cultured milks, are frequently used to enhance
the flavor of cottage and processed cheese as well as ice cream, margarine, butter, yogurt,
snack foods, and certain types of candy. Hence, in connection with the study just described,
El-Gazzar and Marth [ 1551 also examined the fate of listeriae in a commercially available
starter distillate that was inoculated to contain 102- I O6 L. monocytogenes strain CA, Scott
A, or V7 CFU/mL and held at 7C. Overall, strain CA decreased to nondetectable levels
in all samples after 2-7 days of storage depending on the initial inoculuni, whereas 7-
28 days of incubation were required to eliminate strains Scott A and V7 from similar
samples. Therefore, barring inadvertent contamination in the cheese factory, the time in-
volved in shipping and distributing these starter distillates should be more than sufficient
to eliminate any inadvertent Listeria contaminants.

Mold-Ripened Cheeses
Mold-ripened cheeses can be divided into two categories: (a) white mold cheeses, which
are surface-ripened by either Penicillium camemberti, Penicillium caseicolum, or Penicil-
lium candidum (i.e., Brie and Camembert), and (b) blue-mold or blue-veined cheeses in
which ripening results from growth of Penicillium roqueforti or P. glaucum throughout
the cheese (i.e., Roquefort, blue, Gorgonzola). The relatively high moisture content of
these surface-ripened cheeses, along with a nearly neutral pH in fully ripened cheese,
allows rapid growth of L. monocytogenes as well as other foodborne pathogens that would
normally be inhibited in more acidic cheeses. Since mold-ripened cheeses also are highly
susceptible to surface contamination during ripening, it is not surprising that Brie and
Camembert were among the first varieties of cheese in which L. monocytogenes was de-
tected and the behavior of the organism studied.

Camembert Cheese
Pasteurized milk was inoculated to contain approximately 500 L. monocytogenes strains
Scott A, V7, CA, or OH CFU/mL and manufactured into Camembert cheese by Ryser
-
and Marth [ 2601. Following 10 days of storage at I5"C/95% relative humidity (RH) to
permit proper growth of P. camemberti on the cheese surface, all cheeses were wrapped
in foil and ripened at 6C. Wedge (pie-shaped), surface, and interior samples of cheese
were diluted in Tryptose Broth and analyzed for listeriae at appropriate intervals using
both direct plating and cold enrichment.
Populations of L. monocytogenes increased 5- to 10-fold during the first 24 h after
manufacture; however, this increase probably did not result from growth of the organism
454 Ryser

during cheesemaking. Numerous studies have shown that bacterial populations typically
increase 5- to 10-fold during curd formation as a direct result of entrapment of organisms
in curd particles, with the exact level of increase dependent on the moisture content of the
cheese. In all likelihood, L. monocytogenes was similarly concentrated during formation of
Camembert cheese curd. Entrapment of L. monocytogenes in the curd is further supported
by the fact that, in this study, only 1.3% of the original Listeria inoculum in the milk was
lost in the whey. Yousef et al. [308] later demonstrated that the failure to observe L.
monocytogenes population increases of approximately 5- to 10-fold after formation of
Camembert as well as Cheddar and cottage cheese curd was probably related to the method
of sample preparation. Their improved procedure in which curd samples were homoge-
nized in warm (45C) Tryptose Broth containing 2% trisodium citrate was subsequently
used to examine behavior of L. monocytogenes during manufacture and storage of brick
[264], Colby [306], feta [238], blue [237], and Parmesan cheese [307].
During the initial 17 days of cheese ripening, the first 10 days of which occurred
-
at 15"C, populations of three of four L. monocytogenes strains decreased 10- to > 1000-
fold, with lowest numbers generally being observed in surface samples (Fig. 4). On further
ripening at 6"C, all four Listeria strains grew (particularly between 25 and 30 days of
storage) and attained maximum populations of 106- 108CFU/g in wedge and surface sam-
ples from fully ripened cheese; however, maximum listeriae populations were generally

8.0 8.0

s!
3
/
.-c
E
;3

35.0

ULLld 4 . 5
" 0 "5 10 15 20 75 30 35 40 45 50 55 60 65
Days

FIGURE4 Behavior of L. monocytogenes strain CA (solid symbols) and pH (open

symbols) during ripening of Camembert cheese. Solid symbols at <1.0 log,, strain
CA/g indicate results for cold enrichment. Numbers indicate the week at which strain
CA was found, whereas an "x" signifies that the pathogen was not detected after 8
weeks of cold enrichment. (Adapted from Ref. 260.)
L. monocytogenes in Fermented Dairy Products 455

10- to 100-fold lower in interior samples from the same cheeses. Although growth of L.
monocytogenes clearly paralleled the increase in pH of the cheese during ripening, with
growth usually commencing after the cheese attained a pH value of 5.75-6.25, decreased
viability of three of four Listeria strains in surface samples having pH values of 6.25-
6.50 suggests that factors other than pH, including the presence of potentially inhibitory
surface bacteria and yeasts, may also be involved in controlling growth of this pathogen
in Camembert cheese.
Results from a subsequent study by Ryser and Marth [ 2621 showed greater growth
of L. monoc-ytogenesin filter-sterilized Camembert cheese whey previously cultured with
P. camemberti than in uncultured whey adjusted to pH values of 5.60-6.80 and thus
suggest that P. camemberti is not involved in reducing Listeria populations on the surface
of Camembert cheese. In support of these findings, Geisen et al. [ 1731 also failed to ob-
serve any antilisterial activity among several strains of P. camemberti that were tested
against L. monocytogenes in vitro. However, in the work by Ryser and Marth [260], possi-
ble antilisterial activity from yeasts and non-lactic acid bacteria (i.e., micrococci, coryne-
forms) that are naturally present on the cheese surface during initial stages of ripening
was not precluded.
To simulate contamination of cheese in the ripening room, Ryser and Marth [260]
also inoculated surfaces of 10-day-old wheels of Listeria-free Camembert cheese to con-
tain 2-40 L. monocytogenes (four strains tested separately) CFU/20 cm2.All cheeses were
then ripened at 6C for 60 days, during which time 10-g surface samples were analyzed
for listeriae. Three of four L. monocytogenes strains grew on the surface of the cheese
-
and attained maximum populations of 10'- 105CFU/g (Fig. 5 ) . Although the remaining
Listeria strain failed to grow on the cheese surface after 60 days of storage, the pathogen
was routinely detected throughout the ripening period using cold enrichment. These find-
ings, along with the unfortunate recall of over 300,000 tons of French Brie cheese, stress
the importance of manufacturing surface-ripened soft cheeses from high-quality, Listeria-
free milk and observing good sanitary practices in the ripening room. Nonetheless, even
under ideal manufacturing and ripening conditions, such cheeses may still inadvertently
become contaminated with listeriae.
Since Vacherin Mont d'Or and Brie de Meaux (a raw milk cheese) were directly
involved in two major outbreaks of listeriosis in Europe (see Chap. lO), scientists in both
Europe and North America have been exploring various means of eliminating this patho-
gen from such cheeses that are surface ripened with mold and/or bacteria. Not surprisingly,
Banks [ 1 101 reported that L. monocytogenes grew rapidly in Camembert cheese prepared
from raw milk, reaching a population of 106 CFU/g in fully ripened cheeses. Although
heat treating this Listeria-contaminated milk at subpasteurization temperatures (i.e., 62.8
or 65.6"C) led to markedly lower Listeria populations in the cheese immediately after
manufacture, the pathogen was not completely inactivated, with some growth being re-
ported during 7 weeks of cheese ripening. In another report [ 1451, addition of lactoperoxi-
dase system components to the surface of soft bacterial smear-ripened French cheese con-
taining 102--1 O6 L. monocytogenes CFU/g led to complete inactivation of the pathogen
following 4 days of storage at 15C. In 1989, Hughey et al. [ 1931 showed that lysozyme
was only bacteriostatic to L. monocytogenes in Camembert cheese. Incorporating 2- 10%
carrot juice into homogenized Brie cheese was also effective in minimizing growth of L.
monocytogenes in refrigerated samples [ 1 171. Although several additional reports also
attest to the usefulness of x-ray [ 12I] and gamma irradiation [ 1621 in eliminating high
456 Ryser

./
/

Days

FIGURE5 Growth and survival of L. rnonocytogenes on the surface of Camembert

cheese. Half-solid and solid symbols at < I log,o Listeria/g indicate that the organism
was detected in one of t w o or two of t w o samples, respectively, using cold enrich-
ment. Numbers indicate the week at which L. rnonocytogenes was found. (Adapted
from Ref. 260.)

populations of L. monocytogenes from Camembert and other soft surface-ripened cheeses,

such treatments do not appear to be very practical, since ripening of the cheese will be
adversely affected.
Although initial attempts by Asperger et al. [ 1051 failed, results from several subse-
quent studies yielded more promising findings concerning the use of nisin and nisin-pro-
ducing starter cultures to eliminate chance Listeria contaminants from soft surface-ripened
cheese. When Maisner-Patin et al. [216] used a nisin-producing strain of L. lactis subsp.
lactis to manufacture Camembert cheese from milk inoculated to contain 101, 103,or 105
L. monocytogenes strain V7 CFU/mL, numbers of listeriae decreased dramatically 6-9 h
into cheesemaking because of the presence of -700 IU nisin/g of curd. Richard [252]
also reported that inactivation of L. monocytogenes can be further enhanced by directly
adding as little as 25 IU nisin/mL to the milk at the start of cheesemaking.
Inhibition of Listeria continued during the first 2 weeks of ripening, however, re-
growth of survivors was then reported in the presence of 250-300 IU nisin/g, first on the
cheese surface and later in the interior, with the rate and extent of regrowth again parallel-
ing the increase in cheese pH during ripening. Addition of other inhibitory organisms,
including Enterococcus faecalis and Luctobacillus paracasei, to milk failed to arrest L.
monocytogenes growth in Camembert cheese during extended ripening. However, a differ-
ence of 2.4 log CFU/g between numbers of L. monocytogenes in cheeses made with and
without nisin-producing starter cultures was maintained throughout 6 weeks of ripening.
L. monocytogenes in Fermented Dairy Products 457

Nisin was most effective when the milk for cheesemaking contained 10' L. monocytogenes
CFU/mL, with the pathogen being absent in 25-g samples even after 6 weeks of ripening.
These findings agree with those of Sulzer and Busse [285], who used a different nisin-
producing strain of L. lactis subsp. lactis. Although the nisin system has limits to pre-
venting regrowth of Listeria during cheese ripening, use of nisin-producing starter cultures
appears to be a relatively simple and effective means of minimizing Listeria growth during
Camembert cheese manufacture provided that the cheese milk is of good hygienic quality
and contains < 103L. monocytogenes CFU/mL.
At least five additional studies have addressed the fate of L. rnonocytogenes during
manufacture and ripening of Camembert cheese [ 108,225,282,284,2891.Regardless of the
method of inoculation (i.e., milk, surface, brine), the basic conclusions from these reports
were similar to those reached by Ryser and Marth [260] years earlier in that (a) L. monocy-
togenes failed to grow during cheesemaking, (b) low numbers of listeriae were recovered
during the period of rapid growth of Penicillium candidum on the cheese surface, (c)
populations of listeriae in cheese increased rapidly after 21 to 28 days of ripening, and
(d) maximum Listeria populations of >106 CFU/g were detected in surface slices from
fully ripened cheese. However, Terplan et al. [289] found that interior samples from 4-
to 56-day-old Camembert cheeses ripened at 5C consistently contained < 100 L. monocy-
togenes CFU/g and never attained pH values >5.6 even after 56 days of ripening. Hence,
under these conditions, growth of the pathogen was probably suppressed or severely re-
tarded. However, some cells may have been sublethally injured during continuous expo-
sure to this acidic environment, which in turn would have probably decreased the number
of Listeria colonies observed on selective plating media. Two of these studies also ad-
dressed the influence of cheese ripening temperature on Listeria growth [ 108,2841. As
expected, growth of L. monocytogenes was enhanced as the cheese storage temperature
was increased from 3 to 15C in response to more rapid ripening of the cheese and a
concomitant increase in pH.
Current evidence suggests that this pathogen behaves similarly in naturally contami-
nated, conimercially produced soft and semisoft mold-ripened cheese. While conducting
a survey of soft/semisoft cheese sold in Canada, Farber et al. [ 1661 discovered eight 4-
month-old French cheeses, presumably of the BrieKamembert variety, that contained
- 104-105L. monocytogenes CFU/g. Following 1 year of continuous storage at 4"C, Liste-
ria populations remained constant in one cheese and decreased only 10- to 100-fold in
the seven remaining cheeses. In view of these results, it is easy to understand why this
pathogen has been most frequently detected in soft/sernisoft cheeses that have been surface
ripened by molds.
Blue Cheese
Blue-veined cheeses such as blue, Roquefort, and Gorgonzola that are ripened internally
and sometimes externally with P. roqueforti or P. glaucum also have been examined for
their ability to support growth and survival of listeriae. Papageorgiou and Marth [237] used
the modified Iowa method to manufacture blue cheese from pasteurized milk inoculated to
contain approximately 1000 L. rnonocytogenes (strains Scott A or CA) CFU/mL. All
cheeses were ripened 84 days at 9-12C (90-98% RH) and then held an additional 36
days at 4C.
Numbers of listeriae increased by an average of 1.50 log,, CFU/g during the first
24 h of manufacture, with increases of 0.62 and 0.71 log,, CFU/g being attributed to
entrapment of the organism within curd particles and growth, respectively. Growth of L.
458 Ryser

monocytogenes occurred primarily during the first 9 h of manufacture and ceased when
the pH of the cheese dropped below 5.0. As expected, somewhat less growth occurred in
two lots of cheese with particularly rapid acid production.
Unlike the behavior of L. monocytogenes in Camembert cheese, the pathogen not
only failed to grow during ripening of blue cheese, but decreased in numbers by two to
nearly three orders of magnitude during the first 56 days of storage at 5C (Fig. 6). These
decreases, which occurred despite favorable pH values that developed during ripening
from growth of P. roqueforti, were most likely caused by formation of free fatty acids
[27 11, with listeriostatic/listeriocidal levels of caproic, caprylic [204], lauric, and other
medium-chain fatty acids [205] produced as by-products of P. roqueforti growth during
ripening of blue-veined cheeses. However, at least eight different P. roqueforti strains can
also produce listeriocins in laboratory media [ 1731. Nonetheless, combined effects of a
relatively high pH and low storage temperature were probably responsible for both Listeria
strains surviving at least 120 days in all lots of blue cheese. Although additional tests

f +T r i a l
__t_ Trial 2
I

._f_ l r i a l 3

0 20 40 60 80 100 120 140

Days
7

?i
5
-* Trial 1
Trial 2
Trial 3
4
0 20 40 60 80 100 120 140
Days

FIGURE6 Changes in population of L. monocytogenes strain Scott A and pH during

ripening of blue cheese. (Adapted from Ref. 237.)
L. monocytogenes in Fermented Dairy Products 459

showed that strain Scott A was evenly distributed throughout blocks of 120-day-old blue
cheese, strain CA was far less tolerant to environmental conditions on the cheese surface
and was detected in such samples only after cold enrichment. The lengthy survival of L.
rnonocytogeizes in blue cheese, coupled with the recall of Danish blue cheese and isolation
of L. rnonocytogenes from Italian Gorgonzola cheese, all stress the importance of preparing
blue-mold cheeses from properly pasteurized milk under good hygienic conditions to pre-
vent a possible public health problem involving Listeria.

Bacterial Surface-Ripened Cheeses

This group of cheeses consists of soft and semisoft varieties that are ripened under condi-
tions which induce a progression of microbial growth on the cheese surface. Examples
of such cheeses include brick from the United States, Pont 1'EvEque and Saint Paulin from
France, Tilsiter from Germany, Trappist from Yugaslavia, Havarti from Denmark, Be1
Paese from Italy, and Limburger from Belgium. Differences between these varieties result
from the shape of the cheese as well as the amount and/or type of surface growth. Microor-
ganisms are not normally added as pure cultures but rather develop naturally on the cheese
surface, since ripening conditions promote growth of organisrns that are normally present
in the ripening room.
Proper aging of these surface- or smear-ripened cheeses results from the sequential
growth of halotolerant yeast, lactic acid-metabolizing bacteria (Micrococcus spp.), and
Brevibacteriurn linens, the last-named organism being essential for proper flavor develop-
ment. As in mold-ripened cheeses, a pH gradient also develops during aging of bacterial
surface-ripened cheeses, which in turn creates a more favorable environment for growth
of contaminating microorganisms, including Listeria.
Brick Cheese
Using the washed curd procedure, Ryser and Marth [264] prepared brick cheese from
-
pasteurized milk inoculated to contain 102- 1O3 L. rnonocytogenes (strain OH, Scott A,
V7, or CA) CFU/mL. Following manufacture, cheeses were smeared with a culture of B.
linens and ripened at 15"C/95% RH for 2, 3, and 4 weeks to simulate production of mild,
aged, and Limburger-like brick cheese, respectively. Since a natural pH gradient develops
as brick cheese ripens, three types of cheese samples-surface, interior, and slice (surface
and interior)-were analyzed for numbers of listeriae during 20-22 weeks of additional
storage at 10C.
Populations of strains OH, Scott A, CA, and V7 increased approximately 64.6-,
37.2-, 7.4-, and 6.8-f0ld, respectively, on completion of brining approximately 32 h after
the start of cheesemaking. Since a population increase of approximately 10-fold can be
attributed to entrapment of listeriae within curd particles, with relatively few organisms
appearing in the whey, growth of L. rnonocytogenes during the latter stages of cheesemak-
ing before brining was confined to strains OH and Scott A. Numbers of listeriae remained
relatively stable at 103-104CFU/g of cheese during brining; however, a few organisms
were leached from the cheese into the 22% NaCl brine solution. Information on behavior
of Listeria in salt brine solutions appears at the end of this chapter.
Strains OH (isolated from Liederkranz, a bacterial surface-ripened cheese formerly
produced in Ohio) and Scott A grew rapidly during the initial 2 weeks of smear develop-
ment required to manufacture mild brick cheese and generally attained maximum popula-
tions of approximately 6.20 and 6.60, 6.90 and 7.0, and 5.10 and 5.60 log,,,CFU/g in 4-
460 Ryser

week-old slice (pH 6.0-6.5), surface (pH 6.5-6.9), and interior (pH 5.6-6.2) samples,
respectively. During the remaining 20 weeks of ripening at 1O"C, numbers of strains OH
and Scott A generally decreased only 1- to 7-fold in mild brick cheese. Both strains also
behaved similarly in aged and Limburger-like cheese during smear development and ex-
tended storage at 10C.
In contrast, strains CA and V7 failed to grow appreciably during or after smear
development, despite favorable pH values of 6.8-7.4 in fully ripened cheese. Although
strains CA and V7 were detected only sporadically in 4- to 26-week-old samples of mild,
aged, and Limburger-like cheese at levels ranging between 2.7 and 4.6 log,, CFU/g, both
strains were routinely recovered from 24- to 26-week-old slice, surface, and interior sam-
ples after cold enrichment. Hence, all four L. monocytogenes strains survived beyond the
normal shelf life of brick cheese. Subsequent experiments [26 11 dealing with possible
antilisterial effects of several sulfur compounds (i.e., methyl sulfide, dimethyl disulfide,
and methyl trisulfide) produced during ripening of brick cheese failed to explain the inabil-
ity of strains CA and V7 to grow in mild, aged, and Limburger-like brick cheese. Addi-
tional possibilities include (a) inhibition of strains CA and V7 by smear-ripening organ-
isms such as Geotrichum candidum [2 151, Lactobacillus plantarum [ 1631, Brevibacterium
linens [2 181, enterococci [ 178,2661, coryneform bacteria [266], and/or certain staphylo-
cocci [266], all of which can reportedly produce bacteriocin-like substances active against
listeriae or (b) heightened sensitivity of these L. monocytogenes strains to the inhibitory
effects of certain listeriocidal fatty acids (i.e., linoleic) and monocglycerides [301] pro-
duced during cheese ripening.
In conjunction with the previously mentioned European study involving Camembert
cheese, Terplan et al. [289] also assessed the behavior of Listeria during manufacture and
ripening of red smear-ripened ("brick-like") cheese. When this cheese was produced from
pasteurized milk inoculated to contain 95 L. monocytogenes CFU/mL, numbers of listeriae
increased 10-fold after the coagulum was cut as a result of entrapment within the curd;
however, no growth of the pathogen was detected during the remainder of cheese manufac-
ture. In fact, unlike the study by Ryser and Marth [264], Listeria populations decreased
10-fold by the time the cheese (pH 4.9) was ready for brining, with the cheese containing
only 9 L. rnonocytogenes CFU/g after brining. Following 8 days of smear development
at 16SC/93% RH, all cheeses were ripened at 5C for an additional 62 days. Listeria
populations close to the cheese surface increased from 2.5 X 10' CFU/g immediately
after smear development to 1.5 X 104 CFU/g in 14-day-old cheese, during which time
the pH increased from 4.9 to 5.1. Continued ripening of brick-like cheese at 5C led to
development of stable L. monocytogenes populations of 2.5 X 105CFU/g in 1-cm thick
surface slices of 42-day-old cheese. However, unlike the study by Ryser and Marth [264],
the pathogen was never detected in interior cheese samples that were more than 4 days
old despite pH values of 5.7 in interior samples of 56-day-old cheese. Although results
of Ryser and Marth [264] suggest that L. monocytogenes should at least have been isolated
occasionally from interior samples of brick-like cheese, the FDA procedure used in this
study was unable to detect listeriae in these samples, possibly because of acid injury which
may have occurred during exposure to pH values 5 5.2 for as long as 6 months.
Ti lsiter Cheese
In 1995, Bachmann and Spahr [ 1071 manufactured Tilsiter cheese (a semifirm, slightly
yellow, smear-ripened variety similar to brick cheese) from milk inoculated to contain
104 L. monocytogenes CFU/mL. Overall, their findings were similar to those observed
L. monocytogenes in Fermented Dairy Products 461

for brick cheese containing strains L. rnonocytogenes CA and V7 [264], with Listeria
populations varying between 103 and 104 CFU/g in Tilsiter cheese during 90 days of
ripening at 10- 13C.

Trappist Cheese
The ability of L. monocytogenes to survive the Trappist cheesemaking process and persist
during 90 days of ripening was investigated by Kovincic et al. [206]. Cheeses were pre-
pared from pasteurized milk inoculated to contain I 02- 1O5 L. rnonocytogenes CFU/mL
and a 1% starter culture inoculum of L. lactis subsp. Zactis and L. Zactis subsp. crernoris.
After rennet coagulation, the curd was cooked at 39C for 45 min, hooped, drained, and
pressed for 10-12 h. Thereafter, the cheese was brine salted (18% NaC1,4 h), dried (5 days
at 16-18Q waxed, and aged at 10C for up to 90 days. Populations of L. rnonocytogenes
increased -10-fold in the finished cheese during the first 30 days of ripening, stabilized
over the next 30 days, and then gradually decreased to levels approaching the original
inoculum after 90 days of storage. Similar results were also obtained when L. rnonocyto-
genes was added to the curd/whey mixture rather than the pasteurized milk during cheese-
making. The limited growth and extended survival of L. rnonocytogenes in Trappist cheese
(pH 4.9, 30% moisture, 1.4% NaCI) is generally similar to what has been observed for
several common varieties of semisoft/hard cheeses to be discussed shortly.

Soft Italian Cheeses

Most of the Italian soft cheeses are classified as pasta filata or plastic curd cheeses
and include such varieties as mozzarella, Provolone, Caciocavallo, and Scamorze with
mozzarella clearly being the most economically important. Traditionally, fresh mozzarella
has been produced from the high-fat milk of the water buffalo, whereas mozzarella cheese
for use on pizza is manufactured from cows milk and aged several weeks to develop
proper elasticity and meltability. Production of these pasta filata-type cheeses is similar
in that the resulting curd is always stretched in hot (160-190F) water and then kneaded
and molded into a characteristically shaped mass of curd which is hardened in cold water,
brine salted, and ripened for various times.

Mozza re1la
The severe heat treatment that the cheese curd receives and the reported thermal tolerance
of listeriae led Buazzi et al. [125] to assess the fate of L. rnonocytogenes during manufac-
ture of mozzarella cheese. When this cheese was prepared from pasteurized milk inocu-
lated to contain 104- 105L. rnonocytogenes strain OH, CA, or V7 CFU/mL, Listeria popu-
lations of 104-105 CFU/mL were reported in the curd after cutting, cooking, and
cheddaring. However, immersing and stretching the curd 3 to 4 min in hot water (77C)
led to the complete demise of the pathogen. Given that the temperature of the curd was
maintained at 65C for at least 2 min and that L. monocytogenes has a reported D-value
of 28.1 s at 65C [214], mozzarella cheese should be Listeria-free even if small numbers
of the pathogen are present in the curd before stretching. These findings are generally
similar to those reported during manufacture of traditional mozzarella cheese from buffalo
milk [299], with L. rnonocytogenes populations decreasing at least 100-fold during brief
stretching of the curd at 90-95C. Although a few survivors remained after curd stretching
and molding, all cheeses prepared from milk inoculated to contain 1O3 and I O5 L. rnonocy-
462 Ryser

togenes CFU/mL were free of this pathogen after 24 and 48 h of refrigerated storage,
respectively.
The heat treatment given to mozzarella cheese curd is clearly sufficient to inactivate
small numbers of listeriae that might be present. However, ample opportunity exists for
postprocessing contamination as evidenced by recent surveys and a Class I recall that was
issued in early 1991 for over 89,000 lb of mozzarella cheese harboring L. monocytogenes.
Stecchini et al. [283] addressed the issue of postprocessing contamination by inoculating
the surface and packaging fluid of mozzarella cheese with L. monocytogenes and then
storing the product at 5C for up to 21 days. Under these conditions, numbers of listeriae
increased about 10,000-fold during 21 days of storage, with inclusion of a crude heat-
treated bacteriocin preparation from L. lactis subsp. lactis yielding final populations only
10-fold lower as compared with untreated controls. Thus, manufacturers of mozzarella
cheese must adhere to good manufacturing and sanitary practices to prevent contamination
and growth of L. monocytogenes to potentially hazardous levels in this product during
storage.

Semisoft and Hard Cheeses

By definition, hard cheeses are those which contain 5 40% moisture. Cheeses in this
category include such varieties as Edam and Gouda (which can also be classified as semi-
soft cheeses) as well as Colby, Cheddar, Swiss, Emmentaler, Gruykre, Romano, and Par-
mesan, the last two of which are very hard grating cheeses. Transformation of chalky, acid-
tasting curd into a ductile, full-flavored cheese is accomplished during ripening through the
action of milk enzymes, rennet, and various microorganisms in the cheese, including the
starter culture. The biochemical changes which occur during cheese ripening are complex
and involve hydrolysis of fats and proteins with subsequent decarboxylation, deamination,
and/or dehydrogenation as well as production of carbonyls, nitrogenous compounds, fatty
acids, and sulfur compounds, all of which contribute to the overall flavor of the final
product. The amount of aging needed to obtain a fully ripened cheese is directly related
to moisture content, with a minimum of 2 and 10 months of ripening being required for
Edam (-40% moisture) and Parmesan cheese (-30% moisture), respectively. The current
popularity of many of these cheeses, along with the ability of L. monocytogenes to survive
in acidic environments during refrigerated storage, has prompted a series of studies exam-
ining the behavior of L. monocytogenes during manufacture and storage of at least seven
semisoft and hard cheese varieties.
Gouda and Maasdam Cheeses
Beginning with semisoft/hard cheeses, Northolt et al. [235] prepared Gouda and Maasdam
cheese in The Netherlands from pasteurized milk inoculated to contain approximately
500 L. monocytogenes CFU/mL. Gouda cheese was manufactured according to standard
procedures, with the exception that one lot was prepared using 0.3 rather than 0.6% starter
culture to obtain a cheese with an unusually high moisture content of -45%. Maasdam
cheese was prepared using a culture of propionic acid bacteria in combination with 0.6%
mesophilic lactic starter. After brine salting, Gouda cheese was ripened 6 weeks at 13OC,
whereas Maasdam cheese was ripened 2 weeks at 13"C, 2 weeks at 18OC, and then stored
at 4C for an additional 2 weeks.
As in previous studies, entrapment of listeriae within curd particles during cheese-
making resulted in population increases of approximately 10-fold as compared with the
L. monocytogenes in Fermented Dairy Products 463

original level in pasteurized milk. However, some Listeria growth was noted during manu-
facture, with populations increasing an additional fourfold in normal Gouda and Maasdam
cheese before brining. Six hours after manufacture, slightly higher Listeria populations
were detected in Gouda cheese of high rather than normal moisture. Although numbers
of listeriae in interior samples from both Gouda and Maasdam cheese remained relatively
constant at 104CFU/g during the first 2 weeks of ripening, the pathogen was not detected
I-

in cheese samples taken at or near the surface. After 6 weeks of ripening, L. monocytogenes
reappeared in surface samples from all cheeses at levels between 102and 104CFU/g. In
contrast, numbers of listeriae in interior samples from 6-week-old cheese were only four-
to eightfold lower than populations in the same cheeses immediately after brining. Al-
though L. monocytogenes survived best in high-moisture Gouda cheese which had a pH
of 6.0, the selective plating medium used in this study, TrypaBavine Nalidixic Acid Serum
Agar, proved to be less than optimal for recovery of stressed or acid-injured listeriae that
probably were present in fully ripened Gouda and Maasdam cheese having pH values of
5.48 and 5.44, respectively.
Colby Cheese
Yousef and Marth [306] prepared Colby cheese from pasteurized milk inoculated to con-
tain 102-103L. monocytogenes (strain V7 or CA) CFU/mL. Following manufacture, all
blocks of cheese were held at 4C for 140 days.
During cheesemaking, most Listeria cells were trapped in curd, with an average of
only 2.4% of the original inoculum escaping in whey. Populations of L. monocytogenes
in cheese increased an average of 1.27 orders of magnitude after pressing-about 29 h
after the start of manufacture (Fig. 7). Since an increase of no more than one order of
magnitude can be attributed to entrapment of listeriae within curd particles after cutting,
these findings suggest that slight growth of the organism did occur, particularly during

"1

Time

FIGURE7 Behavior of L. rnonocytogenes during manufacture and ripening of Colby

cheese. (Adapted from Ref. 306.)
464 Ryser

the later stages of cheesemaking and pressing. Numbers of both Listeria strains remained
relatively constant in cheese during the first 40 days of ripening, after which populations
decreased almost linearly (Fig. 7). Viability of L. rnonocytogenes was strongly influenced
by moisture content with strain V7 decreasing more than twice as fast in cheese containing
38.5% (D-value of 54 days) rather than 42.3% (D-value of 124 days) moisture, which is
well above the maximum allowable moisture content of 40.0% for Colby cheese.
Behavior of L. rnonocytogenes in cheese of normal moisture content also was strain
dependent, with strain CA being less stable than strain V7. However, strains V7 and CA
were still detected in 140-day-old Colby cheese by direct plating, with cold enrichment
results from a follow-up study [308] indicating that both strains were still viable in 5- to
8-month-old Colby cheese stored at 4C. According to the FDA, Colby and other selected
cheeses can be manufactured from raw or heat-treated (subpasteurization) milk provided
that the finished cheese is held a minimum of 60 days at or above 1.7"C (35F) before
sale in an attempt to eliminate pathogenic microorganisms. These results (and those for
Cheddar cheese to follow) have recently prompted the FDA to reconsider the adequacy
of this aging requirement for cheeses prepared from raw milk.
Cheddar Cheese
Normal stirred-curd Cheddar cheese, which has a moisture content only slightly less than
Colby cheese (i.e., 36-38%), was manufactured by Ryser and Marth [259] from pasteur-
ized whole milk inoculated to contain approximately 5 X 102L. rnonocytogenes (strain
Scott A, V7, or CA) CFU/mL. The resulting 10-lb blocks of cheese were ripened at 6
and 13C and assayed for numbers of listeriae at appropriate intervals.
All curd samples examined during manufacture contained approximately 5 X 102
L. rnonocytugenes CFU/g, which suggests that the organism was only minimally concen-
trated in the curd and failed to grow during cheesemaking. However, since only 6.4% of
the initial Listeria inoculum was recovered in the whey, the expected 10-fold increase
from entrapment of the organism in curd particles probably went unnoticed because of
inadequate sample preparation methods which have since been improved in our laboratory
[308]. Numbers of listeriae increased slightly in cheese during pressing, with all three
-
strains attaining maximum populations of 3.50-3.75 log,, CFU/g after 14-35 days of
ripening at 13 (Fig. 8) and 6C (Fig. 9). This population increase, which was approximately
10-fold higher than that of the original inoculum in milk, probably resulted because of
enhanced recovery of the pathogen from older cheese which was easier to homogenize
rather than from actual growth, as shown by Yousef et al. [308]. After 35 days of storage
at either temperature, Listeria populations in cheese began to decrease, with all cheeses
maintaining pH values of 5.04-5.09 throughout ripening. Strains Scott A, V7, and CA
survived 70-224, 126-196, and 70-126 days in cheese ripened at 13"C, respectively,
whereas the same strains remained viable for 70-154, 126-434, and 70-154 days in
cheese aged at 6C. Thus, except for strain V7, which was still present in one block of
434-day-old cheese at a level of 30 CFU/g, the remaining two strains survived equally
well in cheeses ripened at either temperature. These findings suggest different acid toler-
ances among the L. rnonocytogenes strains tested, as has also been reported by Gahan et
al. [172]. Additional experiments with strains V7 and CA demonstrated that L. rnonocyto-
genes was uniformly distributed in Cheddar cheese during at least the first 98 days of
ripening at 6C. Working in England, Banks [ 1101 also reported that L. rnonocytogenes
persisted 8-9 months and up to 7 months in Cheddar cheese prepared from Listeria-
contaminated raw and subpasteurized (i.e., 62.8 or 65.6"C) milk, respectively. Hence,
L. monocytogenes in Fermented Dairy Products 465

4.0 r

2.01
-- * Trial 4

-A Trial 5

\
--4 Trial 6
b
\

\
\

\
1 .o L

t
0 0
A A2 A A
0 0

FIGURE 8 Survival of L. monocytogenes strain V7 in Cheddar cheese ripened at 13C.

Open symbols at < I log,,, Listeria/g indicate that the organism was not detected after
8 weeks of cold enrichment, whereas half-solid or solid symbols at < I log,, Listeria/
g indicate that the pathogen was detected in one of two or two of two samples, respec-
tively, using cold enrichment. Numbers indicate the week at which L. monocytogenes
was found. (Adapted from Ref. 259.)

these data provide some of the strongest evidence for the inadequacy of the 60 day/? 1.7"C
minimum holding period for cheeses manufactured from raw milk.
Several subsequent studies examined the effect of Cheddar cheese compositional
changes on Listeria survival during cheese ripening. Mehta and Tatini [226] assessed the
behavior of L. monocytogenes strains Scott A and V7 in stirred-curd Cheddar cheese
containing 1.3 or 2.5% NaCl or an equal molar mixture of NaCl and KCl. Lowering the
level of NaCl enhanced destruction of Listeria, with 1.3% NaC1, 2.5% Na/KCl, and 2.5%
NaCl decreasing populations 4.3-, 2.3- and 0.4-orders of magnitude in cheese, respec-
tively, after 10 weeks of aging at 7C. Thus, in addition to being healthier, low-sodium
Cheddar cheese appears to be safer in regard to listeriae. When these same investigators
[227] prepared stirred-curd Cheddar cheese from whole milk and reduced fat milk (1.55
or 2.0% milkfat), L. monocytogenes strains Scott A and V7 persisted in the finished cheese
during 20 months of storage at 7OC, with no survival differences being observed between
full fat (28.9% fat) and reduced fat (20.7%) cheese. However, Listeria survival in Cheddar
cheese is strongly influenced by milk fat composition and release of free fatty acids during
cheese ripening. Schaffer et al. [271] increased the levels of both long-chain (c18, c18.2)
and unsaturated fatty acids in milk by feeding cows a diet of extruded soybeans or sun-
flower seeds and then using this milk to manufacture stirred-curd Cheddar cheese con-
taining L. monocytogenes strains Scott A and V7 as previously described. During manufac-
ture, numbers of listeriae increased 1.O- 1.5 orders of magnitude as previously reported
466 Ryser

-- Trial 4

+Trial 5
-4 Trial 6

'w-- <
\
\
\
\
I
L

FIGURE 9 Survival of L. monocytogenes strain V7 in Cheddar cheese ripened at 6C.

See Figure 8 for explanation of symbols. (Adapted from Ref. 259.)

by Ryser and Marth [259]. More important, after 120 days of ripening at 7C L. monocyto-
genes populations were five- to eight orders of magnitude lower in cheeses prepared from
modified fat milk as compared with unmodified milk. Although some inconsistences were
noted in performance of sunflower-and soybean-modified milk, inactivation of L. monocy-
togenes always occurred most rapidly in cheeses containing the highest levels of free fatty
acids, with oleic, linoleic, lauric, and myristic acids shown to be major contributors to
Listeria destruction during cheese ripening.

Swiss Cheese
Unlike the aforementioned cheeses, manufacture and ripening of Swiss cheese involves
several decidedly different steps, including cooking of the curd at 50-53C and ripening
the finished cheese at an elevated temperature for "eye" development. These observations
prompted Buazzi et al.[ 1261 to examine the fate of L. monocytogenes during manufacture
and ripening of Swiss cheese. When rindless Swiss cheese was prepared from pasteurized
milk inoculated to contain 104-105 L. monocytogenes strain V7, CA, or OH CFU/mL,
the pathogen was generally unable to grow during cheesemaking, with populations increas-
ing 43% during the early stages of cooking owing to physical concentration and curd
shrinkage. Thereafter, about 57% of the population in the curd was inactivated after 30-
40 min of cooking at 50C. After pressing, the curd contained 50% fewer listeriae, with
this population decreasing most sharply after 30 h of brining at 7C. Storing the finished
cheese (pH 5.2-5.4) 10 days at 7C reduced the Listeria population to very low numbers.
Complete inactivation of the pathogen occurred after 66-80 days of ripening at 24"C,
L. monocytogenes in Fermented Dairy Products 467

with production of propionate by eye-forming bacteria likely contributing to the death of

listeriae. Two studies conducted in Switzerland [ 107,20I ] demonstrated that the environ-
ments within Emmenthaler and Gruykre cheese (i.e., other varieties of Swiss cheese) also
are not conducive to Listeria survival, with the pathogen no longer being present in 24-
hour-old cheeses (pH 5.2-5.4) prepared from raw milk inoculated to contain 104L. mono-
cytogenes CFU/mL.
Parmesan Cheese
Parmesan cheese, a hard cheese with a low moisture content, was prepared by Yousef
-
and Marth [ 3071 from pasteurized milk inoculated to contain 104- 1O5 L. monocytogenes
(strains V7 or CA) CFU/mL. Unlike the cheeses discussed previously, a lipolytic enzyme
(lipase) is often added to cheesemilk to produce the characteristic flavor of fully ripened
Parmesan cheese. In addition, the coagulum is cut into very small particles which are
cooked at -52C (125F) for 45 min until the pH decreases to 6.1. This step serves to
expel whey, thus producing a dry, rice-like curd which can be pressed to form a very
dense, low-moisture cheese. Following manufacture, the cheese produced in this study
was brine salted (22% NaC1) for 7 days at 13OC, dried 4-6 weeks in a humidity-controlled
chamber at I3"C, vacuum-packaged, and ripened at 13C for a minimum of an additional
9 months.
During the first 2 h of cheesemaking, populations of both Listeria strains increased
approximately 6- to 10-fold, largely from entrapment of the organism within curd particles
(Fig. 10). Although Listeria counts remained relatively stable during cooking of the curd
at 52C ( I 25F) for 45 min, populations decreased appreciably during pressing of the
curd. During brining, drying, and ripening at 13C numbers of both Listeria strains de-
creased almost linearly, with estimated D-values ranging between 8 and 36 days. Using
direct plating, strains V7 and CA were no longer detected in cheese after 21-1 12 and
14-63 days of ripening at 13"C, respectively. Despite large differences in survival of L.
monocytogenes between different batches of cheese, both Listeria strains decreased at a
faster rate in Parmesan than in Gouda, Maasdam, Cheddar, and Colby cheese during ripen-
ing. Decreased viability of the pathogen in Parmesan cheese is probably related to a combi-
nation of factors, including (a) action of lipase added to the milk, (b) heat treatment that
the curd receives during cheesemaking, and (c) lower moisture content (and water activity)
of the fully ripened cheese. To decrease the moisture content and develop proper flavor,
the present regulation in the United States requires that Parmesan cheese be aged a mini-
mum of 1 0 months regardless of whether or not the cheese is prepared from raw or pasteur-
ized milk, According to the results of this study, such an aging process should be sufficient
to produce Listeria-free Parmesan cheese.
Hard Italian-Type Cheese
Working in Italy, Comi and Valenti [ 1351 inoculated the surface and interior of three
freshly prepared hard Italian-type cheeses (a, 0.95-0.98, pH 5.2-5.5) to contain 104- 105
L. monocj~togenesCFU/g. As was true for Parmesan cheese, the pathogen failed to grow
in this relatively hard cheese, with Listeria populations decreasing 10- to 100-fold in both
surface and interior samples from all three cheeses during the first 28 days of ripening at
4C. Although the pH of surface and interior samples increased to 5.4-5.5 and 5.8-6.0,
respectively, after 35 days ripening, Listeria populations continued to decrease, with the
pathogen only being detected by cold enrichment (i.e., populations < 10' CFU/g) in sam-
468 Ryser

E
107
Manufacture

106

lofi

104
-
-
Batch # 1
103 --.I Batch # 3
Batch H6

-
102 -
--
-
-
-
10 J L
0 1 2 3 0 40 80 120
Days
Hours

FIGURE10 Survival of Listeria rnonocytogenes strain V7 during manufacture and

ripening of Parmesan cheese. (Adapted from Ref. 306.)

ples from 35-day-old cheeses. Compositional analysis of these cheeses suggested that the
amount of moisture lost after 35 days of ripening (a, 0.95-0.96) may have offset the
benefit for Listeria growth caused by the increase in pH.

Hispanic Cheeses
Traditional Hispanic-type cheeses comprise a wide range of white cheeses produced in
Mexico and in Central and South America. Some of the most popular varieties, including
Queso Blanco, Queso Fresco, and Queso de Puna, are high-moisture fresh cheeses con-
sumed shortly after manufacture, whereas others, such as Queso Anejo, Queso de Bola,
Queso de Crema, Queso de 10s Ibores, and Queso de Prensa, are lower in moisture and
undergo various degrees of aging. Although 30 min of heating at 80-85C is more than
adequate to inactivate L. monocytogenes during manufacture of Queso Blanco cheese
[ 1791, typical production practices for Hispanic-type cheeses involve extensive curd mani-
pulations, including hand stirring, salting, and molding, any of which can easily lead to
L. monocytogenes in Fermented Dairy Products 469

product contamination. Queso Blanco and Queso Fresco cheese pose a particular threat
to the industry given the involvement of these cheeses in the 1985 listeriosis outbreak in
California.
Queso Blanco Cheese
In 1995, Glass et al. [179] reported on the behavior of L. monocytogenes in starter culture-
free Queso Blanco cheese containing citric, malic, or acetic acid as acidulants and ALTA
(a commercial bacteriocin preparation resembling pediocin AcH) as an antilisterial agent.
After the finished product (pH 5.2) was inoculated to contain 106L. monocytogenes CFU/
g, populations increased about 10-fold in cheeses containing citric or malic acid during
42 days of storage at 4C, whereas numbers of listeriae decreased slightly in cheeses
prepared with acetic acid. These findings are consistent with those of other investigators
who used various laboratory media acidified with malic, citric, or acetic acid (see Chap. 6).
Addition of 0.6% ALTA to these cheeses yielded slightly lower Listeria counts as com-
pared with cheeses without ALTA. Using an L. monocytogenes inoculum level of 102
CFU/g, these workers concluded that acetic acid was significantly more effective than
malic or citric acid in reducing numbers of L. monocytogenes in Queso Blanco cheese
and that addition of ALTA provided added protection against this pathogen. Similar bene-
fits also were reported when Queso Blanco cheese was prepared using a nisin-producing
starter culture, with L. monocytogenes populations being about 1000-fold lower in such
cheeses (pH 5.3) after 21 days of storage at 4 or 12C than in nisin-free controls [144].
Although direct addition of Nisaplin (1000 AU/mL) to the cheese milk yielded Listeria
populations 100-fold lower in 1-day-old cheeses than in Nisaplin-free controls, the patho-
gen recovered to control levels within 21 days at 12C. Hence, incorporating a nisin-
producing starter culture was superior to direct addition of Nisaplin for minimizing sur-
vival of L. monocytogenes in Queso Blanco cheese during storage.
Queso de 10s lbores Cheese
The fate of L. monocytogenes in Queso de 10s Ibores cheese (a hard, ripened cheese of
pH -5) also was indirectly determined by Mas and Gonzalez-Crespo [219] using commer-
cially available cheeses of various ages. Overall, detecting Listeria spp. in 5 of 10, 2 of
10, and 1 of 10 cheeses that had been aged for 7, 30, and 60 days, respectively, suggests
that this hard, low-moisture cheese will not support long-term survival of listeriae.

Pickled Cheeses
The terms pickled and white-brined are often used to describe a group of soft/semisoft,
white curd cheeses to which large quantities of salt are added as a preservative. Cheeses
belonging to this group are principally manufactured in countries bordering the Mediterra-
nean Sea and include such varieties as feta (Greece), Turkish white-brined cheese (Tur-
key), Teleme (Bulgaria), Domiati (Egypt), and Kareish (Egypt). Some of these cheeses
are frequently prepared from ewes, goats, or buffalos milk. Depending on the cheese
variety, salt either can be added directly to the milk or curd, or the finished cheese can
be stored in salt brine, salted whey, salted skim milk, or dry salt. The extreme tolerance
of L. monocytogenes to high concentrations of salt, along with the organisms ability to
grow at refrigeration temperature, has made these cheeses of particular interest to food
microbiologists working with Listeria.
470 Ryser

Feta and Turkish White-Brined Cheese

In 1989, Papageorgiou and Marth [238] described the fate of L. monocytogenes during
manufacture, ripening, and storage of feta cheese. During the course of this work, there
was an unconfirmed report of a woman in New York who delivered a stillborn infant in
December of 1987 after consuming feta cheese contaminated with L. monocytogenes.
Hence this study, which will now be discussed, took on added importance.
According to these authors, cow's milk was inoculated to contain approximately 5
X 103L. monocytogenes (strain Scott A or CA) CFU/mL. After warming milk to 35"C,
a 1% commercial starter culture of S. thermophilus/L. bulgaricus was added. Forty min
after addition of rennet, the coagulum was cut and the resulting curd was transferred to
metal hoops. Following 6 h of draining, cheeses were removed from the hoops and placed
in a 12% salt brine solution for 24 h. The following day, all cheeses were transferred to
6% salt brine at 22C for 4 days until the cheese attained a pH of 4.3-4.4. Finally, cheese
in the same 6% brine solution was moved to storage at 4C.
Cells of L. monocytogenes were entrapped in the curd during cheesemaking, with
populations nearly 10-fold greater in curd than in inoculated milk. Only about 3.2% of
the original inoculum was lost in the whey. During whey drainage and the first 1-2 days
of ripening at 22"C, numbers of listeriae increased - 1.5 log,,, CFU/g, with both strains
attaining maximum populations of approximately 1 X 106 CFU/g (Fig. 11). Although
growth of both Listeria strains generally ceased at pH values between 4.6 and 5.0, numbers
of listeriae remained virtually constant in cheese during 2-5 days of storage at 22C in

- 7

Average pIl
%I
. . 6

PH
- s

Cp- Trial 1
- 4
&-- Trial 2
Trial 3

' I . , ' I t , , , , , I
- 3
-20 0 20 40 60 80 100 120 I40
Hours

FIGURE11 Fate of L. rnonocytogenes strain Scott A during manufacture and early

brining of feta cheese. (Adapted from Ref. 238.)
L. monocytogenes in Fermented Dairy Products 471

6% brine solution. Both salt brines in which feta cheese was ripened and/or stored were
positive for listeriae (these details appear in our discussion of brine solutions at the end
of this chapter). Although all feta cheeses older than 5 days maintained a pH of 4.3, both
L. monocytogenes strains survived >90 days in finished cheese stored at 4C (Fig. 12).
However, differences between the two Listeria strains were noted, with populations of
strains Scott A and CA decreasing 1.28 and 3.07 log,, CFU/g in 90-day-old as compared
with 2-day-old feta cheese, respectively. Sarumehmetoglu and Kaymaz [267] reported
that L. monocytogenes behaved similarly in Turkish white-brined cheese prepared from
artificially contaminated raw milk, with numbers of listeriae generally decreasing < 100-
fold in the finished cheese during 90 days of refrigerated storage. Although feta and Turk-
ish white-brined cheese can be prepared from raw milk, ripening such cheese at or above
1.7"C for 60 days will not in any way guarantee that the final product is Listeria-free with
long-term survival of this pathogen highly probable. Hence, it would appear prudent to
manufacture these cheeses only from pasteurized milk under good hygienic conditions to
decrease the chance of a public health problem involving Listeria.
Domiati Cheese
Domiati cheese, a popular fresh white-brined cheese most commonly consumed in Egypt
and other parts of the Middle East, was prepared by Tawfik [286] using a 1 : 1 mixture
of pasteurized cow: buffalo milk to which 7.5% NaCl, 0.5% Lactobacillus casei, and 106
L. monocytogenes were added. Listeria populations increased approximately 10-fold in
the finished cheese as a result of entrapment within the curd and then decreased over time,
with the pathogen surviving 4-8 weeks when the cheese was stored in salted whey at
20-25C. Similar findings were reported by Ahmed et al. [ 5 ] , with L. monocytogenes
strain V7 being inactivated in Domiati cheeses (pH 4.5-5.5) containing 5 and 10% NaCl

Trial 1
&- Trial 2
Trial 3

2 1 8 I I I I I I I I I I 1
0 20 40 60 80 100

Days

FIGURE12 Survival of L. rnonocytogenes strain Scott A during storage of feta

cheese. (Adapted from Ref. 238.)
472 Ryser

during 4 weeks of ripening at 30C. According to Abou-Donia and Al-Medhagi [3], L.

monocytogenes also persisted no more than 8 weeks in naturally contaminated retail sam-
ples of Domiati cheese. A decrease in cheese pH from 6.1 to 3.8 and an increase in salt
content from 6.8 to 11.4% are clearly responsible for limiting growth and survival of L.
monocytogenes in this product.
Bulgarian White-Pickled Cheese
As early as 1964, Ikonomov and Todorov [ 1951 reported manufacturing white-pickled
cheese from ewe's milk containing 102-103 L. monocytogenes CFU/mL. A mixture of
0.1% S. Zactis and Lactobacillus casei served as the starter culture. Although L. monocyto-
genes persisted 15-30 days in white-brined cheese ripened at 18-22OC, the pathogen
survived twice as long when the same cheese was ripened at 12-15C. In addition to
storage temperature, Listeria viability also was partly dependent on the amount of acid
produced in the cheese during ripening, with pH values of approximately 4.3 and 4.6
being reported as lethal to listeriae in cheese ripened at 12-15 and 18-22OC, respectively.
Sudanese White-Pickled Cheese
Working in the United States, Abdalla et al. [l] prepared a traditional Sudanese white-
pickled cheese from Lactococcus starter and Lactococcus starter-free pasteurized milk that
was inoculated to contain 105L. monocytogenes Scott A CFU/mL. During cheesemaking,
Listeria populations increased approximately 10-fold to 106 CFU/g in the cheese, with
growth and acid production by the starter culture being prevented by addition of 8% NaCl
to the milk during cheesemaking. Consequently, the finished product had a pH of 6.5-
7.0 which is optimal for Listeria growth. Relatively stable Listeria populations of approxi-
mately 10' CFU/g developed in the cheese after 30-65 days of refrigerated storage in
brine containing 8.6% NaC1. Similar Listeria populations were observed when the same
starter-free cheese was prepared from pasteurized milk containing 4% NaCl and preserved
in a 4% brine solution with addition of 1% potassium sorbate, nisin (25 pg/mL), or 0.1%
hydrogen peroxide to the milk not affecting Listeria survival [2]. Ineffectiveness of these
antimicrobial agents was attributed to several factors, including loss during processing,
degradation during the early stages of cheese ripening, and suboptimal environmental
conditions. In contrast, when a 1% lactic starter inoculum was used, L. monocytogenes
populations decreased more than 6-orders of magnitude, with the pathogen no longer being
detected in cheese after 50 days of ripening (pH 4.6) at 4C. As was true for the other
pickled cheeses, large numbers of listeriae again leached from the cheese into the brine
solution; these findings are discussed in detail at the end of this chapter.
Yugoslavian White-Brined Cheese
In 1974 Sipka et al. [278] published results from another study in which white-brined
cheese was prepared from naturally infected cow's milk containing 240 L. monocytogenes
CFU/mL. Following manufacture, the pathogen grew rapidly in cheese, reaching popula-
tions of 7.8 X 105 and 1.0 X 106 CFU/g after 7 and 14 days of brining, respectively.
Although maximum Listeria populations were similar to those observed in feta cheese
[238], the pathogen appeared to be less hardy in white-brined than in feta cheese, with
populations decreasing to 1.3 X 105 and 1.2 X 102 CFU/g in 24- and 42-day-old fully
ripened cheese, respectively. Increased inactivation of listeriae in white-brined rather than
feta cheese apparently is not entirely related to acid development, since the pH of the
former cheese, 4.5-5.1, was higher than that of the latter, 4.3.
L. monocytogenes in Fermented Dairy Products 473

Twenty-one years later, Katic [200] prepared a similar white-brined cheese from
artificially contaminated milk containing 0.8% starter culture and found that after 40 days
of storage at 4C, L. monocytogenes populations had increased 100- and 43-fold in cheeses
stored in 10 and 16% brine, respectively. In contrast, numbers of listeriae increased only
4- and 12-fold in identical cheeses that were stored in 10 and 16% brine solutions at 8C,
respectively. Thus, given the same brining conditions, higher storage temperatures were,
as expected, more detrimental to Listeria survival.

Ewes and Goats Milk Cheese

In areas of the world where cows are not plentiful (i.e., northern Scandinavia, Eastern
Europe, Mediterranean, Middle East), ewes and goats milk have been adapted for use
either alone or in combination with cows milk to manufacture different types of cheese.
Representative cheeses in this group include such well-known varieties as French Roque-
fort and Greek feta cheese, both of which are tIaditionally prepared from ewes milk.
Lesser known cheeses typically prepared from ewes and/or goats milk include Egyptian
Kachkaval, Italian Fontina, Spanish Manchego, and Italian Pecorino Romano as well as
many varieties of white-brined and ethnic goats milk cheese, the latter of which are often
produced in mountainous areas of Central Europe and Scandinavia. The occasional pres-
ence of L. monocytogenes in milk from healthy ewes and goats has prompted several
studies dealing with behavior of this pathogen during manufacture and ripening of some
of these less common cheeses.
Kachkaval Cheese
Work with this group of cheeses dates back to 1964 when Ikonomov and Todorov [ 1951
examined the behavior of L. monocytogenes in Kachkaval cheese (a relatively soft, brine-
salted cheese manufactured from ewes milk in Eastern Europe and the Middle East, pH
5.0-5.8) prepared from raw ewes milk inoculated with the pathogen. According to these
investigators, L. monocytogenes survived in curd immersed in 5 6 % salt brine at 71-
76C during cheesemaking and was still present in Kachkaval cheese (pH 5.0-5.4) after
30-50 days of ripening at 18-22C.
Manchego Cheese
The next such study did not appear in the literature until 1987 when Dominguez et al. [ 1491
reported manufacturing four lots of Manchego cheese (a hard aromatic cheese traditionally
prepared from ewes milk in Spain, pH = 5.8) from a blend of pasteurized ewes, goats,
and cows milk (15 :35 :50) inoculated to contain either 4.0 X 103or 1.9 X 105L. monocy-
togenes CFU/mL. To assess growth of listeriae in cheeses having different rates of acid
development, milks were inoculated to contain either 0.1 or 1.0% of a mesophilic lactic
acid bacteria starter culture. The coagulum was cut approximately 45 min after addition
of rennet; the resulting curd was drained, hooped, and pressed for approximately 10 h.
The finished cheese was then brine salted overnight, ripened 10 days at 15C/85-90%
RH, covered with paraffin, and aged an additional 50 days at 15C.
Numbers of L. monocytogenes increased 5 10-fold in all cheeses during the first 10
h of manufacture, primarily from entrapment of the organism within curd particles. The
fact that cheeses prepared with either 0.1 or 1.O% starter culture contained similar Listeria
populations indicates that behavior of the pathogen was not greatly influenced by the rate
of acid production during cheesemaking. After the cheese was brined overnight, popula-
 474 Ryser

tions of listeriae decreased approximately 3- to 100-fold, with additional small decreases

being observed during ripening of unparaffined cheese at 15C. However, numbers of
listeriae remained relatively constant in cheese at pH 5.1-5.8 after paraffining, with popu-
lations in 60-day-old cheese approximating the original inoculum in milk from which the
cheese was manufactured.

Goats Milk Cheese

Working in Sweden, Tham [291] examined the viability of Listeria in cheese prepared
from raw goats milk inoculated to contain 105-106L. monocytogenes CFU/mL. A mix-
ture of mesophilic lactic acid bacteria served as the starter culture. Approximately 45 min
after addition of rennet, the coagulum was cut into cubes which were cooked at 37C,
drained, hooped, pressed for I h, and brine salted for 10 h. The finished cheese was then
ripened at 12C for 22 weeks.
Actual numbers of L. monocytogenes could be determined in cheeses from only two
of six lots using a blood agar/pour plate method. As shown in Fig. 13, Listeria populations
decreased approximately 10-fold in goats milk cheese during the first 14 weeks of ripen-
ing at 12C. Extended survival, along with slight growth of listeriae in cheeses ripened
longer than 14 weeks, probably is related to an increase in pH from 5.55 to 6.20 during
ripening as well as a decrease in numbers of competitive microorganisms that were initially
present in the raw milk. Although large numbers of enterococci and other microbial com-
petitors interfered with the quantitative recovery of L. monocytogenes in the remaining
four cheeses, the pathogen survived 10-16 weeks in three of these cheeses as determined
by cold enrichment. The impact of an active starter culture on the fate of Listeria in
Swedish raw milk goat cheese was also later confirmed by Eilertz et al. [152], with L.

-0 L . m o n o c y t o g e n e s in cheese VI
-4 L . m o n o c y f o g e n e s in cheese V
-A Total aerobic count in cheese V

;- ;
9.0 - 4 Total aerobic count in cheese VI
I
Added I, m o n o c y t o g e n e s per ml milk

-
8.0 *

-L-
7.0 .

6.0 .

5.0 -

L
I I I 1 I I 1 I I I
0 2 4 6 8 10 12 14 16 18
Weeks

FIGURE13 Survival of L. rnonocytogenes and total aerobic flora during manufacture

and ripening of raw goats milk cheese. (Adapted from Ref. 291.)
L. monocytogenes in Fermented Dairy Products 475

rnonocytogenes populations increasing 10- and 10,000-fold in cheeses prepared with and
without starter culture, respectively, after 4 weeks of ripening at 4C.
As you will recall from Chapter 10, a middle-aged English woman developed liste-
rial meningitis in February of 1988 after consuming 2-3 oz of commercially produced
Anari-type goats milk cheese containing 107L. rnonocytogenes CFU/g [63]. During a
series of follow-up investigations [224], L. monocytogenes populations of < 10 CFU/g
were discovered in one 2- to 3-day-old Anari cheese and in three 2- to 3-day-old Halloumi
cheeses produced by the same manufacturer. After these naturally contaminated cheeses
were stored at 4C for 4-5 weeks Listeria populations as high as 8 X 104 and 1 X 106
CFU/g developed in Anari (pH 5-6) and Halloumi cheese (pH 6), respectively. Assuming
a lag time of zero and an original L. rnonocytogenes population of 1-9 CFU/g, these
authors calculated generation times of 47-56 and 32-37 h for this pathogen in Anari and
Halloumi cheese, respectively. Both of these generation times are similar to those previ-
ously reported for L. rnonocytogenes in refrigerated fluid milks (see Chapter 1 1, Table 7).
Assuming that these cheeses were on sale for up to 3 months after distribution, potentially
hazardous levels of listeriae easily could have developed in such products during retail
storage. Hence, as with cheeses prepared from cows milk, it is imperative that goats
milk and ewes milk cheeses also be manufactured from high-quality pasteurized milk
under the best possible hygienic conditions.

Soft Unripened Cheese

Soft unripened cheeses include such high moisture, white-curd varieties as cottage,
bakers, cream, and American-type Neufchitel cheese. Unlike the groups of cheeses dis-
cussed thus far, milk to be manufactured into soft unripened cheese is coagulated through
production of acid by the starter culture (or alternatively, by direct acidification of milk
to pH 4.6-4.7 with gluconic acid, glucono-delta-lactone, or a mineral acid plus the lactone)
rather than by the addition of a coagulant. Hence, these products are sometimes referred
to as acid-curd cheese. Since the refrigerated shelf life of most soft unripened cheeses is
typically less than 60 days, these varieties must be prepared from pasteurized milk or
cream in the case of cream cheese. Although pH values of 4.6-5.0 provide an unfavorable
environment for microorganisms that may contaminate soft unripened cheese before, dur-
ing, or after manufacture, the fact that these cheeses can be consumed immediately after
production may pose a public health risk, particularly if psychrotrophic, acid-tolerant or-
ganisms such as L. rnonocytogenes are present.
Cottage Cheese
Nearly 1 year before the famed cheeseborne listeriosis outbreak in California, Ryser et
al. [265] used the short-set procedure to prepare cottage cheese from pasteurized skim
milk inoculated to contain l 04- 105L. rnonocytogenes CFU/mL. Following manufacture,
half the curd was creamed to contain 2 4 % milk fat and half remained uncreamed. Both
products were examined for numbers of listeriae during 28 days of storage at 3C.
Numbers of L. rnonocytogenes remained relatively constant during the initial 5-6
h of cheesemaking, during which the pH of milk decreased from 6.65 to 4.70. These
findings agree with those of Schaack and Marth [269], who later demonstrated that growth
of L. rnonocytogenes in slum milk at 30C is completely suppressed by a 5% inoculum
of S. crernoris. After increasing the temperature of the curd/whey mixture to 57.2C
(135F) over 90 min and cooking the curd at this temperature for an additional 30 min,
476 Ryser

L. monocytogenes was not detected in samples of curd or whey that were directly plated
on McBride Listeria Agar. However, following cold enrichment in Tryptose Broth, L.
monocytogenes was detected in four of eight, two of eight, one of eight, and two of eight
samples of cooked curd, whey, wash water, and washed curd, respectively, which suggests
that some Listeria cells were only sublethally injured during cooking of the curd at pH
4.6-4.7. As indicated in Chapter 7, such injury may preclude growth on McBride Listeria
Agar which contains both lithium chloride and phenylethanol as selective agents.
Examination of the finished product indicated that L. monocytogenes survived in
both creamed and uncreamed cottage cheese at levels generally <100 CFU/g during 28
days of refrigerated storage. Although there was no evidence for growth of listeriae in
either cheese during storage, probably because of pH values generally <5.5, the pathogen
was recovered more frequently and at higher numbers in creamed rather than uncreamed
cottage cheese. Higher pH values in 3-day-old creamed (pH 5.32-5.45) rather than un-
creamed (pH 5.12-5.22) cottage cheese may have been responsible for increasing the
repair rate of injured cells, thereby increasing recovery of listeriae.
Although behavior of L. monocytogenes in cheese failed to gain widespread attention
until 1985, a search of the scientific literature has uncovered an earlier study by Stajner
et al. [281] which examined the viability of Listeria in unsalted small-curd skim milk
cheese (similar to uncreamed cottage cheese) manufactured from naturally infected milk
containing approximately 5 X 105L. monocytogenes CFU/mL. Results from these Yugo-
slavian investigators support the findings of Ryser and Marth [265] in that the pathogen
survived at least 7 days in finished cheese (pH 4.55-4.75) stored at 3-5C.
More recently, El-Shenawy and Marth [160] studied the behavior of listeriae in
cottage cheese prepared from pasteurized skim milk that was inoculated to contain 106
L. monocytogenes CFU/mL and then coagulated over a period of 3 h using hydrochloric
acid, gluconic acid, or bovine rennet rather than a lactic acid bacteria starter culture during
which time the temperature of the milk was gradually increased from 2 to 32C. The
resulting coagulum was then cut and cooked using the aforementioned procedure of Ryser
et al. [265].
As might be expected, acidification of the milk to a pH of 4.7-4.8 followed by
heating was again detrimental to survival of listeriae during manufacture of cottage cheese.
Overall, L. monocytogenes populations decreased -4.5 and >6.0 orders of magnitude in
fully cooked (57.2"C/30 min) curd obtained by adding hydrochloric and gluconic acid,
respectively (Fig. 14). However, using cold enrichment, Listeria was recovered from sam-
ples of fully cooked gluconic acid curd. Numbers of listeriae decreased faster in acidified
whey than curd, with cold enrichment results indicating that the pathogen was eliminated
from gluconic but not hydrochloric acid whey after 30 min of cooking at 57.2"C. Nonethe-
less, direct acidification of milk (pH 4.7-4.8) for cottage cheesemaking followed by cook-
ing the resultant curd at 572C for 30 min should be more than sufficient to eliminate
expected numbers of listeriae (<10 CFU/mL) that might inadvertently enter pasteurized
milk as postprocessing contaminants.
In contrast to acid curd/whey, populations of listeriae in freshly cut rennet curd and
whey were virtually identical to those initially observed in milk (Fig. 14). Furthermore,
slight increases in numbers of listeriae were noted midway through manufacture with fully
cooked rennet curd (pH 6.6) and whey still containing 104 and 103 L. monocytogenes
CFU/g or CFU/mL, respectively. Thus, listericidal effects associated with cooking were
greatly enhanced under acidic conditions. Subsequent experiments with selective plating
media confirmed that substantial numbers of L. monocytogenes cells were sublethally in-
L. monocytogenes in Fermented Dairy Products 477

3,
0 - Rennet Curd
0 - -- Rennet Whey
2, A HC1 Curd
A - - HC1 Whey
I- GACurd
1,
0 - - GA Whey

0 1 1 I I
A B C D E F

FIGURE 14 Survival of L. monocytogenes in curd and whey obtained during prepara-

tion of cottage cheese made with rennet, HCI, or gluconic acid (GA). A: Immediately
after cutting; B: after temperature was increased to 48.9"C; C: after temperature was
increased to 54.4"C; D: after temperature was increased to 57.2"C; E: after 15 minutes
of cooking; and F: after 30 minutes of cooking. (Adapted from Ref. 160.)

jured during manufacture of cottage cheese, as was suggested by Ryser and Marth [265]
five years earlier. Sublethal injury was far more evident in whey rather than curd samples,
probably because curd afforded some thermal protection to listeriae. Not surprisingly, the
degree of sublethal injury was also closely related to coagulant type (i.e., acidity) and
cooking temperature, with less injury being observed in rennet rather than acid curd/whey
and partially rather than fully cooked samples of curd and whey. Heat alone was primarily
responsible for rennet-associated injury, whereas the combined effects of heat and acid
led to injury of listeriae in acid curd and whey.
With the exception of cottage cheese prepared from milk acidified with gluconic
acid, both of these studies demonstrated limited survival of L. monocytogenes in cottage
cheese. However, the fact that the pathogen failed to grow in the product and decreased
drastically in numbers during manufacture suggests that cottage cheese poses far less of
a public health threat than do those varieties that are surface ripened with molds or bacteria.
Lower health risks associated with consumption of cottage cheese are also supported by the
extremely low incidence of L. monocytogenes in commercially produced cottage cheese
examined in American and European surveys.
The likelihood of L. monocytogenes entering cottage cheese during creaming and/
or packaging of the product is far greater than having listeriae present in pasteurized milk
at sufficiently high levels to survive in the cooked curd. Although most recently published
work has addressed the fate of L. monocytogenes in cottage cheese as a postmanufacturing
contaminant, results from these efforts have been somewhat conflicting. Based on the
findings of three studies conducted in the United States [240] and England [169,192],L.
478 Ryser

monocytogenes failed to grow in artificially contaminated, commercially prepared creamed

cottage cheese (pH 4.5-5. I), with populations generally decreasing 0.5- 1.5 orders of mag-
nitude during 1-5 weeks of storage at refrigeration or abusive temperatures. According to
Moir et al. [230], numbers of L. monocytogenes remained relatively stable in commercial,
Australian-produced creamed cottage cheese during 1 month of storage at 15C. This
behavior is similar to that observed by Hicks and Lund [ 1921 when creamed cottage cheese
was inoculated with an acid-adapted strain of L. monocytogenes previously cultured in
Tryptose Phosphate Broth at pH 5.5. In contrast to these findings, at least four additional
studies attest to growth of L. monocytogenes [ 132,143,1761and L. innocua [ 1681 in similar
samples of creamed cottage cheese, with populations increasing 0.5-3 .O orders of magni-
tude during refrigerated storage. Although not readily apparent, such behavioral differ-
ences in creamed cottage cheese may be related to differences between Listeria strains
as well as differences in acid tolerances [ 1721 and abilities to readily compete with the
native microflora.
Since L. monocytogenes can persist beyond the normal shelf life of cottage cheese,
several options also have been examined for minimizing growth and/or survival of Listeria
in this product during refrigerated storage. A few chemical additives, including sorbate
[240], 3% sodium lactate [240], 3% calcium lactate [240], and 0.04% lysozyme, were
shown to be, at best, only minimally effective, with Listeria populations in inoculated
creamed cottage cheese decreasing 5 10-fold during the products normal refrigerated
shelf life.
Addition of various bacteriocins, including PA- 1 (an inhibitory substance produced
by one strain of Pediococcus acidilactici) and nisin (produced by certain strains of S.
Zactis), appears to be a far more promising means of ridding cottage cheese of viable
listeriae that may have inadvertently entered the product after cooking. Using commer-
cially prepared dry cottage cheese curd to which 7.5 X 103 L. monocytogenes CFU/g
were added during creaming, Pucci et al. [245] found that the product (pH 5.1) still con-
tained 1 X 102 L. monocytogenes CFU/g after 7 days of storage at 4C. In contrast,
addition of bacteriocin PA-1 powder to identical samples led to complete inactivation of
the pathogen within 24 h. However, inability of bacteriocin PA-1 to prevent growth of
the pathogen in commercially prepared cheese sauce (pH 6.0) and half-and-half (pH 6.6)
shows that listericidal activity of this bacteriocin is strongly dependent on the pH of the
food system.
Using a different brand of dry cottage cheese curd that was inoculated to contain
3 X 105L. monocytogenes CFU/g during creaming, Benkerroum and Sandine [ 1161 de-
tected very few viable listeriae in the product (pH 4.9-5.0) after l or more days of storage
at 4C. This antagonistic effect of cottage cheese (presumably from natural flora or by-
products in cottage cheese) toward listeriae was enhanced by adding nisin (2.5 X 103IU/
g), with viable listeriae completely being eliminated from such cheese after I day of
refrigerated storage. The efficacy of nisin has since been confirmed by Ferreira and Lund
[ 1691, who reported that L. monocytogenes populations decreased about 1000-fold in
creamed cottage cheese (pH 4.6-4.7) containing 2000 IU nisin/g after 3 days of storage
at 20C.
In these studies, nisin and bacteriocin PA-1 remained active in creamed cottage
cheese during prolonged storage. Thus it appears that these bacteriocins may also prove
useful in limiting Listeria survival in other fermented dairy products, including natural
cheeses, cold-pack cheese food, and various cheese spreads [65,72] during the normal
shelf life.
L. monocytogenes in Fermented Dairy Products 479

Additionally, modified atmosphere packaging also has been explored as a means

for both extending the refrigerated shelf life of cottage cheese and minimizing Listeria
growth. According to Chen and Hotchkiss [ 1321, L. monocytogenes populations in
creamed cottage cheese (pH 5. I ) packaged under 35% CO2remained stable during 9 weeks
of storage at 4"C, whereas the pathogen increased 1000-fold in aerobically packaged
cheese. At 7"C, numbers of listeriae failed to change in CO:-packaged cheese during the
first 4 weeks of storage. However, rapid growth occurred within 16 days in aerobically
packaged cheese. Fedio et al. [ 1681 also reported that L. innocua failed to grow in creamed
cottage cheese (pH 5) during 28 days of storage at 5C when the product was packaged
under 50 or 100% CO2. However, this organism began growing rapidly in identical sam-
ples packaged aerobically or under 100% N2 after 7 days of incubation. Although modified
atmospheric packaging appears to be useful in minimizing Listeria growth in cottage
cheese, inactivation of the pathogen will not occur under such conditions, with the results
of Chen and Hotchkiss [132] also indicating that the effectiveness of CO2 is strongly
temperature dependent.
Cream Cheese
In the only other study dealing with the behavior of listeriae in soft unripened cheese,
Cottin et al. [ 1401 prepared cream cheese from a chemically acidified mixture of milk and
cream that was inoculated to contain 10'- 10' L. monocytogenes CFU/mL. Using the low-
est inoculum, Listeria grew in the finished product (pH 5 6 ) and attained a stable popula-
-
tion of 10' CFU/g within 2 days of storage at 4C. Thus, unlike cottage cheese, the pH
and moisture content of cream cheese are both sufficiently high to permit limited growth
of listeriae in the product during refrigeration.

Whey Cheeses
A few cheeses such as ricotta, Broccio, and Ricotone are prepared from sweet whey de-
rived from the manufacture of mozzarella, Cheddar, Swiss, Tilsiter, and feta cheese. Manu-
facture of these whey cheeses is based on the direct acidification of whey, whey/milk,
and whey/cream mixtures to pH 5.9-6.0 using food-grade acids (i.e., citric, acetic), lactic
starter cultures, or acid whey powder followed by cooking at 180-190F to precipitate
the whey protein. The fine precipitate which eventually rises to the vat surface is then
removed, drained, matted, and either marketed as whey cheese or a dairy ingredient. Sev-
eral additional extremely low-moisture ( I 3 to 18%) whey cheeses, including Gjetost, My-
sost, and Gudbrandsdalsost, are unique to Norway and are prepared by thermally concen-
trating and then boiling a blend of goat's and cow's milk whey until the mixture carmelizes
and becomes viscous. This plastic mass is then cooled, extruded, and cut into extremely
dense blocks for marketing.
As was true for mozzarella cheese, L. monocytogenes will be completely inactivated
during manufacture of these whey cheeses. However, the potential still exists for contami-
nation during packaging as is evidenced by at least one Class I recall of ricotta cheese in
July of 1991 and the report of a small cluster of listeriosis cases traced to Anari whey
cheese produced in Cyprus (see Chap. 10).Consequently, Papageorgiou et al. [239] exam-
ined the fate of L. monocytogenes as a postprocessing contaminant in Greek Myzithra
(identical to Anari cheese), Anthotyros, and Manouri cheese, all of which are starter cul-
ture-free, soft (50-70% moisture), low-acid (pH 6.0-6.5) whey cheeses. Immediately after
commercial manufacture, these cheeses were inoculated to contain -500 L. monocyto-
480 Ryser

genes strain Scott A or CA CFU/g. Regardless of cheese type or Listeria strain, the patho-
gen grew rapidly and attained maximum populations of 107-108CFU/g after 24-30 days,
5-12 days, and 56-72 h of storage at 5, 12, and 22OC, respectively. Consequently, strict
hygienic practices must be followed to prevent Listeria contamination during packaging,
with postpackaging pasteurization of the product also being recommended as an added
safeguard.
Cold-Pack C h e e s e Food
Unlike the natural cheeses discussed thus far, cold-pack cheese food is typically prepared
by comminuting and blending aged Cheddar cheese (or another variety) with nonfat dry
milk, dried whey, water, cream, plastic cream (composition similar to butter), salt, acidu-
lants (i.e., lactic and/or acetic acid), preservatives (i.e., potassium sorbate and/or sodium
propionate), and other optional ingredients into a homogeneous mass without heating.
Since all of the dairy ingredients and some of the optional ingredients used in manufactur-
ing cold-pack cheese food can potentially harbor L. monocytogenes, Ryser and Marth
[263] investigated the behavior of this pathogen in nine different formulations of cold-
pack cheese food inoculated to contain approximately 500 L. monocytogenes strains Scott
A, V7, CA, or OH CFU/g.
During 182 days of storage at 4OC, populations of all four Listeria strains decreased
less than 10-fold in nonacidified, (pH 5.20) preservative-free cheese food, with the patho-
gen surviving throughout the product's entire 6-month shelf life (Fig. 15).In sharp contrast
to these findings, addition of preservatives with or without acidifying agents led to the

--a Scott A

1.0 - --0 v7

- * CA
-5 -
--+ OH

10

FIGURE15 Survival of four strains of L. monocytogenes in nonacidified cold-pack

cheese food manufactured without preservatives. (Adapted from Ref. 263.)
L. monocytogenes in Fermented Dairy Products 481

eventual demise of listeriae in cheese food stored at 4C (Fig. 16). In nonacidified cheese
food (pH 5.20) preserved with 0.3% sodium propionate, L. monocytogenes survived an
average of 142 days as compared with 118, 103, and 98 days in the same product adjusted
to pH 5.0-5.1 with lactic, acetic, and lactic plus acetic acid, respectively. Using 0.3%
sorbic acid in place of sodium propionate, the pathogen survived an average of 130 days
in nonacidified cheese food (pH 5.45) as compared with 112, 93, and 74 days in cheese
food acidified to pH 5.0-5.1 with lactic, lactic plus acetic, and acetic acids, respectively.
Thus, sorbic acid was consistently more antagonistic to listeriae than sodium propionate.
In addition, antilisterial effects of both preservatives were more pronounced in cheese food
acidified with acetic rather than lactic acid. Since organic acids are far more bactericidal in
the undissociated than dissociated state, increased inactivation of listeriae in the presence
of acetic acid probably resulted from the higher proportion of undissociated acetic (-36%)
rather than lactic acid (-5.9%) in cheese food acidified to pH 5.0-5.1. These findings
indicate that it would be prudent to consider (a) adding preservatives, particularly sorbic
acid, to cold-pack cheese food and (b) reducing the pH of the product to 5.0 by adding
small amounts of lactic and/or acetic acid to minimize L. rnonocytogenes survival in the
finished product. Additional information on conditions leading to inhibition and/or inacti-
vation of this pathogen by sorbic, propionic, lactic, and acetic acids can be found in
Chapter 6.

150 -
140 -
S SorbicAcid
130 - p NaPropionata
A Acetic Acid
120 - L LacticAcid
I I

01
110 -
6
h

100-

? -
'$
90

vI 80 -
70 -
60 -
50 I
P S P+L S+L P+A P+L+A S+L+A S4A

Additive

FIGURE16 Maximum length of survival of L. monocytogenes in acidified and nona-

cidified cold-pack cheese food containing preservatives.Each bar represents the aver-
age maximum length of survival of all four Listeria strains in one of eight different
formulations of cheese food manufactured in duplicate. Any two differing by >20.24
days (length of bar) are significantly different ( p < 0.05). (Adapted from Ref. 263.)
482 Ryser

Behavior of L. monocytogenes in Cheese as Affected by

Cheese Composition
As suggested earlier, the fate of L. rnonocytogenes and other foodborne pathogens during
cheese ripening is determined by the microbiological, biochemical, and physical properties
of the particular cheese. Thus cheese is a very complex system, with the following fac-
tors acting simultaneously to determine the behavior of L. monocytogenes during ripening:
(a) type, amount, and activity of the starter culture; (b) pH as determined by concentrations
of lactic, acetic, formic, and other acids; (c) presence of hydrogen peroxide, diacetyl, and
various antimicrobial agents (i.e., nisin, diplococcin, and other bacteriocins); (d) levels
of nutrients, salt, moisture, and oxygen; and (e) the temperature at which the cheese is
ripened.
All of these factors act together to produce a particular outcome, however, a few
conclusions concerning the ability of L. monocytogenes to grow and/or survive in some
of the aforementioned cheeses prepared from contaminated milk can be drawn by examin-
ing the behavior of this pathogen in relation to the combined effects of moisture content,
water activity (a,), and salt in the moisture phase as well as the pH of the cheese and the
temperature(s) at which the cheese is ripened (Table 15).
Although fully ripened Camembert and feta cheese have widely differing pH values
of 7.5 and 4.4, respectively, both cheeses are very similar in terms of moisture content,
water activity, percentage of salt in the water phase, and ripening temperature. Thus rapid
growth of L. rnonocytogenes in Camembert cheese can be largely attributed to the increase
in pH of the cheese during ripening, whereas a pH value of 4.4 appears to be responsible
for preventing growth of the bacterium during ripening of feta cheese. Inability of L.
rnonocytogenes to multiply in blue cheese during ripening and storage is probably related
to the high concentration of salt in the water phase (which results in a low a,), since other
workers have confirmed that this organism will not grow in laboratory media [275] and
skim milk [236] containing > I 0 and 12% salt, respectively. As with Camembert cheese,
growth of two of four L. rnonocytogenes strains in brick cheese appears to be directly
related to the high pH that the cheese attained during extended ripening. However, a
general inability of the remaining two strains to grow in brick cheese of similar composi-
tion is as yet unexplained.
When comparing the behavior of L. monocytogenes in Cheddar and Colby cheese,
the initial inactivation rate for the pathogen was somewhat slower in the latter cheese. At
first glance, it appears that increased viability of Listeria in Colby cheese during the early
stages of ripening may be related to the lower percentage of salt in the water phase in
this cheese than in Cheddar cheese. However, data in Table 15 show that L. monocytogenes
was inactivated at similar rates in Colby cheese and cold-pack cheese food, the latter is
compositionally similar to Colby cheese except for a higher concentration of salt in the
water phase. Hence, factors other than a low concentration of salt in the water phase also
must be involved in enhancing the viability of listeriae during ripening of Colby cheese.
Lack of growth and decreased survival of L. rnonocytogenes in Parmesan cheese
and in an unidentified hard Italian cheese as compared with other varieties in Table 15
correlate well with the lower moisture content/water activity of these cheeses during ripen-
ing. Barring thermal and/or acid injury of L. rnonocytogenes, which is likely to occur
during manufacture of cottage cheese and other varieties that undergo substantial heat
treatments during manufacture (i.e., mozzarella, Swiss), factors outlined in Table I5 are
L. monocytogenes in fermented Dairy Products 483

TABLE
15 Behavior of L. monocytogenes During Cheese Ripening as Affected by Cheese Composition
Estimated
salt in Ripening Log,, of L. monocytogenes CFU/g
PH temp. Survival
Moisture Estimated water phase
Cheese @) a, (%) Initial" Final ("C) Initiala Maximum Final (days) Ref.

Camembert 54.4 0.975 4.72 4.6 7.5 15/6 3.1-3.6 6.7-7.5 6.7-7.5 65 260
Blue 38.9 0.950 11.52 4.6 6.3 9-12/4 4.0-5.0 4.0-5.0 1.0-2.3 120 237
Brick 43.0 0.990 1.89 5.3 7.3 15/10 3.0-4.7 4.6-6.7 2.7-6.1 168 264
Feta 54.7 0.975 4.57 4.7 4.4 22/4 5.2-6.2 5.7-6.2 2.8-4.6 90 238
Cheddar 37.2 0.975 4.61 5.1 5.1 6 2.5-3.2 2.6-3.8 0-1.5 70-434 259
Cheddar 37.2 0.975 4.61 5.1 5.1 13 2.6-3.4 3.0-3.7 0 70-224 259
Colby 40.0 0.975 3.91 5.1 5.1 4 3.5-4.5 3.6-4.6 2.3-4.1 112-140 306
Parmesan 32.0 0.935 4.96 5.1 5.1 13 3.3-4.3 3.3-4.3 1.0-1.3 21-1 12 307
Hard Italian NR' 0.950 2.12d 5.3 5.7 4 4.5-5.1 4.5-5.6 2.0 35 I35
Cold-pack 41.4 0.975 4.90 5.3 5.1 4 2.4-2.8 2.4-2.8 1.1-2.0 180 263
cheese foodb
-

Approximately 24 h after the start of cheesemaking.

Prepared without preservatives or acidifying agents.
Not reported.
Percentage of salt in solid and water phase.
484 Ryser

useful in predicting whether or not this pathogen will grow in other cheeses having similar
microbiological, biochemical, and physical characteristics.
In addition to being present in milk at the time of cheesemaking, Listeria also can
easily contaminate the finished cheese during packaging, ripening, and storage. Conse-
quently, Genegeorgis et al. [ 1761 evaluated the fate of Listeria as a postprocessing contam-
inant by inoculating 49 retail cheeses (24 typed28 brands) with L. monocytogenes and
then storing these cheeses at 4-30C for up to 36 days. As expected, Listeria growth was
primarily confined to high-moisture varieties, including Brie, Camembert, ricotta, and the
soft Hispanic cheeses, all of which had a pH 2 6 and low to moderate levels of salt in
the moisture phase (Table 16). Back et al. [ 1081 also reported temperature-dependent
surface growth of L. monocytogenes on several additional retail European soft cheeses
including Cambazola, English Brie, Blue Lymeswold, and White Lymeswold, with Liste-
ria populations remaining relatively stable on Blue Stilton, White Stilton, Mycella, and
Chaume cheese during short-term storage. In addition, commercial [ 1311 and experimen-
tally produced [170] Arzua cheese (a soft, low-acid Spanish cheese prepared from raw
cow's milk) supported growth of Listeria as evidenced by populations >loo0 CFU/g in
the finished product. All of these findings again point to the high-moisture, low-acid
cheeses as being of primary public health importance.

Feasibility of Preparing Cheese from Raw Milk

According to current FDA regulations, milk pasteurization or use of a similar heat treat-
ment during cheesemaking is required for the manufacture of 16 cheese varieties, including
Brie, cottage, cream, Neufchgtel, Monterey, mozzarella, Scamorza, Muenster, Gammelost,
Koch Kaese, and Sapsago [197]. Seven varieties of manufacturing cheese (i.e., for use in
pasteurized processed cheese, cheese foods, cheese spreads) require neither pasteurization
of the cheesemilk nor a 60-day minimum ripening at 21.7"C (235"F), whereas the 34
remaining varieties of cheese recognized under current standards of identity must either
be manufactured from pasteurized milk or held a minimum of 60 days at 2 1.7"C (135F)
to eliminate pathogenic microorganisms. Although statistics on milk pasteurization for
cheesemaking are scarce, available evidence indicates that a major portion of the natural
cheeses sold in the United States are prepared from pasteurized milk.
The mandatory holding period of 60 days at 21.7"C (235F) for cheeses manu-
factured from raw milk was adopted in 1949 [6,197] after researchers demonstrated
that viable Brucella abortus, the causative agent of brucellosis, was eliminated from
cheese by such an aging process. Although this 60-day holding period was generally
deemed adequate to eliminate most foodborne pathogens, later studies demonstrated
that Salmonella typhimurium and other hazardous microorganisms can survive such a
cheese-ripening process [ 183,1981.Furthermore, results in Table 15 indicate that L. mono-
cytogenes can survive well beyond 60 days in many natural cheeses held at 21.7"C
(235OF).
In keeping with the grave nature of listeriosis as compared with most other food-
borne illnesses, the FDA has continued to maintain a policy of zero tolerance for L. mono-
cytogenes in all ready-to-eat foods. Thus far no well-documented cases of listeriosis have
been associated with consumption of cheeses that were legally prepared from raw milk
and held a minimum of 60 days at 21.7"C (235F). However, since -4% of the raw
milk supply can be expected to contain L. monocytogenes, it would be prudent to manufac-
ture cheeses from pasteurized milk whenever possible. Although Yousef and Marth [307]
L. monocytogenes in Fermented Dairy Products 485

TABLE
16 Growth and Inactivation of L. monocytogenes in Surface-
Inoculated Retail Cheeses During Storage at 4-30C

% NaCl in
Cheese category and type PH moisture phase Growth
Soft mold ripened
Brie 6.0-7.7 2.5-3.6 +
Camembert 7.3 2.5 +
Blue 5.1 6.1
Bacterial surface ripened
Limburger 7.2 4.8
Muenster 5.5 3.8
Soft Italian
Provolone 5.6 4.6
String cheese 5.5 4.4
Semisoft and hard ripened
Monterey Jack 5.0-5.2 1.O-3.0
Colby 5.5 4.9
Cheddar 4.9-5.6 2.6-5.4
Swiss 5.5 2.7
Hispanic
Queso Fresco 6.5-6.6 4.5-6.6 +/-
Queso Rancher0 6.2 4.1 +
Queso Panella 6.2-6.7 2.5-3.9 +
Cotija 5.5-5.6 9.6- 12.5 -
Pickled cheese
Feta 4.2-4.3 2.2-7.5
Ewes milk cheese
Kasseri 4.8-5.3 5.5-5.8
Soft unripened
Cottage Cheese 4.9-5.1 1.0-1.2 +/-
Cream Cheese 4.8 <0.9 -

Whey cheeses
Ricotta 5.9-6.1 <0.7 +
Processed cheese
American 5.7 2.1
Monterey Jack 5.7 4.4
Piedmont 6.4 5.1
Source: Adapted from Ref. 176.

demonstrated that ripening Parmesan cheese for 10 months, as legally required, is suffi-
cient to produce a high-quality, Listeria-free product, desirable flavor and texture charac-
teristics are not easily attainable in sharp Cheddar and Swiss cheese prepared from pasteur-
ized milk. Hence, alternative means should be developed to enhance the safety of these
products. Such methods might include cold sterilization of the milk via microfiltration or
addition of various flavor- and texture-enhancing enzymes (or microorganisms) to pasteur-
ized milk, which would allow the cheesemaker to obtain a higher quality product [199].
However, as important as it is to manufacture cheese from Listeria-free milk, it is equally
important to prevent contamination of the product during manufacture, ripening, and
storage by using good manufacturing practices. Information concerning problem areas
486 Ryser

and safeguards during manufacture of dairy products and other, foods can be found in
Chapter 17.

Whey
Listeria monocytogenes has not yet been isolated from commercially produced cheese
whey. However, studies have shown that when various cheeses were experimentally pro-
duced from pasteurized milk inoculated with L. monocytogenes, between 1-5% of the
original inoculum was lost in the whey during cheesemaking (Table 17). These findings
again demonstrate that the pathogen is concentrated 8- to 10-fold in curd during milk
coagulation. Unlike other wheys, populations of L. monocytogenes in acid whey (pH 4.6)
obtained from the manufacture of cottage cheese were reduced more than 10,000- fold
after cooking the curdlwhey mixture at 57.2"C (1 35F) for 30 min. However, as previously
noted, Listeria was detected in several whey samples after 6 weeks of cold enrichment
[265], which, in turn, suggests that some cells were only sublethally injured during cooking
of the curd/whey mixture.
These observations prompted Ryser and Marth [262] to examine the behavior of L.
monocytogenes in wheys from Camembert cheese that were filter-sterilized and adjusted
to pH values of 5.0-6.8. All whey samples were then inoculated to contain -100-500
L. monocytogenes strains Scott A, V7, CA, or OH CFU/mL and incubated at 6C.
Although the four L. monocytogenes strains failed to grow in wheys having pH
values 15.4, the pathogen survived in all samples with populations decreasing 110-fold
during 35 days of refrigerated storage. In contrast, L. monocytogenes grew in all remaining
samples after a 3-day lag period and attained average maximum populations of 7.48, 7.87,
and 7.84 log,, CFU/mL in wheys adjusted to pH 5.6, 6.2, and 6.8, respectively, following
35 days of incubation. As previously noted, these Listeria populations in whey were
slightly higher than those that would be expected to develop in skim or whole milk during
refrigerated storage. Generation times for L. monocytogenes in wheys adjusted to pH 5.6,
6.2, and 6.8 ranged between 25.2-31.6 h, 14.8 and 21.1 h, and 14.0 and 19.4 h, respec-
tively, depending on the individual strain. Although doubling times were similar for all
strains at the same pH value, generation times were significantly longer at pH 5.6 than

TABLE17 Number of Listeriae Recovered from Whey

During Manufacture of Various Cheeses Prepared from
Pasteurized Milk Inoculated to Contain -500-5000 L.
rnonocytogenes CFU/mL

% of original
L. monocytogenes inoculum in
Cheese CFU/mL of whey whey Ref.
Camembert 8 1.3 26 1
Blue 43 3.6 237
Brick 12 2.5 264
Feta 15 3.2 238
Gouda 5 1 .o 235
Colby 21 2.4 306
Cheddar 22 5.0 259
Average 18 21.7
L. monocytogenes in fermented Dairy Products 487

at pH 6.2 and 6.8. Interestingly, Hughey et al. [193] observed that L. monocytogenes could
be inactivated in similar samples of demineralized whey by adding lysozyme. However,
this enzyme did not decrease viability of the pathogen in normal whey, which in turn
suggests that lysozyme activity is neutralized by whey minerals and/or proteins.
Using a different approach to examine the behavior of Listeria in whey, Northolt
et al. [235] inoculated heat-treated (68"C/lO s) wheys (pH 6.5) to contain 500-1000 L.
monocytogenes CFU/mL and incubated the samples at temperatures between 7-30C.
Following a 6- to 24-h lag period, the pathogen grew in all samples with doubling times
of 12 h, 6 h, 4 h, and 40 min in wheys incubated at 7, 12, 20, and 30"C, respectively.
Although incubation of all whey samples was terminated before L. monocytogenes reached
the stationary growth phase, the pathogen did attain populations of 104-106 CFU/mL at
the conclusion of the experiment.
In the only study thus far reported dealing with nonsterile whey, researchers in
France [ 1131 produced whey containing 2-7 L. monacytogenes CFU/mL from previously
inoculated milk and examined samples for listeriae after 101, 156, and 25 1 days of storage
at 4C. Moderate growth and extended survival of the pathogen were observed in whey
collected immediately after coagulation of the milk (pH 5.4), with 156- and 251-day-old
whey samples at pH 4.8 containing 2.5 X 104 and 7.0 X 102 L. monocytogenes CFUI
mL, respectively. As predicted by Ryser and Marth [262], the pathogen also failed to
grow in more acidic wheys (pH 5.2-5.3) collected after hooping with listeriae no longer
observed in 101- and 156-day-old wheys having pH values of 3.28 and 4.26, respectively.
Not surprisingly, increasing the incubation temperature to 6, 14, and 20C led to faster
demise of listeriae in 21 similar wheys (pH 3.75-5.72) initially containing 60-96 L. mono-
cytogenes CFU/mL, with the pathogen being eliminated from all but one sample (pH
5.72) examined after 114 days of incubation at 6C. These findings demonstrate that L.
monocytogenes can grow to high numbers in fluid wheys having pH values of 25.4 and
remain viable in more acidic wheys during many months of refrigerated storage.
Given results of a study described in Chapter 11 in which numbers of L. monocyto-
genes decreased only 1-5 orders of magnitude during manufacture of nonfat dry milk
[ 1501, it appears that this organism also is likely to survive the spray-drying process used
in converting fluid whey into whey powder. However, to our knowledge, no Listeria spp.
have yet been isolated from dried whey manufactured commercially in Europe or the
United States. Although Gabis et al. [171] failed to find any Listeria spp. in 23 environ-
mental samples from whey processing factories, these authors did isolate L. monocyto-
genes from a floor drain that was located within a raw milk receiving room of a dry milk
processing factory. Additionally, Listeria spp. other than L. monocytogenes were isolated
from several drains and trenches in the powder production area of a second factory that
manufactured dry milk products. Considering the widespread use of dried whey (and non-
fat dried milk) as an ingredient in numerous products including cheese food, ice cream,
sherbet, candy, beverages, and baked goods, strict enforcement of good manufacturing
practices for dried whey and nonfat dry milk should be continued to prevent a possible
public health problem involving listeriae.

Brine Solutions
Since L. monocytogenes is quite halotolerant, it is not surprising to learn that brine solu-
tions in which cheeses are salted and/or ripened also can serve as potential reservoirs for
this organism. Current evidence suggests that these brine solutions may become contami-
488 Ryser

nated with L. monocytogenes through directhdirect contact with the cheese factory envi-
ronment (i.e., equipment, condensate on walls, floors, and ceilings) as well as actual shed-
ding of L. monocytogenes into the brine solution from Listeria-contaminated cheese. Breer
[ 1231 and Terplan [28] reported isolating L. monocytogenes from commercial brine solu-
tions in Europe. In one instance, the pathogen was detected in brine tanks 4 days after
soft/semisoft cheeses were removed from the salt brine. Since one 1991 recall of mozza-
rella cheese in the United States [80] was presumably traced to a contaminated brine tank,
interest in the incidence of listeriae in brine solutions will likely increase.
Migration of L. monocytogenes into brine solutions and salted whey during salting
of artificially contaminated cheese has been well documented. Ryser and Marth [26] brine
-
salted brick cheese containing 103-104 L. monocytogenes CFU/g at 10C. Cold enrich-
ment of membrane filters through which 50-mL portions of 22% brine solution were fil-
tered indicated that the pathogen leached from the cheese into the salt solution during 24
h of brining. Furthermore, viable listeriae were detected in samples of 22% brine stored
at 10C at least 5 days after blocks of cheese were removed from the brine. When Abdalla
et al. [ l ] brine salted Sudanese white pickled cheese in whey containing 8% NaCI, L.
monocytogenes once again leached from the cheese into the salted whey, with populations
increasing 2.5-3.5% orders of magnitude during 65 days of storage at 4C.
In conjunction with their study on the fate of L. monocytogenes during manufacture,
ripening, and storage of feta cheese, Papageorgiou and Marth [238] also examined Listeria
viability in the brine solution in which cheese was salted and stored. After 1 day of salting,
a 12% brine solution contained an average Listeria population of 2.63 log,, CFU/g, which
again indicates that the pathogen leached from cheese into the brine (Fig. 17). However,
no growth of L. monocytogenes was observed in the 12% brine solution despite ample
migration of cheese nutrients into salt brine as well as favorable values for temperature
and pH. After transferring feta cheese to a 6% brine solution, the pathogen leached from
cheese into salt brine and grew rapidly, with similar populations being observed in both
cheese and 6% brine after 6 days of incubation at 22C. Although numbers of listeriae

I I I I I I I I , I I ,<' 1 I I I
0 1 2 3 4 5 20 34 48 62 76
Days

FIGURE17 Average populations of L. rnonocytogenes strain CA in 12 and 6% salt

brine during ripening and storage of feta cheese. (Adapted from Ref. 238.)
L. monocytogenes in Fermented Dairy Products 489

decreased in both cheese and salt brine during 90 days of refrigerated storage, the pathogen
was inactivated slower in 6% salt brine than in feta cheese.
Larsen et al. [208] reported that L. monocytogenes began growing in salt-free whey
(pH 5.6) and whey containing 3.6% NaCl and/or KC1 after 7-14 days of storage at 4"C,
with Listeria growth generally being unrelated to the type of salt used. However, L. mono-
cytogenes populations remained unchanged in identical samples containing 4.8% NaCl
and/or KCI. When similar whey samples were incubated at 25"C, L. monocytogenes popu-
lations increased 3-4 orders of magnitude, with more rapid growth being observed in
wheys containing 3.6% rather than 4.8% NaCl and/or KCI. However, Listeria growth at
25C could be prevented by adding a L. l a d s subsp. lactis or L. lactis subsp. cremoris
starter culture before incubation. In 1994, Rajkowski et al. [249] also reported that addition
of 4.5% NaCl to ultrahigh temperature (UHT)-sterilized milk decreased the growth rate
of L. monocytogenes in samples incubated at 12-37C. However, fortification of these
same samples with 0.5 or 1 .O% polyphosphate did not alter Listeria growth characteristics.
Since feta and other white-pickled cheeses such as Teleme, Halloumi, and Domiati
are frequently cured in whey or skim milk containing 6 or 12% salt, Papageorgiou and
Marth [2361 examined behavior of Listeria in salted whey and skim milk. Autoclaved
samples of skim milk (pH 6.0-6.2) and deproteinated whey (pH 5.5-5.7) containing 6
-
and 12% NaCl were inoculated to contain 103 L. monocytogenes (strains Scott A or
CA) CFU/niL and incubated at 4 and 22C.
After a lag period of 5- 10 days, L. monocytogenes grew rapidly in 6% salted whey
and 6% salted skim milk, with the pathogen attaining maximum populations of 107-10R
CFU/mL following 50-55 days of refrigerated storage (Table 18). In the study discussed
earlier, Ryser and Marth [262] reported that these same Listeria strains had shorter lag
periods and generation times but achieved similar maximum populations in unsalted, filter-
sterilized whey (pH 5.6) after 24 days of incubation at 6C. Hence these findings suggest
that addition of NaCl or KCI to whey and milk plays a major role in decreasing the growth
rate of Listeria.
Increasing the incubation temperature to 22C resulted in lag periods of 6-12 h for
both Listeritr strains in whey and skim milk containing 6% salt. Generation times were

TABLE
18 Generation Times (GT) and Maximum Populations
(MP) of L. monocytogenes Strains Scott A and CA in 6% Salted
Whey and Skim Milk Incubated at 4 and 22C
MP
Strain Product GT (h) (logl,,CFU/mL)
Incubation at 4C
Scott A Whey 46.8 1 7.97
Scott A Skim milk 45.23 7.58
CA Whey 37.49 8.04
CA Skim milk 49.43 7.69
Incubation at 22C
Scott A Whey 3.67 8.02
Scott A Skim milk 4.3 1 7.70
CA Whey 3.56 8.10
CA Skim milk 4.42 7.89

Source: Adapted from Ref. 236.

490 Ryser

5 -
ScottA Whey

Scott A Skim milk

4 -

f
0

3 -

2 -

1 - CAWhey

@- CA Skim milk

0 1 I I I I I I I I I I I
-25 0 25 50 75 100 125 150

Days

FIGURE18 Behavior of L. monocytogenesstrains Scott A and CA in 12% salted whey

and skim milk during extended storage at 22C. (Adapted from Ref. 236.)

similarly reduced with both strains exhibiting faster growth rates and higher maximum
populations in salted whey rather than salted skim milk (see Table 18). These findings
agree with those of other researchers who also observed that L. monocytogenes grew faster
and attained higher maximum populations in unsalted whey [262] than in skim milk [255].
Unlike the previous findings obtained with 6% salt, growth of L. rnonocytogenes
was completely inhibited in 12% salted whey and skim milk, with populations of both
strains decreasing < 10-fold during 130 days of storage at 4C. Although strain Scott A
also persisted >130 days in 12% salted whey and skim milk incubated at 22"C, strain
CA proved to be less salt tolerant, surviving only 80 and 105 days in 12% salted skim
milk and whey, respectively (Fig. 18). Increased destruction of L. monocytogenes in salt
solutions held at ambient rather than refrigeration temperatures has been well documented.
Results from several of these studies dealing with viability of listeriae in salted Tryptose
Broth 112761 and cabbage juice [ 1391 are discussed in Chapter 6. Based on these observa-
tions, acidification of brine solutions used in cheesemaking to pH values <5.0 has been
recommended to prevent growth of L. monocytogenes, particularly if such solutions con-
tain 510% salt. In 1989, Hughey et al. [193] also noted that addition of 0.35% H 2 0 2to
a 23% brine solution caused numbers of L. monocytogenes to decrease by six orders of
magnitude within 24 h. However, unlike organic acids, the bactericidal activity of H 2 0 2
dissipates fairly rapidly. Hence addition of H202to salt brine provides only temporary
protection against listeriae that might be present within the cheesemaking environment.

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L. monocytogenes in Fermented Dairy Products 493

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494 Ryser

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502 Ryser

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 13
Incidence and Behavior of
Listeria monocytogenes
in Meat Products

JEFFREY
M. FARBER
AND PEARL
1. PETERKIN
Health Canada, Ottawa, Ontario, Canada

INTRODUCTION
Only within the past 10 years have there been cases of human listeriosis traced to
meat, with evidence for transmission of listeriosis through consumption of contaminated
meats and meat products (see Chap. 10). Products such as piit&, turkey frankfurters,
and sausages have been implicated [ 11 1,1151. In a valuable review, Jay [ 1 111 has
summarized reports on the prevalence of listeriae in meat products from 1971 to 1994
worldwide.
Recently, leading researchers in the United States [13] and the United Kingdom
[ 1441 have stated that all Listeria monocytogenes strains should be considered as poten-
tially pathogenic. With this in mind, and given the ubiquity of this pathogen within slaugh-
terhouse and meat-packing environments, it is not surprising that the incidence and behav-
ior of L. monocytogenes in meat products are receiving increased attention worldwide. In
the beginning of this chapter, results are given for both the IJ.S. Department of Agricul-
ture-Food Safety Inspection Service (USDA-FSIS) Listeria-testing program for cooked
and ready-to-eat meat products and the Agriculture Canada Listeria monocytogenes-moni-
toring program for ready-to-eat meat products. Thereafter results from nonregulatory, in-
dependent surveys of raw meat and of various meat products marketed in North America,

505
506 Farber and Peterkin

Europe, and elsewhere will be discussed. Information regarding behavior of L. monocyto-

genes in raw meat, cooked meat, and various sausage products can be found in the second
half of this chapter.

INCIDENCE OF L/STR/A IN MEAT PRODUCTS

Results from USDA-FSIS Listeria-Monitoring Programs
The USDA-FSIS monitoring/verification program for L. rnonocytogenes in meat products
began in September, 1987, with sampling of domestic corned beef, cooked corned beef,
and massaged corned beef, as well as imported cooked meats. This program was later
expanded to include a much wider range of products (Fig. l), with meat/poultry salads
and spreads being added most recently [93].
Under the revised Listeria policy (Fig. 2), which appeared in the May 23, 1989,
issue of the Federal Register [55], 25-g rather than 1-g monitoring samples obtained from
intact retail packages (e.g., frankfurters, luncheon meat, sausages) are now examined for
L. monocytogenes. If any of these samples contain L. rnonocytogenes, the entire lot manu-
factured on the day or during the shift the positive monitoring sample was taken is consid-
ered adulterated. Unlike the previous policy, which required identification of L. rnonocyto-
genes in one or more verification samples before initiation of further action, the present
policy grants USDA-FSIS officials power to request firms to issue an immediate Class I
recall for all tainted lots in the marketplace. However, manufacturers can avoid a formal
recall by holding all sampled lots until results of Listeria testing are known. In either event,
government officials also will (a) initiate a hold-test program until consistent production of
Listeria-free product has been achieved; (b) collect and analyze other potentially contami-
nated products manufactured at the same facility; (c) encourage firms to review their opera-

I Plans for LIsteda testlng program announced - December 1985

I
4
I Developmentof USDA method and sampltngscheme for detectinglkWa in meat prod= 1986
I
.c
I Announcement of I.M&I monitoringhrerlficationprogram- March 1987 I
1
I inmate various samplingprograms 1

IJerky - October 1988to present1 I

I
products - September
1987 to present

I
Cooked beef, roast beef.
and cooked c o r m beef -
September 1987 to present sausage - September

FIGURE1 Chronological development of the USDA/FSIS testing programs for Liste-

ria in cooked and ready-to-eat meat products. (Adapted from Ref. 93.)
Listeria monocytogenes in Meat Products 507

MONITORING: MONITORING:
Intact retail packages - e.g., Non-intact samples from a large
frankfurters, sausage, luncheon unpackaged product - e.g., roast beef,

1
corned beef
meat
Homogenize 25-g sample in 225 ml
1
Homogenize 25-8 sample in 225m1

+
enrichment broth

L. monocytogenesdetected
enrichment broth
4
L. monocytogenesdetected using
using USDA Method USDA Method
+
1
1. FSIS rgquests immediate Class I
recall of the sample production
VERTFICATTON:
Intact sample fiom subsequent

lots 1
Homogenize 25-g sample in
lot 225 ml enrichment broth

2. FSIS initiates
+
L. monocytogenes detected using
(a) Hold-test program for USDA Method
contaminated product

(b) Testing of other potentially

contaminated products

(c) Microbiological Incident

Surveillance Sampling Program

FIGURE2 USDA/FSIS Monitoring/Verification Program for L. rnonocytogenes in

cooked and ready-to-eat meat products. (Adapted from Ref. 55.)

tions for conditions that may allow growth of listeriae, and (d) take any additional appro-
priate in-factory action to prevent production of contaminated product.
The current policy regarding large products that are not normally available in retail-
sized packages (e.g., roast beef, corned beef) (see Fig. 2) more closely resembles the
September 1 987 version of the monitoring/verification program in that USDA-FSIS offi-
cials will not request an immediate Class I recall if L. rnonocytogenes is detected in one
or more monitoring samples. In such instances, verification samples (intact samples from
subsequent lots) will be collected and analyzed for L. rnonocytogenes using current USDA
methodology. Any verification sample that is positive for L. monocytogenes will automati-
cally trigger action similar to that just described for products in intact retail-sized packages
weighing 1 3 lb; included is the issuance of an immediate Class I recall in the unlikely
event that the firm failed to hold all sampled lots until results of Listeria testing became
known.
Raw Meat
In January 1987, the USDA-FSIS began a monitoring and sampling program to determine
the incidence of L. rnonocytogenes in domestic raw beef. This program was not undertaken
to initiate regulatory action against particular firms but rather to provide the agency with
critical background information. During the 26-month period from January 1987 to Febru-
ary 1990, L. rnonocytogenes was isolated from 122 of 1726 (7.1%) 25-g samples of domes-
508 Farber and Peterkin

tically produced raw beef [64]. In 1995, the FSIS reported on the levels of bacteria, includ-
ing L. monocytogenes, in ground beef [21]. The survey, based on 600 1-lb samples of
ground beef from 661 plants, reported an 18% incidence for the organism.

Cooked a n d Ready-to-Eat Meat Products

The USDA-FSIS monitoring/verification program, started in September of 1987, is de-
signed to determine the extent of L. monocytogenes contamination in both domestic and
imported cooked and ready-to-eat meat products. These products now include beef jerky,
cooked sausage (both large and small diameter), cooked/roast/corned beef, meat salads/
meat spreads, and sliced canned ham/luncheon meat (see Fig. 1). Each sample collected,
consisting of one to six subsamples, represents one lot of product. A portion of each
subsample is then pooled to form a composite, which is analyzed. If a positive result is
obtained for a lot, the composite is not reanalyzed to determine the number of positive
subsamples in the lot.
Results from this program for the years 1993-1996 (Table I ) show that, in general,
there is a low incidence of L. monocytogenes in cooked and ready-to-eat meat products
in the United States, with overall incidences from 0 to 8.1%. The organism was usually
absent from beef jerky, as its presence was reported in this product only once, in 1994,
at a 2.2% incidence. Cooked small-diameter sausage showed a higher prevalence of the
organism than cooked large-diameter sausage, with the incidences ranging from 3.7 to
5.3%, and 1.O to 2.1 %, respectively. L. monocytogenes was present in only 2.1-3.4% of
samples of domestic/imported cooked beef, roast beef, and cooked corned beef over this
4-year period. In most instances, these products were removed from their packages after
cooking and then repackaged for sale, thus suggesting that the pathogen most likely en-
tered the product through direct contact with the factory environment, knives, and/or
gowns worn by workers [ 1071. At least two other firms handled raw and finished product
in the same production area of the factory but at different times, which, in turn, indicates
the possibility of cross contamination between raw and finished product.
In December 1987, USDA-FSIS officials added sliced canned ham and sliced canned
luncheon meat to the monitoring/verification program for the presence of L. monocyto-
genes (see Fig. 1). From 1993 to 1996, the pathogen was detected in 5.1-8.1% of samples,
with these products exhibiting some of the highest incidences among the ready-to-eat meat

TABLE
1 Incidence of Listeria rnonocytogenes in USDA-FSIS Monitoring Samples
of Cooked and Ready-to-Eat Meat Products, 1993-1996
Number of lots sampleda (% positive)

Meat product 1993 1994 1995 1996

Beef jerky 39 (0) 45 (2.2) 50 (0) 43 (0)
Cooked sausage-large diameter 328 (2.1) 438 (1.1) 438 (1.1) 420 (1.0)
Cooked sausage-small diameter 472 (5.3) 602 (4.8) 611 (4.1) 562 (3.7)
Cooked/roast/corned beef 426 (3.1) 479 (2.1) 560 (2.7) 507 (3.4)
Salads/spreads 273 (2.2) 580 (2.4) 597 (4.7) 554 (2.2)
Sliced ham/sliced luncheon meats 149 (8.1) 232 (5.6) 99 (5.1) 91 (7.8)
aEach sample, consisting of one to six subsamples, represents one lot of product.
Source: From Ref. 25.
Listeria monocytogenes in Meat Products 509

products examined (see Table 1). In June 1988, monitoring of meat/poultry salads and
spreads was begun, with incidences of 2.2-4.7% being reported from 1993 to 1996.
After a request from the food industry that the FDA reconsider its policy of zero
tolerance for the presence of L. rnonocytogenes in cooked and ready-to-eat foods, the
FDA stated that it would need the support of scientific data on infectious dose to justify
any change in its regulation that the pathogen should not be present in such foods [ 111.
Later, the FDA announced that its pathogen-monitoring program would concentrate on
selected high-risk foods, including prepared sandwiches [22]. The agency stated that if a
prepared sandwich is found to be positive for L. monocytogenes, then a comprehensive
follow-up inspection of the firm is warranted, including sampling of raw materials, food
processing surfaces, and finished product.

Recalls and Other Regulatory Actions

Between 1990 and 1997, there were at least 12 separate Class I or voluntary recalls of
cooked and ready-to-eat meat contaminated with L. rnonocytogenes (Table 2). Of these,
seven recalls were for prepared sandwiches, three were for ham products, and the re-
maining two for frankfurters. Thus, over half of all recalls issued for cooked and ready-
to-eat meat products since 1990 have involved prepared sandwiches, with at least 60,000
sandwiches being removed from the marketplace. In all but one of these recalls of prepared
sandwiches, the distribution involved at least four states. Product losses may be higher
than indicated, since firms holding tested products until results of Listeria analyses become
known can recall all contaminated lots internally, thereby avoiding a formal Class I
recall. Considering the wide variety of tainted sandwiches that have been withdrawn from
sale, it appears that L. monocytogenes entered these products during cutting, slicing, and/
or packaging rather than being initially present in the many different types of sandwich
fillings.
The two recalls of ham salad in 1991, one originating in Minnesota and the other
in West Virginia, were each confined to one state (see Table 2). The quantities involved
were smaller than those for recalls of prepared sandwiches, with 460 and 600 Ib of products
being recalled, respectively. The frankfurter recalls in 1991 and 1992 were also confined
to one state each; namely, Michigan and Connecticut. These quantities were larger than
for the ham salad, with over 3000 Ib of product being recalled in both instances.

Results from Agriculture Canada Listeria monocytogenes-

Monitoring Programs
Cooked and Ready-to-Eat Meat Products
Agriculture Canada started its L. rnonocytogenes monitoring program on processed and
ready-to-eat meat products from registered processing plants in 1987. At the same time,
the Health Protection Branch (HPB), Health Canada, started a similar testing program for
L. rnonocytogenes in ready-to-eat meat products from nonregistered establishments [7 11.
Five subsamples of 150 g (or a convenient package size) each were collected. The quantity
analyzed depended on the product: For meats supporting growth of Listeria spp., 25 g
was tested, whereas for meats not supporting growth of Listeria spp., 5 g was tested.
During 1989- 1990, the incidence of L. monocytogenes in the ready-to-eat meat
samples tested by the Agriculture Canada monitoring program was high: an average of
about 24% (Table 3). During this period, samples taken at establishments with Listeria-
510 Farber and Peterkin

TABLE
2 Chronological List of Recalls (Class I or Voluntary) Issued in the United States for Cooked and Ready-to-Eat Meat Products
Contaminated with Listeria rnonocytogenes, 1990-1997

Date recall
Product initiated Origin Distribution Quantity Ref.
Sandwiches-BBQ beef, ham and cheese, etc. 1991 LA LA, FL,AL, MS -4860 units 16
Ham salad 1991 MN MN 460 lb 16
Sandwiches-roast beef, etc. 07/03/9 1 LA LA, FL, AL, MS -8057 units 16
Skinless hot dogs 1991 MI MI 3700 lb 14
Ham salad 1991 wv wv 600 lb 15
Frankfurters-beef, pork 03/20/92 CT CT 3578 lb 17
Sandwiches-roast beef, turkey, subs, ham and cheese, BBQ 06118/92 LA LA, FL,AL, MS NA 18
beef, etc.
Sandwiches-beef, ground beef, pork, ham and cheese, ham 09/25/92 TN TN, NC, SC, GA, AL, NA 19
salad, hot dog, BBQ beef, etc. MS, KY, VA, AS
Sandwiches-ham and cheese, ham salad, etc. 09/08/95 MI MT, IN, OH, IL, WI 20, 563 units 16
Sandwiches-roast beef, hot dog, burgers, sausage, meat ball, 02/16/96 LA LA, FL, AL, MS 20-25,000 units 16
ham and cheese, etc.
Sandwiches-hot dog, ham and cheese, sausage, beef, salami, 0 1122197 MS MS NA 23
etc.
Smoked ham 07131/98 NH NH, VT NA 25a
NA. not available.
Listeria monocytogenes in Meat Products 51 1

TABLE
3 Incidence of Listeria monocytogenes in Agriculture Canada Testing
Samples of Domestic and Imported Ready-to-Eat Meat Products and in Processing
Plants, 1989- 1994
Domestic Imported products
products
no. of samples
no. of samples (% positive) Plant environment
Year (% positive) USA Others no. of samples (% positive)
1989 396 (12) 50 (4) 5 (0) 677 (17)
1990 66 (35) 1279 (2) 3 (0) 2267 (8)
1991 19 (0) 984 (2) 14 (0) 3314 (13)
1992 35 (3) 469 (3) 9 (0) Phase Ia Phase I1 Phase I11
~- ~ ~

3808 (10) 430 (9) 164 (13)

1993 NDb 417 (3) 1 (0) 2522 (6) 124 (19) 20 (0)
1994 ND 382 (5) 1 (0) 3629 (2) 138 (6)
a Phase 1-10 composited postprocess samples of product contact surfaces.
Phase 11-if Phase I positive, 10 individual environmental samples.
Phase 111-if Phase I1 positive, review of plant procedure prior to further individual sampling.
ND, not determined. After 1992, environmental testing only was done for domestic products.
Source: From Ref. 8.

positive products showed an incidence of 46% for meat products (data not shown) and
an average of 12% for environmental samples. The incidence dropped sharply during
1991-1992 to levels of 0-3% for meat products, but the contamination in the factory
environment remained about the same. After 1992, only factory environmental samples
were taken for testing domestic products, but a more rigorous testing procedure involving
three phases was instituted. Phase I of the program consisted of taking 10 postprocess
environmental samples of a product contact surface. These 10 samples were composited
into one sample for analysis. Any positive Phase I analysis triggered entrance into Phase
11, consisting of 10 repeat environmental samples, analyzed individually, and a review of
good manufacturing practices (GMP) throughout the plant. Phase 111 was entered if one
or more analyses in Phase I1 were positive, in which case a thorough review of all manufac-
turing procedures was undertaken before collecting further environmental samples for
individual analysis. Between 1992- 1994, the incidence of L. monocytogenes in food pro-
cessing environments dropped sharply, indicating the value of this approach.
The presence of L. monocytogenes in imported ready-to-eat meat products during
this period remained low, with incidences ranging from 0 to 5% (see Table 3). The impor-
tance to Canadian consumers of imports from the United States compared with other
countries is evident from the the relatively large number of American samples tested.

Recalls and Other Regulatory Actions

The current Canadian policy on control of L. monocytogenes in foods is risk based. Thus,
for high-risk meats such as liver pit6 and jellied pork tongue, which have been causally
linked to listeriosis, a Class I recall would be initiated at the retail level. However, all
other ready-to-eat meats that support growth of L. monocytogenes and have a refrigerated
shelf life of >10 days would only be subject to a Class I1 recall. In Canada between
5 12 Farber and Peterkin

TABLE
4 Class II Recalls issued in Canada for Processed and Ready-to-Eat Meat
Products Contaminated with Listeria monocytogenes, 1989-August 1997
Number of recalls

dry
fermented sandwiches1 sliced cooked
Year wieners sausages subs roast beef ham miscellaneousa
1989 4 2
1990 1 1 2
1991 1 8 3
1992 1 1
I993 1
1994 1
1995 1 2 4 3
1996 15
1997 2 1 1 2
Totals 7 20 19 12 9 8
aPizza, burrito, cretons, pork ribs, ham salad
Source: From Refs. 9 and 24.

1989-1997, 20 recalls were ordered for dry, fermented sausages, some of which were
smoked (Table 4). An almost equal number of recalls were initiated for sandwiches, in-
cluding submarine sandwiches, containing meat. Sliced cold meats of various types also
were found to contain L. monocytogenes during this period, possibly because of contami-
nation from retail slicers. Wieners and miscellaneous products comprised the balance of
the recalls.

Incidence of Listeria spp. in Raw Meat

Slaughter animals are a recognized reservoir of human pathogens, including L. monocyto-
genes. Numerous studies have shown that, in general, the incidence of listeriae is low (0-
9%), both in feedlot cattle [166] and in pork carcasses [2,82,150,159]. However, a 96%
incidence for listeriae was reported on pork carcasses from one slaughterhouse [82], thus
suggesting poor sanitation. The incidence of listeriae on slaughterhouse equipment was
also low (0-3%) [82,154] except for the factory mentioned above where the incidence
was reportedly 42% [82]. Levels of the organism were low (from 10 to 100 CFU/g) [82].
The only factory area yielding L. monocytogenes was a cold room maintained at 5"C, at
which temperature this psychrotrophic organism is able to grow.
North America
In addition to the government monitoring programs in North America, there are several
reports of nonregulatory surveys of both raw and ready-to-eat meat products (Table 5).
Other authors also found that in raw meat products such as roasts and steaks sampled in
the United States, the incidence of Listeria spp. ranged from 0 to 6%, with L. monocyto-
genes being in the same range (Table 5). In contrast, comminuted raw meats showed a
much higher incidence of listeriae, from 24 to 1OO%, with L. monocytogenes ranging from
0 to 25%. Reported levels of listeriae ranged from 4.1 X 103to 2.1 X 104 CFU/g.
In raw meat products sampled in Canada (principally ground meats), the incidence
Listeria monocytogenes in Meat Products 513

TABLE
5 Incidence of Listeria monocytogenes in R a w M e a t a n d Ready-to-Eat M e a t
Products in t h e USA a n d Canada, 1990-1997

Percentage of
Number of positive samples Levels of
samples - Listeria spp.
Product analyzed Listeria spp. LM (CFU/g) Ref.
Raw meats-USA
beef roast 50 116
pork roast 50 LM- 10 1 I6
lamb roast 10 116
ground beef 39 4-560 192
ground pork 20 4-240 192
pork sausage 17 240-2 1000 192
lamb patty 2 4-56 192
beef 658 111
pork loin 135 159
Raw meats-Canada
ground beef 22 73
ground pork 19 73
ground veal 3 73
ground meat 11 135
meat cuts 18 135
wild animal meat- 10 135
moose, deer, bear
RTE products -USA
wieners 93 181
wieners 24 181
wieners 30 20
RTE products -Canada
fermented sausages 30 73
cooked meat 16 170
wieners 38 I70
luncheon meats 67 170
sausages 9 170
LM, L. monocytogenes; ND, not determined; RTE, ready-to-eat.

of Listeria spp. is high (ranging from 66 to 100%).The incidence of L. monocytugenes

ranged from 44 to loo%, indicating that contamination of raw meat with this pathogen
is relatively common (see Table 5). This may be the result of contaminated equipment at
either the wholesale or retail level. It is noteworthy that there was a much lower incidence
of listeriae in wild rather than domesticated animals.
Europe
Since 1990, many reports have been published on the prevalence of Listeria spp. and L.
monocytogenes in raw meat and raw meat products produced in various European coun-
tries (Table 6). In the United Kingdom, MacGowan et al. [ 1361 reported incidences of
Listeria spp. ranging from 59 to 88% in beef, lamb, and pork, with L. monucytugenes
being preseni in 28-40% of these samples. Based on a larger sampling of beef, lamb, and
pork in Northern Ireland, Wilson [184] reported an incidence of about 4% for listeriae.
514 Farber and Peterkin

TABLE
6 Incidence of Listeria monocytogenes in Raw Meat in European and Other
Countries, 1990-1997
% of positive
Number of samples
samples
Country Product analyzed Listeria spp. LM Ref.
United Kingdom Pork sausage 59 79
Beef 26 136
Lamb 20 136
Pork 32 136
Sausage 23 136
Meat 15 96
Beef 1295 184
Lamb 37 184
Pork 794 184
Ireland Beef 20 163
Ground beef 85 163
Pork 20 163
Lamb 20 163
Sausage 20 163
Frozen beef burgers 94 163
Norway Ground meat 40 157
Germany Ground meat 21 111
Rinderhack 59 111
Italy Calf 19 138
Horse 19 138
Pork 19 138
Mutton 18 138
Sausage 156 43
casing surface 116 43
Pork 67 179
Beef 99 179
Ground beef 148 52
Pork 153 52
Ground meat 308 53
Beef 174 53
Raw meat 82 131
Sausage 30 131
Switzerland Ground meat 85 38
Beef 18 38
Pork 31 38
Sausage meat 102 38
Dried meat 44 38
Ham-uncooked 19 38
Spain Ground meat 168 61
Ground pork 42 139
Ground beef 41 139
Poland Pork 245 126
Beef 114 126
Bosnia/Hercegovina Beef 20 133
Pork 50 133
Listeria monocytogenes in Meat Products 515

TABLE
6 Continued
~-

% of positive
Number of samples
samples .
Country Product analyzed Listeria spp. LM Ref.
Bulgaria Beef and pork 234 151
Trinidad Beef 76 1
Ground beef 35 1
Mutton 66 1
Goat meat 70 1
Pork 71 1
United Arab Emirates Beef 15 86
Goat 17 86
Sheep 24 86
Camel 14 86
Australia Beef 50 110
Lamb 50 110
Pork 50 110
Malaysia Beef 12 26
India Cattle 54 37
Buffalo 54 37
Sheep 54 37
Goat 54 37
Japan Beef 15 158
Ground beef 5 158
Pork 18 158
Ground pork 6 158
China Pork 25 182
Beef 10 182
Lamb 14 182
Taiwan Beef steak 25 187
Pork 34 187
LM, L. rnonocytogenes; ND, not determined.
a 5-500 Listerra CFU/g.

5- 10,000 Lisferia CFU/g.

<loo Listeria CFU/g.
<I00 L. monocytogenes CFU/g.

According to Gilbert 1791, 49% of raw pork sausage harbored L. monocytogenes, with
MacGowan et al. [136] reporting a similar incidence of 35%.
Working in Ireland, Sheridan et al. [ 1631 observed high incidences of listeriae rang-
ing from 45 to 85% in 20 samples each of beef, pork, and lamb, with L. monocytogenes
being present in 1 5 5 0 % of the samples. Interestingly, less contamination was observed
in comminuted meats (94 samples of frozen beef burgers, 85 samples of ground beef, and
20 samples of sausage), with L. monocytogenes being present in only 5- 18% of the sam-
ples. An incidence of 5% for L. monocytogenes in ground meat was found in Norway
[157], with the pathogen belonging to serotype 1 .
In Germany, about 50% of ground beef and raw beef products reportedly contain
L. rnonocytogenes 11113. Since raw minced meat is a popular dish in Germany, the German
5 16 Farber and Peterkin

Ministry of Health decided to restrict the sale of contaminated product [7]. For samples
containing <100 CFU/g, the factory or retail outlet undergoes further surveillance, and
a public warning is issued if numbers of listeriae reach 2 1000 CFU/g.
Workers in Italy have examined many types of raw meat for listeriae. Maini et al.
[138] reported that in 19 samples each of calf, horse, pork, and mutton, the incidences of
listeriae were 63, 26,53, and 56%, respectively. Cantoni et al. [43] showed that the preva-
lences of the organism on sausages and their casing surfaces were roughly equal; that is,
60 and 47% for listeriae and 13 and 12% for L. monocytogenes, respectively. Other surveys
of fresh pork and beef showed incidences ranging from 21 to 47% for listeriae and 13 to
22% for L. monocytogenes, with serotype 4 being found in 7% of these samples [43].
Comminuted meats and sausages showed higher incidences for Listeria spp. (44-54%)
than for L. monocytogenes (9- 19%) [52,53,13I], with serotype 1/2c predominating [52].
Listeria-positive samples generally contained < 100 CFU/g.
Breer and Schopfer [38] reported that listeriae were recovered from 11-45% of 209
samples of beef, pork, and pork products collected in Switzerland, with an incidence of
0- 15% for L. monocytogenes. In comminuted meat products, the corresponding incidences
were 40-65% and 8- 15%, respectively. Reports from Spain showed that of 25 1 samples
of ground meat, 63-80% contained listeriae, with L. monocytogenes being recovered from
17-29% of the samples [61,139].
In Eastern European countries, workers have observed similar levels (8-20%) of
listeriae contamination in 613 samples of fresh beef and pork to those found in Western
Europe [ 126,133,1511. The incidence of L. monocytogenes in these samples ranged from
7 to 10%, with a prevalence (94%) of serotypes 1 to 3 and the remaining being serotype
4 [126].
Other Countries
Reports from elsewhere indicate that the problem of Listeria-contaminated meat is world-
wide and deserves the serious attention of the industry. In general, the prevalence of Liste-
ria spp., and more particularly, L. monocytogenes, is in the same range as that already
discussed for North America and Europe.
In Trinidad, Adesiyun [ I ] identified listeriae in 0- 10% of samples of fresh beef, mut-
ton, and goat meat, with L. monocytogenes being present in 0-4% of the samples. The organ-
isms were prevalent in ground beef samples at similar levels: 1 1 and 6%, respectively. L.
monocytogenes serotypes 1/2c and 4 were found in both local and imported meat.
Similar incidences of listeriae have been reported in the Asian countries. In the
United Arab Emirates, Gohil et al. [86] examined fresh beef ( I 5 samples), goat (17 sam-
ples), sheep (24 samples), and camel (I4 samples) and found that listeriae were present
in 0-21% of the samples. No samples contained L. monocytogenes. Among 50 samples
each of fresh beef, lamb, and pork examined in Australia, listeriae were found in 34, 40,
and 30% and L. monocytogenes in 24, 16, and 10%, respectively [ 1 101. In Malaysia, 50%
of 12 beef samples contained L. monocytogenes [26]. Examination of 54 samples each of
cattle, buffalo, sheep, and goat in India by Erahmbhatt and Anjaria [37] yielded a low
incidence of L. monocytogenes (4-6%). Ryu et al. [ 1581 reported high incidences for
listeriae in Japanese meats, with 40 and 13% of fresh beef and and 61 and 39% of fresh
pork yielding Listeria spp. and L. monocytogenes, respectively. In ground beef and pork
samples, the prevalence was higher, with 80 and 60% and 100 and 67% of the samples
being positive, respectively.
A study done by Wang et al. [182] in China showed a high incidence (from 43 to
Listeria monocytogenes in Meat Products 517

70%) of listeriae in pork, beef, and lamb. However, the incidence of L. monocytogenes
was much lower, ranging from 0 to 28%, with serotypes 1/2a, 1/2b, and 1/2c being identi-
fied. In contrast, meats from Taiwan showed a high incidence, with 24 and 59% of all
beef steak and pork samples respectively being positive for L. monocytogenes [ 1871.

Incidence of Listeria spp. in Sausage and Ready-to-Eat

Meat Products
The prevalence of L. rnonocytogenes in processed, ready-to-eat meats which may be con-
sumed without further heating is obviously of greater concern than contamination of raw
meats. Given that one survey of retail meat slicers revealed an L. monocytogenes contami-
nation rate of 13%, such machines can easily serve to cross contaminate sliced meats
[ 1071. Two factors, namely, development of improved Listeria isolation methods and a
heightened concern about foodborne listeriosis, have led scientists to examine sausage,
piti, and other ready-to-eat meat products for listeriae. Consequently, numerous world-
wide surveys have been conducted since 1990 to determine the incidence of these organ-
isms in such products.
North America
Since the association of sporadic listeriosis with consumption of uncooked frankfurters
[ 1611, several nonregulatory United States surveys were initiated to determine the inci-
dence of listeriae in packages of retail wieners (see Table 5). After examining 20 brands
of retail wieners, Wang and Muriana [ 18 11 reported that 19 brands (93 samples) showed
a 10% incidence of listeriae, with an 8% incidence of L. monocytogenes. However, one
brand contained listeriae in 83% of its 24 packages, with a 7 1% incidence of L. monocyto-
genes. Most listeriae were found in the liquid exudate at a level of 1-3 CFU/mL, rather
than in the meat itself, thus indicating probable postprocessing contamination. A survey
commissioned by the Los Angeles Times found that, in a sample of 30 packages of retail
wieners, 20% contained listeriae, with a 17% incidence for L. rnonocytogenes [20].
A Canadian study [ 1701 reported that wieners (38 samples) and sliced meats (67
samples) showed incidences of 13-26% for Listeria spp., and 13-21% for L. monocyto-
genes (see Table 5). In contrast, Listeria spp. were not isolated from cooked meat (16
samples) anti sausages (9 samples). However, fermented sausages (30 samples) studied
by Farber et al. [73] yielded a 20% incidence for L. rnonocytogenes.
Europe
Numerous surveys have been conducted to determine the incidence of listeriae in ready-
to-eat meat and sausage products manufactured in Europe (Table 7). Public Health Labora-
tory Service workers in England and Wales surveyed a large number of samples to deter-
mine the incidence of listeriae in various foods. McLauchlin and Gilberts report [145]
on pit6 (696 samples) and ready-to-eat meat and poultry (3939 samples) showed listeriae
present in 20 and 15% and L. monocytogenes present in 17 and 8% of the samples, respec-
tively. A report on 605 samples of sliced meats showed a lower incidence for listeriae
(6%) and L. monocytogenes (3%), with levels of <200 to <500 CFU/g [ 1771. Most (91%)
L. rnonocytogenes isolates were serotype 1/2, with the remainder being serotype 4. In an
extensive study conducted in Yorkshire 121, cooked meats (1 55 1 samples), pit6 (239
samples), meat salad (1 5 samples), and meat sandwiches (237 samples) revealed inci-
dences of 13, 11, 33, and 20% for listeriae and 5, 7, 13, and 8% for L. rnonocytogenes,
518 Farber and Peterkin

TABLE
7 Incidence of Listeria monocytogenes in Sausages and Ready-to-Eat Meat
Products in European and Other Countries, 1990-1997
% of positive
No. of samples Levels of
samples Listeria spp.
Country /Product analyzed Listeria spp. LM (CFUlg) Ref.
United Kingdom
pit6 696 145
RTE meat and poultry 3939 145
sliced meat
ham 303 177
salami 128 177
tongue 28 177
corned beef 27 177
mixed meats 119 177
prepared sandwiches 91 108
cooked meats 1551 <20- 1000 12
pit6 239 <20- 100 12
salad with meat 15 <20-100 12
sandwiches with meat 237 20- 100 12
cook-chill-meat and 736 79
poultry
salami 67 79
pit6 1834 LM-<200-106 80
pit6 626 LM-<200- 105 80
cook-chill food-meat 992 LM-< 10- 103 101
cook-chill food-meat 854 LM-< 10-103 101
pit6 216 79
cured or smoked meat 29 10
cooked tripe 44 109
Denmark
sliced ham 80 156
sliced rolled sausage 80 156
sliced smoked pork loin 78 156
frankfurters 67 156
Norway
VP processed meat 35 157
France
sausages, pitis, ham 990 128
Italy
pork sausage 55 132
sausage-mixed meat 20 132
ground beef rissoles 45 132
sausage 82 47
wiirstel 118 LM- 10-224 47
pit6 48 47
Switzerland
sliced cured dried beef 26 LM-20 172
salami 59 LM-20 172
mettwurst 14 172
Listeria monocytogenes in Meat Products 5 19

TABLE
7 Continued
% of positive
No. of samples Levels of
samples - Listeria spp.
Country/Product analyzed Listeria spp. LM (CFU/g) Ref.
Spain
cured sausilge 17 30
cooked ham or sausage 15 30
luncheon meat 60 1-100 127
pit6 36 1-100 127
Hungary
dry cured sausage 136 124
fermented sausage 21 124
smoked sausage 23 124
Yugoslavia
fermented sausage 21 40
hot smoked sausage 15 40
VP hot smoked sausage 14 40
South Africa
Vienna sausage 47 180
ham 43 180
cervelat 44 180
Australia
piit6 7 162
luncheon meat 28 162
pit6 25 98
processed meat 25 98
luncheon meat 20 171
VP salami 19 91
VP corned beef 72 91
VP ham 71 91
VP luncheon meat 13 91
salami 132 176
ham 90 176
corned beef 39 176
pit6 7 176
luncheon meat 16 176
New Zealand
RTE pork 34 103
RTE beef 18 103
RTE lamb 3 103
LM, L. monocytogenes; RTE, ready-to-eat; ND, not determined; VP, vacuum-packed.

respectively. Levels of the organism in this study ranged from <20 to 1000 CFU/g. An-
other report on 91 samples of preprepared sandwiches showed a 17% incidence for L.
rnonocytogenes [ 1OS]. Of 13 strains examined, 10 were serotype 1 /2 and 3 were serotype
4. A survey of 1834 and 626 pi% samples in 1989 and 1990 showed incidences of 10
and 4%, respectively, for L. rnonocytogenes with levels of the organism up to 106CFU/
g [80]. Both serotypes 1/2 and 4 were present. In another study, L. rnonocytogenes was
520 Farber and Peterkin

present in 16% of 67 salami samples [79]. Cooked tripe, which is often eaten without
further heating, showed a 9% incidence of L. monocytogenes [ 1091. In cook-chill catering,
often used by hospitals, food is prepared and cooked in a traditional manner, cooled very
rapidly, and maintained chilled (0-3C) for up to 5 days until reheated for use. In three
surveys of cook-chill foods containing meat (736, 992, and 854 samples), the incidence
of L. monocytogenes was 2, 3, and 9%, respectively [79,101]. Of 28 strains typed, 7 were
serotype 1/2, 17 were serotype 3, and 4 were serotype 4 [ l o l l . These results must give
rise for concern in light of the many highly vulnerable patients in hospitals.
In Denmark, 305 samples of wieners and sliced meats showed similar incidences,
10% of which contained L. monocytogenes, at a level of < 100 CFU/g [ 1561. A Norwegian
study of vacuum packaged processed meat showed that 11% of 35 samples contained L.
monocytogenes [ 1571.
In France, 22% of 18 dry sausages harbored L. monocytogenes, whereas in Germany,
9% of 11 mettwurst samples contained the organism [ 1111.
Several surveys of sausages in Italy showed similar incidences to the above. Levrk
et al. [132], reporting on 120 samples of pork sausage/mixed chicken, turkey, pork
sausage/beef rissoles, found that overall as many as 90% of these samples contained lister-
iae, with 33% being positive for L. monocytogenes. In another study carried out over a
2-year period (1990- 1991), Casolari et al. [47], demonstrated similar incidences in sau-
sage, with 88% and 28% of 82 samples showing the presence of Listeria and L. monocyto-
genes, respectively. Additionally, 36% of 166 meat product samples (wurstel, p2t6) con-
tained listeriae, with L. monocytogenes being present in 4% of these samples at a level
of 10-224 CFU/g.
A survey in Switzerland taken during the production of cured and air-dried meat
products [ 1721 uncovered a substantial number of Bundnerfleisch (cured, air-dried beef),
salami, and mettwurst samples that contained listeriae. Overall, of samples taken during
production, Bundnerfleisch (19 samples), salami (30 samples), and mettwurst (3 samples)
contained 26, 50, and 100% of listeriae and 11, 10, and 0% of L. monocytogenes, respec-
tively (data not shown). The endproducts showed listeriae at incidences ranging from 15
to 93%, in contrast to incidences ranging from 0 to 5% for L. monocytogenes. The organ-
ism was present at levels of <20 CFU/g. Listeriae were isolated only from the surface
of Bunderfleisch. L. monocytogenes isolates were of serotype 1/2 (86%), and serotype 4
(14%). Since most L. monocytogenes isolates from human listeriosis patients in Switzer-
land belong to serotype 4b, these data suggest that transmission of the pathogen via meat
is relatively uncommon.
Surveys of ready-to-eat meat products in Spain showed a 13% incidence for Listeria
spp. in cured or cooked sausages and ham (a total of 32 samples) [30]. Twelve percent of
the cured sausages contained L. rnonocytogenes,with the pathogen being absent in ham and
cooked sausage [30]. Among sliced meat (60 samples) and pi%&(36 samples), 42 and 14%
of samples contained Listeria spp., with 22 and 3% of samples being positive for L. monocyto-
genes, respectively [ 1271. Both organisms were found at levels of 1- 100 CFU/g.
In 1991, a Hungarian researcher, KovAcsn6 Domjan [ 1241, recovered listeriae from
37 of 136 (27%), 7 of 21 (33%), and 15 of 23 (65%) samples of dry-cured, fermented,
and smoked pork sausage, respectively. L. monocytogenes was present in 10, 10, and 13%
of these samples, respectively. Working in Yugoslavia, Buntid [42] found that 28 and
19% of 2 1 fermented sausage samples contained listeriae and L. monocytogenes, respec-
tively. In contrast, listeriae were not recovered from hot, smoked sausage. Of 14 surface
samples of vacuum-packed hot smoked sausage, 35 and 2 1% yielded listeriae and L. mono-
cytogenes, respectively, indicating recontamination before and during vacuum packaging.
Listeria monocytogenes in Meat Products 52 1

Overall, the results from Table 7 indicate that a substantial portion of European
processed meat is contaminated with listeriae, including L. monocytogenes. The presence
of listeriae other than L. monocytogenes in processed as well as raw meat may be indicative
of possible contamination with L. rnonocytogenes.
Other Countries
A recent report by Vorster et al. [ 1801 in South Africa showed a low prevalence for listeriae
in 134 retail samples of processed, ready-to-eat meats, with an overall incidence of 8%
in Vienna sausage, ham, and cervelat (see Table 7). None of the samples yielded L. mono-
cytogenes.
Several surveys from Australia show incidences in processed meats which are simi-
lar to those reported from North America and Europe. According to Robertson and co-
workers [162,171], l of 20 (5%) and 3 of 28 (1 l%) sliced meat samples yielded Listeria
spp., with L. monocytogenes being recovered at rates of 0 and 7%, respectively. No Listeria
spp. were recovered from seven samples of pgt6. Hobson et al. [98] reported that in 25
samples each of pgt6 and processed meat, L. monocytogenes was present in 8 and 4%,
respectively. When 175 samples of vacuum-packaged processed meat were examined,
Grau and Vanderlinde [91] reported that 60 of 72 (88%) siimples of corned beef, 29 of
71 (41%) samples of ham, 3 of 13 (23%) samples of luncheon meats, and 1 of 19 ( 5 % )
samples of salami yielded listeriae, with 72,34, 15, and 0% of these samples being contam-
inated with L. monocytogenes, respectively. Sixteen corned beef samples had listeriae
counts >50 CFU/g, and, of these, six counts were >I000 CFU/g. Listeria counts for
salami were <50 CFU/g, and no L. rnonocytogenes was found. In another survey involving
284 samples of ready-to-eat meat products, Varabioff [ 1761 found that except for pit&,
which was free of listeriae, the incidence of listeriae ranged from 5 to 41% (see Table
7); L. rnonocytogenes was present in 4-19% of the samples. Of the isolates recovered,
62% were serotype 1 and 36% were serotype 4. Additional testing of cutting utensils and
cutting boards showed that most of the contamination occurred at the retail level.
A New Zealand survey of 55 samples of ready-to-eat meat products showed listeriae
incidences ranging from 23 to 67% [ 1031. Interestingly, even though mixed-source prod-
ucts had a lower incidence (23%) of listeriae than products made from a single meat (50-
67%), at least half of the isolates from mixed meats were L. monocytogenes, whereas the
pathogen was seldom recovered from single meat products. The authors also reported a
significantly higher incidence for L. monocytogenes in smoked products than in meats
preserved by other methods.
Additional information concerning the various habitats, niches, and relative inci-
dence of listeriae in all facets of the meat industry is needed, as it is now evident that the
presence of L. rnonocytogenes in processed meat products may pose a serious health hazard
to certain individuals. Therefore, it is necessary to control all listeriae in meat-processing
facilities and to design procedures and treatments that will eliminate L. rnonocytogenes
from ready-to-eat processed meat products.

BEHAVIOR OF L. MONOCYTOGENES IN MEAT PRODUCTS

Although L. monocytogenes was found during the 1950s in European meat products des-
tined for human consumption, a general failure to positively link cases of human listeriosis
to foods other than raw milk provided little incentive to examine the behavior of listeriae
in meat. This situation was further complicated by a lack of reliable and convenient meth-
ods to isolate listeriae selectively from heavily contaminated samples of animal origin,
522 Farber and Peterkin

including organ as well as muscle tissue. Thus, the first definitive studies on the behavior
of L. monocytogenes in raw meat were not begun until the 1970s. Even then, to quantitate
this organism in meat products during extended storage accurately, it was generally
deemed necessary to use specially treated sterile meat rather than raw meat products
containing a normal microbial background flora.
Outbreaks of foodborne listeriosis linked to consumption of contaminated cheese
prompted concerns about the microbiological safety of raw and, particularly, ready-to-eat
meat products marketed in North America and Europe. These outbreaks also demonstrated
an urgent need for methods to detect listeriae rapidly and accurately in a wide range of
foods. Subsequent development of the USDA procedure to detect listeriae in meat and
poultry products provided researchers with a method by which to determine the growth
and survival of L. monocytogenes in raw and ready-to-eat products. Recent meat-oriented
epidemiological studies along with several large listeriosis outbreaks linked to meat prod-
ucts suggest that the behavior and control of L. monocytogenes in meat products is likely
to remain an active area of research for some time to come. As in the previous section
of this chapter, information concerning the behavior of L. monocytogenes in raw meat
will be presented first, followed by a discussion of the fate of this pathogen in processed
meat products.

Listerial Infections in Domestic Livestock

As you will recall from Chapter 3, domestic farm animals such as cows, sheep, and pigs
not only succumb to listerial infections but also asymptomatically shed L. monocytogenes
in their feces for many months. Although virtually all meat from animals exhibiting obvi-
ous signs of listeriosis will be condemned and destroyed immediately after slaughter, meat
from subclinically infected animals will likely be passed by inspectors as being fit for
consumption. Since between 2 and 16% of all healthy cows, sheep, and pigs passively
shed L. monocytogenes in their feces, ample opportunity exists for contamination of mus-
cle tissue during slaughter, evisceration, and dressing of the animals.
In all likelihood, meat from domestic livestock will first be exposed to listeriae in
the slaughterhouse environment. However, various organs from apparently healthy ani-
mals can also occasionally contain L. monocytogenes. In 1972, Hohne [99] reported that
approximately 13% of the parotid glands from clinically healthy pigs contained L. monocy-
togenes. Three years later, Hohne et al. [IOO] detected L. monocytogenes in intestinal
lymph nodes from eight apparently healthy slaughtered animals (five small ruminants,
two pigs, one cow) destined for human consumption. Subsequently, Cottin et al. [54]
identified L. monocytogenes in 15 of 514 (3.1%) spleen and/or lung tissue samples ob-
tained from apparently healthy cattle. According to Amtsberg et al. [3], L. monocytogenes
was isolated from the spleen and muscle tissue of an apparently healthy animal. These
findings suggest that L. monocytogenes might be transported to tissue via the blood stream
in animals suffering from symptomatic as well as asymptomatic septicemic listerial infec-
tions. Hence, to understand the behavior of L. monocytogenes in meat products, it is fitting
to begin this section by first examining the localization of L. monocytogenes in organ and
particularly muscle tissue from infected animals.

Localization In Tissues
In 1988, Johnson et al. [ 1 141 reported results from a study in which samples of muscle,
organ, and lymphoid tissue as well as feces and blood from several Holstein cows were
Listeria monocytogenes in Meat Products 523

examined for L. monocytogenes 2, 6, or 54 days after intravenous inoculation (lO'o-lO1l

L. monocytogenes cells) using a combination of direct plating and cold enrichment. As
expected, recovery of listeriae varied among animals and was strongly influenced by the
time that elapsed between inoculation and slaughter. Overall, 94% of all samples obtained
from cows slaughtered 2 days after inoculation contained L. monocytogenes, with 23 of
32 (72%) samples being positive by direct plating. More important, the pathogen was
routinely detected in muscle tissue from the same animals, frequently at levels of 120-
280 CFU/g. Despite a marked decrease in recovery of L. monocytogenes from animals
-
examined 6 and 54 days postinoculation, the pathogen was still present at levels of 140-
675 CFU/g in kidney tissue as well as in mesenteric and mammary lymph nodes, with
populations being about two orders of magnitude lower in liver and spleen tissue. Follow-
ing 2 weeks of cold enrichment, the pathogen also was detected in plate-flank tissue taken
from an animal 6 days after slaughter. These findings suggest that consumption of animal
organs may constitute a greater health hazard than muscle tissue, and they also readily
explain how evisceration of domestic animals can lead to surface contamination of muscle
tissue.
Assuming that most contamination occurs during handling of carcasses after slaugh-
ter, Chung et al. [49] investigated the ability of L. monocytogenes to attach to (or become
entrapped) and proliferate on muscle and fat tissue both alone and in the presence of
Pseudomonas aeruginosa. Immersion of lean muscle and fat tissue in broth cultures con-
taining 10' or 108 L. monocytogenes CFU/mL for various times followed by thorough
rinsing resulted in large numbers of listeriae being attached to (or entrapped in) both types
of samples within the first 10 mins. Despite large differences in hydrophobicity, pliability,
and surface qualities, L. monocytogenes adhered (entrapped) equally well to lean muscle
and fat tissue during 50-60 min of incubation at ambient temperature. According to an
earlier report by Herald and Zottola [97], attachment of L. monocytogenes to stainless
steel and presumably meat surfaces is related to flagellae, fibrils, and exopolymeric sub-
stances (i.e., polysaccharides), all of which are readily produced by Listeria during ex-
tended incubation at room temperature. However, lean muscle tissue supported faster
growth of the pathogen than fat tissue when samples were stored 1 day at ambient followed
by 7 days at refrigeration temperature. Following attachment of L. monocytogenes and P.
-
aeruginosa to lean meat at a concentration of 106 CFU/4 cm2, Listeria populations
increased approximately 100-fold during 24 h of incubation at room temperature and re-
mained at this level during 7 days of refrigerated storage regardless of the presence or
absence oft'. aeruginosa. In contrast, populations of P. aeruginosa on lean meat increased
> 100-fold during initial storage at room temperature, but then decreased with levels fre-
quently 10 times lower than the Listeria population following 7 days of refrigerated
storage.
Dickson [62] simulated contamination of raw beef during processing, handling, and
storage by placing surfaceF of heavily inoculated lean and fat beef tissue (-2 X 106 L.
monocytogenes CFU/cm2) in direct contact with uninoculated tissue. Overall, transfer of
listeriae was largely dependent on the type of tissue, with minimum and maximum transfer
being observed from fat-to-fat and lean-to-fat tissue, respectively. However, bacterial
transfer was also influenced by adsorption time of the original inoculum and contact time
with uninoculated tissue, Adsorption times of <60 min generally led to the highest Listeriu
transfer rates, particularly between inoculated and uninoculated lean beef tissue. These
findings are readily explained when one considers that most listeriae are likely to be freely
suspended (unattached) in water films on tissue surfaces shortly after inoculation. Follow-
524 Farber and Peterkin

ing an adsorption time of 30 min, approximately 30 and 50% of the original Listeria
inoculum migrated from lean and fat tissue, respectively, to lean tissue after 5 min of
direct contact at ambient temperature. When contamination between fat and lean tissue
was simulated using shorter contact times of 15-60 s, a greater percentage of listeriae
migrated from inoculated fat to uninoculated lean tissue, which in turn likely reflects the
transfer of cells in unadsorbed water from hydrophobic fat to hydrophilic lean tissue. More
important, the fact that bacterial transfer also occurred at 5C with 0.6-9.5% of the original
Listeria population migrating from inoculated to uninoculated lean and/or fat tissue after
an 18-h adsorption period provides a reasonable explanation for the spread of this pathogen
to Listeria-free meat during storage in walk-in coolers.
These findings attest to the hardy nature of L. monocytogenes on the surface of raw
meats and to the need for effective means of reducing surface contamination on carcasses.
Regarding the latter, Chung et al. [50] reported that wash solutions containing nisin effec-
tively delayed growth of L. monocytogenes on surfaces of raw meats, particularly when
such products were incubated at refrigeration, rather than ambient temperatures. Although
nisin-producing bacterial starter cultures have been used in the dairy industry for many
years, with the exception of certain types of cheese spread, present laws in North America
still forbid direct addition of nisin to most foods, including raw meat.
Populations of enteric pathogens (i.e., Salmonella spp., enteropathogenic Esche-
richia coli, and Yersinia spp.) on raw meats can be sharply reduced by exposing the
water phase of meat surfaces to 0.2 M lactic acid (pH 2.5) at 21C [ 1141. Although L.
monocytogenes is generally recognized as being more acid tolerant than the previously
mentioned enteric pathogens, this organism is nevertheless inactivated at pH values <4.
Hence, provided that L. monocytogenes is exposed to lactic acid for sufficient time, acid
washes may be somewhat helpful in decreasing Listeria populations on the surface of
animal carcasses.
As noted by Johnson et al. [ 1141, L. monocytogenes was routinely detected in muscle
tissue from cows that were killed 2 days after being inoculated intravenously with the
pathogen. Although some contamination of muscle tissue might have occurred during
sampling, results from this study suggest that L. monocytogenes can enter muscle tissue
via the blood stream.
To further investigate this hypothesis, Johnson et al. [ 1121 examined muscle, liver,
and spleen tissue from two lambs and one calf that had been inoculated intravenously with
L. monocytogenes. Microscopic examination of tissues stained with immunoperoxidase or
Azure A revealed L. monocytogenes cells at levels of 103-104CFU/g in muscle tissue
and 103-106 CFU/g in both liver and spleen tissue. Although L. monocytogenes appeared
to be associated with phagocytes in liver and spleen tissue, the pathogen was observed
in loose connective tissue between muscle fibers and also within the muscle fibers
themselves.
Using the USDA enrichment method, Johnson et al. [ 1 161 subsequently detected L.
monocytogenes serotype 1/2a at 5 10 CFU/g in aseptically removed interior samples from
2 of 50 (4%) and 3 of 50 (6%) retail whole muscle beef and pork roasts, respectively.
One beef sample also was positive for L. innocua and L. welshimeri. Although the presence
of these two nonpathogenic (i.e., noninvasive) Listeria spp. within a whole muscle roast
suggests possible contamination during sampling, other results from Johnson et al. [ 114-
1161 strongly support at least limited transmission of L. monocytogenes from the blood
stream into muscle tissue.
Listeria monocytogenes in Meat Products 525

Raw Beef
Growth and Survival
Interest in behavior of Listeria in raw meat products dates back to at least 1966 when
Sielaff [ 165al inoculated beef, pork, and rabbit with L. rnonocytogenes immediately after
slaughter and examined these samples for listeriae during extended storage at 3-4C.
Under these conditions, the pathogen survived at least 15 days in all three products.
Although this study was among the first to examine the ability of L. rnonocytogenes
to survive in raw beef, information concerning actual growth of this pathogen in raw beef
was not available until 1978. In that year, Gouet et al. [87] reported results from a study
which examined multiplication of L. monocytogenes in sterile minced beef alone and
in combination with a defined microflora. L. monocytogenes failed to grow alone in minced
beef (pH 5.8) stored at 8C, and populations decreased < 10-fold during 17 days of incuba-
tion. In contrast, numbers of listeriae decreased approximately 100-fold in samples of
minced beef (pH 5.8) that were simultaneously inoculated with Lactobacillus plantarurn
and held at 8C for 17 days. These researchers also found that higher concentrations of
L. rnonocytogenes (106 CFU/g) enhanced growth of L. plantarurn. When samples of
minced beef were simultaneously inoculated to contain equal numbers of L. rnonocyto-
genes, Pseudornonasjluorescens, and Escherichia coli, Listrria populations decreased ap-
proximately 10-fold after 24 h of incubation at 8OC, with numbers rapidly increasing after
day 7 (Fig. 3). Rapid growth of listeriae during the latter half of incubation was likely
caused by proteolysis of meat proteins by P. jluorescens, which in turn gradually increased
-
the pH of the meat from 5.8 to 6.8. With a complex microflora consisting of 103CFUI
g each of L. plantarurn, P. jluorescens, E. coli, Micrococcus sp., Clostridiurn perjhkgens,
and Enterococcus (Streptococcus)faecalis, behavior of L. rnonocytogenes was similar to
that previously observed for the pathogen in the presence of P.jluorescens and E. coli, with
listeriae populations reaching approximately 6 X 105CFU/g in minced beef following 17
days at 8C (Fig. 4). A rapid increase in numbers of P. jluorescens before growth of L.
plantarurn again appeared instrumental in raising the pH from 5.8 to 7.2 which in turn
stimulated growth of listeriae. Hence, results from this early study suggested that L. rnono-
cytogenes can grow in temperature-abused retail ground beef, since the microbial composi-
tion of this product was fairly similar to that found in the ground beef inoculated with
the seven different organisms.
In a similar investigation completed 11 years later, Kaya and Schmidt [ 1201 also
found that growth of L. rnonocytogenes in artificially contaminated sterile minced meat
during extended incubation at 8-20 C was suppressed by adding 106Lactobacillus CFUI
g but was unhindered in the presence of 1O6 Pseudornonas sp. CFU/g. However, in contrast
to the previous study by Gouet et al. [87], this L. monocytogenes strain readily grew in
the absence of other microorganisms, with populations increasing approximately 2 and
2 4 orders of magnitude in sterile minced meat after 10 and 1-5 days of storage at 4
and 8-2OoC, respectively. In this study, proteolysis of meat proteins by pseudomonads
apparently was not a prerequisite for growth of L. monocytogenes in sterile minced meat
having an initial pH value of 5.8-6.0.
During 1988 and 1989, three additional studies were done to determine the behavior
of listeriae in ground beef; however, unlike previous investigations, the meat was not
pretreated to eliminate the normal background flora. When ground beef at pH 5.6-5.9
was inoculated to contain 105and 106L. rnonocytogenes Scott A or V7 CFU/g, packaged
526 Farber and Peterkin

- 7.0
l90 1 4

7iI /
/
/
- 6.5

'd
=e

- 6.0

'1
0 ' I I I I - 5.5
1 3 7 11 17
Days

FIGURE3 Behavior of L. monocytogenes alone (H) and in combination with fsche-

richia c o l i ( 0 )and Pseudomonas fluorescens (+) in minced beef stored at 8C. Dashed
line (-A-)designates the change in pH during storage. (Adapted from Ref. 87.)

in oxygen-permeable or impermeable film and examined for numbers of listeriae during

extended storage at 4OC, Johnson et al. [ 1131 found that the populations and pH remained
relatively constant in both products during 14 days of incubation regardless of the film's
degree of permeability. In contrast to the previous study by Gouet et al. [87] in which
the pH of minced beef containing a complex microflora increased from 5.8 to 7.2 during
extended incubation at 8"C, all packaged ground beef samples in this study remained in
the range of pH 5.6-5.9 throughout storage. Thus, the lower pH of the meat in this study
along with a lower incubation temperature (4 vs 8OC) are likely to have been at least
partly responsible for inhibiting growth of listeriae in packaged samples of "retail-like"
ground beef.
In keeping with these findings, Shelef [163] also reported that L. monocytogenes
failed to grow in artificially contaminated ground beef or ground liver during 1 and 40
days of incubation at 25 and 4OC, respectively. However, in contrast to the findings of
Johnson et al. [ 1131, the pathogen failed to grow in ground beef despite a final pH value
of 7.8. Although the reason(s) for this behavior remain(s) obscure, inability of L. monocy-
togenes to multiply in ground beef under these conditions is likely related to the type and
load of inherent microflora in the product.
This hypothesis is supported by a West German study in which Kaya and Schmidt
[120] showed that an increase in the natural bacterial flora of ground beef from 105 to
Listeria monocytogenes in Meat Products 527

10

1
1 I I I I
0 1 1' 3 7 11 17
Days

FIGURE 4 Behavior of L. monocytogenesalone (m) and in combination with Lactoba-

cillus plantarum (O), Pseudomonas fluorescens (+I, Escherichia coli (A),Microoccus
sp. (A),Clostridium perfringens ( O ) ,and Streptococcus faecalis (U) in minced beef
stored at 8C. (Adapted from Ref. 87.)

1O7 CFU/g led to increased inhibition of L. rnonocytogenes in artificially contaminated

-
meat. When ground beef was inoculated to contain 10s L. rnonocytogenes CFUlg, the
pathogen grew after 1-5 days at 7-20C in samples harboring -105 non-Listeria
contaminantslg but failed to multiply in corresponding samples containing higher levels
of naturally occurring organisms. Although a similar growth pattern was observed for
samples naturally contaminated with 1O2 Listerialg and 1O 5 non-Listerialg, growth was
markedly hindered in naturally rather than artificially contaminated samples, with Listeria-
populations remaining constant in the former during 14 days at 8C. However, behavioral
differences were no longer observed at lower temperatures, with the organism failing to
grow during 14 days of incubation at 4C in both artificially and naturally contaminated
ground beef containing similar background populations.
Work done by Barbosa et al. [29] with vacuum-packaged ground beef supports pre-
vious findings that the organism grows better in beef having a pH > 6. This study also
showed that besides pH, strain type also can affect the fate of Listeria in ground beef.
The authors used four different strains of L. monocytogenes for inoculation and observed
slightly different responses with each organism. For example, in normal pH (5.47) ground
beef, L. rnonocytogenes serotypes 3a and 3b increased 2.3 and 1.8 logs, respectively after
35 days of storage at 4"C, whereas after 56 days, the levels of strains 1/2a and Scott A
(serotype 4b) remained constant and decreased 1 log in number, respectively. In general,
the organism multiplied slowly on all vacuum-packaged samples, but growth was better
528 Farber and Peterkin

on ground beef of pH 6.14 than of pH 5.47. For example, after 28 days of storage at 4"C,
serotypes 3b, 3a, and 1/2a had increased by 2.87, 2.64, and 2.24 logs, respectively, in
high-pH ground beef, whereas strain Scott A did not change significantly in numbers [29].
The authors felt that the antimicrobial effect of low pH may also impact Listeria indirectly,
since altering the natural microbial flora could possibly promote growth of bacteriocin-
producing lactic acid bacteria.
L. monocytogenes appears to behave similarly on the surface of fresh intact beef
muscle. In two studies conducted shortly before L. monocytogenes emerged as a serious
foodborne pathogen, Lee et al. [129,130] dealt indirectly with the incidence and subse-
quent behavior of Listeria spp. along with many other psychrotrophic and mesophilic
organisms present on the surface of hot-boned and conventionally boned beef. In this
study, hot-boned beef was obtained from five steers 2 h after slaughter, vacuum packaged,
and cooled from 32 to 21C. In contrast, conventionally processed beef was obtained from
carcasses that were hung in a cold room at 2C for 2 days after slaughter. Both types of
beef were then examined for mesophilic and psychrotrophic organisms at day 0 when the
surface temperature had decreased to 2 1"C (<1 h for conventionally processed beef) and
again following 14 days of storage at 2C. Nearly 1250 bacterial isolates were subse-
quently identified by computer analysis of 116 miniaturized testdisolate. Although Liste-
ria spp. were never isolated from slow or moderately chilled, hot-boned beef at day 0,
9.1-13.5% of all microorganisms present on the surface of such beef after 14 days were
identified as Listeria spp., some isolates of which were likely L. monocytogenes (Table 8).
Overall, only one isolate from conventionally processed beef was identified as belonging to
the genus Listeria. From these data one can infer that Listeria can grow on the surface
of vacuum-packaged hot-boned beef but not on the surface of unpackaged conventionally
processed beef during 2 weeks of storage at 2C. Since the high water-binding properties

TABLE
8 Generation (GT) and Lag Times (LT) of L.
monocytogenes in Meats

Roast beef - 1Sa 100.0 173.7 104

3a 26.7 59.0 104
3b 80.9 477.1
Corned beef 0 110.0 91
Cooked meat 5 44-6 1 167
Ham 5 33.2 91
10 13.4 91
15 6.1 91
Cooked beef 5 18.6-22.6 80.6-83.4 102
10 8.5-9.0 22.6-30.4
Piit6 7 19.7 48 60
12Sa 1.6 24
Piit6 10 a 9.12 27.6 75
aVacuum packs.
CO2 packs.
Source: Adapted from Refs. 46, 104, and 167.
Listeria monocytogenes in Meat Products 529

of hot-boned beef make this product particularly well suited for sausage making, wide-
spread use of hot-boned beef by the processed meat industry may be partially related to
the relatively high incidence of listeriae in ready-to-eat meat products.
In a more definitive Australian study reported in 1988, Grau and Vanderlinde [90]
examined the ability of L. monocytogenes to grow on the surface of artificially contami-
nated (102--l O3 CFU/cm2) vacuum-packaged, nonsterile beef striploin during extended
incubation at 0 and 5.3"C. Although L. monocytogenes populations increased in all sam-
ples (and in product exudates that developed in packages) during storage, the extent of
Listeria growth was markedly influenced by incubation temperature, pH of the sample
(5.6 vs 6.01, and type of tissue (lean vs fat). Overall, higher Listeria populations consis-
tently developed on fat tissue, with growth also being more rapid at the higher of the two
incubation temperatures and pH values. Numbers of listeriae on fat tissue of pH 5.6 in-
creased from 5 X 103to 3 X 107 CFU/cm2 during 16 days of incubation, whereas the
pathogen was just beginning to grow on corresponding samples after 7- 14 days of storage
-
at 0C. These researchers also noted that Listeria populations increased < 10- and 1000-
fold on vacuum-packaged meats of pH 5.6 and 6.0, respectively, after 10-1 1 weeks of
storage at 0C. Thus, it appears that two conditions, (a) a storage temperature of 0C and
(b) a product pH value of 55.6, must be met simultaneously to prevent significant growth
of L. monocytogenes in vacuum-packaged raw meats destined for export. Grau and Vand-
erlinde [92] extended their studies by using two models: the modified Arrhenius and
square-root model to examine aerobic growth of L. monocyiogenes on lean and fatty raw
beef tissue. For both lean and fatty tissue, the modified Arrhenius model gave better fits
and estimates of the growth rates. The effect of temperatures between 0 and 30C on the
growth rate could be described by a modified Arrhenius equation: Ln (gen/h) = -205.73
+ 1.2939 X 105/K - 2.0298 X 107/K2,where K = OK. The combined effect of tempera-
ture and pH on the growth rate of the organism on lean beef was best described by the
following equation: Ln (gen/h) = -232.64 + 1.4041 X 105/K - 2.1908 X 107/K2 +
1.1586 X 102/pH - 4.0952 X 102/pH2.For lean meat at pH values of about 5.5-5.6 and
6.0-7.0, the latter equation applied at 2.5 to 35C and 0 to 35"C, respectively. There was
considerable scatter in the measured lag periods for both types of meat, and therefore a
poor fit was observed for both models. In two trials, both models predicted growth on
lean tissue where no growth was observed experimentally. In the first, L. monocytogenes
failed to grow on lean tissue with a mean pH of 5.61 during 13 weeks of storage at OOC,
whereas in the other no growth was observed after 48 h at 43.2"C and pH 5.46. However,
growth of the organism was observed on lean tissue having a mean pH of 5.61 at 2.5"C,
on lean meat of pH values 16.0, and all fatty tissue (pH 5.5-5.7) regardless of storage
temperature. One should be aware, however, that these investigators used only one strain
of L. monocytogenes (which was not a meat strain), and that meats where the contaminat-
ing flora exceeded 10% of the Listeria count were discarded, thus partially eliminating
the effects of background microflora on growth of the organism.
Growth rates predicted by the best equations for lean tissue were compared with
literature data for aerobic growth of L. monocytogenes on several foods. Predicted growth
rates for lean meat were higher than those for corn, clarified cabbage juice, and milk,
whereas food supporting growth rates similar to lean beef included UHT milk, raw and
cooked chicken, and cooked ground meat [92].
Other researchers have found that some models derived from the growth of L. mono-
cytogenes in broth cannot reliably predict growth of the organism in raw pork [83]. For
530 Farber and Peterkin

example, L. monocytogenes grew on pork fat tissue without lag at -0.3"C. At higher
temperature, growth rates were greater than those in unacidified Tryptic Soy Broth (TSB)
held at lower temperatures. Faster growth of the organism on fat tissue as compared with
TSB suggests that fat tissue may contain micronutrients which are not present in TSB
[83]. In addition, the organism grew slower on pork muscle tissue at temperatures 2 15.4"C
than in acidified (pH 5.5) TSB, implying that pork muscle tissue may contain growth
inhibitors which are either absent or present in very low levels both in TSB and fat tissue.
All models published thus far predict that the organism will grow in broth at pH 5.5 and
5C in contrast to the failure of L. monocytogenes to grow on normal pH raw pork muscle
even at temperatures as high as 15C. Only one published study has shown that the organ-
ism can grow on raw meat (beef muscle) of normal pH at low temperatures [92].

Raw Lamb and Pork

Most investigations have focused almost exclusively on the behavior of Listeria in raw
beef; however, several reports on the fate of this pathogen in raw lamb and pork can be
found in the scientific literature. As early as 1973, Khan et al. [121] published results
from a study in which aseptically obtained "sterile" raw lamb meat was inoculated to
-
contain 105CFU/g of L. monocytogenes, packaged in gas-permeable or gas-imperme-
able film, and examined for numbers of listeriae during extended storage at 0 and 8C.
Although Listeria populations remained relatively constant in lamb meat packaged in gas-
permeable film during 20 days at OOC, numbers of listeriae decreased approximately 10-
fold in corresponding samples that were packaged in gas-impermeable film. These results
are similar to those obtained by Johnson et al. [ 1 131, who concluded that L. monocytogenes
also was unable to grow in refrigerated ground beef that was packaged in gas-permeable
or gas-impermeable film. Following 12 days of storage at 8 rather than OOC, populations
of listeriae in lamb increased > 1000-fold; however, unlike meat packaged in gas-perme-
able film, the pathogen exhibited a 2-day lag period and grew markedly slower in gas-
impermeable packages. Various physical differences of the meat, combined with an in-
creased concentration of CO2 (and presumably a lower pH) in gas-impermeable packages,
may have been responsible for partially inactivating the pathogen at 0C and delaying the
onset of growth at 8C.
Two years later, Khan et al. [ 1221 examined the behavior of L. monocytogenes in
preparations of sarcoplasmic pork, beef, and lamb protein that were inoculated to contain
- 104CFU/mL of L. monocytogenes and then held at 4C. According to these investiga-
tors, the pathogen grew readily in preparations of pork and beef protein, reaching levels
of approximately 105and l O7 CFU/mL, respectively, after 12 days of refrigerated storage.
Although Listeria populations remained relatively constant in corresponding preparations
of lamb protein held at 4"C, the pathogen grew readily in lamb meat stored at 8C. Hence,
it appears that raw pork, beef, and lamb can support rapid growth of listeriae, particularly
when these products have undergone temperature abuse, as frequently occurs at the retail
level.
These early observations were confirmed by Lovett et al. [134], who found that L.
monocytogenes reached levels of at least 108CFU/g in inoculated samples of retail lamb,
pork, and beef after 14 days at 7C. L. monocytogenes exhibited both a 2-day lag period
and a slower rate of growth in beef and pork than in lamb. Such variations in growth rate
might be related to differences in concentrations of various amino acids (particularly ly-
Listeria monocytogenes in Meat Products 531

sine, serine, and valine), which are reportedly essential for growth of L. monocytogenes,
as first suggested by Khan et al. [ 12 1 ] in 1973.

Cooked and Ready-to-Eat Meats

Cured Ham
Investigators in Europe and the United States determined the behavior of L. monocyto-
genes in ham during processing and extended storage. Working in The Netherlands, Stege-
man et al. [ 1681 examined the thermal resistance of listeriae in experimentally produced
hams to which 2 or 3% NaCl and 120 or 180 ppm sodium nitrite were added during
-
manufacture. After inoculating the product to contain 1O4 CFU/g of L. monocytogenes,
all hams were canned, heated to an internal temperature of 68.9-71.0 (or 64.0"C) within
5 h to simulate normal and underprocessing, respectively, cooled, and sampled after 5
days of storage at 4C. According to these authors, three different enrichment procedures
failed to recover viable listeriae from any samples.
Results from the study just described indicate that standard thermal treatments are
more than sufficient for producing Listeria-free ham. However, once removed from protec-
tive packaging, all cooked/ready-to-eat meats can become contaminated with listeriae dur-
ing slicing and further handling. To simulate postprocessing contamination, Glass and
Doyle [84] inoculated the surface of commercially produced ham slices and five other
meat products (to be discussed shortly) to contain approximately 0.2 or 500 CFU/g of L.
monocytogenes. All samples were then vacuum packaged and periodically examined for
numbers of listeriae during prolonged incubation at 4.4"C. Regardless of the original inoc-
ulum, L. monocytogenes attained populations of 1OS- 1Oh CFU/g on organoleptically ac-
ceptable ham (pH 6.3-6.5) after 4 weeks of refrigerated storage, indicating that manufac-
turers cannot rely on the combination of vacuum packaging and refrigeration for control
of listeriae on ham.

Cooked Roast Beef

Glass and Doyle [84] evaluated the potential for growth of L. monocytogenes on the sur-
face of vacuum-packaged samples of cooked roast beef having initial pH values of -5.90.
Unlike sliced ham, cooked roast beef supported far less growth of listeriae, with popula-
tions increasing 2 2 orders of magnitude on organoleptically acceptable product after 4
weeks of refrigerated storage. Slower growth of the pathogen on precooked roast beef
correlated well with a decrease in product pH to 55.15 in 4-week-old samples.

Luncheon Meats
Since previous studies found that luncheon meat, cooked ham, and cooked breast meat
were the most frequently contaminated cooked meats in The Netherlands, these products
were used in a study to determine the survival and growth of L. monocytogenes [34].
Products were inoculated with low levels (10 CFU/g) of the organism and then stored
under vacuum or an atmosphere of 30% c o 2 / 7 0 % N 2 at 7C for 4-6 weeks. Growth on
vacuum-packed product was similar to that of modified-atmosphere packaged (MAP)
stored meats, with counts increasing up to 108CFU/g after 35 days (Fig. 5). High numbers
of lactic acid bacteria were present but did not affect growth of the organism. However,
the pathogen decreased in number on saveloy (fermented sausage; pH 5.5-5.7) and raw
532 Farber and Peterkin

chicken breast
8 A luncheon meat
0 cooked ham
6
--.
bD
z
$ 4
c(

I I I I I I I
0 5 10 15 20 25 30 35
Time (days)

FIGURE5 Growth of Listeria rnonocytogenes on MAP (30% CO2/7O% N 2 )luncheon

meat, cooked ham, and chicken breast at 7C. (Adapted from Ref. 34.)

Coburger ham (pH 4.3-4.5), most likely as a result of the acidity of these products. Grau
and Vandelinde [91] examined growth of L. monocytogenes on both naturally contami-
nated and artificially inoculated processed corned beef and ham. For corned beef stored
at O"C, L. monocytogenes grew at about half the rate of the other microflora, whereas at
9"C, both groups of organisms grew at similar rates. On ham stored at 5OC, the organism
grew at only one third the rate of the other flora. Meat composition, that is, pH, salt, and
residual nitrite, played a role in determining the growth potential for this pathogen. For
example, at OOC, the organism failed to grow on ham containing 170 ppm residual nitrite,
but it did grow on ham with 11 ppm nitrite. Although L. monocytogenes grew at similar
rates on ham stored at 15"C, as the storage temperature decreased, the organism again
grew slower on ham containing the higher level of residual nitrite. Fastest growth was
observed on corned beef of pH 6.2, a, 0.97, and <5 ppm nitrite, with slowest growth
being seen on ham of pH 6.6, a, 0.97, and 170 ppm nitrite. From these inoculated pack
studies, equations were developed to describe the growth of both L. monocytogenes and
the other flora; that is, lactic acid bacteria and Brochothrix thermospacta on ham and
corned beef (Fig. 6). For naturally contaminated corned beef, good agreement was ob-
tained between predicted and actual growth of the organism. As predicted, L. monocyto-
genes could not grow on naturally contaminated ham stored at 0.loC, although slight
growth (i.e., from -0.3 to 6.2 CFU/g) of the organism was observed on product stored
at 4.8"C. Juneja et al. [ 1171 examined the potential for outgrowth of various foodborne
pathogens on cooked ground beef during cooling from 54.4 to 7.2"C within 6, 9, 12, 15,
18 or 21 h. L. monocytogenes was inoculated into ground beef at a level of 103 CFU/g,
the meat was then heated linearly to 60C within 1 h, and then cooled as described above.
The organism was not detected in any beef sample examined. Presumably, the slight heat-
ing and cooling regime was sufficient to reduce levels of the organism below the detectable
level; levels at which the organism remained during the duration of the cooling period
[117]. The fate of L. monocytogenes on unirradiated and irradiated cook-chill roast beef
Listeria monocytogenes in Meat Products 533

I I

7.5

p
4

6
3 2 5.5
3
I
J
bD

.-cd

0 3.5 sB
0

-2 1.5
0 10 20 30 20 40 60
Time (Days)

FIGURE6 Changes in the number of L. monocytogenes (II) and of other (lactic acid
bacteria and B. thermosphacta) flora ( 0 )o n the lean tissue of commercial vacuum
packs of corned beef stored at (a) 4.8"C or (b) 0.1"C. Note the different vertical scales
for Listeria and other flora. Off odors first detected (?I. (Adapted from Ref. 91.)

and gravy was examined at 5 and 10C [89]. The organism grew well on both products,
increasing 5 logs in number on unirradiated beef and gravy over 15 days at 5C and by
6 logs in irradiated products stored at 10C for 23 days. Although the observed lag phase
for L. rnonocytogenes was longer on irradiated as compared with unirradiated product,
the specific growth rates were similar at each storage temperature, suggesting that the
background microflora of beef and gravy did not interfere with growth of L. rnonocyto-
genes. The authors concluded that there would be no increased risk of listeriosis if cook-
chill roast beef and gravy were to be irradiated with 2 kGy [89]. L. rnonocytogenes also
grows well on cooked beef [102] (Table 8). Interestingly, the organism grew at similar
rates both aerobically and anaerobically, although the authors claimed that samples stored
under aerobic conditions probably became anaerobic during the course of the experiment
[102]. L. rnonocytogenes also survived pasteurization (e.g., 91 and 96C for 3 or 5 min)
of precooked beef roasts and then grew when samples were stored at 4 and 10C for up
to 56 and 12 days, respectively [94]. Products stored at 10C supported far better growth
of the surviving listeriae than those stored at 4C; that is, at IOOC, L. rnonocytogenes
reached levels in 12 days that took up to 56 days to attain at the lower storage temperature.
It is well known that the organism can repair itself much better at higher temperatures.
Van Laack et al. [ 1751 examined the effect of three packaging treatments, that is, vacuum
packed directly after hot boning (hot packaged), vacuum packed after chilling for 1 day
(cold packaged), or unpackaged, on survival of L. rnonocytogenes on pork loins stored at
1C for up to 9 days. Populations increased about 1 log (Table 9), demonstrating that the
organism can grow on raw refrigerated pork in the presence of numerous competitors.
Although the data were not analyzed statistically, the type of packaging did not appear
to greatly influence growth.
534 Farber and Peterkin

TABLE
9 Influence of Packaging Treatment on
Numbers of L. rnonocytogenes on Pork Loins During
9 days storage at 1 1C *
Average log,,, CFU/cm2a
Sampling Packaging
day treatment Trial 1 Trial 2
HP 2.68 (5/8)h 2.42 (8/8)
CP 2.48 ( 5 / 8 ) 2.86 (8/8)
cu 2.65 (618) 2.86 (8/8)
HP 2.98 (6/8) 2.86 (818)
CP 2.48 (5/8) 2.86 (8/8)
cu 2.91 (7/8) 2.73 (8/8)
HP 3.36 (8/8) 3.1 1 (8.8)
CP 2.68 ( 5 / 8 ) 2.77 (7/8)
cu 2.64 (6/8) 2.91 (7/8)
HP 3.77 (7/8) 3.1 1 (8/8)
CP 2.92 (7/8) 3.36 (8/8)
cu 2.73 (8/8) 3.19 (7/8)
HP 3.73 (8/8) 3.23 (8/8)
CP 3.19 (7/8) 3.61 (7/8)
cu 3.48 (5/8) 3.23 (5/8)

HP, hot packaging; CP, cold packaging; CU, cold unpackaged.

aAverage of MPN values from positive samples.
The number between brackets indicates the proportion of samples
from which a given pathogen was recovered.
Source: Adapted from Ref. 175.

The issue of whether L. rnonocytogenes can grow on raw and cooked meats is com-
plex. As seen in this section, factors such as pH, a,, background microflora, sodium nitrite
and NaC1, length and temperature of storage, strain type, and history all play a role in
determining the fate of Listeria on a meat surface. Another seemingly important factor
which is often overlooked is the initial inoculum on the product. Although early work
from modeling experiments suggested that initial numbers of organisms present on a food
surface had little or no influence on subsequent outgrowth and growth rate, more recent
work does not substantiate this theory. For example, Farber and Daley [70] found that
when L. monocytogenes was present in very low numbers on meats such as sliced ham,
turkey breasts, wieners, and pSt6 stored at 4OC, its numbers did not increase. Similar results
have been observed for foods other than meats and poultry (J.M. Farber, unpublished
results).

Unfermented Sausage
Even though potentially contaminated raw meats find their way into enormous quantities
of sausage products, with over 200 varieties manufactured in the United States alone, no
information pertaining to the behavior of L. monocytogenes during manufacture and stor-
age of these popular meat products appeared in the scientific literature before 1988. Al-
though the California listeriosis outbreak of 1985 eventually led to the aforementioned
surveys in which Listeria was detected in raw and processed meats, including sausage,
Listeria monocytogenes in Meat Products 535

the early consensus was that consumption of such products did not pose a serious threat
to public health, as shown by a lack of any confirmed cases of meatborne listeriosis.
However, this situation changed in December, 1988, following the report of a breast cancer
patient who developed listerial meningitis and eventually died after consuming turkey
frankfurters that were contaminated with L. monocytogenes.
Unfermented sausages are best classified according to the following five categories,
which are based on the method of manufacture: (a) fresh sausage (e.g., fresh pork sausage,
bratwurst), (b) cooked sausage (e.g., liver sausage, Braunschweiger), (c) cooked smoked
sausage (e.g., frankfurters, bologna), (d) uncooked smoked sausage (e.g., Mettwurst,
smoked country-style pork sausage, kielbasa), and (e) cooked meat specialty items (e.g.,
head cheese). Research efforts have dealt primarily with the behavior of Listeria in sau-
sages belonging to the first three categories.
Fresh Sausage
Although fresh pork sausage is by far the most widely manufactured type of fresh sausage,
this category also includes other well-known varieties such as fresh Italian, breakfast, and
beef sausage as well as fresh bratwurst, Thuringer, and bockwurst. The last two are most
popular in Germany. All varieties of fresh sausage are normally prepared from coarse or
finely comminuted pork, beef, or veal to which water is added along with an array of
spices which varies with the type of sausage. In the United States, certain varieties of
fresh sausage also may contain binders and/or extenders (e.g., cereal, vegetable starch,
nonfat dry milk, dried whey) at levels not exceeding 3.5% by weight. After being
stuffed into natural or artificial casings, the product is twisted and cut to form individual
sausage links which are cooled rapidly to preserve freshness and flavor. Unlike cooked
and fermented sausages, fresh sausages have a short shelf life and must be kept
refrigerated to prevent growth of spoilage organisms, including lactic acid bacteria
and micrococci.
When commercially prepared fresh bratwursts were surface-inoculated to contain
approximately 0.1 or 600 L. monocytogenes CFU/g, vacuum packaged, and stored at
4.4OC, Glass and Doyle [84] found that the pathogen attained populations of 106CFU/g
on organoleptically acceptable 4-week-old bratwursts regardless of the initial inoculum.
As with ham, profuse growth of listeriae on fresh bratwurst was attributed to a pH value
>6 which was maintained by the product throughout the first 4 weeks of refrigerated
storage.
In another study involving fresh sausage, Hughey et al. [ 1061 investigated the ability
of lysozyrne to prevent growth of L. monocytogenes in bratwurst prepared from coarsely
ground pork. After addition of commercial bratwurst spice, distilled water was added
with or without 100 ppm lysozyme and 5 mM ethylenediaminetetraacetic acid (EDTA),
a generally recognized as safe chelating agent that enhances the antibacterial activity of
lysozyme. This meat mixture was then inoculated to contain -4 X 103 CFU/g of L.
monocytogenes and stuffed into natural hog casings which were subsequently linked, sepa-
rated, vacuum packaged, and stored at 5C for 45 days.
As expected from the previous study, Listeria populations increased rapidly in fresh
bratwurst (pH z 6) without added lysozyme or EDTA, reaching levels of > 106CFU/g
following 10 days of refrigerated storage (Fig. 7). L. monocytogenes also behaved similarly
in bratwurst containing lysozyme alone; however, the presence of EDTA alone resulted
in a 15-day lag period, thus preventing the pathogen from reaching populations of 10'
536 Farber and Peterkin

t t
/
/
/
/

/ CI
/
/
/
/

4.......+
/

1 1 I I I

FIGURE7 Effect of lysozyme (Lys) and EDTA on growth of L. monocytogenes in fresh

bratwurst. (Adapted from Ref. 106.)

CFU/g until nearly 30 days of storage. In contrast, lysozyme and EDTA acted synergisti-
cally to retard growth of L. monocytogenes in fresh bratwurst. Under these conditions,
the pathogen exhibited a lag period of nearly 2 1 days, that is, approximately 7 days beyond
the normal shelf life of the product, and reached populations of <105 CFU/g following
44 days of refrigerated storage. Although only listeriostatic, the combined use of lysozyme
and EDTA appears to be an effective means of controlling Listeria growth during the
normal shelf life of fresh bratwurst. Furthermore, once growth is prevented, low levels
of L. monocytogenes that occasionally appear in fresh bratwurst (<103CFU/g) should be
readily eliminated by proper cooking.

Cooked Smoked Sausage

This group of sausages, which includes the ever-popular frankfurter (hot dog) as well as
bologna and various luncheon meats, is prepared from mixtures of comminuted beef and/
or pork to which salt, sugar, sodium nitrite, and spices are normally added. When making
frankfurters, this meathgredient mixture, commonly referred to as the sausage emulsion,
is stuffed into natural or artificial casings, which are then twisted to form sausage links.
This string of frankfurter links is cooked to an internal temperature of 7 1.1"C (160'F) to (a)
coagulate protein, (b) fix the color, and (c) pasteurize the product. Although not absolutely
Listeria monocytogenes in Meat Products 537

necessary, frankfurters and other similar sausages are frequently hung in smoking rooms
either before or after cooking. Alternatively, commercially available liquid smoke products
can be added to the sausage emulsion or applied directly to the frankfurter surface before
or during heating. In either event, beside imparting a pleasant smoked flavor to the finished
product, some smoke components (i.e., formaldehyde, acetic acid, creosote, and phenols
with high boiling points) possess bacteriostatic and/or bactericidal properties. After cook-
ing, frankfurters are carefully cooled, packaged, and refrigerated during shipment to
wholesale and retail markets. Skinless frankfurters, which are very popular, are produced
in a similar manner except that the artificial casings are mechanically peeled from the
sausage after cooking or smoking.
Epidemiological data from the Centers for Disease Control and Prevention (CDC)
showing an apparent association between listeriosis and undercooked frankfurters
prompted several thermal resistance studies. Zaika et al. [ 1931 prepared frankfurters from
-
a sausage emulsion inoculated to contain 1O8 CFU/g of L. monocytogenes. After stuffing,
all frankfurters were thermally processed (without smoke) according to a standard com-
mercial heating schedule. These USDA officials found that L. monocytogenes populations
decreased approximately 1000-fold in frankfurters that were heated to an internal tempera-
ture of 7 1.1"C (160F). Based on these data, cooking frankfurters to an internal tempera-
ture of 7 1,I "C would probably eliminate maximum levels of L. monocytogenes (<103
CFU/g) that could conceivably occur in raw frankfurter eniulsions.
Data gathered by the American Meat Institute in 1988 pointed to frankfurters as
being likely carriers of L. monocytogenes and also suggested that poor environmental
conditions before packaging could play a major role in contaminating the finished product
[4]. Moreover, Glass and Doyle [84] reported that this pathogen can proliferate on vacuum-
packaged, artificially contaminated (-0.0 1 L. monocytogenes CFU/g) retail frankfurters
at 4.4"C with populations two to five orders of magnitude higher on organoleptically
acceptable samples after 4 weeks of refrigerated storage. Similarly, L. monocytogenes
increased in number from 5 X 10' to 2.1 X 105MPN/g on vacuum-packed frankfurters
stored at 4C for 20 days. Interestingly, uninoculated control samples which were initially
negative for Listeria contained 1.2 X 102MPN/g after the 20-day storage period [40]. In
a more detailed study examining growth of L. monocytogenes on vacuum-packaged all-
beef, poultry or beef/pork wieners at 5C for up to 28 days, McKellar et al. [ 1431 found
that of 61 wieners analyzed, 40 (65.6%) supported growth of L. monocytogenes. For those
samples supporting growth, an average increase of 1.26 logs was observed within a 14-
day period. Unlike NaCl levels, concentrations of phenol, nitrite and lactic acid bacteria
varied considerably during storage. In addition, average pH levels decreased significantly
by 0.19 pH units during storage. Several statistical models were derived in an attempt to
describe growth and death of the organism in all wiener samples. Although no single
model was completely adequate, the best model implicated initial and final lactic acid
bacteria counts and initial pH as factors influencing growth of L. monocytogenes. Pre-
venting Listeria contamination and subsequent growth is further complicated by present
consumer demands for reduced levels of salt and preservatives, along with longer shelf
life, smaller packages, and greater convenience, all of which will require the processed
meat industry to develop even stricter requirements for processing, cooking, handling,
packaging, and refrigeration of products.
Several studies were initiated by the food industry to examine the feasibility of using
heat to eliminate L. monocytogenes from the surface of finished frankfurters. In one such
study [6], frankfurters were dipped in a broth culture of L. monocytogenes ( 106-108 CFU/
538 Farber and Peterkin

mL) to simulate postprocessing contamination. Listeria populations on the surface of the

frankfurters decreased only 100-fold after 8 min of heating at 86.1-873C (1 87- 190F).
Furthermore, this heat treatment rendered the sausages organoleptically unacceptable for
most consumers. Hence postprocess pasteurization may not be a viable means of elimi-
nating L. monocytogenes from the surface of frankfurters that have been contaminated
after manufacture.
Additional efforts to control Listeria contamination on the surface of frankfurters
have focused on the bactericidal properties of commercially available liquid smoke prod-
ucts. In one study [147a], beef frankfurters were immersed in a culture containing 1 X
103CFU/mL of L. monocytogenes, removed, thoroughly air dried, and then dipped in full-
strength commercially available liquid smoke solution (CharSol C- 10). Although Listeria
populations were unchanged in control frankfurters that were dipped in phosphate buffer,
vacuum packaged, and analyzed after 72 h at 4C, numbers of listeriae had decreased 60
to 299.9% 15 min after the frankfurters were treated with liquid smoke. Furthermore,
the pathogen was never detected in smoke-treated sausage following 72 h of refrigerated
storage. However, although dipping frankfurters in full-strength liquid smoke eliminated
L. monocytogenes, this treatment produces an extremely intense smoke-flavored product
that is no longer organoleptically acceptable to most consumers.
Results from several additional experiments dealing with the antilisterial effects of
less concentrated liquid smoke solutions were reported by Wendorff [ 1831. Initially, beef
frankfurters were dipped in a concentrated broth culture of L. monocytogenes, removed,
thoroughly dried, dipped into aqueous liquid smoke solutions containing 10-40% CharSol
C- 10 or CharSol Poly- 10 (concentrations normally used in frankfurter production) and
then analyzed for listeriae after 72 h of refrigerated storage using the Gene-Trak DNA
Hybridization assay. Although L. monocytogenes was eliminated from the surface of
frankfurters dipped in 40% solutions of CharSol C- 10 or Poly- 10, the pathogen was still
detected on frankfurters treated with 10 and 25% solutions of CharSol C-10.
After demonstrating that these liquid smoke compounds lost their activity against
Listeria on the surface of frankfurters when added directly to sausage emulsions before
stuffing, Wendorff [ 1831 assessed inactivation of listeriae on surface-inoculated skinless
frankfurters that were sprayed with five levels of CharSol Poly-10 and CharSol Supreme
(twice the strength of CharSol C-10) just before vacuum packaging (Table 10). When
used at organoleptically acceptable concentrations, CharSol Supreme was more effective
than CharSol Poly- 10, with Listeria populations on the surface of frankfurters decreasing
>40% following 72 h of refrigerated storage. Using a realistic L. monocytogenes inoculum
level (- 10 CFU/g), approximately 89% of the Listeria population was inactivated on
frankfurters treated with CharSol Supreme or CharSol Poly-10 at levels of 2 and 4 oz./
100 lb of frankfurters, thus indicating that none of these treatments can guarantee a Liste-
ria-free product. Hence, to avoid contamination of finished product, efforts must be made
to develop microbial monitoring, sampling, and hazard analysis critical control point
(HACCP) programs that can effectively address problems pertaining to a lack of separation
between raw and finished processing areas, as well as procedures used to clean and sanitize
the factory environment and equipment such as grinders, mixers, and particularly sausage
peelers.
In the only other published study involving cooked smoked sausage, Glass and
Doyle [84] examined the behavior of L. monocytogenes on vacuum-packaged, artificially
contaminated slices of commercially produced bologna. As was true for ham and brat-
wurst, the pathogen also grew well on bologna (pH 6.1 -6.4), with populations generally
Listeria monocytogenes in Meat Products 539

TABLE
10 Fate of L. monocytogenes o n the Surface o f Beef
Frankfurters Sprayed with CharSol Poly-I0 or CharSol
Supreme Liquid Smoke and Stored at 4C for 72 h

L. monocytogenes
Level Initial
(oz/ 100 lb inoculum Inactivation
Treatment frankfurters) (CFU/g) after 72 h (%)
Control 0 5.28 0
CharSol Poly - 10 1.7a 5.30 Growth
3.4a 5.28 0
5.1a 5.16 23.6
8.5 4.84 63.1
12.0 4.67 75.2
CharSol supreme 1 5.02 44.7
3.6a 5.04 42.1
5.4 4.73 71.5
9.0 4.4 1 86.3
12.6 4.30 89.5
aOrganoleptically acceptable concentration of liquid smoke.
Source: Adapted from Ref. 183.

three to four orders of magnitude above initial levels on organoleptically acceptable sam-
ples after 4 weeks at 4.4"C. These findings stressed the importance of following good
manufacturing practices, which will in turn greatly reduce the possibility of listeriae con-
taminating ready-to-eat meats during slicing and packaging.

Uncooked Smoked Sausage

According to incidence data in Table 7, one variety of western European uncooked smoked
sausage, namely Mettwurst, appears to be particularly prone to contamination with Listeria
spp., including L. monocytogenes. These observations prompted a 1989 study by Triissel
and Jemmi [ 1731 in which an experimentally produced Mettwurst emulsion (pH 5.3) con-
taining -2.6% NaCl and 100 ppm sodium nitrate was inoculated with L. monocytogenes
at levels of 103or 107CFU/g and examined for numbers of listeriae during manufacture
after 7 days of ripening at 14"C, and after 3 weeks of subsequent storage of 4C. Overall,
Listeria populations remained relatively constant during manufacture and ripening, which
in turn reflects the apparent inability of this organism to multiply in ready-to-eat meat
products having pH values <5.5. As predicted from the European surveys, this pathogen
also survived well in Mettwurst during the product's entire refrigerated shelf life, with 4-
week-old samples still containing approximately 102and 1O6 CFU/g of L. monocytogenes.
Given these findings, it would be prudent for Mettwurst producers to review their manufac-
turing practices and develop procedures for decreasing Listeria contamination during all
facets of production.

Cooked Meat Specialty Items

A significant amount of beef jerky is consumed annually in the United States, with its
popularity primarily resulting from the product's ease of preparation and stability at ambi-
540 Farber and Peterkin

ent temperature. However, some health concerns have arisen with beef jerky following
linkage to several outbreaks of salmonellosis [48].
This situation prompted Harrison and Harrison [95] to examine the fate of E. coli
0 157 :H7, Salmonella typhimurium, and L. monocytogenes in beef jerky during prepara-
tion and storage. Half of the inoculated beef loin strips were marinated at 4C overnight
and then dried at 60C for 10 h. The remaining samples were heated in marinade to 7 1.7"C
and then dried. L. monocytogenes populations decreased by 1.8 and 6.0 logs after 3 and
10 h of drying, respectively. Cooking to 7 1.7"C before drying led to a 4.5-log decrease
in numbers of the organism, with a further 2-log reduction in numbers occurring during
the 10-day drying period (Fig. 8). After 8 weeks of storage at 25"C, none of the beef
jerky samples yielded pathogens.
Much attention has been given to p2t6 following its incrimination in at least two
foodborne listeriosis outbreaks. However, the growth potential of L. monocytogenes in
p2t6 still remains controversial. De Boer and van Netten [60] reported that inoculated
retail pit6 was a good growth menstruum for L. monocytogenes, with the pathogen reach-
ing levels as high as >8.3 log,, CFU/cm2 after 7 days at 12.5"C when the background
microflora was low (<2.3 CFU/cm2). An inverse correlation was observed between Liste-
ria growth and the presence of lactic acid bacteria. When high numbers of lactic acid
bacteria were present, the pH was low (average of 4.9), and L. monocytogenes increased
slightly or decreased in numbers after 1 week of storage at 7 or 12.5"C. Morris and Ribeiro
[ 1481 also found that L. monocytogenes grew on some naturally contaminated pgtis, reach-
ing levels as high as 2 X 108CFU/g after 21 days of refrigerated storage. However,
other samples failed to support growth. Several other investigators also reported that L.
monocytogenes failed to multiply on retail inoculated p2t6 stored at 4C for up to 3 weeks
[70]. To more fully assess the potential health hazards posed by L. monocytogenes in liver
pit6 products, multifactorial design experiments were conducted to examine the influence
of temperature (4 and lO"C), NaCl (1 and 3%), sodium nitrite (0 and 200 ppm), sodium
erythrobate (0 and 550 ppm), and spice (0 and 0.4%) on growth of L. monocytogenes on
experimental pit6 [75]. A total of 16 different liver p2t6 formulations were prepared and

7
0 Lm (unheated)
6 1
5- Lm (heated)
Y
.3

Y 4-
a
E
3-
5 2-

1-
3
- -

0 "
6 10
Drying time (h) in dehydrator

FIGURE8 Survival of L. monocytogenes on beef jerky during drying at 60C (140F).

(Adapted from Ref. 95.)
Listeria monocytogenes in Meat Products 54 1

stored at 4 and 10C. When analysis of variance was usecl to assess the impact of the
various factors on maximum growth rate, temperature was the only factor that affected
the growth rate, with L. monocytogenes growing well in all experimental piit6s.
The generation and lag times for the organism in piit6 can be seen in Table 8. Overall,
potential growth of L. rnonocytogenes in pgt6 appears to be related to pSt6 composition
and pH, initial numbers of lactic acid bacteria, and storage temperature and time.
Fermented Sausage
Sausages classified as fermented undergo a controlled lactic acid-type fermentation, usu-
ally through the action of a commercially produced starter culture added to the meat.
Although all fermented sausages can be further classified according to moisture content
as either semidry or dry, manufacturing procedures for both types are generally similar
until the point of drying. Fermented sausages are normally prepared from comminuted
beef and/or pork to which sugar and various spices are added along with sodium or potas-
sium nitrate and/or nitrite. This meat preparation, known as a mix rather than an emulsion,
is inoculated with a commercial lactic acid bacteria starter culture, which frequently in-
cludes species of Pediococcus (particularly P. cerevisiae and P. acidilactici), Lactobacil-
lus, and Leuconostoc. After stuffing the inoculated sausage mix into natural or artificial
casings, the strings of sausage links are hung in ripening or "green-rooms" at 27-40" C/
80-90% RH. Within 2-3 days, sugar added to the mix is fermented to lactic acid by the
starter culture, which in turn decreases the pH to -5.1 and produces the characteristic
tangy flavor found in fermented sausages. As in cheese making, controlled lowering of
the sausage pH to levels near the isoelectric point of meat protein is crucial for proper
removal of water during later stages of sausage manufacture. Following fermentation,
sausages destined to become semidry varieties containing -50% moisture (e.g., Cervelat-
type sausages and Lebanon bologna) zre normally placed in smokehouses where they are
smoked and cooked to internal temperatures of 60-68C. In contrast, dry sausages which
will ultimately contain -35% moisture (e.g., pepperoni, Genoa, and Milano salamis) are
moved to drying rooms (10-17"C/65-80% RH) where they remain for various times,
depending on the type and size of sausage. Some varieties also may be exposed to cool
smoke before drying; however, unlike semidry varieties, dry sausages are never cooked.
Although fermented sausages keep well because of their relatively high salt content, low
pH, and low moisture (a,) content, both varieties, particularly semi-dry types, should be
refrigerated.
Semidry Fermented Sausage
The relatively severe heat treatment that semidry sausages receive during manufacture is
generally sufficient to eliminate most commonly encountered non-spore-forming food-
borne pathogens. Hence, despite concerns regarding the presence of listeriae in meat prod-
ucts, behavior of L. monocytogenes during manufacture and storage of semidry fermented
sausage has received relatively little attention.
Although L. monocytogenes is unlikely to survive during manufacture of semidry
sausage, ample opportunity exists for this pathogen to contaminate the finished product
during slicing and packaging. Hence, to simulate postprocessing contamination, Glass and
Doyle [84] inoculated slices of commercially produced, fermented semidry sausage to
contain approximately 0.01 or 100 L. monocytogenes CFU/g, after which all samples were
vacuum-packaged and quantitatively examined for listeriae during prolonged incubation
542 Farber and Peterkin

at 4.4"C. Unlike ham, bologna, and frankfurters, the pathogen failed to grow on fermented
semidry sausage of pH 4.8-5.2, with populations generally decreasing 5 10-fold on organ-
oleptically acceptable samples after 6- 12 weeks of refrigerated storage.
Recognizing the likelihood of listeriae being introduced into semidry sausage during
slicing/packaging and surviving throughout the normal shelf life of the product, Cirigliano
et al. [5 11 investigated the possibility of eliminating L. monocytogenes from inoculated
slices of German-type and Polish-type beef sausage ( 104- 105 L. monocytogenes strain
Scott A or V7 CFU/g) by exposing vacuum-packaged product to temperatures of 32.2-
5 1.7"C (90- 125F) for up to 72 h. According to the authors, L. monocytogenes populations
failed to change in product stored at 32.2"C (90F); however, numbers of both Listeria
strains decreased approximately 100-fold after product was held at 373C (100F) for 72
h, with a slightly faster rate of inactivation being observed in Polish-type than German-
type sausage. Although increasing the temperature to 43.3"C (1 10F) eliminated strain
V7 from Polish-type and German-type sausage after 8 and 48 h, respectively, strain Scott
A was not eliminated from either product until completion of a 72-h heat treatment. When
exposed to 48.9 and 51.7"C (120 and 125"F), strain V7 was inactivated in Polish-type
and German-type sausages within 4 and 24 h, respectively. Although somewhat more heat
resistant, strain Scott A was eliminated from both products after 24 h at 51.7"C (125F).
In some instances, objectionable fat losses were observed for both product types; however,
the authors concluded that mild heat treatments can be used to salvage Listeria-contami-
nated German-type or Polish-type sausage without seriously affecting product quality. In
fermented "tea" sausages inoculated with 8 X 106MPN/g of L. monocytogenes, the organ-
ism decreased about 1.5 logs during the initial 4-day ripening period where the pH dropped
from 5.47 to 4.80, decreased another 1.5 logs during the 9-day drying period, and then
remained constant in number during the 20 days of storage at 18-22C [41]. Similar
results were obtained when sausages were inoculated with lower levels of the organism.
The initial a, value as well as those after drying and after storage were 0.974, 0.933, and
0.861, respectively.
Dry Fermented Sausage
Unlike semidry varieties, dry fermented sausages are never exposed to Listeria inactivation
temperatures. Consequently, dry sausages have attracted more attention, as shown by sev-
eral studies that examined the fate of L. monocytogenes during the fermentation, drying,
and storage of hard salami, pepperoni, and several other sausages.
In the first such study, Johnson et al. [ 1141 prepared hard salami from naturally
contaminated (i.e., meat from cows inoculated intravenously with L. monocytogenes) as
well as artificially inoculated ground beef, both of which contained -104 CFU/g of L.
monocytogenes. Glucose, spices, sodium nitrite, and salt were added to the ground beef
along with a glucose-fermenting strain of Pediococcus acidilactici. After stuffing the mix
into casings, all sausages were fermented at 40C for 24 h, dried at 13C for 9 days,
vacuum packaged in gas-impermeable film, and stored at 4C for 12 weeks. As shown in
Fig. 9, listeriae populations decreased approximately 10- and 100-fold during fermentation
(40C/24 h) of hard salami prepared from naturally contaminated and artificially inocu-
lated ground beef, respectively. Inactivation of listeriae during fermentation was primarily
attributed to production of lactic acid (or other metabolites) by the starter culture, with
pH values decreasing from approximately 5.7 to 4.4 by the end of fermentation. Although
numbers of listeriae remained relatively constant in naturally contaminated hard salami
following 9 days of drying (13"C/65% RH), populations in product prepared from artifi-
Listeria monocytogenes in Meat Products 543

5.0

Ferm , Drying Refrigerated Storage

I
I
I

4.0
t-- Naturally Contaminated

&- -- Artificially Inoculated

3.0

\ \

2.0

a
1.0 e----.
a 0
A A A A A A A
t -0- , ' 1 1 I I I-

0 1 2 4 6 8 1 0 14 28 42 56 70 84

Days

FIGURE9 Behavior of L. rnonocytogenes during fermentation (Ferm), drying, and

storage of hard salami prepared from naturally contaminated and artificially inocu-
lated ground beef. Solid (
.,A) and open (0) symbols at 1 log,, CFU/g represent posi-
tive and negative results, respectively, after 8 weeks of cold enrichment. (Adapted
from Ref. 114.)

cially inoculated ground beef decreased nearly 100-fold during drying. L. monocytogenes
was detected in both products during 8 weeks of refrigerated storage; however, higher
levels of listeriae were recovered from naturally contaminated rather than artificially inoc-
ulated hard salami, thus suggesting that behavior of this pathogen is best studied using
sausage prepared from naturally contaminated rather than artificially inoculated ground
beef. Compositionally, both products were very dry, having a, values of 0.79-0.81 as
compared with -0.9 1 for commercially produced hard salami. Although L. monocyto-
genes might be expected to survive more readily in higher moisture commercial products,
growth of the pathogen in retail hard salami appears unlikely given the presence of 5-
7% NaCl and 100- 150 ppm sodium nitrite combined with a pH of 4.3-4.5 and a relatively
low storage temperature.
In contrast to the study just described, Triissel and Jemmi [I721 reported that L.
monocytogenes populations in salami prepared from a mix inoculated to contain approxi-
mately 103or 107CFU/g of L. monocytogenes decreased 5 10-fold in product of pH 5 5 . 6
during 7 days of ripening at 12-22"C/82-95% RH. After 8 weeks of drying at 10- 17"Cl
78-82% RH, numbers of listeriae in salami of pH 5.4-5.7 decreased to <10 CFU/g
regardless of the initial inoculum. However, using an enrichment procedure, the pathogen
was detected in these sausages after an additional 6-1 1 weeks of drying at 10-17"C/35-
50% RH to a, values of 0.68-0.69. Thus, as was true for certain fermented dairy products
discussed in Chapter 12, small numbers of L. monocytogenes cells also can persist in
fermented dry sausages for at least 14-19 weeks. Farber et al. [74] examined the fate of
544 Farber and Peterkin

L. monocytogenes during production of uncooked German, American, and Italian-style

fermented sausages. Similar results were obtained for both German and American sausage,
with Listeria populations decreasing about 2-3 logs after fermentation and smoking and
a further 1-2 logs after drying. In contrast, Listeria populations in Italian-style fermented
sausage prepared without starter culture increased slightly during fermentation, remained
constant during drying, and then decreased slightly during the 4-week holding period at
4C. This study points to the importance of using starter cultures for production of dry
fermented sausages and emphasizes the necessity of having a well-designed and operating
HACCP plan for each type of sausage.
In another study examining the fate of L. monocytogenes in fermented sausage pre-
pared from poultry [ 1421, meat supplemented with two levels (2.0 and 2.5%) of sodium
-
nitrite and glucono-delta-lactone (GdL) was inoculated to contain 104CFU/g of L. mo-
nocytogenes, and then fermented at 20C both with and without a starter culture. L. mono-
-
cytogenes survived the 28-day ripening period, with numbers decreasing 1 log in formu-
lations containing 2.5% sodium nitrite and either GdL or starter culture. Except for a
possible 2-log decrease in batches containing 2% sodium nitrite, L. monocytogenes popula-
tions remained constant in all other formulations. Thus, the microbiological hurdles imple-
mented, including preservatives, starter culture, a, (0.90-0.92), and pH (final pH ranged
from 4.8 to 5.9), failed to completely inactivate Listeria [142].
After recognizing that L. monocytogenes may survive the typical manufacturing
process for hard salami, Glass and Doyle [85] attempted to identify various heat treatments
that could be used to inactivate L. monocytogenes during manufacture of dry fermented
sausage. Work with "beaker sausage" prepared from ground beef/pork containing 3.5%
salt, 103 ppm sodium nitrite, and approximately 5 X 103 CFU/g of L. monocytogenes
indicated that Listeria populations decreased > 10-fold after fermentation (32.2"C/ 16 h)
with P. acidilactici during which the pH decreased from approximately 6.3 to 4.8. Prolific
growth of L. rnonocytogenes in a similar lot of beaker sausage prepared without P. acidi-
lactici confirms the importance of an active starter culture in preventing growth of listeriae
in fermented sausage. Furthermore, these findings suggest that 3.5% salt and 103 ppm
sodium nitrite are of virtually no value in preventing growth of listeriae in sausage mix.
Subsequent holding of fermented beaker sausage at 46.1"C for 8 h or heating to an internal
temperature of 5 1.7 or 57.2"C failed to eliminate listeriae, with the pathogen still being
present in all samples examined by the USDA enrichment method. Although holding
samples at 5 1.7"C for 8 h or 57.2"C for 4 h reduced Listeria populations > 100-fold, the
pathogen was still detected by enrichment in one of two samples that received each of
the two heat treatments. Only after heating beaker sausage to an internal temperature of
623C was the pathogen no longer detected either by direct plating or enrichment.
Subsequently, Glass and Doyle [85] investigated the fate of L. monocytogenes in
pepperoni during normal processing and storage and during heating to an internal tempera-
ture of 5 1.7"C for 4 h immediately after fermentation or drying. After inoculating commer-
cially prepared pepperoni mix to contain 104 CFU/g of L. monocytogenes, populations
of listeriae decreased approximately 100-fold following fermentation (35.6"C/ 12 h) by P.
acidilactici which caused the pH to decrease from 6.0 to 4.7. These findings are similar
to those of Johnson et al. [ 1141 (Fig. 9), who found Listeria populations decreased 10-
to 100-fold during fermentation of hard salami. Following 5 days of drying at 12.8"C,
numbers of listeriae decreased to < 10 CFU/g in normally processed pepperoni; however,
with the USDA enrichment procedure, L. monocytogenes could still be detected in 82-
day-old refrigerated samples of vacuum-packaged pepperoni. Heating the same pepperoni
to an internal temperature of 5 1.7"C between fermentation and drying had relatively little
Listeria monocytogenes in Meat Products 545

effect on 1,. monocytogenes, with viable populations decreasing only about 10-fold. Al-
though holding pepperoni for 4 h at 5 l .7"C reduced Listeria populations to undetectable
levels (as determined by direct plating and enrichment), the pathogen was sporadically
recovered from 5- to 22-day-old sausage using the USDA enrichment procedure. Subse-
quent holding of the same pepperoni (pH 4.6) at an internal temperature of 51.7"C for 4
h immediately after 26 days of drying at 123C completely inactivated the pathogen as
determined by direct plating and enrichment procedures. Additional experiments con-
ducted on pepperoni containing 5.3 X 103CFU/g of L. monocytogenes after 19 days of
drying verified that a minimum heat treatment of 4 h at 51.7"C was required to obtain a
Listeria-free product. Thus, although normal processes used to manufacture pepperoni
will not eliminate L. monocytogenes from heavily contaminated product, holding pep-
peroni and possibly other dry sausages at an internal temperature of 5 1.7"C for at least
4 h may prove to be a viable means of salvaging contaminated product.
Although the antibotulinal properties of nitrate, and particularly nitrite, have been
recognized for many years, much remains to be learned concerning the effect of these
preservatives on Listeria behavior in dry fermented sausage. Junttila et al. [ 1 181 examined
the ability of L. monocytogenes to survive in dry Finnish sausage containing various levels
of potassium nitrate, sodium nitrite, and salt. All sausage was prepared from a mixture
of ground beef and pork to which sugar, spices, and 3.0 or 3.5% salt were added along
with 50- 1000 pprn potassium nitrate and/or sodium nitrite. After inoculation to contain
- 105CFU/g of L. monocytogenes and a starter culture consisting of Staphylococcus car-
nosus and Lactobacillus plantarum, the sausage mix was stuffed into casings. All sausage
links were fermented 2 days at 23"C, smoked 5 days at 20--22"C, and then dried 1 week
each at 18 and 10C. L. monocytogenes populations in sausage containing commonly used
levels of salt (3.0%) and sodium nitrite ( I 20 ppm) decreased 1.14 orders of magnitude
over 21 days (Fig. 10). Similar findings also were reported when 3.5 rather than 3.0%
salt was used. Increasing the levels of sodium nitrite (200 ppm) and potassium nitrate
(330 ppm) to those commonly used 30 years ago led to somewhat faster inactivation of
listeriae in dry fermented sausage, with inactivation again being most pronounced during
the later stage of drying. Over the same 21-day period, Listeria populations decreased
approximately 3.3 orders of magnitude in sausage containing 3.5% salt and 1000 ppm
potassium nitrite; however, this concentration of potassium nitrate is no longer permitted
in dry fermented sausage. Growth of L. monocytogenes in this product was apparently
suppressed by the combination of salt, sodium nitrite, and a pH of 4.7; however, given
the pathogen's known tolerance to salt, acid, and low temperatures, addition of commonly
used levels of sodium nitrite to fermented sausage was only marginally effective in inacti-
vating listeriae. Thus, although this and other studies have provided valuable information
concerning the behavior of L. monocytogenes in sausage products, an understanding of
interactions between various factors such as starter cultures, food additives, and various
heat treatments is still needed to develop suitable methods to eliminate L. monocytogenes
from fermented sausage and other processed meat products.

Modified-Atmosphere Packaging
Modified-atmosphere packaging (MAP) can extend the shelf life of many perishable foods
such as meats and poultry. The C02-enriched atmosphere which is created within a meat
pack can inhibit normal spoilage flora and select for certain groups of organisms such as
the lactic acid bacteria [69]. Concerns have been raised about the ability of L. monocyto-
genes to outgrow the normal spoilage flora on MAP foods. In addition, MAP foods have
546 Farber and Peterkin

,
8
Dryingat 18C ,
I
Drying at 10C
I

3.0 I
1 I I I I I 1
I,
N I 1
0 1 2 3 4 5 6 7 14 21

Days

FIGURE10 Fate of L. monocytogenes during fermentation (Ferm),smoking, and dry-

ing of Finnish sausage prepared from ground beef/pork containing 3% NaCl and vari-
ous concentrations of NaNO, and/or KN03.(Adapted from Ref. 118.)

a relatively long shelf life, which in turn can give extra time for psychrotrophic foodborne
pathogens such as L. monocytogenes to grow to high levels. Although Listeria can grow
on vacuum-packed meats such as beef, lamb, and pork, as discussed earlier, the effect of
intermediate to high levels of CO2 on survival and growth of this pathogen on meat and
poultry is not clear [77].
Beef
Growth of L. monocytogenes was observed on samples of vacuum-packaged high-pH (>6)
beef stored at 0, 2, 5, and 10C but not on those vacuum-packs stored at -2C. However,
long lag periods were usually observed, with the organism growing at a slower rate than
the spoilage flora [81]. When samples were packaged under CO2, Listeria only grew at
10C and not at any of the lower storage temperatures tested. However, when normal
ultimate-pH beef (pH 5.3-5.5) was tested, L. monocytogenes was unable to grow on sam-
ples stored in CO2 packs at 5 or 10C [27]. Hence, the lower pH of normal as compared
with dark firm dry (DFD) meat, combined with the high CO2environment, was probably
sufficient to inhibit growth and partially inactivate the organism. As in the findings of
Grau and Vanderlinde [ 8 3 ] ,L. rnonocytogenes grew well on vacuum-packaged meat stored
at 5 and 10C. It is interesting that in the study by Avery et al. [27], L. monocytogenes
outgrew the spoilage flora on vacuum-packaged beef, which is in contrast to the results
obtained by Gill and Reichel [81]. Perhaps L. monocytogenes can compete better with
spoilage organisms at a lower pH. A follow-up study by Avery et al. [28] was designed
to assess the effects of previous high CO2 exposure of Listeria to its subsequent growth
Listeria monocytogenes in Meat Products 547

during abusive retail display. Beef steaks of normal pH were inoculated with L. monocyto-
genes, individually packaged in CO2 packs, and then stored at - 1.5"C for <3 h and 5
or 8 weeks. At the end of each storage period, samples were removed, overwrapped, and
placed on retail display at 12C for up to 140 h. Even after only a brief (<3 h) exposure
to COz, L. rizonocytogenes grew slightly, if at all, during retail display, with demonstrated
lag phases of >75 h. However, no comparative controls were used to confirm growth of
the organism following inoculation and storage at 12C. Experiments were also done
whereby steaks were removed from storage and then rinsed to remove some cells of L.
monocytogmes, which were reinoculated onto freshly cut beef steaks to simulate cross
contamination. Under these conditions, Listeria cells previously exposed to CO, for 5 or
8 weeks did not grow on cross-contaminated steaks. Consequently, it was concluded that
(a) prior exposure of L. monocytogenes-contaminated beef steaks to high CO, environ-
ments will not increase the likelihood for growth of the organism when the steaks are
placed on retail display and possibly temperature abused; and (b) the risk of growth is
minimal when cross contamination occurs between high-C02 stored beef and fresh raw
beef before retail display. Additional experiments have examined survival and growth of
this pathogen on sliced roast beef stored under vacuum or CO2 at - I .5 and 3.0"C. Al-
though unable to grow under CO, at - l SoC, L. monocytogrwes did grow under all other
test conditions. At 3"C, the organism grew three times faster on vacuum-packaged as
compared with CO2-stored roast beef (see Table 8). When growth occurred, maximum
populations were attained only at the end of product shelf life.
Sausages
Experiments were done to determine survival of L. monocytogenes on sliced frankfurters
incubated under 20-80% CO, at 4,7, and 10C for up to 6 weeks [ 1251. Although numbers
of L. mono~ytogenesin vacuum packs, 20% CO,, and 30% CO2 increased by 2.5, 1.0,
and 0.5 logs, respectively, during the commercial minimum shelf life of 3 weeks at 4"C,
the pathogen was inhibited in the presence of both 50 and 80% CO,. During an additional
3 weeks of storage, the organism grew in the presence of 50% but not 80% COz. However,
increasing the storage temperature to 7C permitted growth of the organism during this
3-week storage period even in the presence of 80% COz. Therefore, under commercial
conditions (4-10C, 3-week shelf life), only 80% CO2prevented growth of the organism.
However, since this level of CO2,caused undesirable organoleptic changes in the product,
CO, levels between 50 and 80% may be more appropriate [ 1251.
Lamb
Sheridan et al. [165] examined growth of L. monocytogenes on both raw minced lamb
and lamb pieces stored at 0 and 5C under various atmospheres (vacuum; 80% 02:20%
CO,; 50% 0,:50% CO,; and 100% CO,). No growth was observed on lamb stored at
0C. At 5"C, L. monocytogenes grew on aerobically packaged and vacuum-packaged lamb
pieces but not on minced lamb (Table 1 1 ) . Disregarding aerobically packaged samples,
highest Listcria populations were observed on pieces and minced lamb stored under 80%
0,:20% CO1 and 50% Oz:5O%CO2,respectively, after 42 days of storage at 5C. Again,
L. monocytogenes failed to grow on lamb stored under 100% CO2.
Pork
When the microbial ecology of fresh MAP pork was assessed at various storage tempera-
tures, listeriae were one of the predominant organisms on product stored at - I "C but not
on samples stored at 4.4 or 10C [ 1461. In fact, most bacteriocin-producing organisms
548 Farber and Peferkin

TABLE11 Mean Total Aerobic and L.

monocytogenes Counts (loglo CFU/g) at 42 Days on
Lamb Pieces and Mince Packaged i n Different Gas
Atmospheres at 5C
Pieces Mince
-
Meat type TA LM" TA LM
Gas atmosphere:
Air 8.86 6.07 9.20 5.37
Vacuum pack 7.53 3.81 8.65 1.74
80% 02/20% CO2 8.91 4.91 8.97 2.68
50% C02/50% N2 8.15 4.22 7.75 3.68
100% CO2 7.55 1.35 8.07 0.58
TA, total aerobic: LM, L. rnonocytogenes.
a Although not clearly stated, the initial total LM count appears to be

2.8 X 10' CFU/g.

Source: Adapted from Ref. 165.

were isolated from samples stored at the two higher temperatures. No growth of L. rnonocy-
togenes was observed on fresh pork longissimus dorsi at 1C regardless of storage atmo-
sphere. At 7"C, L. rnonocytogenes grew on aerobically stored samples but not on those
stored under 100% N,, 80% 02:20% CO, or 60% 0,:40% CO, [140]. No additional
hazards were identified using modified atmosphere (MAs) for packaging fresh pork of
normal pH. According to Davies [59], under an atmosphere of 80% 02:20%CO,, growth
of L. rnonocytogenes on cooked ham was no greater than that observed in aerobically
stored control samples. Manu-Tawiah et al. [ 1411 examined the influence of 20 and 40%
CO2on growth of L. rnonocytogenes and Yersinia enterocolitica on fresh pork chops stored
at 4C for 35 days. Levels of L. rnonocytogenes on air- or vacuum-packaged pork generally
did not differ significantly from the numbers on chops packaged in 20 and 40% CO2,
with populations increasing 1.5 to 2.0 logs (from 3.7-106 CFU/cm2) after 35 days. The
growth rates for Listeria and the aerobic psychrotrophic spoilage flora were faster in air
as compared with C02-packaged samples, with the aerobic psychrotrophic spoilage flora
always outgrowing L. rnonocytogenes. In contrast to the results of Wimpfheimer et al.
[185], no differences were observed in the numbers of L. rnonocytogenes on chops pack-
aged in 60% N2:40% CO, or 50% N,:lO% 02:40% CO2. Y. enterocolitica outgrew L.
rnonocytogenes in all MA-packaged samples, increasing to nearly 108CFU/cm2 after 35
days of storage at 4C. The fact that Yersinia grew much better in MA-packaged than in
aerobically stored chops is disconcerting from a public health standpoint.
The application of additional hurdles in addition to MAP is a strategy that will be
more commonly used in the future. As a case in point, the combined effect of nisin and
MAP on growth and survival of L. rnonocytogenes on cooked pork tenderloin was exam-
ined during storage at 4 and 20C [67]. The organism grew on pork tenderloin packaged
under 100% CO, at both storage temperatures. However, when nisin ( 104 IU/mL) was
added, the combination proved to be bactericidal (Table 12). Although a lower concentra-
tion of nisin ( 103IU/mL) also was listericidal in pork stored at 4O, but not at 20"C, simulta-
neous growth of Pseudornonas fragi, a spoilage organism, was observed. Fewer pseu-
domonads were generally seen in MA-stored samples, with these levels being unaffected
Listeria monocytogenes in Meat Products 549

TABLE12 Cumulative Changea in Log CFU/g for L. monocytogenes on Cooked

Tenderloin Stored in MAP at 4 and 20C
MAP

100% air 100% CO;! 100% air 100% CO2

with nisinb with nisin with nisinb with nisin
Time Timed -
(days) 0 104 0 104 (days) 0 104 0 104
6 2.78 -2.11 0.65 -2.18 1 4.20 -2.10 1.90 -2.10
12 4.28 -2.10 2.16 -2.18 2 5.24 -2.11 4.32 -2.11
18 5.03 -2.12 4.97 -2.19 4 5.36 -2.13 5.57 -2.10
24 4.97 -2.15 4.74 -2.18 7 5.64 -2.14 5.67 -2.13
30 4.81 -2.18 4.67 -2.18 10 6.07 -2.15 5.53 -2.14
MAP, modified-atmosphere packaging.
aLog CFU/g at day X -log CFU/g at day 0 for two samples per treatment per day. The concentration of L.
rnonocytogenes on the cooked tenderloin surface at 0-day was 3.18 log,, CFU/g.
The unit for nisin activity was IU/mL.
Storage temperature of 4C.
Storage temperature of 20C.
Source: Adapted from Ref. 67.

by nisin. The authors used the general concept of a safety index, which compares the
relative numbers of spoilage organisms with pathogens. In the MAP samples containing
nisin, numbers of P. fragi increased during storage relative to the growth of L. monocyto-
genes. However, such growth was not observed in nisin-free samples. MAP also has been
used in conjunction with irradiation to minimize growth of foodborne pathogens on raw
pork [88]. In nonirradiated pork stored under an atmosphere of 75% N2: 25% COz, L.
monocytogenes populations increased about 2 logs after 9 days of storage at 10C, with
the pathogen being undetected in irradiated (1.75 kGy) control samples. Similar results
were obtained using a higher initial inoculum (106 versus 103 CFU/g). After 9 days of
incubation at 10C, Listeria counts numbered about 4.5 X 104 and 1 X 10s CFU/g in
irradiated and nonirradiated treated samples, respectively. Potential benefits of using irra-
diation after meat packaging were also evident, since lactic acid microflora outgrew L.
monocytogenes and several other pathogens tested during storage [88]. Since Lactobacillus
sake usually predominates the microflora of irradiated MAP pork, sakacin A (a bacteriocin
produced by this organism) may have been responsible for inhibiting the growth of L.
monocytogenes. However, L. monocytogenes grew to similar levels in both irradiated and
nonirradiated samples.
Given these findings, L. monocytogenes will likely grow in the presence of up to
50% CO2, with growth at higher CO2 concentrations mainly dependent on the interplay
between the gas atmosphere, pH, temperature, and other microbial competitors.

Thermal Inactivation in Meats

Interest in the heat resistance of listeriae in raw meat products dates back to at least 1980
when Karaioannoglou and Xenos [ 1191 reported on survival of Listeria in grilled meat-
balls. In their experiments, minced beef inoculated to contain 102, 103, 104, or 105 L.
rnonocytogenes CFU/g was combined with eggs, bread, onion, garlic, salt, and spices and
then fashioned into meatballs weighing 35-40 g each. Meatballs were then placed on a
550 Farber and Peterkin

coal-fired grill at 110-120C and cooked for 15 min to an internal temperature of 78-
85C. After grilling, L. monocytogenes was isolated from all meatballs that originally
contained 104-1OS listeriae/g. However, the pathogen was recovered from only one of
four meatballs inoculated to contain 103listeriae/g and was absent from meatballs that
originally contained 1O2 listeriae/g. Since data from the European surveys discussed earlier
indicate that retail raw beef may occasionally contain up to 103Listeria CFU/g (some of
which are likely to be L. monocytogenes), thorough cooking of raw meat is presently
advised to eliminate L. monocytogenes as well as E. coli 0157:H7, salmonellae, Campylo-
bacter spp., and other organisms that have been associated with foodborne illness. Al-
though these findings attest to the hardy nature of listeriae in fresh ground beef, L. monocy-
togenes also was equally tenacious in artificially contaminated frozen ground beef, with
populations remaining unchanged at 10' CFU/g during 6 months of storage at - 18C.
Concern about possible resistance of L. monocytogenes to pasteurization of milk,
along with recovery of this pathogen from cooked meats, prompted interest in the possibil-
ity of L. monocytogenes surviving thermal processing steps commonly used to convert
raw meat into ready-to-eat products. Boyle et al. [36] investigated the thermal destruction
of L. monocytogenes strain Scott A in ground beef (-20% fat) by submerging sealed
tubes containing ground beef with 10' listeriae/g in a water bath at 75C until the internal
temperature of samples reached 50, 60, 65, or 70C. Samples then were examined for
listeriae by direct plating as well as selective and cold enrichment. According to these
researchers, Listeria levels remained constant in samples heated to an internal temperature
of 50C over 6.2 min. Numbers of listeriae decreased 4.4-6.1 orders of magnitude in
samples of ground beef during 8.4 and 10.6 min of heating to 60 and 65"C, respectively,
with similar results also being reported in 1988 by Farber et al. [72]. Heating ground pork
to 62C over a period of 25 min was sufficient to inactivate 5.8-7.35 log CFU/g of L.
monocytogenes depending on the pork formulation. Most additives, such as kappa-carra-
geenen, sodium lacate, and algin/calcium binders used in the ground pork formulations,
did not influence thermal resistance of the organism [188]. Similarly, Yen et al. [190]
found that sodium phosphate, sodium erythorbate, and added water had little or no effect
on survival of various L. monocytogenes strains in ground pork during heating. Interest-
ingly, although an added cure decreased Listeria inactivation by 2.0-2.2 logs in ground
pork cooked to 62C as had been noticed previously [69a, 1891, this protective effect was
only seen at temperatures below 67C.
Several studies were done to determine the D- and z-values for L. monocytogenes
in various ground and whole meat products (Table 13). D,,,o,-values for most products
range from 1.8 to 8.3 min. Carlier et al. [45] examined destruction of L. monocytogenes
in whole hams cooked to an internal temperature of 583C. When stored for 2 months
at 9"C, survivors were found among hams inoculated to contain 4 X 10s CFU/g of L.
monocytogenes but not in those inoculated with <10 CFU/g. It was suggested that a
minimum core temperature of 65C be attained in these products, with an F700c-value of at
least 40 min. Another study examined survival of L. monocytogenes in vacuum-packaged,
nitrite-free beef roasts prepared with brines containing selected antimicrobial agents [ 1741.
The brines used included sodium chloride, sodium tripolyphosphate, Brifisol 4 14, acetic
acid, sodium lactate, Lauralac, and potassium sorbate. Meats were cooked in a bag once
or twice to 623C under conditions simulating product contamination from pumping
brines or from slicing/cutting after cooking. Survival of L. monocytogenes on the surface
of beef roasts was surprisingly high considering that cells had possibly been exposed to
temperatures 280C for more than 30 min. Irrespective of the number of cookings, Liste-
Listeria monocytogenes in Meat Products 551

TABLE
13 Heat Resistance of L. rnonocytogenes in Meats
Temp. D-value z-value
Product ("C) ("C; min.) ("Cl Ref.
Ground beef 60 3.12 5.3 72
Fermented sausage mix 60 16.7 4.6
Ground beef roast 60 4.47 160
Ground beef 60 1.62
Beaker sausage 60 9.13
Meat slurry 60 2.54 36
70 0.23
Meat slurry 60 7.3 6.8 155
Beef 60 3.8 7.2 137
70 0.14
Beef steak 60 8.32, 6.27 5.98, 5.98 78
70 0.20, 0.14
Liver sausage slurry 60 2.42 6.2 35
Lean ground beef 62.8 0.6 9.3 68
Fatty ground beef 1.2 11.4
Meats (predicted value) 60 3.82 6.8 I37
70 0.13
Ham 60 1.82 5.05 44
60 3.48a 6.74
Ground pork 62 6.5-7.7 123
Ground pork 60 1.14- 1.7 5.05-5.45 152
aCells heat shocked at 42C for 1 h.

ria survival rates in internally cooked inoculated roasts were similar for all brine treatments
except NaC1-sodium tripolyphosphate, raising the possibility that sublethal heating may
have induced a heat-shock response in these organisms. The highest number of Listeria-
positive samples was seen among roasts processed with standard NaC1-phosphate brines,
which most closely resembles product currently being marketed. In contrast, greatest de-
struction of the organism was observed with brines containing a phosphate blend and
sodium lactate or glycerol monolaurin in combination with one, and especially two, cook-
ings [174].
Steam pasteurization is gaining acceptance as a means of reducing surface levels
of pathogenic microorganisms on meats. Some advantages of using steam pasteurization
include its ability to uniformly heat the entire carcass surface, and uniformly cover irregu-
larly shaped surfaces. Furthermore, waste water accumulation is not an issue, and, by
virtue of automation, the process is not subject to operator misuse. In one study, frankfurt-
ers were inoculated with L. innocua and steam pasteurized in a small pasteurizer designed
in-house. The heating chamber was evacuated for 15 s after which the product was steam
treated at a set time, temperature, and pressure. Treatment times of 32 and 40 s at 136
and I15"C, respectively, led to a &log reduction in counts of L. innocua on the meat
surface while only slightly affecting color and weight [58]. Another study compared steam
pasteurization (S; 15-s steam pasteurization) to traditional methods for reducing pathogens
on meat surfaces [ 1531. The latter methods, which were tested both individually and in
combination, included knife trimming (T), water washing (W; 35"C), hot water/steam
vacuum spot cleaning (V), and spraying with 2% vol/vol lactic acid (pH 2.25 at 54C)
552 Farber and Peterkin

TABLE
14 Effectiveness of Combination and Individual Decontamination
Treatments in Reducing L. monocytogenes on Surfaces of Freshly Slaughtered
Beef
Mean Mean
Treatmenta Initialh reductionc Treatmenta Initialb reductionc
TW 5.52 f. 0.22 4.96 f. 0.34d T 5.26 f 0.33 2.54 f. 0.33'
TWS 5.57 f 0.15 4.56 t 0.34d' W 5.27 t 0.35 1.28 2 0.33g
ws 5.46 f. 0.03 4.40 -t 0.34def V 5.37 f 0.20 3.33 f. 0.33ef
vw 5.56 k 0.12 3.49 2 0.34f S 5.38 f. 0.16 3.44 k 0.33ef
vws 5.49 t 0.04 3.84 ? 0.34" VWLS*5 5.18 f. 0.35 4.51 k 0.33d
TWLS 5.51 k 0.19 5.07 f. 0.34d VWLS*lO 5.21 f. 0.33 4.23 -+ 0.33de
VWLS 5.51 & 0.13 5.01 t 0.34d

a Order of treatment within abbreviation indicates order of application: T, trim; W, 35C water wash; S, 15-s
steam pasteurization; V, hot water/steam vacuum spot cleaning; L, 2% lactic acid spray; S* 5 and S* 10, 5-
and 10-s exposure time, respectively, for steam pasteurization.
Mean initial pathogen population (log CFU/cm') from four replications * standard error of mean.
Mean reduction in pathogen population (log CFU/cm') from four replications 5 standard error of mean.
d,ef.g Means having the same superscript within columns are not significantly different ( P < .05).

Source: Adapted from Ref. 153.

(L). All combination treatments reduced numbers of L. monocytogenes on meat surfaces,

with TWLS, VWLS, and TW (the order of treatment within the abbreviation indicates
the order of application) being most effective and VW and VWS the least effective (Table
14). The authors concluded that greater pathogen reduction can be attained by using combi-
nations of decontamination treatments, with knife trimming and/or steam vacuum spot
cleaning (to remove visible contamination), followed by steam pasteurization, being a
very effective intervention strategy for the meat industry [ 1531. Dorsa et al. [63] also
examined the efficacy of using steam vacuuming (SV) and a hot water spray wash of 74
t 2C (W) to eliminate L. innocua from the surface of beef carcass tissue. Initial reduc-
tions of 2.0, 2.2-2.5, and 2.6-2.7 CFU/cm2 were observed after the SV, W, and SV +
W treatments, respectively. Although numbers of L. innocua on all treated meat samples
were the same as those on the untreated control following 21 days of storage at 5C (Fig.
1 l), it should be noted that the initial loads of listeriae on meat were high (106 CFU/cm2)
and that with lower initial levels, outgrowth of the organism may not have occurred or
been minimal.
Regarding heat resistance in meat and poultry, we can conclude that L. monocyto-
genes is about four times more heat resistant than salmonellae in meats. Attention should
be given to those meats which are heated slowly, since such processes may induce produc-
tion of heat-shock proteins which would in turn enhance thermal resistance of the patho-
gen. D-values obtained for meats can vary quite widely depending on the prior history
of the food and organism, recovery method, and strain type; however, in general, L. mono-
cytogenes exhibits higher D-values in meat rather than dairy products: Cured meats require
greater heating than noncured products to assure the same level of protection, with multiple
decontamination treatments working best to eliminate this pathogen from carcass surfaces.

Bacteriocins for Controlling Listeriae in Meat

Several articles have reviewed the potential benefits of using bacteriocins and/or lactic
acid bacteria to control Listeria spp. in foods [149] and more specifically in meats
Listeria monocytogenes in Meat Products 553

.o
2*o*
1Before

treatment
After
treatment
2 7

Time (Days)
14 21

FIGURE11 Effects of moist heat interventions on the initial levels and subsequent
outgrowth of Listeria innocua (least squares means, log CFU/cm2:error bars denote
standard error of the mean) during a combination of aerobic and vacuum storage at
5C for 21 days. (Adapted from Ref. 63.)

[147,169]. Since the general use of bacteriocins to control listeriae has been discussed
earlier in this book (Chap. 6), the following discussion of bacteriocins will be confined
to meat applications.
Raw Ground Meat
In one of the first bacteriocin-related studies, Buchanan and Klawitter [39] assessed the
ability of Curnobacterium piscicola strain LK5 to inhibit growth of L. monocytogenes in
a variety of different foods. Foods were inoculated with 103CFU/g of L. monocytogenes
either with or without 104CFU/g of strain LK5. In sterile raw ground beef, strain LK5
inactivated L. monocytogenes at 5C and prevented its growth at 19C. C. piscicola had
no effect on L. monocytogenes in nonsterile ground beef (or chicken roll), with no growth
of the pathogen being observed in control samples. The bacteriocin-producing strain gener-
ally was most effective in foods containing a background microflora. Similar studies using
sterile and nonsterile ground beef were done with Lactobacillus casei and its associated
bacteriocin, lactocin 705 [ 1781. Meat was inoculated with either L. casei or the pure bacte-
riocin and then stored at 20C for 24 h. In general, inactivation of L. monocytogenes was
greatest at the highest level of lactocin 705 tested ( 1 6,80OAU/mL), with the fewest listeriae
being recovered from a meat slurry and autoclaved ground beef.
Inhibition of L. monocytogenes by starter cultures also has been assessed in mini-
mally heat-treated vacuum-packaged beef cubes either with or without gravy and/or glu-
cose. For these experiments, Lactobacillus bavaricus strain MN was inoculated into beef
at 10sor 103CFU/g, along with 102CFU/g of L. monocytogenes, then vacuum sealed,
and stored at 4 or 10C [ 1861. Strain MN grew and produced bacteriocin during the early
stages of growth, with inhibition of L. monocytogenes being most pronounced at the higher
MN inoculum level and lower incubation temperature. Bacteriocin production was inde-
pendent of the presence of glucose in the meat. Addition of sugar enhanced the antilisterial
activity even though the pH was not greatly reduced in those meats with gravy and glucose.
554 Farber and Peterkin

Greater antilisterial activity was seen in meats containing gravy with glucose, suggesting
that the gravy may have enhanced diffusion of the bacteriocin.
Bacteriocin-producing lactic acid bacteria also have been used to minimize growth
of L. rnonocytogenes on frankfurters. In these experiments, either high ( 107CFU/g) or
low ( l 03- 1O4 CFU/g) levels of a bacteriocin-producing strain of Pediococcus acidilactici,
as well as its plasmid-cured, bacteriocin-negative derivative (bac- ), were inoculated sepa-
rately onto frankfurters along with a five-strain cocktail of L. rnonocytogenes. Frankfurters
were stored both aerobically and anaerobically at 4 and 15C. The presence of P. acidilac-
tici on the frankfurters inhibited listeriae to various degrees depending on the pediococci
levels, storage temperature, and packaging atmosphere [32]. Yousef et al. [ 1913 reported
that L. rnonocytogenes grew in two of five wiener exudates tested, with the best growth
being observed in exudates containing the lowest concentration of phenols, the lowest
indigenous levels of lactic acid bacteria, and the highest pH. Those exudates which did
not support growth of the organism, including those at 4OC, proved to be listericidal. Glass
and Doyle [85] previously showed that L. monocytogenes could grow on wieners stored
at 4.4"C. These conflicting results could be explained by the loss of wiener exudate during
manipulation, uneven distribution of exudate on the wiener surface, or variations in the
intensity of smoking within and among different brands of wieners [ 19I]. Both P. acidilac-
tici H and pure pediocin AcH inactivated listeriae that had been inoculated into one brand
of wiener exudate and stored at 25C for 8 days. In control samples, L. rnonocytogenes
populations increased from about 104to nearly 107CFU/g within 4 days. In a follow-up
study, the latter authors assessed the ability of the same lactic acid bacteria to limit growth
of L. rnonocytogenes in temperature-abused vacuum-packaged wieners stored at 4 and
25C. At 25OC, the presence of P. acidilactici strain LB42 inhibited but did not completely
inactivate L. rnonocytogenes. However, P. acidilactici strain JBL 1095 was listericidal,
decreasing counts of L. rnonocytogenes by 2.7 logs. This inactivation was not solely caused
by pH, since pH values in wieners inoculated with both strains of pediococci were similar.
The only difference between the two strains was that production of pediocin AcH was
confined to JBL 1095, strongly suggesting that this bacteriocin enhanced the antilisterial
activity of lactic acid bacteria in vacuum-packaged wieners.
Fermented Sausages
Similar work also has been done to examine the effects of starter cultures and/or their
bacteriocins on survival and growth of L. rnonocytogenes in sausages. Foegeding et al.
[76] used pediocin-producing strains of P. acidilactici (along with an isogenic mutant as
a control) to minimize growth of L. rnonocytogenes on dry fermented American-style
sausages. Recovery of the organisms during fermentation was made easier by using antibi-
otic-resistant strains of pediococci and listeriae. The study was unique in showing that in
combination with other fermentation endproducts, inhibition of L. rnonocytogenes was
enhanced by bacteriocin production in situ during fermentation and drying. Berry et al.
[31] had also previously shown the benefits of using P. acidilactici as a starter culture to
minimize growth of listeriae during sausage fermentation. According to these authors, a
commercial summer sausage mix was inoculated to contain - 106CFU/g of L. rnonocyto-
genes and fermented with either a bacteriocin-producing or non-bacteriocin-producing
strain of P. acidilactici. Following a 12- to 14-h fermentation at 37.8"C, populations of
listeriae decreased approximately 100-and 10-fold in summer sausage fermented with bac-
teriocin-producing and non-bacteriocin-producing strains of P. acidilactici, respectively.
The pathogen also was inactivated in sausages with slower acid production (pH >5.5),
Listeria monocytogenes in Meat Products 555

which suggests that bacteriocin production occurred independent of carbohydrate fermen-

tation. However, L. monocytogenes was still detected in 9 of 90 (10.0%)sausage samples
that had been heated to an internal temperature of 64C and then refrigerated for up to 2
weeks. Thus, although use of this bacteriocin-producing starter culture led to a dramatic
decrease in numbers of viable listeriae in summer sausage, it did not completely inactivate
the pathogen in finished product prepared from sausage mix containing 106CFU/g of L.
monocytogenes. However, since the presence of > I O3 L. monocytogenes/g in commer-
cially prepared sausage mix is highly unlikely, it appears that current heat treatments are
adequate to produce Listeria-free semidry sausage. Another starter culture, Lactobacillus
sake, was found to be capable of minimizing growth of L. monocytogenes in German-
style fresh sausages. Results varied depending on the initial pH of the fresh Mettwurst,
with bacteriostatic and bacteriocidal ( 1-log reduction) effects on L. monocytogenes ob-
served at initial pH values of 6.3 and 5.7, respectively. In another study [ 1051, a similar
bacteriocin-producing (sakacin K) strain of L. sake isolated from a naturally fermented
sausage was able to reduce the numbers of L. monocytogenes in dry fermented sausage
by 1.25 logs as compared with the isogenic bac- control strain. The fate of L. monocyto-
genes in naturally and artificially contaminated salami also has been assessed using Lucto-
bacillus plantarurn as the starter culture [42]. In this study, the starter culture prevented
growth of listeriae but was not listericidal. In inoculated samples, little difference was
observed between samples containing bac+ or bac- strains of L. plantarurn. However,
among naturally contaminated samples, only those inoculated with the bacteriocin-produc-
ing strain of L. plantarurn were free of listeriae.
Bacteriocins also have been used in studies aimed at inactivating or preventing at-
tachment of L. monocytogenes to muscle surfaces. El-Khateib et al. [65] investigated the
ability of lactic acid, nisin, and pediocin to inactivate L. monocytogenes on beef muscle,
as well as to prevent its attachment after short-term exposure and during 48 h of refriger-
ated storage. The latter experiments were done to simulate secondary contamination of
meat during refrigerated storage. Lactic acid (2%), nisin (4 X 104IU/mL), and pediocin
(3.2 X 103AU/mL) decreased the numbers of listeriae on the meat surface by 1.7, 1.1, and
0.6 log 1o CFU/6 cm2 (cubical piece), respectively. Interestingly, compared to the control,
treatment with lactic acid caused a greater percentage of listeriae cells to attach to the
meat surface. This was partially attributed to the fact that acidic conditions can sometimes
enhance adhesion of bacteria to surfaces [65]. Using nisin and pediocin, the percentage
of L. monocytogenes cells that attached to beef muscle in the presence of both bacteriocins
either did riot change significantly or decreased slightly. However, after 1 h, 30 and 17%
of the cells were attached to the nisin- and pediocin-treated samples, respectively, as com-
pared with 6.3 and 12% for the controls. Other work done by Cutter and Siragusa [56,57]
has shown nisin to be effective in reducing (by 2.0-2.83 log,,, CFU/cm2) the numbers of
L. innocua on beef. In the latter study, the combination of vacuum-packaging and nisin
spraying suppressed growth of L. innocua to the point where at the end of the 4-week
incubation period at 4OC, counts were lower than in the corresponding nontreated controls.
In summary, bacteriocins, especially pediocin, appear to be good candidates for
controlling growth of listeriae in meat and poultry, with their effectiveness having been
proven in wieners and wiener exudates, semidry and dry sausages, and fresh beef and
pork. The main obstacles to their present use in foods are the development of bacteriocin-
resistant strains, activity loss over time, inactivation by proteases, and/or adsorption to
meat and fat particles resulting in inactivation, limited diffusion, especially in minced
meats, and poultry, and finally regulatory acceptance.
556 Farber and Peterkin

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modified atmosphere and in air. Int. J. Food Microbiol. 11:205-214.
186. Winkowski, K., A.D. Crandall, and T.J. Montville. 1993. Inhibition of Listeria monocyto-
genes by Lactobacillus bavaricus MN in beef systems at refrigeration temperatures. Appl.
Environ. Microbiol. 59:2552-2557.
187. Wong. H.-C., W.-L. Chao, and S.-J. Lee. 1990. Incidence and characterization of Listeria
monocytogenes in foods available in Taiwan. Appl. Environ. Microbiol. 56:3 101-3 104.
188. Yen, L.C., J.N. Sofos, and G.R. Schmidt. 199 1. Effect of meat curing ingredients on thermal
destruction of Listeria monocytogenes in ground pork. J. Food Prot. 54:408-4 12.
189. Yen, L.C., J.N. Sofos, and G.R. Schmidt. 1992. Destruction of Listeria monocytogenes by
heat in ground pork formulated with kappa-carrageenan, sodium lactate and the algin/calcium
meat binder. Food Microbiol. 9:223-230.
190. Yen, L.C., J.N. Sofos, and G.R. Schmidt. 1992. Thermal destruction of Listeria monocyto-
genes in ground pork with water, sodium chloride and other curing ingredients. Lebensm.
Wiss. Technol. 25:61-65.
191. Yousef, A.E., J.B. Luchansky, A.J. Degnan, and M.P. Doyle. 1991. Behavior of Listeria
monocytogenes in wiener exudates in the presence of Pediococcus acidilactici H or pediocin
AcH during storage at 4 or 25C. Appl. Environ. Microbiol. 57:1461-1467.
192. Yu, L.S.L., R.K. Prasai, and D.Y.C. Fung. 1995. Most probable number of Listeria species
in raw meats detected by selective motility enrichment. J. Food Prot. 58:943-945.
193. Zaika, L.L., S.A. Palumbo, J.L. Smith, F. Del Corral, S. Bhaduri, C.O. Jones, and A.H. Kim.
1990. Destruction of Listeria monocytogenes during frankfurter processing. J. Food Prot. 53:
18-21.
Incidence and Behavior of Listeria
monocytogenes in Poultry and
Egg Products

NELSON
A. Cox AND J. STANLEY
BAILEY
Agricultural Research Service, U.S. Department of Agriculture,
Athens, Georgia

ELLIOTT. RYSER
Michigan State University, East Lansing, Michigan

INTRODUCTION
Avian listeriosis was first recognized in 1932 when TenBroeck isolated Listeria monocyto-
genes (then Bacterium monocytogenes) from diseased chickens [ 1 1 1,1121. Chickens have
remained a common avian host for L. monocytogenes since avian listeriosis was first recog-
nized. Listeriosis also has been observed in at least 22 other avian species, including
such frequently consumed fowl as turkeys [25,59,94], ducks [55,113], geese [55,96], and
pheasants [55].Large outbreaks of listeriosis in domestic fowl are relatively rare [93];
however, sporadic cases occur much more frequently and are often accompanied by
asymptomatic shedding of Listeria in feces. According to one report, 4.7% of cecal sam-
ples from Danish broiler chickens harbored L. monocytogenes [96]. Hence, as was true
for beef, pork, and lamb, poultry meat destined for human consumption also is at risk of
becoming contaminated with L. monocytogenes, particularly when birds are slaughtered,
defeathered, and eviscerated. Several early studies suggested that poultry- and egg-pro-
cessing workers can contract Listeria infections by handling contaminated birds

565
566 Cox et al.

[40,71,721. Additionally, several reports from England during the 1970s indicated that L.
monocytogenes could be isolated with some frequency from raw chicken as well as turkey,
duck, and pheasant. Nevertheless, consumption of poultry products has been only recently
linked to listeriosis in humans. An added concern with poultry relates to eggs that might
become contaminated with this pathogen during collecting and processing.
The emergence of L. rnonocytogenes as a bonafide foodborne pathogen following
the Mexican-style cheese outbreak of 1985 prompted immediate concern about presence
of Listeria in dairy products and also generated a parallel interest in the incidence and
behavior of L. rnonocytogenes in meat and poultry products; the latter is the topic of this
chapter. Interest in this area has increased as a result of listeriosis cases that were directly
linked to consumption of turkey frankfurters and ready-to-eat/cook-chill poultry products
in the United States and England, respectively. A discussion of the incidence and behavior
of L. monocytogenes in egg products appears toward the end of this chapter.

USDA-FSIS LISTERIA-MONITORING/VERIFICATION
PROGRAM FOR COOKED/READY-TO-EAT POULTRY
PRODUCTS
Public health concerns about Listeria-contaminated raw and, particularly, processed ready-
to-eat poultry products sold in the United States also stem directly from the 1985 listeriosis
outbreak in California associated with consumption of Mexican-style cheese. Soon thereaf-
ter U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) offi-
cials announced their intentions to develop Listeria-monitoring/verification programs for
cooked and ready-to-eat meat as well as poultry products. Since these monitoring/verifica-
tion programs for meat and poultry products developed in parallel and were both similar
in terms of sampling scheme, methodology, and action taken when L. rnonocytogenes is
found in a product, the following discussion focuses on the various products tested and
pertinent results rather than on an in-depth analysis of this program.
A Listeria-monitoring/verification program for cooked/ready-to-eat poultry was
first suggested in December 1985 and was to cover all such products prepared in federally
inspected establishments as well as those produced by certified foreign manufacturers [60].
However, actual testing of poultry sausage, that is, the first category of ready-to-eat poultry
products examined, did not begin until September 1988, 1 year after the Listeria-
monitoring/verification program was first instituted for cooked beef, roast beef, and
cooked corned beef.
Before April 1989, the USDAs Listeria policy, which gave firms the opportunity
to clean up their facility before additional verification samples were analyzed under hold-
test procedures, was consistent with the fact that listeriosis had not yet been linked to
consumption of poultry products. However, the official USDA-FSIS position regarding
the presence of L. monocytogenes in cooked and ready-to-eat poultry products changed
radically on April 14, 1989, when Centers for Disease Control and Prevention (CDC)
investigators directly linked consumption of contaminated turkey frankfurters to a case
of listerial meningitis in a breast cancer patient in Oklahoma [ 141. After isolating L. mono-
cytogenes serotype 1/2a of the same electrophoretic enzyme type from the woman and
opened, as well as unopened, retail packages of turkey frankfurters, USDA-FSIS officials
requested that the manufacturer issue an immediate Class I recall for approximately
Listeria monocytogenes in Poultry and Egg Products 567

600,000 pounds of Texas-produced turkey frankfurters that were marketed by retail and
institutional establishments in Alaska, Arizona, Arkansas, California, Florida, Georgia,
Idaho, Illinois, Indiana, Kentucky, Louisiana, Mississippi, Missouri, New Jersey, New
York, Ohio, Oklahoma, Pennsylvania, Tennessee, Texas, Utah, and Washington. As ex-
pected, this recall immediately prompted an intensified monitoring/verification program
to determine the extent of Listeria contamination in a far wider range of cookedheady-
to-eat poultry products marketed in the United States.
Despite pleas by the National Turkey Association to adopt tolerance levels for L.
rnonocytogenes in cooked and ready-to-eat poultry products [9], USDA-FSIS officials
maintained that since an acceptable level of L. monocytogenes in such products could
at that time not be determined, the only acceptable alternative was to adopt a policy of
zero tolerance for this pathogen in cookedheady-to-eat poultry and meat products [ 101.
Consequently, under the program [36] which is identical to that developed for cooked and
ready-to-eat red meat products, USDA-FSIS officials request firms to issue a Class I recall
for all lots of cooked and ready-to-eat poultry products in which L. rnonocytogenes is
detected in monitoring samples taken from intact packages of product. However, Listeria-
positive lots that are still under direct control of the manufacturer can be recalled internally,
thus avoiding adverse publicity. If the pathogen is initially detected in monitoring samples
from unpackaged products, USDA-FSIS officials do not request firms immediately to re-
call the sampled lot. Instead, government officials will analyze subsequent lots and, if
necessary, initiate further steps (i.e., hold-test programs) to prevent distribution of contam-
inated products.
Our knowledge concerning the incidence of Listeria in cookedh-eady-to-eat poultry
products marketed in the United States has come primarily from the USDA-FSIS Listeria-
-
monitoringherification programs with results indicating that 1.5-2.0% of all such prod-
ucts are contaminated with L. rnonocytogenes [7,18,31]. Poultry products in which L.
rnonocytogenes has been found include chicken patties [7], chicken thighs [7], chicken
salad [7], diced poultry [7], poultry salad [18], poultry spread [18], poultry frankfurters
[7], poultry bologna [7], and turkey sausage [7]. In all likelihood, the pathogen entered
these products during the later stages of manufacture and/or packaging. Since most manu-
factures now retain all sampled lots of product until results of Listeria testing become
known, formal Class I recalls for such products are quite limited and include the aforemen-
tioned recall of turkey frankfurters [14], two recalls of chicken salad [4,6], and one addi-
tional incident involving 13,000 pounds of chicken spread produced by a Virginia-based
firm [ 151. However, numerous Class I recalls have been issued for prepared delicatessen-
type sandwiches, with at least two of these recalls [ 11,171 involving items that also con-
tained processed chicken and/or turkey.

INCIDENCE OF LISTERIA SPP. IN RAW POULTRY

MARKETED IN THE UNITED STATES
Immediately after the 1985 listeriosis outbreak in California was announced, public con-
cerns were raised about safety of dairy products and other potentially contaminated foods,
including meat and poultry products. Interest in the safety of raw poultry soon escalated
following a nationwide telecast which informed the general public that -50% of all raw
chicken marketed in the United States was contaminated with Salmonella. Consequently,
568 Cox et al.

several surveys were initiated to determine the extent to which raw chicken and turkey
meat are contaminated with Listeria and Salmonella.

Chicken
The aforementioned concerns prompted USDA-FSIS officials to initiate a poultry back/
neck testing program for L. monocytogenes, Salmonella, and Escherichia coli serotype
0157 :H7 in January of 1989. L. monocytogenes and Salmonella were detected in 508 of
2686 (18.9%) and 792 of 2739 (28.9%) samples, respectively, with E. coli serotype 0157 :
H7 being absent from 2696 samples [ 181. Bailey et al. [20] determined the incidence of
L. monocytogenes and other Listeria spp. on the surface of broiler carcasses processed in
the southeastern United States. They also compared L. monocytogenes serotypes isolated
from raw chicken with those that are commonly associated with human cases of listeriosis.
Using an enrichment procedure together with three selective plating media, Listeria spp.
were detected in rinse samples from 34 of 90 (37.8%) chicken carcasses; however, recov-
ery of Listeria varied widely with three lots of 10 birds each being reported as negative.
More important, 21 of 90 (23.3%) carcasses contained L. monocytogenes, with 64, 18, 6
and 12% of the isolates being identified as serotypes I /2b, 1/2c, 3b, and nontypable strains,
respectively. Although only 7 of 1 15 (6.1%) L. monocytogenes isolates from listeriosis
victims in the United States were of serotype 1/2b or 1/2c, the fact that most L. monocyto-
genes strains isolated from chickens were pathogenic to mice, suggests chicken meat as
a possible vehicle in human cases of listeriosis.
Between June 1988 and May 1989, Genigeorgis et al. [45,46] conducted two large
surveys which examined the incidence of Listeria spp. on fresh and/or semifrozen, chicken
and turkey parts obtained from retail and slaughterhouse sources. According to their results
for chicken, 12.5% of fresh wings, 16.0% of fresh legs, and 15.0% of fresh livers purchased
at three supermarkets in northern California contained detectable levels of L. monocyto-
genes (Table 1). Furthermore, with the exception of fresh chicken liver, L. innocua was
generally two to three times more prevalent in the remaining samples than was L. monocy-
togenes. Overall, the highest incidence of Listeria spp. was observed for fresh legs (54.0%)
followed by fresh wings (42.5%) and fresh livers (32.5%). In contrast to fresh products,
only 10% of semifrozen chicken wings, legs, and livers contained Listeria spp. However,
finding L. monocytogenes alone in 1 of 10 semifrozen legs and livers points to the ability
of this pathogen to survive in semifrozen raw chicken and turkey, as also was observed
by Palumbo and Williams [98].
In addition to these efforts, Genigeorgis et al. [45] also attempted to trace the route
of Listeria contamination on fresh chicken wings, legs, and livers by examining samples
at various stages of production and storage. Although all chicken parts from the beginning
of the production line were free of L. monocytogenes, results in Table 2 indicate that most
contamination occurred during the latter stages of production when carcasses came in
direct contact with Listeria-laden fecal material, since at the time of packaging, 70, 30,
and 33% of chicken wings, legs, and livers contained L. monocytogenes, respectively. Not
surprisingly, L. innocua, which was virtually absent from chicken parts at the beginning
of production, also was routinely isolated from wings, livers, and particularly legs at the
end of production. Despite these relatively high contamination rates, both Listeria spp.
failed to grow on all three packaged products during the first 4 days of refrigerated storage.
Wimpfheimer et al. [ 1321 observed a 3- to 4-day lag phase for L. monocytogenes when
inoculated samples of raw minced chicken were held at 4C. Given this information, the
Listeria monocytogenes in Poultry and Egg Products 569

TABLE
1 Incidence o f Listeria spp. on Fresh and/or Semi-Frozen Chicken a n d Turkey Parts Purchased f r o m Three California
Supermarkets Between J u n e 1988 a n d M a y 1989

No. (%) of positive parts

Type and part No. of parts L. mono- Total
of poultry examined cytogenes L. innocua L. welshimeri Listeria spp.
Chicken
wings Fresh 40 5 (12.5) 14 (35.0) I (2.5) 17a (42.5)
Semifrozen 10 0 1 (1 0.0) 0 1 (10.0)
legs Fresh 50 8 (16.0) 19 (38.0) 0 27 (54.0)
Semifrozen 10 1 (10.0) 0 0 1 (10.0)
livers Fresh 40 6 (15.0) 8 (20.0) 1 (2.5) 13a (32.5)
Semifrozen 10 1 (10.0) 0 0 1 (10.0)
Turkey
wings Fresh 60 12 (20.0) 3 (5.0) 12 (20.0) 27 (45.0)
legs Fresh 60 8 (13.3) 0 9 (15.0) 17 (28.3)
tails Fresh 60 7 (11.7) 0 7 (11.7) 14 (23.2)
a Some chicken parts contained both L. monocytogenes and L. innocua or L. innocua and L. welshimeri.

Source: Adapted from Refs. 48 and 49.

570 Cox et al.

TABLE
2 Incidence of Listeria spp. on Commercially Produced Fresh Chicken and
Turkey Parts Before and After Being Packaged and/or Stored at 4C
4-day -old
packaged
Production line product
Type and part stored at
of poultry Listeria sp. beginning end 4C
Chicken
wings L. monocytogenes 0/20a 21/30 (70) 18/25 (72)
L. innocua 0/20 6/30 (20) 4/25 (16)
legs L. monocytogenes 0/20 11/30 (37) 13/25 (52)
L. innocua 0/20 19/30' (67) 17/25 (68)
livers L. monocytogenes 0/3 1 5/15 (33) 6/15 (40)
L. innocua 2/31 (6.5)b 4/15 (27) 4/15 (27)
Turkey
wings L. monocytogenes 1/30 (3.3) 0/30 NDd
L. welshimeri 1/30 (3.3) 4/30 (13.3) ND
legs L. monocytogenes 0/30 2/30 (6.7) ND
L. welshimeri 1/30 (3.3) 1/30 (3.3) ND
livers L. monocytogenes 0/30 0/30 ND
L. welshimeri 1/30 (3.3) 5/30 (16.7) ND

ND, not determined.

a Number of positive partdnumber of parts examined.

Percentage positive.
Strain of L. welshimeri also detected.
Source: Adapted from Refs. 48 and 49.

failure of Genigeorgis et al. [45] to detect growth of L. rnonocytogenes and L. innocua

on naturally contaminated packaged chicken parts is not surprising.
In more recent surveys, 91% of raw poultry samples and 8% of cooked samples
contained listeriae. Although L. rnonocytogenes was isolated from 59% of raw samples,
all cooked samples were negative [76]. Among the chicken parts examined (i.e., drum-
sticks breasts, wings, livers), drumsticks were most frequently contaminated with listeriae
and also harbored the highest populations. In a survey of seven Danish abattoirs [96], 0.3-
18.7% of processing line and final product samples contained L. rnonocytogenes. Based on
serotyping, phage typing, pulsed-field gel electrophoresis, and ribotyping, 62 distinct L.
rnonocytogenes strains were identified from the 247 isolates tested, several of which pre-
dominated in poultry-processing samples. Subsequently, Ryser et al. [ 1081 identified Liste-
ria spp. in 34 of 45 retail samples of chicken pieces, with 11 different L. rnonocytogenes
ribotypes, including three ribotypes associated with previous foodborne listeriosis cases,
being detected among the Listeria-positive samples. These findings suggest the presence
of multiple incoming sources of contamination and/or heavily contaminated single sites
within the poultry-processing environment. Hence, improved disinfection procedures may
be necessary to effectively control Listeria spp. in the processing environment.
In a study by Cox et al. 1351 to determine how often L. rnonocytogenes enters poultry
processing plants on live chickens, L. rnonocytogenes was infrequently found in hatchery
samples and on the exterior of fully grown birds. Although L. rnonocytogenes was not
Listeria monocytogenes in Poultry and Egg Products 571

recovered from the intestinal tract of broiler chickens at the time of slaughter, 25% of
postprocessing and retail-level carcasses contained L. monocytogenes. In another study
involving perorally dosed chicks, Husu et al. [65] reported that L. monocytogenes was
generally eliminated within 9 days, which again suggests that intestinal carriage of L.
monocytogenes is most often transient.
It is important to remember that like other meats, poultry products also can be used
for purposes other than human consumption. Al-Sheddy and Richter [ 11 determined the
incidence of L. monocytogenes in frozen ground meat that contained raw chicken together
with chopped beef by-products. Although not conclusive, recovery of L. monocytogenes
from all five samples examined and the fact that this pathogen is more commonly found
in chicken rather than beef products leads one to conclude that raw chicken was the most
likely source of contamination. Hence, considering the high incidence of L. monocytogenes
in raw chicken, it may be prudent to eliminate raw poultry products from the diet of zoo
animals to curb the number of listeriosis cases occurring in zoological parks.
The scientific literature relating to pathogens commonly associated with processed
poultry is extensive, and a review of this literature has been published [129]. In another
review paper [69] covering the years 1971-1994, the prevalence of L. monocytogenes in
meats appears to be highly variable, with approximately 16?6 of products being positive.
In general, the highest numbers of L. monocytogenes have been found in processed meat
and poultry products, with fresh meats generally containing much lower numbers. Al-
though serotypes 1/2a, 1/2b, and 1/2c are most frequently isolated from meats, most
human outbreaks have been traced to serotype 4b, thus suggesting that poultry products
play a relatively minor role in foodborne listeriosis.

Turkey
Since chickens and other types of domesticated fowl are similarly processed, one would
expect various Listeria spp., including L. monocytogenes, superficially to contaminate
other raw poultry products, including turkeys, ducks, and pheasants. After completing the
aforementioned survey of California chicken parts for Lisleria [45], Genigeorgis et al.
[46] initiated a similar study to determine the prevalence of various Listeria spp. on fresh
turkey parts obtained from retail sources and slaughterhouses.
Listeria contamination rates were generally similar to those previously observed for
fresh chicken, with 45.0% of fresh turkey wings, 28.3% of fresh legs, and 23.3% of fresh
tails obtained from three local northern California supermarkets harboring various Listeria
spp. (see Table 1). Although their findings further demonstrate that L. monocytogenes is
equally common on fresh chicken and turkey parts, with isolation rates of 10.0-16.0%
and 11.7-20.0%, respectively, the same cannot be said for I,. innocua and L. welshimeri.
In fact, L. innocua, the Listeria sp. most commonly detected on fresh chicken, was recov-
ered from only 3 of 180 (1.7%) fresh turkey parts. Similarly, L. welshimeri, the dominant
Listeria sp. on fresh turkey, was only rarely observed on fresh chicken. Although both
surveys were confined to fresh chicken and poultry parts available from three local super-
markets, these findings still suggest the interesting possibility that chickens and turkeys
may be preferential hosts for L. innocua and L. welshimeri, respectively. However, addi-
tional data need to be gathered from other parts of the country to prove or disprove this
theory.
In a subsequent survey [33], 9 of 42 turkey skin samples harbored L. monocytogenes
with L. monocytogenes contamination rates apparently unrelated to the incidence in the
572 Cox et al.

flock before processing or after defeathering. As was true for fresh chicken, additional
testing at a local slaughterhouse once again demonstrated that fresh turkey parts are most
likely to become contaminated with Listeria during later stages of processing (i.e., eviscer-
ation, chilling) (see Table 2). This scheme, mentioned earlier as the route by which fresh
poultry becomes contaminated, was further confirmed by identifying various Listeria spp.,
including L. monocytogenes, in 4 of 15 (26.7%) samples of mechanically deboned raw
turkey meat obtained from the same slaughterhouse. Ryser et al. [ 1081 further stressed
the importance of postprocessing contamination when they reported that 33 of 45 (73%)
retail samples of ground turkey contained Listeria spp., including a diverse group of L.
monocytogenes strains belonging to nine different ribotypes.

INCIDENCE OF LISTERIA SPP. IN POULTRY PRODUCTS

MARKETED IN WESTERN EUROPE AND ELSEWHERE
Raw Poultry
European scientists have been aware of the possible relationship between listeriosis in
fowl and humans for nearly 40 years, as evidenced by several reports in which infected
poultry was found in the immediate vicinity of human cases. Considering the fecal carriage
rate for L. monocytogenes in domestic and wild fowl as well as the mechanized slaughter-
ing practices that result in heavy fecal contamination of carcasses during defeathering,
evisceration, and subsequent chilling in spin-chillers, it is not surprising that interest in
the incidence of Listeria in poultry has escalated. However, given that the first European
case of avian listeriosis was diagnosed in England during 1936 [99], and that no additional
cases were recorded in England over the next 22 years [55], one would probably not
expect to learn that our first knowledge concerning the incidence of Listeria in European
raw poultry products originates from surveys made in England and Wales during the
1970s.
After identifying L. monocytogenes in human stool samples from 32 of 5 100 (0.6%)
asymptomatic individuals who resided in an area of Wales in which clinical cases of
listeriosis had not been identified for 15 years, Kwantes and Isaac [74] postulated that the
fecal carriage rate may be related to food consumption and began surveying both fresh
and frozen chicken for L. monocytogenes. Following a 1971 preliminary report [74],
Kwantes and lsaac [75] published final results of their study in 1975 when they reported
detecting L. monocytogenes on the internal/external surfaces of 27 of 51 (52.9%) raw
chickens obtained from a local processor (Table 3), with 23 (85.2%) and 4 (14.8%) L.
monocytogenes isolates being identified as serotype 1 and 4b, respectively. To determine
the publics actual exposure to contaminated poultry, these investigators went to homes
of poultry consumers in Wales and swabbed the externalhnternal surfaces of locally pur-
chased fresh and frozen chicken, turkey, duck, and pheasant carcasses. Overall, L. monocy-
togenes was isolated from 50% of fresh chickens sampled from home refrigerators, and
also from 64% of frozen chickens stored in home freezers, thus demonstrating the ability
of this pathogen to persist on frozen carcasses. However, unlike chickens obtained directly
from processors, L. monocytogenes isolates of serotype 4b outnumbered those of serotype
1 on fresh and, particularly, on frozen chickens obtained from consumers homes. Al-
though relatively few samples were examined, isolation of L. monocytogenes from the
internal/external surface of one turkey, three ducks, and one wild pheasant (see Table 3)
Listeria monocytogenes in Poultry and Egg Products 573

3 Incidence of L. rnonocytogenes in or o n Raw Poultry Carcasses Marketed

TABLE
in Western Europe and Elsewhere Between 1971 and 1994

No. (%) of
No. of positive
Origin Type of poultry carcasses examined carcasses Ref.
Denmark Fresh chicken 17 8 (47.1) 120
England/ Fresh chicken 51 27 (52.9) 75
Wales
Fresh chicken 38 19 (50.0) 75
Fresh chicken 50 33 (66.0) 102, 103
Fresh chicken 6 2 (33.3) 51
Fresh chicken 100 60 (60.0) 104
Fresh chicken 30 15 (50.0) 63
Fresh chicken 16 10 (62.5) 88
Fresh chicken 32 21 (65.6) 83
Frozen chicken 64 41 (64.0) 75
Frozen chicken 50 27 (54.0) 102, 103
Frozen chicken 56 7 (12.5) 51
Turkey 4 1 (25.0) 75
Fresh turkey 1 0 51
Frozen turkey 3 0 51
Duck 3 3 (100.0) 75
Frozen duck 2 1 (50.0) 51
Wild pheasant 2 1 (50.0) 75
Italy Fresh chicken --200 0 34
Fresh chicken 50 18 (36) 44
Sweden Fresh chicken 45 0 123
Switzerland Fresh chicken 24 5 (20.8) 29
West Germany Fresh/frozen chicken 100 85 (85.0) 110, 122
Unspecified poultry 30 6 (20.0) 97
Unspecified poultry 11 3 (27.3) 122

indicates that improperly handled poultry products other than chicken also may pose a
potential threat to consumers.
One year later, Gitter [51] published results from a similar study which examined
the incidence of L. monocytogenes on surfaces of various oven-ready poultry products
purchased at 26 different shops and supermarkets in southern England. Using a combina-
tion of direct plating and cold enrichment, L. monocytogenes was identified on 7 of 56
(12.5%) frozen and 2 of 6 (33.3%) fresh chickens as well as on 1 of 2 frozen ducks (see
Table 3). Although the incidence of L. monocytogenes on raw poultry products was mark-
edly lower than that previously found by Kwantes and Isaac [75], L. monocytogenes iso-
lates identified as serotype 4 again outnumbered those of serotype 1/2.
Following emergence of L. monocytogenes as a serious foodborne pathogen in June
1985, Pini and Gilbert [ 1031 determined the prevalence of this pathogen in uncooked fresh
and frozen chickens obtained from retail outlets throughout metropolitan London. Unlike
previous studies, which relied on swab samples from carcasses, these researchers exam-
ined two different samples from each chicken carcass whenever possible-one sample
574 Cox et al.

consisting of edible offal (trimmings and/or viscera) and the other a composite sample
of skin and carcass remnants. Using cold enrichment in conjunction with the Food and
Drug Administration (FDA) procedure, L. rnonocytogenes was recovered from 33 of 50
(66%) fresh and 27 of 50 (54%) frozen chickens. These results are similar to those reported
from other 1987- 1994 surveys [49,63,83,88,104] in which L. rnonocytogenes was detected
on 10 of 16 (62.5%), 15 of 30 (50%), 60 of 100 (60%) and 21 of 32 (66%) fresh chicken
carcasses marketed in England and Wales (see Table 3). According to a second report by
Pini and Gilbert [102], other Listeria spp., including L. innocua, L. seeligeri, and L.
welshirneri, also were detected either alone or together with L. rnonocytogenes in 26 and
30% of the fresh and frozen chicken samples tested, respectively. Overall, 74 L. rnonocyto-
genes strains representing serotypes 1/2, 3a, 3b, 3c, 4b, 4d, and two nontypable strains,
with serotype 1/2 predominating, were isolated from 160 samples consisting of 60 edible
offal and 100 composite samples. Composite samples yielded more isolates of L. rnonocy-
togenes (57%) than did edible offal samples (22%) and also a higher percentage of other
Listeria spp. (23%) than did edible offal samples (15%). Despite differences in methodolo-
gies and types of samples analyzed in the above-mentioned studies, averaging the results
in Table 3 indicates that 187 of 323 (58%) fresh chickens and 75 of 170 (44.1%) frozen
chickens marketed in England and Wales between 1971 and 1994 contained L. monocyto-
genes. That the 1986 findings of Pini and Gilbert [103,104] are similar to those obtained
in both American and European surveys as far back as the mid 1970s underscores the
continuing need for proper kitchen hygiene, cooking of raw poultry products, and continu-
ous inspection of carcasses (e.g., identification of liver and heart lesions), along with use
of good manufacturing and sanitizing practices in poultry-processing facilities. Since 152
of 214 (71%) clinical L. rnonocytogenes isolates obtained from British patients between
November 1986 and 1987 [87] were of serotype 4b, poultry products, in which L. rnonocy-
togenes serotype 1/2 predominates, may be a less common vehicle for listeriosis than
other foods. However, pit&, which are in essence poultry spreads prepared from chicken
or goose liver, may be an exception.
European concern about the incidence of L. rnonocytogenes in raw poultry products
consumed outside of England/Wales dates back to at least 1982 when two Swedish work-
ers, Ternstrom and Molin [ 1231, examined 45 chickens obtained from two local slaughter-
houses (see Table 3). Although these researchers failed to isolate L. rnonocytogenes from
any of the chickens examined, it appears that the Listeria isolation/detection methods used
in this study were primarily responsible for their lack of success, since listeriosis in Swed-
ish poultry is relatively common, with 112 cases being diagnosed in the 10-year period
between 1948 and 1957 [94]. In view of the high incidence of L. rnonocytogenes in raw
poultry marketed in the United States and England, inadequate isolation/detection methods
of the early 1980s also were likely responsible for the inability of Comi and Cantoni [34]
to recover this pathogen from approximately 200 chicken samples (i.e., carcass, skin,
entrails) obtained from slaughterhouses in northern Italy, with more recent Italian surveys
[44,84] showing L. monocytogenes contamination rates of 10.6 and 36% for raw poultry.
After the 1985 cheeseborne listeriosis outbreak in California, western European
scientists began to determine the incidence of Listeria in a wide range of foods, includ-
ing fresh poultry products. In the first of these studies, which was published in 1988,
Skovgaard and Morgen [ 1201 visited two large Danish poultry slaughterhouses and exam-
ined chilled chicken carcasses for evidence of Listeria contamination. According to these
authors, Listeria spp. were detected in neck-skin samples from 16 of 17 (94.1 %) chicken
carcasses, with L. innocua being identified in all but two Listeria-positive samples. Al-
Listeria monocytogenes in Poultry and Egg Products 575

though most of the poultry processed at these two facilities was heavily contaminated
with L. innocua, 8 of 17 (47.l%), 1 of 17 (5.9%), and 2 of 17 (1 1.8%) carcasses also
contained detectable levels of L. monocytogenes (see Table 3), L. innocua, and other
Listeria spp. (L. welshimeri, L. murrayi, and/or L. denitrificans), respectively. Thus the
L. monocytogenes contamination rate for chickens processed in Denmark was only slightly
lower than the average (56.7%) for fresh chicken carcasses marketed in England and
Wales. These Danish researchers also identified Listeria spp., including L. monocytogenes,
in chicken feces and transport cage material, further supporting the widespread belief that
poultry carcasses most likely become contaminated with Listeria during evisceration and
subsequent handling, as also was suggested by Genigeorgis et al. [45,46] and Cox [35]
based on results of surveys of poultry-processing facilities in California and Georgia.
Interest in the incidence of Listeria in European fresh poultry again intensified fol-
lowing reports that 34 individuals in Switzerland died after consuming contaminated
Vacherin Mont dOr soft-ripened cheese. Breer [29] isolated L. monocytogenes, L. inno-
cua, and 1,.seeligeri from 5 of 24 (20.8%), 6 of 24 (25.0%), and 1 of 24 (4.2%) raw
chickens purchased in Switzerland, (see Table 3), respectively. Using a modified version
of the FDA procedure, German researchers [ 110,121], found Listeria spp. and L. monocy-
togenes in 94 of 100 (94%) and 85 of 100 (85%) chicken carcasses, respectively. However,
results from two smaller surveys of German poultry [97,121] suggested far lower L. mono-
cytogenes contamination rates, with only 20.0-27.3% of unspecified fresh poultry car-
casses (presumably chicken) containing this pathogen (see Table 3). Similarly, Rijpens
et al. [ 1051 more recently recovered Listeria spp. and L. monocytogenes from 35.5% and
15.5%, respectively, of poultry samples examined in Belgium, with the incidence of Liste-
ria spp. markedly higher in unpackaged (41.7%) than in prepackaged (1 1.1%) poultry. L.
monocytogenes also has been detected in French poultry since 1988 [26]. During a subse-
quent large-scale survey of poultry carcasses in France and Belgium, Uyttendaele et al.
[ 1271 reported that the L. monocytogenes contamination rate decreased from 32.1% in
1992 to 9.2% in 1995, with 87%-98% of the carcasses tested yielding < I L. monocyto-
genes CFU/cm2. Contamination of poultry parts also decreased from 25.8% to 3% during
this same 4-year period, with L. monocytogenes-positive samples primarily being confined
to chicken legs and wings. In another survey similar to that conducted in the United States
by Genigeorgis et al. [45], Franco et al. [43] identified Listeria spp. on 96, 84, 80, and
0% of the fresh chicken legs, wings, breasts, and livers, respectively, that were processed
at a Spanish facility, with Listeria counts ranging from <2.0 to 2.8 log CFU/g on chicken
wings. Follow-up testing of the processing environment again showed that most of the
contamination occurred during the later stages of production as carcasses entered the quar-
tering room and exited on conveyor belts. In a Danish survey [96], L. monocytogenes was
not observed in cecal samples from over 2000 broilers representing 90 randomly selected
broiler flocks. However, L. monocytogenes was isolated from 0.3 to 18.7% of all poultry-
processing environmental samples examined. Using several strain-specific typing meth-
ods, L. monocytogenes contamination was shown to be primarily localized in the pro-
cessing facility, with the incidence reducible through improved hygiene.
Elsewhere, 10 of 30 domestically grown fresh chickens from the United Arab Emir-
ates [54] contained Listeria spp., with 1 of these carcasses being positive for L. monocyto-
genes. However, Listeria spp. were detected on 32 of 39 (82%) imported frozen chickens,
18 (46%) of which contained L. monocytogenes. Similarly, Rusul et al. [47] identified L.
monocytogenes in 4 of 16 (25%) poultry samples collected from six local Malaysian mar-
kets, with one of these markets subsequently yielding L. monocytogenes in 15 of 24
576 Cox et al.

(62.5%) samples. In a South African study [38] of Listeria in poultry-processing facilities,

L. innocua was recovered from the rubber fingers of a defeathering machine and on all
neck skin samples after evisceration. L. monocytogenes strains were detected on the rubber
fingers, packaging funnel, and all neck skin samples after chilling.

Cooked/Ready-to-Eat Poultry
Increasing evidence indicates that contamination of ready-to-eat foods, including cooked
chicken, is most likely to originate from the processing environment. With this premise
in mind, Lawrence and Gilmour [76] examined the incidence of Listeria spp., including
L. monocytogenes, in one poultry-processing facility, along with raw and cooked chicken
processed at this same facility in Northern Ireland between March and August 1992.
Within the raw and cooked poultry-processing environments, 36 of 79 (46%) and 51 of
173 (29%) samples harbored Listeria spp., whereas 21 of 79 (26%) and 27 of 173 (15%)
yielded L. monocytogenes, respectively. Contamination rates were fairly uniform, with
several environmental sites yielding L. monocytogenes throughout the study. Among raw
and cooked products tested, 53 of 58 (91%) and 8 of 96 (8%) contained Listeria spp.,
whereas 34 of 58 (59%) and none of 96 cooked samples yielded L. monocytogenes, respec-
tively. Although L. monocytogenes was absent from all cooked products tested, the pres-
ence of other Listeria spp. and a subsequent report by the same authors [77] attesting to
isolation of identical L. monocytogenes strains from raw poultry, cooked poultry, and the
processing environment (some environmental strains of which persisted for up to 1 year)
confirms the importance of minimizing postprocessing contamination.
An association between human listeriosis and consumption of cooked/cooked-
chilledheady-to-eat poultry products which are cooked, rapidly chilled, and frequently
held refrigerated for at least 5 days before being consumed without further heating has
been observed in both the United Kingdom and the United States. According to one
survey conducted by the Public Health Laboratory Service in London [49,50,104], L.
monocytogenes was present in 63 of 527 (1 2.0%) precooked ready-to-eat poultry products
collected from London-area retail establishments between mid November 1988 and mid
January 1989. Little information is available concerning actual numbers of L. monocyto-
genes present in cooked poultry products; however, 14 samples that were examined quanti-
tatively contained < 100 CFU/g. In addition L. monocytogenes was isolated from 13 of
74 (18%) retail chilled meals, most of which were poultry products given to hospital
patients. The pathogen was also discovered in 6 of 24 (25%) cook-chilled poultry products
[13], 7 cook-chilled poultry dishes at levels up to 700 L. monocytogenes CFU/g, and 2
cooked chicken products labeled ready-to-eat which contained up to 400 L. monocyto-
genes CFU/g.
Working at the Cardiff Public Health Laboratory Service in Wales, Morris and
Ribeiro [90] determined the potential Listeria-related risks associated with consumption
of pit&,an appetizer-type poultry spread that is typically prepared from chicken or goose
liver. According to their report, 14 varieties of pit& (primarily imported from Belgium)
were obtained in bulk from area delicatessen counters or in unopened packages from
supermarket refrigerators and examined for L. monocytogenes using established methods.
Overall, this pathogen was isolated from 37 of 73 (50.4%) pitis, with 28 (75.7%) and 9
(24.3%) of these positive samples originating from delicatessens and supermarket refriger-
ators, respectively. These pit& were subsequently withdrawn from the market after offi-
cials discovered dangerously high L. monocytogenes populations of l 0 4 - r 1O5 CFU/g in
Listeria monocytogenes in Poultry and Egg Products 577

seven samples [8]. L. monocytogenes strains of serotype 4b, the serotype responsible for
-80% of all human listeriosis cases in England and Wales, were isolated from 36 of 37
positive samples, with one strain of L. monocytogenes serotype 4b matching clinical iso-
lates from a 1987 cluster of listeriosis cases in which the exact origin of illness could
never be determined.
These findings prompted Public Health Laboratory officials greatly to expand their
survey of pihi. By March 1990 [48,104], workers at 48 of 53 (90.6%) participating labora-
tories in England and Wales isolated L. monocytogenes from 187 of 1834 ( 10.2%) samples
of imported and domestic pit& As in the previous survey, -10% of all positive samples
contained 104-> 106L. monocytogenes CFU/g, with over half of all isolates belonging to
serotype 4. Following this pitk-related outbreak discussed in Chapter 8, the number of
listeriosis cases reported in England and Wales decreased to approximately half the level
reported in 1988. Although some individuals expected to see a further decline [48,104],
the incidence of human listeriosis in the United Kingdom has since stabilized, with about
100-125 cases, being reported annually over the last 5 years.
In one other European survey of cookedheady-to-eat poultry products, Lieval et al.
[80] isolated L. monocytogenes, L. seeligeri, and L. innocua from one of nine, two of
nine, and one of nine chicken sandwiches obtained from cafes in and around Paris. Al-
though identical efforts to recover Listeria from 20 fast-food fried chicken items ended
in failure, the ability of such foods to harbor Listeria, including L. monocytogenes, has
been well established by the previously discussed surveys in England.

BEHAVIOR OF L. MONOCYTOGNS IN RAW AND

COOKED POULTRY PRODUCTS
Although L. monocytogenes was first detected on European raw chicken nearly 20 years
ago, behavior of Listeria in raw and processed poultry products received no attention
until this organism was recognized as a bonafide foodborne pathogen in the mid 1980s.
Subsequent research efforts have provided the poultry industry with valuable information
concerning growth of L. monocytogenes in raw and cooked chicken products, including
the levels of heat and microwave/gamma irradiation needed to destroy this pathogen in
raw chicken. However, our present-day knowledge of Listeria behavior in raw and pro-
cessed chicken products is still incomplete. Although results from these efforts will now
be summarized, several listeriosis cases that were positively linked to consumption of
processed poultry products in the United States and England have prompted additional
work in thiq area. Results from these studies should add much to our knowledge about
behavior of Listeria in poultry products and aid in development of processing methods
that will decrease the incidence of this pathogen in raw and cooked-chilled poultry
products.

Growth-Raw Chicken
Wimpfheimer et al. [ 1321 examined the behavior of L. monocytogenes in raw chicken. In
their study, raw minced chicken meat was inoculated to contain 10, L. monocytogenes
CFU/g, packaged anaerobically (75% CO2:25% N2), microaerobically (72.5% CO,:
22.5% N 2 : S % O,), or aerobically (air) and examined for numbers of L. monocytogenes
as well as aerobic spoilage organisms during storage at 4, 10, or 27C. Neither L. monocy-
togenes nor aerobic spoilage organisms grew in anaerobically packaged raw chicken dur-
578 Cox et al.

ing extended storage at any of the three temperatures, with both populations decreasing
to <10 CFU/g after 6 days of storage at 4C (Fig. 1). When packaged microaerobically
under conditions more closely simulating commercial practices, numbers of L. monocyto-
genes in raw chicken increased rapidly during extended storage at 4"C, whereas growth
of aerobic spoilage organisms was strongly inhibited (see Fig. 1). Under these conditions,
L. monocytogenes can rapidly proliferate in normal unspoiled raw chicken during refriger-
ated storage. Zeitoun and Debevere [133] later reported on the successful use of a 10%
lactic acid/sodium acetate buffer (pH 3) in conjunction with modified atmosphere packag-
ing (90% CO2, 10% 0,) to prevent growth of L. monocytogenes on uncooked chicken
legs and extend the product's shelf life at 6C to 17 days. According to Wimpheimer et
al. [132], the ability of L. monocytogenes to grow in microaerobically packaged raw
chicken was not affected by initial levels of Listeria (<10' or 102CFU/g) or aerobic
spoilage organisms ( l O4 or 1O8 CFU/g). However, the ratio of Listeria to spoilage organ-
isms was strongly temperature dependent, with both organisms reaching populations of
107-10Rand 109-10'" CFU/g in microaerobically packaged chicken following <2 and 8
days of storage at 10 and 27"C, respectively. Neither L. monocytogenes nor aerobic spoil-
age organisms were inhibited in aerobically packaged raw chicken, with Listeria and spoil-
age organisms attaining populations > l O7 and 1O9 CFU/g, respectively, in products stored
at 4, 10, and 27C. Thus with exception of the microaerobically packaged product, raw
chicken would likely become overtly spoiled before L. monocytogenes could proliferate

L. monocytogenes 0
c.v

Aerobic Spoilage
98 1 Organisms /

67l P'
/

0 2 4 6 8 10 12 14 16 18 20 22

Days

FIGURE1 Growth of L. rnonocytogenes and aerobic spoilage organisms in aerobi-

cally ( O), microaerobically (01, anaerobically ( 0 )packaged raw chicken during
and
incubation at 4C.(Adapted from Ref. 132)
Listeria monocytogenes in Poultry and Egg Products 579

to the point where the pathogen might be detectable in minimally cooked chicken. Never-
theless, it is important to remember that consumers must take special precautions to pre-
vent cross contamination between raw chicken that may contain L. monocytogenes and/
or Salmonella spp. and ready-to-eat products including cooked chicken.

Growth-Cooked / Ready-to-Eat Poultry Products

Confirmation of cooked-chilled chicken and turkey frankfurters as vehicles of Listeria
infection in England and the United States during 1988 and 1989 prompted immediate
international efforts to assess the potential hazards associated with growth of L. monocyto-
genes in a wide range of retail cookedheady-to-eat poultry products, six of which have
been briefly summarized in Table 4. Since all of these studies differ in experimental design,
sampling times and initial inoculum levels of L. monocytogenes, these findings cannot be
compared directly. However, it is evident that numbers of Listeria increased 1-6 orders
of magnitude in all six artificially contaminated products after 6-28 days of storage at
3-7"C, with higher populations being observed in aerobically packaged as opposed to
vacuum-packaged or modified-atmosphere-packaged products. Equally important, sen-
sory acceptability of these products was not altered by growth of this pathogen. Overall,
L. monocytogenes grew most abundantly in vacuum-packaged sliced chicken and in one
brand of sliced turkey, both of which were similar in pH (6.3, 6.4) and in contents of
moisture (7 1.3, 74.0%) protein (18.9, 22.6%) carbohydrate (1.3, 0.9%) and salt (1.7,
1.4%). These authors attributed decreased growth of the pathogen in a second brand of
sliced turkey to higher levels of salt (2.7%) and carbohydrate (1.7%); the latter was largely
responsible for the eventual decrease in pH of this product to 4.97.
In another study by Ingham et al. [68], cooked/sterilized chicken loaf was inoculated
to contain 1 O3 L. rnonocytogenes and l O3 Pseudomonasfragi CFU/g, packaged aerobically
(air), microaerobically (10% O,), or anaerobically and examined for both organisms during
6 days of incubation at 37 and 11C. Regardless of incubation temperature, P. fragi at-
tained maximum populations of approximately 4 X 109 CFU/g in all aerobic samples
after 6 days of storage (Fig. 2). However, growth of Listeriu was totally or partially sup-
pressed in similar samples stored at 3C for up to 15 days [67]. Although both organisms
attained lower maximum populations in cooked chicken loaf following 6 days of microaer-
obic or anaerobic incubation at all three temperatures, these conditions led to Listeria
populations that were 1-2 orders of magnitude higher than those attained by P. fragi.
Thus, as was true for raw poultry (see Fig. l), microaerobic and anaerobic refrigerated
storage both appear to selectively favor growth of L. rnonocytogenes over P. fragi and
possibly other spoilage organisms which, in turn, could potentially yield an organolepti-
cally acceptable product with dangerously high numbers of Listeria.
Identification of turkey frankfurters as the infectious vehicle in a 1988 case of listeri-
osis involving an Oklahoma breast cancer patient eventually led to a safety assessment
of poultry sausage. According to McKellar et al. [86], L. monocytogenes grew on the
surface of I3 of 27 (48%) artificially contaminated retail poultry weiners, with populations
on some vacuum-packaged samples increasing nearly 4 orders of magnitude during 21
days of storage at 5C. Wederquist et al. [131] reported sirnilar growth of L. monocyto-
genes when vacuum-packaged slices of turkey bologna were stored at 4C. However,
incorporating 0.5% sodium acetate, 2.0% sodium lactate, or 0.26% potassium sorbate into
the product completely suppressed growth of the pathogen during the first 35 days of
refrigerated storage, with populations in 3-month-old samples remaining 2-5 orders of
580 Cox et al.

TABLE
4 Populations of L. monocytogenes (log,, CFU/g) in Artificially Contaminated Cooked/Ready-to-Eat Poultry Products of
Acceptable Organoleptic Quality During Extended Refrigerated Storage

Length of incubation (days)

Incubation Initial
Product temp. ("C) inoculum 3 6 8 10 14 20 28 Ref.
Sliced chickena 4.4 2.79 -b 6.94
4.4 0' 5.90
Sliced turkey 4.4 3.04 5.04
(brand A)a 4.4 - 1.30 2.38
Sliced turkey 4.4 2.87 6.70
(brand B)a 4.4 - 1.70 4.79
Chicken homogenate 4 6.7
4 2.7 -
Breaded chicken fillets 5 2.7 3.6
5 1.7 - 3.6 - -
Chicken casserole 3 2.7 2.9 3.5 3.3 3.6
6 2.7 3.6 5.3 6.3 7.5 -
Chicken nuggets 3 4.8 4.8 5.2 5.7 6.1 6.3
7 4.8 5.3 6.0 6.1 7.4 -
Chicken nuggetsd 3 4.7 4.8 4.8 5.0 5.2 6.0
7 4.7 4.9 5.1 5.3 5.3
Vacuum packaged.
Not tested.
1 CFU/g.
Modified atmosphere--80% CO,: 20% Nz
Listeria monocytogenes in Poultry and Egg Products 581

10
I 1
9

6
m
3
U-
5
* I
r 4
cs,
0
J
.-a 3
5
*-.
0
a 2
m
1

I 1 I
1 4 6
Days

FIGURE2 Growth of L. monocytogenes and Pseudomonas fragi in aerobically (O),

microaerobically (01,and anaerobically ( 0 )packaged cooked chicken loaf during in-
cubation at 7C. (Adapted from Ref. 68.)

magnitude lower than those in the controls. Two additional studies on fermented summer
sausage prepared from ground chicken [ 191 and turkey [8 I] also demonstrated the impor-
tance of an active starter culture in limiting growth of L. rnonocytogenes during fermenta-
tion. When these sausages were prepared from a chicken or turkey batter (pH 6.6) con-
taining a pediococcal starter culture and L. rnonocytogenes at a level of 1O4--1O7CFU/g,
numbers of Listeria decreased 0.9- 1.8 orders of magnitude after an I 1-h fermentation
(pH 5). Replacing this pediococcal starter culture with a pediocin-producing strain of
Pediococcus acidilactici essentially doubled the inactivation rate of L. rnonocytogenes
during fermentation. However, regardless of the starter culture used, all remaining listeriae
were subsequently inactivated during 45 min of cooking to an internal temperature of
66.5"C. Thus, an active fermentation combined with normal thermal processing before
packaging should result in a Listeria-free product.
Finally, several investigations also have assessed behavior of Listeria in inoculated
samples of chicken gravy and chicken broth during cooling and/or refrigerated storage.
According to Huang et al. [62], L. monocytogenes populations in individual 1000-g sam-
ples of artificially contaminated chicken gravy (prepared from poultry stock, spices, waxy
maize wash, and chicken base) increased by 2 orders of magnitude as the product cooled
from 40 to 9C during 24 h of storage in a refrigerator at 7C (Fig. 3). Even though
generation times for L. rnonocytogenes approximately doubled after the chicken gravy
stabilized at 7"C, the pathogen still attained a maximum population of 109CFU/g in 8-
day-old gravy. Huang et al. [61] subsequently reported a reduction in L. rnonocytogenes
582 Cox et al.

+ L. monocvtoaenes
0- - Temperature

0 1
-- 2
- -0- -- -0- - -e- -a-- - 0 - - -0
3 4
I

5
1

6
I

7 8
I

Days

FIGURE3 Behavior of L. monocytogenes in chicken gravy during cooling to 7C and

extended storage. (Adapted from Ref. 62.)

when similar chicken gravy was held at 65C for 1.3 min; however, such a heat treatment
is clearly inadequate to inactivate higher Listeria populations that can develop in chicken
gravy during prolonged refrigerated storage. Walker et al. [ 1301 also demonstrated the
ability of three L. monocytogenes strains to multiply in artificially contaminated sterile
chicken broth (pH E 6.4) held at heat-freezing temperatures, with Listeria populations in
this product increasing 100-fold during extended incubation at 03C (Fig. 4). In fact,
growth of this pathogen was also evident in samples of chicken broth that were held at
temperatures as low as -0.4"C, below which the broth froze and was no longer sampled.
Results from these investigations stress the importance of cooling foods as rapidly
as possible and show that hazardous situations can easily develop if refrigerated foods
are subjected to mild temperature abuse, that is, holding at temperatures above 4C. In
an effort to increase the safety of cooked, cooked-chilled, and ready-to-eat poultry, USDA
officials lowered the recommended long-term storage temperature for such products from
4.4 (40F) to 1.7"C (35F) and also have developed stricter guidelines that require faster
(than previously recommended) cooling of warm products at the end of manufacture [3].
Continued attention to rapid cooling of finished products and avoidance of postprocessing
contamination are both essential to decrease the incidence of this psychrotrophic pathogen
in cookedh-eady-to-eat poultry products.
Listeria monocytogenes in Poultry and Egg Products 583

I
I I I I 1
0 10 20 30 40 50
Days

FIGURE4 Growth of L. monocytogenes in sterile chicken broth during extended incu-

bation at 8.7 (01,3.5 (m), 1.5 (01
and
, 0.8C (0).(Adapted from Ref. 130.)

Thermal Inactivation
Heating is the most obvious means of destroying L. monocytogenes and other foodborne
pathogens i n any raw food, including poultry. However, numerous reports attesting to
unusual thermal tolerance of L. monocytogenes in various foods, coupled with discovery
of L. monocytogenes in several cooked poultry products that were directly linked to cases
of listeriosis, have raised questions about the exact thermal processing times and tempera-
tures required to eliminate this pathogen completely from raw poultry products. In re-
sponse to these concerns, Carpenter and Harrison conducted three studies in which raw
-
chicken breasts were surface inoculated to contain 1OS- l O7 L. monocytogenes CFU/g
and cooked to internal temperatures of 65.6, 71.1, 73.9, 76.7, or 82.2C using dry heat
[32], moist heat [57], and microwave radiation [58].All cooked chicken breasts were then
vacuum-packaged or wrapped in oxygen-permeable film and analyzed for numbers of
Listeria during 4 weeks of storage at 4 and 10C.
Overall, L. monocytogenes was recovered from chicken breasts cooked to all five
internal temperatures, using dry heat, moist heat, and microwave radiation. As expected,
the magnitude of lethality was directly related to cooking temperature. Since chicken
breasts contained somewhat different levels of L. monocytogenes before heating, one can-
not directly compare the effectiveness of the three cooking methods used in these studies.
However, assuming that L. monocytogenes populations in these chicken breasts decreased
linearly during heating (admittedly, some tailing of the survivor curve likely occurred
at the three highest temperatures using dry heat, moist heat, and microwave irradiation),
then the number of survivors in chicken breasts that contained any initial inoculum can
be estimated. Thus, if Carpenter and Harrison had used an initial population of 1 .O X 106
L. monocytogenes CFU/g in all three studies, one would expect their results to have been
similar to the estimated number of survivors shown in Table 5. Considering these approxi-
mations, it appears that L. monocytogenes was generally more tolerant of microwave radia-
tion than dry or moist heat, with numbers of Listeria decreasing less than 4 orders of
magnitude on chicken cooked to an internal temperature of 822C. Of greater importance
is the fact that Listeria populations decreased 1 2 orders of magnitude on chicken breasts
cooked to an internal temperature of 7 1.1 C, the minimum internal temperature to which
poultry must be heated to designate the product as fully cooked in the United States [2].
Although numbers of Listeria decreased approximately 5 orders of magnitude when
584 Cox et al.

TABLE
5 Estimated Decrease in Numbers of L. rnonocytogenes o n the Surface of
Chicken Breasts Inoculated to Contain 6.00 log,, CFU/g and Cooked t o Various
Internal Temperatures Using Dry Heat, Moist Heat, or Microwave Radiation
NO.^ of L. monocytogenes (log,, CFU/g) decrease after cooking to
internal temp. of
~ ~

Cooking method 65.6"C 71.1"C 73.9"C 76.7"C 82.2"C

Dry heat 2.42 2.00 5.05 5.24 5.04
Moist heat 2.08 1.83 3.46 5.08 4.96
Microwave radiation 0.82 1.95 2.50 3.77 3.26
aInitial inoculum of 6 log,, L. rnonocyrogenes CFU/mL.
Source: Adapted from Refs. 32, 57, and 58.

chicken was cooked to higher internal temperatures using either dry or moist heat, these
authors [56] later demonstrated that moist heating of surface-inoculated chicken breasts
to an internal temperature of 73.9"C also failed to completely inactivate more realistic L.
monocytogenes populations of 102-104CFU/g. Overall, microwave heating was less effec-
tive than either dry or moist heating, with Listeriu populations decreasing less than 4
orders of magnitude on chicken breasts cooked to an internal temperature of 82.2"C (see
Table 5). In 1989, researchers in England [82] also reported that microwave heating was
less effective than other forms of cooking for eliminating L. monocytogenes from the
surface and interior (stuffing) of whole stuffed chickens (- 1.7 kg each) inoculated to
contain 106and 107 Listeria CFU/g of skin and stuffing, respectively. Uneven heating,
which is an inherent problem in microwave cooking, accounted for the 20 min of additional
standing time that was required after 38 min of cooking (final skin temperature of 80-
99C) to completely inactivate the pathogen on the surface of whole chickens. However,
low levels of L. monocytogenes (<10 CFU/g) were still detected in stuffed samples from
one of two similarly treated whole chickens that attained temperatures of 72-85C after
20 min of standing. Thus, these findings serve as a warning to people who regularly cook
large stuffed birds (particularly turkeys) in microwave ovens, and they also stress the
importance of postcooking standing time for further inactivation of Listeriu and other
foodborne pathogens in poultry products after microwave cooking.
Not surprisingly, follow-up work by Carpenter and Harrison [32,57,58] demon-
strated that L. monocytogenes survivors (likely sublethally injured during heating) can
persist and multiply on both oxygen-permeable film-wrapped and vacuum-packaged
cooked chicken during extended storage at 4 and 10C. As shown in Figure 5, growth of
Listeriu on chicken breasts packaged in oxygen-permeable film was most evident after 2
weeks of refrigerated storage with larger populations generally developing on products
that were cooked using microwave radiation, followed by those given moist or dry heat.
Most important, L. monocytogenes was recovered via direct plating from all 4-week-old
aerobically packaged chicken breasts except those that were cooked to an internal tempera-
ture of 82.2"C using moist heat. In addition, higher numbers of Listeriu also developed
on aerobically packaged chicken breasts that were exposed to less severe heat treatments.
As expected, increasing the incubation temperature also led to much faster growth of
Listeria, with the pathogen generally attaining populations of 106-107CFU/g on aerobi-
cally packaged chicken breasts after only 7 days at 10C.
Behavior of L. monocytogenes on chicken breasts was influenced by the heating
Listeria monocytogenes in Poultry and Egg Products 585

P
3
U.
0

I I 1 I
0 1 2 3 4

Weeks

FIGURE5 Effect of three different heating methods on behavior of L. rnonocytogenes

on inoculated chicken breasts that were cooked to internal temperatures of 71.1 (O),
76.7 (A),or 82.2"C (U), packaged in oxygen-permeable film, and stored at 4C.
(Adapted from Refs. 32, 57, and 58.)

methodkreatment, temperature at which the cooked product was ultimately stored, and
type of packaging material. According to these authors, Listeria populations were gener-
ally 1 to 2 orders of magnitude lower in vacuum-packaged than in aerobically wrapped
product following 4 weeks of storage at 4C; however, the pathogen was present in all
4-week-old samples except those that were originally cooked to an internal temperature
of 82.2"C using moist or dry heat (Fig. 6). Although numbers of Listeria were again
generally 1-2 orders of magnitude lower in vacuum-packaged than in aerobically wrapped
chicken breasts following 1 week of storage at 10C, populations were as much as 5 orders
of magnitude lower in vacuum-packaged chicken breasts that were cooked to an internal
temperature of 82.2"C using moist heat.
Since raw chicken normally contains <loo0 L. monocytogenes CFU/g [88] and
Listeria populations generally decrease approximately 3-5 orders of magnitude in fully
cooked poultry heated to an internal temperature of 63.9"C, as specified in the U.S. Code
of Federal Regulations, it appears that present cooking temperatures are, at best, only
marginally adequate to eliminate this pathogen from raw poultry products. In fact, Harrison
 Cox et al.

I 1 I I
1 2 3 4

Weeks

FIGURE6 Effect o f three different heating methods o n behavior o f L. monocytogenes

o n inoculated chicken breasts cooked t o internal temperatures of 71.1 (O), 76.7 (A),
or 82.2"C (W), vacuum-packaged, and stored at 4C.(Adapted f r o m Refs. 32, 57, and
58.)

and Carpenter [56] reported that moist heating of inoculated chicken breasts to an internal
temperature of 73.9"C failed to completely inactivate L. monocytogenes surface popula-
tions of 102-104CFU/g, with the pathogen reestablishing itself at levels equal to or greater
than the original inoculum level after 4 weeks at 4C. Nonetheless, the adequacy of current
poultry-processing methods was maintained by another report [5] which indicated that a
turkey meat emulsion (containing salt, sodium tripolyphosphate, carrageenan, and water)
inoculated to contain 5.78 L. monocytogenes log,,, CFU/g was free of the pathogen after
holding the product at 7 1.1 "C for 2 min (estimated D71,10C= 0.28 min). However, in view
of at least three human listeriosis cases linked to cooked-chilled chicken meat and turkey
frankfurters (the latter was reportedly warmed 45-60 s in a microwave oven before con-
sumption), and that small numbers of L. monocytogenes survived a wide range of heat
treatments given to chicken breasts and frequently grew in these products during refrig-
eration, it is prudent for poultry processors to cook their products to somewhat higher
internal temperatures (76.7-82.2"C) than is now routinely practiced until the adequacy
of the current minimum heat treatment can be firmly established. Future studies should
address the effect of processed poultry ingredients (i.e., salt, preservatives) on thermal
resistance of Listeria during conventional as well as microwave heating, since Harrison
and Carpenter [58] found that the latter cooking method was less successful in eliminating
L. monocytogenes from the surface of chicken breasts than either dry or moist heating.
Listeria monocytogenes in Poultry and Egg Products 587

Chemical Treatments
Various food additives have been evaluated for their ability to inactivate Listeria spp. on
fresh poultry. Dipping inoculated chicken wings in 10% trisodium phosphate (TSP) at
10C for 15 s and hot water (95C) for 5 s resulted in a 79.49% reduction in numbers of
L. monocytogenes, with minimal changes in subcutaneous temperature [ 1061. Hwang and
Beuchat [66] also found that washing chicken skin in 1% TSP or 1% lactic acid signifi-
cantly reduced viable populations of L. monocytogenes compared with washing in water.
According to Shelef and Yang [ 1 161, growth of L. rnonocytogenes in sterile comminuted
chicken was slowed during refrigerated storage by adding 4% sodium or potassium lactate.
When used at levels 10.3%, sodium diacetate also was inhibitory to L. rnonocytogenes
in poultry slurries [ 1091, with the antilisterial activity of sodium diacetate being further
enhanced by supplementing the product with 0.25% ALTA, a commercially available
microbially produced shelf life extender.

Susceptibility to Gamma Radiation

Survey results indicate that up to -60% of all raw poultry products sold in retail stores
may be contaminated with L. rnonocytogenes and Salmonella spp. These statistics have
prompted development of various processes to eliminate such pathogens from raw poultry.
Exposure to the bactericidal effects of gamma radiation appears to be among the most
effective means of reducing populations of both pathogenic and spoilage organisms. Al-
though presently allowable gamma radiation doses of 2.5-7.0 kGy in England [92] and
<3.0 kGy in the United States [16] are generally regarded as sufficient for eliminating
these organisms from raw poultry [ 1241, readers should be aware that exposing cooled
poultry to levels >2.5 kGy may adversely affect product odor, color, and flavor [88].
In 1989, Patterson [ 1011 examined sensitivity of Listeria to gamma radiation using
-
radiation-sterilized raw minced chicken meat that was inoculated to contain 1O6 L. rnono-
cytogenes CFU/g. After exposing the product to gamma radiation doses of 0, 0.5, 1.0,
1.5,2.0, and 2.5 kGy, L. monocytogenes exhibited a DIo-value(i.e., radiation dose required
to decrease the population 10-fold) of 0.417-0.553 kGy, depending on the bacterial strain
and the type of plating medium used to quantitate the pathogen in minced poultry. In
support of these findings, Huhtanen et al. [64] also reported that a gamma radiation dose
-
of 2 kGy was sufficient to inactivate an L. monocytogenes population of 104 CFU/g
(average D-value of 0.45 kGy) in artificially contaminated, mechanically deboned chicken.
Similar D-values also have been published for Salmonella spp. in fresh poultry [91].
Although all of the aforementioned studies support the use of 2.5 kGy of gamma
radiation to eliminate < 1O4 L. monocytogenes CFU/g from raw poultry meat, researchers
in England [ 881 recovered this pathogen from 1 of I2 (8.3%) fresh chicken carcasses that
had been surface inoculated to contain approximately 102or 1O4 L. rnonocytogenes CFU/
cm2 and exposed to 2.5 kGy of gamma radiation. More important, after extended storage
at 5-10C, the pathogen was recovered from 1 of 18 (5.6%) and 7 of 18 (38.8%) irradiated
carcasses that originally contained low and high inoculum levels, respectively. When poul-
try carcasses were inoculated to contain > 1O4 L. rnonocytogenes CFU/g, several investiga-
tors [79,128] also confirmed that small numbers of the pathogen survived irradiation at
2.5 kGy and eventually grew, particularly in the absence of air, in samples stored at 4C.
However, Shamsuzzaman et al. [ 1141 reported that combined use of 3.1 kGy irradiation
and sous-vide cooking to an internal temperature of 71.1"C was sufficient to reduce L.
588 Cox et al.

monocytogenes populations in chicken breast meat from 1O6 CFU/g to undetectable levels,
with no growth of the pathogen being observed during 5 weeks of storage at 8C. Hence,
in the absence of multiple treatments, these findings suggest that small numbers of Listeria
may either escape sublethal injury during irradiation or undergo repair and grow on these
carcasses during refrigerated storage.
From this information, it appears that a gamma radiation dose of 2.5 kGy may be
only marginally sufficient to inactivate levels of Listeria that one might reasonably expect
to find on naturally contaminated raw poultry. Nevertheless, provided that irradiated poul-
try products are properly packaged to prevent recontamination (and subsequent growth)
with Listeria and other foodborne pathogens, this procedure should markedly decrease the
risk of contaminating ready-to-eat foods (e.g., salads, raw vegetables) when raw poultry is
prepared by the consumer. Unfortunately, although the scientific community generally
contends that foods exposed to such low levels of radiation are safe for human consump-
tion, irradiated foods have not yet gained full acceptance by consumers. Perhaps the con-
tinued outpouring of scientific evidence will eventually curb the remaining unfounded
fear of irradiated foods in the mind of the general public.

EGG PRODUCTS
Listeria monocytogenes is most frequently isolated from heart, liver, and spleen tissue of
poultry suffering from listeriosis; however, according to the early scientific literature, this
pathogen also has been detected in necrotic oviduct lesions of several infected hens [55]
and in follicles of one artificially infected chicken [72]. These observations prompted a
large-scale survey in 1958 [70] in which 600 intact hens eggs were examined and found
to be negative for L. monocytogenes. In keeping with these findings, consumption of eggs
and egg products also has not yet been linked to a single case of listeriosis. However, the
possible presence of L. monocytogenes on egg shells which may contain minute cracks
along with the ability of this pathogen to survive 90 and > 14 days on eggs stored at 5 [22]
and 10C [24], respectively, persist on inoculated eggs treated with sodium hypochlorite
containing 100 ppm available chlorine [24], and grow in artificially contaminated eggs
stored at refrigeration as well as ambient temperatures [22] suggests that eggs cannot be
ignored as a possible source of listerial infection. Hence, it is not surprising that recent
concerns about contaminated poultry products also have prompted efforts to define both
the incidence and behavior of L. monocytogenes in eggs and egg products, including pas-
teurized liquid and dried egg.

Incidence
As mentioned in the preceding paragraph, isolation of L. monocytogenes from intact whole
eggs has not yet been documented; however the same is not true for broken eggs. Ac-
cording to Leasor and Foegeding [78], Listeria spp. were isolated from 15 of 42 (36%)
previously frozen samples of raw commercially broken solids-adjusted liquid whole egg
(21 samples), natural-proportion liquid whole egg (20 samples), and yolk (1 sample) ob-
tained on several occasions from 6 of 11 (54%) commercial manufacturers located
throughout the United States. On closer examination of the data, L. innocua and L. monocy-
togenes were identified in 15 of 15 (100%) and 2 of 15 (13.3%) positive samples, respec-
tively. Twelve of 15 (80%) and 3 of 15 (20%) positive samples, including one sample
each with L. monocytogenes, were classified as solids-adjusted and natural-proportion
Listeria monocytogenes in Poultry and Egg Products 589

liquid whole egg, respectively. Increased handling during manufacture and, as suggested
by the authors, a higher solids content which may enhance growth and survival of Listeria
are just two of many possible reasons why a higher incidence of Listeria was observed
in solids-adjusted rather than natural-proportion liquid whole egg. Although results from
direct plating indicated that the two L. monocytogenes-positive samples contained approxi-
mately 1 and 8 L. monocytogenes CFU/g at the time of analysis, the fact that these samples
were held frozen for up to 4.5 months and subjected to two freeze-thaw cycles suggests
that numbers of Listeria likely decreased by at least 50% during storage (see Chap. 6).
Hence, both samples probably contained < 100 L. monocytogenes CFU/g before being
frozen.
Working in the United Kingdom, Moore and Madden [23] recovered Listeria from
125 of 173 (72%) in-line filters used to remove shell fragments from raw blended whole
egg; 78 and 47 samples of which contained L. innocua and L. monocytogenes, respectively,
generally at levels <400 CFU/mL. However, since 500 pasteurized egg samples yielded
negative results for listeriae, pasteurization at 64.4"C for 2.5 min, as required in the United
Kingdom, appears to afford a high degree of safety. During a subsequent survey of
three Australian egg factories in New South Wales, Desmarchelier et al. [37] identified
L. innocua in 9 of 13 (69%) samples of unpasteurized liquid whole egg, with no other
Listeria spp. being observed. Floors, drains, and mobile equipment in raw processing areas
of these factories were later confirmed as major sources of contamination through strain-
specific typing of environmental Listeria isolates. Although 7 samples of raw sugared
yolk and 26 peeled boiled eggs were Listeria free, L. innocua was recovered from 1 of
51 samples of pasteurized liquid whole egg, with this organism also being detected in 2
of 14 peeled boiled eggs following 3 weeks of modified-atmosphere storage at 4C. Hence,
under certain conditions, Listeria can multiply to detectable levels in egg products during
extended cold storage.

Growth
The ability of Listeria to grow in hen's eggs was first recognized in 1940 when Paterson
[ 1001 inoculated a laboratory culture of L. monocytogenes into the chorioallantoic mem-
brane of a chicken embryo. This procedure was traditionally used to determine virulence
of L. rnonocytogenes isolates [ 1211. Information concerning growth of this pathogen in
nonfertile eggs and egg products is limited. A search of the scientific literature has uncov-
ered only two studies pertaining to growth of L. monocytogenes in raw whole eggs or egg
components. According to results from the first such paper published in 1955, Urbach
and Schabinski [ 1251 found that populations of L. monocytogenes in intact experimentally
infected nonfertile eggs increased nearly 6 orders of magnitude during 10 days of storage
at ambient temperature. Following this study, 20 years passed until viability of L. monocy-
togenes was again examined in artificially contaminated raw, as well as cooked (1 2 1"C/
15 min) whole egg, albumen, and egg yolk during extended storage at 5 and 20C [73].
Results for raw whole and separated egg showed that growth of L. monocytogenes was
primarily confined to egg yolk (Fig. 7), with the pathogen exhibiting respective generation
times of 1.7 days and 2.4 h at 5 and 20C. Overall, Listeria populations in raw whole
egg generally varied less than 1 order of magnitude from the original inoculum during
extended storage at either temperature; however, numbers of Listeria in raw albumen (pH
8.9) decreased 3 and 5 orders of magnitude during prolonged incubation at 5 and 2OoC,
respectively. Despite the reported ability of L. monocytogenes to grow in laboratory media
590 Cox et al.

0 10 20 30
Days

FIGURE 7 Behavior of L. rnonocytogenes in raw and cooked whole egg (A),albumen

(HI, and egg yolk ( 0 )during extended incubation at 5C. (Adapted from Ref. 73.)

having pH values as high as 10 [ 1131, loss of Listeria viability in raw albumen was pH
related, with numbers of Listeria decreasing less than 2 orders of magnitude in samples
that were preadjusted to pH 7 and held at 5C. Unlike raw whole egg, albumen, and egg
yolk, Listeria grew rapidly in corresponding cooked samples. Generation times for the
pathogen in cooked whole egg, egg yolk, and albumen were 1.9,2.3, and 2.4 days, respec-
tively, at 5"C, and 2.6, 2.6, and 3.5 h at 20C. These authors speculated that loss of the
aforementioned antilisterial properties of raw albumen resulted from inactivation of one
or more binding proteins (i.e., ovotransferrin, ovoflavoprotein, avidin) during heating.
Since L. monocytogenes can grow rapidly in cooked whole as well as separated eggs,
investigators of foodborne outbreaks should not overlook these products as potential vehi-
cles of infection.
In 1990, Sionkowski and Shelef [ I171 provided the first information concerning
growth of L. monocytogenes in pasteurized egg products. To simulate postpasteurization
contamination, pasteurized (64.4OU2.5 min) samples of liquid egg and reconstituted dried
-
egg were inoculated to contain 104- I O5 L. monocytogenes CFU/mL and examined for
numbers of Listeria during 7 days of storage at 4C. As shown in Fig. 8, the pathogen grew
-
similarly in both products, reaching populations 1O7 CFU/mL after 7 days of refrigerated
storage. Although transmission of L. monocytogenes by pasteurized egg products has not
yet been documented, these findings suggest that a possible public health problem could
develop if L. monocytogenes enters pasteurized liquid egg, particularly since the shelf life
of some of these refrigerated products now has been extended to several months.
Growth of L. monocytogenes in commercially processed, liquid whole egg was first
assessed by Foegeding and Leasor [41]. In this study, commercially broken, liquid whole
egg was ultrapasteurized (68"C/ I 18 s), homogenized, inoculated to contain one of five
L. monocytogenes strains (Scott A [clinical isolate], F5069 [milk isolate], ATCC 191I 1
[poultry isolate], NCF-U2K3, and NCF-FI KKr [raw liquid whole egg isolate]) at a level
of 5 X 102to 1 X 103 CPU/mL, overlaid with mineral oil to prevent oxygen transfer,
and examined for numbers of Listeria during extended incubation at 4, 10, 20, and/or
30C.
Generation times and maximum populations were generally similar to those previ-
Listeria monocytogenes in Poultry and Egg Products 591

107

106

Tl
A Iiquidegg
0 Reconstituted dried egg

1 0 5 1 1

I I I I I I I

0 1 2 3 4 5 6 7
Days

FIGURE8 Growth of L. monocytogenes in liquid and reconstituted dried egg incu-

bated at 4C. (Adapted from Ref. 117.)

ously observed in fluid milk products (see Chap. 11) with the exception that strain Scott
A failed to grow in liquid whole egg during prolonged incubation at 4 and 10C (Table
6). Although growth of strain Scott A in fluid milk, cheese, and cabbage juice during
refrigeration is well documented, Buchanan et al. [30] recently reported that this strain
failed to grow in meat and poultry products incubated at 4C. As shown in Table 6,
generation times for the five L. monocytogenes strains ranged from 24.0 to 51.0, 8.0 to
31.0, 7.5 to 26.0 and 4.3 to 15.0 h at 4, 10, 20, and 30"C, respectively. Maximum popula-
tions ranged from 5.0 to 7.0, 5.48 to 8.48, 6.85 to 8.0, and 7.0 to 8.0 L. monocytogenes
log,, CFU/g in liquid whole eggs incubated at 4, 10,20, and 3OoC,respectively. However,
Sheldon and Schuman [ 1151 reported that when stored at 4"C, L. monocytogenes popula-
tions in an inoculated commercially available reduced-cholesterol liquid whole egg prod-
uct could be reduced as much as 3.9 orders of magnitude by adjusting the pH of the
product to 6.6 with citric acid and adding nisin at a level of 1000 IU/mL. Considering
current distribution and marketing practices, it is likely that perishable products such as
liquid whole egg will occasionally encounter periods of temperature abuse. Hence, from
these data, it follows that even brief exposure to temperatures 210C can lead to a dra-
matic increase in both growth rate (i.e., decreased generation time) and maximum popula-
592 Cox et al.

TABLE 6 Generation Times and Maximum Populations of L. rnonocytogenes in

Ultrapasteurized Liquid Whole Egg Incubated at 4, 10, 20, and 30C
Incubation temperature ("C)
Strain of
L. rnonocytogenes 4 10 20 30
Generation time (h)
F5069 24 12 7.5 4.3
Scott A NGa NG 26 7.1
ATCC 19111 51 31 22 15
NCF-U2K3 26 7.8 NDb ND
NCF-F1KK4 25 8.0 ND ND
Maximum population (log 1o CFU/g)
F5069 6.70 7.OO 8.00 8.00
Scott A NG ND 7.00 7.85
ATCC 19111 5.00 5.48 6.85 7.00
NCF-U2K3 7.00 8.48 ND ND
NCF-F1KK4 6.48 8.30 ND ND
ND, not determined.
a No growth.

Source: Adapted from Ref. 41.

tions of L. monocytogenes attained in ultrapasteurized liquid whole egg. Since most L.

monocytogenes strains can proliferate in ultrapasteurized liquid egg products at refrigera-
tion temperatures, postpasteurization contamination should be avoided and the product
should be stored at temperatures close to, or preferably below, 0C.
The behavior of L. monocytogenes has been assessed in several additional egg and
egg-related products. Brackett and Beuchat [27] showed that L. monocytogenes can sur-
vive throughout the normal shelf life of powdered and frozen egg products. The survival
characteristics of L. monocytogenes on shell eggs and after cooking raw whole and scram-
bled eggs by frying also were determined by Brackett and Beuchat [28]. On the surface
of egg shells, L. monocytogenes popultions decreased from 104to <10 CFU/egg after 6
days of storage at 5 and 20C. Frying scrambled eggs reduced L. monocytogenes popula-
tions >3 orders of magnitude, whereas frying eggs sunny side up did not significantly
reduce levels of L. monocytogenes. In commercial mayonnaise products, Erickson and
Jenkins [39] showed that L. monocytogenes inactivation rates were directly correlated with
aqueous phase acetic acid concentration as follows: sandwich spread 2 real mayonnaise
> cholesterol-free reduced calorie mayonnaise dressing > reduced calorie mayonnaise
dressing. The higher antilisterial activity in the cholesterol-free formulation was attributed
to egg white lysozyme. Additionally, Glass and Doyle [53] reported that L. monocytogenes
populations in two types of commercially produced low-calorie mayonnaise containing
0.7% acetic acid in the aqueous phase decreased from an initial inoculum of -106 CFU/
g to nondetectable levels following 10-14 days of ambient storage. These studies docu-
ment that commercial mayonnaise products represent a negligible consumer safety risk.
In the only other egg-related growth study thus far reported, Notermans et al. [95] exam-
ined the viability of several foodborne pathogens, including L. monocytogenes, in an egg-
nog-like product prepared from raw whole egg and sugar (25%, w/v) with/without ethanol
(7%, v/v). When samples of ethanol-free product were inoculated to contain 104-105 -
L. monocytogenes CFU/g, numbers of Listeria generally decreased 10-fold during the first
Listeria monocytogenes in Poultry and Egg Products 593

2 days of incubation at 4C and then slowly increased to levels near or slightly above
the original inoculum level after 5 additional days of refrigerated storage. Although L.
monocytogenes generally exhibited similar behavior patterns in nonalcoholic samples in-
cubated at 22OC, initial population decreases were far more abrupt, with the pathogen then
increasing to populations 1 to 3 orders of magnitude lower than the original inoculum after
7 days of incubation. Unlike alcohol-free samples, L. monocytogenes was slowly inactivated
in product containing 7% ethanol, with populations typically 1-2 and more than 4 orders of
magnitude lower in 7-day-old samples held at 4 and 22"C, respectively, than were present
initially. Hence, given the normal 2-week refrigerated shelf life of similar commercially
available nonalcoholic eggnog-like products, recontamination of these beverages during
packaging could lead to potential public health problems involving Listeria and other foodb-
orne pathogens, with Salmonella enteritidis and S. typhimurium reportedly also remaining
viable in artificially contaminated samples during 63 days of refrigerated storage.

Thermal Inactivation
Interest in possible heat resistance of L. monocytogenes in eggs is of recent origin; how-
ever, concerns by European scientists regarding potential transmission of Listeria through
eggs prompted a 1955 study by Urbach and Schabinski [ 1251, which examined the ability
of this pathogen to survive in artificially infected eggs that were fried. According to these
authors, L. monocytogenes was isolated from fried eggs (congealed white, soft yolk) pre-
pared from inoculated raw eggs in which the pathogen had previously grown to levels
>5 X 105CFU/g.
Whereas the aforementioned work appears to be fairly crude by current standards
and is now primarily only of historical interest, Foegeding and Leasor [41] conducted a
more sophisticated study in which D-values were determined for five strains of L. monocy-
togenes (Scott A [clinical isolate], F5069 [milk isolate], ATCC 19111 [poultry isolate],
NCF-U2K3 and NCF-FlKK4 [raw liquid whole egg isolates]) in sterile raw egg. Inocu-
lated samples of raw liquid whole egg were added to glass capillary tubes which were
heat-sealed and immersed in a water or oil bath at 51.0, 55.5, 60.0, and 66.0C. After
various times, tubes were removed and contents examined for survivors. Numbers of
Listeria decreased linearly in raw egg during all four heat treatments, with D-values for
the five L. monocytogenes strains ranging from 14.3 to 22.6, 5.3 to 8.2, 1.3 to 1.7, and
0.06 to 0.20 min at 51.0,55.5,60.0, and 66.OoC,respectively. Strain Scott A was generally
less heat resistant than were the other four strains, particularly at the two lower tempera-
tures; however, strain F5069 and the two isolates from raw egg exhibited moderate thermal
tolerance at all four temperatures. Muriana et al. [92] subsequently reported similar
D-values at 60C for L. monocytogenes strain Scott A when inoculated samples of liquid
whole egg were tested using either capillary tubes (D-value of 1.8 min) or a flow injection
system (D-value of 1.95 min). Although this pathogen appears to exhibit a similar degree
of heat resistance in both raw whole milk (see Chap. 6) and raw liquid whole egg, survival
of L. monocytogenes is enhanced by supplementing liquid whole egg or egg yolk with
-
10% NaCl [89]. At 64"C, L. monocytogenes exhibited D-values of 10 and 10.5 min in
-
salted liquid whole egg and egg yolk, respectively, as compared with 1 .O and 1.8 min
for unsalted samples, with increased thermal tolerance attributed to a decrease in water
activity. In contrast, adding 10% sucrose to unsalted samples neither increased thermal
resistance of listeriae nor altered the product's water activity.
In more practical terms, USDA officials currently require that liquid whole egg be
594 Cox et al.

pasteurized at a minimum of 60C for 3.5 min to effect a 9-order of magnitude (9-D)
kill of Salmonella spp. [ 1261. Although results from the study just discussed indicate that
minimum pasteurization of liquid whole egg would yield only a 2.1- to 2.7-D kill of
L. monocytogenes, one must remember that current estimates place L. monocytogenes
populations in liquid whole egg at < 100 CFU/g [4 11. Hence, as is true for milk pasteuriza-
tion, current minimum pasteurization requirements for raw liquid whole egg appear ade-
quate to inactivate normal levels of Listeria that might be present in the product. However,
it is important to stress that current minimum pasteurization requirements for liquid whole
egg, as specified in the USDA Egg Pasteurization Manual, indicate that the margin of
safety is approximately 6 orders of magnitude lower for L. monocytogenes than for most
Salmonella spp. Furthermore, such pasteurization treatments appear to be inadequate for
salted liquid whole egg and egg yolk.
In 1987, Ball et al. [2 11 documented that ultrapasteurization (i.e., pasteurization
at>60"C for <3.5 min) in combination with aseptic processing and packaging can be
used to produce liquid whole egg with a refrigerated shelf life of 3-6 months. After results
from this study were published, FDA officials issued a temporary permit allowing a North
Carolina firm to market ultrapasteurized liquid whole egg [ 12,1071. Although two of the
four objectives of the process were to render the product free of Salmonella and L. monocy-
togenes, the exact time/temperature requirements to completely inactivate L. monocyto-
genes in liquid whole egg were not specified in the FDA temporary permit.
Based on extrapolations from the aforementioned survivor curves which showed no
evidence of tailing, Foegeding and Leasor [4 11 predicted that the ultrapasteurization pro-
cesses used by Ball et al. [21] would effect a 1- to 34-D (average of 14-D) kill of L.
monocytogenes in liquid whole egg. Assuming that the ultrapasteurization times and tem-
peratures used in conjunction with the temporary FDA permit are those values that were
previously determined by Ball et al. [21], Foegeding and Leasor [41] went on to speculate
that four of the 10 thermal treatments used by Ball and coworkers may not conform
to the definition of ultrapasteurization in the temporary permit, depending on how one
views the necessity for a 9-D reduction in numbers of Listeria. However, it appears that
Listeria-free ultrapasteurized liquid whole egg having a refrigerated shelf life of 1 to sev-
eral months can be produced, provided that the raw product is processed using one of the
six more severe timehemperatwe treatments proposed by Ball et al. [21] and then is asep-
tically packaged to eliminate postpasteurization contamination.
Foegeding and Stanley [42] verified the previous predictions concerning heat resis-
tance of Listeria by determining the thermal death time (F-value) for L. monocytogenes
strain F5069 in liquid whole egg. Using their previously described submerged capillary
tube method [39], they found L. monocytogenes was eliminated from inoculated samples
(1.0 X 10' to 4.0 X 108CFU/mL) of sterile liquid whole egg after processing at 62, 64,
66, 69, and 72C for 16.0, 8.0, 4.5, 1.6, and 0.6 min, respectively.
Although results from this study confirm that minimum pasteurization (60C/3.5
min) will not result in a Listeria-free product if initial populations are large, the need for
a 9-D kill as currently required by the USDA may not be appropriate for L. monocytogenes,
since present estimates place the population of this pathogen at < 100 CFU/g in raw liquid
whole egg. Hence, based on maximum expected L. monocytogenes levels in raw liquid
whole egg, pasteurization by current standards should render such products free of
Listeria. The situation regarding ultrapasteurization appears to be somewhat different since
the thermal death-time data obtained by Foegeding and Stanley [42] indicate that 4 of
the 10 ultrapasteurization processes proposed by Ball et al. [21] (63.7OU26.2 s, 63.8*C/
Listeria monocytogenes in Poultry and Egg Products 595

92.0 s, 67.7OU9.2 s, and 71.5OC12.7 s) would likely fail to produce a 9-D decrease in
numbers of Listeria in raw liquid whole egg. Nonetheless, the >9-D kill effected by
the remaining six ultrapasteurization processes proposed by Ball et al. [2 I ] indicates that
ultrapasteurization processes can be designed to produce Listeria-free liquid whole egg
with an anticipated refrigerated shelf life of 3-6 months.

REFERENCES
1. Al-Sheddy, and E.R. Richter., 1989. Microbiological quality/safety of zoo food. Annual
Meeting of the Institute of Food Technologists, Chicago, June 26-29, Abstr. 476.
2. Anonymous. 1988. Code of Federal Regulations, Title 9, Section 381.150.
3. Anonymous. 1988. FSIS recommends 35F for long-term storage of meat, poultry, Food
Chem. News 30( 12):25-28.
4. Anonymous. 1989. Chicken salad recalled in New England due to Listeria. Food Chem.
News 3 1(42):65-66.
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 15
Incidence and Behavior of Listeria
monocytogenes in Fish and
Seafood*

KARENC. JINNEMAN, M. WEKELL

AND MARLEEN
Seafood Products Research Center, U.S. Food and Drug
Administration, Bothell, Washington

MELW. EKLUND**
U.S.National Marine Fisheries Service, Northwest Fisheries
Science Center, Seattle, Washington

INTRODUCTION
Listeria monocytogenes is ubiquitous in nature. Many aquatic creatures, including fin fish,
oysters, shrimp, crabs, lobsters, squid and scallops, are harvested from natural environ-
ments; therefore, fish and seafood have been targeted as potential sources of Listeria in
the human diet. Many of these products also undergo various processing procedures, some
of which can inactivate Listeria present on the raw product. Listeria also can enter the

* The views expressed here are those of the authors and are not necessarily endorsed by the U.S. Food and
Drug Administration, National Marine Fisheries, or the Government of the United States.
** Retired.

60 1
602 Jinneman et al.

product both during and after processing by poor sanitation conditions or manufacturing
practices. The psychrotrophic nature of L. monocytogenes allows survival or even multipli-
cation of this potential pathogen during refrigerated storage or temperature abuse situa-
tions. This is a special concern for those products which receive minimal or no heat treat-
ment before consumption. Since the first time L. monocytogenes was isolated from
imported cooked crabmeat in 1987, at least 112 Class I recalls (i.e., a situation where
reasonable probability exists that the use of or exposure to a violative product will cause
serious adverse health consequences or death) have been issued by the U S . Food and
Drug Administration (FDA) for more than 250,000 pounds of ready-to-eat domestidim-
ported fish and seafood, with this pathogen routinely being found in 8.7% of all such
products marketed in the United States. The first of several cases of listeriosis positively
linked to consumption of fish or seafood was not reported until 1989 when a 54-year-old
woman in Italy contracted listerial meningitis 4 days after consuming steamed fish from
which L. monocytogenes was later isolated [36]. This case and the potential hazard associ-
ated with consumption of other Listeria-contaminated ready-to-eat food such as cooked
crabmeat, cooked shrimp, and smoked salmon has prompted studies to determine the inci-
dence and control of Listeria in various seafoods. The incidence and behavior of L. mono-
cytogenes in fish and seafood have been addressed in several review papers [ 13,26,
32,391.
In this chapter, data reviewed are from a series of FDA surveys from I987 to 1996.
These were designed to determine the incidence of L. monocytogenes in domestic and
imported shrimp, crab, and various other fish and seafood products. As in previous chap-
ters, Class I recalls that have been issued for Listeria-contaminated fish and seafoods also
will be mentioned. Surveys of fish and seafood products for Listeria conducted by many
other international groups will be reviewed. The behavior of L. monncytogenes in these
foods, data concerning growth and thermal resistance of L. monocytogenes in seafoods,
as well as measures used such as the application of lactic acid for controlling growth of
Listeria in seafood also will be covered.

FDA SURVEYS OF L. MONOCYTOGENES IN DOMESTIC

AND IMPORTED SEAFOOD
Immediately after the June 1985 outbreak of cheeseborne listeriosis in California, FDA
officials focused their attention on urgent problems that confronted the dairy industry.
Despite a lack of evidence linking consumption of meat and poultry products to cases of
human listeriosis before 1988, as early as December 1985 U.S. Department of Agricul-
ture-Food Safety Inspection Service (USDA-FSIS) officials began taking an active inter-
est in determining the incidence of L. monocytogenes in meat and poultry products.
Increased concern about the potential hazard of Listeria-contaminated seafood to
public health began in the spring of 1987 after a private testing laboratory in the United
States isolated L. monocytogenes from frozen cooked crabmeat obtained from a Mexican
supplier [4]. L. monocytogenes was confirmed in this product by the FDA in Baltimore,
Maryland, in May of 1987. In maintaining FDAs zero-tolerance policy for L. monocyto-
genes in ready-to-eat foods, the first in a series of Class I recalls was issued for nearly 4
tons of tainted crabmeat that was marketed in four states. These events also prompted an
import alert on June 17, 1987 [ 5 ] ,which called for automatic detention and testing for
Listeria monocytogenes in Fish and Seafood 603

Listeria and Escherichia coli in all frozen crabmeat shipped to the United States from
Mexico.
Less than 1 month after this product was recalled, the FDA in Seattle, Washington,
detected L. monocytogenes in samples of imported frozen raw shrimp [3] and lobster tails
[15]. Although no recalls were issued for these products, which are almost invariably
cooked before consumption, confirmation of Listeria in these seafoods together with the
finding in cooked crabmeat noted previously prompted the FDA to initiate two surveys
in July of 1987.
In the first of these surveys, six imported samples of frozen raw shrimp were col-
lected monthly and examined for Listeria at each district office. The samples represented
as many different countries as possible (Table 1) [3]. Additionally, each district also was
requested to collect three domestic samples of frozen raw shrimp per month at the whole-
sale or retail level. Using the original FDA method [69], Listeria spp. were detected in
18 of 74 (24.3%) samples of frozen raw shrimp imported from 10 different countries
between July and October of 1987 (see Table 1). L. monucytugenes also was isolated from
4 of 74 imported samples of frozen raw shrimp, with all positive samples originating from

TABLE1 Results from an FDA Survey of Imported Frozen Raw

Shrimp, July-October, 1987.
No. of positive samples
(%)
No. of Other
samples L. mono- Listeria
Country of origin analyzed cytogenes SPP.
Brazil 4 1 (25)
Ecuador 8 1 (12.5)
Guyana 1 1 (100)
Honduras 5 l a (20)
Hong Kong 1 0
India 4 0
Indonesia 1 0
Macau 1 0
Mexico 10 0
Nigeria 3 0
Norway 1 0
Pakistan 4 0
Panama 7 0
Peoples Republic of China 4 0
Peru 1 0
Philippines 3 0
Taiwan 9 0
Thailand 4 0
Venezuela 3 0
Total 74 4 (5.4)
a One sample contained L. monocyotgenes and other Listeria spp.

Source: Adapted from Ref. 40.

604 Jinneman et al.

Central or South American countries. Subsequently, three lots of raw shrimp, imported
from Ecuador and Honduras, were found to contain 103-105L. monocytogenes or L. inno-
cua CFU/g [76]. However, since shrimp are normally not consumed raw in the United
States, FDA officials did not request the recall of any of these contaminated lots.
In the second FDA survey, domestic and imported samples of cooked, frozen, and
refrigerated crabmeat (i.e., picked or extracted) were examined for the presence of L.
monocytogenes, Staphylococcus aureus, Vibrio cholera, V. parahaemolyticus, V. vulniJ-
cus, and Yersinia enterocolitica and numbers of E. coli [3]. Again, samples of imported
crabmeat from as many different countries as possible were collected. As of January 1988,
6 of 98 (6.1 %) domestic samples of cooked crabmeat contained Listeria, with L. monocyto-
genes and L. innocua being recovered from 4 and 2 samples, respectively (Table 2). Simi-
larly, Listeria spp. were detected in 3 of 24 (12.5%) imported samples of cooked crabmeat,
with L. monocytogenes being discovered in 2 of 24 (8.3%) samples of product marketed
in the United States.
Weagant et al. [106] formally published the first results of a survey dealing with
the incidence of Listeria spp. in imported/domestic frozen seafood products analyzed at
the FDA District Laboratory in Seattle during the second half of 1987; 31 of 50 (62%)
imported and 4 of 7 (57%) domestic samples of frozen seafood tested positive for Listeria
spp. using the FDA method [69]. The only Listeria spp. detected were L. monocytogenes
(15 of 57,26.3%) and L innocua (26 of 57,45.6%); both L. monocytogenes and L. innocua
were isolated from several of the samples. Although the number of samples examined
from the various product categories was limited, results suggested that frozen seafood
more frequently contains L. innocua than L. monocytogenes. Hence, as was true for raw
milk, meat, and poultry products, both organisms also likely occupy similar niches in
seafood-processing environments. Therefore, the presence of L. innocua in raw and partic-
ularly in cooked seafood should not be ignored but rather should be viewed as an indicator
of possible contamination with L. monocytogenes.

TABLE
2 Results from an FDA Survey of Domestic/lmported
Refrigerated or Frozen Cooked Crabmeat, July 1987 to January
1988
No. of positive samples (%)
No. of Other
samples L. mono- Listeria
Country of origin analyzed cytogenes SPP.
~~

United States 98 4 (4.1) 2 (2.0)

Canada 3 0 1 (33.3)
Chile 2 0 0
Korea 11 2 (18.2) 2a (18.2)
Japan 2 0 0
Mexico 3 0 0
Venezuela 3 0 0
Total (imported) 24 2 (8.3) 3 (12.5)
~ ~~~~~~~

aOne sample contained L. monocytogenes and L. innocua.

Source: Adapted from Ref. 15.
Listeria monocytogenes in Fish and Seafood 605

Discovery of Listeria in raw shrimp, crabmeat, and other seafood products, coupled
with an increased concern about the general safety of seafood, prompted FDA officials
in October 1987 to include analysis for L. monocytogenes in a compliance program for
domestic/imported shrimp [7] and to increase testing of many other domestically produced
seafoods for Listeria spp. under the program for pathogen monitoring of select high-risk
foods (CPGM 7303.030) [ 11,15,40]. This increased sampling effort sought to determine
the geographical distribution of Listeria in domestic/imported seafood and to identify the
incidence of Listeria spp. in such products. In March 1988, a processed seafood assignment
was issued [42]. The purpose of the processed seafood compliance program (CPGM
7303.036) was to test for several bacterial pathogens, including Listeria, in imported and
domestic processed seafood that receives minimal to no processing by the consumer. Prod-
ucts selected for Listeria analyses under the processed seafood compliance program in-
cluded crabmeat (cooked or pasteurized), crayfish/crawfish, lobster, langostinos (cooked,
parboiled), molluscan shellfish, processed imitation seafood (surimi), seafood salads,
shrimp (cooked), smoked or salted fish, and other processed seafood. In addition, the
National Advisory Committee on Microbiological Criteria for Foods (NACMCF) in April
1988 began the laborious task of developing microbiological criteria for cooked shrimp
and crabmeat [8]. During the FDA surveys from October 1988 through September 1990,
L. monocytogenes was recovered from domestic samples of crabmeat, lobster, shrimp,
smoked salmon, and surimi. Imported fish, lobster, shellfish, shrimp, smoked fish, squid,
and surimi also tested positive for L. monocytogenes during the same time period [42].
Three FDA compliance programs in effect since 1991 have surveyed the incidence
of Listeria in fish and seafood products and reported analytical data into the FDA Microbi-
ological Information System. The Domestic Fish and Fisheries Products Compliance Pro-
gram (CPGM 7303.842) covers investigations and sampling of domestic fish and fishery
products [45]. Imported seafood and seafood products are surveyed for presence of Listeria
under the Import Seafood Products Compliance Program (CPGM 7303.844) [44]. The
Processed Seafood Compliance Program (CPGM 7303.036) was in existence through 1994
and covered both domestic and imported processed seafood and seafood products [42,43].
Since 1994 the items covered by this program have been incorporated into the CPGM
7303.842 and CPGM 7303.844 programs for domestic and imported products, respec-
tively. Ready-to-eat food products that require no further or minimal processing by the
consumer or products collected as a follow-up to suspected cases of foodborne illness are
identified for Listeria analyses in each of these compliance programs. The domestic fish
and fisheries product compliance program also includes analyses for Listeria of in-line
and swab samples collected during processor establishment investigation reviews.
Among these three compliance programs a total of 7158 samples of fish, seafood
products or seafood processing in-line samples were analyzed by the FDA between 1991
and 1996. Listeria monocytogenes was detected in 622 of these samples for an overall
incidence of 8.7%. The breakdown of samples analyzed and those in which L. monocyto-
genes was detected is given by year and origin (domestic or import) in Table 3. There is
no significant difference based on a heteroscedastic T-test ( P = .OS) between the incidence
of L. monocytogenes in imported compared with domestic products. The 8.7% incidence
found by the FDA is comparable to the 4-12% incidence of L. monocytogenes in seafood
and seafood products from temperate areas as reported by Embarek [32]. Surveys of other
food products have indicated a 4-60% incidence in raw meat, 23-60% in fresh poultry,
and 2.2% in raw milk [32,39,64].
606 Jinneman et al.

3 Seafood Product and Seafood Processor In-line Samples Analyzed for

TABLE
Listeria monocytogenes by the FDA from 1991 through 1996
~

Samples 1991 1992 1993 1994 1995 1996 Overall

Domestic product (CPGM) (1,3) ( 193) (1,3) (193) (1) (1)
positive 20 63 89 94 67 41 374
negative 403 50 1 558 623 48 I 382 2948
total 423 564 647 717 548 423 3322
% positive 4.7% 1 1.2% 13.8% 13.1% 12.2% 9.7% 11.3%
Import product (CPGM) (3) (273) (293) (273) (2) (2)
positive 32 51 65 42 41 17 248
negative 362 643 80 1 737 589 456 3588
total 394 694 866 779 630 473 3836
% positive 8.1% 7.3% 7.5% 5.4% 6.5% 3.6% 6.5%
Overall
positive 52 114 154 136 108 58 622
negative 765 1144 1359 1360 1070 838 6536
total 8 17 1258 1513 1496 1178 896 7158
% positive 6.4% 9.1% 10.2% 9.1% 9.2% 6.5% 8.7%
Source: Data compiled from the FDA Microbiological Information System. Samples collected from the following
Compliance Programs (CPGM) [42-451: 1 . CPGM 7303.842 Domestic Fish and Fisheries Products (1991-
1996); 2. CPGM 7303.844 Import Seafood Products (1992-1996); 3. CPGM 7303.036 Processed Seafood
(1991- 1994).

Samples found positive for L. monocytogenes in the FDA compliance programs

represent a wide range of fish and seafood products (Table 4). Among the crustacean
products, (crab, shrimp/prawns, lobster, and crawfish), 2 18 samples were positive, with
crab accounting for 142 positive samples. Fifteen samples were positive for L. monocyto-
genes from the shellfish category which includes mussels, oysters, clams, scallops, and
snails. The fin fish category had 231 samples positive for L. monocytogenes, with 164 of
these from a smoked seafood product. The remaining positive L. monocytogenes samples
represent a diverse group of products, including squid/calamari, 3 samples; eel, 9 sam-
ples; roe/caviar, 19 samples; imitation seafood, 17 samples; seafood salad/spread/p$tb or
mousse, 13 samples; or processor in-line or swab samples, 94 samples.
Overall, the two fish or seafood products identified in the FDA compliance programs
which account for the highest incidence of L. monocytogenes are crab and smoked fin
fish. Together these two product categories represent nearly half (306 of 622 positive
samples) of all the fish or seafood product samples in which L. monocytogenes was de-
tected between 1991 and 1996 by the FDA. A closer look at the FDA Microbiological
Information System data for these two products appears in Table 5. For crab products,
1886 samples were analyzed for Listeria. Of these, 142 (7.5%) were positive for L. mono-
cytogenes. This is within the 0-29.2% L. monocytogenes incidence rate reported in several
other surveys [20,30,37,84,94,106]. It is presumed that the presence of L. monocytogenes
in ready-to-eat crab is the result of postprocess contamination of the product.
In the FDA studies, a total of 1210 smoked fin fish products were analyzed for
Listeria, with 164 (13.6%) samples being positive for L. monocytogenes. Between 1991
and 1995 for those smoked seafood samples in which the smoking process was known,
the incidence of L. monocytogenes was higher in cold smoked 21.3% (5 1 of 240) compared
 Listeria monocytogenes in Fish and Seafood 607

TABLE4 Fish and Seafood Products from which L. monocytogenes Was Isolated
by FDA from 1991 to 1996.
Product/Sample 1991 1992 1993 1994 1995 1996 Total
Crustacean
crab 12 34 37 28 25 6 142
shrimplprawns 7 1 7 11 2 4 32
lobster 8 9 9 7 4 1 38
crawfish 1 0 2 1 2 0 6
Shelljish
mussels 0 1 0 1 0 2 4
oysters 0 1 0 0 0 0 1
clams 1 0 3 1 3 0 8
scallops 0 0 0 0 1 0 1
snails 0 0 1 0 0 0 1
Fin Fish
smoked 16 32 38 33 23 22 164
other 2 12 10 25 11 7 67
Other seafood products
squid/calamari 1 0 1 0 1 0 3
eel 0 5 4 0 0 0 9
roelcaviar 1 1 9 2 5 1 19
imitation seafood 0 2 7 2 2 4 17
seafood (salad, spread, 0 4 3 4 1 1 13
pit& mousse)
processor in-line or swabs 3 12 22 19 28 10 94
not specified 0 0 0 1 0 0 1

Source: Data compiled from the FDA Microbiological Information System. Samples collected from the following
Compliance Programs (CPGM) [42-451: CPGM 7303.842 Domestic Fish and Fisheries Products (1 99 I - 1996);
CPGM 7303.843 Import Seafood Products (1992- 1996); CPGM 7303.036 Processed Seafood (1991-1994).

TABLE
5 Crab and Smoked Fin Fish Samples Analyzed for L. monocytogenes by
FDA from 1991 Through 1996
1991 1992 1993 1994 1995 1996 Total
Crab
positive 12 34 37 28 25 6 142
negative 248 324 363 320 272 217 1744
total 260 358 400 348 297 223 1886
% positive 4.6% 9.5% 9.3% 8.0% 8.4% 2.7% 7.5%
Smoked fin fish
positive 16 32 38 33 23 22 164
negative 117 175 193 23 1 154 176 1046
total 133 207 233 264 177 198 1210
% positive 12.0% 15.5% 16.3% 12.5% 13.0% 11.1% 13.6%
~ ~~

Source: Data compiled from the FDA Microbiological Information System. Samples collected from the following
Compliance Programs (CPGM) [42-451: CPGM 7303.842 Domestic Fish and Fisheries Products (1 99 I - 1996);
CPGM 7303.844 Import Seafood Products (1 992- 1996); CPGM 7303.036 Processed Seafood ( I 99 1- 1994).
608 Jinneman et al.

TABLE
6 Class I Recalls Issued i n the United States for Domestic and Domestic/
Import Ready-to-Eat Seafood Products Contaminated with L. monocytogenes Since
1987
No. of Class I
recalls since
Product 1987 lb affected Location of manufacturer
Crustacean
crab 46 >141197 AL, FL, GA, ME, NC, OR, TX,
VA, WA, Chile, Mexico
shrimp 7 >3 1332 FL, GA, MA, NY, WA
lobster 2 >264 Canada
ShellBsh
mussels (marinated) 1 Unknown MA
mussels (smoked) 1 Unknown New Zealand
snails 1 1455 CO, FL, GA, IL, KS, LA, NH, NJ,
NY, OR, PA, TX, WA
Fin Fish
hot smoked 6 >253 KY, MD, ME, NY, WA
cold smoked 22 >93722 CA, MA, ME, NJ, NY, OR, WA,
United Kingdom
smokeda 16 >9292 CA, FL, IL, MD, ME, NC, NY, SC,
TN, VA, WA
salted 1 Unknown Canada
Other
imitation seafood 5 > 1773 ID, NV, OR, UT, VA, WY, Japan,
Korea
seafood salad or spread 3 >42 FL, ME, WA

aHot or cold smoking process not identified.

Source: Data compiled from FDA Enforcement reports, Refs. 14 and 41.

with hot smoked 8.8% (19 of 215) samples [57]. Several other investigations have evalu-
ated the prevalence of L. monocytogenes in smoked fin fish products, with incidence rates
ranging from 0 to 75% [32]. In surveys with over 100 samples, the incidence of L. monocy-
togenes also tended to be greater in cold smoked fin fish products (1 1.3-24.0%) [60-621
compared with hot-smoked fin fish products (8.4-8.9%) [61,62]. Similar to ready-to-eat
crab products, postprocess contamination likely accounts for the presence of L. rnonocyto-
genes when it occurs in hot-smoked fin fish products. However, with cold-smoked fin
fish, the process may not eliminate L. monocytogenes present on the raw product; neverthe-
less, it is also possible that contamination could occur during or after processing of the
product [29].
Overall, there have been 112 Class I recalls for domestic and domestichm-
ported ready-to-eat seafood products resulting from presence of L. monocytogenes
during 1987 through August 1998 in the United States [14,41]. Recalls only have been
issued when L. monocytogenes was found in seafood or seafood products which are ready-
to-eat and would therefore receive no subsequent or minimal heat treatment by the
consumer before consumption. The number of recalls by product category is shown in
Table 6. Products which resulted in the greatest number of recalls reflect the types of
products which were most frequently identified as being positive for L. monocytogenes
Listeria monocytogenes in Fish and Seafood 609

in compliance program surveys. Crab accounted for 47 and smoked fin fish for 16 of the
112 recalls.

OTHER SURVEYS FOR L. MONOCYTOGNS IN FISH AND

SEAFOOD PRODUCTS
The documented presence of Listeria in fish and seafood products and subsequent product
recalls prompted several surveys to determine the incidence of Listeria spp. in various
products from many geographical locations (Table 7). Results from these studies have been
extensively reviewed [ 13,26,32,62]. Sampling strategies, number of samples analyzed, and
detection methods varied, so that although it is useful to note the results from these studies,
the data from them cannot always be directly compared.

Crustaceans
Listeria spp., including L. monocytogenes, have been recovered from cooked and picked,
ready-to-eat crabmeat, and, as noted earlier, this product has been the subject of several
recalls. Since crab meat is heat processed to eliminate or reduce microorganisms, the
presence of L. monocytogenes on the finished product most likely represents postpro-
cessing contamination. Several surveys have included small numbers of crab samples. L.
monocytogenes was detected in 7 and L. innocua in 12 of 24 cooked imported crab prod-
ucts [ 1061. Although L. monocytogenes was not detected in another study, L. welshirneri
was found in one of two cooked crab samples [20]. Listeria spp. were recovered in two
of five crab samples collected as part of a survey in Alexandria, Egypt [30], and L. monocy-
togenes was isolated from one of seven crab samples from the United States and China
[37]. Two larger U.S. studies of cooked and processed crab also have been published. In
the first, 3 1 of 138 (22.5%) processed crab samples contained Listeria spp., identified only
to the genus level [84]. In the second study which examined 126 cooked and picked blue
crab samples, 10 samples (7.9%) were positive for L. monocytogenes and 3 samples (2.4%)

TABLE
7 Incidence o f Listeria spp. in Fresh, Frozen, and Processed Seafood
% positive for

No. of L. mono-
Product (country) samples Listeria spp. cytogenes Ref.
Crab
crab (c) (multiple countries) 24 29.2 106
crab (c) (USA) 2 50 0 20
crab (r) (China) 7 14 37
crab (c or p) (USA) 138 22.5 84
crab (QY Pt 1 5 40 0 30
blue crab (c) (USA) 126 10.3 7.9 94
Shrimp/prawn s
shrimp (f), (multiple countries) 7 28.6 106
shrimp (f and fr) (USA) 4 25 0 20
shrimp (f) (Japan) 70 8.6 1.4 75
shrimp (f) (Trinidad) 41 5 1
shrimp (raw, fr) (France) 17 23.5 11.8 93
shrimp (c and p) (multiple countries) 8 25 106
610 Jinneman et al.

TABLE
7 Continued
% positive for
No. of L. mono-
Product (country) samples Listeria spp. cytogenes Ref.
shrimp (r) (multiple countries) 49 8.2 37
shrimp (f and p) (India) 19 10.5 0 73
shrimp (Canada) 20 20 37
shrimp in brine (r) (Norway) 16 18 97
shrimp (c and f ) (Iceland) 11 9 9 56
prawn (r) (Japan) 38 15.8 2.6 99
shrimp (Egypt) 5 40 20 30
prawns/shrimp/cockles (c) (UK) 40 0 95
Lobster
lobster tail (fr) (multiple countries) 2 50 106
Mussels
mussels (sm) (New Zealand) 14 35.7 59
mussels (f) (Spain) 40 22.5 7.5 101
mussels (f) (Australia) 15.4 102
Oysters
oysters (fr) (multiple countries) 1 0 0 106
oysters (p) (USA) 2 0 0 20
oysters (f) (Japan) 84 0 0 75
oysters (f) (Egypt) 2 0 0 30
oysters (f) (Australia) 15.4 102
Clams
clam (f) (India) 1 0 0 49
clam (f) (USA) 1 0 0 20
Scallops
scallops (fr) (multiple countries) 2 50 0 106
scallops (raw) (USA) 1 0 0 20
Other shellfish and invertabrates
shellfish (c) (Iceland) 11 0 0 56
non-oyster shellfish (f) (Japan) 147 11.6 1.4 75
shellfisha 25 44 25 59
Donax spp. (coquina) (f) (Egypt) 6 16.7 16.7 30
Ruditapes spp. (clam) (f) (Egypt) 4 50 25 30
Fin Fish
fish (f) (USA) 4 50 50 20
catfish (f) (USA) 1 100 0 20
fish (f) (Trinidad) 61 14.8 2 1
minced fish (f) (Norway) 8 12 97
fish (f) (Japan) 382 12.6 2.4 75
fish (f) (India) 51 3.9 0 73
fish (f) (Egypt) 39 25.6 12.8 30
fish (fr) (Egypt) 17 17.6 5.9 30
fish (f) (India) 4 25 49
fish (fr) (India) 10 20 49
fish (r) (New Zealand) 25 52 32 59
fish minced (raw, r) (Japan) 37 43.2 8.1 99
fish (trout) (f) (Iceland) 2 0 0 56
Listeria monocytogenes in Fish and Seafood 611

TABLE7 Continued
% positive for

No. of L. mono-
Product (coutitry) samples Listeria spp. cytogenes Ref.
fish (dried haddock) (Iceland) 5 0 0 56
fish (fr) (multiple countries) 4 25 106
fish (ceviche) (Peru) 32 75 9 48
fish (lightly pickled) (Switzerland) 89 25.8 61
fish (gravad) (Iceland) 22 63.6 22.7 56
fish (cold-sm, salmon) (Switzerland) 100 24 60
fish (cold-sm, salmon) (Switzerland) 64 6.3 52
fish (cold-sm, fish) (Switzerland) 324 13.6 61
fish (cold-sin, fish) (Switzerland) 434 11.3 62
fish (cold-sin, salmon) (Norway) 33 9 97
fish (sm,-salmon) (Iceland) 31 29 3.2 56
fish (cold-sin, salmon) (Canada) 32 31.2 37
fish (cold-srn, salmon) (Italy) 37 0-80 0 104
fish (cold-srn, salmon) (New Zealand) 12 75 59
fish (cold-sm, salmon) (USA) 61 78.7 29
fish (sm)-(New Zealand) 12 66.7 59
fish (sm fish) (Canada) 71 11.3 27
fish (sm fish) (Canada) 20.4 4.4 25
fish (hot-sm fish) (Switzerland) 496 8.9 61
fish (hot-sm fish) (Switzerland) 69 1 8.4 62
fish (sm and/or salted) (Egypt) 11 18.2 5.6 30
fish (f) (Denmark) 232 14.2 2
fish (cold-sm, cured) (Denmark) 335 10.8 2
Other fish and seafood products
seafood (squid, langostinos) (multiple countries) 2 I00 0 106
seafood (f arid fr) (Taiwan) 57 10.5 105
seafood (f and p) (Iceland) 26 3.9 3.9 56
seafood (India) 200 8 0 65
seafood ( f ) (USA) 59 49.2 84
seafood (p) (USA) 14 0 84
seafood (other) (Iceland) 5 20 20 56
seafood raw, (r) (Japan) 28 7.1 10.7 99
seafood (r) (New Zealand) 50 48 26 59
seafood (c) (Japan) 5 0 0 99
seafood (other) (Japan) 6 0 0 99
seafood salad (p) (USA) 2 0 0 20
fish salads (r) (Iceland) 37 32 16 56
seafood (past ) (pasta with minced fish) (Iceland) 3 0 0 56
seafood (surirni) (multple countries) 7 28.6 106
seafood (surirni) (USA) 1 0 0 20
seafood (surirni) (Canada) 46 2 37
~ ~-

c, cooked; f, fresh: fr, frozen; p, processed; r, ready-to-eat; sm, smoked.

Includes the 14 smoked mussel samples listed in mussles catagory.

Included in the 25 ready-to-eat fish samples in the New Zealand study listed above.
Source: Adapted in part from Ref. 32.
672 Jinneman et al.

positive for L. innocua [94]. Very few studies have enumerated L. monocytogenes in
naturally contaminated cooked and processed crab. Among the 10 samples positive for
L. monocytogenes in one study [94], one sample contained 1100 Listerialg, but in the
remaining samples, <100 Listeridg were detected, indicating a generally low level of
contamination.
Listeria spp. have been detected in fresh or raw shrimp. Two of seven samples
contained L. monocytogenes [ 1061; one of four samples contained Listeria innocua 1201
in two U.S. studies. In a survey of fresh shrimp in Japan, Listeria spp. were recovered
from 6 of 70 samples (8.6%), with one (1.4%) being positive for L. monocytogenes [70].
In France, L. monocytogenes was isolated from 2 of 17 (1 13%) uncooked shrimp samples
and Listeria spp. from 4 samples (23.5%) [93]. In Trinidad where shrimp are consumed
in a nearly raw state, Listeria spp. were recovered from 2 of 41 ( 5 % ) fresh, uncooked
shrimp samples [I].
Despite cooking and other heat-processing steps which should eliminate Listeria
spp. present on raw product, several investigators have recovered Listeria spp. from
cooked and ready-to-eat shrimp/prawns. L. monocytogenes was detected in 2 of 8 (25%)
cooked and processed shrimp in a U.S. study [106], 4 of 49 (8.2%) ready-to-eat shrimp
in Canada [37], and 1 of 38 (2.6%) ready-to-eat shrimp products in Japan 1993. No L.
monocytogenes was recovered from 40 retail samples of cooked prawns, shrimp, and cock-
les sold in England and Wales between 1987 and 1989 [95]. Ready-to-eat shrimp in brine
had L. monocytogenes in 3 of 18 (16.7%) samples, tested in Norway [97].
As with the surveys of cooked and processed crab, there have been few studies where
numbers of L. monocytogenes were determined in cooked shrimp products. However, low
levels of L. monocytogenes in three lots of naturally contaminated ready-to-eat shrimp
(0.54,5.5, and 0.04 most probable number (MPN)/g)and lobster (2.0,0.23, and 0.4 MPN/
g) were reported in a Canadian study [37].

She1Ifish
Smoked mussels have been associated with several listeriosis cases in Australia and New
Zealand [35,77] and with recalls in the United States. L. monocytogenes was isolated from
5 of 14 smoked mussel samples in a survey [59] in New Zealand, whereas fresh Spanish
mussels yielded L. monocytogenes from 3 and other Listeria spp. from 9 of 40 samples
[loll.
The incidence of Listeria in other shellfish products, including oysters, clams, and
scallops has been remarkably low. No Listeria spp. were isolated from oysters in two
studies in the United States and one in Egypt which included one or two samples
[20,30,106]. In a study in Japan, no Listeria spp. were recovered from 84 oyster samples
[75]. No Listeria spp. were found in a single clam sample in each of two studies [20,49];
however, in a study in Egypt, Listeria spp. were found in two of four Ruditapes spp.
(clam) samples, with L. monocytogenes being present in one of these samples [30]. Only
a limited number of samples of scallops have been tested; however L. innocua was isolated
from one of two frozen U.S. samples [ 1061, but no Listeria spp. were recovered from a
single sample in a later study [20]. No Listeria were found in 11 Icelandic cooked shellfish
samples [56]. In a survey of fresh seafood samples purchased from markets in Alexandria,
Egypt, one of six Donax spp. (coquina) was positive for L. monocytogenes [28]. In Japan,
L. monocytogenes was isolated from 2 samples and Listeria spp. were recovered from 17
samples of a total of 147 non-oyster shellfish samples [75,81].
Listeria monocytogenes in Fish and Seafood 613

Fin Fish
In the United States, raw fresh or frozen fish are generally not consumed without further
processing; therefore, surveys for Listeria spp. have included very few fresh or frozen
fish samples. In surveys conducted in the United States, L. monocytogenes was isolated
from two of four fresh fish samples, L. innocua from one catfish sample [20] and L.
monocytogenes from one of four frozen fish samples [106]. In India, Fuchs and Surrendan
[49] reported Listeria spp. in 1 of 4 fresh and 2 of 10 frozen fish samples. In a larger
survey of fresh fish in India, Listeria spp. were isolated from 2 of 51 (3.9%) samples, but
L. monocytogenes was not recovered [73]. In Alexandria, Egypt, Listeria spp. were present
in 10 of 39 (25.6%) fresh and 3 of 17 (17.6%) frozen fish samples, with L. monocytogenes
being isolated from 5 of 39 (12.8%) and 1 of 17 (5.9%) of these fresh and frozen fish
samples, respectively [30]. In Norway, L. monocytogenes was isolated from one of eight
(12.5%) minced fresh fish samples [97]. The practice of consuming fresh seafood in an
almost raw state is common in Trinidad where Listeria spp. were detected in 9 of 61
(14.8%) fresh fish samples [I]. In Japan, 3 of 37 (8.1%) raw ready-to-eat minced fish
samples were positive for L monocytogenes and 16 of 37 (43.2%) for Listeria spp. [99].
In a similar Japanese survey, L. monocytogenes was found in 9 (2.4%) and Listeria spp.
in 48 of 382 (12.6%) samples [75]. However, it is not clear if all these samples were
ready-to-eat.

Smoked Fish Products

The presence of Listeria in smoked and lightly processed fish products is often a concern,
because many of these products are commonly eaten without further heating. The cold-
smokmg process does not generate sufficient heat to inactive Listeria organisms which
may be present on fish [29,53,71]. In Switzerland, L. monocytogenes was recovered from
24 and 6% of cold smoked salmon [52,60] and 13.5 and 11.3% of cold smoked fish
samples [61,62]. In studies in Norway [96], Canada [37], and New Zealand 1591, the
organism was isolated from 9, 3 1, and 66%, respectively, of cold smoked salmon samples
(see Table 7). A detailed study on the incidence and sources of L. monocytogenes in
several processing facilities producing cold-smoked salmon showed the primary sources
of L. monocytogenes were surface areas of frozen or fresh raw fish coming into the plant.
As the processing of fish progressed, this pathogen was transferred to other processing
areas and these became secondary sources of the bacterium [29]. In those studies which
specifically identified hot smoked fish samples, L. monocytogenes also was recovered from
8.9 and 8.4% of samples despite the heat processing these products received [61,62].

Lightly Processed Fish Products

Other lightly processed ready-to-eat fish products also can harbor L. monocytogenes. These
include lightly pickled fish from which L. monocytogenes was isolated in 25.8% of samples
surveyed in Switzerland [61]. Cerviche is a lightly acidified ready-to-eat fish product,
which is popular in several South American countries. Listeria spp. were recovered from
75% and L. rnonocytogenes was recovered from 9.4% of cerviche samples examined in
Peru [45]. In Iceland, Listeria spp. were identified in 32.4% and L. rnonocytogenes in
16.2% of ready-to-eat fish and seafood salads tested [56]. No Listeria spp. were recovered
from two seafood salad samples included in a U.S. survey [20] or three pasta salads with
minced fish in the Icelandic study [56].Imitation seafood made from surimi is another
614 Jinneman et al.

processed fish-based product from which L. monocytogenes was found, with an incidence
of 28.6% in the United States [I061 and 2.2% in Canada. No Listeria were recovered
from a single sample in a second U.S. survey [20].

HUMAN LlSTERlOSlS ASSOCIATED WITH FISH AND

SEAFOOD PRODUCTS
Despite the high prevalence of L. monocytogenes, there have been few reported human
listeriosis outbreaks associated with consumption of fish and seafoods. The first case of
listeriosis positively linked to consumption of fish or seafood was not reported until 1989
when a 54-year-old woman in Italy contracted listerial meningitis 4 days after consuming
steamed fish from which L. monocytogenes was later isolated [36]. The fact that the two
L. monocytogenes isolates from the patients cerebrospinal fluid and leftover portion of
fish both were of serotype 4 and were identical in terms of phage type and DNA restriction
analysis, confirms fish as the vehicle of infection in this case of listerial meningitis. How-
ever, the mode by which this fish became contaminated remains unknown.
In 1991, three previously healthy people, aged 83, 37, and 10 years, became ill
in two separate incidents in the State of Tasmania, Australia, after consuming smoked
mussels [77]. Symptoms included malaise, chills, fever, and headache followed by diar-
rhea. Samples of implicated mussels from both incidents contained over I million L.
monocytogeneslg. In addition, L. monocytogenes also was isolated from feces of the pa-
tients. Mussels had been imported from New Zealand, repackaged illegally in Australia
by a retail outlet, and labeled with a code date that overestimated their shelf life by 3
months or more.
Newborn twins died from a L. monocytogenes infection in Auckland, New Zealand,
in 1992. Their deaths were attributed to consumption by their mother of smoked mussels
contaminated with L. monocytogenes. Reports from New Zealand have indicated that the
company producing the smoked mussels had detected Listeria in their product several
months before the deaths of the infants. In 1993, the owner of the company and a consul-
tant to the company were charged with manslaughter by New Zealand police [ 19,351.
Since these outbreaks, one report indicating a contamination rate of 15.4% for Aus-
tralian oysters and mussels [ 1021, and concerns by the Australian Quarantine and Inspec-
tion Service [74], the Australian National Health and Medical Research Council [82,83]
has issued two bulletins providing special dietary advice for pregnant women, transplant
patients, and other immunocompromised patients and information for medical prac-
titioners on diagnosis, treatment, and advice to patients.

REGULATORY ASPECTS OF L. MONOCYTOGENES IN FISH

AND SEAFOODS
Although discussion of proposed criteria for L. monocytogenes and other pathogens in
foods appears in Chapter 17, the reader should be aware that the NACMCF has recom-
mended a zero tolerance for L. monocytogenes in cooked shrimp and crabmeat [ 10,121.
The regulatory policies of different countries vary concerning allowable levels of L. mono-
cytogenes in food products. In 1994, Madden stated, The policy of the [U.S.] FDA re-
mains what has been commonly referred to as the zero tolerance policy, which is very
conservative. No L. monocytogenes organisms are permitted in a food which was not
intended for further heat treatment. [72]. Canadian regulations for presence of L. monocy-
Listeria monocytogenes in Fish and Seafood 615

togenes in ready-to-eat foods have been based on the ability of L. monocytogenes to grow
in a given food product [38].
There still is not complete agreement among the countries which compose the Euro-
pean Economic Community (EEC) for criteria regarding L. monocytogenes in various
foods [ 1031. Criteria for L. monocytogenes developed by the EEC are only within the Milk
Hygiene Directive. Several public health approaches to food safety including addressing
industry groups about HACCP-based hygiene plans and educating the most susceptible
groups, (e.g., pregnant women and immunocompromised individuals) have been used by
individual European countries. A quantitative approach setting limits at the point of sale
or at the end of product shelf life, also has been explored by several countries. For example,
German regulations categorize foods into four risk levels and set specific L. monocyto-
genes action levels based on the risk category. Group I foods have the most restrict-
ive limit (absence of L. monocytogenes in 25 g or 25 mL of food). Within the German
approach, seafood products like heat-treated shrimp or prawns would be categorized
as Group 111. For products in this category, low-level contamination (<loo L.
monocytoguneslg) would require a food establishment hygiene check. Higher levels of
Contamination would cause the product to be classified as unfit for human consumption
or a health hazard in substantiated cases [ 1031.

BEHAVIOR OF L/STR/A IN FISH AND SEAFOOD

Before 1987, very little was known about the incidence and behavior of Listeria spp. in
fish and seafoods. Since this time, a number of studies have focused on the growth, inhibi-
tion, and thermal resistance of L. monocytogenes in different products. The results from
these studies and the means by which this pathogen may be transmitted to various forms
of aquatic life are discussed in this section.

Modes of Transmission
Current data point to cross contamination as the major source of Listeria in cooked or
otherwise processed seafood, as evidenced by the recovery of healthy, noninjured cells of
L. monocytogenes from the surface of many heat-processed/ready-to-eat seafood products.
However, a small percentage of aquatic creatures may become contaminated through
direct/indirect contact with Listeria in their natural environment. This appears even more
plausible when one recalls the salt-tolerant nature of L. monocytogenes and that this patho-
gen has been isolated from sewage effluent entering the North Sea [24] and also from
crustaceans that were harvested from stream water in which L. monocytogenes was previ-
ously identified [86]. In 1989, Fuad et al. [47] evaluated the ability of L. monocytogenes
to survive in the estuarine environment. Since there appears to be a higher incidence of
Listeria in chitinous seafood (i.e., shrimp, crab, lobster), samples of filtered and unfiltered
seawater with and without chitin and chitin-free filtered and unfiltered stream water were
inoculated with various strains of L. monocytogenes, many of which possessed chitinase
activity. Although Listeria populations decreased in chitin-free filtered and unfiltered sea-
water, adding chitin to both types of seawater stimulated growth of Listeria. Moreover,
the pathogen grew in filtered stream water.
These findings, along with those from a report in which L. monocytogenes was found
on the exoskeleton but not in the digestive tract of shrimp that were exposed to high
levels of L. monocytogenes in aquaculture tanks [ 1051, suggest that this pathogen may be
676 Jinneman et al.

ecologically adapted to chitin. If this is true, then it is imperative that holding tanks for
chitinous marine animals be properly set up and maintained to avoid potential microbiolog-
ical problems involving L. monocytogenes and other foodborne pathogens including Vibrio
spp. and Aeromonas hydrophilia.
It is well established that Listeria spp. are often associated with wild animals and
birds which can serve as reservoir hosts. Fenlon et al. [46] demonstrated the role that
scavenging birds can play in the Listeria cycle. In that study, a definite association between
gulls feeding on sewage and fecal carriage of Listeria (26.3% positive) was shown, which
compared with a carriage rate of only 8% for gulls feeding in less polluted areas. This
higher carrier rate suggests that L. monocytogenes is a part of the normal microflora of
the near estuarine environment [ 131. Two studies of estuarine waters, shrimp, and oysters
along the northern Gulf of Mexico [80] and of freshwater tributaries, sediments, bay water,
and oysters in the Humboldt-Arcata Bay of Northern, California 1221, reached similar
conclusions. In one study [79], 5% of 78 saltwater samples were positive for Listeria spp.
In comparison, 11% of 74 shrimp samples were positive for L. monocytogenes and no
Listeria were isolated from oysters. Listeria species and L. monocytogenes were found in
8 1 and 62% (37 samples), respectively, of freshwater or low-salinity waters in tributaries
draining into Humboldt-Arcata Bay. The incidence of Listeria spp. and L. monocytogenes
in sediment (46 samples) from the same tributaries was 30.4 and 17.4%, respectively. One
of three bay water samples contained Listeria spp. (including L. monocytogenes), whereas
L. innocua was recovered from only 1 of 35 oyster samples [22].Both of these studies indicate
that the estuarine environments are continuously subjected to potential contamination with
Listeria spp. from, for example, processing effluents, agricultural runoff, and sewage efflu-
ents. Listeria spp. can be recovered from nonpolluted environments, and the source of these
bacteria may very well be from avian species, especially sea-gulls [79].

Growth and Survival

Raw and processed seafoods have been long regarded as excellent substrates for growth
of most common agents of foodborne disease, particularly if seafoods are held at improper
temperatures; however, interest in behavior of L. monocytogenes in these products is of
recent origin [32]. According to data gathered by Lovett et al. [70) in 1988, L. monocyto-
genes grew readily (generation time G 12 h) in inoculated samples of raw shrimp, crab,
surimi and whitefish, with the pathogen attaining maximum populations of > 10' CFU/g
in all four products following 14 days of storage at 7C. Two years later, Brackett and
Beuchat [16] also reported that L. monocytogenes grew and retained similar levels of
pathogenicity on artificially contaminated crabmeat during 14 days of storage at 5 to 10C.
In similar studies, Rawles et al. 1941 examined both the incidence and growth of L.
monocytogenes in blue crab meat held at refrigeration temperatures. Of the 126 samples
analyzed, 10 were positive for L. monocytogenes and 3 were positive for L. innocua.
Populations of Listeria found in fresh-picked blue crabmeat were usually < 100 CFU/g.
Based on these data, an inoculum level of 50 CFU/g was added to commercially pasteur-
ized crabmeat and the growth rate determined at 1.1, 2.2, and 5C. Calculated generation
times were 68.7 h at I.1"C; 31.4 h at 2.2OC, and 21.8 h at 5C. At 5"C, there was a 7
log 10 increase in L. monocytogenes population but only a 2.5 log,,, at I . 1"C after 2 I days.
The authors therefore concluded that blue crab meat needs to be stored at 5 1.1"C. How-
ever, in contrast to what Lovett et al. [70] observed for shrimp and whitefish, Harrison
et al. [54] and Shineman and Harrison [ 1001 found that L. monocytogenes failed to grow
in overwrapped/vacuum-packaged raw shrimp and fin fish, with numbers of Listeria gen-
Listeria monocytogenes in Fish and Seafood 617

erally decreasing by approximately I log after 2 I days of storage in an ice chest. When
catfish were stored at 4C the L. monocytogenes population increased slowly ( 1 .O- 1.5
log,,,) during the first 12 days and then decreased I .5 log,,, by day 16 [68]. During storage,
psychrotrophic populations increased from 1O3 to > 1 O7 CFU/g, thus reinforcing the notion
that L. monocytogenes can readily survive in refrigerated raw foods even when greatly
outnumbered by other natural contaminants. Since L. monocytogenes was recovered from
laboratory-contaminated shrimp (initial inoculum 2 10' CFU/g) after 90 days at -20C
[76], it is evident that this pathogen also is fairly resistant to subfreezing temperatures.
Unlike the aforementioned products, preliminary results from Kaysner et al. [66]
suggest that L. monocytogenes was unable to grow in artificially contaminated oysters,
with Listerill populations remaining constant in shucked oysters after 21 days at 4C.
Apparent inability of Listeria to grow in raw oysters may be related to difficulties in
isolating Listeria from retail raw oysters. According to Farber [37], L. monocytogenes
(inoculum level of 2 X 103CFU/mL) grew fairly well on cooked lobster, shrimp, crab,
and smoked fish and in most instances increased about 2-3 log,,, within 7 days at 4C.
When these same products were temperature abused for a short time (6 h) at room tempera-
ture, levels of L. monocytogenes increased by 1 log on shrimp. crab, and lobster, and only
0.2 log on smoked salmon. In a survey for the incidence of this pathogen on shrimp and
lobster meat at the wholesale level, 13 of 113 samples were positive and were contami-
nated at a level of < 10 MPN/g. Storage of these naturally contaminated products at 4C
resulted in L. monocytogenes populations of <I00 MPN/g after 1 week, with numbers
increasing to 2.5 X 103MPN/g after 2 weeks [37]. In these studies, the growth rate of
different strains of L. monocytogenes was comparable in shrimp, crab, and cooked lobster.
However, the difference in growth rate of two strains in smoked salmon probably reflected
a greater tolerance of one strain to sodium chloride and/or the antimicrobial compounds
present in smoke.
The behavior of L. monocytogenes in cold-smoked salmon has been studied at sev-
eral laboratories. Guyer and Jemmi [53] and Jemmi [62] determined growth of L. monocy-
togenes during fabrication and storage of cold-smoked salmon. During the preparation of
cold-smoked salmon, L. rnonocytogenes did not change when inoculated onto the surface
levels. Populations increased to 2.3 X 106 MPN/g at 10C and up to I .5 X 10s at 4C
during 20 days of storage. After 30 days, L. monocytogenes had increased to 107MPN/
g in samples stored at both temperatures. A pH of 5.8 and a, of 0.93 did not prevent
growth of L. nionocytogenes. Eklund et al. [29] demonstrated that with injection of recircu-
lated brines into the interior of cold-smoked salmon, L. monocytogenes also was inoculated
into these sites. During processing at 17.2-2 1.1 "C, L. rnonocytogenes increased 2- to
6-f0ld, and at 22.2-30.6"C this pathogen increased up to 100-fold. These authors thus
emphasized the importance of eliminating or reducing the L. rnonocytogenes population
on the outside of the fish before they are filleted. In addition, they also recommended that
brines that drain off fillets during the injection process not be collected, recirculated, and
injected into the product.
In a similar study by Hudson and Mott [ 5 8 ] ,L. rnonocytogenes populations increased
rapidly on colcl-smoked salmon within the shelf life of the product. At 10C, L. monocyto-
genes growth was comparable in samples packaged in either oxygen-permeable or oxygen-
impermeable films. However, at 5 C the lag phase was longer in vacuum-packaged prod-
uct, but once growth was established, generation times were again comparable. The effect
of inoculum level on the growth of L. monocytogenes in cold-smoked salmon stored at
4C was reported by Rorvik et al. [96]. Starting with an initial population of 6 or 600
CFU/g, levels increased about 4.8 log,(]for the higher inoculum and 2.1 log,,, for the 6
6 78 Jinneman et al.

CFU/g inoculum. In these same studies, the growth rate of L. monocytogenes also was
determined in products with different initial bacterial counts. When the inoculum level
was 6 CFU/g, L. monocytogenes populations increased faster in samples with the lower
rather than higher initial total bacterial populations.

Inhibition
Of the processes used to prepare smoked fishery products, the cold-smoking operation has
been of special interest, because the temperatures used are not lethal to L. monocytogenes.
Hence, the following interventions have been recommended to reduce the risks associated
with L. monocytogenes in these products: (a) eliminate or reduce numbers of L. monocyto-
genes on the outside surfaces of frozen or fresh fish before filleting, (b) prevent recontamina-
tion and growth of L. monocytogenes during all stages of processing, and (c) inhibit growth
of any possible survivors or recontaminants during processing and distribution 1291.
Several papers have been published on inhibition of L. monocytogenes in cold-
smoked fish processed with sodium chloride, sodium nitrite and sodium lactate. In these
studies, smoke was not applied to the products, so that the efficacy of the different forms
of inhibition could be addressed. Peterson et al. [91] studied behavior of L. monocytogenes
(150 CFUI15 g) in cold-processed salmon containing 3.5 or 6.0% water phase sodium
chloride. The products were packaged in either oxygen-permeable film or vacuum sealed
in impermeable film and stored at 5 and 10C. After the second week at 1OOC, L. monocytu-
genes populations increased to the range of 106-108 CFU/g, with no difference being
attributed to the sodium chloride concentration. Vacuum packaging suppressed growth of
L. monocytogenes by 10- to 100-fold in samples with 3 or 5% sodium chloride. Inhibition
related to salt concentration was most apparent at 5"C, with L. rnonocytogenes populations
being held below 10' CFU/g by 6% water phase salt, but increased to 104CFU/g in
products with 5% water phase salt and to 10' CFU/g with 3% water phase salt. Brown
sugar is often used in processing of cold-smoked salmon; use of the sugar in the product,
however, did not influence growth of L. monocytogenes. In these same studies, growth
of the clinical isolate Scott A and two L. monocytogenes strains isolated from salmon
were comparable in cold-smoke salmon stored at 5 and 10C.
Given the salt tolerance of L. monocytogenes and consumer unacceptability of
smoked fish products with water phase NaCl concentrations much above 3 or 4%, it was
concluded that other inhibitors, in addition to NaCl, were needed to control growth of
this bacterium. Pelroy et al. [90] therefore studied the behavior of L. monocytogenes (150
or 4.9 X 103CFU/15-g sample) in relation to sodium nitrite (190-200 ppm) combined
with sodium chloride in cold-processed salmon stored at 5 and 10C. The combination
of NaCl and NaN0, was most effective at 5C. With an initial inoculum of 150 CFU/l5 g,
L. monocytogenes was held below IS CFU/g by a combination of 190-200 ppm NaN02
and 3% water phase salt and below 20 CFU/g with NaNO, and 5% NaC1. Packaging in
oxygen-permeable or oxygen-impermeable (vacuum-sealed) films had little effect on
growth of L. monocytogenes when NaN02 was included in the process. Increasing the
storage temperature to 10C markedly reduced the efficacy of both NaCl and NaCl +
NaN0, treatments. There was little difference in inhibition between 3 or 5% water phase
NaCl at 10C, and the combined effect of NaCl and NaNO, was only slightly greater than
than that of NaCl alone. However, the packaging method had the most pronounced effect
on growth of L. monocytogenes at 10C. Growth was consistently greater in samples
packaged in oxygen-permeable film than in the vacuum-sealed impermeable film pack-
Listeria monocytogenes in Fish and Seafood 619

ages. L. monocytogenes populations had increased from 10 CFU/g to about 108 CFU/g
in samples packaged in oxygen-permeable film and 106 CFU/g in impermeable film.
Differences in inhibition attributable to inoculum level and NaCl + NaN02concentrations
were obvious in products stored at 5C. In comparison, when the initial inoculum level
was increased to 327 CFU/g, L. monocytogenes reached a population of l 06- 107CFU/g
after 20 days of storage.
Of the different inhibitors studied by Pelroy et al. [89], sodium lactate used in combi-
nation with salt or salt plus sodium nitrite was most effective in controlling growth of L.
monocytogenes (150 CFU/l5 g) on vacuum packaged cold-smoked salmon stored at 5
and 10C [89]. A concentration of 3% lactate in combination with 3% salt or 3% salt +
125 ppm nitrite prevented any increase in L. monocytogenes populations during 40-50
days of storage at 5 or 10C (Fig. 1). Addition of 2% sodium lactate prevented growth
of L. rnonocytogenes in all samples stored at 5C. At 10C, however, a combination of
sodium lactate (2%) and NaCl (3%) inhibited growth for 14 days, but then the pathogen
reached populations about 1-2 logs less than those of the control samples with NaCl only.
When the products contained NaN02 (125 ppm), sodium lactate (2%), and NaCl (3%),
growth of L. monocytogenes was totally suppressed at 10C except in one of the four
samples where populations reached 9.3 X 102 CFU/g.
Certain spices and herbs also can be used to minimize growth of Listeria, as previ-
ously discussed in Chapter 6. According to Rorvik et al. [98], L. monocytogenes popula-
tions remained unchanged on vacuum-packaged gravad salmon (i.e., an unsmoked salmon
product containing dill with a similar pH, salt content, and a, to that of smoked salmon)

10C 5C

7 E 3% NaCl

$ 7
0 No Lactate
A 2% Lactate

"0 10 20 30 40 500 10 20 -30 40 50

Days stored

FIGURE1 Growth of L. monocytogenes (150 Scott A/15-g sample) in comminuted

salmon containing 0, 2, or 3% water phase NaCI, with or without 125 ppm NaN02,
during storage at 10" or 5C in vacuum-sealed impermeable film packages. Symbols
on baseline indicate L. monocytogenes was detectable by enrichment only. (Adapted
from ref. 89.)
620 Jinneman et al.

during 4 weeks of storage at 4C. Since addition of as little as 0.5% dill prevented growth
of Listeria in laboratory media, dill was most likely responsible for inhibiting L. monocyto-
genes in gravad salmon during extended cold storage.
Fermentation products from lactic acid bacteria have been studied to determine their
efficacy in controlling L. monocytogenes growth in blue crab meat [23]. In these studies,
steam-sterilized crab meat was inoculated to contain 5.5 log,, CFU/g of a three strain
mixture of L. monocytogenes and then washed with various lactic acid bacteria fermenta-
tion product at levels of 2000-20,000 arbitrary units (AU) per milliliter of wash water.
L. monocytogenes populations remained relatively constant in control samples stored for
6 days at 4C. In comparison, numbers of L. monocytogenes on crabmeat washed with
Perlac 1911 or Micro-Gard (10,000-20,000 AU) initially decreased (0.5-1 .O log,, unit/g)
and then recovered to original levels within 6 days. When crab meat was washed with
10,000-20,000 AU of Alta 234 1, enterocin 1083, or nisin per milliliter, L. monocytogenes
populations initially decreased by I .5-2.7 log units/g. Thereafter, counts increased by
0.5- 1.6 log 1o units within 6 days.
Degnan et al. [23] also showed that when the crabmeat was washed with food-grade
sodium acetate (4 M), sodium diacetate (0.5 to 1 M), and sodium lactate ( I M) or sodium
nitrate (1.5 M), there was only a modest reduction in L. monocytogenes population (0.4-
0.8 log,, unit/g). However, Listeria counts decreased 2.6 logl0/gwithin 6 days when the
sodium diacetate concentration was increased to 2 M. Trisodium phosphate (1 M) also
reduced L. monocytogenes counts from 1.7 to >4.6 log 10/gwithin 6 days. Hence, L. mono-
cytogenes populations on crabmeat can be reduced by washing with selected antimicrobial
agents.
Potential use of certain strains of Enterococcus faecium to control L. monocytogenes
was suggested by Embarek et al. [34]. E. faecium isolates from sous-vide cooked fish
fillets were tested on different strains of L. monocytogenes and other pathogenic bacteria
using Brain Heart Infusion broth with added CaC03 to avoid decreases in pH during
growth of E. faecuim. Of the 19 isolates tested, 14 produced proteinaceous substances
inhibitory to different strains of L. monocytogenes. An inoculum of 107CFU E. faecium/
mL reduced L. monocytogenes populations of 102CFU/mL to lO/mL after 14 days at
3C and to I CFU/mL after 35 days. With a lower inoculum of 1 O4 CFU/mL, L. monocyto-
genes was only slightly inhibited at 15C and not at 3 or 5C. However, after 11 days at
15"C, spontaneous resistance was observed and L. monocytogenes reached I X I O8 CFU/
mL. All of these L. monocytogenes isolates were resistant to E. faecium.
The antibacterial effects of 209 psychrotrophic Pseudoinonas strains isolated from
spoiled iced fish and newly caught fish were assessed by screening L. monocytogenes and
other organisms using agar diffusion assays [51]. Only eight strains inhibited growth of
L. monocytogenes. Inhibitory action was most pronounced among Pseudomonas strains
producing siderophores which chelate iron; addition of iron sometimes eliminated the
antibacterial effect. Some strains of Pseudomonas enhanced growth of L. monocytogenes,
with dense growth being observed around wells containing these Pseudomonas strains.
These strains may have created a more advantageous nutritional environment for L. mono-
cytogenes by increasing the supply of iron or the availability of low molecular weight
nutrients.

Inactivation
As was true for dairy, meat, and poultry products, the rash of Class I recalls during the
past decade involving ready-to-eat seafoods has prompted concerns about the adequacy
Listeria monocytogenes in Fish and Seafood 621

of thermal processing treatments used for raw seafood. Hence, in early 1988, Pace et al.
[88] reported results from a study in which freshly shucked oysters were exposed to 150
ppm chlorine for 30 min, pasteurized at an internal temperature of 72-74C for 8 min,
and then periodically examined for major bacterial groups during 5 months of refrigerated
storage. According to these authors, chlorination reduced initial aerobic plate counts of
4.5 X 1O5 C'FU/g by 40-90%, with pasteurization reducing the population by an additional
99.9%. Despite these large reductions in microbial flora, survivors classified in eight differ-
ent genera of aerobic or facultatively anaerobic bacteria were present in the product. How-
ever, at this point, the oysters were unfit for consumption, as evidenced by profuse gas
production and swelling of plastic pouches in which the product was pasteurized.
In response to public and industry concerns, the FDA 1761 also examined the thermal
resistance of Listeria in raw shrimp tails that were inoculated internally to contain approxi-
mately 104--1OS L. rnonocytogenes CFU/g. Using a combination of cold (with/without
broth) enrichment and warm (selective) enrichment, these investigators failed to recover
the pathogen from shrimp tails that were boiled longer than 5 min. Although appreciable
numbers of heat-stressed cells were detected in inoculated shrimp tails that were boiled
for 3 min, frozen storage of the product at -20C eventually led to complete inactivation
of the pathogen. More important, when this study was repeated using naturally contami-
nated frozen shrimp from Ecuador and Honduras in which 1 03-1OS L. monocytogenes or
L. innocua CFU/g were presumably present only on the chitinous exoskeleton, all Listeria
were eliminated after 1 min of boiling. Hence, since shrimp are more likely to be contami-
nated externally than internally with relatively low levels of Listeria, these findings sug-
gest that present cooking methods are adequate to eliminate these organisms from raw
shrimp.
Since these initial studies, the thermal death time of L. monocytogenes has been
determined in different seafoods and fish. The results of these studies are summarized in
Table 8. In an effort to determine if the presence of L. rnonocytogenes in processed lobster
could be the result of undercooking or postcooking contamination, Budu-Amaoko et al.
[21] determined the thermal death time of 107cells of L. monocytogeneslg of product
using 25-g samples. The observed D-values at 51.6, 54.4, 57.2, 60.0, and 62.7"C were
97.0, 55.0, 8.3, 2.39, and 1.06 min, respectively, with a z-value of 5.0"C (Table 8). After
isolating this pathogen from plants using good manufacturing practices, the authors specu-
lated that the presence of L. monocytogenes in the final product was probably the result
of underprocessing .
Blue crab is a popular seafood item, with more than 50% of picked meat being
pasteurized to offset seasonal fluctuations in some regions. The lack of information about
the growth and survival of this pathogen in blue crabmeat prompted Harrison and Huang
[55] to determine the heat resistance of the Scott A strain in this product. The crabmeat
was inoculated with 107 cells/g before distributing 7.5 g into sausage casings (1.6 X 4
cm) for thermal processing. D-values were 40.43, 12.0, and 2.6 I min at 50, 55, and 6OoC,
respectively, with a z-value of 8.40"C as determined by using Trypticase Soy Agar (see
Table 8). Use of Vogel Johnson agar, a selective plating medium that is less able to support
repair and subsequent growth of sublethally injured Listericz, yielded lower D-values
(34.48, 9.18, and 1.31 min) and a lower z-value (6.99"C) at the same temperatures.
Heightened interest in foodborne listeriosis has also led to intense efforts toward
determining whether current industry practices for cooking crawfish are adequate to inacti-
vate L. rnonocytogenes. In 1993, Dorsa et al. [28] first determined the growth rate of L.
monocytogenes in crawfish tail meat stored at 0.6 and 12C. Exponential growth began
with no apparent lag phase and 109CFU/g were observed after 10 and 4 days at 6 and
622 Jinneman et al.

8 Thermal Death times of L. rnonocytogenes in Different Seafood and Fish

TABLE
Product
Lobster Blue Crawfish Mussels
Temperature meat crab meat tail meat brine soaked Salmon Cod
("C) (21) (55) (28) (18) (33) (33)
D-value (min)
50.0 40.43
51.6 97.0
54.4 55.0
55.0 12.00 10.23
56.0 48.09
57.2 8.3
58.0 16.25 10.73 7.28
59.0 9.45
60.0 2.39 2.6 1 1.98 5.49 4.48 1.98
62.0 1.85 2.07 0.87
62.7 1.06
65.0 0.19 0.87 0.28
68.0 0.15 0.15
70.0 0.07 0.03
z-values, lobster meat 5.0"C; blue crabmeat 8.40"C in Trypticase Soy Agar; crawfish tail meat 5.5"C; mussels
4.25"C, salmon 5.6"C; cod 5.7"C.

12"C, respectively. Rapid growth of L. monocytogenes at these temperatures further em-

phasized the need for information on thermal resistance of the bacterium in crawfish tail
meat. In precooked crawfish meat, D-values for L. monocytogenes were 10.23, 1.98, and
0.19 min at 55, 60, and 65"C, respectively, with a z-value of 5.5"C. Since commercially
processed crawfish are normally boiled for 5-10 min before hand peeling, most Listeria
contamination occurs postboiling during peeling and packaging. Therefore, in addition to
strict in-plant sanitation programs, postpicking or postpackaging heat treatments, as de-
scribed by Dorsa et al. [28], can be used to produce Listeria-free product.
Based on the psychrotrophic nature of L. monocytogenes and the lack of information
on the minimum oral infective dose, the New Zealand Department of Health has taken a
conservative approach and recommended a policy of "zero tolerance" for L. monocytu-
genes in a 25-g sample of ready-to-eat seafood. Hot water blanching at 68-72C has
been used by several New Zealand mussel harvesters to inactivate L. monocytogenes in
greenshell mussels [87]. Bremer and Osborne [ 181 also determined the thermal death time
of seven strains of L. monocytogenes in green shell mussels that were brined in preparation
for hot smoking. Brined mussels were blended, inoculated to contain 106CFU/g, and heat
resistance was determined in plastic pouches. The D-values at 56, 58, 59, 60, and 62C
were 48.09, 16.25, 9.45, 5.49, and 1.85 min, respectively, with a z-value of 4.25"C (see
Table 8). When the D-values for different seafoods at 60C are compared, values for
lobster meat, crawfish tail meat, and crabmeat are similar. The D-value for brined mussels,
however, were two to three times higher (see Table 8). The authors indicated that addition
of salt and brown sugar (used in the smoke products) may have enhanced the thermal
resistance of L. monocytogenes in mussels during hot smoking. These differences suggest
that product form and composition should be considered when determining the heat resis-
Listeria monocytogenes in Fish and Seafood 623

tance of a pathogen. Furthermore, these data emphasize the importance of determining

the D-value for each product experimentally.
Minimally processed foods, also called refrigerated pasteurized foods, of extended
durability [78] that can be prepared using the sous-vide technology have raised new con-
cerns regarding survival of Listeria. Embarek and Huss [33] studied the heat resistance
of two strains of L. monocytogenes (062 and 057) in fatty fish (salmon) and nonfatty fish
(cod), with the former strain being slightly more heat resistant. For sous-vide processing,
the fish were vacuum packaged and then heated at 58, 60,62, 65, 68, and 70C. For cod,
D-values were 7.28, 1.98, 0.87, 0.28, 0.15, and 0.03 min, respectively, with a z-value of
5.7C for L. monocytogenes strain 062 (see Table 8). Both strains of L. monocytogenes
were one to four times more heat resistant in salmon than in cod. The D-values for salmon
were 10.73, 4.48, 2.07, 0.87, 0.2, and 0.07 min, respectively, with a z-value of 5.6C for
L. monocytogenes strain 062 (see Table 8). The authors attributed the protective effect in
salmon to its higher fat content. They also emphasized the importance of product form
and ingredients in determining the heat resistance of L. monocytogenes.
Embarek [311 subsequently assessed the thermal resistance of L. monocytogenes in
sous-vide fish fillets containing various levels of NaCl and also the potential growth of
this pathogen in the product during cold storage. When cod fish fillets were heated at 58-
68OC, destruction of Listeria took up to five times longer in fillets containing 5% added
NaCl as compared with product prepared without salt. Thus, although the present time/
temperature treatments recommended by the NACMCF will easily inactivate expected
numbers of L. monocytogenes in sous-vide fish prepared without salt, identical processing
will likely produce some survivers in product containing added salt. Growth experiments
also showed that L. monocytogenes populations increased 2 and 4 log,, CFU/g in salt-
free sous-vide fish after 14 days of storage at 3 and 5-10C, respectively. These findings
stress the importance of eliminating L. monocytogenes from the product during sous-vide
processing.
If the aforementioned timekemperature treatments for the different seafoods had
been followed, then the recent Class I recalls likely resulted from postprocessing contami-
nation, as has already been implied by Kvenburg [9,67] and other FDA officials [6,105].
However, considering the possibility for errors during thermal processing, members of
the seafood working group of NACMCF agreed it would be prudent to consider certifying
or licensing persons who are directly involved in thermally processing the different sea-
foods, as has been done for many years in the canned food industry [9].
Recognizing that L. monocytogenes is more likely to contaminate the surface rather
than the interior of most fishery products, several studies have concentrated on using
different treatments to inhibit or inactivate L. monocytogenes on the surface of processed
seafoods. Noel et al. [85]investigated the possibility of using various lactic acid treatments
to inactivate L. monocytogenes on processed peeled and unpeeled shrimp. The shrimp
were immersed in a broth culture of L. monocytogenes, drained, and then immersed in
an aqueous solution of 1.5,3.0, or 6.0% lactic acid for 1, 10, or 120 min. The untreated and
treated shrimp were examined for survivors during 28 days of storage at 20C. Although all
lactic acid treatments decreased the numbers of Listeria, exposure to 1.5% lactic acid for
10 min was deemed most appropriate, since the treatment did not adversely affect the
products organoleptic quality. Overall, initial L. monocytogenes populations of 2.6 X 103
CFU/g on inoculated shrimp decreased to <3.2 X 102CFU/g following 10 min of expo-
sure to 1.5% lactic acid. Although Listeria populations continued to decrease in all sam-
ples, fewer Listeria were observed in lactic acid-treated (-10 CFU/g) than in untreated
shrimp (-40 CFU/g) after 28 days of frozen storage.
624 Jinneman et al.

In a similar investigation, decimal reduction times (D-values) were determined for

a mixture of seven strains of L. monocytogenes exposed to marinades in the presence
and absence of greenshell mussels [ 171. When an acetic acid (1.5% wt/vol) marinade was
used, the calculated D-values in the presence and absence of mussels were 77.3 and
33.3 h, respectively. When a combination of acetic acid (1.5%) and glucono-delta-lactone
(0.2%)-based marinades were used, the D-values increased to 86.3 h in the presence of
mussels and decreased to 19.3 h without the mussels. Likewise, for acetic acid (0.75%)
and lactic acid (0.75%) marinades the D-values increased to 125.5 h in the presence of
mussels and 26.9 h in the absence of mussels. Through enrichment experiments, L. mono-
cytogenes was detected after 26 days for acetic acid ( I .5%) and after 53 days for the acetic
acid, lactic acid (0.75% each) combination, but no survivors were found after 29 days
with the acetic acid (1 S % ) and the glucono-delta-lactone (0.2%) combination. Their re-
sults prompted the authors to emphasize that care must be taken in extrapolating from
results of a model broth system to a "real food." Thus despite the inability of organic
acids to eliminate completely L. monocytogenes from seafoods, storage of marinated prod-
ucts before release is an effective method for further decreasing the possibility of L. mono-
cytogenes being associated with these products. This also was demonstrated with inocu-
lated gravad salmon processed with dill and stored at 4C. Dill at 0.5 and 5.0% prevented
growth of L. monocytogenes during storage of the salmon. These studies also indicate that
dipping products such as shrimp, mussels, lobster, crab, and scallops in organic acids
could prove useful in decreasing the levels of Listeria before and during frozen storage.
During the past decade, there have been several recalls of smoked fish products
because of L. monocytogenes contamination. To estimate the potential health hazard for
the consumer eating such products, Jemmi and Keusch [63] determined the heat resistance
and growth of L. monocytogenes in artificially inoculated heat-smoked trout. In these
experiments, two strains of L. monocytogenes were surface inoculated ( 106MPN/g) onto
raw trout which then were kept in a salt and spice marinade (10% NaCl and 0.7% spices,
pH 6.4) for 12 h before smoking in a kiln. The trout were surface dried at 60C for 30 min,
the kiln was heated to 1lO"C, and fish were held at this temperature for 20 min after an
internal temperature of 65C was reached. Then the product was smoked for 45 min,
cooled, and stored at 8-10C for up to 20 days. L. monocytogenes decreased from an
initial population of 106MPN/g to nondetectable levels after hot smoking and throughout
20 days of storage at 4-10C. However, when the smoked trout were inoculated with 30
or 45 MPN/g after processing and stored at 4 and 8-10C, L. monocytogenes increased
to 107 MPN/g.
The presence of L. monocytogenes on hot-smoked fishery products typically sug-
gests postprocessing contamination. Thus Poysky et al. [92] assessed various processing
parameters required to inactivate L. monocytogenes during the hot-smoking process. Since
L. monocytogenes contamination is most likely to occur on the surface of raw fish fillets
or steaks [29], the effectiveness of using a combination of heat and smoke to inactivate
L. monocytogenes on the surface of brined salmon steaks was investigated. When brined
salmon steaks were heat processed without smoke, L. monocytogenes survived on steaks
processed to an internal temperature of 181F (823C) for 30 min. Application of gener-
ated smoke reduced the minimum lethal temperature to 153F (67.2"C). In contrast, when
smoke was applied during the last half of the process after surface drying and formation
of the surface pellicle, L. monocytogenes was recovered from steaks heated to an internal
temperature of 176F (8O.OOC) for 30 min.
Poysky et al. [92] also reported that by using undiluted liquid smoke at the beginning
Listeria monocytogenes in Fish and Seafood 625

of the process, the inactivation temperature could be decreased to as low as 138F

(58.9"C). Diluting liquid smoke to 50% reduced the effectiveness and increased the lethal
temperature to 150F (65.5"C). The oil soluble fraction of CharSol C-10, CharOil, was
less effective and L. monocytogenes survived in samples processed to an internal tempera-
ture of 166F (74.4"C), the highest temperature tested with this liquid smoke fraction. In
these studies, preenrichment was used to enhance repair and recovery of injured cells.
The processed product was stored at 5C for 4 days and then preenriched in Trypticase
Soy Broth for 6 h at 30C before adding selective agents to UVM enrichment broth.
Length of enrichment also played an important role in detection of survivors. Of the 245
samples tested, 57 that were negative for L. rnonocytogenes after 1 day at 30C turned
positive after 6-9 days of incubation. These studies of Poysky et al. [92] demonstrated
that inactivation of L. monocytogenes is dependent on the interaction between heat and
smoke. For best results, smoke should be applied before pellicle formation, since this
pellicle on the fish surface serves to protect L. monocytogenes by limiting absorption of
smoke.

ACKNOWLEDGMENTS
The authors thank Cecilia Wolyniak (FDA, CFSAN), Pat Pinkerton (FDA, Seattle Dis-
trict), Stephanie Dalgliesh (FDA, Seattle District), and Daryl Thompson (FDA, Atlanta
Regional Office) for retrieval and assistance with the FDA recall information. Maxine
Heinitz (FDA, Midwest Laboratory for Microbiological Investigations, FDA Microbiolog-
ical Information Systems Manager) and Jan Johnson (FDA, Seattle District) provided valu-
able assistance with accessing data from the FDA Microbiological Information System.
Literature searches were conducted by Walter E. Hill and Jim Hungerford (FDA, Seattle
District) for which we are grateful. We would also like to thank Walter E. Hill and Nancy
Hill (FDA, Seattle District) for assistance with building and finalizing a data base for the
reference list.

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 16
Incidence and Behavior of Listeria
monocytogenes in Products of
Plant Origin

ROBERTE. BRACKETT
The University of Georgia, Griffin, Georgia

INTRODUCTION
Use of adequate isolation procedures, enough time, and a little perseverance by investiga-
tors makes it possible to isolate Listeria spp., including L. rnonocytogenes, from most
forms of animal life. A similar situation also exists with products of plant origin. Chapter
3 describes the apparent association between consumption of silage and occurrence of an
illness resembling listeriosis in ruminants, which was observed as early as 1922; however,
this link between silage consumption and listeriosis in domestic livestock was not con-
firmed until 1960 [43]. Although several papers published during the next 15-year period
documented the presence of L. rnonocytogenes in vegetation grown primarily for consump-
tion by animals [75-771 (see Chap. 2), scientists at the time were generally unconcerned
about the incidence of listeriae in produce destined for human consumption, primarily
since such products had not been positively linked to human listeriosis. In fact, the only
instance in which listeriae were recovered from raw retail produce before the 1981 listeri-
osis outbreak in Canada involving coleslaw occurred in 1975 when successful isolation
of three untypable Listeria strains from lettuce marketed in Brazil was reported [50].
Although 18 of 41 Canadians died of listeriosis in 1981 after consuming coleslaw
from which L. rnonocytogenes was isolated and positively identified [69], it was not until

631
632 Brackett

the 1985 cheese-related listeriosis outbreak in California that researchers began to develop
more than a passive interest in the public health significance of L. monocytogenes in
vegetables, fruits, and other products of plant origin. Nevertheless, with the exception of
one isolated listeriosis case in Finland involving homemade salted mushrooms [53] and
a recent cluster of five cases traced to frozen broccoli and cauliflower in Texas [70], no
additional cases of listeriosis have been positively linked to consumption of plant products
produced in North America or elsewhere.
This chapter is devoted to food products and it will specifically address the incidence
of Listeria spp. in raw retail vegetables and fruits. As in earlier chapters, information
concerning behavior of L. monocytogenes in fresh produce and related products (orange
juice/serum, soy milk, pasta, beet pigment) will also be presented along with some possible
means by which listeriae can be inactivated in some of these products.

INCIDENCE OF LISTERIA O N RAW VEGETABLES

Unless fertilized with human and/or animal waste or irrigated with water containing such
waste, raw vegetables (and fruits) normally should be free of most human and animal
enteric pathogens. Although the presence of soilborne spore-forming organisms such as
Clostridiurn perfringens and Bacillus cereus are of little consequence on raw vegetables,
these organisms can pose potential health problems in cooked vegetables that have been
held at inappropriate temperatures. Although fewer than 1 % of documented outbreaks of
foodborne illness during the last 10 years have been associated with consumption of vege-
tables, such outbreaks are now beginning to occur with greater frequency. L. monocyto-
genes is among the foodborne pathogens most often associated with these foods. This
may result, in part, from the variety of ways that L. monocytogenes can contaminate fresh
vegetables, as was depicted [10,11] (Fig. 1 ) by Beuchat.

\ soil (cross contamination)

plants- silage, feed - meat, milk, eggs

FIGURE 1 Mechanism b y which fresh produce can become contaminated with patho-
genic microorganisms and serve as vehicles of human disease. (Adapted from Ref.
11.)
Listeria monocytogenes in Products of Plant Origin 633

When one considers the enormous variety and quantity of produce being marketed
annually, routine microbiological examination of raw vegetables (and fruits) seems highly
impractical and probably unnecessary if good agricultural practices are used in growing
crops along with acceptable hygienic practices while harvesting, packaging, and trans-
porting raw produce to market. Although consumption of coleslaw prepared from contami-
nated cabbage was linked to a large Canadian outbreak of listeriosis in 1981 [68], in
retrospect, it appears that this outbreak could have been easily avoided if the coleslaw
manufacturer had realized that the cabbage farmer had fertilized the cabbage with sheep
manure from a flock that was previously diagnosed as having listeriosis. However, van
Renterghem et al. [74] suggested that L. rnonocytogenes dies quickly in fecal matter, and
therefore animal manure may not be as important in the spread of L. monocytogenes as
once thought. (The role of manure and sewage as vehicles for listeriae is discussed in
Chapter 2. ) However, they were able to demonstrate that L. rnonocytogenes could be trans-
ferred from contaminated soil to vegetables. In these experiments, carrots and radishes
were planted in soil which had been inoculated with L. rnonocytogenes (10 CFU/g soil).
They found that three of six radishes but none of the carrots grown in the inoculated soil
contained L. monocytogenes.
In spite of the Canadian outbreak, which resulted in 18 fatalities, it is not surprising
that, unlike dairy, meat, poultry, and seafood, no surveillance and/or regulatory programs
have been initiated until relatively recently to assess the incidence of listeriae in raw
vegetables (or fruits) marketed in the United States, Canada, or elsewhere. Nevertheless,
inadvertent isolation of L. rnonocytogenes from potato salad in April 1990 prompted a
Virginia-based manufacturer to recall 5700 pounds of product that had been distributed
in the southeastern United States [2]. During 1997 and the first half of 1998, six Class 1
recalls were issued for fresh frozen coconut [2c], hummus with red peppers and vegetables
[2a,2d,2e], sprouts [2f], and potato salad [2b], the last of which involved over 5.5 million
pounds of product. Although the risk of contracting listeriosis from such products is gener-
ally thought to be quite low, lack of routine microbiological analysis of raw produce
should not be interpreted to mean that fresh vegetables and fruits will always be free of
listeriae, including L. monocytogenes.

United States
In response to heightened concern about foodborne listeriosis, several small surveys were
conducted after the 1985 outbreak in California to determine the extent of Listeria contam-
ination in raw fresh and frozen vegetables destined for human consumption. During 1986
and 1987, Petran et al. [65] used the U.S. Food and Drug Adniinistration (FDA) procedure
in an attempt to isolate Listeria spp., including L. rnonocytogenes, from 23 retail samples
of vegetables, including fresh beet peels, broccoli, cabbage (outer leaves), carrot peels,
cauliflower sterns, corn husks, head lettuce, leaf lettuce, mushroom stems, potato peels,
and spinach as well as frozen green beans, pea pods, green peas, and spinach. Using
FDA and Centers for Disease Control and Prevention (CDC) procedures along with direct
plating, officials at the CDC [3] tried to isolate listeriae from 22 samples of broccoli,
carrots, celery, lettuce, green peppers, and potatoes in conjunction with several clusters
of listeriosis cases in Los Angeles County, California, and Philadelphia, Pennsylvania.
Finally, as part of a much larger survey dealing with the incidence of Listeria spp. in
retail meat, poultry, and seafood products, Buchanan et al. 1231 used an MPN (most proba-
ble number) method to examine two samples of potato salad for listeriae. As already
634 Brackett

implied, no Listeria spp. were recovered from any samples examined in the three surveys
just described. However, when one considers the small number of samples examined,
these findings cannot assure consumers that these products will always be free of listeriae.
In the first truly definitive survey reported, Heisick et al. [48] used the FDA proce-
dure to determine the incidence of various Listeria spp., including L. monocytogenes, in
10 different varieties of raw unwashed vegetables (total of 1000 samples) obtained from
two Minneapolis-area supermarkets between October 1987 and August 1988. As shown
in Table 1, Listeria spp. were detected in one or more samples of cabbage, cucumbers,
lettuce, mushrooms, potatoes, and radishes, but were never found in broccoli, carrots,
cauliflower, or tomatoes. Although L. monocytogenes, L. innocua, L. welshimeri, and L.
seeligeri were recovered from 5.0,2.6,0.8, and 1.3% of all raw produce examined, respec-
tively, with 41 of 50 (82%) and 9 of 50 (18%) L. monocytogenes strains classified as
serotypes l a and 4a/4ab, respectively, the overall incidence of Listeria spp. as well as L.
monocytogenes was markedly higher in radishes and potatoes than in other types of vegeta-
bles. Given that carrots recently were shown to possess some inherent antilisterial activity
[6,14,19,59,60], it appears that root crops such as potatoes and radishes more frequently
carry viable listeriae than other vegetables because of their close association with soil.
Interestingly, contamination rates for most raw vegetables were fairly consistent through-
out the year, and this reinforces the belief that listeriae populations remain relatively con-
stant in soil. These findings also are supported by those from a similar study in which
Heisick et al. [48] used four procedures to ultimately identify Listeria spp. in 19 of 70
(27.1%) and 25 of 68 (36.8%) potato and radish samples, respectively, with no listeriae
being detected in mushrooms, carrots, cabbage, broccoli, cauliflower, lettuce, tomatoes,
or cucumbers obtained from the same two Minneapolis supermarkets.
Use of an adequate isolation procedure and sufficient time to examine large numbers
of samples, has made it clear that a small percentage of raw vegetables marketed in
the United States is likely to harbor Listeria spp., including L. monocytogenes, with the
incidence of this pathogen being highest in root crops. Hence, the inability to detect
listeriae in raw vegetables examined in the three aforementioned surveys probably resulted
because insufficient numbers of samples were examined. In support of this observation,
Steinbruegge et al. [72] isolated L. monocytogenes from only 2 of 43 retail samples of
head lettuce purchased in Nebraska. Although the occasional presence of listeriae in retail
raw vegetables should not be viewed with alarm, careful handling and washing of all
produce to be consumed raw is recommended, particularly for pregnant women, the
elderly, and other individuals at greater than normal risk of developing listeriosis.
Others in the United States have similarly found L. monocytogenes to be an infre-
quent contaminant of fresh vegetables. Lin et al. [57] determined occurrence of L. monocy-
togenes and other foodborne pathogens in vegetable salads served in 3 1 food service estab-
lishments in Florida. Of the 63 vegetable salad samples tested, only one was contaminated
with L. monocytogenes. The vegetable salad from which the bacterium was isolated con-
sisted of iceberg lettuce, red cabbage, carrots, cucumbers, and tomatoes. Interestingly, this
salad and others yielding potentially pathogenic bacteria other than Listeria were pur-
chased from only 5 of the 31 establishments. Moreover, several of these implicated facili-
ties were apparently guilty of selling contaminated salads on more than one occasion,
with contamination most likely a result of product mishandling by workers.
The importance of proper sanitation and handling in minimizing L. monocytogenes
contamination of salads and vegetables was mentioned by Harvey and Gilmour [47]. They
suggested that systematic contamination of vegetable salads by L. monocytogenes was
Listeria monocytogenes in Products of Plant Origin 635

TABLE1 incidence of Various Listeria spp. in Unwashed Raw Retail Vegetables Marketed in the Minneapoiis, Minnesota, Area
Between October 1987 and August 1988
No. of positive samples (%)
No. of
samples L. rnono-
Type of vegetable analy zed cytogenes L. innocua L. welshimeri L. seeligeri Total
Broccoli 92 0 0 0 0 0
Cabbage 92 1 (1.1) 0 0 1 (1.1) 2 (2.2)
carrots 92 0 0 0 0 0
Cauliflower 92 0 0 0 0 0
Cucumbers 92 2 (2.2) 5 (5.4) 2 (2.2) 0 9 (9.8)
Lettuce 92 0 1 (1.1) 0 0 1 (1.1)
Mushrooms 92 0 11 (12.0) 0 0 1 1 (12.0)
Potatoes 132 28 (21.2) 5 (3.8) 1 (0.8) 0 34 (25.8)
Radishes 132 19 (14.4) 4 (3.0) 5 (3.8) 12 (9.1) 40 (30.3)
Tomatoes 92 0 0 0 0 0
Total 1,000 50 (5.0) 26 (2.6) 8 (0.8) 13 (1.3) 97 (9.7)

Source: Adapted from Ref. 47

636 Brackett

more likely a result of improper handling by food service workers rather than from natural
contamination of the raw product.

Canada
Despite the Canadian coleslaw outbreak of 1981, the incidence of listeriae in fresh produce
has received relatively little attention in Canada, with only one formal Canadian publica-
tion on the subject presently recorded in the scientific literature. Using the original FDA
procedure, Farber et al. [37] failed to recover any Listeria spp. from lettuce (50 samples),
celery (30 samples), or tomatoes (20 samples) purchased in Ottawa during 1988. However,
L. innocua was detected in 1 of 10 radish samples, which again suggests that the incidence
of listeriae may be somewhat higher in root crops than in other vegetables.

Western Europe
When the first edition of this book appeared in 1991, knowledge concerning the incidence
of listeriae in raw vegetables marketed in western Europe was confined to a few scattered
reports. Since then much more information has been published.
An increase in the number of listeriosis cases in England, along with the possibility
that some of these cases may have been food-related, prompted several surveys to deter-
mine the incidence of listeriae in various foods including dairy, meat, poultry and seafood
products, raw vegetables, and prepackaged salads. Working at Cambridge, Sizmur and
Walker [71] examined 10 different varieties of prepackaged salads obtained from two
leading area supermarkets. Overall, L. monocytogenes serotype 1/2 was isolated from 4
of 60 (6.7%) samples, with L. monocytogenes serotype 4b also being present in one of
these positive samples. Prepackaged salads from which the pathogen was recovered con-
sisted of two varieties that contained either (a) cabbage, celery, sultanas, onions, and car-
rots or (b) lettuce, cucumbers, radishes, fennel, watercress, and leeks. Both of these salad
varieties contained cabbage, cucumbers, and/or radishes-three of four raw vegetables
from which L. monocytogenes (predominantly serotype la) was isolated in the United
States (see Table 1). Although no Listeria spp. were recovered from plain bean sprout
salads or those that contained nuts, possibly because of a low pH, L. innocua was detected
in 13 of 60 (21.7%) samples representing five different varieties of mixed vegetables and/
or fruit salad. In addition to these findings, English investigators [41] also have isolated
L. monocytogenes from coleslaw. Thus, raw salad vegetables can serve as a potential
source of L. monocytogenes in the human diet.
Bending and Strangeways 171 proposed that a 74-year-old postoperative patient in
a London hospital may have acquired listerial septicemia and meningitis from consuming
contaminated lettuce. Although different serotypes of L. monocytogenes (1/2a and 1/2c)
were isolated from the patient and 1 of 11 (9.1 %) samples of washed English round
lettuce, recovery of the pathogen from washed lettuce prepared in the hospitals kitchen,
but not from 44 other food samples examined, suggests that consumption of washed raw
vegetables may pose a potential health threat to hospital patients, many of whom are
debilitated and/or immunocompromised.
Consumption of homemade uncooked salted mushrooms containing 1O6 L. monocy-
togenes serotype 4b CFU/g has been positively linked to a nonfatal case of listerial septice-
mia in an 80-year-old apparently healthy Finnish man [53] (see Chap. 10). (Working in
the Netherlands, van Netten et al. [73] also isolated L. monocytogenes from 2 of 20 raw
mushroom samples obtained from area markets.) During this investigation, low levels
Listeria monocytogenes in Products of Plant Origin 637

(<102CFU/g) of an unrelated L. monocytogenes strain belonging to serotype 1/2a also

were detected in carrots that were stored in the same cow barn with the tainted mushrooms,
thus making this the first account in which this pathogen has been recovered from raw
carrots, a vegetable that reportedly possesses listericidal properties [6,14,19,59,60].
As was true for surveys in England, preliminary data from Switzerland indicated
the presence of Listeria spp. in a small percentage of raw vegetable salads [21]. Although
27 raw vegetable samples obtained from a group of retail markets in Switzerland were
free of listeriae, L. monocytogenes was detected in 3 of 64 (4.7%) raw salad (2 mixed
salads and 1 parsley salad) obtained from the same group of markets. Similarly, L. innocua
was recovered from 4 of 64 (6.3%) raw salads. These values are essentially the same as
those reported 4 years later by Breer and Baumgarner [22] for German salads.
Earlier reports documenting the presence of L. monocytogenes in fresh produce and
the fact that this bacterium is ubiquitous in the environment has prompted Harvey and
Gilmour [47] to conduct an extensive survey of foods in Northern Ireland. Their results
were similar to previous reports in that low incidences of L. monocytogenes occurred in
fresh salad vegetables (8.9%) and prepared salads (7.5%). However, the rates of contami-
nation were not consistent. Vegetables and prepared salads obtained from one source never
yielded Listeria, whereas one third of products obtained from a different source were
contaminated. The authors used these results to suggest that, despite previous reports of
L. monocytogenes contamination of vegetables, producing a Listeria-free product was
possible.
Several studies have been published reporting the incidence of L. rnonocytogenes
in Spanish fresh produce. De S i m h et al. [31] surveyed 103 samples of raw vegetables
and reported an overall listeriae contamination rate of 18%. Listeria monocytogenes
was the most common Listeria species found (7.8%), followed by L. innocua (5.8%), L.
welshimeri (2.9%), and L. seeligeri (1.9%). The most common serovars of L. monocyto-
genes isolated were 1/2a (62.5%), 1/2c (25%), and 4b (12.5%).
More recently, Garcia-Gimeno et al. [39] also examined the prevalence and growth
of L. monocytogenes in vegetable salads produced in Spain. Unlike other market sample
surveys, these authors followed the presence and development of L. monocytogenes in
vegetable salad containing lettuce (75%),carrot (15%), and red cabbage (10%) produced
at a specific Spanish food processing plant. During processing, the individual ingredients
were cut, washed in potable water containing 100 mg/L active chlorine, placed in barrier
bags, and then stored or distributed. L. monocytogenes was isolated from 21 of the 70
samples examined. Isolates primarily belonged to serotype 3a, although 3b also was found.
The authors pointed out that the number of positive samples was much greater than previ-
ously published [37,49,65,7I] but offered no explanations for the high percentages found.
Most European surveys for the presence of listeriae in vegetables yielded similar
results. Except for samples from Spain [31,39], the incidence of listeriae in European
vegetables generally appears to be less than 10%. Moreover, the levels of L. monocyto-
genes are usually even lower.

Asia and the Middle East

Although the presence of L. monocytogenes in food has received most attention in Europe
and North America, scientists in the Middle East and in Asia also have conducted surveys
for the bacterium. Wong et al. [79) were among the first to publish results of an extensive
survey of foods, including a variety of vegetables, in Taiwan. They analyzed 49 different
638 Bracketf

vegetables and found L. monocytogenes present in 12.2% of vegetables sampled; the or-
ganism was only recovered from lettuce, Chinese cabbage, and green onions. On further
characterization, all isolates obtained from vegetables were of serotypes other than 1 and
4. In contrast, more than 90% of isolates from turkey and beef were serotype 1, with these
strains and those from seafood having greater hemolytic activity than isolates obtained
from vegetables. Consequently, these authors hypothesized that characteristics of L. mono-
cytogenes might be related to their food origins.
A similar survey of occurrence of L. monocytogenes in foods sold in Tokyo was
published by Ryu et al. in 1992 [66]. Their survey also included a variety of plant products,
including fresh vegetables, potato salad, and pickled vegetables. L. monocytogenes was
frequently isolated from meat (34%) and fish products (6.1%); however, no listeriae were
recovered from vegetable products or from ready-to-eat vegetable foods such as fermented
soybeans, cooked bamboo shoots, or coleslaw.
Arumugaswamy et al. [4] surveyed various fresh and ready-to-eat foods in Malaysia
and found the highest incidence of L. monocytogenes contamination yet reported. About
22% of leafy vegetables analyzed contained the bacterium, with similar proportions of
bean cakes and peanut sauces also reported as being positive. Although these values are
higher than those reported for other countries, they were lower when compared with other
Malaysian vegetable products tested. Eighty percent of ready-to-eat cucumber slices and
80% of bean sprouts tested positive for L. monocytogenes. The authors suggested that a
high percentage of positive samples may have resulted because many of the samples came
from street vendors or small processors, which often employed less than desirable sanita-
tion practices. They also suggested that the results indicate a need for health agencies to
put greater priority on street-vended foods.
Salamah [67] conducted an extensive survey for the presence of L. rnonocytogenes
in various fresh market vegetables sold in Riyadh, Saudi Arabia. A summary of their
results is given in Table 2. In general, all samples analyzed contained L. monocytogenes,
with incidence rates varying from 1.3 to 16.3%. Salamah confirmed the observations of
Heisick et al. [46] that root crops were more frequently contaminated with L. monocyto-
genes than vegetables grown above ground.
Finally, Gohil et al. [42] examined 183 imported and locally grown vegetables in
the United Arab Emirates for the presence of L. monocytogenes. Unlike other surveys,

TABLE 2 Presence of L. rnonocytogenes in Market

Vegetables in Saudi Arabia
No. of No. of
samples positive Incidence
Vegetables examined samples (%)
Cabbage 70 2 2.8
Carrot 120 16 13.3
Cucumber 110 4 3.6
Lettuce 80 I 1.3
Potato 80 13 16.3
Radish 75 8 10.7
Source: Adapted from Ref. 67
Listeria monocytogenes in Products of Plant Origin 639

however, they were unable to detect any L. monocytogenes in vegetables. However, they
isolated L. innocua from two samples each of imported and locally grown vegetables.

BEHAVIOR OF L. MONOCYTOGNS ON VEGETABLES

Despite the 1981 listeriosis outbreak in Canada directly linked to consumption of contami-
nated coleslaw [69] and an earlier cluster of listeriosis cases in Massachusetts that appears
to have been epidemiologically linked to raw celery, lettuce, and tomatoes, until recently
scientists were generally unconcerned about behavior of L. monocytogenes in raw produce.
This lack of concern probably existed because vegetables grown in modern industrialized
nations only rarely have been associated with any type of bacterial foodborne illness.
Furthermore, the source of contamination in the Canadian coleslaw outbreak was readily
traced to a farmer who fertilized cabbage with untreated manure obtained from a flock
of sheep that was previously diagnosed as having listeriosis. Consequently, most individu-
als in the scientific community probably viewed this outbreak as an isolated incident which
could have been easily prevented if better agricultural practices had been followed in
growing cabbage.
The fast changing global market and increase in consumption of fresh produce has
challenged the above assumptions. Many consumers do not realize that vegetables are
frequently imported from other countries. Consequently, such products can sometimes be
plagued with the same microbiological hazards as the countries in which they were grown.
Hence, a more global perspective on potential microbiological hazards, including Listeria
is warranted.
Interest in L. monocytogenes as a serious foodborne pathogen increased in 1985
following reports from California that at least 40 individuals died after consuming contam-
inated Mexican-style cheese. This outbreak sparked a renewed interest in the Canadian
coleslaw outbreak and also prompted a series of investigations to determine both the inci-
dence and behavior of L. monocytogenes on raw vegetables including cabbage. Results
from these studies will now be reviewed along with some data concerning thermal inacti-
vation of L. rnonocytogenes on cabbage and the effect of modified atmospheric storage,
chlorine, and lysozyme on growth and survival of this pathogen on various raw vegetables.
As was true for meat, poultry, and seafood, behavior of listeriae on raw produce has
become an active area of research. Hence, additional information regarding various means
to inactivate Listeria (e.g., chlorine dioxide, ozone) on raw vegetables and particularly
prepackaged refrigerated salads will probably be available in the near future.

Growth and Survival

Coleslaw was the first vegetable that was directly linked to an actual outbreak of listeriosis
in humans. Consequently, it is not surprising that growth and survival of L. monocytogenes
in cabbage was initially investigated. In the first such study published in 1986, Beuchat
et al. [ 181 determined behavior of L. monocytogenes strains Scott A (clinical isolate) and
LCDC 81-861 (Canadian cabbage isolate) on inoculated samples of shredded raw and
autoclaved (121"C/20 min) cabbage as well as in autoclaved (12l0C/15 min) salted
and unsalted cabbage juice during extended storage at 5 and 30C. According to the au-
thors, both test strains exhibited similar patterns of behavior on sterile cabbage, with popu-
640 Brackett

lations decreasing from approximately 107to 1O4 or 1O5 CFU/g during 42 days of refriger-
ated storage.
This apparent inability of L. monocytogenes to grow on heat-sterilized cabbage at
5C suggests that heating either decreases the availability of essential nutrients or leads
to development of toxic and/or inhibitory constituents in cabbage. In sharp contrast, L.
monocytogenes competed well with the normal aerobic flora and lactic acid bacteria of
raw cabbage, with Listeria populations increasing approximately 4 orders of magnitude on
raw cabbage during the first 25 days of refrigerated storage (Fig. 2). Thereafter, numbers of
listeriae failed to decrease appreciably on raw cabbage stored up to 64 days. Similar results
also were observed in subsequent studies by Hao et al. [44] and Lovett et al. [58]. Thus,
these findings demonstrate that L. rnonocytogenes can grow under conditions normally
encountered during shipping and distribution of cabbage. Although both Listeria strains
failed to grow in autoclaved cabbage juice containing >5% NaCl during 2 weeks of stor-
age at 3OoC, L. monocytogenes increased to 106CFU/g in the aforementioned homemade
salted mushrooms (-7.5% NaCl) during 5 months of cold storage [53]. Hence, behavior
of listeriae in raw vegetables appears to be greatly affected by incubation temperature as
well as the concentration of salt and various growth constituents [64].
Subsequently, Conner et al. [29] more closely examined the influence of tempera-
ture, NaCI, and pH on growth of L. monocytogenes in autoclaved (121"C/ I5 min) clarified
and unclarified cabbage juice. As in the previous study, salt-free unclarified cabbage juice
again was an excellent growth medium for L. monocytogenes, with initial populations of
- -
104CFU/mL increasing to I09CFU/mL after 8 days of incubation at 30C. Beyond
8 days, populations in cabbage juice containing low levels of salt decreased rapidly and
viable cells were no longer detected after 20 days of incubation at 30C. Growth rates of

M
0, 6-1 I
el

0 16 32 48 61
Days

FIGURE2 Behavior of L. monocytogenes (O), lactic acid bacteria (m), and total
aerobic microorganisms (A)o n raw cabbage incubated at 5C. (Adapted from
Ref. 18.)
Listeria monocytogenes in Products of Plant Origin 64 7

both Listeria strains at 30C decreased markedly in cabbage juice containing low levels
of salt, with inactivation of strains Scott A and LCDC 81-861 occurring in the presence
of 1 1 . 5 and 12.5% NaCI, respectively. As expected, behavior of both strains was strongly
influenced by acid production, with the pH of samples in which growth had occurred
decreasing from 5.6 to 5 4 . 3 after 8 days of incubation at 30C. Although numbers of
both Listeria strains failed to increase in salted and unsalted cabbage juice when the experi-
ment was repeated at 5"C, populations remained relatively stable, generally decreasing
only 10- to 100-fold during 70 days of refrigerated storage of cabbage juice contain-
ing 3.5-5.0% NaCI. Results from another study [28] suggest that viability of
Listeria in similar samples of salted and unsalted cabbage juice can be reduced by adding
extracts from several Chinese medicinal plants.
Interestingly, growth patterns for L. monocytogenes differed dramatically in clarified
cabbage juice, with both strains growing well at 30C in the presence of up to 2% NaCl.
Since similar changes were observed between pH and growth of listeriae in clarified and
unclarified cabbage juice, these findings suggest that particulate matter in unclarified cab-
bage juice may be partly inhibitory to L. monocytogenes in the presence of salt.
Using pH-adjusted unclarified cabbage juice, these researchers [25] further demon-
strated that L. monocytogenes failed to initiate growth at pH values <5 when inoculated
samples were incubated at 30C. Although more acidic environments (pH 14.8) were
lethal, complete inactivation of both Listeria strains did not occur until the pH was reduced
to -4.1. When the incubation temperature was lowered to 5"C, L. monocytogenes popula-
tions gradually decreased in cabbage juice adjusted to pH (5.2, with the pathogen surviv-
ing >63, 49, <21, and <21 days in samples adjusted to pH values of 5.2, 5.0, 4.8, and
4.6, respectively. In contrast, numbers of Listeria remained relatively constant during ex-
tended cold storage of cabbage juice adjusted to pH values >5.2. Since inactivation rates
were markedly slower at 5 than at 3OoC, it appears that lower temperatures help protect
L. monocytogenes from the harmful effects of low pH, as noted in Chapter 6.
Concern about behavior of L. rnonocytogenes in fresh produce has extended beyond
cabbage and now includes an ever increasing variety of fresh salad vegetables. In 1988,
Steinbruegge et al. [72] first reported results of a study which examined the ability of L.
monocytogenes to survive and grow on inoculated ( 103- l O5 CFU/g) samples of washed
retail head lettuce during storage in sealed and unsealed plastic bags at 5, 12, and 25C.
Although behavior of L. monocytogenes on lettuce was somewhat variable, the pathogen
generally grew under conditions simulating proper refrigeration, normal handling, and
ambient serving temperatures, with the pathogen increasing 1-4 orders of magnitude fol-
lowing 2 weeks of storage. Similar results were observed when inoculated samples of fresh
lettuce juice were held at 5C for 2 weeks. Salamah [67] later reported similar increases in
lettuce juices held at 26C and up to a 2-log increase at 4C.
Several more recent reports have appeared regarding the fate of L. monocytogenes
on various types of salad greens. According to Carlin and Nguyen-the [24], butterhead
lettuce (Lnctuca sativa L.) supported growth of L. monocytagenes better than did endive
(Cichoriumendivia L.), but the bacterium was unable to grow on lamb's lettuce (Valeria-
nella olitoria L.). In contrast, populations of total aerobic microorganisms were unaffected
by salad type. The authors offered no hypothesis as to why lamb's lettuce failed to support
growth of L. monocytogenes.
Carlin et al. [25] followed up on their previous work by more closely examining
several factors that affected growth of L. monocytogenes on endive, with emphasis on
storage temperature, age, and quality of the endive leaves, role of epiphytic microflora,
642 Brackett

and the strains and initial concentration of L. monocytogenes present. Overall, the growth
rate of L. monocytogenes was essentially the same as that of the natural aerobic microflora
at 10 and 20C but slower than the native microflora at 30 and 6C. Furthermore, the
bacterium grew faster when initially present at lower (10-1000 CFU/g) rather than at
higher ( 105CFU/g) populations. In accord with earlier results of Beuchat and Brackett
[ 151, Carlin et al. [25] detected no differences in growth among the various strains tested.
Carlin and coworkers [26] subsequently investigated in detail the role of indigenous
microflora on growth of L. monocytogenes in unsanitized endive leaves and on leaves
treated with 10% hydrogen peroxide to reduce or eliminate the indigenous microflora.
These investigators also challenged L. monocytogenes with individual strains of pseu-
domonads and Enterobacteriaceae isolated from endive. The authors observed that reduc-
ing the native microflora by disinfection resulted in higher populations of L. monocyto-
genes on endive leaves. Moreover, they also observed that high populations ( 106- 107
CFU/g) of some strains of indigenous microorganisms reduced growth of L. monocyto-
genes on endive. A complex mixture of various microorganisms isolated from endive
completely inhibited growth of L. monocytogenes in a medium composed of endive leave
exudate.
In addition to lettuce, tomatoes are among the most popular ingredients in fresh
salads. Beuchat and Brackett [16] demonstrated that tomatoes are not a good substrate
for growth of L. monocytogenes, probably because of their acidity. Although some growth
of Listeria was evident on whole tomatoes after 10-2 1 days of storage at 10 and particu-
larly 21"C, the pathogen was inactivated in chopped tomatoes (-pH 4.1) held at these
same temperatures. Additionally, when commercial tomato products were inoculated to
-
contain 1O6 L. monocytogenes CFU/g, populations remained reasonably stable in tomato
sauce and tomato juice during 14 days of storage at 21 and particulary 5C. However,
the pathogen survived only 4 and 8 days in samples of ketchup held at 21 and 5"C, respec-
tively, with Listeria inactivation being attributed to higher levels of acetic acid in ketchup
as compared with the other tomato products.
Information regarding the fate of L. monocytogenes in or on other types of salad
vegetables also is limited; however, results from several studies indicate that with the
exception of raw carrots [ 14,221, fennel, red cabbage, and Savoy cabbage [22], and beets
[2 11, this pathogen will grow and/or survive on most other types of fresh produce including
asparagus [9], broccoli [9], cauliflower [9], corn [52], green beans [52], lettuce [15,52],
certain types of cabbage [22,29], celery [21], potato juice [67], and radishes [58] during
the normal refrigerated shelf life of the product. Gianfranceschi and Aureli [40] similarly
noted that survival of L. monocytogenes in spinach was essentially unaffected by freezing
at -50C and extended storage at - 18C. In addition, Sizmur and Walker [7 11 reported
that L. monocytogenes populations in several naturally contaminated vegetable salads pur-
chased from two supermarkets in England increased approximately twofold after 4 days
of refrigerated storage. Given the apparent ability of L. monocytogenes to survive and/or
grow on most raw salad vegetables and the possible presence of this pathogen on many
types of raw produce, health officials need to consider raw vegetables as another possible
source of listerial infections.
The observation that L. monocytogenes can thrive in various fresh vegetables also
led to questions regarding its growth and survival in products prepared from these vegeta-
bles. In 1995, Lee et al. [56] published a study on growth and survival of L. monocytogenes
in kimchi, a traditional Korean fermented vegetable product. This product can be prepared
from various ingredients, but the most common type contains Chinese cabbage and various
Listeria monocytogenes in Products of P h t Origin 643

flavoring agents such as red pepper, garlic, ginger, NaCl, and pickled seafood. These
ingredients are mixed together and subjected to a natural lactic acid fermentation, with
the product ultimately reaching a mildly acidic pH [4-51. The authors found that popula-
tions of L. monocytogenes Scott A increased during the first 2 days of kimchi fermentation
but then decreased. Although the bacterium was eventually inactivated by kimchi ingredi-
ents and low pH, the pathogen still persisted after 10 days of fermentation. However, the
authors concluded that kimchi could be safely produced by using ingredients of good
microbiological quality.

Modified Atmosphere Storage

The widespread practice of packaging and/or storing fresh produce in a modified atmo-
sphere has led to dramatic increases in types of produce available to consumers and in
their shelf life so that most fresh vegetables (and fruits) are now available throughout the
year. However, given the occasional presence of L. monocytogenes on raw produce, the
ability of this pathogen to multiply relatively rapidly under microaerobic conditions at
refrigeration temperatures and the present popularity of modified-atmosphere packaging,
legitimate questions have been raised about the safety of refrigerated produce during long-
term storage with modified atmosphere [78].
In response to these concerns, Berrang et al. [9] investigated behavior of L. monocy-
togenes on inoculated ( 103- l O5 CFU/g) samples of fresh asparagus, broccoli, and cauli-
flower during extended refrigerated storage in glass jars containing (a) 15% 0 2 : 6 %
c o 2 : 7 9 % N2, (b) 11% 0,: 10% C02:79% N2, (c) 18% 02:3% c o 2 : 7 9 % N2, or (d) air.
L. monocytogenes behaved similarly on each vegetable when the product was stored in
a modified atmosphere or air. All three vegetables supported growth of L. monocytogenes
at 15"C, with the pathogen attaining populations of -106 to nearly 109/g when these
products were first deemed to be unfit for human consumption 6-10 days after the start
of incubation. Although storage in a modified atmosphere at 15C did not appreciably
affect growth of listeriae on any of the three vegetables examined, the ability of such
storage conditions to increase the shelf life of these products by 2-4 days beyond that of
the products packaged in air led to higher Listeria populations when these vegetables were
first declared inedible by subjective evaluations. In contrast, only asparagus supported
growth of L. monocytogenes at 4"C, with initial numbers being approximately 10- to 100-
fold higher at the end of the product's 21-day shelf life. However, numbers of listeriae
remained relatively constant on broccoli and cauliflower during 21 days at 4C regardless
of the storage atmosphere. Since these findings and those of the previous study indicate
that L. monocytogenes is basically unaffected by controlled-atmosphere storage, the ex-
tended shelf life gained with such storage conditions provides additional time for growth
of this pathogen which, in turn, increases the public health hazard associated with con-
sumption of' raw vegetables. Similarly, Garcia-Gimeno et al. [39] and Kalander et al. [54]
observed that use of modified atmosphere with salads neither enhanced nor repressed
growth of L. monocytogenes but that the extended shelflife afforded by modified-atmo-
sphere packaging increased the risk of foodborne illness.
Subsequently, Beuchat and Brackett observed that L. monocytogenes behaved simi-
larly when inoculated samples of iceberg lettuce [ 151 and tomatoes [ 161 were stored at
5" or 10C (lettuce) or 10" and 21C (tomatoes) in 3% 02:97% N 2 or air. As with cabbage
and asparagus, the pathogen grew on lettuce, reaching populations of 1ON- 1O9 CFU/g after
10 days of storage at IOOC, with only slight growth observed in identical samples held
644 Braekett

at 5C. However, the bacterium only achieved populations of about 105-106 CFU/g in
tomatoes regardless of storage atmosphere.
The term modijied atmosphere usually refers to systems where atmosphere in which
a product is stored is intentionally changed to the desired gas composition. One such
example is vacuum packaging of produce. This action has the effect of reducing O2 and
thereby slowing respiration and senescence. Aytac and Gorris IS] investigated the effect
of a moderate vacuum on growth of L. monocytogenes in chicory endive and mung-bean
sprouts; the organism responded differently depending on the product in question. Growth
of L. monocytogenes at 5.6"C was enhanced by 400 mB of vacuum in the endive but was
repressed in sprouts. The authors pointed out the need for additional barriers when such
techniques are used to extend the shelf life of fresh vegetables.
The atmosphere also can be changed as a result of metabolic processes of fresh
fruits and vegetables. In this instance, gas-permeability characteristics of the packaging
material can often drastically affect the atmosphere within the package and, consequently,
the microflora in the food. Omary et al. [6 1 ] investigated the influence of packaging mate-
rial on growth of L. rnonocytogenes in shredded cabbage packaged in films having oxygen
transmission ratios (OTR) of 5.6, 1500, 4000, and 6000 cc 02/m2/24h. They found that
the type of packaging material used had a significant effect on growth of L. innocua (as
a substitute for L. monocytogenes) in cabbage. Populations of L. innocua decreased in all
samples after 14 days of storage regardless of packaging film used. However, populations
of L. innocua then increased 3.5 logs CFU/g in cabbage packaged in all but the film with
the highest ORT. In that instance, populations only increased by about 2 logs CFU/g. In
the latter sample, CO2 concentrations had equilibrated at near ambient concentrations,
whereas concentrations reached from 30 to 90% when films of lower OTR were used.
Although most publications to date indicate that only low populations of L. monocy-
togenes infrequently contaminate fresh produce, the chance of this pathogen multiplying
in products with extended shelf life is significant. Therefore, it appears prudent for handlers
of raw produce to store their product in a modified atmosphere and institute sanitation
and quality control programs that will decrease the incidence of listeriae in incoming raw
vegetables. It also may be necessary to shorten the marketable period for such products
even though the food may appear to be acceptable.

Inactivation
As afollow-up to the aforementionedstudies dealing with growth and survivalof L. monocyto-
genes in raw vegetables, scientists also examined different methods by which this pathogen
can be eliminatedfrom raw vegetables. Although these methods, which will now be discussed,
primarily involve use of heat, chlorine, and lysozyme, information concerning the effect of
other methods such as ozone, chlorine dioxide, and irradiation should be forthcoming.
In response to the 1981 listeriosis outbreak in Canada involving consumption of
contaminated coleslaw, Beuchat et al. [ 181 investigated thermal inactivation of L. monocy-
togenes in cabbage juice. Flasks of sterile, clarified cabbage juice adjusted to pH 4.0, 4.6,
and 5.6 were inoculated to contain approximately 4 X 106 L. monocytogenes CFU/mL;
placed in a shaking water bath at 50, 52, 54, 56, or 58C; and sampled for listeriae at
10-min intervals for up to 60 min. As expected, thermal inactivation rates for Listeria in
cabbage juice at 50, 52, 54, and 56C were faster at lower pH values, with D-values of
25, 14, 6.7, and 3.6 min at pH 4.6 as compared with D-values of 60, 34, 8.4, and 6.8 min
at pH 5.6, respectively. No viable cells were detected in cabbage juice held at 58C for
Listeria monocytogenes in Products of Plant Origin 645

10 min. Although inactivation rates were unaffected by addition of 1 or 2% NaCl to

cabbage juice, sublethally injured cells were more sensitive to NaCl on a nonselective
plating medium (Tryptic Soy Agar) than were uninjured cells. As shown by data in Figure
2, L. monorytogenes can multiply on raw cabbage during extended refrigerated storage;
however, results from the study just discussed suggest that normal pasteurization treat-
ments given to cabbage juice and sauerkraut are probably sufficient to eliminate any viable
listeriae that may be present. Hence, unlike unfermented raw vegetables, the risk of con-
tracting listeriosis from sauerkraut and other pasteurized fermented vegetable products
appears to be minimal.
Brackett 1201 investigated the possibility of using hypochlorite solutions to inacti-
vate L. monocytogenes on the surface of Brussels sprouts. In this study, fresh retail
-
Brussels sprouts were inoculated to contain 1O6 L. monocytogenes CFU/g, immersed
in a hypochlorite solution containing 200 mg of chlorine/L, removed, air dried for 30 min,
and examined for numbers of surviving listeriae. The procedure just described decreased
populations of L. monocytogenes on Brussels sprouts approximately I 00-fold. However,
since dipping inoculated Brussels sprouts in sterile chlorine demand-free water reduced
the number of viable listeriae by approximately 10-fold, the author concluded that many
cells were simply washed from the surface rather than inactivated by chlorine. In a follow-
up study with fresh retail lettuce, Beuchat and Brackett [15] also found that L. monocyto-
genes was still present at levels of 1OS- 1O6 and I 07- 1 Ox CFU/g after extended storage at
5 and 10C, respectively, regardless of whether or not the lettuce was pretreated with a
sodium hypochlorite solution to reduce the population of naturally occurring microflora.
Zhang and Farber [801 evaluated several sanitizers and sanitizer combinations, in-
cluding sodium hypochlorite, chlorine dioxide, trisodium phosphate, and lactic and acetic
acids, for inactivation of L. monocytogenes on lettuce and cabbage. Their results generally
were similar to those of Brackett [ 181 in that disinfectants were ineffective at reducing
populations of L. monocytogenes. None of the treatments tested provided more than a
1.7-log reduction and most were under a 1-log reduction. Trisodium phosphate had no
effect on listeriae and surfactants actually reduced efficacy of sanitizers. As indicated in
Chapter 6, hypochlorite can be used very effectively to inactivate L. monocytogenes in
water supplies and on the surface of previously cleaned equipment. However, current
evidence indicates that chlorine dips are relatively ineffective for eliminating L. monocyto-
genes from contaminated raw vegetables.
After demonstrating that egg white lysozyme, a GRAS (generally recognized as
safe) food additive, inhibited growth of several foodborne pathogens, including L. monocy-
togenes, in laboratory media and phosphate buffer [51], Hughey et al. [55] investigated
the possibility of using this enzyme during refrigerated storage to inactivate L. monocyto-
genes on various retail vegetables, including fresh lettuce, cabbage, sweet corn, green
beans, and carrots as well as previously frozen corn and green beans. In this study,
1.8-kg portions of coarsely shredded or cut fresh and thawed frozen vegetables were
treated to contain 100 mg of lysozyme/kg of vegetables and/or 5 mM of ethylenediamine
-
tetraacetic acid (EDTA), inoculated to contain 103-104L. monocytogenes CFU/g, mixed
by hand, and examined for numbers of listeriae during extended storage at 5C.
Overall, lysozyme was fairly effective in decreasing populations of L. monocyto-
genes on the surface of fresh vegetables, particularly when used in conjunction with
EDTA, which presumably facilitated cell lysis by increasing contact between lysozyme
and peptidoglycan in the cell wall. Listericidal effects from the combined use of lysozyme
and EDTA were most pronounced on lettuce, with the pathogen no longer being detected
646 Brackett

after 12 days of refrigerated storage (Fig. 3). Although lysozyme alone was listeriostatic,
use of EDTA alone failed to prevent growth of L. monocytogenes, with the pathogen
eventually attaining levels only slightly lower than those observed in untreated lettuce.
Listeria behaved similarly on fresh green beans and sweet corn with two exceptions: (a)
growth occurred on lysozyme-treated sweet corn and (b) combined use of lysozyme and
EDTA never completely eliminated the pathogen from either product. Unlike fresh lettuce,
green beans, and sweet corn, numbers of listeriae on EDTA-and lysozyme-treated raw
cabbage increased during the first 20 days of refrigerated storage and then decreased
4-5 orders of magnitude during 28 days of incubation at 5C. Although combined use
of lysozyme and EDTA again was most listericidal, 41 days of refrigerated storage were
required to rid this lettuce of listeriae. Unlike other fresh vegetables, the pathogen was
eliminated within 9 days from untreated raw carrots as well as from those that were treated
with lysozyme alone or in combination with EDTA (Fig. 4). Hence, these findings support
the notion that carrots probably contain one or more naturally occurring listericidal sub-
stances [ 141.
In contrast to fresh vegetables, numbers of listeriae remained relatively constant
on previously frozen green beans and corn that were treated with lysozyme alone or in

"1

3i
\ 0 EDTA
Control

2i\
0 5 15
Days

FIGURE3 Behavior of L. rnonocytogenes on fresh lettuce treated with lysozyme (Lys)

and EDTA. (Adapted from Ref. 58.)
Listeria monocytogenes in Products of Plant Origin 647

J K 0 Control

FIGURE4 Behavior of L. rnonocytogeneson fresh carrots treated with lysozyme (Lys)

and EDTA. (Adapted from Ref. 58.)

combination with EDTA. This apparent failure of lysozyme to inactivate listeriae on frozen
vegetables may be related to loss of certain lysis-enhancing substances during processing
of vegetables. These findings, together with the current use of lysozyme to prevent growth
of gas-producing spore-forming bacteria in certain European cheeses, suggest that com-
mercial use of lysozyme in combination with other previously discussed measures should
help to inhibit Listeria and other foodborne pathogens on fresh vegetables.
Various plant components have long been known to possess antimicrobial proper-
ties. Specifically, the essential oils of herbs and spices have been studied most extensively
in this regard. Kim et al. [55] demonstrated the antilisterial activity of various essential
oil components in vitro and suggested that these components might also be incorporated
into foods as barriers to microbial growth. Hao and Brackett [45] and Hao et al. [46]
tested this suggestion by determining the efficacy of plant extracts in inhibiting growth of
L. monocytogenes in beef and chicken, respectively. They found that eugenol and pimento
extracts significantly inhibited growth of the bacterium on cooked chicken breasts during
refrigerated storage [46]. However, they also noted that the type of food in which extracts
were used was important. Unlike chicken, none of the spice extracts tested effectively
inhibited growth of L. monocytogenes in refrigerated, cooked beef [45]. Moreover, some
extracts contributed strong odors and flavors which would need consumer approval if
actually used for commercial products.
As mentioned previously, carrots reportedly possess some inhibitory antilisterial
factor(s). Beuchat and Brackett [ 141 were the first to document this when they attempted
to artificially inoculate carrots by dipping in suspensions of L. monocytogenes. They noted
that populations of the bacterium decreased on both raw whole and shredded carrots but
not cooked carrots. Moreover, numbers of L. monocytogenes decreased in the inoculating
648 Brackett

suspensions after dipping of shredded carrots. Based on these results, they suggested that
the antilisterial component(s) of carrots is heat sensitive and released when carrot tissue
is damaged. The authors suggested that phytoalexin 6-methoxymellein might be one poten-
tial compound responsible for the antilisterial action. The results of Beuchat and Brackett
were later confirmed by Nguyen-the and Lund [59], who also noted that maceration of
carrots in a high-speed blender or in liquid nitrogen likewise destroyed the antilisterial
activity.
The obvious potential for using carrot juice or its antilisterial component(s) as a
natural antimicrobial agent in foods prompted the same two groups of researchers to inves-
tigate the mechanism of antilisterial activity. Nguyen-the and Lund [60] found that the
antilisterial effect of carrots was suppressed by anaerobiosis, thiol compounds, bovine
serum, and the free-radical scavengers histidine and diazabenzocyclooctane. However, the
activity was not affected by sodium ascorbate, propyl gallate, catalase, superoxide dismu-
tase, or chelating agents but was enhanced by Tween 20. Despite these results, these
authors were unable to determine a specific compound or compounds responsible for the
antilisterial activity.
Beuchat et al. [ 171 likewise attempted to characterize the antilisterial component(s)
of carrots. They found that the lethal and inhibitory effects were greatest in the pH range
of 5.0-6.4 and that the optimum concentration of carrot juice needed to inhibit L. monocy-
togenes growth was about 10%. In addition, they observed that NaCl at concentrations
up to 5% protected the listeriae from the antimicrobial action of carrot juice, especially
in 10% juice incubated at 5 or 12C. Despite these observations, they were unable to
identify the compound(s) responsible for antilisterial activity or predict the effectiveness
of carrot juice as an antilisterial ingredient in food. However, Babic et al. [6] suggested
that dodecanoic acid and methyl esters of dodecanoic and pentadecanoic acids identified
in purified active extracts of carrots may be responsible for antimicrobial activity.
Looking at the potential of using carrot juice as an antilisterial treatment for foods,
Beuchat and Doyle [19] determined the influence of dipping shredded lettuce in 20 or
50% carrot juice or adding up to 10% carrot juice to Brie cheese and frankfurter homoge-
nates. Overall, both concentrations of carrot juice significantly repressed growth of L.
monocytogenesin shredded lettuce stored at 5 and 12C but not at 20C. It is also notewor-
thy that the carrot juice was rather specific in its activity in that it had no discernible effect
on growth of other aerobic microorganisms. In contrast to lettuce, addition of carrot juice
to Brie cheese was less effective and was completely ineffective in frankfurter homoge-
nates. Hence, it appears that carrot juice may be of value as an antilisterial agent in some
foods.

INCIDENCE OF LISTERIA IN FRUITS

Unlike raw vegetables, information concerning the incidence of Listeria spp. on raw fruit
is virtually nonexistent. The first documentation was a preliminary report [3] in which
CDC officials failed to recover listeriae from seven fruit samples (e.g., cherries, pears,
peach, avocado, and tomato) while investigating two clusters of presumed foodborne liste-
riosis in Los Angeles County, California, and Philadelphia, Pennsylvania. More recently,
Schlech [68] mentioned that blueberries, strawberries, and nectarines were implicated in
outbreaks of listeriosis. One of the few culture-confirmed cases of listeriosis resulting
from consumption of a fruit product occurred in Italy. In this case, DNA fingerprint-
Listeria monocytogenes in Products of Plant Origin 649

ing was used to link consumption of pickled olives with a sporadic neonatal case of liste-
riosis [27].
Although scientific evidence is lacking, two observations, namely, (a) infrequent
association between consumption of fruit and listeriosis and (b) most fruits grow well
above ground and are therefore not subject to frequent contact with Listeria-contaminated
soil or feces, lead one to speculate that the incidence of listeriae on fruit may well be as
low or lower than that observed for raw vegetables.
Given the probable low incidence of listeriae on raw fruits, it may seem somewhat
surprising to learn that FDA officials prompted an Oregon firm to issue a Class I recall
for over 500,000 flavored frozen ice and juice bars that were contaminated with L. monocy-
togenes during the latter stages of manufacture [ 11, which suggests postpasteurization
contamination. Since raw milk also was routinely processed into frozen dairy products at
this same facility, L. monocytogenes was most likely introduced into the factory environ-
ment through the raw milk supply rather than fruit juice. If this is true, then it follows
that the incidence of listeriae in highly acidic fruit juice is likely to be extremely low.
This view is further supported by results from a recent survey in which Parish and Higgins
[62] failed to detect any Listeria spp. in 100 retail samples of reconstituted single-strength
orange juice (pH 3.63-3.84) that were pasteurized at 30 geographically distinct dairy and
nondairy facilities located across the United States and Canada.

BEHAVIOR OF L. MONOCYTOGENES IN FRUIT JUICES

Since we currently lack appreciable information on the incidence of listeriae in raw fruits,
it is not surprising that our knowledge of Listeria behavior in these products also is ex-
tremely limited. As of January 1997, results from only two studies dealing with viability
of listeriae in orange serum and juice have appeared in the scientific literature.
In 1989, Parish and Higgins [63] published data from the first of two studies in which
orange serum was adjusted to pH values of 3.6-5.0 with hydrochloric acid, inoculated to
contain -106 L. monocytogenes CFU/mL, and examined for numbers of listeriae during
prolonged incubation at 4 and 30C. As was true for cabbage juice [29], behavior of
listeriae in orange serum also was markedly influenced by incubation temperature and
pH. Overall, L. monocytogenes failed to grow in refrigerated orange serum adjusted to
pH 5 4 . 6 and was completely eliminated from these samples after 18 to 70 days of storage,
with lowest pH values proving most detrimental to survival of Listeria (Fig. 5). However,
modest growth of listeriae was observed in orange serum samples at pH 5 , with the patho-
gen still being present at levels of 102-103CFU/mL in the two least acidic samples after
90 days of refrigerated storage. Incubation at 30C led to Listeria increases of approxi-
mately 10- and 100-fold in orange serum samples adjusted to pH values of 4.8 and 5.0,
respectively. As was true for unclarified cabbage juice [29], overall viability of listeriae
again was greatly reduced by raising the incubation temperature, with the pathogen gener-
ally being eliminated from orange serum samples at pH 3.6-4.0 and 4.2-5.0 after 5 and
8 days of incubation at 3OoC, respectively.
These same authors [62] subsequently used several enrichment procedures to deter-
mine viability of listeriae in single-strength reconstituted samples of commercial frozen
-
concentrated orange juice that were inoculated to contain 1-- 10 L. monocytogenes CFU/
mL. Although numbers of inherent microorganisms (primarily lactic acid bacteria and
yeast) increased from - 102to 10' CFU/mL after 4 weeks of incubation at 4OC, L. monocy-
togenes was eliminated from reconstituted frozen orange juice (average pH of 4.06) after
650 Brackett

10 20 30 40 50 60 70 80 90
Days

FIGURE5 Behavior of L. monocytogenes in pH-adjusted orange serum during ex-

tended incubation at 4C.(Adapted from Ref. 63.)

42 days of refrigerated storage. These findings appear to be consistent with those from
the previous study involving orange serum.

BEHAVIOR OF L. MONOCYTOGNS IN OTHER

PRODUCTS OF PLANT ORIGIN
Increased consumer demand for precooked, long shelf life, ready-to-eat foods containing
a minimum of preservatives is making development of microbiologically safe products
increasingly difficult. Not too many years ago, it was thought that no foodborne pathogen
could multiply in properly refrigerated food. However, this belief has been proven invalid
by emergence of L. monocytogenes and Yersinia enterocolitica, in particular, as causes
of foodborne illness. Thus public health officials have become concerned about the safety
of many cook-chilled and ready-to-eat foods of animal origin, including fermented and
unfermented dairy products, luncheon meats, sausage, and precooked chicken as well as
peel-and-eat shrimp. These concerns have since spread to products of plant origin, includ-
ing soy milk, precooked delicatessen products such as ravioli (prepared in part from flour),
and food colorants derived from red beets.
Since there is an increased use of soy milk by individuals who cannot tolerate cow's
milk, the ability of Listeria to proliferate in this product should be known. Hence, Ferguson
and Shelef [38] inoculated commercially available pasteurized and sterile soy milk to
contain 1 O2 or l O4 L. monocytogenes CFU/mL and incubated the products at 5 and 22C.
The pathogen attained maximum populations similar to those previously observed in cow's
milk, reaching levels of 7 X 10'- 3 X 109and 8 X 10' CFU/mL of soy milks following
3 and 30 days of incubation at 5 and 22"C, respectively. Not surprising, generation times
for L. monocytogenes in soy milk, 1.55 h at 22C and 37.68 h at 4OC, are similar to those
previously reported for the same strain of L. monocytogenes in cow's milk (see Chap. 10).
No one has yet investigated the incidence of Listeria spp. on soybeans; however, since
listeriae are relatively common in soil, the potential exists for these organisms to find their
way into soy milk-processing factories and also into the finished product as a postpasteur-
ization contaminant. If this is true, then rapid growth of L. monocytogenes in soy milk at
Listeria monocytogenes in Products of PIant Origin 65 7

refrigeration temperatures suggests that this product should not be overlooked as a possible
vehicle of human listerial infections.
Concern about behavior of Listeria in delicatessen products marketed in England
and the United States prompted Beuchat and Brackett [13] to investigate viability and
thermal inactivation of L. monocytogenes in commercially prepared meat, cheese, and egg
ravioli purchased from Atlanta-area delicatessens. The growth portion of this study in-
volved quantitation of L. monocytogenes in inoculated (- 104and 1 O6 CFU/g) samples
of ravioli during 14 days of incubation at 5C. For thermal inactivation tests, the three
types of ravioli were inoculated to contain 3 X 105L. monocytogenes CFU/g, stored 0
or 9 days at 5OC, and then boiled up to 7 min using cooking procedures that might be
practical in the home. Overall, numbers of viable listeriae decreased < 10-fold during the
9-day refrigerated shelf life of the three types of ravioli. Results of thermal inactivation
studies indicated that normal cooking procedures (7 min of boiling) were adequate to
-
destroy L. monocytogenes populations of 105CFU/g in all three types of ravioli regard-
less of whether or not ravioli was refrigerated 0 or 9 days before cooking. Although this
study provides valuable information concerning behavior of Listeria in ravioli, there ap-
pears to be an urgent need for more work of this type to address the microbiological safety
of precooked and/or ready-to-eat delicatessen products such as sandwiches, filled rolls,
pizza, garlic bread, desserts, confectionery products, and chocolate, since work in England
[41] and elsewhere has shown that all of these products can harbor L. monocytogenes.
Increased use of plant-based food colorants prompted El-Gazzar and Marth [35] to
investigate behavior of Listeria in a commercial aqueous extract from the red beet root
(Beta vulgaris) to which vitamin C, citric acid, and sodium propionate ( 51.5%) were
added as preservatives. As in their previous work with milk coagulants [33,34] and annatto
colorants [32], samples of beet extract were inoculated to contain 103-107L. monocyto-
genes strain CA, V7, or Scott A CFU/mL and examined for numbers of survivors during
prolonged storage at 7C. Not surprisingly, the combined effect of a relatively low pH
of 4.3-4.8 and sodium propionate prevented growth of listeriae in all samples of beet
colorant. However, although 42-56 days of incubation at 7C was sufficient to rid these
extracts of 10'-104 strain CA CFU/mL, this strain was still detected in 56-day-old samples
that contained larger initial populations. In contrast, strains V7 and Scott A were far more
resistant to the listericidal action of beet extract, with both strains being recovered at levels
of 10'-104 CFU/mL, depending on initial inoculum, following 56 days of storage. Hence,
unlike highly alkaline annatto extracts in which L. monocytogenes was inactivated almost
instantaneously (see Chap. 12), prolonged survival of listeriae in beet colorants makes it
imperative that these extracts be processed and handled carefully to prevent their contami-
nation with this pathogen.
The sporadic nature of listeriosis suggests that L. monocytogenes can be an infre-
quent problem in unusual foods of plant origin as well as fruits and vegetables. Whereas
this viewpoint is supported by one sporadic case of listeriosis in Canada that was linked
to alfalfa tablets [30,36], the fact that L. monocytogenes can be present in virtually any
ecological niche suggests that occasional cases of listeriosis are likely to be associated
with unusual as well as common plant-based foods.

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Hightower, S.E. Johnson, S.H. King, E.S. Nichols, and C.V. Broome. 1983. Epidemic listeri-
osis: evidence for transmission by food. N. Engl. J. Med. 308:203-206.
70. Simpson, D.M. 1996. Microbiology and epidemiology in foodborne disease outbreaks: the
whys and why nots. J. Food Prot. 59:93-95.
71. Sizmur, K.I., and C.W. Walker. 1988. Listeria in prepackaged salads. Lancet i: 1167.
72. Steinbruegge, E.G., R.B. Maxcy, and M.B. Liewen. 1988. Fate of Listeria monocytogenes on
ready to serve lettuce. J. Food Prot. 5 1596-599.
73. Van Netten, P., I. Perales, A. van de Moosdijk, G.D.W. Curtis, and D.A.A. Mossel. 1989.
Liquid and solid selective differential media for the detection and enumeration of L. monocyto-
genes and other Listeria spp. Int. J. Food Microbiol. 8:299-3 16.
74. Van Renterghem, B., F. Huysman, R. Rygole, and W. Verstraete. 1991. Detection and preva-
lence of Listeria monocytogenes in the agricultural ecosystem. J. Appl. Bacteriol. 71 :211-
217.
75. Weis, J. 1975. The incidence of Listeria monocytogenes on plants and in soil. In M. Woodbine,
ed. Problems of Listeriosis. Surrey, UK: Leicester University Press, pp. 61-65.
76. Weis, J., and H.P.R. Seeliger. 1975. Incidence of Listeria monocytogenes in nature. Appl.
Microbiol. 30:29-32.
77. Welshimer, W.J. 1968. Isolation of Listeria monocytogenes from vegetation. J. Appl. Bacte-
ri01. 95:300-303.
78. Willcox, F., P. Tobback, and M. Hendrickx. 1994. Microbial safety assurance of minimally
processed vegetables by implementation of the hazard analysis critical control point (HACCP)
system. Acta Aliment. 23:221-238.
79. Wong, H.-C., W.-L. Chao, and S.-J. Lee. 1990. Incidence and characterization of Listeria
monocytogenes in foods available from Taiwan. Appl. Environ. Microbiol. 56:3 101-3 104.
80. Zhang, S., and J.M. Farber. 1996. The effects of various disinfectants against Listeria monocy-
togenes on fresh-cut vegetables. Food Microbiol. 13:31 1-321.
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17
Incidence and Control of Listeria
in Food-Processing Facilities

ROBERTGRAVANI
Cornell University, Ithaca, New York

INTRODUCTION
Overwhelming evidence indicates that when L. monocytogenes and other Listeria spp. are
present in commercially processed foods, this happens primarily because the product was
contaminated after processing rather than because these organisms survived heat treat-
ments that normally render the product safe. This view is strongly supported by the lack
of scientific evidence indicating that minimum required heat treatments given to dairy,
meat, poultry, seafood, and other products are inadequate to inactivate levels of listeriae
that might be reasonably expected to occur in such products before heat processing. Al-
though L. monocytogenes is clearly more heat tolerant than most other non-spore-forming
foodborne pathogens, to date, no recalls of commercially prepared, Listeria-contaminated
products have been unequivocally linked to the inadequacy of minimum required heat
treatments. However, the clearest indication that L. monocytogenes and other Listeria spp.
enter commercially processed foods as postprocessing contaminants comes from the fact
that apparently healthy, non-thermally injured cells have been routinely recovered from
many thermally processed dairy, meat, poultry, and seafood products and that these organ-
isms have been found in the working environments of virtually all processing facilities
that have produced foods involved in Listeria-related recalls.
L. monocytogenes is a particularly difficult organism to control in food-processing
facilities [61d]. Refrigerated food plants, in particular, provide conditions which allow for

657
658 Gravani

L. rnonocytogenes survival and growth. The organism can adhere to food-contact surfaces
and form a biofilm or coating which impedes the effectiveness of sanitation procedures
[4 lc]. The refrigerated, moist environment, coupled with organic soil deposition, allows
L. rnonocytogenes to survive and grow. L. rnonocytogenes is also a frequent contaminant
of raw materials used in processing plants, so there is constant reintroduction of the organ-
ism into the plant environment [41b]. To control this pathogen, every potential avenue of
entry and cross contamination must be controlled.
This final chapter has been specifically designed for plant managers, sanitation work-
ers, and quality control/quality assurance personnel employed in the food industry. In
keeping with the format of Chapters 11 through 16, results from recent American and
European surveys concerning the incidence of listeriae in environments of dairy-, meat-,
poultry-, seafood-, vegetable-, and fruit-processing facilities as well as household kitchens
will be described first. This will be followed by some general guidelines for reducing
levels of listeriae and other microbial contaminants in working areas that are common to
virtually all food-processing facilities.
The wide variations in microbial load and types of microorganisms present in similar
raw and finished products manufactured at different facilities along with the fact that no
two factories are exactly alike in terms of design, equipment, maintenance, product flow,
sanitation practices and procedures, distribution patterns, and managerial policies suggest
that a discussion of current cleaning and sanitation programs used at particular food-pro-
cessing facilities would be of little benefit. Instead specific problem areas within pro-
cessing plants such as pasteurizers, fillers, sausage peelers etc., associated with the manu-
facture of particular products will be identified. Then a brief discussion of how good
manufacturing practices (GMPs), prerequisite programs, and the Hazard Analysis Critical
Control Point (HACCP) programs can all be used to decrease sharply the microbial load
in any food, thereby reducing the possibility of producing a product contaminated with
L. rnonocytogenes or any other foodborne pathogen.

INCIDENCE OF LISTERIA SPP. IN VARIOUS TYPES OF

FOOD-PROCESSING FACILITIES IN THE UNITED STATES
Interest in the extent of Listeria contamination in various food-processing facilities is of
recent origin, since L. rnonocytogenes was not identified as a serious foodborne pathogen
until 41 cases of listeriosis in Canada, including 17 deaths, were linked to consumption of
contaminated coleslaw in 1981. Despite further evidence 2 years later suggesting possible
involvement of pasteurized milk in an outbreak of listeriosis in Massachusetts, public
health officials in the United States and elsewhere did not yet regard the presence of L.
rnonocytogenes in food as a major threat to public health. However, this situation changed
dramatically in June of 1985 when up to 300 cases of listeriosis, including 40 deaths,
were eventually linked to consumption of contaminated Mexican-style cheese in southern
California. This listeriosis outbreak, along with Americas major outbreak of salmonellosis
in which over 16,000 individuals in the Chicago area became ill during March and April of
1985 after consuming a particular brand of pasteurized milk contaminated with Salmonella
typhirnuriurn [59], prompted U.S. Food and Drug Administration (FDA) officials to begin
testing various types of domestic and imported cheese for Listeria. FDA officials also
developed the Dairy Safety Initiative Program, which included microbiological surveil-
lance of finished dairy products and the factory environment in which they were produced
Listeria in Food-Processing Facilities 659

along with in-depth inspection of fluid milk factories and eventually all types of dairy-
processing facilities located throughout the United States. With the subsequent discovery
of L. monocytogenes in cooked, ready-to-eat meat, poultry, and seafood products by FDA
and U.S. Department of Agriculture (USDA) officials, manufacturers of foods other than
dairy products also became concerned about the incidence of listeriae in their products
and processing facilities.
Once FDA and USDA officials announced their plans to review GMPs that were
presumably being used by most American firms, the food industry launched a Herculean
effort to identify Listeria spp. and eliminate such problems within the food-processing
environment before governmental inspectors arrived. Considering the adverse publicity
and potential monetary losses that could result from discovery of L. monocytogenes in the
finished product and the factory environment, it is not surprising that very little information
concerning the incidence of listeriae and other microbial contaminants in food (except
Class I recalls) and food-processing facilities has been released to the scientific commu-
nity. Hence, although vast amounts of data have been generated since 1985 by local, state,
and federal government inspectors as well as private microbiological testing and con-
sulting laboratories and the food manufacturers themselves, much of the information which
now follows is either of a general nature describing particular niches within food-pro-
cessing facilities from which listeriae have been isolated or consists of limited results
from academic surveys which describe the actual incidence of Listeria contamination in
a relatively small number of food-processing facilities.

Dairy-Processing Facilities
Following several dairy-related outbreaks of salmonellosis and listeriosis, FDA officials
in cooperation with state governments and the dairy industry intensified surveillance of
various types of dairy-processing facilities under the Dairy Safety Initiative Program
which began April 1, 1986 [57]. Under this program, state officials were requested to
sponsor a series of statewide meetings to discuss foodborne illness associated with Grade
A and non-Grade A dairy products and to intensify their surveillance and inspection
efforts in dairy-processing facilities. Nationally, FDA officials vowed to (a) conduct inten-
sified check ratings in every interstate milk shipment (IMS) milk pasteurization plant, (b)
conduct similar inspections at non-Grade A (non-IMS) milk pasteurization plants, (c)
initiate a microbiological surveillance program designed to detect pathogenic microorgan-
isms in finished product (see Chaps. 11 and 12), (d) intensify and upgrade training and
standardization practices for federal and state milk specialists, rating officers, and sanitari-
ans, and (e) regularly prepare national reports which summarize the status of the United
States dairy industry.
In the first of these reports [4] covering the 6-month period from April to September
1986, 9 of 357 (2.5%) milk pasteurization factories examined produced various dairy
products contaminated with L. monocytogenes. A subsequent report in February 1987
indicated generally similar contamination rates with 16 of 620 (2.6%) and 3 of 620 (0.48%)
dairy-processing facilities manufacturing finished products containing L. monocytogenes
and L. innocua, respectively [ 5 ] .Eight months later, FDA officials reported that 11 of
604 (1 3%)IMS and 18 of 412 (4.4%) non-IMS milk pasteurization factories had produced
products contaminated with Listeria spp., principally L. rnonocytogenes [6].
Extensive follow-up efforts in milk-processing plants producing Listeria-contami-
nated products uncovered various defects in factory design and pasteurization equipment.
660 Gravani

Nevertheless, FDA officials have maintained that listeriae entered these products as post-
pasteurization contaminants. This view is strongly supported by FDAs success in isolating
Listeria from numerous floor drains in processing and other areas, wooden (porous) walls,
floors and ceilings, wooden pallets, external surfaces of milk cartons, and sweetwater
(refrigerated water) from leaking pasteurizer plates. Although not clearly identified in
FDAs list of environmental samples that harbored Listeria, FDA officials [75] noted
the following problem areas related to environmental, postpasteurization contamination
of dairy products with listeriae: (a) improperly operating high-temperature short-time
(HTST) and/or vat pasteurizers, (b) leaking and/or cracked storage tanks, jacketed vessels,
and valves, (c) inadequate sanitizing regimens, (d) cross-connecting pipes which allow
commingling of raw and pasteurized product, (e) use of contaminated rags and sponges,
(f) exposure to contaminants in unfiltered air and condensate, (g) filling and packaging
operations, (h) conveyor belts, (i) use of returned product and reclaiming operations, (i)
walls, floors, and ceilings particularly in walk-in refrigerators, (k) formation of aerosols,
(1) traffic patterns within the factory, (m) entrances and floor mats, and (n) personal cleanli-
ness of employees and others in the factory. In reality, L. monocytogenes and other food-
borne pathogens have been detected in environmental samples from many of these problem
areas as indicated in the following surveys of dairy factories in California and Vermont.
In response to these federal programs, officials of the Milk and Dairy Foods Control
Branch of the California Department of Food and Agriculture published [38] results from
a statewide survey in which 597 environmental samples were collected from 156 milk-
processing facilities during the first half of 1987 and analyzed for listeriae. Overall, Liste-
ria spp. were identified in the working environment of 46 (29.5%) milk-processing facili-
ties with 31 of these 46 (67.4%) Listeria-positive factories being contaminated with L.
monocytogenes (Table 1). Furthermore, L. monocytogenes and other Listeria spp. were
most frequently observed in factories producing fluid milk products followed by those
that manufactured frozen dairy products (i.e., ice cream and novelty desserts) and cultured
milk products (i.e., yogurt and cottage cheese), with lowest contamination rates being
associated with production of miscellaneous products and cheese. In all likelihood, this
unusually low incidence of listeriae in California cheese factories was a direct result of

TABLE
1 Incidence of Listeria in Various Types of Milk-Processing
Facilities in California, January to July 1987

No. of No. (%) of positive facilities

facilities
Type of facility examined L. rnonocytogenes All Listeria spp.
Fluid milk 63 19 (30.2) 27 (42.9)
Frozen milk products 30 7 (23.3) 11 (36.7)
Cheese 41 2 (4.9) 4 (9.8)
Cultured milk products 9 2 (22.2) 3 (33.3)
Miscellaneous productsa 13 l b (7.7) lb (7.7)
Total 156 31 (19.9) 46 (29.5)
aIncludes butter, nonfat dry milk, whey products, and condensed milk.
Positive sample from a butter factory.
Source: Adapted from Ref. 38.
Listeria in Food-Processing Facilities 66 I

massive clean-up efforts that were instituted following the 1985 listeriosis outbreak in the
Los Angeles area involving Mexican-style cheese.
A comparison of the incidence of listeriae in different milk-processing areas and
sample sites (Table 2) supports the widespread belief that listeriae most frequently enter
products after rather than before pasteurization, with the prevalence of these organisms
in the factory environment increasing as the product passes through processing, filling,
packaging, and storage areas. This apparent movement of listeriae through milk-processing
facilities is most readily seen in results obtained from sampling conveyor belts and floor
drains. However, sporadic isolation of listeriae from condensate as well as wooden blocks,
pallets, case dollies, and utility tables points to additional routes by which these organisms
can be disseminated in dairy processing plants. Although the low incidence of listeriae
in raw milk receiving rooms as compared to other areas of the factory may at first seem
surprising, these findings most likely reflect difficulties encountered in adequately cleaning
and sanitizing equipment in processing, filling, and packaging areas of factories rather
than what could be interpreted as a near absence of listeriae in California raw milk.
In an environmental survey of 39 frozen milk product plants in California, Walker
et al. [77a] collected 922 samples and found 111 (12%) positive for Listeria spp. Listeria
monocytogenes was the only species recovered from 5 (12.8%) plants and L. innocua was
the only species recovered from 13 (33.3%) plants. Both species were isolated from 9
(23.1%) plants. The highest recovery rates of Listeria were found in the batch flavoring,
freezing, ingredient blending, and packaginglfilling areas of plants surveyed.
Working at the University of Vermont, Klausner and Donnelly [56] made a large-

TABLE
2 Incidence of Listeriae in Different Milk-Processing Areas
and Sample Sites
No. of No. (%) of positive samples
Facility working area samples
and sample site examined L. monocytogenes ,411 Listeria spp.
Raw milk receiving room
Drain 30 1 (3.3) I (3.3)
Condensate 32 0 1 (4.5)
Othei 1 0 0
Processing room
Drain 150 4 (2.7) 14 (9.3)
Condensate 76 1 (1.3) 3 (3.9)
Other" 21 3 (14.3) 6 (28.6)
Filling/Packaging room
Drain 60 7 (11.7) 12 (20.0)
Condensate 36 1 (2.8) 1 (2.8)
Conveyor 15 5 (33.3) 7 (20.0)
Othei 10 0 2 (20.0)
Cold storage room
Drain 105 12 (11.4) 17 (16.2)
Condensate 44 0 1 (2.3)
Conveyor 14 4 (28.6) 9 (64.3)
a Includes wooden blocks, pallets, case dollies, and utility tables.

Source: Adapted from Ref. 38.

662 Gravani

scale survey to identify sources of Listeria (and Yersinia) contamination in fluid milk-,
cheese-, and non-cheese-processing facilities. Overall 66.7, 9.5, and 23.8% of samples
collected from floors and other non-food-contact surfaces at 34 fluid milk, cheese, and
non-cheese factories were positive for Listeria spp., with L. rnonocytogenes and L. inno-
cua being identified in 1.4 and 16.1%, respectively, of 361 samples examined. As ex-
pected, the percentage of Listeria-positive samples was higher among those from floors
(12.0-27.9%) than from other non-food-contact surfaces (8.1%) (Table 3) and wet
(85.7%) rather than dry (14.3%) areas. According to these investigators, paper filler beds,
whey drainage pans on cheese presses, and case-washing areas were particularly prone
to contamination with Listeria and Yersinia. The fact that 20.9% of all positive samples
contained both Listeria and Yersinia suggests that yersiniae might be somewhat useful as
a potential indicator of Listeria contamination within the dairy-processing environment.
As noted, L. rnonocytogenes, Yersinia spp., and most other foodborne pathogens are
more commonly found in wet than dry processing areas. However, the fact that listeriae
(a) were recovered from whey drainage pans, (b) were routinely shed in whey during
cheese-making experiments, (c) grew in samples of refrigerated milk and whey, and (d)
survived the typical spray-drying process used to manufacture nonfat dry milk suggests
that these organisms should be of concern to manufacturers of dry dairy products.
In the light of these concerns, Gabis et al. [44] determined the incidence of Listeria
in the working environment of 18 dry milk- and whey-processing facilities throughout
the United States. The authors supplied environmental sampling kits containing sterile
cellulose sponges, fabric-tipped swabs, and other necessities to all firms participating in
the survey along with instructions as to how and where to collect samples. All samples
were then sent to a central laboratory and within 48 h of collection were analyzed for
listeriae according to the FDA procedure. Overall, only 2 of 410 (0.24%) samples exam-
ined were positive for Listeria spp., with L. monocytogenes and a species other than L.
rnonocytogenes being isolated from floor drains in a raw milk receiving area and from a
composite sample from several floor drains and trenches in a powder production area,
respectively (Table 4). Allowing factory employees to choose specific sampling sites as
well as the number of samples to be analyzed may have somewhat biased these results;

TABLE
3 Incidence of Listeria and Yersinia on Floors and Non-
Food-Contact Surfaces of 34 Fluid Milk, Cheese, and Non-Cheese
Factories i n Vermont

No. of No. (%) of positive samples

samples
Type of sample analyzed Listeria Yersinia
Floor areas
Coolers 43 12 (27.9) 9 (20.9)
Processing 117 21 (17.9) 14 (12.0)
Entrances 64 1 1 (17.2) 18 (27.7)
MatdFootbaths 25 3 (12.0) 1 (4.0)
Other areas 38 10 (26.3) 6 (15.8)
Non-food-contact surfaces 74 6 (8.1) 4 (5.4)
Total 36 1 63 (17.5) 52 (14.4)
~~ ~

Source: Adapted from Ref. 56.

Listeria in Food-Processing Facilities 663

TABLE4 Incidence of Listeria in the Working Environment of 18 Nonfat

Dry Milk- and Whey-Processing Facilities Located Throughout the
United States
No. of No. (%) of positive samples
samples
Work area analyzed L. monocytogenes Other Listeria spp.
Raw milk receiving 62 1 (1.6) 0
Wet processing 151 0 0
Cheese factory 22 0 0
Whey factory 23 0 1 (4.3)
Dryer room 38 0 0
Bagging room 27 0 0
Heating, ventilating and air 53 0 0
conditioning system
Miscellaneous 34 0 0
Total 410 1 (0.24) I (0.24)
Source: Adapted from Ref. 44.

however, the incidence of listeriae and hence the risk of postprocessing contamination
appears to be many times lower in dry rather than wet dairy-processing facilities. Neverthe-
less, since manufacturers of nonfat dry milk and dry whey are not immune to the Listeria
problem, they should take appropriate action to eliminate this organism from the pro-
cessing environment, thereby greatly reducing the chance of producing a contaminated
product.
In a follow-up study, Pritchard et al. [67a] sampled 30 dairy processing plants in
Vermont. Of the 346 sites tested, 122 (35.3%) contained one or more species of Listeria.
Coolers and freezers had the highest rate, with 14 of 30 sites (46.7%) being positive for
Listeria (Table 5). Other sites that resulted in high positive rates included dry storage
areas (39.6%) and raw milk receiving and storage areas (39.4%). Pritchard et al. [67a]

TABLE5 Evaluation of Listeria Species Isolates Based on

Area of Processing Plant
No. of No. of
Area designation sites positive % Positive
Processing 145 52 35.9
Entrances to processing 53 21 39.6
Entrances not to processing 37 11 29.7
Raw milk receiving/storage 33 13 39.4
Coolers and freezers 30 14 46.7
Dry storage 16 7 43.8
Othera 31 4 12.9
Total 346 122 --

a Includes areas such as common hallways, testing laboratories, and wheels of fork-
lifts.
Source: Adapted from Ref. 67a.
664 Gravani

also noted that plants producing dairy ingredients, frozen milk products or fluid milk, had
significantly higher incidence rates of Listeria than expected. Facilities producing cultured
dairy foods or a combination of cultured dairy foods and fluid milk were found to have
significantly lower incidence rates of Listeria than expected. These researchers also ob-
served that when dairy farms were contiguous to the processing facilities, these plants
were more likely to be contaminated than plants without on-site dairy farms.

Meat-Processing Facilities
Unlike dairy-processing facilities in which raw milk is pumped into the factory, pasteur-
ized, and then either packaged immediately or pumped to closed vats for processing into
cream, butter, ice cream, cheese, or other dairy products, meat-processing factories are in
actuality open-air disassembly line operations in which animals are slaughtered, eviscer-
ated, and broken down to obtain various cuts of meat, hides for leather, and other items
of commercial value. Considering that domestic cattle, sheep, and pigs frequently shed
L. monocytogenes asymptomatically in fecal material, it is not surprising that surveys have
shown this pathogen to be not only ubiquitous but also endemic to slaughterhouses and
meat-packing facilities.
Initiation of the USDA-FSIS testing program for listeriae in cooked and ready-to-
eat meat products in September of 1987 (see Chap. 13) prompted immediate action by the
meat industry. However, even before government testing began, meat processors became
concerned about the incidence of listeriae in the working environment. In June 1987,
results from a large-scale survey were reported in which nearly 2300 environmental sam-
ples were collected from over 40 meat-processing facilities nationwide and analyzed for
listeriae [9]. Fourteen processing areas within these factories yielded evidence of being
contaminated with L. monocytogenes or other Listeria spp. Overall, listeriae were recov-
ered from -21% of all environmental samples examined. (These results also compare
favorably with those of a much smaller survey [15] in which Listeria spp. were detected
in 9 of 27 (33%) meat-processing environmental samples.) Problem areas in which 220%
of the samples were positive included drains, trenches, floors, exhaust hoods, cleaning
aids (sponges, brooms, hoses, and mops), product-contact surfaces (peelers, conveyors,
and slicers), and wash areas. Sampling of surfaces in contact with sliced luncheon meat
revealed Listeria contamination rates of 9.3, 32.3, and 23.6% before, during, and after
production, respectively. Similarly, listeriae were recovered from 2.8, 14.5, and 25.5%,
respectively, of food-contact surfaces examined before, during, and after production of
frankfurters.
From September 1987 to October 1991, USDA-FSIS inspectors sampled over
15,000 processed meat products, including cooked beef, sliced hams from cans, cooked
sausage, jerky, cooked poultry, salads and spreads, and imported meats [74a]. The overall
incidence of L. monocytogenes during this sampling period was 1.6%, with 235 products
testing positive for L. monocytogenes This led to 25 recalls of product from the market
during 1989-1991 [74a].
Data from the USDAs microbiological monitoring of over 13,000 lots of ready-to-
eat meat products, from January 1, 1993 to September 30, 1997, indicated an incidence
of 2.9% of the lots testing positive for L. monocytogenes (Levine, personal communication
1998). Results from a large-scale 1987 survey sponsored by the American Meat Institute
[2,15] support the notion that Listeria spp. are widely distributed within the environment
of many meat-processing facilities, and as in the earlier study by Flowers [9], also point
Lister ia in Food-Processing Facilities 665

to floors, drains, cleaning aids, wash areas, sausage peelers, and food-contact surfaces as
significant problem areas, with between 20 and 37% of such samples harboring listeriae
(Table 6). With the identification of listeriae in condensate and compressed air and on
walls and ceilings, there can be no doubt that these organisms are ubiquitous in at least
some meat-processing facilities.
Recognizing the potential opportunity for Listeria to contaminate meat during pack-
aging, one major manufacturer of processed meat products attempted to obtain near-
operating room conditions in its packaging room by cleaning the area for 3 days and
then fogging the entire packaging room with 200 ppm quaternary ammonium compound
[8,9]. In spite of these efforts, listeriae were still detected in 1 of 19 environmental samples
obtained from the packaging room. After this exercise, the firm packaged processed meat
products in this room over a 2-week period. Despite adherence to normal cleaning and
sanitizing procedures at the end of each working day, the overall incidence of listeriae in
the packaging room increased, with 3 of 20 (15%), 6 of 20 (30%), and 8 of 20 (40%)
samples being positive for Listeria spp. 3,6, and 8 days after the room was initially cleaned
and fogged, respectively.
Owing to the increased concern for L. monocytogenes in meat products, there has
been a concerted effort to minimize the risk of postprocess contamination during the pro-
duction of processed meats. In one study, cited by Tompkin et al. [74a], swab samples
were collected from packaging lines and floors where exposed ready-to-eat product was
transported, chilled, stored, or packaged. The incidence of Listeria at these locations, from
August 1989 to January 1992, is summarized in Figure 1. The overall trend is toward
improved control of Listeria with fewer positive samples being evident following the
inception of a Listeria control program. The results show a strong seasonal effect for the
presence of Listeria in the finished product environments, with fewer positive samples
being detected during the winter months. In addition, Tompkin et al. [74a] also reported
results of a three-year study in which about 100 packaging lines were tested for Listeria
(Table 7). The percentage of Listeria-negative samples increased from 44 in 1989 to 64
in 1991. The percentage of lines that exceeded the companys established criterion of 5%
of samples positive for Listeria decreased from 29 in 1989 to 13 in 1991. As improvements
were made, attention was given to chronically positive lines. Examples of contaminated

TABLE
6 Incidence of Listeria spp. in
Post-Heat-Processing Areas of 41 Meat
Factories Examined in the United States
During 1987
Area Positive samples (%)
Floors 37
Drains 37
Cleaning aids 24
Wash areas 24
Sausage peelers 22
Food contact surfaces 20
Condensate 7
Walls and ceilings 5
Compressed air 4

Source: Adapted from Ref. 2.

666 Gravani

30

25

%
20
P
0
15
I
T
I
v 10
E

n
D J D J D J D
E U E U E U E
C N C N C N C
1989 1990 1991

FIGURE1 Incidence of Listeria on packaging lines and in the environment (floors)

from August 1989 to January 1992 inclusive. (From Ref. 74a.)

sites on packaging lines included hollow rollers for conveyors, on/off valves and switches,
rubber seals around doors, fibrous conveyor belts, and areas of equipment which were
inaccessible to cleaning. The authors also noted that occasional lapses in cleaning and
sanitizing procedures resulted in a fairly rapid loss of control. They observed that a certain
sequence of events can lead to periodic contamination of packaging lines. The floor is
particularly difficult to render Listeria-negative, and this situation provides a ready source
of organisms to contaminate the packaging line during production or while cleaning,
allowing the establishment of sites for microbial multiplication. This sequence of events
can be prevented by striving for Listeria-negative floors, effectively cleaning and sanitiz-
ing the packaging lines at the end of each days production, eliminating inaccessible sites
in the equipment, and by providing adequate preventive maintenance of the equipment.

TABLE
7 Listeria Contamination on Packing Lines
from 1989 to 1991
% of Lines positive for Listeria at
No. of
Year lines 0% 15% >5%a
1989 96 44 27 29
I990 I06 59 27 14
1991 97 64 23 13
aExceeds company guideline.
Source: Adapted from Ref. 74a.
Listeria in Food-Processing Facilities 667

The authors summarized their report with the comment, ". . . for the present, it must be
concluded that existing technology cannot eliminate Listeria from the cooked product
environment of processing plants."
Since Listeria spp., including L. monocytogenes, have been found in up to 50% of
raw beef, pork, and lamb marketed in the United States, complete elimination of listeriae
from meat-processing environments appears highly improbable. However, the American
Meat Institute has developed a series of interim guidelines [2], which, if followed, will
reduce the incidence of listeriae and decrease the overall microbial load in the working
environment. A detailed description of these guidelines appears later in this chapter.

Poultry-Processing Facilities
Reports have shown that up to 50% of all raw poultry sold in the United States contains
various Listeria spp., including L. monocytogenes, with fecal material from infected flocks
cited most frequently as the source of contamination. Unfortunately, information concern-
ing the incidence of listeriae in American poultry-processing facilities is presently limited
to results from two California surveys. In these studies, researchers at the University of
California-Davis investigated the prevalence of listeriae in processing samples from one
chicken [46] and one turkey slaughterhouse [47] during three or four separate visits. Ac-
cording to these investigators, no Listeria spp. were isolated from feathers, incoming
chiller water, or scalding water, the latter of which aids in feather removal (Table 8).
Nonetheless, L. monocytogenes and L. innocua were identified in samples of overflow
chiller water and feather picker drip water obtained from the chicken slaughterhouse, with
both organisms being detected in recycled water used to clean gutting equipment. Inci-
dence rates for L. monocytogenes in chicken- and turkey-processing facilities were gener-
ally similar, with the percentage of Listeria-positive samples increasing approximately
2- to 2.5-fold during the latter stages of processing. However, L. welshimeri and L. innocua
were absent from most chicken- and turkey-processing samples, respectively. Although
only two poultry slaughterhouses were examined in this survey, inability of these research-
ers routinely to detect L. welshimeri in fresh chicken meat and L. innocua in fresh turkey
meat processed at these facilities suggests that L. welshimeri and L. innocua might be
able preferentially to colonize the gastrointestinal tract of turkeys and chickens, respec-
tively. These findings, along with the ability of these investigators to further demonstrate
an increasing incidence of Listeria spp. on the gloves and hands of poultry workers from
the beginning to the end of processing (Table 9) confirms that these contaminants move
along the processing line with the raw product.
Unfortunately, neither the USDA nor the poultry industry have released any data
regarding the incidence of listeriae within the general working environment of poultry-
processing facilities. However, considering the fecal carriage rate for listeriae in domestic
birds, the current assembly line methods for processing poultry, and the fact that Listeria
spp. (including L. monocytogenes) and salmonellae have be.en isolated from up to about
half of all raw chickens marketed in the United States, one can speculate that the poultry
and meat industries face similar problems in controlling the spread of listeriae and other
organisms in the work environment. If one draws a parallel between methods used to
process meat and poultry, then floors, drains, cleaning aids, wash areas, and food-contact
surfaces emerge as likely niches for Listeria spp., including I,. monocytogenes, in poultry-
processing facilities. These predictions may be supported by published scientific data in
the future.
668 Gravani

TABLE
8 Incidence of Listeria spp. in One Chicken and One Turkey Slaughterhouse in California
No. of
chickenhurkey No. (%) of positive samples
slaughterhouse
Sample samples analyzed L. monocytogenes L. innocua L. welshimeri Total
o/o o/o o/o
~~~~~~~ ~ ~ ~ ~

Scalding water overflow 16/15 010

Feather picker drip water 16/15 0/1 (6.7) 3 (18.8)/0 0/1 (6.7) 3 (18.8)/2 (13.3)
Incoming chiller water 16/0 o/o o/o o/o 010
Overflow chiller water 16/15 2 (12.5)/0 010 0/1 (6.7) 2 (12.5)/1 (6.7)
Recycled water for cleaning gutters 16/15 1 (6.3)/2 (13.3) 5 (31.3)/0 0/3 (20.0) 6 (37.5)/5 (33.3)
Source: Adapted from Refs. 46 and 47.
Listeria in Food-Processing Facilities 669

TABLE
9 Incidence of L. rnonocytogenes and L. innocua on the Hands and Gloves of Poultry Meat Processors Assigned to Three
Different Stations in a Slaughterhouse
No. of
chickedturkey No. (%) of positive samples
slaughterhouse
Sample samples analyzed L. monocytogenes L. innocua L. welshimeri Total
Postchilling handlers 20/30 2 (10.0)/3 (10.0) 2 (10.0)/0 012 (6.7) 4 (20.0)/5 (16.7)
Leg/wing cutters 11/30 4 (36.4)/3 (10.0) 1 (9.1)/0 0/7 (23.3) 5 (45.5)/10 (33.3)
Leg/wing packers 44/30 20 (45.5)/5 (16.7) 1 1 (25.0)/0 0/7 (23.3) 31 (70.5)/12 (40.0)
Source: Adapted from Refs. 46 and 47.
670 Gravani

Egg-Processing Facilities
The discovery of L. innocua and, to a lesser extent, L. monocytogenes in 15 of 42 (36%)
samples of frozen, raw, commercial liquid whole egg obtained from 6 of 11 manufacturers
located throughout the United States suggests that listeriae-as well as salmonellae-contam-
inated poultry feces may contaminate the surface of eggs before breaking, and that these
organisms in turn may be spread to various areas within the egg-processing environment.
Fortunately, the Egg Products Inspection Act of 1970 led to regulations which now require
that all egg products be pasteurized to eliminate salmonellae (and L. monocytogenes).
However, as is true for fluid milk, there is ample opportunity for recontamination of liquid
egg products with listeriae, salmonellae, and nonpathogenic organisms after pasteurization
which can greatly decrease the shelf life and/or microbial quality of the finished product.
Although Listeria spp. have not yet been recovered from commercially prepared pasteur-
ized egg products or the associated manufacturing environment, prudent producers of such
products should be certain that floors, drains, cleaning aids, wash areas, and food-contact
surfaces as well as egg-breaking and egg-separating, pasteurization, and packaging equip-
ment are thoroughly cleaned and sanitized on a regular basis to eliminate potential prob-
lems involving listeriae, salmonellae, and high levels of spoilage organisms.

Seaf ood-Processing FaciIities

After L. monocytogenes was recovered from fresh frozen crabmeat in May of 1987, FDA
officials began testing a wide range of domestic and imported fish and seafood products
for listeriae and other organisms of public health significance. The results from these
analyses led to numerous Class I recalls of Listeria-contaminated products, and govern-
ment officials also released additional findings that were obtained during visits to various
seafood-processing facilities.
Between January and April of 1988, inspectors from the Oregon Department of
Agriculture analyzed 480 environmental swab samples from 17 seafood-processing facili-
ties located throughout Oregon [ 10,43,77]. Although only 4% of all samples were positive
for Listeria spp., 10 of 17 (60%) factories yielded evidence of Listeria contamination in
the work environment. Specific locations from which listeriae were isolated included (a)
a fiberglass tote in a walk-in cooler, (b) a drain in a walk-in cooler, (c) a phosphate recircu-
lation system on a shrimp-processing line, (d) an ice tote in a cold room, (e) a floor gutter
near a shrimp peeler, (f) a wooden door frame in a crab-freezing room, (g) tires on heavy
machinery, (h) a cold saturated (-23%) brine solution, (i) the framework of a fish dumps-
ter, (j) floor and wall junctions in a cooler, and (k) seagull droppings on an office manag-
ers window. Additional environmental niches within processing plants that are strongly
suspected of harboring listeriae include walls, floors, ceilings, condensate, pooled water,
and processing wastes. Hence, this information along with other observations that virtually
all Listeria cells recovered from processed seafoods have been healthy rather than ther-
mally or otherwise injured suggest that the presence of listeriae in processed seafood is
almost exclusively the result of recontamination after processing. Although L. monocyto-
genes and other Listeria species have been isolated from different types of raw and pro-
cessed seafood, the main source of contamination is unknown. Several studies [41a,41d]
have been conducted to detect the potential sources of this pathogen in seafood-processing
plants so product contamination could be minimized. Eklund et al. [41d] surveyed cold-
smoked salmon-processing plants to determine the occurrence and sources of L. monocy-
togenes. These authors observed that cleaning and sanitizing procedures adequately elimi-
 Listeria in food-Processing facilities 671

nated L. monocytogenes from the processing line and equipment, but recontamination
occurred soon after processing was resumed. They also identified the external surfaces of
fresh and frozen fish as the primary source of L. monocytogenes in cold-smoked fish-
processing plants (Table 10). During the filleting, rinsing, and brining operations, the
bacterium is transferred to the exposed flesh, and as the product moves through the pro-
cessing steps, the equipment, personnel, and other surfaces which the product contacts
become contaminated and these then serve as secondary sources of contamination.
Destro et al. traced the transmission of L. monocytogenes in a shrimp-processing
plant [41a 1, using two molecular typing methods: random amplified polymorphic DNA
(RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Of the 115 L. monocyto-
genes isolates examined, 25 were recovered from the plant environment (floors, walls,
and pipes); 15 were from equipment and utensils, including,tables, plastic boxes, knives,
and trays; 9 were found in water used in shrimp processing; 7 were isolated from the
hands of employees; and 59 were from the shrimp. The results from this interesting study
indicated that environmental strains all fell into composite groupings unique to the envi-
ronment, whereas strains from both water and utensils shared another composite profile
group. The L. monocytogenes isolates from fresh shrimp belonging to one profile group
were found in different areas of the processing line. This same profile group was also
present on the hands of employees from the processing and packaging areas of the plant.
This study showed that there were many different sources of L. rnonocytogenes in the
shrimp-processing plants. Information on preventing postprocessing contamination of fish,
seafood, and other fishery products is presented in the second half of this chapter.

Vegetable- and Fruit-Processing Facilities

Although consumption of coleslaw prepared from contaminated cabbage was directly
linked to the first documented outbreak of foodborne listeriosis in 1981, the incidence of

TABLE
10 incidence of Listeria in a Cold-Smoked
Sal mon-Processi n g Pia nt
Area in Plant L. monocytogenes L. innocua
~~~~ ~ ~~

Raw Product and Processing Area

Thawing water for fish (from tank) 11/59 15/59
Rack from bottom of thawing tank 212 012
Filleting table 1 /9 019
Rinse water 616 316
Skins from raw salmon 317 417
Slime from raw salmon 414 3 /4
Drip from raw salmon 819 3I9
Trimming from raw salmon 15/26 14/26
Finished Product and Processing Area
Salmon sides from smokehouse 919 719
Trim table 1/8 018
Trim machine 6/15 2/15
Skins from skinning machine 29/30 8/30
Fillet midline trimmings 8/20 0120
Product trimmings from slicers 17/35 20135

Source: Adapted from Ref. 41d.

672 Gravani

listeriae in raw vegetables and fruits and particularly the prevalence of these organisms
in work environments of vegetable- and fruit-processing facilities have received relatively
little attention. Nevertheless, the long-recognized association of listeriae with soil and the
discovery of Listeria spp., including L. monocytogenes on raw vegetables suggest that
these organisms are almost certainly in vegetable- and fruit-processing facilities. Unfortu-
nately, the extent of Listeria contamination in such facilities in the United States is cur-
rently unknown. However, soil and production-area samples from one potato-processing
factory in the Netherlands have yielded L. monocytogenes, L. innocua, and L. seeligeri
(see Tables 14 and 15).

INCIDENCE OF LISTERIA SPP. IN WESTERN EUROPEAN

AND AUSTRALIAN FOOD-PROCESSING FACILITIES
As is true for the United States, information concerning the extent of Listeria contamina-
tion in European food-processing facilities also is limited. However, existing information
indicates that European and American food companies are experiencing similar problems
regarding listeriae in the manufacturing environment. Furthermore, since similar food pro-
duction, processing and packaging methods as well as cleaning and sanitation practices are
employed in both Western Europe and North America, much of the following information
regarding the incidence of Listeria contamination within Western European food-pro-
cessing facilities is probably applicable to manufacturers of similar products in the United
States and Canada.

Western Europe
Interest in the incidence of listeriae within European food-processing facilities has devel-
oped in parallel with the discovery of these organisms in foods destined for human con-
sumption. As noted in Chapter 12, large quantities of French Brie cheese were contami-
nated with L. monocytogenes in 1986. Therefore, emphasis was first placed on determining
the prevalence of listeriae in cheese factories. The results of one small-scale environmental
survey of French cheese factories [32] identified L. monocytogenes in one floor sample
and L. innocua was recovered from boards, wheels, and equipment (7 of 22 samples),
brushes (1 of 6 samples), and filtered air (1 of 19 samples). From 1988 to 1990, a French
cheese factory was sampled for Listeria contamination [53a]. Of the 344 samples collected
and analyzed for Listeria, 61 strains (44 L. monocytogenes and 17 L. innocua) were iso-
lated from four varieties of cheese, cheese brines, processing equipment, and the plant
environment. The L. monocytogenes strains were recovered from the ripening and rind
washing stages and not before, so Jacquet et al. [53a] theorized that the cheese contamina-
tion occurred at these points in the manufacturing process. During a survey of German
factories producing soft smear-ripened cheese, Terplan 1741 also isolated nonpathogenic
Listeria spp. from smear liquid, various pieces of machinery (especially smearing ma-
chines), and floor drains, with L. monocytogenes being detected far less frequently than
other listeriae (Table 11). Hence, opportunity exists for contamination of both mold and
bacterial surface-ripened cheese during the latter stages of manufacture and storage.
Although such published information is limited, some unpublished data are available
on the prevalence of listeriae in other Western European cheese factories. As mentioned
in Chapter 12, Swiss officials who were tracing the source of contamination in the 1987
listeriosis outbreak involving consumption of Vacherin Mont dOr soft-ripened cheese
Listeria in Food-Processing Facilities 673

TABLE 11 Prevalence of L. rnonocytogenes and Nonpathogenic Listeria spp.

Within the Working Environment of German Factories Producing Soft Smear-
Ripened Cheese
~ ~~

No. (%) of positive samples

No. of
samples Nonpathogenic
Environmental sample analyzed L. rnonocytogenes Listeria spp. Total
Smear liquid and smearing machines 2 10 2 (0.9) 33 (15.7) 35 (16.7)
Other machinery 25 1 12 (4.8) 31 (12.3) 43 (17.1)
Ripening boards 69 0 2 (2.9) 2 (2.9)
Condensate and cooling water 36 1 (2.8) 2 (5.6) 3 (8.3)
Floor drains 74 3 (4.1) 29 (39.2) 32 (43.2)
Source: Adapted from Ref. 74.

recovered the epidemic strain of L. monocytogenes from smear brine, curing brine, waste-
water sinks, wooden cheese hoops, and wooden boards used in 10 different cheese facto-
ries that manufactured Listeria-contaminated cheese [37]. Additionally, nearly half of the
12 cellars used to ripen cheese contained listeriae, with the pathogen being detected on
6.8% of the wooden shelves and 19.8% of the brushes used in the ripening cellars. Al-
though not noted in the report, one would suspect that L. mc,nocytogenes also was present
in commonly recognized environmental niches such as drains, floors, stagnant water, and
various food-contact surfaces within cheese factories and ripening cellars. Thus brushing
cheese with saltwater and ripening hooped cheese on wooden shelves appear to be two
important means for dissemination of listeriae within cheese factories.
In 1988, Cox [39,40] presented some preliminary data concerning the prevalence
of Listeria spp. within one blue and six soft cheese factories in Western Europe as well
as in one ice cream factory and eight chocolate factories. As expected, listeriae generally
occupied similar environmental niches in both soft and blue cheese factories; however,
Listeria contamination was far more common in ripening than production areas of the
one blue cheese factory examined (Table 12). Ripening practices for blue cheese, including
maintenance of a relatively moist environment, appear to be the likely reason for higher
rates of Listeria contamination in ripening than production areas. Although some environ-
mental niches in this blue cheese factory were not sampled, results for soft cheese factories
point to walls, air coolers, stagnant water, and condensate as possible problem areas in
blue cheese factories as well.
During a similar investigation, samples from at least half of the drains, conveyors,
stagnant water, floors, and residue and waste products from one Western European ice
cream factory contained populations of Listeria spp. ranging from 10 to >106 CFU/g or
mL (Table 13). This factory manufactured all of its ice cream from commercially produced
reconstituted powdered milk (a product from which Listeria has not yet been isolated)
rather than fresh milk. Hence, these findings strongly suggest that Listeria contamination
in dairy-processing facilities is not always linked to incoming raw milk or milk haulers.
Listeria spp., including L. monocytogenes, also have been detected in commercially
produced chocolate that was marketed in England [48]. Furthermore, a 1988 report by
Cox [39] indicated that 8 of 32 (25%) and 10 of 59 (17%) samples obtained from damp,
wet, and dry areas of eight Western European chocolate factories were positive for Listeria
spp. Although growth of listeriae in chocolate is very unlikely, contamination of the fin-
674 Gravani

TABLE
12 Incidence of Listeria spp. in Several Western
European Blue and Soft Cheese Factories
~~~ ~

Percentage of samples yielding

Listeria spp.
Soft cheese Blue cheese
Environmental sample factory factory
Drains 22 71
Floors 20 5/83
Residues NA' 23/46
Equipment 0 O/NA
Walls 33 NA/NA
Air coolers 22 NAINA
Stagnant water 14 NAINA
Condensate 5 NAINA
Brine NA O/NA
Miscellaneous 19 NAINA
NA, not applicable.
a Production areas.

Ripening areas.
Not analyzed.
Source: Adapted from Refs. 39 and 40.

ished product during packaging is clearly possible. The relatively low risk of producing
Listeria-contaminated chocolate can be further reduced by development of adequate clean-
ing and sanitation programs and by maintaining production and packaging areas as dry
as possible.
In one of the largest European surveys reported thus far, Cox et al. [41], during the
latter half of 1986, investigated the incidence of Listeria spp. in the processing environ-
ment of 17 establishments in the Netherlands that produced fluid dairy products, ice cream,
Italian-style cheese, frozen food, potato products, and dry culinary foods. A total of 608
samples were collected from drains, floors, condensed and stagnant water, residues, pro-
cessing equipment, and/or other areas and were analyzed for listeriae using the original

TABLE 13 Incidence of Listeria spp. i n the Production

Environment of One Western European Ice Cream Factory
Percentage of
samples yielding Listeria populations
Environmental sample Listeria spp. (CFU/g or ml)
Drains 100 ?lob
Conveyors 75 1o2
Stagnant water 66 NR
Floors 63 10-1O6
Residuedwaste products 50 10-104

NR, Not reported.

Source: Adapted from Refs. 39 and 40.
Listeria in Food-Processing Facilities 675

USDA or FDA method with or without modification. All presumptive Listeria isolates
were then speciated according to results from conventional biochemical tests.
Despite use of GMPs in these factories, Listeria spp. were recovered from all types
of food-processing facilities examined with the exception of two that produced dry culi-
nary products. Overall, 181 of 608 (29.8%) samples yielded Listeria spp. with L. innocua,
L. monocytogenes, and L. seeligeri being identified in 87.3, 14.9, and 0.5% of all positive
samples, respectively. Although only five samples contained both L. monocytogenes and
L. innncua, the actual number of such samples is probably somewhat greater, since a
limited number of presumptive Listeria isolates from each sample were chosen for con-
firmation. As shown in Table 14, L. innocua was most prevalent in establishments that
produced processed potato products followed by those that produced ice cream, frozen
food, Italian-style cheese, and fluid dairy products, with the organism generally being
isolated most frequently from drains, floors, and condensed and stagnant water.
In contrast, L. monocytogenes was detected in 11.8% of all environmental samples
obtained from one ice cream factory but was found in 2.9, 3.0, 3.3, and 3.7% of similar
samples from establishments that manufactured fluid dairy products, potato products, fro-
zen food, and Italian-style cheese, respectively (Table 15). Although only one ice cream
factory was examined in this survey, the results are as expected when one recalls that
Cox [39,40] previously found that listeriae were widespread in another Western European
ice cream factory and also were present in very large numbers, particularly in floor drains
(Table 13). Given such populations of listeriae in ice cream factories and the current
extruding, niolding, and freezing methods used to produce ice cream, and particularly ice
cream novelties, one can easily postulate many routes whereby listeriae may recontaminate
the finished product, as has been reported in the United States.
Results concerning the incidence of Listeria spp. as well as L. innocua and L. muno-
cytogenes in various work environments of all 15 food-processing facilities are summa-
rized in Table 16. Overall, these findings are comparable to what has been previously
noted for similar food-processing facilities in the United States; for example, Listeria spp.
and L. innocw were most frequently recovered from drains followed by condensed and
stagnant water, floors, residues, and processing equipment. With a few minor exceptions,
which probably resulted from the number of samples analyzed, this same trend is readily
apparent for all five types of food-processing facilities listed in Table 14. Thus a logical
pattern emerges in which L. innocua moves from floor drains to pools of condensed and
stagnant water, which then come into direct contact with floors and residues. Once present
in open areas of the work environment, L. innocua is spread by employees to processing
equipment that comes into direct contact with the product. Unlike L. innocua, L. monocyto-
genes was far less prevalent in all types of food-processing facilities and was distributed
fairly evenly within the factory environment with incidence rates ranging between 2.3 and
7.7%. Although L. innocua is by definition nonpathogenic, the fact that L. innocua and
L. monocyto,qenes (and possibly other Listeria spp.) occupy similar environmental niches
indicates that detection of listeriae anywhere within the manufacturing environment should
prompt immediate corrective action, the details of which will be discussed shortly.
In one of the remaining few Western European surveys reported, Hudson and Mead
[511 determined the incidence of Listeria spp. at 10 different sites within one large English
poultry-processing facility. According to these authors, scald water, feathers, and chill
water as well as swab samples from defeathering machines and conveyors leading to
the chiller were free of listeriae; however, L. monocytogenes was routinely isolated from
automatic carcass openers and also was present in samples from evisceration-line drains,
676 Gravani

TABLE
14 Incidence of L. innocua in Working Environments of 15 Food-Processing Facilities in the Netherlands
No. of positive samples/No. of samples analyzed (%)
Italian-style Frozen food Potato-processing
Fluid dairy factory Ice cream factory cheese factory factory factory
Environmental sample na = 5 n = l n= 5 n = 3 n = l
Drains 2/4 (50.0) 4/4 (100.0) 19/42 (45.2) 2/3 (66.7) 7/13 (53.8)
Condensed/stagnant water 2/5 (40.0) 4/8 (50.0) 7/20 (35.0) NA 7/10 (70.0)
Floors 0/2 8/16 (50.0) 14/44 (31.8) 2/4 (50.0) 9/13 (69.2)
Residues NAb 4/12 (33.3) 16/71 (22.5) NA 5/15' (33.3)
Processing equipment o/ 10 7/20 (35.0) 6/68 (8.8) 1/6 (16.7) NA
Miscellaneous 0/13 2 B d (25.0) 12/103' (11.7) 15/78 (19.2) 4/ 17' (23.5)

Total 4/34 (11.8) 29/68 (42.6) 74/348 (2 1.3) 20/91 (22.0) 32/68 (47.1)

NA, not applicable.

a Number of factories analyzed.

Not analyzed.
Includes one sample positive for L. seeligeri.
Conveyor belt (two of two positive).
Raw milk (two of two positive), untreated effluent.
Potato delivery soil (two of three positive), sand from effluent treatment (two of two positive).
Source: Adapted from Ref. 4 1.
Lister ia in Food- Processing Facilities 677

TABLE15 Incidence of L. monocytogenes in Working Environments of 15 Food-Processing Facilities in the Netherlands

No. of positive samples/No. of samples analyzed (%)
Italian-style Potato-processing
Fluid dairy factory Ice cream factory cheese factory Frozen food factory factory
Environmental sample na = 5 n = l n= 5 n = 3 n = l
Drains 0/4 0/4 2/42 (4.8) 1/3 (33.3) 0/13
Condensed/stagnantwater 0/5 0/8 0/20 NA o/ 10
Floors 0/2 1/16 (6.3) 2/44 (4.5) 0/4 1/13 (7.7)
Residues NAb 0/12 7/71 (9.9) NA 0/15
Processing equipment 0/10 6/20 (30.0) 2/68 (2.9) 0/6 NA
Miscellaneous 1/13 (7.7) 1/8' (12.5) O/ 103 2/78 (2.6) 1/17d(5.9)
Total 1/34 (2.9) 8/68 (1 1.8) 13/348 (3.7) 3/91 (3.3) 2/67 (3.0)
NA, not applicable.
Number of factories analyzed.
Not analyzed.
Sponge (one of one positive).
Potato delivery soil (one of three positive).
Source: Adapted from Ref. 4 1 .
678 Gravani

TABLE
16 Overall Incidence of Listeria spp. in Working Environments
of 15 Food-Processing Facilities in the Netherlands

No. of No. (%) of positive samplesa

samples
Environmental sample analyzed Listeria spp. L. innocua L. rnonocytogenes
Drains 66 36 (54.5) 34 (51.5) 3 (4.5)
Condensed/stagnant water 43 20 (46.6) 20 (46.5) 0
Floors 79 36 (45.6) 33 (41.8) 4 (5.1)
Residues 97 32a (33.0) 24 (24.7) 7 (7.2)
Processing equipment 104 20 (19.2) 14 (13.5) 8 (7.7)
Miscellaneous 219 37 (16.9) 33 (15.1) 5 (2.3)
Total 608 181h(29.8) 158 (26.0) 27 (4.4)
One sample yielded L. seeligeri.
Five samples yielded both L. monocytogenes and L. innocua.
Source: Adapted from Ref. 4 1.

neck-skin trimmers, and conveyors on which carcasses travel to the packing area (Table
17). Although only one to three samples from each site were analyzed in three successive
visits, the areas from which L. monocytogenes was recovered in this poultry-processing
facility are generally similar to those observed by Genigeorgis et al. [46,47] for chicken
and turkey slaughterhouses in California (see Table 8 ) .

Australia
Information concerning the prevalence of listeriae in food-processing facilities located in
other parts of the world is currently limited to a few Australian studies. Following the
isolation of L. monocytogenes from ricotta cheese in 1987, the Victorian Dairy Industry
Authority and the Department of Agriculture and Rural Affairs conducted a joint survey
to determine the extent of Listeria contamination in the working environments of 5 2 Mel-
bourne-area factories producing pasteurized milk and different types of cheese [76]. Over-
all, various Listeria spp. were detected in 141 of 763 ( 1 8.5%) environmental samples from
21 of 5 2 (40.4%) factory environments, with L. monocytogenes, L. seeligeri, and L. iva-

TABLE17 Incidence of Listeria spp. in the Working Environment

of One Poultry-ProcessingFacility in England
No. of No. (%) of positive samples
samples
Type of sample analyzed L. monocytogenes L. innocua
Transport crates 9 0 I (11.1)
Automatic carcass opener 3 3 (100) 0
Evisceration-line drain 3 2 (66.7) 0
Neck-skin trimmer 3 2 (66.7) 0
Conveyor to packing area 3 1 (33.3) 0
Source: Adapted from Ref. 5 I .
Listeria in Food-Processing Facilities 679

novii being identified in 132 (93.6%), 8 (5,7%), and 1 (0.7%) of these Listeria-positive
samples, respectively. More important, L. rnonocytogenes was present in all but one of
the ListeriLz-positive factories. As expected from other surveys conducted in the United
States and Western Europe, factory sites most frequently contaminated with listeriae once
again included drains and floors in coolers, surfaces of manufacturing and packaging
equipment, and conveyors. Even though strict cleaning and sanitizing programs were im-
plemented at many of these facilities, Listeria spp. were very difficult to eliminate from
the working environment, with these organisms being continuously isolated from one fac-
tory over a period of 5 months.
Sutherland and Porritt [73a] conducted a 3-year study in 12 Australian dairy-pro-
cessing facilities to assess the environmental diversity and identify the major environmen-
tal niches for L. rnonocytogenes. A total of 565 environmental samples were collected
and tested. The overall incidence of Listeria-positive samples was 21% (Table 18). Ap-
proximately half of these samples (12%) were positive for L. monocytogenes.
Cheese, ice cream, and mixed-product plants all had similar incidences of L. rnono-
cytogenes and Listeria spp. The incidence of L. rnonocytogenes in mixed-product factories
(18%) was comparable to the higher levels found in milk factories. Sutherland and Porritt
[73a] also highlighted four major ways that L. rnonocytogenes enters a dairy-processing
facility, including:

1. Ingredients-especially raw milk

2. Inward goods-including milk crates and crate washers, vehicles (trucks, road,
and rail tankers), and wooden pallets
3. Environment-including air and internal air quality
4. Personnel-especially outside contractors and visitors

Once L. rnonocytogenes is inside the processing plant, these authors [73a] found numerous
areas in which this pattern can survive, grow, and potentially contaminate product. Con-
veyor systems, drains, and floors were the most common isolation sites. Other areas of
concern related to were traffic flow, cooking units, and internal air quality.
Complete elimination of listeriae from dairy-processing facilities may, in some in-
stances, be nearly impossible; however, the likelihood of producing Listeria-contaminated
products can be greatly reduced by following GMPs, which include implementation of
rigorous cleaning and sanitizing programs for equipment used at critical points during
manufacture and packaging of the foods in question.

INCIDENCE OF LISTERIA IN HOUSEHOLD KITCHENS

Thus far, this chapter has dealt exclusively with Listeria contamination in commercial
food-processing facilities; however, because of the relatively high incidence of Listeria
spp. (including L. monocytogenes), salmonellae, and other foodborne pathogens in fresh
beef, pork, lamb, and poultry available to the general public at butcher shops and supermar-
kets, safe home preparation of these foods must be reemphasized. In 1989, Cox et al. [41]
isolated nine strains of listeriae from 7 of 35 (20%) household lutchens surveyed in the
Netherlands. Overall, L. rnonocytogenes was recovered from four dishcloths and one re-
frigerator, with two dishcloths and two dustbins from two other households yielding L.
innocua and L. welshirneri, respectively. Considering results from commercial food-pro-
cessing facilities, one might expect to recover Listeria spp. from such household kitchen
680 Gravani

TABLE
18 Three-Year Study in 12 Australian Dairy-Processing Plants to Determine Environmental Diversity and Identify Major
Environmental Niches of L. rnonocytogenes

No. of No. of Listeria spp. L. monocytogenes L. innocua L. grayii Mixed

Factory type factories samples (%) (%o) (%) (%) (%I
Cheese 7 319 34 (11) 26 (8)
Milk 2 87 51 (59) 20 (23)
Ice cream 1 53 9 (17) 3 (6)
Mixed product 2 106 22 (21) 19 (18)
Total 12 565 116 (21) 68 (12)
Source: Adapted from Ref. 30a.
Listeria in Food-Processing Facilities 681

areas as drains, U-tubes, and drain boards. If this is true, then garbage disposal systems
could conceivably lead to problems from production of aerosols. Although further work
is needed to clarify the public health significance of listeriae in the kitchen environment,
you may recall from Chapter 13 that L. rnonocytogenes was found in many refrigerated
foods belonging to an Oklahoma woman who contracted listeriosis after consuming con-
taminated turkey frankfurters that were eventually recalled nationwide.
Centers for Disease Control and Prevention (CDC) officials also isolated L. rnonocy-
togenes from 15 of 25 (60%)refrigerators that were used by apparent victims of foodborne
listeriosis [25]. Hence, consumers should regularly clean and sanitize kitchen areas, sinks,
and refrigerators. Such efforts should help prevent potential problems involving listeriosis
and other forms of foodborne illness in the home.

CONTROL OF LISTERIA IN FOOD-PROCESSING FACILITIES

The discovery of Listeria spp., including L. rnonocytogenes, in various fermented and
unfermented dairy products, raw and ready-to-eat meats, poultry products, seafoods, and
vegetables has prompted food manufacturers to renew their concern about factory hygiene
and product safety. Although failsafe procedures for the production of Listeria-free foods
largely do not yet exist, specific guidelines have been developed for controlling listeriae
and other microbial contaminants within American dairy- [4,18,36,49,57,68,73,75],meat-
[ I ,2], poultry- [ 191, and seafood- [43,45,77] processing facilities with Denmark [21], En-
gland [53,58,67], France [ 171, and Australia [30a] also addressing the elimination of lister-
iae from fluid milk and cheese operations during all facets of production, distribution, and
retail sale.
In response to the discovery of L. rnonocytogenes in ready-to-eat foods and delicates-
sen products, European public health officials have expressed particular concern about
contamination of these products during retail slicing and storage. They also have warned
grocery store managers to give particular attention to storage temperatures for refrigerated
foods in display cases and the potential sale of products beyond their normal code dates.
Most of these guidelines stress the need to (a) decrease the possibility that raw products
will contain listeriae, (b) minimize environmental contamination in food-processing facili-
ties, and (c) use processing methods that will eliminate listeriae from food. Following
these proposed guidelines, which will be discussed in detail shortly, will decrease the
possibility of producing foods contaminated with L. monocytogenes and other foodborne
pathogens. I11 addition, diligent attention to cleaning and sanitation and overall GMPs will
lead to lower microbial populations in processed foods which will in turn increase the
shelf life of the finished product.
Any approach to controlling the spread of listeriae and other microorganisms in
food-processing facilities is complicated by the enormous variety of foods being processed
today along with variability in quality of incoming raw products, design of the factory,
sanitary design of the processing and packaging equipment, and processing methods. How-
ever, this subject can be simplified by first focusing on problem areas such as factory
design, general factory environment, heating and air-conditioning systems, traffic patterns,
and personnel cleanliness that are common to all food-processing facilities. Once Listeria-
control measures for these problem areas are understood, attention can be given to specific
processing steps which are unique to the dairy, meat, poultry, seafood, and vegetable
industries.
682 Gravani

General Guidelines
Factory Design
Every food processor should be firmly committed to the long-term production of safe,
wholesome food. The first step toward such a goal is an adequately designed factory
to produce the particular product. Although newly constructed buildings offer countless
advantages in that they can be designed for production of specific products, existing build-
ings also can be used for safe production of food provided that such facilities have been
properly modified to meet certain basic requirements.
Design features that are widely considered to be essential for all types of food-
processing facilities include (a) a raw product receiving area that is completely isolated
from processing and packaging areas of the factory; (b) tight-fitting exterior windows and
doors that will prevent animals and insects from entering processing and packaging areas;
(c) easily cleaned and sanitized walls, floors, and ceilings that are constructed of tile,
metal, or concrete and not porous materials such as wood; (d) floors designed to drain
rapidly and prevent pooling of water; (e) floor drains located away from packaging equip-
ment, especially if processed foods are exposed to factory air; (f) proper screens, debris
baskets, and traps on floor drains; (g) a quality control and/or quality assurance laboratory
that is well isolated from other areas of the factory; and (h) proper means of waste disposal
outside the factory to discourage congregation of insects, rodents, birds, and other animals
that may harbor Listeria and other pathogenic microorganisms.
In addition to these concerns, the heating, ventilating and air-conditioning (HVAC)
system also must be properly designed to minimize airborne contamination [68]. Features
considered to be essential for such a system include (a) intake air vents on the roof of
the building that are located upwind from prevailing air currents but away from dumpsters,
raw product receiving areas, and vents that are discharging factory air; (b) installation of
screens and filters inside incoming air vents to remove particulate matter and condensate;
(c) easily cleanable HVAC systems; and (d) proper location of dehumidifiers and air-
conditioning systems so that these units drain away from processing and packaging areas.
Most important, all HVAC systems must be designed to produce a higher positive air
pressure in processing and packaging rather than in receiving areas. This design readily
prevents movement of airborne contaminants from raw product areas to the cleanest areas
of the factory where foods are processed and packaged.
Factory Environment
Various bacteria, yeasts, and molds can be found in most food-processing areas other than
those associated with aseptic packaging, with populations normally being many times
higher in receiving than in processing and packaging areas. Furthermore, most of these
microorganisms will grow in the factory environment if given a suitable temperature and
enough time along with an adequate supply of nutrients and water. Although microbial
contamination will always occur in food-processing facilities, eliminating microbial
growth by altering (a) temperature, (b) time that the organism is present in the environ-
ment, (c) availability of nutrients, and/or (d) availability of water will sharply decrease
the incidence of L. rnonocytogenes and other foodborne pathogens as well as spoilage
organisms in the factory environment. Hence, production of a safe food product with a
long shelf life depends largely on control of timehemperature constraints and elimination
of available nutrients and/or water through the concerted effort of everyone involved.
Since air, water, waste products, and anything else that comes in contact with the
Listeria in Food-Processing Facilities 683

finished product must be considered as a potential source of contamination, food proces-

sors must strive to prevent the spread of microbial contaminants from heavily contami-
nated raw product receiving areas to processing and packaging areas. In addition to con-
struction of physical barriers between such areas in food-processing plants, all incoming
cases, pallets, containers, forklifts, and cleaning materials such as brushes and other equip-
ment must be assumed to harbor listeriae along with other microbial contaminants and
therefore should never be allowed to enter processing and packaging areas. Ideally, sepa-
rate equipment, including tools employed by maintenance persons, should be available
for use in raw and finished product areas. If this is not possible, then all equipment should
be cleaned and sanitized before entering processing and packaging areas.
As previously stressed, all areas within food-processing facilities should be kept dry
and as free as possible from processing waste to minimize microbial growth. Also, floor
drainage problems that lead to pooling of water must be eliminated as well as cracks and
holes in floor tiles and grouting in which water and food particles can accumulate. Since
L. monocytogenes has been recovered from condensate in dairy factories, it is imperative
to keep all processing and packaging equipment and walls, floors, and ceilings as conden-
sate-free as possible. In the event that dripping condensate cannot be prevented by manipu-
lation of temperature and humidity in processing and packaging areas, deflector shields
should be installed to prevent direct contact between exposed product and dripping con-
densate. Aerosols provide another ready means for disseminating listeriae and other micro-
bial contaminants throughout critical areas of food-processing facilities [55],with L. rno-
nocytogenvs surviving 3.42 h in experimentally produced aerosols of reconstituted skim
milk [7 11. Therefore, high-pressure sprays should never be used in processing and packag-
ing areas for cleaning floors or drains, since both are major sources of listeriae and other
microbial contaminants and resulting aerosols can contaminate food-contact surfaces of
equipment. Operation of unshielded centrifugal pumps in such areas also is discouraged.
In addition to the building itself, all equipment within the factory should be designed
to minimize cross contamination between the factory environment and product and also
should be constructed of stainless steel or other easily cleaned and sanitized nonabsorbent,
nontoxic materials such as certain types of bonded rubber and plastic. All piping in food-
processing facilities should be free-draining and designed to eliminate trapping of food
and cleaning and sanitizing solutions used in clean-in-place (CIP) systems. It also is impor-
tant that equipment such as product conveyors is positioned high enough above the floor
to minimize cross contamination from floors and drains.
The air supply within the factory must be considered as a potential source of Listeria
and other microbial contaminants. Hence, all HVAC ducts and accompanying air filters
should be kept in good repair and cleaned regularly to eliminate excessive dust and dirt.
Compressed air lines and filters should also be inspected regularly and be free of moisture,
oil, and debris.
Cleaning a n d Sanitizing
Cleaning can be defined as the physical removal of visible dirt, impurities, and other
extraneous matter (i.e., nutrients for growth of microorganisms, including Listeria)
through proper use of solutions of soaps, detergents, surfactants, and abrasive agents. In
contrast, sanitizing causes inactivation of most microorganisms left on cleaned surfaces
by exposing them to heat or chemical agents such as chlorine, iodine (iodophor), acid
anionic, or quaternary ammonium compounds. Hence, the routine use of good cleaning
and sanitizing practices is of utmost importance in controlling microbiological safety and
684 Gravani

quality of finished products. In establishments such as those that produce fluid milk and
ice cream, adherence to good cleaning and sanitation practices that involve both equipment
and the factory environment is the only means of preserving product quality beyond initial
pasteurization of ingredients.
Each food-processing facility needs to institute and enforce an effective cleaning and
sanitizing program that will ensure production of safe products. As part of this program,
management personnel need to develop standard operating procedures for every job in
the factory along with master schedules with the frequency of cleaning and sanitizing
procedures so that the workers will recognize their individual responsibilities and will
maintain accurate records regarding routine sanitation practices. Management personnel
also need to instill in their employees the great importance of good cleaning and sanitizing
practices through the use of continuing education programs that deal with current issues
such as Listeria. Such cleaning and sanitizing responsibilities should never be assigned
to new untrained employees.
Floors, drains, walls, ceilings, and each piece of equipment in the factory should
be cleaned and/or sanitized on a regular basis with the frequency of cleaning and sanitizing
being dependent on the extent to which the particular item becomes contaminated during
normal operation and whether or not a product is likely to come in contact with the item
during processing and/or packaging. All food-contact surfaces such as tables, peelers,
slicers, collators, overhead shielding, conveyors, conveyor belts, chain rollers, supports,
and other intricate equipment directly associated with processing, filling, and packaging
operations need to be cleaned and sanitized daily and in some instances more often, partic-
ularly around filling and packaging operations. A regular cleaning and sanitizing schedule
also must be adopted for non-food-contact surfaces such as floors, walls, ceilings, floor
drains, pipes, blowers, HVAC ducts, coils and pans from dehumidifying and air-condition-
ing units, light fixtures, material handling equipment, and wet and dry vacuum canisters.
As indicated in the first half of this chapter, Listeria spp., including L. monocytogenes,
have been most frequently isolated from floor drains and floors, thus suggesting that these
areas may function as reservoirs for listeriae in food-processing facilities. Although all
floors and drains, including drain covers and baskets, in production and refrigerated storage
areas should be thoroughly cleaned and sanitized daily, high-pressure hoses should never
be used in these areas, since such practices readily promote the spread of listeriae to nearby
equipment and other areas of the factory through splashing and the production of aerosols.
Managers of food-processing facilities must be sure that proper equipment is avail-
able for daily cleaning and sanitizing operations. Absorbent articles such as sponges and
rags should never be used in the factory environment, since these items function as virtual
microbial zoos. Various types of metal scrappers can be used for removing hard mineral
deposits, with disposable paper towels being best suited for eliminating excess moisture
and accidental spills. Unlike sponges and rags, brushes are readily cleaned and sanitized
and are therefore suitable for widespread use in the factory. However, to avoid cross
contamination, separate color-coded brushes with nonporous plastic or metal handles
should be used for scrubbing (a) exterior and interior surfaces of equipment, (b) raw and
finished product areas, (c) food-contact and non-food-contact equipment surfaces, and
(d) floor drains. Brushes, particularly those used to scrub floor drains, are best cleaned
and stored in sanitizing solution after use.
Sanitizing is the final step in eliminating L. monocytogenes, other foodborne patho-
gens, and the myriad of spoilage organisms present in the production environment. Since
the presence of organic debris, particularly if proteinaceous, readily decreases the effec-
Listeria in Food-Processing Facilities 685

tiveness of sanitizing agents against most microorganisms, including listeriae [33],it is

important to remember that every item must first be thoroughly cleaned before it is sani-
tized.
Research has demonstrated that L. monocytogenes is sensitive to sanitizing agents
commonly employed in the food industry. According to several authors [6 1,651, chlorine-
based, iodine-based, acid anionic, and/or quaternary ammonium-type sanitizers were ef-
fective against L. monocytogenes when used at concentrations of 100 ppm, 25-45 ppm,
200 ppm, and 100-200 ppm, respectively. Although these concentrations may have to be
adjusted to compensate for in-plant use as well as oxidation and reduction factors relating
to water quality and hardness, recommended concentrations should not be markedly ex-
ceeded, since the use of extremely concentrated sanitizing solutions heightens the danger
to employees, increases the risk of chemical contamination of food, and in some instances
causes corrosion of equipment. Since foaming chlorine-based sanitizers are corrosive, their
use should be primarily confined to floors, floor drains, walls, and ceilings. Alternatively,
these areas can be flooded or foamed with quaternary ammonium-type sanitizers (-300
ppm); however, fogging exterior surfaces with quaternary ammonium-type sanitizers is
frequently regarded as being ineffective and dangerous for employees. Quaternary ammo-
nium-based sanitizers also are not recommended for use on food-contact surfaces and
should never be used in cheese or sausage factories, since lactic acid starter culture bacteria
are rapidly inactivated by small residues of these sanitizers. In contrast, acid anionic and
iodine-type sanitizers are best suited for equipment surfaces, with the former readily neu-
tralizing excess alkalinity from cleaning compounds and preventing formation of alkaline
mineral deposits. Although also effective, the use of steam should be confined to closed
systems because of potential hazards associated with aerosol formation. Sanitizing with
hot water is not advised, since sufficiently high water temperatures cannot be easily main-
tained.
Custom-designed CIP systems have been installed in many food-processing facili-
ties, particularly dairies, for automated cleaning and sanitizing of pipelines, tanks, vats,
heat exchangers, homogenizers, and other equipment in processing lines. Although pre-
sumably adequate by design, CIP systems also should be reviewed for proper timing, flow
rate, temperature, pressure, and sanitizer strength as recommended by chemical suppliers.
Furthermore, proper operation of the entire system should be verified from data collected
on recording charts, which can be stored for future reference.
Regardless of how well these recommendations for cleaning and sanitizing are fol-
lowed, every food-processing facility should verify the effectiveness of its cleaning and
sanitation program through daily microbiological analysis of both product and environ-
mental samples gathered from all areas of the facility. During environmental sampling,
the efficacy of cleaning and sanitizing procedures can be easily determined through the
use of ATP bioluminescence monitoring systems that are available from a number of
manufacturers [42d]. Particular attention should be given to floor drains, floors, filling
and packaging areas, and any processing equipment that is difficult to clean. Although
environmental samples are most easily collected using swabs or sponges, only polyure-
thane or expanding cellulose sponges should be used for such a purpose, since other types,
including retail cellulose sponges, contain inhibitory agents that not only prevent recov-
ery of L. monocytogenes and Staphylococcus aureus but also interfere with recovery of
Brochothrix thermosphacta, Aerornonas hydrophila, Pseudornonus putrefuciens, and P.
jhorescens as well as Escherichia coli, Serratia marcescens, and Enterobacter cloacae
[60]. It is important to stress that laboratory personnel should never attempt to isolate
686 Gravani

pathogenic microorganisms from such samples unless the laboratory is in a separate build-
ing and completely removed from the factory. Although analysis of environmental and,
if necessary, food samples for microbial pathogens is best left to outside commercial
testing laboratories that are FDA approved or otherwise certified, coliform and standard
aerobic plate counts should be obtained for samples from the factory environment and
the food during all stages of production to monitor the extent of postprocessing contamina-
tion and thus to quickly identify any problems associated with inadequate cleaning and
sanitizing. Coliform organisms are commonly regarded as being indicators of postpro-
cessing contamination and the possible presence of pathogens; however, the presence or
absence of coliforms in food or environmental samples does not guarantee the presence
or absence of foodborne pathogens. In fact, often little if any correlation has been observed
between the presence of coliforms and Listeria in finished product. Therefore, routine
testing of environmental samples for Listeria spp. and other foodborne pathogens by out-
side laboratories remains a critical component of any sanitation verification program.
Traffic Patterns
Employee movement within food-processing facilities also can have a major impact on
the microbiological quality of finished products. Therefore, traffic patterns need to be
developed that restrict or preferably eliminate movement of workers between raw, pro-
cessing, filling, packaging, and shipping areas. Managers need to educate employees about
the spread of Listeria and other microbial contaminants from clothing, boots, and tools
to all areas of the factory, and they need to situate locker rooms, changing areas, and
lunch and break rooms to minimize traffic through production areas. Issuing different-
colored outer garments to workers in various areas of the factory has proven helpful in
monitoring employee movement. Since L. monocytogenes and other microbial pathogens
are commonly associated with raw products of both plant and animal origin, employees
working in raw product receiving areas (including maintenance personnel) and individuals
who deliver raw products, particularly milk haulers, should be denied access to all pro-
cessing areas. When necessary, employee movement between raw product and processing
areas of the factory should only be allowed after completely changing outer garments as
well as scrubbing and disinfecting boots. All workers should be encouraged to use disinfec-
tant-containing footbaths that should be placed in all doorways leading into the factory
as well as between raw product and production areas. These footbaths need to be monitored
daily for sanitizer strength and cleanliness. Since a great variety of microorganisms are
carried on street clothing, it also may be prudent for managers to consider limiting the
number of visitors and tour groups going through the factory. Large glass observation
windows provide ample opportunity for visitors to view processing areas while at the
same time prevent introduction of additional microbial contaminants.
Personnel Clean Iiness
Factory managers and supervisors must stress good employee hygiene and also set a good
example for other workers. All individuals with obvious illnesses, infected cuts, or abra-
sions need to be excluded from working in processing areas or from doing other tasks
that may lead to contamination of food, food-contact surfaces, or packaging materials or
equipment. Furthermore, the use of tobacco and chewing gum as well as the consumption
of food should be banned in processing areas along with the wearing of hairpins, rings,
earrings, watches, and other jewelry. Above all, employees should always wash their hands
thoroughly before starting work, on returning to work, and after touching floors, walls,
Listeria in Food-Processing Facilities 687

light switches, any other unclean surface, and garbage. To further promote their use, hand-
washing facilities should be properly designed and conveniently located near work sta-
tions. All factory workers need to be provided with hair and/or beard nets as well as clean
clothes, suitable footwear, and disposable gloves. Special attention also is needed to assure
that street clothes do not enter processing areas and that factory clothing, including foot-
wear, remain inside the factory. All factory clothing should be changed daily or more
often if soiled, with the responsibility of laundering being left to the employer. Although
these recommendations may, in some instances, be difficult for food processors to follow
and enforce, this task will be made much easier if management can instill in workers the
conviction that each employee is personally responsible for both the quality and safety
of the foods that are produced and ultimately consumed by the public.

INDUSTRY-SPECIFIC EQUIPMENT, PROCESSING

METHODS, AND PRODUCTS
It now is appropriate to briefly examine some of the industry-specific equipment and pro-
cessing methods, many of which have been cited as critical control points for the produc-
tion of Listeria-free dairy, meat, poultry, seafood, and vegetable products. Although this
information will be useful to enhance the effectiveness of preexisting cleaning and sanita-
tion programs, the reader is reminded that food-processing facilities, even though they
manufacture similar products, are all unique in terms of factory design, raw product qual-
ity, and product flow, handling, and processing methods. Therefore, no universally accept-
able cleaning and sanitation program can be developed for the safe production of a given
product.

Dairy Industry
Farm Environment
Since listeriae are widespread in the environment, any quality control program should first
contain a plan to minimize contamination of raw milk with Listeria and other microorgan-
isms on the dairy farm. Along with good animal husbandry practices, including the use
of only high-quality feed and silage, farm workers also should give attention to cleanliness
of the milkhouse and milking equipment. Most important, teats and udders of all cows
should be properly sanitized and dried before milking equipment is attached. Bulk tanks
in which raw milk is stored also need to be properly maintained and inspected regularly.
CIa rif iers and Separat0 rs
All raw milk should be filtered and subsequently clarified and separated by centrifugation
to remove extraneous matter and somatic cells (i.e., leukocytes) before pasteurization.
Since L. monocytogenes is sometimes found in leukocytes, clarifiers and separators should
be well isolated from the pasteurizer and all finished product areas of the factory. Sealed
containers should be used to dispose of all clarifier and separator waste, both of which
may contain high levels of listeriae. Special care also should be used in cleaning and
sanitizing separators, clarifiers, and surrounding areas.
Pasteurization
Proper pasteurization using a vat or high-temperature short-time (HTST) pasteurizer is
the only commercially practical means by which all non-spore-forming pathogens, includ-
688 Gravani

ing L. monocytogenes, can be inactivated in raw milk. Thus it is imperative that all pasteur-
ization equipment be designed, installed, maintained, and operated properly.
Although continuous-flow HTST pasteurization is used to process virtually all fluid
milk and ice cream mix, vat (or batch) pasteurization is employed by many smaller firms,
particularly those involved in cheesemaking, when the volume of incoming raw milk is
too small to justify the use of a continuous-flow HTST system. If vat pasteurization is
used, raw milk must be heated to a minimum of 623C (145F) and then held at that
temperature for at least 30 min. In theory, vat pasteurization is a relatively simple process
with raw milk being pumped into a steam- or hot water-jacketed vat and held for the
prescribed time. However, FDA inspections conducted as part of the Dairy Initiative Pro-
gram mentioned earlier have uncovered numerous problems with vat pasteurizers, includ-
ing improper equipment design, the absence of proper outlet valves and air space thermom-
eters, and improperly operated air space heaters. The latter problem is particularly critical,
since the air space temperature above the product in the vat must be at least 23C (5F)
higher than that of the product at all times to assure proper Pasteurization. Operators of
such pasteurizers should be made accountable for proper performance as well as proper
cleaning and sanitizing of the equipment. In addition, recording charts showing time and
temperature relationships along with other data for each vat of product pasteurized should
be kept for at least 3 months.
As mentioned earlier, continuous-flow HTST pasteurization at 71.7"C (161OF) for
a minimum of 15 s is the principal method for processing raw milk. Although an in-depth
discussion of the many intricate problems associated with HTST pasteurization equipment
is beyond the scope of this book, a basic knowledge of HTST pasteurization is essential
to appreciate the seriousness of some of the recently identified problems that have been
linked to faulty maintenance and/or operation of the equipment. Interested readers may
consult the HTST Pasteurizer Operation Manual [50] for more detailed information on
HTST pasteurization.
All HTST pasteurizers consist of five basic components, as shown in Figure 2:
(a) plate heat exchanger-a series of thin, gasketed stainless steel plates divided into
three sections (heater, regenerator, and cooler) for heating incoming raw milk and cooling
outgoing pasteurized milk; (b) constant level tank-provides a constant level of raw milk
to the HTST system; (c) timing pump-a positive displacement pump that establishes the
holding time of the time and temperature relationship for pasteurization; (d) holding
tube-a length of pipe in which fully heated milk is held for the required holding time;
and (e) flow diversion valve-a three-way valve that will allow properly pasteurized milk
to enter the regenerator section of the plate heat exchanger or divert improperly pasteurized
milk to the constant level tank for repasteurization. In addition to these five components,
a source of steam and/or hot water is required to heat incoming raw milk, a safety thermal
limit recorder is needed to activate the flow diversion value in the event of improper
pasteurization, and a cold milk recorder is required to record the temperature of outgoing
pasteurized milk. Finally, auxiliary components that may be added to HTST units for
additional processing of milk or milk products include a booster pump, homogenizer as
a timing pump, stuffing pump, and flavor treatment or vacuum units.
Inspections of HTST pasteurizers conducted in conjunction with the FDA Dairy
Initiative Program uncovered numerous problems relating to proper installation and main-
tenance of these units. Problems most commonly associated with HTST pasteurization
equipment have included stress cracks and/or pinholes in the heat exchanger plates, leak-
ing gaskets, improper flow diversion valves, and inadequate cleaning and sanitizing of
Listeria in Food-Processing Facilities 689

Raw Milk
Pasteurized Milk - --- Diversion Line
Flow-Diversion valve

Cold Past,
-I
Holding Tube

Cooler Regenerator Heater

Timing Pump

Raw Milk Constant

Level Tank

FIGURE2 Schematic diagram o f milk flow through an HTST pasteurizer. (Adapted

f r o m Ref. 50.)

the pasteurization unit. Although not a strict regulatory requirement, positive pressure
should be maintained between the product and heating medium as well as the product
and cooling medium (sweetwater) to prevent Listeria-contaminated raw milk or sweetwa-
ter from mixing with pasteurized product in the event that some of the heat exchanger
plates contain stress cracks or pinholes. Operators should examine all pasteurization plates
for defects every 6 months using the standard dye test. Sweetwater and glycol solutions
also should be routinely examined for microbial contaminants, since these coolants may
harbor L. monocytogenes, Yersinia enterocolitica, and Salmonella typhimurium for ex-
tended periods along with large populations of psychrotrophs [ 18,661; the latter are particu-
larly detrimental to product shelf life. As was true for vat pasteurization, operators of
HTST pasteurizers must be responsible for proper operation of these units and retain accu-
rate records and chart recordings for each lot of pasteurized product for at least 3 months.
Although the inability of L. monocytogenes to survive the minimum allowable HTST heat
treatment given to commercially available raw milk ( 7 1 . 7 W 1 5 s) is now generally ac-
cepted, most fluid milk processors in the United States are pasteurizing milk at -76.7"C
for 20 s, which is well above the minimum requirements established in the Pasteurized
Milk Ordinance. This more severe heat treatment markedly extends the shelf life of the
finished product by inactivating larger numbers of spoilage organisms than does minimal
HTST pasteurization. However, the psychrotrophic nature of L. monocytogenes increases
the need to prevent introduction of listeriae into the product after pasteurization.
Pipeline and Cross Connections
Many large dairy processors have installed up to several miles of pipeline in the factory
to handle movement of raw milk from storage tanks to the pasteurizer and pasteurized
milk from the pasteurizer to various holding tanks, mixing tanks, and product areas located
690 Gravani

throughout the factory. Considering the enormous quantities of product that can be manu-
factured at such facilities during one production period, careful attention must be given
to each stage of manufacture, since an error made during these operations could adversely
affect thousands of people, as was true for the 1985 outbreak of milkborne salmonellosis
in Chicago.
FDA inspections have uncovered numerous violations related to pipelines, including
cross connections between raw and pasteurized milk lines and/or storage and holding
tanks as well as cross connections between CIP and product lines and other potentially
hazardous circuits. Since many of these lines allow easy bypass of raw product around
the pasteurizer thus permitting postpasteurization contamination in the event of equipment
failure or operator error, factory managers, engineers, or other qualified people need to
walk through the factory and construct an up-to-date detailed blueprint of raw and pasteur-
ized product flow throughout the entire factory. Once the blueprint is constructed, any
unwanted piping, dead ends, illegal cross connections, or unauthorized changes made to
initial installations should be promptly identified and eliminated. Most important, all pas-
teurized product lines need to be separated from raw and CIP lines by a physical break.
In many plants, pipes are physically labeled with the type of product (raw or pasteurized)
that flows through them. To be of continued use, blueprints must be routinely updated
and reviewed for accuracy by walking the blueprints through the factory. Finally, no
piping changes should ever be made without prior review by qualified authorities.
Filling a n d Packaging
Postpasteurization contamination frequently occurs during filling and packaging opera-
tions when products are exposed to difficult-to-clean surfaces on equipment, the manufac-
turing environment, and airborne contaminants [54]. Areas associated with product con-
tamination have included mandrels, drip shields, bottom and top breakers, prefilling coding
equipment, deflecter bars, and cutting blades as well as overhead shielding, conveyors,
conveyor belts, chain rollers, supports, and lubricants. Product extruder heads are particu-
larly prone to contamination and therefore should be sanitized frequently during filling
operations. Such practices will lead to the production of safe products with markedly
increased shelf lives.
Reclaimed a n d Reworked Product
Salvage programs, by their nature, are high-risk operations that can put an entire company
in jeopardy if not done in a sanitary manner. Potential hazards associated with such salvage
operations include (a) failure to repasteurize returned product before reuse; (b) inadver-
tently pumping returned but not repasteurized product through pasteurized product lines
without proper cleaning and sanitizing between use; (c) accidental reuse of outdated prod-
uct; (d) reuse of product returned from retail stores that may have been temperature abused,
tampered with, or exposed to chemical or biological contamination; and (e) the use of
product from contaminated, leaking, or otherwise damaged containers. Therefore, any
product that left the possession and control of the processor or has been mishandled,
inadequately protected from Contamination, or exposed to temperatures of 27.2C (45F)
should be discarded. Dairy processors also should seriously consider confining the use of
reclaimed and repasteurized milk to dairy products prepared from non-Grade A milk.
According to the Pasteurized Milk Ordinance, American dairies involved in re-
claiming programs now must have separate areas or rooms isolated from Grade A milk
operations for receiving, handling, and storing all returned products. Outdated products
Listeria in Food-Processing Facilities 691

and those which have left the control of the processor and later are returned to the dairy
for disposal should never reenter the factory. Given the recent isolation of L. monocyto-
genes and other microbial contaminants from the external surface of cartons containing
returned product, along with the proven ability of L. monocytogenes and Salmonella spp.
to survive up to 14 days on the external surface of both waxed cardboard and plastic-type
milk containers [72], the process of opening containers and emptying reclaimed product
into vats for reprocessing will likely introduce many new unwanted microbial contami-
nants into the factory environment. Therefore, it is imperative that all returned products
be handled similar to raw milk and be repasteurized, preferably using times and tempera-
tures well above the required minima. After reprocessing, all equipment including tanks,
pumps, and pipelines used in the reclaiming operation should be thoroughly cleaned and
sanitized. In view of the problems associated with salvage operations, each dairy processor
needs to reevaluate the advantages and disadvantages involved in reclaiming products and
then decide whether or not the monetary benefits gained by such practices will outweigh
the potential public health and other risks.
Frozen Dairy Products
Although few bacterial species can grow at temperatures below OOC, most microorgan-
isms, including listeriae, can survive for long times in frozen dairy products such as ice
cream, ice cream novelties, and sherbet. Unlike fluid milk, frozen dairy products are partic-
ularly susceptible to microbial contamination during freezing and filling operations. All
barrel freezers used to make frozen dairy products should be thoroughly sanitized before
use, since hand assembly of the many intricate freezer parts is likely to introduce numerous
contaminants. The source of air for the barrel freezer is another likely source of contamina-
tion. Hence, in addition to maintaining positive air pressure in this area and keeping the
surrounding area as clean and sanitary as possible, all air lines connected to the barrel
freezer should be equipped with dryers and bacterial filters to prevent airborne contami-
nants from entering the product.
Ingredient feeders are perhaps the greatest source of Contaminants in frozen dairy
products. Therefore, fruits, nuts, candy, and other ingredients that are added directly to
frozen ice cream mix need to be closely monitored for coliforms, pathogens, and other
microbial contaminants. Exposure of ingredients to the factory environment also should
be minimized. Strict adherence to GMPs is necessary during the production of molded,
extruded, and/or dipped ice cream novelties, since many such products have been recalled
because of contamination with L. monocytogenes (see Table 5 in Chapter 11). Condensate
in and around hardening rooms as well as conveyor belts appears to be a likely source
for such contaminants.
Finally, handling of product rerun exiting the freezer needs to be assessed at each
factory. Although rerun product should never be added directly back to tanks containing
unfrozen mix, frozen rerun product can be reclaimed by blending it with fresh mix, which
is then repasteurized. Any rerun that is not reclaimed should be clearly separated from
reclaimable material and properly disposed.
Fermented Dairy Products
Fortunately, the incidence of Listeria contamination in yogurt, cultured cream, cultured
buttermilk, and other fermented fluid milk products appears to be quite low with very
few recalls being issued for these products. The species of lactic acid bacteria used in
manufacturing these products as well as the bacteriostatic and bactericidal effects of vari-
692 Gravani

ous organic acids produced during fermentation and the resultant lowering of pH are un-
doubtedly responsible for the near-absence of such recalls. However, since bacterial patho-
gens (including L. monocytogenes) and various spoilage organisms may inadvertently
contaminate fermented milk products during any stage of manufacture, producers of such
products need to follow GMPs and be readily aware of potential problems regarding im-
proper cleaning and sanitizing of equipment and the processing plant environment as well
as potential sources of postpasteurization contamination (e.g., filling and packaging areas)
discussed earlier in this chapter.
The production of Listeria-free cheese, particularly soft and semisoft varieties
surface-ripened with mold (e.g., Brie, Camembert) or bacteria (e.g., brick, Limburger), is
difficult, since environmental conditions required for proper cheese ripening also promote
the growth of L. monocytogenes and other unwanted organisms. Swiss officials who inves-
tigated the 1987 listeriosis outbreak involving consumption of Vacherin Mont d' Or soft-
ripened cheese (see Chapter 12) eventually isolated the epidemic strain of L. monocyto-
genes from wooden shelves and cheese hoops found in over half of the caves used to
ripen the tainted cheese. Thus the basic problem associated with soft cheese manufacture
is to prevent postprocessing contamination by eliminating L. monocytogenes from the
ripening room and particularly the shelves on which such cheese must be ripened. Consid-
ering the ability of L. monocytogenes to grow very rapidly both inside and on the surface
of Brie, Camembert, brick, Limburger, and other similar cheeses during ripening, manu-
facturers of such products should test a portion of each lot for listeriae before releasing
the product for sale.
In addition to these concerns, several studies have demonstrated that L. monocyto-
genes can survive well beyond 60 days in brick, Cheddar, and other varieties of cheese
that were prepared from pasteurized milk inoculated with the pathogen. Certain cheeses,
primarily hard and semihard varieties, can be manufactured from raw milk in the United
States and elsewhere if the finished product is aged a minimum of 60 days at or above
1.7"C (35F) to eliminate pathogenic microorganisms. However, since experimental evi-
dence has indicated that this process is inadequate to free contaminated cheese from viable
cells of L. monocytogenes, cheesemakers should consider preparing cheese from pasteur-
ized milk whenever possible.

Meat Industry
Since Listeria spp., including L. rnonocytogenes, are virtually endemic to slaughterhouse
environments, meat processors are faced with an almost impossible challenge of producing
Listeria-free raw meats. Direct application of lactic and/or acetic acid to animal carcasses
is one of the few economically feasible means by which meat processors can effectively
reduce populations of listeriae and other surface contaminants, including common spoilage
organisms [ 16,28,62]. Nevertheless, although adoption of this procedure and following the
general guidelines for controlling listeriae in food-processing establishments will benefit
slaughterhouse operators, it appears unlikely that rigid enforcement of even the most strin-
gent slaughter, dressing, cleaning, and sanitizing procedures will completely eliminate L.
monocytogenes from wholesale and retail cuts of raw beef, pork, and lamb. Therefore,
consumers of such products need to understand the potential health hazards associated
with consumption of less than thoroughly cooked meats and also must follow appropriate
hygienic practices in the kitchen to prevent the spread of listeriae from raw meats to ready-
to-eat foods.
Firms producing processed meat products must assume that all incoming raw meat
Listeria in Food-Processing Facilities 693

is potentially contaminated with listeriae, including L. monocytogenes. Since most Listeria

contamination of finished product appears to result from postprocessing contamination
rather than from the organism surviving various processing treatments, it is essential to
segregate raw and finished products as well as employees working in raw and finished
product areas of the factory. Although there is no magic bullet for Listeria control,
the incidence of listeriae in all areas of the factory can be greatly reduced through conscien-
tious enforcement of a stringent cleaning and sanitation program. One six-step program
that has been recommended for cleaning food contact surfaces [7] includes (a) an initial
dry clean-up step to remove as much product residue as possible, followed by (b) a warm
water rinse (with minimum splashing) to mobilize fat and remove product; (c) cleaning
with an appropriate foaming detergent; (d) warm or hot water rinse with minimum splash-
ing; (e) disinfecting with an appropriate sanitizing agent (i.e., chlorine or quaternary am-
monium compound); and finally (f) thorough drying of the cleaned and sanitized area.
According to Boyle et al. [34,35], L. monocytogenes populations in inoculated samples
of carcass rinse fluid, Hobart meat grinder rinse fluid, and floor drain waste water obtained
from a beef- and lamb-processing facility increased one to four orders of magnitude during
24 h of incubation at 8 and 35C with the pathogen exhibiting shorter generation times
in waste fluids containing 3.1 rather than 5 I .4% protein. Hence, although the procedure
just described may seem adequate, routine random testing for Listeria and coliforms as
well as an estimation of the general microbial load on cleaned and sanitized food-contact
surfaces should be done as an integral part of any sanitation program.
In 1987, the American Meat Institute published some interim guidelines for control-
ling the incidence of listeriae and other pathogenic and nonpathogenic microbial contami-
nants during production of ready-to-eat meat products [2]. Although the recommendations
in this report regarding facility requirements, factory environment, food-contact and non-
food-contact surfaces, cross contamination, airborne contamination, condensation control,
cleaning and sanitizing, traffic patterns, and personnel cleanliness are generally similar to
those already presented in this chapter as General Guidelines, this report also outlined
some of the critical operations associated with the production of specific categories of
ready-to-eat meat products
Roast Beef, Corned Beef, a n d Other Rebagged Products
Products such as roast beef and corned beef that are repackaged after cooking are particu-
larly prone to contamination with listeriae and other microorganisms. Therefore, attention
must be given to proper sanitation and prevention of cross Contamination when these
products are removed from bags in which they were cooked. The outside surface of all
bags should be thoroughly washed and sanitized before the bags are opened. In addition
to a sanitary working environment, repackaging of cooked product requires use of clean
clothing as well as frequently sanitized utensils and gloves. Trimming and cutting of
cooked product just before rebagging are two more critical steps where listeriae and other
contaminants can enter and compromise the integrity of the final product. Therefore, con-
tact between cooked product and unsanitized surfaces must be avoided during rebagging
operations. Since repackaging is by nature a wet process, this operation also needs to be
well isolated from other processing areas to reduce cross contamination.
Frankfurters and Other Link Products
Sausages such as frankfurters and other link varieties are typically prepared from a finely
ground mixture (or emulsion) of beef and/or pork, which is stuffed into artificial or nat-
ural casings. After twisting the casing at approximately 6-inch intervals, the links are
694 Gravani

cooked using steam or hot water and then hung for smoking. To obtain skinless frankfurt-
ers, the artificial casing must be mechanically peeled from the congealed meat mixture.
Although prompt attention to cleaning, sanitizing, and cross-contamination problems is
required during all stages of frankfurter production, the product is particularly vulnerable
to contamination with listeriae and other microorganisms during the peeling process. It
is imperative to keep the area around peeling machines as dry and as free from meat
scraps and juices as possible. Peeling machine operators also need to change protective
garments and gloves frequently. Hoods on peeler machines have been cited as a source
of listeriae and should therefore be eliminated if at all possible.
Manufacturing practices also should be reviewed to ensure that losses from floor
contamination and reworked product are minimized. Although unpeeled frankfurters that
touch the floor or other unclean surfaces can be reworked (i.e., washed and peeled after
all other frankfurters have been peeled), any peeled frankfurters that come in contact with
the floor or other unclean surfaces should be destroyed. This latter recommendation is
supported by data indicating that L. monocytogenes is difficult to destroy on the surface
of frankfurters during cooking without making the product organoleptically unacceptable
[ 131. In addition to these concerns, brine chillers also have been cited as a potential source
of listeriae, thus leading to contamination of casings and product surfaces. Finally, all
packaging and heat-shrinkmg equipment should be cleaned and sanitized daily to avoid
spreading contaminants from steam and water to packaging lines.
Luncheon Meats
Concerns regarding control of listeriae and other contaminants in luncheon meats are
generally similar to those just discussed for frankfurters and other link products. However,
in addition, slicing equipment should be kept dry and free of scraps and juices that may
serve as potential nutrients for microbial contaminants, including listeriae.

Poultry Industry
Potential sources of listeriae contamination during processing of raw poultry are in many
ways similar to those just discussed for the meat industry. Since a substantial percentage
of birds harbor Listeria spp. (including L. monocytogenes) and Salmonella in their intesti-
nal tract, enforcement of proper clean-up (i.e., elimination of water, condensate, and waste)
and cleaning and sanitizing programs will likely decrease the incidence of contamination
but will never completely eliminate these pathogens from raw poultry-processing facilities
or the raw product.
Most modern poultry-processing facilities are continuous line operations in which
incoming birds are shackled, electrically stunned, bled, scalded to facilitate feather re-
moval, plucked of feathers, eviscerated, inspected, washed, chilled, dried, and packaged
for sale. Processing steps during which L. rnonocytogenes, Salmonella spp., and other
pathogens are most likely to contaminate the product include scalding, defeathering, evis-
ceration, and chilling [63,64]. In 1988, USDA officials proposed processing changes that
may be helpful in decreasing the incidence of Salmonella (and presumably Listeria) in
raw poultry [ 191. These changes included (a) segregating and processing pathogen-infected
flocks at different times from noninfected flocks; (b) examining the potential benefits of
adding bactericidal concentrations of organic acids to chill water tanks; (c) experimenta-
tion with different scalding methods (e.g., hot water sprays, steam scalders, or scald addi-
tives); (d) routine sanitizing of all equipment and utensils with hot water or bactericidal
Listeria in Food-Processing Facilities 695

agents; (e) reemphasis of employee hygiene programs with routine handwashing and sani-
tizing required by all evisceration line workers; (f) elimination of off-line processing; and
(g) installation of equipment designed automatically to transfer carcasses from the picking
line to the evisceration line. Additional work is needed to streamline further processing
of poultry carcasses and minimize cross contamination during their processing.
With increasing consumption of poultry both in and outside the home, persons pre-
paring these products must take special precautions to prevent the spread of L. monocyto-
genes, Salmonella spp., and other foodborne pathogens from raw poultry to other products
(e.g., fruits and vegetables) that are frequently consumed without heating. The common
practice of washing and rinsing raw poultry before coohng has been questioned, since
this step fails to reduce microbial populations markedly on poultry skin and also leads to
increased contamination of kitchen sinks, faucets, and other food preparation areas [79].
Since all foodborne pathogens commonly associated with raw poultry (including L. mono-
cytogenes) are readily susceptible to heat, thorough cooking appears to be the best means
of assuring that such products are free of hazardous microorganisms.
An Oklahoma breast cancer patient contracted listeriosis in December of 1988 after
consuming Listeria-contaminated turkey frankfurters. Thus producers of processed poultry
products (e.g., turkey and chicken frankfurters and rolls) need to take precautions similar
to those previously described for the manufacture of roast beef, corned beef, frankfurters,
link sausage, and luncheon meats with special attention being given to the cleanliness of
rebagging operations and sausage peelers.

Egg Industry
As stated earlier, the contents of intact whole eggs are normally sterile unless the laying
hen infects the yolk with Salmonella enteritidis. Foodborne pathogens, including L. mono-
cytogenes and S. enteritidis, have frequently been isolated from commercially broken,
raw liquid whole egg, with contamination most likely resulting from the presence of the
organisms in the manufacturing environment or on eggshells. Although pasteurization as
required for commercially broken, raw liquid whole egg is likely sufficient to eliminate
normally encountered populations of L. monocytogenes and salmonellae in raw liquid egg,
all egg-breaking operations need to be well isolated from pasteurization and filling and
packaging areas to minimize recontamination of finished product. Since L. monocytogenes
and other foodborne pathogens probably enter egg-processing facilities as eggshell con-
taminants, egg receiving and washing sections of the factory also should be segregated
from other processing areas. Considering the potential for postpasteurization contamina-
tion, many of the previously described guidelines for cleaning and sanitizing dairy facto-
ries also appear to be applicable to manufacturers of pasteurized liquid egg products.

Fish and Seafood Industry

L. monocytogenes and other foodborne pathogens such as Vibrio, Salmonella, Shigella,
Staphylococcus aureus, Clostridium botulinum, Aeromonas hydrophila, and certain strains
of Escherichia coli have been isolated from raw and/or cooked finfish, shrimp, crab, lob-
ster, oysters, and scallops. An integrated approach to product safety is needed to minimize
contamination of seafood from harvest to the time of consumption.
On December 18, 1997, the FDA adopted the final regulations to ensure the safe
and sanitary processing of fish and fishery products [75a]. The regulations mandate the
application of HACCP principles to the processing of seafood. Seafood processors and
696 Gravani

importers need to evaluate the kinds of hazards that could affect their products, institute
controls to keep these hazards from occurring, or significantly to minimize their occur-
rence, monitor the performance of those controls, and maintain records of this monitoring
as a matter of routine practice.
Limiting postharvest contamination of freshly caught fish and seafood is the first
step toward producing a safe, high-quality endproduct. Adherence to good sanitation and
hygienic practices aboard fishing vessels is imperative. Contact between freshly caught
seafood and waterfowl such as pelicans and seagulls should be minimized, because these
birds are intestinal carriers of L. monocytogenes and other foodborne pathogens. All sea-
food should be either frozen or refrigerated immediately after harvest to stop or retard
growth of microbial contaminants, including spoilage organisms.
Two observations, namely, (a) the routine recovery of healthy rather than thermally
or otherwise injured listeriae from processed seafood and (b) the discovery of L. monocyto-
genes in the manufacturing environment of all American factories that have been involved
in Listeria-related recalls, indicate that this pathogen enters the product primarily after
processing through improper handling. Inadequate separation between raw and finished
product resulting from faulty factory design and indifferent attitudes of employees toward
proper sanitation have been most frequently cited as factors that promote postprocessing
contamination.
The general guidelines that were discussed previously regarding factory design, pro-
cessing environment, proper cleaning and sanitizing, employee traffic patterns, and person-
nel cleanliness also are valid for the seafood industry. In addition to these recommenda-
tions, seafood processors also are urged to (a) eliminate processing waste, pooled water,
and condensate from walls, floors, and ceilings as well as from processing and refrigerated
areas; (b) eliminate the use of high-pressure sprays; (c) reduce airborne contamination;
(d) cover outside dumpsters to decrease problems involving seagulls and other wildlife;
(e) assign specific equipment (i.e., product totes) for use in either raw or cooked product
areas of the factory; and (f) if possible, replace wooden totes with fiber totes, which can
be easily cleaned and sanitized.
Listeria spp., including L. monocytogenes, have been isolated most frequently from
crabmeat and cooked and peeled shrimp (see Table 4 in Chapter 15). This observation is
not surprising if one considers how these products are processed and packaged for the con-
sumer. Processing of Dungeness crab generally begins by immersing and cooking either
sections of or the entire crab in boiling water for approximately 7-9 or 17-20 mins, re-
spectively. Although current information indicates that such a heat treatment is sufficient
to destroy listeriae [43], underprocessing may lead to survivors. After cooking, the crab
is cooled in a water bath and either picked immediately or iced and refrigerated in a
walk-in cooler until the meat can be hand picked from the shell. Extensive handling of the
product by workers during picking, subsequent inspection, and packaging affords many
opportunities for postprocessing contamination. Although lactic acid dips appear to be
somewhat useful in reducing populations of L. monocytogenes and other microorganisms on
the surface of crabmeat as well as fresh and frozen shrimp, such treatments will not com-
pletely eliminate listeriae from the finished product [43]. Therefore, strict adherence to
GMPs, which include proper employee hygiene and cleaning and sanitizing of picking equip-
ment, must be observed in and among picking areas to avoid negating the benefits of cooking.
Unfortunately, crab processing varies widely with the species of crab-Dungeness,
blue, stone, king, and golden crab. Hence, some of the critical control points discussed
for Dungeness crab are not applicable to other species. For example, blue crabmeat is
Listeria in Food-Processing Facilities 697

typically removed from the animal in the raw state, placed in sealed containers, and then
pasteurized (85"C/ 1 min) to eliminate L. monocytogenes and other microbial pathogens.
Since pasteurization of blue crabmeat becomes a critical control point in processing, it
may be prudent to certify and/or license crabmeat pasteurization operators or their supervi-
sors, as has been required for operators of retorts in the canning industry for many years.
Problems regarding postprocessing contamination also are encountered during the
production of cooked and peeled shrimp. After shrimp are cooked, those destined for
breading are mechanically peeled and sometimes deveined by splitting and removing the
vein-like intestine. Unfortunately, many mechanical shrimp peelers have design flaws
which necessitate almost continuous movement of the operator between both raw and
cooked sides of the equipment, thus affording ample opportunity for postprocessing con-
tamination. Proper cleaning and sanitizing of the equipment (particularly protective covers
over flumes and gutters) and the surrounding area are essential for producing high-quality
microbiologically safe products.
Even when handled under the best possible conditions, raw seafood such as crab,
shrimp, lobster, clams, oysters, and the myriad of finfish currently available to consumers
probably will never be completely free of L. monocytogenes or other foodborne pathogens.
Considering that many individuals are not ' 'seafood-smart,' ' processors and marketers of
seafood have an obligation to educate the general public and provide consumers with
proper haridling and cooking instructions. Individuals who insist on consuming unpro-
cessed fish (e.g., sushi) and seafood (e.g., oysters) also should be made aware of potential
health problems associated with consumption of such products.

Fruit and Vegetable Industry

Despite a limited amount of information concerning incidence of L. monocytogenes in
raw fruits, the pathogen has been recovered from raw vegetables including cabbage, cu-
cumbers, inushrooms, potatoes, and radishes. Other than the 198 1 Canadian listeriosis
outbreak involving coleslaw and one isolated case in Finland linked to consumption of
raw salted mushrooms, no additional confirmed cases of vegetableborne listeriosis have
been documented in the literature. Thus the scientific community and the public at large
have been, until recently, somewhat less concerned about Listeria contamination in vegeta-
bles than in dairy, meat, poultry, and seafood products.
Routine examination of raw vegetables for L. monocytogenes and other foodborne
pathogens is unlikely to reduce the risk of foodborne illness to any great extent. However,
since raw sheep manure was the probable source of L. monocytogenes in the Canadian
coleslaw outbreak, vegetable processors should have some assurance that incoming raw
vegetables have been grown, irrigated, fertilized, harvested, packaged, and transported to
the firm using hygienically sound agricultural practices. Vegetable processors should con-
sider rejecting raw vegetables that probably will be consumed without cooking if the
grower fails clearly to demonstrate the use of good agricultural practices.
Consumption of vegetables that will be adequately cooked before eating is of little
concern, since L. monocytogenes and other non-spore-forming pathogens are destroyed
during cooking. Although routine washing of raw vegetables in potable water is recom-
mended for commercial establishments and homes, this practice generally fails to reduce
the microbial load on raw vegetables by more than 10-fold. Therefore, persons handling
and preparing raw produce and salad vegetables should follow good hygienic practices
during slicing, dicing, chopping, and grating operations to prevent the spread of potentially
698 Gravani

hazardous microorganisms to other foods. Finally, all knives, cutting boards, and other
food-contact surfaces should be thoroughly cleaned and sanitized after use to inactivate
organisms inadvertently introduced into the kitchen environment during preparation of
raw produce.

PRACTICAL APPROACHES TO FOOD SAFETY

Traditional Approaches
The traditional approaches to controlling microbiological hazards associated with food
products involve the simultaneous use of employee education and training programs, fre-
quent inspection of facilities and operations, extensive microbiological testing of raw in-
gredients, and unfinished and finished products. Employee education and training pro-
grams should be directed toward a thorough understanding of food hygiene, factory
cleaning and sanitation requirements, and various causes of microbial contamination, in-
cluding growth and survival patterns of potential contaminants such as listeriae. Trained
employees also should be able to select and apply control methods that will provide con-
sumers with safe, high-quality products.
The second means of controlling microbiological hazards, frequent inspection of
facilities and operations, is necessary to ensure that GMPs (i.e., procedures that consis-
tently yield safe products of acceptable quality) are being followed. GMPs to produce
specific foods have been outlined in both advisory and regulatory documents such as GMP
guidelines and the various codes of hygienic practice developed by the Codex Alimentarius
Committee on Food Hygiene. The final means of controlling microbial hazards in finished
products is through rigorous microbiological testing of ingredients as well as unfinished
and finished product. Analysis of samples for pathogens or, more commonly, indicator
(coliforms, fecal streptococci) or spoilage organisms is crucial to ascertaining that good
manufacturing, handling, and distribution practices are being followed.

Hazard Analysis Critical Control Point Concept

Although the traditional approaches for controlling microbial contaminants are being used
by many food companies around the world, cases of foodborne illnesses still occur. The
need for a modified approach to food safety assurance led to the development of the
Hazard Analysis Critical Control Point Concept (HACCP) which can be used to identify
and control biological, chemical, and physical hazards in foods from raw material produc-
tion, procurement, and handling to manufacturing, distribution, and consumption of the
finished product. Although a detailed discussion of HACCP is beyond the scope of this
book, a brief explanation will assist the reader in understanding how the concept, along
with GMPs and prerequisite programs, can be used to reduce the level of L. monocytogenes
in food processing facilities and subsequently in cooked, ready-to-eat foods.
The HACCP concept was developed by the Pillsbury Company with the cooperation
and participation of the National Aeronautics and Space Administration (NASA), the Na-
tick Laboratories of the U.S. Army, and the U.S. Air Force Space Laboratory Project
Group [32a]. The development of the HACCP system began in 1959 when Pillsbury was
asked to produce a food that could be used in the space program. There needed to be
assurance (as close to 100% as possible) that the food produced for the space program
would not be contaminated with bacterial or viral pathogens, toxins and chemical or physi-
Listeria in Food-Processing Facilities 699

cal hazards that could cause illness or injury. After much research and evaluation, the
HACCP concept was developed and first presented to the scientific community at the 1971
Conference for Food Protection [74b]. The HACCP concept was first used in the acidified
and low-acid canned food industry and was then adopted by a number of companies during
the 1970s and early 1980s. After an important 1985 National Academy of Sciences publi-
cation strongly recommended the HACCP concept, the food industry expressed consider-
able interest in the application of HACCP.
The National Advisory Committee on Microbiological Criteria for Foods
(NACMCF) was established and embraced the HACCP concept. In 1989, the committee
developed a HACCP document as a guide for maintaining uniformity of the principles
and definitions of terminology [61a]. Since then, the NACMCF has made several refine-
ments and improvements in the HACCP concept and published revisions in 1992 [61b]
and 1997. The 1997 document, entitled HACCP Principles and Guidelines [61c], contains
many additions and includes a section on prerequisite programs. Prerequisite programs
are essential to the successful development and implementation of a HACCP plan [70b]
and form the foundation upon which a HACCP plan is built. Many of the prerequisite
programs are based on the current GMPs in the Code of Federal Regulations [43a] and
in the Codex Alimentarius General Principles of Food Hygiene [53b] for foods intended
for international trade. In addition to specific items in the GMPs, prerequisite programs
can include other activities such as ingredient specifications, supplier approval programs,
ingredient-to-product traceability, and consumer complaint management programs. A
summary of prerequisite program activities is presented in Table 19 [70b].
Prerequisite programs are not part of the formal HACCP system and are established
and maintained separately. There are some circumstances where the existence of a prereq-
uisite program does not preclude the use of specific activities with a HACCP system [70b].
For example, although sanitation procedures are normally part of a prerequisite program,
some manufacturers manage selected sanitation procedures as critical control points
(CCPs) in their HACCP systems. This has been done frequently in the meat and dairy
industries where sanitation procedures for meat slicers, ice cream fillers, and other pieces
of equipment were established as CCPs to help prevent recontamination of processed
products by L. rnonocytogenes [70b].
The existence and effectiveness of prerequisite programs should be assessed during
the design and implementation of each HACCP plan. Well-developed and consistently
performed prerequisite programs can simplify the HACCP plan, so it is imperative that
all food processors establish, document, and maintain effective prerequisite programs to
support their HACCP plans [70b].
HACCP is a management system that is designed for use in all segments of the
food industry from production agriculture to consumption of the finished product. The
HACCP approach for controlling biological hazards in food is based on seven principles
[61c]:

1. Conduct a hazard analysis

2. Determine the critical control points
3. Establish critical limits
4. Establish monitoring procedures
5. Establish corrective actions
6. Establish verification procedures
7. Establish record-keeping and documentation procedures.
700 Gravani

TABLE
19 Summary of Prerequisite Program Activities
-~

Facilities
Adjacent properties
Building exterior
Building interior
Traffic flow patterns
Ventilation
Waste disposal facilities
Sanitary handwashing facilities
Water, ice, culinary steam
Lighting
Raw Materials Controls
Specifications
Supplier approval
Receipt and storage
Temperature control
Testing procedures
Sanitation
Master schedules
Pest control program
Environmental surveillance activities
Chemical control programs
Training
Personal safety
GMPs
HACCP
Production Equipment
Sanitary design and installation
Cleaning and sanitation
Preventive maintenance
Calibration of equipment
Production Controls
Product zone controls
Foreign material control
Metal protection program
Allergen control
Glass control
Storage and Distribution
Temperature control
Transport vehicle cleaning and inspection
Product Controls
Labeling
Product traceability
Customer and consumer complaint investigations

Source: Adapted from Ref. 70b.

Listeria in Food-Processing Facilities 70 1

Principle 1: Conduct a Hazard Analysis

The hazard analysis is the key element in developing an effective HACCP plan. The
purpose of the hazard analysis is to determine which of the potential hazards associated
with a food or a manufacturing process presents a significant risk to consumers. The hazard
analysis involves two stages. The first stage, hazard identification, involves the review of
ingredients used in the product, the activities conducted at each step in the process and
the equipment used, the final product and its method of storage and distribution, and the
intended use and consumers of the product. Hazard identification focuses on developing
a list of potential food hazards associated with each process step. In stage two, the hazard
evaluation, each potential hazard is evaluated based on severity and its likelihood of occur-
rence.
It is important that food processors conduct a hazard analysis on all existing and any
new products to be manufactured, since ultimate microbiological safety of nonthermally
processed foods is directly related to the quality of raw materials. Any hazard analysis
must begin with identification of hazards associated with raw materials, with particular
attention being given to raw products of animal origin (i.e., milk, meat, poultry, and sea-
food), all of which may harbor L. monocytogenes and other foodborne pathogens. Al-
though heat. treatments, acidulation, fermentation, salting, and drying are designed to de-
stroy or inhibit growth of pathogenic and spoilage microorganisms, other operations such
as slicing and dicing, cooling of cooked products, and filling and packaging may allow
pathogenic organisms to contaminate the final product. Therefore, all hazards associated
with manufacturing procedures and postprocessing contamination, as previously dis-
cussed, must be fully understood along with the consequences of processing failures and/
or errors. Food processors should also be familiar with the effect of various physicochemi-
cal factors (i.e., pH, water activity, preservatives, and type of packaging with or without
modified atmosphere) on the behavior of pathogenic organisms, including L. monocyto-
genes, in the product during processing, distribution, storage, and use by the consumer.
The National Advisory Committee HACCP document [61c] contains a series of questions
regarding ingredients, intrinsic factors of the food during and after processing, processing
procedures, microbial content of the food, faulty design, equipment design and use, and
packaging sanitation that can be used when conducting a hazard analysis. It should be
noted that any change in raw materials, product formulation, processing, packaging, distri-
bution, or intended use of the product should prompt an immediate reassessment of haz-
ards, since these changes have the potential to affect product safety adversely.

Principle 2: Determine CCPs

A CCP is a step at which control can be applied and is essential to prevent or eliminate
a food safety hazard or reduce it to an acceptable level. Complete and accurate identifica-
tion of CCPs is fundamental to controlling food safety hazards, and CCPs must be carefully
developed and documented. Examples of CCPs may include thermal processing, chilling,
and producl formulation control.

Principle 3: Establish Critical Limits

A critical limit is a boundary of safety and is used to distinguish between safe and unsafe
operating conditions at a CCP. Each CCP will have one or more control measures to
assure that the identified hazards are prevented, eliminated, or reduced to acceptable levels.
Critical limits may be based on factors such as temperature, time, physical dimensions,
702 Gravani

humidity, moisture level, water activity (aw), pH, titratable acidity, salt concentration,
available chlorine, viscosity, or preservatives. Critical limits must be scientifically based
and may be obtained from regulatory standards and guidelines, the scientific literature,
experimental results, and experts.
Principle 4: Establish Monitoring Procedures
Monitoring is a planned sequence of observations or measurements to assess whether a
CCP is under control and to produce an accurate record for future use in verification.
Monitoring facilitates the tracking of an operation and is used to keep the process in
control. Monitoring is also used to determine when there is loss of control and a deviation
occurs at a CCP (i.e., exceeding or not meeting a critical limit). Monitoring procedures
must be effective to determine deviations and then corrective actions must be taken. Most
monitoring procedures need to be rapid and often include visual observations and measure-
ment of temperature, time, pH, and moisture level. Microbial tests are seldom effective
for monitoring owing to their time-consuming nature and problems with assuring detection
of contaminants.
Principle 5: Establish Corrective Actions
When there is a deviation from an established critical limit, corrective actions are neces-
sary. Through the establishment of corrective actions, foods that may be hazardous are
prevented from reaching consumers. When there is a deviation from critical limits, correc-
tive actions are needed to:
Determine and correct the cause of noncompliance.
Determine the disposition of noncompliant product.
Record the corrective actions that are taken.
Specific corrective actions should be developed for each CCP and included in the HACCP
plan.
Principle 6: Establish Verification Procedures
Verification determines the validity of the HACCP plan and is used in evaluating whether
the facilitys HACCP system is functioning according to the HACCP plan. An effective
HACCP system requires little endproduct testing, since sufficient validated safeguards are
built in early in the process. Firms should rely on frequent reviews of their HACCP plan,
verification that the plan is being correctly followed, and review of CCP monitoring and
corrective action records.
Another important aspect of verification is the initial validation of the HACCP plan
to determine that the plan is scientifically and technically sound, that all hazards have
been properly identified, and that if the HACCP plan is properly implemented, these haz-
ards will be effectively controlled. Subsequent validations are performed and documented
by the HACCP team or independent expert as needed. Validations are conducted when
there is an unexplained system failure, when a significant product, process, or packaging
change occurs, or when new hazards are recognized.
Principle 7: Establish Record-Keeping and
Documentation Procedures
The establishment of an effective record-keeping system is an integral part of a HACCP
system. Records are the only reference available to trace the production history of a fin-
Listeria in Food-Processing Facilities 703

ished product. If questions arise concerning the safety of a product, a review of records
may be the only way to prove that the product was prepared and handled in a safe manner
in accordance with the company's HACCP plan [51a].
A well-developed and implemented HACCP plan built on a strong foundation of
GMPs and prerequisite programs can reduce the level of L. monocytogenes in food-
processing facilities and in cooked, ready-to-eat foods.

SAMPLING PLANS FOR L. MONOCYTOG/VSIN FOODS

Prevention of microbiological hazards is clearly of considerable importance when one
considers the serious health problems that may develop in certain individuals who consume
Listeria-contaminated foods. Consequently, the International Commission on Microbio-
logical Specifications for Foods (ICMSF) proposed that L. monocytogenes be placed in
the same category with Brucella, Clostridiurn botulinurn, C. pe$ringens type C, Salrno-
nella typhi, Shigella dysenteriae, Vibrio cholera, and hepatitis A virus [ 121, all of which
pose severe health hazards.
In February 1988, the ICMSF considered application of its sampling plans to assess
acceptability of foods with respect to L. monocytogenes. (The reader must be cautioned
from the start that no microbiological sampling plan other than one which involves total
destructive sampling of all products manufactured can ever provide complete consumer
protection. j According to terminology developed previously by the ICMSF, sampling
plans for L. monocytogenes would follow the recommendations made for cases 13, 14,
and I5 [52]. Case 13 applies when conditions under which the product is normally handled
and consurned after sampling reduce the degree of hazard associated with the product,
whereas cases 14 and 15 refer to situations in which hazard levels remain constant or
increase, respectively. Using a statistically based two-class attribute sampling plan, n (i.e.,
number of sample units to be examined from a particular lot) would equal 15, 30, or 60
for cases 13, 14, and 15, respectively, and c (i.e., the maximum allowable number of
sample units containing L. rnonocytogenes) would equal 0 for all three cases.
A three-class attribute sampling plan also was proposed in the United States by a
working group of the National Advisory Committee on Microbiological Criteria for Foods
[22]. According to this plan, which was developed for ready-to-eat shrimp and crabmeat,
n (i.e., number of samples for foods produced in facilities employing HACCP and GMP
systems) would equal 10, whereas c (i.e., mandatory standard for L. monocytogenes that
should not be exceeded) would equal 0. Thus, with the exception of n, this plan is similar
to the two-class attribute plan proposed by the ICMSF.
Before recommending any Listeria-sampling plan, there must be good epidemiologi-
cal evidence indicating that the product or product group to be sampled has been impli-
cated in foodborne listeriosis. In addition, there must be good reason to believe that intro-
duction of a sampling program will substantially reduce the risk of contracting listeriosis
from consumption of such products.
Based on information collected in 1988, the ICMSF made a series of recommenda-
tions concerning sampling plans for listeriae in milk, soft cheeses, and vegetables [ 121.
Although I,. monocytogenes is commonly found in raw milk, minimum required pasteur-
ization (7 1.7"C/ 15 s) should eliminate this hazard. Therefore, the ICMSF recommended
that manufacturers institute monitoring programs to prevent postpasteurization contamina-
tion rather than routine sampling plans of pasteurized endproducts as the most appropriate
means of protecting the consumer.
704 Gravani

Many varieties of Listeria-contaminated cheese also have been identified since 1985,
with contamination most frequently being reported in surface-ripened cheeses. Since L.
monocytogenes can grow rapidly in Brie and Camembert cheese during the late stages of
ripening, a two-class attribute sampling program should be considered if such cheeses are
destined for consumption by pregnant women, immunocompromised adults, or the elderly.
However, since no sampling program can ensure that such products are completely free
of L. monocytogenes, public health interests are far better served by application of HACCP
principles during cheese manufacture and ripening. Although the ICMSF recognized that
raw vegetables also may become contaminated with L. monocytogenes, routine testing of
raw vegetables is unlikely to markedly reduce the risk of contracting listeriosis. Hence,
consumers of raw vegetables are urged to wash all such products vigorously before con-
sumption.
Endproduct-sampling programs are not the answer to protecting consumers from
listeriosis or other types of foodborne illness. However, microbiological sampling is
recommended by many regulatory agencies and the World Health Organization as part
of the HACCP approach to prevent opportunities for contamination by, survival, and
growth of L. monocytogenes as well as other microbial pathogens in raw materials,
factory environments, and food products during manufacture, storage, distribution, sale,
and use.

Status of L. monocytogenes in Foods

The status of L. monocytogenes in cooked, ready-to-eat foods is still being discussed and
debated in scientific communities and regulatory agencies around the world. In the United
States, public health and regulatory agencies have a zero tolerance for this organism, that
is based on its ability to produce a life-threatening illness at a presumably low, but as yet
unknown infectious dose and can grow at refrigeration temperatures. France, Germany,
and the Netherlands accept up to 100 cfu/g [69a, 69bl. In Germany, if the level of L.
monocytogenes is in the range of 100- 1000 cfu/g, the product is reprocessed. In the Nordic
countries, a working group has recommended action be taken for food containing > 100
L. monocytogenes cfu/g [69a]. Denmark has established a zero tolerance for foods that
have received a listericidal treatment after packaging. If > 10 organismdg are found, cor-
rective HACCP actions are required. If the level of L. monocytogenes is > 100 organisms/
g, the product is recalled. The Danish position also states that if the organism can grow
in a product and the shelf life exceeds 1 week, then there is also a zero tolerance with
specific sampling plans. If the organism cannot grow, levels between 10 and 100 may be
acceptable [69b]. (This is a simplification of the Danish position, as there are six food
categories with associated sampling plans and acceptance criteria.) Italy has a zero
tolerance policy in effect. In the United Kingdom, < 100 cfu/g of L. monocytogenes
is considered fairly satisfactory, whereas 100- 1000 cfu/g is unsatisfactory and > 1000
cfu/g is unacceptable [69b]. In Australia, the Australia, New Zealand Food Authority
(ANZFA) is developing a food standards code that specifies a zero tolerance for L.
monocytogenes in ready-to-eat foods such as meat pastes, piit&,smoked fish, marinated
smoked mussels, and cheese made from thermized milk that has a moisture content
>40% and a pH >5 (V.N. Scott, personal communication, 1998). In Canada, the
compliance criteria for L. monocytogenes in ready-to-eat foods are composed of three
categories [42a]:
Listeria in Food-Processing Facilities 705

Category 1. Ready-to-eat foods causally linked to listeriosis, including soft cheeses,

liver piite, coleslaw mix with a shelf life > 10 days, and jellied pork tongue. Action
Level >O cfu/50 g
Category 2. All other ready-to-eat foods supporting growth of L. rnonocytogenes
with refrigerated shelf life of >10 days. Action Level >O cfu/25 g
Category 3. Ready-to-eat foods supporting growth of L. rnonocytogenes with refrig-
erated shelf life < 10 days and all ready-to-eat foods not supporting growth. Ac-
tion Level 5 1 0 0 cfu/g depending on the plants GMPs
In Brazil, for cheeses with a moisture content of >36%, no L. rnonocytogenes is allowed
in a 25-g sample [69a]. In Asian countries, there are no policies for the level of L. rnonocy-
togenes in foods as yet [69a].
Owing to the ubiquitous nature of L. rnonocytogenes, most developed countries
around the world are strongly emphasizing the use of GMPs and HACCP systems for
reducing the levels of this organism in food-processing facilities. There is still considerable
debate among scientists in industry, academia, and regulatory agencies on developing
acceptable levels of L. rnonocytogenes in foods [70a], since the infectious dose is not
yet known. At the present time, all intervention strategies should be used to reduce the
level of this organism in foods.
In conclusion, to reduce the incidence of L. rnonocytogenes in the food supply,
food processors must develop and implement HACCP plans that are built on a strong
foundation of GMPs and prerequisite programs which in turn will decrease the incidence
of listeriosis.

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Appendix
Media to Isolate and Cultivate
Listeria monocytogenes
and Lisferia spp.

FLUID MEDIA FOR ENRICHMENT OF LISTERIA SPP.

Demi-Fraser broth*
Proteose peptone 5.0 g
Tryptone 5.0 g
Lab-Lemco powder 5.0 g
Yeast Extract 5.0 g
NaCl 20.0 g
KHZPO, 12.0 g
Na2HP04 12.0 g
Esculin 1.0 g
Ferric ammonium citrate 0.5 g
Lithium chloride 3.0 g
Nalidixic acid 10.0 mg
Acrifl avine 12.5 mg
Distilled H 2 0 1000 ml

FDA enrichment broth*

Trypticase Soy Broth 30 g
Yeast 6g
Acrifl avine 15 mg
Nalidixic acid 40 mg
Cycloheximide 50 mg
Distilled H 2 0 1000 ml

Fraser broth
Proteose peptone
Tryptone
Lab-Lemco powder
Yeast extract
NaCl
KH2IO,
71 1
7 12 Appendix

Na2HP04 12 g
Esculin 1g
Nalidixic acid 20 mg
Lithium chloride 3g
Acriflavine 25 mg
Ferric ammonium citrate 0.5 g
Distilled H 2 0 1000 ml

IDF pre-enrichment broth

Peptone 10 g
NaCl 5g
Na2HP04. 12 H 2 0 9g
K2HP04 1.5 g
Distilled H 2 0 1000 ml

IDF enrichment broth*

Tryptone soy broth 30 g
Yeast extract 6g
Acriflavine . HCl 10 mg
Nalidixic acid 40 mg
Cycloheximide 50 mg
Distilled H 2 0 1000 ml

Listeria repair broth [LRB]

Trypticase soy broth 30.0 g
Yeast Extract 6.0 g
Glucose 5.0 g
Magnesium sulfate 2.46 g
Ferrous sulfate 0.3 g
Sodium pyruvate 10.0 g
3-N-morpholinepropanesulfonic acid-free acid 8.5 g
3-N-morpholinepropanesulfonicacid-sodium salt 13.7 g
Distilled H 2 0 1000 ml

LRB with selective agents

LRB 225 ml
After 3-4 h of enrichment at 30C add:
Acriflavine 3.4 mg
Cycloheximide 12.5 mg
Nalidixic acid 9.0 mg

L-PALCAMY broth
Special peptone (Oxoid)
Yeast extract
Lab-Lemco powder
Peptonized milk (Oxoid)
NaCl
D-Mannitol
Esculin
Appendix 713

Ferric ammonium citrate 0.5 g

Phenol red 80 mg
Polyniyxin I3 100,000 IU
Acriflavine . HCl 5 mg
Lithium chloride 10 g
Ceftazidime, Latamoxef, or Moxalactam 30 mg
Egg yolk emulsion 25 ml
Distilled H 2 0 1000 ml

Rodriguez enrichment medium 1

Peptone 5 g
Neopeptone 5 g
Lab-L,emco powder 10 g
Yeast extract 5 g
Glucose 5 g
NaCl 50 g
Disodium phosphate-2-hydrate 53.22 g
Potassium phosphate, monobasic 1.35 g
Nalidixic acid 50 mg
Polymyxin B 8 X 105IU
Trypan blue 80 mg
Distilled H 2 0 1000 ml

Rodriguez enrichment medium 2

Peptone 5 g
Neopeptone 5g
Lab-Lemco powder 10 g
Rhamnose 2g
NaCl 50 g
Disodium phosphate-2-hydrate 53.22 g
Potassium phosphate, monobasic 1.35 g
Nalidixic acid 50 mg
Trypan blue 80 mg
Distilled H 2 0 1000 ml

Rodriguez enrichment medium 3

Protease peptone 5g
Tryptone 5g
Lab-Lemco powder 5 g
Yeast extract 5g
Esculin 1g
NaCl 20 g
Disodium phosphate-2-hydrate 24 g
Potassium phosphate, monobasic 1.35 g
Ferric ammonium citrate 1g
Nalidixic acid 30 mg
Trypan blue 40 mg
Agar 3g
Distilled H 2 0 1000 ml
714 Appendix

USDA Listeria enrichment broth I

UVM broth 1000 ml
Containing nalidixic acid 20 mg

USDA Listeria enrichment broth 11"

UVM broth 1000 ml
Containing nalidixic acid 12.5 mg

UVM
Proteose peptone 5 g
Tryptone 5g
Lab-Lemco powder 5 g
Yeast extract 5 g
NaCl 20 g
Disodium phosphate-7-hydrate 12 8
Potassium phosphate, monobasic 1.35 g
Esculin 1g
Nalidixic acid 40 mg
Acriflavine - HC1 12 mg
Distilled H 2 0 1000 ml
* Commercially available from Difco Laboratories, Detroit, MI; BBL, Cockeysville, MD; Oxoid Ltd., Basing-
stoke, Hampshire, England; Merck, Darmstadt, Germany.

SOLID MEDIA TO ISOLATE OF L/STR/A SPP.

AC agar
Columbia agar base 39.0 g
Acriflavine 10 mg
Ceftazidime 50 mg
Distilled H 2 0 1000 ml

ALPAMY agar
Columbia blood agar base (Oxoid) 39 g
Lithium chloride 15 g
D-Mannitol 10 g
2-Phenylethanol 2.5 g
Ferric ammonium citrate 0.5 g
Esculin 0.5 g
Acriflavine 10 mg
Phenyl red 80 mg
Egg yolk emulsion (Oxoid) 25 ml
Distilled H 2 0 1000 ml

ARS-modified MMLA
Phenylethanol agar (Difco) 35.5 g
Lithium chloride 0.5 g
Glycine anhydride 10 g
Cycloheximide 0.2 g
Nalidixic acid 50 mg
Appendix 715

Moxalactam 5 mg
Bacitracin 20 mg
Distilled H 2 0 1000 ml

FDA-modified McBride Listeria agar (FDA-MMLA)*

Phenylethanol agar 35.5 g
Glycine anhydride 10 g
Lithium chloride 0.5 g
Cycloheximide 0.2 g
Distilled H 2 0 1000 ml

Gum base nalidixic acid medium" '

Tryptone broth (Oxoid) 10 g

Nalidixic acid 50 mg
MgCI2 6 H20
* 0.7 g
Hydrocolloid gum (Merck-Gellan Gum KA40) 8g
Distilled H 2 0 1000 ml

Lithium chloride-ceftazidime agar

Brain-heart infusion agar 52 g
Lithium chloride 5 g
Glycine anhydride 10 g
Ceftazidime pentahydrate 2.5 ml
Distilled H 2 0 1000 ml

LPM agar"
Phenylethanol agar 35.5 g
Glycine anhydride 10.0 g
Lithium chloride 5.0 g
Moxalactam 20 mg
Distilled H 2 0 1000 ml
McBride Listeria agar
Phenylethanol agar 35.5 g
Glycine 10.0 g
Lithium chloride 0.5 g
Sheep blood 50 ml
Distilled H 2 0 1000 ml

Modified McBride Listeria agar*

Phen ylethanol agar 35.5 g
Glycine anhydride 10.0 g
Lithium chloride 0.5 g
Distilled H 2 0 1000 ml

Modified LPM agar

Brain-heart infusion agar 52 g
Lithium chloride 5g
Glycine anhydride 10 g
Ceftazidime 50 mg
Distilled H 2 0 1000 ml
 Appendix
776

Modified Oxford agar*

Columbia blood agar base 39 g
Esculin 1g
0.5 g
Ferric ammonium citrate
Lithium chloride 15 g
1 ml
1% Colistin solution
1 ml
1% Moxalactam solution
Agar 2g
1000 ml
Distilled H 2 0
Modified Vogel-Johnson agar
Vogel-Johnson agar base 60 g
50 mg
Nalidixic acid
20 mg
Bacitracin
Moxalactam 5 mg
20 ml
1% Potassium tellurite solution
980 ml
Distilled H 2 0
Oxford Listeria agar*
Columbia agar base 39 g
Esculin 1g
0.5 g
Ferric ammonium citrate
Lithium chloride 15 g
400 mg
Cycloheximide
20 mg
Colistin sulfate
Acriflavine 5 mg
Cefotetan 2 mg
10 mg
Fosfomycin
1000 ml
Distilled H 2 0
PALCAM agar*
Columbia agar base 39 g
D-Glucose
0.5 g
D-Mannitol 10 8
Esculin 0.8 g
Ferric ammonium citrate
0.5 g
80 mg
Phenol red
Polymyxin B 100,000IU
Acriflavine . HCI 5 mg
Lithium chloride 15 g
20 mg
Ceftazidime, Latamoxef, or Moxalactam
Distilled H 2 0
1000 ml

RAPAMY agar
Columbia blood agar base 39 g
D-Mannitol 10 g
2-Phenylethanol 2.5 g
D-Glucose 1g
Ferric ammonium citrate 0.5 g
Esculin 0.5 g
Appendix 717

Nalidixic acid 40 mg
Acriflavine 10 mg
Phenol red 80 mg
Egg yolk emulsion (Oxoid) 25 ml
Distilled H 2 0 1000 ml

Rodriguez isolation medium I

Peptone 5g
Neopeptone 5g
Lab-Lemco powder 7g
Glucose l g
NaCl 5g
Disodium phosphate-2-hydrate 11.83 g
Potassium phosphate, monobasic 1.35 g
Nalidixic acid 40 mg
Acriflavine . HC1 12 mg
Defibrinated sheep blood 50 ml
Agar 15 g
Distilled H 2 0 1000 ml

Rodriguez isolation medium I1

Peptone 5 g
Neopeptone 5g
Lab-Lemco powder 10 g
Yeast extract 5g
Glucose 5g
NaCl 40 g
Disodium phosphate-2-hydrate 1.83 g
Potassium phosphate, monobasic 1.35 g
Nalidixic acid 40 mg
Polymyxin B 3 x 1041u
Acriflavine - HC1 18.7 mg
Defibrinated sheep blood 50 ml
Agar 15 g
Distilled H 2 0 1000 ml

Rodriguez isolation medium I11

Peptone
Neopeptone
Proteose peptone
Esculin
NaCl
Disodium phosphate-2-hydrate
Ferric ammonium citrate
Nalidixic acid
Acrifavine . HCI
Defibrinated sheep blood
Agar
Distilled H 2 0
718 Appendix

Trypaflavine nalidixic acid serum agar

Peptone 10 g
Lab-Lemco powder 10 g
NaCl 5 g
Inactivated bovine serum 50 ml
Trypaflavine 20 mg
Nalidixic acid 40 mg
Polymyxin B 3 mg
Agar 15 g
Distilled H 2 0 1000 ml
* Commercially available from Difco Laboratories, Detroit, MI; BBL, Cockeysville, MD; Oxoid Ltd., Basin-
gstoke, Hampshire, England; Merck, Darmstadt, Germany.
Index

Acanthumoeba, 100 Aeromenas hydrophila, 133, 188, 189, 615,

Acetic acid, 158, 159, 160, 161, 164, 165, 685, 695
167, 172, 389, 469, 479,480,482, Alcoholism, 79, 321
537, 550, 592, 642, 645 Alfalfa tablets, 346, 65 1
Acid adaptation, 151 Algin, 550
Acid anionic sanitizer, 683, 685 Alkaline phosphatase, 316, 379, 413, 424
Acid tolerance, 478 ALTA, 587, 620
Acidifying agents, 157 (see also individual p-Aminobenzoic acid, 164, 165
acids) Aminoglycosides, 89
acetic acid, 158, 159, 160, 162, 163, 164 Ampicillin, 89
benzoic acid, 164 Anari cheese, 324, 325,417,421, 475, 479
citricacid, 158, 159, 161, 162, 163, 164 Anionic acid sanitizer, 197, 198, 203
formic acid, 164, 165 Anise, 174
hydrochloric acid, 158, 161 Annato, 451, 651
lactic acid, 157, 158, 159, 161, 162, 163, Anthocyanins, 392
164 Anthotyros cheese, 479
malic acid, 158 Antibodies, 262, 263
propionic acid, 161, 164 Antibody-based detection systems, 270
sorbic acid, 162 Antigens, 280
tartaricacid, 161, 162, 163 Antimicrobial susceptibility testing, 282
Acidophilus milk, 445 Antioxidants, 171
Aciduric properties, 151 Antiseptic soaps, 202
Acquired immunodeficiency syndrome Aplastic anemia, 79
(AIDS), 75, 79, 80, 303, 3 11, 321, Arrhenius equation, 529
325 Arthritis, septic, 81
Acridine dye, 23 1 Arzua cheese, 484
Acriflavin, 229, 231, 232, 233, 236, 240, Aseptic processing, 594
242, 250 Asiago cheese, 428
Actin (filaments), 98, 102, 103, 108, 109, Asparagus, 642, 643
110,291 Aspergillus niger, 202
719
720
ATPase, 108
Avidin, 590
Avocado, 648
Bacillus, 4, 108
Bacillus cereus, 347, 632
Bacillus stearothermophilus, 108
Bacillus subtilis, 106, 265
Bacitracin, 230, 232, 236
Bacterial surface-ripened cheeses, 459
Bacteriocin, 182, 183, 186, 187, 281, 447,
462, 469, 478, 482, 528, 549, 552,
553, 554, 555
Bacteriocin typing, 28 1
Bacteriophage typing, 281, 284, 289, 290,
292
Bacterium monocytogenes, 2, 565
Bacterium monocytogenes hominis, 2
Bakers cheese, 475
Bakery products, 651
Bamboo shoots, 638
Bean cakes, 638
Bean sprouts, 636, 638, 644
Beef, 507, 508, 5 14, 5 15, 5 16, 517, 5 19,
525, 528, 529, 530, 54 1, 546, 547,
551, 552, 555, 566, 638, 647, 664,
667, 679, 692, 693
Beef jerky, 508, 539, 540, 664
Beef sausage, 535, 542
Beet pigment, 632, 651
Beet pulp, 341
Beets, 642, 650, 651
Behavior in cheese, 450
Behavior in fermented milks, 440
Behavior in meat products, 521
Behavior in unfermented dairy products, 382
autoclaved fluid products, 386
butter, 399
evaporated milk, 393
growth in mixed cultures, 394
ice cream, 398
intensively pasteurized milk, 385
non-fluid dairy products, 397
pasteurized milk, 385
raw milk, 382
sweetened condensed milk, 393
ultra-filtered milk, 394
Be1 Paese cheese, 459
Benzoic acid, 164, 167
Benzoic acid derivatives, 164
Beta-hemolysis, 238, 264
Beta vulgaris, 651
Biofilms, 169, 195, 196, 197, 201, 658
Biopreservation, 182
Index
Bixia orellana, 451
Bleu de Bresse cheese, 436
Blueberries, 341, 648
Blue (Bleu) cheese, 150, 315, 321, 322, 324,
417,421, 426, 429, 432, 436, 440,
453, 454, 457, 458, 459, 482, 483,
485, 486
Blue cheese factory, 673, 674
Blue Costello cheese, 439
Blue Lymeswold cheese, 484
Blue Stilton cheese, 484
Bockwurst, 535
Bologna, 535, 536, 538, 542
Bonbel cheese, 414
Bordetella pertussis, 118
Bratwurst, 535, 536, 538
Braunschweiger, 535
Breakfast sausage, 535
Brevibacterium linens, 459, 460
Brick cheese, 150, 315,417,451,454, 459,
460, 482, 483,486, 692
Brie cheese, 365, 378, 411, 412, 413, 414,
4 18, 4 19, 420, 432, 436, 453, 455,
457, 484, 485, 648, 672, 692,704
Brie de Meaux cheese, 308, 322, 323, 360,
455
Brine, 330, 487, 488, 617, 622, 624, 672,
673
Broccoli, 345, 632, 634, 635, 642, 643
Brochothrix, 3, 4, 5
Brochothrix thermosphacta, 3, 4, 532, 533
Brocchio cheese, 479
Broiler carcasses, 568, 571
Brucella, 703
Brucella abortus, 484
Brucellosis, 484
Brussels sprouts, 645
Buffalo meat, 515, 516
Bulgarian buttermilk, 445
Bulgarian white-pickled cheese, 472
Bulk starter cultures, 442, 443
Biindnerfleisch, 520
Butter, 307, 369, 370, 37 1, 372, 373, 376,
377,379,399,400,453
Butterine, 376
Buttermilk, 399, 691
Butylated hydroxyanisol, 166, 167, 169, 171
Butylated hydroxytoluene, 171
Butyric acid, 165
Cabbage, 344,633,634,635,636,637,638,
639, 640, 642, 643, 644, 645, 646,
67 1,697
Index 721

Cabbagejuice, 591,639,640,641,644,645, Cheese certification program, 4 11

649 Cheese composition, 482, 483
Caciocavalle cheese, 46 1 Cheese food, 151,414,415,480, 481, 482,
Caciotta cheese, 434 483,484,487
Cadherin, 100, 101, 103, 104 Cheese sauce, 478
Caffeine, 175, 176, 393 Cheese spread, 414,415, 484, 524
Calamari, 606,607 Chemokine, 115
Calcium lactate, 478 Chemotaxonomy,4
Cambazola cheese, 484 Cherries, 648
Camel meat, 5 15, 5 16 Chicken
Camembert cheese, 1 50,3 15,413,414,41 8, breaded fillets, 580
432,436,440,453,454, 455,456, breasts, 570, 575, 583, 584, 585, 586, 588
457,458,460,482,483,484,485, broiler carcasses, 568, 571
486,692,704 broth, 581,582, 583
CAMP test, 10, 11, 264 carcasses, 572, 573, 587
Campylobacter coli, 370,4 17 casserole, 580
Campylobacterjejuni, 182, 370,4 17 drumstick, 570
Campylobacter spp., 550 frozen, 574
Cancer, 79, 80,3 11,321, 337,342, 567,695 gravy, 58 1, 582
Candida albicans, 202 legs, 568, 569, 570, 575
Candy, 453,487 liver, 568, 569, 570, 575, 576
Cane sugar, 390,391,392,393,398 loaf, 579
Capric acid, 168 meat, 333, 334, 346, 532, 566, 586, 647
Caproic acid, 165 mechanically deboned, 587
Caprylic acid, 165 nuggets, 580
Carbon dioxide, 188, 189,479, 530, 545, 547, parts, 568
548,549,577,578,643 patties, 567
Carnitine, 134, 156, 157 raw, growth of L. monocytogenes, 577
Carnobacterium,3, 4 salad, 567
Carnobacteriumpiscicola, 186, 187, 553 sandwiches, 577
Carrageenan, 390,391,392,550,586 skin, 587
Carrots, 633, 634, 635, 636,637,638,642, slaughterhouse, 667,668
645,646,647,648 sliced, 579, 580
Carrot juice, 455,648 spread, 567
Caryophanon,3 summer sausage, 581
Casein, 360, 371, 372, 378, 379, 381, 393, thighs, 567
400 wings, 568,569,570,575,587
Catalase, 113, 114, 146, 648 Chinchilla, 57, 341
Catfish, 6 16 Chinese cabbage, 638,642
Cattle, 40, 47-51,319, 341, 522 Chinese medicinal plants, 64 1
Cauliflower, 345, 632, 634, 635, 642, 643 Chitin, 615
Caviar, 606,607 Chloramphenicol, 89,282
Cefotetan, 235 Chlorine, 621, 637, 639,644, 645, 683, 685
Ceftazidime, 229,233,235 Chlorine compounds, 198, 199,200
Celery 343, 633, 636, 639, 642 Chlorine dioxide, 199, 645
Cell lines, 97, 105, 107, 114 Chlortetracycline,60
Cell-to-cell spread, 108 Chocolate, 651, 673, 674
Cervelat, 5 19, 520. 54 1 Chocolate factory, 673
Cerviche, 613 Chocolate milk, 306, 307, 360, 371, 372, 373,
Chachcaval cheese, 385 377, 379, 381, 382, 386, 387, 388,
Cheddar cheese, 150,314,3 15,324,4 14, 389,390,393,398,418
417,423,438,443,451,454,462, Cholesterol, 105
464,465,466,467,479,480,482, Chromosomal DNA restriction endonuclease
483,485,486,692 analysis, 283, 290, 292
722 Index

Cichorium endivia, 64 1 Corticosteroid therapy, 80

Cinnamon, 173
Cirrhosis, 79
Citric acid, 158, 159, 161, 164, 165, 166,469,
479,591,651
Clams, 606,607,610,612,697
Clean-in-place systems, 685
Clostridium, 4, 176, 183, 188
Clostridium botulinum, 183, 189, 695, 703
Clostridiumperfringens, 182, 525, 527,632,
703
Cloves, 173, 174, 175
Coagulants, 450
Cockles, 6 12
Cocoa, 390,391,392,393
Coconut, 633
Cod, 622,623
Code of Hygienic Practices, 4 18
Colby cheese, 150,315,417,424,451,454,
462,463,464,467,483,485,486
Cold enrichment, 133, 144, 225, 226,227,
228,240, 241, 243, 249, 250,432,
465, 474,476,488, 523, 550, 574,
62 1
Cold shock proteins, 227
Coleslaw, 149,301,305,319,324,341,343,
344, 345, 378, 631, 636, 638,639,
658,671,697,705
Coliforms, 438,439,448
Colistin sulfate, 234
Collagen vascular diseases, 79
Colorants, 45 1,453,650,651
Commercial rapid test systems, 273
Confectionery products, 67 1
Cooked meat specialty items, 539
Cooling system fluids, 205
Combined treatments, 193
Control in food-processingfacilities, 68 1
cleaning and sanitizing, 683,684,685
factory environment, 682
factory design, 682
guidelines, 681
personnel cleanliness, 686
traffic patterns, 686
Conventional subtyping methods, 280
Cooked and ready-to-eat meat products, 508,
509,518,531
Cooked sausage, 508
Cooked smoked sausage, 536
Coriander, 174
Corn, 642,645,646
Corn sweetener, 398
Corned beef, 506,507,5 18,519,521, 528,
532,533,566,695
Corynebacteriaceae, 3
Corynebacterium diphtheriae, 182
Corynebacterium infantisepticum, 2, 302
Corynebacteriumpawulum, 2
Coryneform bacteria, 460
Cotijacheese, 310, 313, 314, 315,414,485
Cottage cheese, 150,151,227,302,309,316,
371,412,418,425,426,429,433,
440,443, 453, 454, 475, 476, 477,
478,479,482,484,485,660
Coxiella burnetti, 145
Crab, 601,606, 607,608,612,615,616, 617,
621,624,695,697
Crabmeat, 602,603,604,605,609,6 14,620,
622,670,696,697
Crawfish, 605,607,621,622
Cream, 140,302,303,307,308,370,371,
372, 379, 381, 382, 386, 412, 475,
480
Cream cheese, 4 15,475,479,484,485
Cr8me de Bleu cheese, 436
Creosote, 537
Crescenza cheese, 429
Cucumbers, 634,635,636,638,697
Cultured buttermilk, 308,418,443,444,445,
448
Cultured cream, 4 12,44 1,445,691
Cultured milks, 44 1, 69 1
Curcuma longa, 45 1
Curing salts, 146
Cutaneous infections, 8 1
Cutting boards, 521
Cycloheximide,235
Cytochalasins, 103
Cytochromes, 7
Cytokine, 114, 115
Cytolysin, 105
Cytoplasm, 97,98, 103, 105, 106, 108, 110,
115,116, 118, 159
Dairy industry
clarifiers and separators, 687
farm environment, 687
fermented dairy products, 69 1
filling and packaging, 690
frozen dairy products, 691
pasteurization, 687
pipeline and cross connections, 689
reclaimed and reworked product, 690
Dairy plant environmental problems, 660,
662, 663, 673, 675, 679
Dairy processing facilities, 659, 660, 661,
662, 663, 664, 678, 680
Index 723

Dairy Safety Initiative Program, 370, 418, [Eggs1

658, 659 scrambled, 592
Decontamination treatments, 552 shells, 588
Diabetes, 79, 80, 321 sugared yolk, 589
Diacetyl, 182, 482 whole, 588, 589, 592
Dill, 619, 620, 624 yolk, 588, 589, 590, 593, 594
Dimethyl sulfide, 460 Electromorphs, 282
Diplococcin, 482 Electrophoretic enzyme type, 320, 325, 334,
Direct fluorescens microscopy , 271 344,567
Direct plating, 225, 226, 238, 432, 584, 589 Ellagic acid, 172
DNA macrorestriction analysis, 287, 305, Emmentaler cheese, 462,467
344 Endive, 641,642,644
DNA restriction endonuclease profile, 3 19 Endocarditis, 80
DNA sequence-based subtyping, 2 91 Endophthalmitis, 8 1
Dogs, 40, 58, 303 Endothelial cells, 100, 1 17
Domestic Soft Cheese Surveillance Program, Enrichment media, 240
412,413,416,417 Enterobacter aerogenes, 444,448
Domiati (Damietta) cheese, 431, 469, 471, Enterobacter cloacae, 685
472,489 Enterobacteriacea, 642
D o m spp., 610, 612 Enterococci, 228,233, 235, 460, 474
Dried beef, 5 18 Enterococcus, 4, 187
Dry fermented sausage, 542 Enterococcusfaecalis, 102, 178,232,235,
Dry heat, 583, 584, 585, 586 456,525,527
Dry milk processing facilities, 662 Enterococcusfaecium, 147,235,620
Dry sausage, 334 Enterocytes, 101
DTH gene, 264, 266 Enzyme-linked immunosorbent assay, 272,
Duck, 565, 566, 571, 572, 573 2 73
D-value, 132, 144, 147, 163, 179, 181, 190, Epidemiology, 279, 280, 281, 282, 283,284,
191, 192,461,464,467, 550, 552, 287, 301, 303, 304, 306, 307, 31 1,
587, 593, 594, 595, 621, 622, 623, 316, 318, 320, 321, 323, 329, 330,
624, 644 333,337, 339, 340, 342, 346, 359,
522,537
Edam cheese, 176,412,417,462 Epithelia1 cells, 100, 101, 104, 115
Eel, 606, 607 Ergo, 449
Egg industry, 695 Erysipelothrix, 3
Eggnog, 592 Erysipelothrix monocytogenes, 2
Egg processing facilities, 670 Escherichia coli, 1 14, 141, 15 1, 164, 172,
Eggs, 566 182, 184, 191, 202,228,232,284,
albumen, 589,590 347,413, 417,418, 419,422,434,
boiled, 589, 590 438,439,444,448, 524, 525, 526,
broken, 588 527,540,603,604,685,695
dried and reconstituted, 590, 591 Escherichia coli 0 157:H7, 182, 192, 550, 568
fried, 593 Esculin, 235, 242, 243
frozen, 592 Esrom cheese, 42 1,439
growth of L. monocytogenes, 589 Essential oils, 174, 647
heat in activation of L. rnonocytogenes, 593 Estilo Casero cheese, 3 14
liquid, 590, 591 Ethanol, 147, 151, 177, 194
liquid whole, 588, 589, 590, 591, 592, 593, Ethylenediaminetetraaceticacid, 167, 169,
594,595 177, 178, 182,535,536,645,646,647
powdered, 592 Eugenol, 647
products, 338, 339, 588 Evaporated milk, 393,394
raw, 590
reduced cholesterol liquid whole, 59 1 Farm-house cheese, 436
salted liquid whole, 593, 594 Fatty acid monoesters, 166
724 Index

Fatty acids and related compounds, 165 Fosfomycin, 234, 235

Fecal material, 24, 46, 50, 53, 54, 56, 302, Fourme de Bresse cheese, 436
319, 326, 335, 338, 339, 344, 345, Fowl-domestic and wild, 40, 53-56
368, 522, 565, 568, 572, 614,616, Frankfurters, 332, 333, 334, 335, 346, 506,
632,633,639,667,670,696,697 509, 510, 512, 513, 517, 518, 520,
Fennel, 636,642 534, 535, 536, 537, 538, 539, 542,
Fermented sausage, 54 1, 554 547,551,554,648,694,695
Ferric ammonium citrate, 235, 243 Free fatty acids, 165, 458, 466
Feta cheese, 150, 3 15, 324, 325,436,454, Fresh sausage, 535
469,470,471,473,479,482,483, Fromage des Burons (cheese), 420
485,486,488,489 Frozen foods, 136
Fibroblasts, 100, 101, 102, 104, 106, 110, Frozen yogurt, 377,418
114,115 Fruit and vegetable industry, 697
Fimbriae, 280 Fruit juice bars, 649
Finnish sausage, 545, 546 Fruit processing facilities, 67 1, 672
Fish (fin), 58, 59,601,610,611,612,616 Fruit salad, 636
dried, 61 1 F-value, 550, 594
pickled, 611,613
processing facilities, 695-697 Gamma irradiation, 190, 191,455, 587, 588
salad, 61 1,613 Gammelost cheese, 484
salted, 605,608,611 Gel electrophoresis, 267, 274
smoked 605,607,608,611,613,617,618, Gelatida products, 373
624 Gemella, 3
steamed, 602,614 Gene expression, 116, 117
thermal death time of L. monocytogenes, Generation time, 387, 392, 398, 475, 486,
622 489, 528, 589, 590, 592, 616
Fish and seafood industry, 695 Genistein, 103
Flagella, 280, 291, 523 Genoa salami, 541
Flavobacterium, 394, 396 Genomic groups, 6
Floor drains, 196 Geotrichum candidum, 460
Flow cytometry, 273,364 Gjetost cheese, 324, 385, 479
Fontina cheese, 42 1,473 Glacee, 374
Food contact surfaces, 195, 658 Glass, 195
Food, Drug and Cosmetic Act, 372 Gluconic acid, 475, 476, 477
Food industry, 90 Glucono-delta-lactone, 475, 624
Foods of plant origin, 341 (see also individual Glucose oxidase, 179, 180, 390
foods) Gluteraldehyde, 204, 205
alfalfa tables, 346 Glycerol rnonolaurate, 165, 56 1
blueberries, 34 1 Glycine, 234, 244
broccoli, 345 Glycine betaine, 134, 156, 157
cabbage, 344 Goat meat, 515, 516
cauliflower, 345 Goat milk cheese, 415, 417, 428, 430, 433,
celery, 343 436,438,473,474,475
coleslaw, 341, 347 Goats, 40, 45, 46, 319, 324
lettuce, 343,346,347 Good manufacturing practices, 412, 485,
mushrooms, 345 621, 658, 659, 675, 679, 681, 691,
nectarines, 34 1 692,698,700,703,705
potato salad, 347 Goose, 565, 576
rice salad, 347 Gorgonzola cheese, 415, 429, 434, 453, 457,
strawberries, 34 1 459
tomatoes, 343 Gouda cheese, 176, 412,417, 436, 462, 463,
Formaldehyde, 537 467, 486
Formic acid, 164, 165,482 Granulocytes, 115
Index
Gravy, 533
Green beans, 633, 642, 645, 646
Green peppers, 633
Ground beef, 508, 510, 513, 514, 515, 516,
526, 527, 528, 530, 542, 543, 550,
551, 553
Ground meat, 332, 334, 335, 514, 516, 553,
57 1
Ground pork, 513, 514, 515, 551
Growth in mixed cultures, 394
Growth temperatures, 132, 146
Gruyke cheese, 436, 462, 467
Gudbrandsdalsost cheese, 479
Gulls, 616
Hafnia,228
Half and half, 371, 372, 373, 400,418,478
Halloumi cheese, 324,325,417,421,438,
475,489
Ham, 332,337,508,510,512,514,518,519,
520, 521, 528, 531, 532, 534, 535,
538,542,550,551,664
Ham salad, 509,5 10
Hard cheese, 321,322,324
Hazard Analysis Critical Control Point, 226,
244, 615, 658, 695, 699, 703, 704,
705
conduct hazard analysis, 701
determine CCPs, 701
establish corrective actions, 702
establish critical limits, 701
establish monitoring procedures, 702
establish record-keeping and documenta-
tion procedures, 702
establish verification procedures, 702
prerequisite program, 700
Head cheese, 535
Heart disease, 80
Heat inactivation, 136,644
Heat resistance, 305, 593
Heat shock, 144, 145, 146, 147, 148,551
Heat-shock protein, 1 16, 145
Heat-treated milk, 4 12
Heating meats, 549
Hemolysin, 11, 105, 264, 265,266,291
Hemolysis, 10, 11, 105, 106, 107, 638
Henrys technique, 237
Heparan-sulfate proteglycan (receptor), 102
Hepatitis A virus, 703
Hepatocytes, 100, 101, 104
Hexanoic acid, 165, 166
Hispanic cheeses, 468,484
Homemade cheese, 325
725
Homogenization, 144, 145, 193
Horsemeat, 5 14, 5 16
Horses, 40, 56
Hot-boned beef, 528,529
Hot dogs (see Frankfurters)
Household kitchens, 679,68 1
Hummus, 633
Hurdle concept, 193, 194
Hybridization assay, 263
Hybridoma technology, 27 1
Hybridomas, 272
Hydrochloric acid, 147, 158, 161, 162, 163,
188,476,477,649
Hydrogen peroxide, 114, 146, 147, 151, 178,
179, 180, 181, 182, 194,482,490,
642
Hydrostatic pressures, high, 183, 189, 192
2-Hydroxy isocaproic acid, 390
Hypochlorite, 197, 199, 200, 201, 204, 645
Hypochlorite ion, 199
Hypothiocyanate, 179
Hypothiocyanous acid, 179
Ice cream, 308,360,369,370,371,372,373,
374, 375, 376, 377, 379, 381, 398,
418,453,487,660,674,679,691
Ice cream factory, 673, 674, 675, 676, 677
Ice cream mix, 140, 373, 374, 398,418
Ice milk, 370, 371, 372, 373, 375, 376
Ice milk mix, 373,418
Ice milk shake mix, 373,374,418
Imitation crab meat, 340, 341
Immunoassays, 262
Immunosuppressive therapy, 79
Incidence in meat products, 506, 5 1 1, 5 13
Incidence in pasteurized milk and other unfer-
mented dairy products, 369
Incidence in raw meats, 5 14
Incidence in sausage and ready-to-eat meats,
517,518,519
Incidence in unfermented dairy products, 360,
380
butter, 38 1
casein, 381
cream, 38 1
dry infant formula, 38 1
ice cream, 38 1
milk, 380
nonfat dry milk, 38 1
raw cows milk, 360, 361, 362, 363, 364,
365,366,367
raw ewes and goats milk, 369
Incubation conditions, 240
 lndex

Industry-specificequipment, processing Lactates, 159, 189

methods and equipment, 687 Lactic acid, 157, 158, 159, 161, 164, 165,
Infectious mononucleosis,2 166, 170, 177, 201, 228, 389, 480,
Injury, cellular, 135, 146, 163, 188, 226, 230, 481, 482, 524, 542, 555, 578, 587,
249, 250,251,253, 262, 274, 372, 602 623,624,645
379, 400, 460, 476, 477, 482, 486, Lactic acid bacteria, 179, 182, 183,228,232,
550,584,657,696 240, 383, 441, 476, 479, 531, 537,
Interferon, 114 540, 541, 549, 552, 554, 620, 640,
Interleukin, 114 649,691
Internal pH-controlled starter media, 442,443 Lactobacillaceae, 3, 4
Internalin, 100, 101, 103, 104 Lactobacillus, 3,4, 541
Interstate milk shipment plant, 659 Lactobacillus acidophilus, 447
Intracellular growth, 104 Lactobacillus bararicus, 187, 553
Intracellular motility, 108 Lactobacillus bulgaricus, 441, 445, 446, 447
Invasion assay, 100 Lactobacillus casei, 47 1, 553
Invasion-associated protein, 264, 266, 29 1 Lactobacillus curvatus, 187
Invasion mechanism, 103 Lactobacillus delbrueckii subsp. bulgaricus,
Iodine monochloride, 203 233,441,447,449 (see also
Iodophors, 198,203,204,683,685 Lactobacillus bulgaricus)
Iron overload, 79 Lactobacillus paracasei, 456
Irradiation, 189, 533, 549,644 Lactobacillus plantarum, 187, 460, 525, 527,
Isobutyric acid, 389 545,555
Isoeugenol, 173 Lactobacillus saM, 549, 555
Isolation media, 233 Lactobacillus salivarius, 187
Isovaleric acid, 390 Lactobacillus viridescens, 172
Italian cheese, 325,421,422,423, 461, 467, Lactocin, 553
482,483,674,675 Lactococcus lactis subsp. cremoris, 44 1,461,
Italian sausage, 535 489 (see also Streptococcus cremor-
Italic0 cheese, 434 is)
Lactococcus lactis subsp. lactis, 183,441,
Jarlsberg cheese, 42 1 456,461,462,489 (see also Strep-
Jellied pork tongue, 302, 322, 324, 326, 329, tococcus lactis)
330,334,5 11,705 Lactoferricin, 182
Jocoque, 3 16 Lactoferrin, 181, 182
Jonesia denitrijicans, 7 (see also Listeria Lactoperoxidasesystem, 179, 180, 181, 383
denitrijkans) Lamb meat, 514, 516, 517, 519, 530, 546,
547,548,566,667,679,692
Lamb patty, 5 13
Kachkaval cheese, 412,473 Lambda gene (bacteriophage), 284
Kareish cheese, 43 1,469 Langostino, 605, 61 1
Kashor cheese, 430 Lantibiotics, 182
Kasseri cheese, 485 Lauric acid, 165, 168
Ketchup, 642 Lebanon bologna, 541
Kidney, 523 Lecithinase, 106, 107, 108, 1 12
Kielbasa, 535 Leeks, 636
Kiln, 624 Legionella pneumophila, 118
Kimchi, 642,643 Lettuce, 343,346,347,631,634,635,636,
Klebsiella pneumoniae, I82 637, 638, 639, 641, 642, 643, 645,
Koch Kaese (cheese), 484 646,648
Kurthia, 3 Lettuce juice, 64 1
Leucocytes, 305
Labneh, 449 Leuconostoc, 54 1
Lactate dehydrogenase, 100 Leuconostoc carnosum, 187
Index 727

Leuconostoc crernoris, 443 [Listeria grgyi]

Leuconostoc dextranicum, 443 sugar utilization, 389, 390
Leuconostoc gelidurn, 186 taxonomy, 6,7
Leuconostoc rnesenteroides, 187 Listeria innocua
Leukemia, 321 antibiotic resistance, 134
Liederkranz cheese, 315,412,413,414,459 in beef, 524,555
Ligase chain reaction, 265, 266 beef carcasses, 552,553
Limburger cheese, 412,415,421,436,459, in cheese, 413,424,425,433,434,435,
485,692 436,438
Linoleic acid, 165 cheese factory, 672,676
Linolenic acid, 165 on chicken meat, 568, 569, 570, 571, 574,
Lipase, 389,467 575,576
Lipoteichoic acid, 4, 7, 103, 280 in chicken sandwiches, 577
Liquid smoke, 172, 173,537, 538, 539,625 chicken slaughterhouse, 667,668, 669
Listerella, 2, 226 cottage cheese, 478
Listerella bovina, 2 in crabmeat, 604, 609
Listerella cunniculi, 2 dairy plants, 661,662,678,680
Listerella gallinaria, 2 dairy products, 37 1 380, 38 1, 659
Listerella heminis, 2 defeathering machine, 575
Listerella gerbilli, 2 in egg products, 588,589
Listerella hepatobytica ,2 expression of ActA, 109
Listerella monocytogenes, 2 expression of inlA, 100
Listeria fatty acids, effects, 165
biochemical characteristics, 9 in fish, 6 13
chemotaxonomy,4 food processing facilities 675, 676,678
culture, 8 frankfurters, 55 1
genomic groups, 6 in frozen seafood, 604
growth temperature, 9 genomic group, 6
hemolysin, 11 growth in autoclaved milks, 389,
hemolysis, 10 growth in cottage cheese, 188
metabolism, 9 growth, low pH, 148
morphology, 8 growth temperature, 133
multitest assays, commercial, 11 high hydrostatic pressure, 192
numerical taxonomy, 3 household kitchens, 679
nutritional requirements, 9 identification, 10
phenotypic markers, 11 induction of NF-KB, 117
phylogentic position, 3 low inoculum effects, 134
rRNA sequencing, 4 in Mexican-style cheese, 3 16
species identification, 10 monoclonal antibody reaction, 272
taxonomy, 5 , 7 no cause of avian listeriosis, 55
Listeria bulgarica, 5,6 not detected with antibodies, 271
Listeria denitrijkans, 7, 271, 575 (see also in oysters, 616
Jonesia denitrificans) pasteurized milk, 379, 380
Listeria grayi potato processing plant, 672,676
in cheese, 431 poultry meat-related illness, 337
dairy plants, 680 raw ewes milk, 369
dairy products, 371 raw milk, 361, 362, 363, 364,365, 366,
growth temperature, 133 367
identification, 10 raw produce, 634,635,636,637,639,644
no antibody reaction, 271 salt tolerance, 155
pasteurized milk, 379 in shrimp, 6 12
raw ewes milk, 369 sugar utilization, 389, 390
raw milk, 363 taxonomy, 5
 Index

[Listeria innocua] [Listeria monocytogenes]

on turkey meat, 569, 571 cell-to-cell spread, 108
turkey slaughterhouse, 668, 669 in cheese composition, effects of, 482
use in food processing tests, 133 chinchilla listeriosis, 57
Listeria ivanovii cold enrichment, 226
ActA-related protein, 110 combined treatments, 193
avian listeriosis, 5 5 commercial rapid test systems, 273
cattle listeriosis, 48 control in food-processing facilities, 68 1
in cheese, 43 1,432 conventional subtyping methods, 280
chlorine inactivation, 199,200 cookedheady-to-eat poultry, 576
DTH gene, 264 cultured cream, 445
genomic group, 6 cultured buttermilk, 443
growth in autoclaved milks, 389 dog listeriosis, 58
growth, low pH, 148 enrichment media, 240
growth prevented on medium, 235 environment affects virulence gene expres-
hemolytic strains, 265 sion, 113
hybridizes, 263 264 excretion, 25
identification, 10 fatty acids and related compounds, 165
intracellular life cycle, 118 fecal carriage, 76-79, 82
irradiation, 190 fecal material, 22, 24
monoclonal antibody reaction, 272 fermented milks, 440,441
polymerizes F-actin, 110 fish and crustaceans, 58, 59
prfA-like gene, 111 food chain, 32
raw ewes and goats milk, 369 freezing, 135
raw milk, 361 gamma irradiation, 587
salt tolerance, 155 gene expression, 116
sanitizer inactivation, 203 genomic group, 6
selective agents, 229 goat listeriosis, 45-47
sheep listeriosis, 45 growth in cookedheady-to-eat poultry pro-
sugar utilization, 389 ducts, 579
taxonomy, 5,7 growth in mixed cultures, 394
Listeria monocytogenes growth and survival in seafood, 6 16
acidity, effects, 148 growth and survival in vegetables, 632
adhesion, 100 growth temperature, 132
animal feed, 22,27 heat inactivation in eggs, 593
antimicrobial food components, 154 hemolysin, 11
antioxidants, 17I history, 1,2,3, 75
apoptosis, 118 horse listeriosis, 56, 57
avian listeriosis, 53-56 host cell respouses, 1 14
behavior of household kitchens, 679
in cheese, 450 human disease, 75-90
in fish and seafood, 6 15 hydrogen perioxide, 178
on food and nonfood contact surfaces, identification of, 10
195 inactivation of in seafood, 620
in fruit juices, 649 incidence of
in meat products, 521 in cheese, 426
in plant products, 650 in eggs, 588
in raw and cooked poultry products, 577 in food processing facilities, 658, 672
in unfermented dairy products, 382 on fruits, 648
on vegetables, 639 in meat products, 506
biopreservation, 182 in raw poultry, 567, 572
catalase, 113 on raw vegetables, 632
cattle listeriosis, 47-52 in unfermented dairy products, 360
Index
[Listeria monocytogenes]
inhibition in seafood, 618
internalin proteins, I00
intracellular growth, 104
intracellular motility, 108
invasion, 100
mechanism of, 103
lactic starter cultures, 441
lactoferrin, 181
lactoperoxidasesystem, 179
liquid smoke, 172
llama listeriosis, 57
lysozyme, 176
modified atmosphere, 187
molecular subtyping methods, 282
monitoringherification program for poultry
products, 566
nisin, 183
nonfermented dairy foods, 307
nonhemolytic mutants, 105
non-human primate listeriosis, 58
nonthermal processing, 189
nucleic acid-based methods, 265
official isolation methods, 244
organic acids and their salts, 157
other bacteriocins, 187
other names, 2
pasteurized milk, 304, 379
pediocin, 185
persistance in environment, 21, 22
phagocytes, invasion, 100-103
phagocytic vacuole, escape, 104
protein ActA, 102
protein p60, 101
rapid detection methods, 262
raw milk, 302,360-368
recalls
domestic cheese, 4 12
imported cheese, 4 18
recovering injured cells, 249
regulation of virulence gene, 111
regulatory aspects, fish and seafood, 614
sampling plans, 703
sanitizers, 198
selective enrichment, 228
selective media, 233
sewage, 26
sheep listeriosis, 4 1-45
signal transduction pathways, 117
silage, 2 I, 27
sodium chloride, 154
sodium nitrite 169
soil, 22, 23
729
[L isteria m onocytogenesJ
spices, herbs, and plant extracts, 173
status in foods, 704
stress adaptation, 145
subtyping methods compared, 29 1
sugar utilization, 389
superoxide dismutase, 113
surveys for in seafood, 602,609
surveys, non-U.S. cheese, 423
swine listeriosis, 52, 53
taxonomy, 5 , 7
thermal inactivation, 1367, 583
thermotolerance, 145
tissue tropism., 103
traditional fermented milks, 449
transmission, 30, 593,615
treatment animals, 59, 60
vegetation, 22
decayed, 21
virulence, 134, 147
water, 22, 26
water activity, 153
yogurt, 447
zoo-animal listeriosis, 57
Listeria murrayi
in cheese, 431
on chicken carcasses, 575
growth temperature, 133
no antibody reaction, 271
raw goats milk, 369
sugar utilization, 389, 390
taxonomy, 6
Listeria seeligeri
in cheese, 425,431,432,440
on chicken meat, 575,575
chicken sandwiches, 577
chlorine inactivation, 195,200
dairy plant, 678
dairy products, 37 1
food processing facilities, 675
genomic group, 6
growth in autoclaved milks, 389
growth, low pH, 148
growth prevented on medium, 235
hemolytic strain, 264,265
identification, 10
irradiation, 190
no cause of avian listeriosis, 55
potato processing facility, 672
raw ewes milk, 369
raw milk, 36 1, 363
raw produce, 634,635,637
salt tolerance, 155
730 Index

[Listeria seeligeri] Listeriosis, cattle, 40

sanitizer inactivation, 203 abortion, 47,48,301,302,360,382
selective agents, 229 encephalitis, 47, 360
sugar utilization, 389 feces, 50
taxonomy, 5 , 6 feed transmission, 47
Listeria welshimeri incidence, 47
in beef, 524 infected tissues, 5 1
in cheese, 43 1 Listeria ivanovii, 48
on chicken meat, 569,570,571,574,575 mastitis, 48, 301, 302, 360
in crabmeat, 609 milk, 48, 49, 301, 360, 368
genomic group, 6 pathology, 47
household kitchens, 679 risk to humans, 51
identification, 10 seasonality-milk,50
monoclonal antibody reaction, 272 septicemia, 47
no cause of avian listeriosis, 55 silage, 47
pasteurized milk, 379, 380 stress-relate immunosuppression, 5 1
poultry slaughterhouse,667,668,669 Listeriosis, foodborne, 30, 3 1, 32
raw ewes milk, 369 Brie de Meaux cheese, 308,322
raw milk, 361, 362, 363 butter, 307
raw produce, 634,636,637 catered food, 83
taxonomy, 5,7 celery, 84
on turkey meat, 569,570,571 cheeseborne, 324 (see also specilfc
Listeriaphages, 198 cheeses)
Listeriolysin, 100, 105, 107, 112, 115, 117, chocolate milk, 82, 83
118, 148, 152, 153,264,265,266, coleslaw, 83, 84
267 common source outbreaks, 300
Listeriosis, animal eggs and egg products, 338
avian, 40,53-56,336,565,572 epidemic listeriosis, 75, 301
cattle, 40,47-51 foods of plant origin, 34 1
fish/shellfish, 58,59 goat cheese, 46
goats, 40,45,46 history, 299
incidence, 39 hot dogs, 87
livestock losses, 40 lettuce, 84
minor species, 40,56,57 Mexican-style soft cheese, 85,302,305,
rabbits, 2 307,309,3 10,
sheep, 28,3 1,40,4 1-44 outbreaks, 84
swine, 40, 52, 53 pasteurized milk, 84,85,301
transmission, 30 pat& 86,302, 305, 327
to humans, 40 pork pat6 rillettes, 33 1
treatment, 59,60 pork tongue in jelly, 86,302,329
Listeriosis, avian, 40, 335,336, 565, 572 poultry products, 87, 335
carriers, 54 raw milk, 49, 88
chick embryo, 55 seafood products, 339, 614
chickens, 53 sporadic listeriosis, 75
feces, 54, 56 Swiss soft cheese, 85,302, 308,3 17
history, 572 turkey frankfurters, 88
incidence, 54, 55 undercooked chicken, 87
meningioencephalitis,55 Listeriosis, goats, 40
poultry products, 56 abortion, 45
secondary infection, 54 bacteremia, 45
septicemia, 54 cheese, 46
transport stress, 56 encephalitis, 45
wild and domestic fowl, 53, 54 feces, 46
Index 731

[Listeriosis, goats] [Listeriosis, human]

intestinal tract, 45 sporadic, 75
mastitis, 369 stillbirth, 302, 303, 335, 343
meninogoencephalits,46 symptoms, 80,8 1,82
milk, 46, 303, 369 transplacental transmission, 82
oral lesions, 46 treatment, 89
septicemia, 45 Listeriosis, minor species
silage, 46 chinchilla, 57
vaccination, 46 dogs, 40,58
Listeriosis, human gazelle, 57
abortion, 343 horses, 40, 56
antibiotic treatment, 89 llama, 57
arthritis-septic, 8 1 non-human primates, 58
breast milk, 303 reindeer, 57
cancer, 70, 567 roe deer, 57
conjunctivitis, 335, 336 zoological animals, 57
cutaneous infection, 8 1 Listeriosis, sheep, 28, 3 1, 40, 345
diagnosis, 88 abortion, 4 1,42
dietary counseling, 8 1, 89, 90 clinical manifestations, 4 1
donated blood, 88 direct entry, 41
in the elderly, 79 encephalitis, 41
encephalitis, 3 17 ewes milk, 44, 303, 369
endocarditis, 80 fetal infection, 42
endophthalmitis, 8 1 infection, 4 1
epidemic, 75 ingestion, 4 1
epidemiology, 83-88 mastitis, 369
fecal carriage, 76, 77, 78, 79, 82, 326 meningoencephalitis,4 1
fetal infection, 81 morbidity/mortality,42
fish associated, 602,614 outbreaks: L. ivanovii, 45
foodborne, 75,82-88,299-358 seasonality, 42,43
food industry, 90 septicemia, 4 1, 42
high-risk groups, 75, 76 silage quality, 43
immunosuppresivetherapy, 79 stress factors, 43
incidence, 87 vaccination, 44
invasive diseasehonpregnant adults 79, 80, Listeriosis, swine, 40
81 abortion, 52
meningitis, 82, 303, 304, 322, 324, 332, age of animals, 52
335, 339, 342, 343, 567, 602, 614, carriers, 53
636 encephalitis, 52
meningoencephalitis,3 17 feces, 53
miscarriage, 335 husbandry practices, 53
mortality rate, 75,76, 82, 304 pork, 53
neonatal disease-early onset, 8 1, 82 seasonality, 52
neonatal disease- late onset, 82 septicemia, 52
oral infective dose, 372 silage, 53
osteomyelitis, 8 1 symptoms, 52
peritonitis, 8 1 tissues, 53
pleural infection, 8 1 Lithium chloride, 229,230, 232,233,234,
poultry associated, 567 235,236,243
pregnancy, 81 Liver, 523, 524
prevention, 89,90 Liver sausage, 535,55 1
public health agencies, 90 Llama, 57
septicemia, 303,304,3 17,322,338,343, Lobster, 601, 603, 605, 607, 608, 610, 612,
344,636 615,617,621,622,624,695,697
sexual transmission, 88 Localization in tissues, 522
732 Index

Low-fat milk, 371, 372, 374, 391 Mettwutst, 5 18, 520, 535, 539, 555
Low pH Mexican-style cheese, 302,307,308,309,
growth, 148 310, 324, 325, 333, 344, 345, 359,
survival, 150 360, 370, 389, 390,411,412,413,
Lubricants, conveyor chain, 204, 205 4 14, 4 15, 468,484, 566, 639, 658,
Luncheon meats, 334,506,508,513,519, 66 1
521,531,532,536,694,695 Microbacterium thermosphactum, 3
Lung, 522 Microbial rennet, 345, 450, 451, 452
Lupus erythematosus, 337 Microbiological Surveillance Program 370,
Lymph nodes, 522,523 371,373
Lymphocytes, 141, 142 Micrococci, 236
Lys Bleu cheese, 436 Micrococcus spp., 235,459,525
Lysozyme, 167, 176, 178, 182, 183, 193,383, Micro-Gard, 620
447, 455,478,487, 535, 536, 592, Microwave radiation, 583, 584, 585, 586
639,644,645,646,647 Milano salami, 541
Milk, 301, 314
Maasdam cheese, 462,463,367 Minas Frescal cheese, 43 1
Macrolides, 282 Mint, 175
Macrophages, 97, 100, 103, 104, 105, 107, Modified atmosphere (packaging), 187, 188,
113, 114, 115, 117, 141, 142,227, 189,479, 531, 532, 545, 548, 549,
265,384 577, 578, 579, 617, 619, 639, 643,
Malic acid, 158, 469 644
Manchego cheese, 150,385,473 Moist heat, 538, 584, 585, 586
Manouri cheese, 479 Mold-ripened cheeses, 453
MAP kinase phosphatase, 116 Monitoring programs, 506, 508, 566, 567
Margarine, 453 Monitoring sample, 506, 507, 567
Marinated seafoods, 624 Monoacylglycerols, 167, 168, 169
Mascarpone cheese, 434 Monocaprin, 166, 168, 169
Mayonnaise Monocaprylin, 166, 169
cholesterol-freereduced-calorie, 592 Monocin, 281, 282
low calorie, 592 Monoclonal antibody, 27 1,272,274, 327
real, 592 Monoglycerides, 460
reduced calorie, 592 Monolaurin, 166, 167, 168, 169, 193, 551
Meatballs, 549, 550 Monolimolein, 166
Meat industry, 692 Monomyristin, 166
frankfurters and other link products, 693 Monoolein, 165
luncheon meats, 694 Monopalmitin, I66
roast beef, corned beef and other rebagged Monostearin, 166
products, 693 Monterey Jack cheese, 325,412,414, 417,
Meat processing environmental problems, 484,485
664,665,666 Morbier Rippoz cheese, 420,422
Meat processing facilities, 664, 665,666,667 Mortality rate, 304, 317, 322, 329, 343
Meat products, 326, 507, 508,664 Moxalactam, 229,230,233,234,236
Meat salads, 508, 509, 5 17, 5 18 Mozzarella cheese, 150, 4 14, 4 17, 429,434,
Meat slicers, 517 443,445,46 1,462,479,482,484
Meat spreads, 508, 509 Mucor miehei, 450
Menaquinones, 7 Muenster cheese, 4 12,419,436,45 1,484,
Meningitis, 3 485
Mercury compounds, 203 Multilocus enzyme electrophoresis, 282, 290,
Mesophilic starter cultures, 44 1, 462, 474 292,313,318,321,330
Metalloprotease, 100, 106, 107, 108, 110, 291 Multitest assays, commercial, 11
Methyl sulfide, 460 Murein Rydrolase, 102
Methyl trisulfide, 460 Murraya grayi, 7
Index 733

Muscle tissue, 522, 523, 524, 530 Oxolinic acid, 23 1

Mushrooms, 345,632,634,635,636,637, Oysters, 339, 340, 601, 606, 607, 610, 612,
640,697 614,616,617,621,695,697
Mussels, 340, 606, 607, 608,610, 612, 622, Ozone, 201
623,624
Mutants, 152 Packaging (see also Modified atmosphere
Mutschli cheese, 436 packaging), 665
Mutton, 514,515,516 Palmitic acid, I65
Mycella cheese, 484 Parabens, 164, 166
Mycobacterium bovis, 145 Parmesan cheese, 150,417,445,454,462,
Mycolic acid, 5 467,468,482,483,484
Myristic acid, 165, 168 Parotid glands, 522
Mysost cheese, 479 Pasta, 632
Myzithra cheese, 479 Pasteurization, 139, 140, 143, 144, 145, 301,
306, 307, 314, 315, 316, 370, 372,
Nalidixic acid, 229,230,231,232, 235, 236, 394,413,484, 533, 550, 551, 552,
242 589, 594, 621, 645, 659, 661, 670,
National Conference on Interstate Milk 687,697
Shipments, 370 Pasteurization processes, 688, 689
Nectarines, 341,648 Pasteurized milk, 301, 302, 304, 305, 316,
Neufchatel cheese, 475, 484 331, 359, 360, 369, 370, 371, 372,
Nisin, 160, 170, 183, 184, 185, 186, 193, 398, 373, 378, 379, 380, 382, 385, 386,
456,457,469,478,482, 524, 548, 387, 390, 398, 412,418,424, 425,
549,555,591,620 432, 435,436,439, 440,449,457,
Nonfat dry milk, 360, 369, 370, 371, 372, 460,461,463,466,467,475,477,
378, 379, 381, 382, 388, 389, 400, 484,485,486,658,692
480,487 Pasteurized Milk Ordinance, 369, 690
Nonfood contact surfaces, 195 P W , 302, 305, 319, 324, 326, 327, 328, 329,
Non-human primates, 58 330. 331, 334, 335, 337, 505, 51 1,
Nucleic acid-based probes, 262, 263, 264, 517, 518, 519, 520, 521, 528, 534,
265 540,54 1,574,576,577, 705
Nucleic acid hybridization assay, 273 Pathogenesis
Nucleic acid sequence-based amplification, adhesion, 100
267,269 apoptosis, I 18
Numerical taxonomy, 3 catalase, 113
Nutmeg, 173, 174 cell-to-cell spread, 108
Nuts, 636 environment affects virulence gene expres-
sion, 113
Oblique lighting, 228, 230, 234,236,237 gene expression, 116
Octanoic acid, 165, 166 host cell responses, 1 14
Official detection methods, 244-249 internalin proteins, 100
Old Heidelberg cheese, 4 14 intracellular growth, 104
Olives, 649 intracellular motility, 108
One-step enrichment, 243 invasion, 100
Onions, 636,638 invasion mechanism, 103
Oral infective dose, 372, 378, 622 phagocytes, invasion, 1100, 101, 102, 103
Orange juice, 632,649 phagocytic vacuole, escape, 104
Orange serum, 632,649,650 protein p60, 101
Organic acids and salts, 157 protein ActA, 102
Oscillating magnetic field, 189 regulation of virulence gene, 111
Osmolytes, 134, 156 signal transduction pathways, 1 17
Osteomyelitis, 81 superoxide dismutase, 113
Ovoflavoprotein, 590 tissue tropism, 103
Ovotransferrin, 590 Peach, 648
734
Peanut sauce, 638
Pears, 648
Peas, 633
Pecorino Romano cheese, 422,429,473
Pediocin, 183, 185, 186, 187, 189, 554, 555
Pediococcus acidilactici, 185, 186, 187, 189,
478,54 1,544,554,58 1
Pediococcus cerevisiae, 54 1
Penicillin, 89
Penicillium camemberti, 453,455
Penicillium candidum, 457
Penicillium caseicolum, 453
Penicillium glaucum, 453, 457
Penicillium roqueforti, 453, 457, 458
Pepper, 174
Pepperoni, 541, 542, 544, 545
Pepsin-rennet extract, 450,45 1, 452
Peptidoglycan, 7, 645
Perfringolysin, 106
Peritonitis, 8 1
Perlac, 620
Permeate, 443
Peroxyautc acid, 204
Phage type, 305,307,313,317,318,319,
321,322,323,329,332,344,345
Phage typing, 281,284,289, 290,292, 327,
330,432,570
Phagocytes 97, 141, 142, 524
Phagocytic vacuole escape, 104
Phagolysosomes, 103
Phagosomal membrane, 106
Phagosome-lysosomefusion, 106
Phagosomes, 103, 107, 118
Pheasant, 565,566,571,572,573
Phenol, 203,204,537
Phenylethanol, 229, 230, 233, 235, 250
Phosphatidylcholine-specificphospholipase
C, 100,106
Phosphatidylinositol-specificphospholipase
C, 100, 106,291
Phosphoinositide-3-kinase,104
Phospholipase, 106, 107, 110, 117
Phospholipase B, 266
Phosphoribosyl-pyrophosphate synthetase,
100
Phytoalixim 6-methoxy mellein, 648
Pickled cheeses, 469
Pickled vegetables, 638
Pimento, 647
Pinene, 175
Pizza, 651
Planktonic cells, 197, 305
Plate pasteurizer, 139, 141, 142
Pleural infection, 8 1
Index
Pneumolysin, 265
Polyclonal antibody, 270,27 1
Polyester-polyurethanebelt, 198
Polymerase chain reaction, 262, 263, 266,
267, 268, 270, 273, 286, 287,288,
289
Polymyxin By228,229,232,233,235,250
Polyphosphate, 160, 163, 170,489
Polypropylene, 195
Polysaccharides, 523
Ponderosa pine needles, 341
Pont Eveque cheese, 436
Pork, 332, 514, 515, 516, 517, 518, 519, 529,
530, 534, 541, 546, 547, 548, 549,
566,667,679,692,693
Pork piit6 rilletes, 326, 33 1, 334
Pork sausage, 332,334,335, 513, 515, 518,
520,535
Port du Salut cheese, 412,459
Post processing contiaminants, 657
Potassium lactate, 587
Potassium sorbate, 161, 162, 163, 166, 167,
177, 189,478,480,. 550, 579
Potassium tellurite, 228, 229, 230, 232
Potassium thiocyanate, 232
Potato juice, 642
Potato products, 674, 675
Potato salad, 347, 633,638
Potatoes, 633, 634, 635, 638, 697
Poultry industry, 694
Poultry processing facilities, 667
Poultry processing plant environmental prob-
lems, 667, 668, 669, 675, 678
Poultry products, 335, 566,679
back and neck testing program, 568
bologna, 567
chicken nuggets, 337
cook-chill products, 336
cooked, 337,566,664
diced, 567
frankfurters, 567
raw meat, 336, 566, 572, 577, 588
ready-to-eat, 517, 566, 567, 579
spread, 567, 664
turkey frankfurters, 337, 338
undercooked, 337
Poultry salads, 509, 664
Poultry sausage, 544,566
Poultry spreads, 509, 576
Practical approaches to food safety, 698
Prepackaged salads, 636
Prerequisite programs, 658,700
Prfa regulon, 1 12
Primary enrichment, 228
Index
Problems in amplification methods, 268
Process cheese, 433,453,484,485
Proline, 156
Propionibacterium thoenii, 187
Propionic acid, 161, 164, 165
Propionic acid bacteria, 462
Propylgallate, 166, 167, 171, 172,648
Propylene glycol, 205, 453
Protein ActA, 102, 103, 108, 109, 110, 118
Protein p60, 10I , 104
Proteus, 228,233
Proteus vulgaris, 182
Provolone cheese, 434,461, 485
Pseudomenus, 204,228,233,394,620
Pseudomonus aeruginosa, 182,202,228,523
Pseudomonusfluorescens, 182,395,396,
397,525,526,527,685
Pseudornonas fragi, 185, 196,395,396,397,
548,549,579,581
Pseudomonas putrefaciens, 685
Pseudomonads, 179,398,642
Public health agencies, 90
Pulsed electric field, 183, 189
Pulsed-field gel electrophoresis, 284, 287,
288, 290, 292, 311, 318, 319, 321,
323,330,331 332,345,570,671
Pulsed light-high intensity, 189, 191, 192
Pyrolysis mass spectroscopy, 3 18,319
Quaternary ammonium sanitizer, 197, 198,
203, 204, 683, 685
Queen A m , 3
Quesito cheese, 314
Queso Anejo cheese, 314, 468
Queso Blanco cheese, 415, 468,469
Queso de Bola cheese, 468
Queso de Crema cheese, 468
Queso de 10s Ibores cheese, 468, 469
Queso de Prensa cheese, 468
Queso de Puna cheese, 468
Queso Fresco cheese, 310, 313, 314,414,
415, 429,468, 469, 485
Queso Panella cheese, 485
Queso Prensado cheese, 415
Queso Ranchero cheese, 485
Queso Sec0 cheese, 314
Rabbit meat, 332
Rabbits, 299, 333
Raclette cheese, 436
Radioimmunoassay, 27 1
Radishes, 633, 634, 635, 636, 638, 642, 697
Ranchero cheese, 3 14
Random amplification of polymorphic DNA,
288,290,292,313,318,344,346,671
Rapid detection methods, 262
Ravioli, 650, 651
Raw beef, 507, 523, 525, 550
Raw fish, 339, 340
Raw meat, 507, 512, 513, 514, 515, 516,
522, 524, 525, 529, 534, 550
Raw milk, 302, 303, 307, 314, 315, 316,
320, 323, 325, 333, 359, 360, 361,
362, 363, 364, 365, 366, 367, 371,
382, 383, 384, 385, 386, 387, 388,
412,413,417, 418, 424, 425,432,
435, 436, 441, 445, 449, 464, 471,
484, 487, 649, 661, 679
Raw milk cheese, 325, 435, 455, 484
Reblochon cheese, 436
Recall, 261, 310, 311, 316, 320, 322, 325,
331, 338, 341, 360, 373, 378, 379,
412, 413, 462, 479, 506, 507, 509,
511, 512, 623, 659, 670, 691
butter, 376, 377
crabmeat, 602, 609
defined, 373
domestic cheese, 412, 414, 415
fish and seafood, 602, 620
frozen yogurt, 376, 377
fruit juice bars, 649
ice cream products, 374, 375, 376, 377
imported cheese, 418, 420, 421
milk, 374
mussels, 612
plant products, 633
poultry products, 567
ready-to-eat meat products, 510, 512
seafood products, 608
sherbet, 376, 377
turkey frankfurters, 567
Recovering injured listeriae, 249
Red pepper, 633
Regulatory actions, 509, 511
Regulatory factor A, 111
Reindeer, 57
Renal disease-chronic, 79, 80
Renibacteriuni , 3
Rennet extract, 450, 45 1, 452
Repetitive element-based subtyping, 289
Restriction fragment length polymorphism
analysis, 283, 367
Retentate, 443
Rhodococcus equi, 11
Ribotypes, 251, 252, 286, 313, 321, 344
Ribotyping, 283, 284, 289, 290, 291, 318,
323, 330, 367, 570
Rice salad, 347
 Index

Rictone cheese, 479 [Sausage]

Ricotta cheese, 325, 379, 414, 417, 440, 536, 537, 538, 539, 541, 542, 543,
443,479,484 544,545,547,550,664,693,695
rRNA sequencing, 4 Sausage casings, 335,514,516,621,693
Roast beef, 507, 510, 512, 513, 524, 528, Saveloy, 531
531, 547, 566, 695 Scallops, 601, 606, 607, 610, 612, 624, 695
Roast lamb, 513 Scamorze cheese, 461,484
Roast pork, 513, 524 Seafood, 602,604,6 11,638
Roe, 606, 607 imitation, 606, 608
Roe deer, 57 meusse, 606,607
Rolls, 651 pasta, 61 1
Romadur cheese, 436 pfititc, 606, 607
Romano cheese, 445, 462 salads, 605, 606, 607, 608, 613
Roquefort cheese, 385, 453, 457, 473 smoked, 606
Rosemary, 173, 175 spread, 606,607,608
Rubber, 195, 196, 197, 201 Seafood processing facilities, 670
Ruditapes spp., 610, 612 Seafood processing plant environmental
problems, 670, 67 1,696
Sage, 173 Secondary enrichment, 228
Sakacin, 549, 555 Selective agents, 229
Salami, 151, 332, 334, 510, 518, 519, 520, Selective enrichment, 225, 226, 228,241,
521,541,542,543,544,555 262,286
Salmide, 200 Selective media, 233
Salmon, 339 Semidry fermented sausage, 54 1
brines, 6 17,624 Semisoft and hard cheeses, 462
gravad, 619 Serotypes, 305,307
heating, 623 Serotyping, 280,290,291,304, 570
packaging, 6 17 Serratia, 204
processing plants, 670, 671 Serratia marcescens, 191,685
smoked, 602,6 17,619 Sewage, 26,59,615,616
thermal death time of L. monocytogenes, Sheep, 40,41,42, 43, 44, 45, 319, 341, 344,
622 385,522,633,639
Salmonella, 104, 119, 192,270,281,347, Sheeps milk cheese, 426,428,430,433,438,
370, 392, 393, 417, 524, 552, 568, 473
587, 594, 667, 670, 679, 691, 694, Shellfish, 58, 59, 339, 340
695 Sherbet, 373, 374, 376, 377,487
Salmonella enteritidis, 164, 175, 182, 192, Shigella, 119, 695
695 Shigella dysenteriae, 703
Salmonella typhi, 703 Shigella Jexneri, 110, 1 18
Salmonella typhimurium, 118, 147, 151, 179, Shredded cheese, 440
182, 184, 190, 192, 202,484, 540, Shrimp, 340,60 1,602,603,604,605,607,
658 608, 609, 612, 614, 615, 616, 617,
Salmonellosis, 370 621,623,624,671,695,696,697
Salvador-style white cheese, 4 14 Siderophores, 620
Sampling plans, 703 Silage, 2, 21, 27,29, 3 1, 40, 43,46,47, 53,
Sandwich hybridization capture assay, 264 56,302,319,341,368,631
Sandwich spread, 592 Skim milk, 371, 372, 387, 388, 389, 391, 393,
Sandwiches, 509, 5 10, 5 12, 5 17, 567, 577, 398,489,490
65 1 Skim milk cheese, 412
Sapsago cheese, 484 Sliced meat, 332
Sacrcoidosis, 79 Smoked meat, 5 18
Sauerkraut, 645 Smoked sausage, 535,536,539
Sausage, 505, 506, 512,513, 514,515, 516, Snails, 606, 607
517, 518, 519, 520, 521, 534, 535, Soaps, 202
Index 737

Sodium acetate, 578, 620 Streptococcus cremoris, 44 1,442,443,444,

Sodium ascorbate, 648 475
Sodium azide, 230 Streptococcusfaecalis, 102,271
Sodium benzoate, 163,451 Streptococcus latis, 441,442,443,444,478
Sodium caseinate, 3 16 Streptococcus mutans, 182
Sodium chloride, 146,151,154,155,1156,159, Streptococcus alivarius ssp. thermophilus,
160,161,167,169,170,184,193,250, 44 1,447,449 (see also Streptococ-
482,487,488,489,490,531,534,536, cus thermophilus),
539,540,542,543,545,546,549,550, Streptococcus thermophilius, 233,44 1,445,
551,586,593,618,619,623,624,640, 446,447
641,645,648 Streptolysin-0, 105, 265
Sodium diacetate, 160, 587, 620 Stress adaptation, 145
Sodium dichloro-s-triazinetrione, 199 Stress response protein, 116
Sodium erythrobate, 540,550 String cheese, 485
Sodium hydroxide, 202 Sublethal injury (see Injury, cellular)
Sodium lactate, 478, 550, 55 1, 587,6 18, 6 19, Subtyping methods compared, 291
620 Sucrose, 146, 593,618,623
Sodium nitrite, 160, 163, 169, 170, 194, 53 1, Sucrose monolaurate, 169
532, 534,536, 537,539, 540, 542, Sudanese white-pickled cheese, 472, 488
543,544,545,546,6 18,619,620 Sultanas, 636
Sodium phosphate, 550 Summer sausage, 554, 555, 581
Sodium propionate, 159, 161, 162, 163,451, Superoxide dismutase, 113, 114, 146,648
480,48 1,651 Superoxide radicals, 114
Sodium tripolyphosphate, 550, 551, 586 Surfactants, 200
Soft cheese factory, 673,674 Surimi, 605,606,611, 613
Soft chive cheese, 324 Sweetened condensed milk, 393,394
Soft unripened cheese, 475 Swine, 40, 52, 53, 522
Soil, 22, 23, 319, 368,633,634,650, 672 Swiss cheese, 150,415,417, 445, 462,466,
Somatic cells, 143 467,479,482,485
Sorbic acid, 162, 48 1
Sour cream, 308 Talleggio cheese, 429,434
Sour milk, 302, 307,412 Tartaric acid, 161
Sour milk cheese, 432 Taxonomy, 5,7
Sous-vide, 147, 188, 587,620,623 T-cell-mediated immunity, 114
Soybeans, 638,650 Teflon, 196,201
Soymilk, 632, 650 Teichoic acid, 7
Spinach, 633 Teleme cheese, 469, 489
Spleen, 522, 523, 524 Tertiary butylhydroxyquinon, 166, 167, 171,
Spray drying, 372 172
Squid, 601,605,606,607,611 Tetracyclines, 60, 89, 282
Staffordshire cheese, 325 Tetrahymena, 100
Stainless steel, 195, 196, 197, 198, 201 Thallous acetate, 232
Staphylococcus, 4,233,460 Theobromine, 175, 176,393
Staphylococcus aureus, 11, 169, 172, 178, Thermal death time, 62 1,622
179, 182, 190, 192, 235,271, 347, Thermotolerance, 145, 146
370,399,417,604,685,695 Thermal inactivation, 136, 657
Staphylococcus carnosus, 545 Thermus aquaticus, 266
Starter cultures, 44 1,479,482, 58 1 Thiocyanate, 179, 180, 181
Starter distillate, 45 1, 453 Thuringer, 535
Stearic acid, 165
St. Paulin cheese, 436,459 Tilsiter cheese, 436,459,460, 479
Strawberries, 34 1,648 Tissue tropism, 103, 104
Streptococci, 23 1, 236 Tomato juice, 642
Streptococcus, 3,4, 81, 82, 109 Tomato sauce, 642
 Index

Tomatoes, 343,634,635,636,639,642,643, Vegetable salads, 634,637,641,642

644,648 Vegetation, 23, 368, 63 1
Tongue, 518 Vegetation, decayed, 2 1, 75
Tourre de 1Aubier cheese, 420 Verification sample, 506, 507, 566
Traditional approaches to food safety, 698 Vibrio, 615, 695
Traditional fermented milk products, 449 Vibrio cholerae, 3 , 4 17, 604, 703
Transmission, 30, 31, 32, 593, 615 Vibrioparahaemolyticus, 4 17, 604
Transposon mutagenesis, 97, 100, 105, 107, Vibrio vulnijkus, 604
108,264 Virulence, 152,589
Trappist cheese, 150,459,46 1 Virulence determinants, 99
Trimethoprim-sulfamethoxazole,89 Virulence gene (cluster), 98, 100, 102, 107,
Tripe, 518, 520 111,112,113,263,266,286,291
Trisodium phosphate, 201, 587,620,645 Virulence gene expression, 113
Trout, 624
Trypaflavin, 229,23 1 Warm enrichment, 144,228, 229, 241, 621
Trypan blue, 242 Water, 22, 26, 59, 75, 632, 675, 682
Tumor necrosis factor, 115 Water activity 153, 154, 394, 482, 534, 543,
Turkey, 565,566,584,638 544,593,619
bologna, 579 Watercress, 636
carcasses, 572, 573 Whey, 149,389,400,455,476,477,486,
frankfurter, 335, 337, 338, 505, 566,567, 487,489,490
586,68 1,695 Whey cheeses, 479
ground, 572 Whey powder, 398,479,480,487
legs, 569, 570, 571 Whey processing facilities, 662
liver, 570 Whipping cream, 373, 374, 377, 387, 388,
meat emulsion, 586 389,400,418
parts, 571,572 Whitefish, 6 16
sausage, 567 White Lymeswold cheese, 484
slaughterhouse, 667, 668 White Stilton cheese, 484
sliced, 579, 580 White pickled cheese, 150, 430, 43 1,436,
summer sausage, 581 469,470,471,472
tails, 569, 571 Whole milk, 387, 388, 389, 390
wings, 569,570,571 Wieners (see Frankfurters)
Turkish white-brined cheese, 470,47 1 Wild animal meat, 5 13
Turmeric, 451 Wiirstel, 5 18, 520
Tyrosine kinase, 103
Tyrosine phosphatase, 103
Xenopus, 108
Ulcerative colitis, 79 X-ray irradiation, 455
Ultrafiltered milk, 394,443
Ultra-high temperature treated milk, 386,489 Yersinia, 119, 524, 662
Ultrapasteurization, 594, 595 Yersinia enterocolitica, 133, 182, 188, 189,
Ultraviolet irradiation, 190, 191 190, 192,202, 370, 417, 548, 604,
Uncooked smoked sausage, 539 650
Unfermented sausage, 534 Yersiniapestis, 3
Yersiniosis, 3 70
Vacherin Mont dOr cheese, 302,308,3 14, Yugoslavian white-brined cheese, 472
317,318, 319,320,321,322,324, Yogurt, 151,308,324,325,377,412,444,
333,344,345,359, 366,378, 412, 445,447,448,453,660,691
434,436,455,575,672,692
Valerianella olitoria, 641 Zero tolerance, 261, 321, 341, 372, 378,484,
Vanadate, 103 509,567,602,614,622,704
Veal, 5 16 Zoological animals, 57, 571
Vegetable processing facilities, 67 1, 672 2-value, 621, 622
Vegetable rennet, 345 Zygosaccharomyces bailii, 202