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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
Part I: Food Chemistry, edited by Owen R, Fennema
Part I I : Physical Methods of Food Preservation, Marcus Karel, Owen R. Fenne-
ma, and Daryl 8.Lund
5. Food Emulsions, edited by Stig E. Friberg
6 . Nutritional and Safety Aspects of Food Processing, edited by Steven R. Tan-
nenbaum
7 . Flavor Research: Recent Advances, edited by R. Teranishi, Robert A. Flath,
and Hiroshi Sugisaw a
8. Computer-Aided Techniques in Food Technology, edited by Israel Saguy
9. Handbook of Tropical Foods, edited by Harvey T. Chan
10. Antimicrobials in Foods, edited by Alfred Larry Branen and P, Michael
Davidson
1 1. Food Constituents and Food Residues: Their Chromatographic Determination,
edited by James F. Lawrence
12. Aspartame: Physiology and Biochemistry, edited by Lewis D. Steghk and L. J,
Filer, Jr.
13. Handbook of Vitamins: Nutritional, Biochemical, and Clinical Aspects, edited
by Lawrence J. Machlin
14. Starch Conversion Technology, edited by G. M. A. van Beynum and J. A.
Roels
15. Food Chemistry: Second Edition, Revised and Expanded, edited by Owen R.
Fennema
16 . Sensory Evaluation of Food: Statistical Methods and Procedures, Michael
0 'Mahony
1 7 . Alternative Sweetners, edited by Lyn O'Brien Nabors and Robert C. Gelardi
18. Citrus Fruits and Their Products: Analysis and Technology, S. V. Ting and
Russell L. Rouseff
19. Engineering Properties of Foods, edited by M. A. Rao and S. S. H. Rizvi
20. Umami: A Basic Taste, edited by Yojiro Kawamura and Morley R. Kare
21, Food Biotechnology, edited by Dietrich Knorr
22. Food Texture: Instrumental and Sensory Measurement, edited by Howard R.
Moskowitz
23. Seafoods and Fish Oils in Human Health and Disease, John E. Kinsella
24. Postharvest Physiology of Vegetables, edited by J. Weichmann
25. Handbook of Dietary Fiber: An Applied Approach, Mark L. Dreher
26. Food Toxicology, Parts A and 6, Jose M. Concon
27. Modern Carbohydrate Chemistry, Roger W. Binkley
28. Trace Minerals in Foods, edited by Kenneth T. Smith
29. Protein Quality and the Effects of Processing, edited by R. Dixon Phillips and
John W. Finley
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
Roger D. Middlekauff and Philippe Shubik
35. Food Additives, edited by A. Larry Branen, P. Michael Davidson, and Seppo
Salminen
36. Safety of Irradiated Foods, J, F. Diehl
37. Omega-3 Fatty Acids in Health and Disease, edited by Robert S. Lees and
Marcus Karel
38. Food Emulsions: Second Edition, Revised and Expanded, edited by Ksre Lar-
sson and Stig E. Friberg
39. Seafood: Effects of Technology on Nutrition, George M, Pigott and Barbee
W. Tucker
40. Handbook of Vitamins: Second Edition, Revised and Expanded, edited by
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41. Handbook of Cereal Science and Technology, Klaus J! Lorenz and Karel Kulp
42. Food Processing Operations and Scale-Up, Kenneth ,J. Valentas, Leon Levine,
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43. Fish Quality Control by Computer Vision, edited by L. F. Pau and R, Olafsson
44. Volatile Compounds in Foods and Beverages, edited by Henk Maarse
45. Instrumental Methods for Quality Assurance in Foods, edited by Daniel Y. C.
Fung and Richard F. Matthews
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
48. Alternative Sweeteners: Second Edition, Revised and Expanded, edited by Lyn
O'Brien Nabors and Robert C. Gelardi
49. Food Extrusion Science and Technology, edited by Jozef L. Kokini, Chi-Tang
Ho, and Mukund V. Karwe
50. Surimi Technology, edited by Tyre C. Lanier and Chong M. Lee
51. Handbook of Food Engineering, edited by Dennis R. Heldman and Daryl B.
Lund
52. Food Analysis by HPLC, edited by Leo M. L. Nollet
53. Fatty Acids in Foods and Their Health Implications, edited by Ching Kuang
Chow
54. Clostridium botulinum: Ecology and Control in Foods, edited by Andreas H. W.
Hauschild and Karen L. Dodds
55. Cereals in Breadmaking: A Molecular Colloidal Approach, Ann- Charlotte
Eliasson and Ksre Larsson
56. Low-Calorie Foods Handbook, edited by Aaron M. Altschul
57. Antimicrobials in Foods: Second Edition, Revised and Expanded, edited by P.
Michael Davidson and Alfred Larry Branen
58. Lactic Acid Bacteria, edited by Seppo Salminen and Atte von Wright
59. Rice Science and Technology, edited by Wayne E. Marshall and James l.
Wadsworth
60. Food Biosensor Analysis, edited by Gabriele Wagner and George G. Guilbault
61. Principles of Enzymology for the Food Sciences: Second Edition, John R,
Whitaker
62. Carbohydrate Polyesters as Fat Substitutes, edited by Casimir C. Akoh and
Barry G. Swanson
63. Engineering Properties of Foods: Second Edition, Revised and Expanded, edi-
ted by M. A. Rao and S. S. H. Rizvi
64. Handbook of Brewing, edited by William A. Hardwick
65, Analyzing Food for Nutrition Labeling and Hazardous Contaminants, edited by
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66. Ingredient Interactions: Effects on Food Quality, edited by Anilkumar G.
Gaonkar
-67. Food Polysaccharides and Their Applications, edited by Alistair M. Stephen
68. Safety of Irradiated Foods: Second Edition, Revised and Expanded, J. F, Diehl
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70. Handbook of Fruit Science and Technology: Production, Composition, Stor-
age, and Processing, edited by D. K. Salunkhe and S. S. Kadam
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edited by D. L. Madhavi, S. S. Deshpande, and D. K. Salunkhe
72. Freezing Effects on Food Quality, edited by Lester E. Jeremiah
73. Handbook of Indigenous Fermented Foods: Second Edition, Revised and Ex-
panded, edited by Keith H. Steinkraus
74. Carbohydrates in Food, edited by Ann-Charlotte Eliasson
75. Baked Goods Freshness: Technology, Evaluation, and Inhibition of Staling,
edited by Ronald E. Hebeda and Henry F. Zobel
76. Food Chemistry: Third Edition, edited by Owen R. Fennema
77. Handbook of Food Analysis: Volumes 1 and 2, edited by Leo M. L. Nollet
78. Computerized Control Systems in the Food Industry, edited by Gauri S, Mittal
79. Techniques for Analyzing Food Aroma, edited by Ray Marsili
80. Food Proteins and Their Applications, edited by Srinivasan Damodaran and
Alain Paraf
81. Food Emulsions: Third Edition, Revised and Expanded, edited by Stig E, Fri-
berg and K&e Larsson
82. Nonthermal Preservation of Foods, Gustavo V. Barbosa-Canovas, Usha R.
Pothakamury, Enrique Palou, and Barry G. Swanson
83. Milk and Dairy Product Technology, Edgar Spreer
84. Applied Dairy Microbiology, edited by Elmer H. Marth and James L. Steele
85. Lactic Acid Bacteria: Microbiology and Functional Aspects, Second Edition,
Revised and Expanded, edited by Seppo Salminen and Atte von Wright
86. Handbook of Vegetable Science and Technology: Production, Composition,
Storage, and Processing, edited by D. K. Salunkhe and S. S. Kadam
87. Polysaccharide Association Structures in Food, edited by Reginald H. Walter
88. Food Lipids: Chemistry, Nutrition, and Biotechnology, edited by Casimir C.
Akoh and David B. Min
89. Spice Science and Technology, Kenji Hirasa and Mitsuo Takemasa
90. Dairy Technology: Principles of Milk Properties and Processes, P. Walstra,
T. J. Geurts, A. Noomen, A. Jellema, and M. A. J. S. van Boekel
91. Coloring of Food, Drugs, and Cosmetics, Gisbert Otterstiitter
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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Current printing (last digit):


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PRINTED IN THE UNITED STATES OF AMERICA


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.
Direct consumption of minimally
processed products i.e. whole fresh
vegetables, cooked carcass cuts of
meat and fish and effectively
4 Consumer at risk 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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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
live and dead intracellular bacteria in vitro. Infect. Immun. 63:720-723.
204. Zychlinsky, A., M.-C. Prkvost, and P.J. Sansonetti. 1992. Shigella Jexneri induces apoptosis
in infected macrophages. Nature 358: 167- 169.
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