Full

Antibiotic resistance in
V Thi Tia An

Front cover: 'Coniugal exchange oI antibiotic resistance between Salmonella and
E. coli and multiple resistance by An T. T. Vo
Back cover: 'Similarity and diIIerence between tulip Irom The Netherlands and
lotus Irom Vietnam by Sylvia Gaastra
Layout: by An T. T. Vo
Printing: by Atalanta Drukwerkbemiddeling. Houten. The Netherlands

ISBN 978-90-393-4529-0
This study was supported by a grant Irom the Vietnamese Government (Proiect 322)

Antibiotic resistance in

Antibioticum resistentie in
(met een samenvatting in het Nederlands)
khang khang sinh cua
(Ban tom tt bng ting Vit)

PROEFSCHRIFT
ter verkriiging van de graad van doctor aan de Universiteit Utrecht
op gezag van de rector magniIicus. proI. dr. W. H. Gispen.
ingevolge het besluit van het college voor promoties in het openbaar te verdedigen
op donderdag 31 mei 2007 des middags te 2.30 uur

door

V Thi Tr An
geboren op 10 Iebruari 1974. te Ha Nam. Vietnam

Promotor: ProI. Dr. W. Gaastra

Co-promotoren: Dr. E. van Duiikeren
Dr. A.C. Fluit

Examining committee:
ProI. Dr. J. P. M. van Putten

Department oI InIectious Diseases and Immunology
Faculty oI Veterinary Medicine. Utrecht University. The Netherlands

ProI. Dr. J. VerhoeI
Eiikman-Winkler Institute
University Medical Center Utrecht. The Netherlands

ProI. Dr. S. Schwarz
Institut Ir Tierzucht
BundesIorschungsanstalt Ir LandwirtschaIt. Germany

ProI. Dr. J. Fink-Gremmels
Department oI Veterinary Pharmacology. Pharmacy and Toxicology

ProI. Dr. F. van Knapen
Institute Ior Risk Assessment Science

"It does not matter how slow you go so long as you do not stop."
(ConIucius)

For me. mv beloved familv. mv dedicated teachers and students
CONTENTS
Chapter 1. Introduction: A review on antimicrobial resistance in non-typhoid
Salmonella and thesis outline
Chapter 2. Distribution oI Salmonella enterica serovars Irom humans. livestock and
meat in Vietnam and the dominance oI Salmonella Typhimurium Phage
type 90
Chapter 3. Antimicrobial resistance. class 1 integrons and a novel variant oI
Genomic Island 1 in Salmonella isolates Irom Vietnam
Chapter 4. Antibiotic resistance. integrons and Salmonella Genomic Island 1 among
non-typhoidal Salmonella serovars in The Netherlands
Chapter 5. Class 1 integrons in Dutch Salmonella enterica serovar Dublin isolates
Irom clinical cases oI bovine salmonellosis
Chapter 6. A novel Salmonella genomic island 1 and rare integron types in
Salmonella Typhimurium isolates Irom horses in The Netherlands
Chapter 7. Characteristics oI extended-spectrum-cephalosporin resistant
Enterobacteriaceae isolates Irom animals.
Chapter 8. Comparison oI the in vitro pathogenicity oI two Salmonella Typhimurium
phage types
Chapter 9. Summarizing discussion
Nederlandse samenvatting voor de leek
Tom tt lun an
Acknowledgements/ Loi cam ta
Curriculum vitae
List oI publications
9

53

61

77

91

103

117

129

137
147
151
155
158
159

IntrnductInn

1. AntImIcrnbIa! rcsIstancc In nnn-tvhnId
1.1. Currcnt status nf nnn-tvhnId sa!mnnc!!nsIs
1.2. AntIbIntIc usc and antIbIntIc rcsIstancc
1.3. AntIbIntIc rcsIstancc - a rnb!cm In thc cnntrn! nf
InfcctInns
1.4. AntImIcrnbIa! rcsIstancc In
1.4.1. Intcgrnn-bnrnc rcsIstancc and mu!tI-drug rcsIstancc
1.4.2. RcsIstancc tn antImIcrnbIa! agcnts
1.4.3. 5rcad nf antImIcrnbIa! rcsIstancc
1.5. PrcvInus studIcs nn antImIcrnbIa! rcsIstancc In nnn-tvhnId
In VIctnam and Thc Ncthcr!ands

2. ThcsIs nut!Inc

Antimicrobial resistance in non-typhoid Salmonella

10
ABSTRACT
Worldwide. antimicrobial resistance among non-typhoid salmonellae is increasing. Many
strains have become multi-resistant. not only against antimicrobial agents that have been in use
Ior decades but also against new antimicrobial agents. This is an alarming development since in
many cases the use oI these agents is critical Ior therapy. InIections with these resistant
Salmonella strains may lead to a poor outcome oI the therapy or patients may be untreatable.
Furthermore. these Salmonella strains can also serve as reservoirs oI resistance determinants Ior
other pathogenic bacteria. Surveillance studies on national. regional and international levels are
necessary to monitor the spread oI multi-drug resistant isolates. Phenotypic resistance studies
may guide clinicians what therapy to apply since they can reveal signiIicant diIIerences and
shiIts in susceptibility to various antimicrobials Ior diIIerent bacterial species. The study oI the
genetic basis oI antibiotic resistance can serve to monitor resistance development by providing
inIormation on the transmission oI resistant strains. the persistence oI resistance in the bacterial
population and the resistance mechanisms involved. From a management point oI view. these
surveillance studies are relevant Ior the implementation oI timely and adequate measures. Many
surveillance programs in the past suIIered Irom several limitations. mainly lack oI resources and
oI standardized methods. Here the current situation with respect to resistance oI non-typhoid
Salmonella serovars to diIIerent antimicrobials and the various mechanisms by which resistance
is obtained are discussed. Methods to detect the resistant phenotype and the resistance
mechanism behind it at the molecular level as well as the acquisition and dissemination oI
resistance determinants will be discussed. Next the aims oI the thesis will be addressed.

Chapter 1
11
1. ANTIMICROBIAL RESISTANCE IN NONTYPHOID SALMOAELLA

1.1. Current status of non-typhoid salmonellosis
In the new millennium. diarrhoeal diseases will remain a maior cause oI morbidity and
mortality worldwide
91
. It is estimated that at a global level. diarrhoeal diseases cause 1.9-2.5
million deaths (mostly in children under Iive years old) annually and no decrease oI the
mortality rate attributable to diarrhoea has been observed
62. 110
. Salmonella is one oI the most
common pathogens associated with acute diarrhoea in humans in developing countries
91. 110
. In
developed countries. it is estimated that up to one third oI the population is aIIected each year
by microbiological Iood-borne diseases and that Salmonella is one oI the most Irequent causes
oI gastroenteritis (Table 1)
66. 77. 122. 127. 128
.

Table 1a. Role oI Salmonella in human Ioodborne outbreaks oI gastroenteritis
77. 122
.
Country Comparison Illness

Hospitalization

Death

Position oI
Salmonella
USA Total bacterial agents
Non-typhoid Salmonella
5.204.934
1.412.498 (27)*
45.826
16.430 (36)
1.458
582 (40)
Second (aIter
Campvlobacter)
France Total bacterial agents
Non-typhoid Salmonella
91.626
43.304 (47)
19.557
10.739 (55)
690
563 (80)
First

Table 1b. Role oI Salmonella in human Ioodborne outbreaks oI gastroenteritis
66
.
Country Comparison Outbreak

Patients

Position oI Salmonella
Korea Total Ioodborne
Salmonellosis
662
137 (20.7)
14.752
3.504 (23.8)
First
Japan Total Ioodborne
Salmonellosis
13.084
1.864 (14.2)
509.472
101.395 (19.9)
Second aIter Vibriosis
* percentage oI total

The maiority oI the 2500 known serovars (or serotypes) oI Salmonella is capable oI
causing inIections in humans and/or animals although only about 50 serotypes are isolated in
signiIicant numbers Irom clinical cases as the causative agent. Salmonella serotypes are
currently divided in three main groups on the basis oI their host range
121
. Host-restricted (or
strictly host-adapted) serotypes are almost exclusively associated with one particular host
species (i.e. Salmonella Typhi. Paratyphi A. Paratyphi C and Sendai with humans; Salmonella
Gallinarum with poultry; Salmonella Typhisuis with pigs; Salmonella Abortusequi with horses
and Salmonella Abortusovis with sheep. Host-adapted serotypes are associated with a particular
host species but can sometimes also cause diseases in other host species. For example.
Salmonella Dublin and Salmonella Choleraesuis are generally associated with severe systemic
disease in cattle and pigs. respectively. but they may also. albeit inIrequently cause diseases in
other mammalian hosts including humans. Non-adapted or ubiquitous serotypes include almost
all the remaining Salmonella serovars.

Non-typhoid Salmonella reIers to any serovar in the genus Salmonella other than
Salmonella enterica Typhi or Salmonella enterica Paratyphi
56
. Non-typhoid salmonellae cause

12
several important clinical syndromes in humans like gastroenteritis. bacteremia. and enteric
Iever. Gastroenteritis is the most common maniIestation oI a Salmonella inIection. Most non-
typhoid Salmonella enterica serotypes can cause primary bacteremia (positive blood culture in
the absence oI diarrhoea) and extra-intestinal inIections like endovascular inIections (the
abdominal aorta is the most common site oI inIection). meningial inIections (in children
younger than one year old). bone inIections (osteomyelitis in young children with sickle cell
disease) or urinary tract inIections (in 5 oI the non-typhoid Salmonella inIections)
34
. In a
study oI 172 cases oI Salmonella bacteremia in Spain (70 caused by S. Enteritidis and 17 by
S. Typhimurium). 16 oI patients developed septic metastases. oI which 16 died. In another
study at Massachusetts General Hospital. 18 oI 45 patients with non-typhoid Salmonella
bacteremia died
56
. Asymptomatic Iaecal excretion in humans is common Ior inIected
individuals who excrete the organisms and remain clinically normal. This 'carrier' condition
may persist Ior months or years
34
.

In animals. non-typhoid salmonellae presents Iour maior Iorms including enteritis.
septicaemia. abortion. and subclinical excretion; sometimes complications like meningitis.
osteomyelitis. and arthritis can occur (Table 2)
98
. Each oI these conditions may be present on its
own or in combination. depending on both the serotype and the host involved.

Table 2. Animal diseases caused by Salmonella serovars
98
.
Host Serovars Diseases
Cattle Dublin

Typhimurium. BovismorbiIicans and others
Enteritis. septicaemia. meningitis.
abortion. osteomyelitis. arthritis.
terminal dry gangrene
Enteritis. septicaemia
Pigs Choleraesuis
Typhisuis
Typhimurium and others
Swine Iever (hog cholera)
Chronic enteritis
Sheep Abortusovis. Montevideo. and Dublin
Typhimurium. Anatum and others
Abortion
Horses Abortusequi
Typhimurium and others
Abortion
Poultry
and
birds
Pullorum
Gallinarum
Arizonae
Enteritidis. Typhimurium and others
Pullorum (bacillary white diarrhea)
Fowl typhoid
Enteritis. septicaemia. swollen wing
ioints

1.2. Antibiotic use and antibiotic resistance
Antimicrobial agents are essential Ior human and animal health and welIare because they can be
used to Iight inIections caused by bacteria. ThereIore. the increasing antimicrobial resistance
seen today is a global public health concern. Antibiotic misuse and excessive antibiotic use in
human and veterinary medicine are important reasons Ior the observed increase and spread oI
Chapter 1
13
antibiotic resistance among the bacterial population. The speciIic causes oI misuse most
commonly cited are: there is actually no inIection. there is a viral inIection. incorrect drugs are
selected and the dose or duration oI the antimicrobial treatment is excessive. The
pharmaceutical industry plays an important role in this excessive and inappropriate use due to
the marketing strategy oI promoting the broadest indication drugs. In addition. a day spend in
hospital Ior intravenous antibiotic therapy can cost US$1000 and patients thereIore preIer
outdoor therapy over hospital therapy. Antibiotic misuse can also occur at home or during
outpatient therapy. Therapy is oIten ended when a person Ieels better and Irequent doses that
have to be taken may be hard to remember
55
.

Use of antimicrobials in human medicine in 1he Aetherlands
In The Netherlands. all antibiotics Ior human use are prescription-only. There is a non-proIit
Ioundation SWAB which publishes guidelines on the prudent use oI antibiotics on the website
http://customid.duhs.duke.edu/NL/Main/Start.asp. The maiority oI the prescribed medicines is
delivered by the community pharmacies. For approximately 10 oI the Dutch population.
mainly in rural areas. direct delivery oI medicines by general practitioners Irom their own
pharmacies takes place
85
. Compared to 25 other European countries. The Netherlands had the
lowest total outpatient antibiotic use (DDD (deIined daily dose) per 1000 inhabitants daily
DID 10. the highest is 32.2 in France) in 2002
46
. DeIined daily dose is a World Health
Organization statistical measure oI drug consumption. DDD is used to standardise the
comparative usage oI various drugs since diIIerent medication can be oI diIIerent strengths and
diIIerent potencies. Formula Ior calculating DDD can be consulted via the link
http://en.wikipedia.org/wiki/DeIinedDailyDoses.

However. in The Netherlands. 40 oI the treatments (4105 Dutch patients studied)
with antibiotics was inappropriate. The association with inappropriate use oI antibiotics was
statistically signiIicant with respect to quinolones. amoxicillin-clavulanic acid. old age patients
and patients on a urology ward
134
. There was a decrease in the number oI prescriptions oI
narrow-spectrum antibiotics (penicillins. tetracyclines. sulphonamides and trimethoprim) and an
increase in the use oI broad-spectrum or new antimicrobials (amoxicillin-clavulanic acid and
Iluoroquinolones) in human medicine in The Netherlands Irom 1992 to 2001
63
.

Use of antimicrobials in animal husbandrv and veterinarv medicine in 1he Aetherlands
In animal husbandry. only Iew antibiotics were still allowed Ior use as antimicrobial growth
promoters (AGP) since 1999. From January 2006 all antibiotics are prohibited Ior use as AGP
as part oI the EU drive to phase out all AGPs Irom livestock production. Local antibiotic
resistance data are available to practitioners since antimicrobial susceptibility results have been
updated annually and are made available on the website http://www.cidc-
lelystad.wur.nl/NL/publicaties/rapporten/maran/
78
. Furthermore. there is a consulting committee
which provides the Iormularies oI chosen antibiotics used Ior certain inIectious diseases in
veterinary medicine. These Iormularies oIten are updated every Iew years when the trend in
resistance to antimicrobials changes signiIicantly. In 2004. the total sales oI antibiotics Ior
therapeutic purposes (453.000 kg) in The Netherlands increased 15 compared to 2003. The

14
use oI penicillins/cephalosporins. tetracycline. macrolides. and quinolones/Iluoroquinolones in
veterinary medicine in 2004 was remarkably increased (18. 19. 33 and 40. higher than in 2003
respectively Ior these antimicrobials). In the same period. the total live weight production oI the
most important Iood production animals (pigs. broilers and veal calves) only slightly increased
(6 compared to 2003). Antibiotics Ior therapeutic purposes in poultry production were mainly
used in turkeys and broilers. Many Ilocks have been exposed to antibiotics; a substantial part
has been exposed to quinolones and Iluoroquinolones. Tetracyclines and
trimethoprim/sulphonamide combinations were most oIten used in pig production as group
medication. Antibiotics were less oIten used in dairy cattle and laying hens
78
. There are no
speciIic data Ior The Netherlands. but the use oI pharmaceutical products in companion animals
and other non-Iood animals in The European Union accounted Ior 36.5 oI the animal health
sales. Anti-inIective products used Ior this group oI animals represented 17. excluding
parasiticides and medicinal Ieed additives
47
. Generally. in The Netherlands antibiotics in
veterinary medicine are registered Ior therapeutic use in speciIic animal species as UDD
(Uitsluitend Door Dierenarts) and UDA (Uitsluitend aan Dierenarts AIleveren). In theory.
UDD-products are prescribed and administered only by veterinarians. UDA-products are
prescribed by veterinarians and are administered by either veterinarians or owners.

Use of antimicrobials in human medicine in Jietnam
Private pharmacies are the Iirst line oI primary health care in many Asian countries. For
instance. in Vietnam. aIter the introduction oI a market economy. the number oI private
pharmacies increased Irom none in 1986 to 6000 in 1996
17
. It is estimated that 80 or more oI
the population when sick. go directly to these pharmacies. to buy drugs without prescription or
adequate user instructions. Regulation 488/BYT-QD was promulgated on the 4
th
oI March 1995
to restrict the sale oI prescription drugs at private pharmacies and private drug outlets.
However. in reality. due to a lack oI enIorcement and strict punishment these sales are still a
problem. The drug sellers. usually proIit-seeking small businesses oIten do not Iollow the rules
with respect to good medical practice. SelI-medication has been observed in 71 oI patients
suIIering Irom inIectious diseases
89
. Clients oIten preIer to buy drugs in private pharmacies due
to the poor administrative procedures in hospitals or the high Iee in private clinics. ThereIore.
prescription-only drugs (mostly antibiotics. and steroids) and sometimes even counterIeit or
low-dose drugs are Ireely sold
17
. Lack oI a desirable attitude towards medicine and oI the
correct knowledge oI good medical practice leads to bad practice at the household level. The
maiority oI antibiotic purchasers i.e. did not buy a Iull course oI treatment
126
.

Use of antimicrobials in animal husbandrv and veterinarv medicine in Jietnam
Food producing animals (pigs. cattle and poultry) receive a large amount oI antibiotics Ior
growth promotion. Feed manuIacturers oIten produce Ieed containing antibiotics as additive
without any description on the label. Antibiotic products which were no longer allowed Ior use
in veterinary medicine in European countries were even Ireely advertised by western veterinary
medicine companies. Healthy animals were given antibiotics Ior prophylaxis in their Ieed or
water. Sick animals received treatment either Irom the owner using medicines sold in veterinary
Chapter 1
15
drug stores or Irom veterinarians. Companion animals are more oIten treated by veterinarians
but the IulIilment oI a course treatment depends on the owners. UnIortunately. there is no
published data on the amount oI antimicrobials used in animal husbandry and veterinary
medicine in Vietnam.

1.3. Antibiotic resistance - a problem in the control of Salmonella infections

Antimicrobial resistance in non-tvphoid salmonellae is a worldwide problem. Human
gastroenteritis due to non-typhoid salmonellae is generally selI-limiting and antimicrobial
therapy is not needed. However. non-typhoid Salmonella inIections in inIants or old age people
or in those with impaired host deIenses can cause systemic inIections which require antibiotic
treatment. The rate oI inIection with non-typhoid Salmonellae in inIants Ior instance is 10-Iold
higher than that in the general population. Young children and inIants are at increased risk oI
developing extra-intestinal inIections
115
. Treatment oI bacteremia caused by Salmonella is
generally undertaken with a single bactericidal drug. However. resistance to diIIerent
antimicrobials in various non-typhoid Salmonella serotypes is currently a challenge. worldwide.
Surveillance data demonstrate a clear increase in the percentage oI Salmonella isolates resistant
to at least one antimicrobial Irom 20-30 in the early 1990s to as high as 70 in some
countries at the turn oI the century. OI particular concern are the Salmonella strains which are
resistant to extended-spectrum cephalosporins and Iluoroquinolones since these antimicrobials
are the drugs oI choice Ior the treatment oI liIe-threatening inIections or inIections caused by
multi-drug resistant Salmonella serotypes
117
.

It is important to keep in mind that the development oI a new antibiotic costs up to
$US300 million and takes about 10 years. Industry and other research institutes have spent large
sums oI money in this respect
54
. However. bacteria have a generation time oI tens oI minutes to
hours. giving them ample opportunity Ior de novo mutation and selection. It took bacteria only
several months or years to become resistant to a new antimicrobial. For example. extended-
spectrum cephalosporins (ESC) were introduced as therapeutics in the 1980s. Less than Iive
years later. new enzymes. which were able to hydrolyze ESC. were reported in Klebsiella
pneumoniae. Escherichia coli and other Enterobacteriaceae
3
. The FDA (Federal Drugs Agency)
issued the Iirst oI several approvals Ior the use oI Iluoroquinolones in poultry Ior the treatment
oI inIections with Escherichia coli and Pasteurella species in 1995. Resistance to
Iluoroquinolone in Campvlobacter species increased Irom 13 in 1997 to up 30 in 1999
115
.

Surveillance on antimicrobial resistance is necessarv. Local. national. regional and global
surveillance oI the development oI antimicrobial resistance is necessary. Local surveillance data
are important Ior the clinician who needs guidance with empirical therapy and Ior the
management oI resistance problems. InIormation on antibiotic resistance at national and
international levels is relevant Ior healthcare-policy makers in order to be able to estimate the
direction in which antibiotic resistance is developing and to implement control measures. This
is especially important since genes encoding Ior antibiotic resistance are no longer conIined to
speciIic area but are able to cross boundaries in the climate oI Ireedom oI travel and trade
7
.

16
Common vehicles oI Salmonella inIection are products oI animal origin and sea-Iood. Food
produced in one country contaminated with Salmonella may cause illness in other countries Iar
away. It is also important to note that people can be inIected with drug-resistant Salmonella
strains by pet animals like horses. dogs. cats. and reptiles
47
. Human-to-human transmission oI
bacteria is common during outbreaks in institutions such as hospitals
59
. Thus. antimicrobial
resistance has to be investigated in speciIic groups that are potential reservoirs oI antibiotic
resistance: hospitalized patients. the community. the veterinary sector. and other sources (water.
vegetables)
40
.

1here are obstacles in antimicrobial resistance surveillance. One oI the main reasons that
makes it sometimes impossible to establish disease control systems in general and antimicrobial
resistance surveillance in particular in some countries is the lack oI Iinancial resources
40
. OIten
these systems are inadequate. For example. the Gross Domestic Product (GDP) per capita/yr in
developing countries ranges Irom 7.374 international dollars (the U.S. dollar converted at
purchasing power parity (PPP) exchange rate
119
) Ior Latin America to 4.327 in Asia and 1.797
in sub-Sahara AIrica
91
. In addition. lack oI awareness oI the importance oI disease control
systems by authorities. lack oI strict policies due to the political corruptive systems. lack oI
competent proIessionals. and lack oI cooperation between laboratories are also obstacles Ior
setting up a regular surveillance system. In countries or regions where surveillance programmes
have been established. several limitations oI these programmes have also been pointed out and
discussed
40
. First. sample bias will have a great impact on the apparent resistance prevalence
since samples Irom patients with persistent inIections can not be considered representative
resistant isolates Ior the community. Second. good-quality susceptibility data is crucial to detect
a trend and new or rare resistance phenotypes but it is oIten questionable since the designation
oI an isolate as either 'resistant. 'susceptible or 'intermediate in a laboratory depends on
how good the susceptibility testing techniques are. Relatively little is known about antibiotic
resistance among isolates oI veterinary and agricultural origin in comparison with human
isolates. Another issue is that the spread oI antibiotic resistance through horizontal transIer is
rarely monitored. The WHO recently identiIied this lack oI inIormation concerning microbial
genetics and ecology in antimicrobial resistant bacteria as a gap in our current knowledge and
hence in need oI Iurther study
40
.

1.4. The study of antimicrobial resistance in Salmonella
Studies on antimicrobial resistance in Salmonella have been perIormed Ior diIIerent purposes
and with diIIerent aims. such as to determine phenotypic resistance to be able to make an
adequate therapeutic decision; to discover new types oI resistance or new mechanisms involved.
to estimate the trends oI resistance development in a certain area. or to study the dissemination
oI antimicrobial resistance by resistant strains. Below the Iollowing three items will be
discussed. Integron-borne resistance and multi-drug resistance in 1.4.1: Basic knowledge
about integrons and associated genetic elements like resistance gene cassettes and Salmonella
Genomic Islands will be addressed. In 1.4.2. Resistance to antimicrobials is the topic in which
general mechanisms oI resistance will be discussed. Focus will be on methods to study and to
determine phenotypic and genotypic resistance to antibiotics in Salmonella used in this thesis.
Chapter 1
17
Other methods which have been described and applied in the literature will also be mentioned
to provide a broader insight in the matter Ior the interested student or investigator. In 1.4.3.
Spread of antimicrobial resistance: the tools available to bacteria which can be used to spread
resistance determinants. like coniugative plasmids or transposons will be discussed.

1.4.1. Integron-borne resistance and multi-drug resistance

Integrons
An integron is a gene capture system Iound on plasmids. on chromosomes and in transposons.
Integrons are genetic elements that contain the genetic determinants Ior the components oI a
site-speciIic recombination system that recognizes and captures mobile gene cassettes.
Integrons themselves are not mobile. An integron is composed oI a gene encoding an integrase
(int) and an attachment site (attI) Ior the gene cassettes. Gene cassettes are not necessarily part
oI the integron (e.g.. the integron In0 lacks gene cassettes and has been observed in clinical
isolates). but they become part oI the integron when integrated
50
. Integrons can be divided into
two maior groups: the resistance integrons and the super-integrons. Resistance integrons carry
less than 10 gene cassettes mostly encoding resistance against antibiotics and disinIectants and
can be located either on the chromosome or on plasmids. Super-integrons contain more than
100 gene cassettes encoding a variety oI proteins with diIIerent enzymatic activities. and are
always chromosomally located
38
. In view oI the topic oI this thesis only the resistance integrons
will be Iurther discussed. Within the resistance integrons three diIIerent classes have been
described. Each class has it own integrase. Integrons belonging to class 1 have identical or
nearly identical sequences in the region containing the intI1 gene and the attI1 site. In contrast.
the sequences oI the region containing the intI2 gene and attI2 site Irom class 2 integrons are
quite diIIerent. as is the corresponding gene oI the class 3 integrons. IntI2 and IntI3 share only
45 and 60 amino acid identity. respectively. with intI1. The attI2 and attI3 sites share little
sequence identity with one another or with attI1. However. each class oI integron appears to be
able to acquire the same gene cassettes
51
. Only class 1 and 2 integrons have been detected in
Salmonella oI which class 1 integrons are the ones most commonly Iound in the various
Salmonella serovars
39
.

The integrases encoded by integrons belong to the tyrosine Iamily oI recombinases.
This Iamily oI enzymes is characterised by the presence oI the conserved amino-acid motiI
RHRY
88
. Three additional conserved motiIs. may be important Ior the secondary structure. have
been identiIied and called patches I-III
16
. The class 1 integron integrase (IntI1) recognizes three
types oI recombination sites: attI1. attC and a non-speciIic site called secondary site. Binding
domains and concensus sequences have been determined Ior all oI them
52
. The attI site is
located in the integron adiacent to the integrase gene and is the preIerred site oI integration Ior
new resistance cassettes. The integrase excises and integrates the gene cassettes Irom and into
the integrons. Integrated cassettes are always oriented in the same orientation. with the 5`-end
oI the coding sequence closest to the integron segment that contains the intI gene and this
segment is designated the 5`-conserved segment (5`-CS). Class 1 integrons (Fig 1) mostly also
contain a quaternary ammonium compound resistance gene. qacE and a sulphonamide

18
resistance gene. sul1. in a conserved region at the 3` end oI the integrated cassettes. This region
is known as the 3`-conserved segment (3`-CS). However. despite it`s name the 3`-CS can be
variable in length and sometimes even absent
39
. Since many integrons possess more than one
antibiotic resistance-conIerring gene cassette and oIten are located on genetic elements that
carry other resistance determinants. integron carrying bacteria are normally multiresistant
36
.

Gene cassettes
A gene cassette is a DNA sequence which can exist by itselI in a circular Iorm. or can be
integrated into an integron. Generally a cassette does not contain promotors that enable
transcription oI the genes in the cassette. Gene cassettes consist oI a coding sequence which is
Iollowed by a palindromic repeat sequence. This sequence is called the 59-base element (59-be)
or attC and is located at the 3`-end oI the gene. The sequence and the size oI the 59-be are
diverse and can vary between 30-130 nucleotides
36
. The attC recombination site is recognized
by the integrase. The integrase cuts the attC site when a circular gene cassette is inserted in an
integron. The gene cassette thus becomes integrated in a linear Iorm. However. the integrase
can also recognize the attC site in integrated gene cassettes and excise them Irom the integron
thereby inducing recirculization oI the gene cassette
8
. At low Irequency. the integrase can also
insert gene cassettes at non-speciIic sites or so-called secondary sites in the integron
101
.
Expression oI genes in a cassette is driven by a promoter located upstream oI the integron
103
.
The promoter region contains two potential promotors called P1 (or P
ANT
) and P2; however. the
latter promoter is Irequently inactive because only 14 nucleotides are present between the -35
and -10 boxes in this promoter instead oI the optimal 17 nucleotides. II more than one cassette
is present. the position oI the cassette in the cassette array inIluences the level oI expression.
Expression is highest and thus the resistance level is highest when the resistance gene cassette is
closest to the promoter
23
.
5` Conserved Segment
P
ant
P P
qacE1 sulI
orI5
int I
P int
59-be
att 1
Gene cassette
an empty integron
59-be
att
P
ant
P P
qacE1 sulI
orI5
int I
P int
59-be
att 1
Gene cassette
an empty integron
59-be
att

Fig 1. Class 1 integron and gene cassette (adapted Irom
50
)
Chapter 1
19
Currently. more than 100 diIIerent gene cassettes are known (Table 3).
Table 3. Characteristics oI class 1 integron gene cassettes
36. 38. 81
.
Gene cassettes Encoded protein
-Lactam resistance
blaP1(bla
PSE-1
). blaP2. blaP3 beta-lactamase
bla
CEF-1.
bla
GES-1.
bla
GES-2.
bla
IBC-1.
bla
IBC-2.
bla
JEB-1.
bla
JIM-3.
oxa1. oxa2a. oxa2b. oxa3.
oxa5. oxa7. oxa9. oxa20. oxa21. oxa-10. oxa-11. oxa-13. oxa-15. oxa-16. oxa-17.
oxa-18. oxa-19. oxa-28. oxa-31. oxa-32
extended-spectrum -
lactamase
bla
IMP-1.
bla
IMP-2.
bla
IMP-3.
bla
IMP-4.
bla
IMP-6.
bla
IMP-7.
bla
JIM-1.
bla
JIM-2.
bla
ESP
carbapenemase
Aminoglycoside resistance
aadA1a . aadA1b. aadA2. aadB. aadA4. aadA5. aadA6. aadA8. aadA10. ant(3)-Ii- aminoglycoside
adenyltransIerase
aac(6)-Ia. aac(6)-Ib. aac(6)-Id. aac(6)-Il. aac(6)-Iq. aacA7. aac(6)-IIa. aac(6)-
IIb. aac(3)-Ia. aac(3)-Ib. aac(3)-JIa. aac29a. aac29b. aacA1b. aac(6)-IId
aminoglycoside
acetyltransIerase
aphA15 aminoglycoside
phosphotransIerase
Trimethoprim resistance
dfrA1. dfrA5. drfA7. dfrA12. dfr13. dfrA14. dfr17. dfrA17. dfrB1. dfrB2. dfrB3 dihydroIolate reductase
Chloramphenicol resistance
catB2. catB3. catB5. catB6. catB8 acetyltransIerase
cmlA . cmlA2. cmlB. cmlA4. cmlA5 eIIlux pump
Rifampicin resistance
arr-2 ADP-ribosylation
Erythromycin resistance
ereA esterase
Quarternary ammonium compounds (disinfectants and antiseptics) resistance
qacE. qacF. qacF. qacG. qacI eIIlux pump
Open Reading Frames (ORFs)
orfA. orfC. orfD. orfE. orfF. orfN. orfO. orfX. orfX. orf"i". orf"ii". orf"iii" unknown

The origin oI resistance genes is still a mystery but there are probably three maior
sources oI resistance genes. (1) Antibiotic producing microbes: they are the prime suspects Ior
the origin oI resistance genes in nature. These microbes have probably developed resistance
genes in order not to become the victims oI their own antibiotics. (2) Another possibility is that
microbes living in close proximity with antibiotic producing Iungi have developed antibiotic
resistance genes in the course oI evolution. (3) The third explanation given in the literature Ior
the appearance oI antibiotic resistance gene is gene duplication and specialization. For example.
genes encoding eIIlux pumps can get duplicated. under the selective pressure oI exposure to
antibiotics. Next the Iunction oI the pump changes through mutation and the eIIlux pump is
now able to pump antibiotics out oI the bacterial cells
27
.

20
The naturally occurring class 1 integrons are named In0. In1. In2. In3. In4 etc. due to
the diIIerences in the inserted gene cassettes
102
. Class 1 integrons with resistance gene cassettes
have been detected worldwide in diIIerent serovars oI Salmonella isolated Irom many diIIerent
reservoirs (Table 4)
1. 2. 4. 10. 18. 24. 25. 28-30. 32. 33. 41-43. 48. 58. 64. 68. 71. 79. 80. 82. 84. 86. 90. 100. 106. 107. 116. 118. 129.
130. 133. 137. 140-142
. In one Salmonella isolate. up to 4 class 1 integrons with 0-4 resistance gene
cassettes can be Iound. The maiority oI the cassettes in these integrons encode resistance to
aminoglycosides. trimethoprim and beta-lactams. Genes encoding resistance to
chloramphenicol can also be present.

Salmonella Genomic Island 1 (SGI1)
The Salmonella Genomic Island 1. a region in the Salmonella genome associated with integrons
and multidrug resistance was Iirst described in Salmonella Typhimurium DT 104 in 2000
13
.
This 43-kilobase long region (SGI1 accession number AF261825) (Fig 2) is located on the
Salmonella chromosome between the thdf and int2 genes. The thdf gene encodes thiopene and
Iuran oxidation. The int2 gene is part oI a retron sequence which today has only been reported
in serovar Typhimurium strains. A retron is deIined as a minimal molecular substructure that
enables transIormation. In other S. enterica serovars SGI1 is located between the thdf and vidY
genes (the vidY gene is related to a putative drug translocase)
11. 12. 29. 30. 68
. All antibiotic
resistance genes are located near the 3`-end oI SGI1 and are part oI a complex class 1 integron
that belongs to the In4 group. The MDR region oI SGI1 consists oI two class 1 integrons.

The Iirst integron carries the aadA2 gene encoding resistance to
streptomycin/spectinomycin and has a sulI gene conIerring sulIamethoxazole resistance. In the
second integron the bla
PSE-1
gene cassette is present. The integrase oI the second integron
probably is not Iunctional because it is a recombinant oI the 5`-end oI a groEL gene and the 3`-
part oI the intI1 gene. The floR gene (also called floSt or cmlA like) encoding resistance to
chloramphenicol and IlorIenicol. the tetracycline resistance gene tet (G) (also known as tetA)
and two open reading Irames (orIs) with unknown Iunction are Ilanked by these two
integrons
13
. Soon aIter the Iirst reports on the presence oI SGI1. variants oI SGI1 (SGI1-A to
SGI1-J) were described in the Salmonella serovars. Agona. Albany. Newport. Meleagridis.
Paratyphi B. Kiambu. InIantis. Derby. Cerro. DusseldorI. and Emek
12. 29. 32. 68
. The diIIerences
among these variants oI SGI1 comprise the presence oI one or two integrons. the characteristics
oI the integrated resistance genes. the presence and genetic organization oI the floR and tet
genes in the MDR region. resulting in the diIIerent resistance phenotypes.

It is important to distinguish between Salmonella Genomic Island 1 (SGI1) and
Salmonella Pathogenicity Island (SPI). The Iormer is associated with antimicrobial resistance in
Salmonella and the latter with Salmonella virulence. At least 5 Salmonella Pathogenicity
Islands have been described. SPI-1 and SPI-2 encode the structural components oI a type III
secretion system. SPI-3 is related to the survival oI the bacteria in a low magnesium
environment (inside the macrophage phagosome). SPI-4 probably also encodes a type III
secretion system and SPI-5 encodes a gene related to Iluid secretion and inIlammatory
exudates
34
.
Chapter 1
21
Table 4. Class 1 integrons and gene cassettes detected in Salmonella serovars worldwide
Size
a
(kb) Gene Serovar Source Country ReI.
0.15 Empty integron 4.5.12.i:- human Spain
48

0.2 Empty integron Worthington pig USA
42

Typhimurium chicken Thailand Vo
b
0.6 sat1 Schwarzengrund imported chilli USA
71

Agona pig USA
140

0.72 dfrJ Virchow human Hungary
86

0.7 dfrA21 Livingstone human Tunisia
10

0.75 aadA Worthington pig USA
42

0.85 aadB Typhimurium pig USA
141

1 aadA Typhimurium pig. cattle. horse.
dog
USA
141

Agona pig. chicken USA
140

Meleagridis poultry. rodent.
reptile. cattle
USA
32

Derby pig USA
42

1 aadA1 Hadar bison USA
71

Virchow human Spain
71

InIantis chicken. human Japan
4

Bareilly human Hungary
86

Agona human Hungary
86

Enteritidis human Spain
116

Newport cattle. swine.
horse. pigeon
USA
100

1 aadA1a Gallinarum chicken Korea
64

1 aadA2 Derby pig Brazil
79

Typhimurium pig Ireland
90

Typhimurium human Hungary
86

Hadar healthy human China
137

Tshiongwe healthy human China
137

Keurmassar na Senegal
41

Newport cattle USA
33

Agona human Argentina
28

aadA21 Dublin cattle USA
33

1 aadA23 Enteritidis human Hungary
86

Bredeney human Hungary
86

Agona pig Brazil
80

1/ 1.2 aadA2/ bla
PSE-1
Typhimurium human France
133

90

Typhimurium Ieed Slovakia
82

Typhimurium river water Japan
1

1/ 1.2 aad/ bla
PSE-1
Typhimurium pig. chicken.
cattle
USA
141

Meleagridis cat. dog. cattle USA
32

1/ 1.2 aadA/dfrA1. orf Typhimurium horse USA
141

1/ 1.2 aadA1/ aadA2 Typhimurium cat USA
141

1/1.2 aadA2/ dfrA1. orfC Derby human Malaysia
68

1/0.8 aadA2/dfrA10 InIantis human Australia
68

1/ 1.2/1.6 aadA/ bla
PSE-1
/ aadB. aadA2 Typhimurium cattle USA
141

1/ 1.2/1.6 aadA/ bla
PSE-1
/ dfrA1. aadA2 Typhimurium cattle Ireland
25

1/1.2/1.7 aadA2/ bla
PSE-1
/ dhfr17. aadA5 Typhimurium human Hungary
86

1/ 1.2/ 1.6 aadA/ bla
PSE-1
/ aadB. aadA2 Kiambu human Australia
68


22
(Cont. Table 4)
Size
a
(kb) Gene Serovar Source Country ReI.
1/1.2/1.45/ 1.6 aadA2/ bla
PSE-1
/-. dhfr1/ aadA2 Typhimurium human Hungary
86

1.2 bla
PSE-1
Derby imported Iish USA
142

90

Typhimurium human Hungary
86

1.2 aadA1 Niakhar cattle USA
118

1.2 dfrA1. orfC Emek sewage eIIluent Australia
68

1.2/ 1.2 dfrA1. orfC/ bla
PSE-1
Cerro human Thailand
68

DussendorI human Malaysia
68

Albany Iish Thailand
29

1.2/ na bla
PSE-1
/ orf513. dhfr10 Agona poultry Belgium
30

1.45 aadB. catB3 Typhimurium human Hungary
86

1.45/2.05 aadB. catB3/ oxa1. aadA1 Typhimurium human Hungary
86

1.5 dhfr1. aadA2 Typhimurium human France
133

1.5 dhfrI. aadA Typhimurium animal Ireland
24

1.5 aadB. catB3 Typhimurium chicken Japan
1

1.6 dhfrI. aadA1 Ohio paprika USA
142

Typhimurium animal. Ieed Slovakia
82

1.6 dhfrI. aadA Virchow human Hungary
86

Hadar human Hungary
86

Saintpaul human Hungary
86

Stanley human Hungary
86

1.6 oxa-2. orfD Typhimurium swine USA
141

1.6 dfrJ. orfD Typhimurium horse USA
141

1.6 aacA5. aadA7 Kentucky spice India
68

1.6 dfrA17. aadA5 Enteritidis water Japan
1

Tshiongwe healthy human China
137

1.6 sat1. aadA1 O8 water Japan
1

1.6 aadB. aadA1b Gallinarum chicken Korea
64

1.8 dfrI. aadA1 InIantis pig Ireland
90

1.7 aac(6)IIc. ereA2 Keurmassar na Senegal
41

1.8 aac(3)Id. aadA7 Newport human France
30

1.9 aadA2.dfrA12 4.5.12. i:- human Spain
48

1.9 bla
OXA-1
. aadA Typhimurium IinIish India
107

2 dhfrXII. orfF. aadA2 Schwarzengrund Iish/squid USA
142

133

Typhimurium pig. chicken.
horse. turkey
USA
141

Choleraesuis human. pig Taiwan
59

Gallinarum chicken Korea
64

2 bla
OXA-30.
aaaA1

133

Typhimurium human. Iood Portugal
2

2 aadA2. bla
OXA-30
Muenchen pig USA
43

2 bla
OXA-53
. aac(6)I30 Agona human Brazil
84

aac(6)Ib. bla
OXA-2.
orfD InIantis human Argentina
28

2.3 sat. smr. aadA1 Virchow human Spain
106

2.2/na aadB. orf513. dfrA23/oxa30.
aadA1
Typhimurium human Italy
130

2.5 aacC4. aadA1. catB3 Enteritidis human Italy
129

2.7 aadA. orf Typhimurium chicken meat USA
18

3.2 dfrA15b. cmlA4. aadA2 Agona pig Brazil
79

a
Size oI cassettes (kb) in integron(s);
b
personal observation; na. not available.
Chapter 1
23

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Y
C
r
y
p
t
i
c

r
e
t
r
o
p
h
a
g
e
C
r
y
p
t
i
c

r
e
t
r
o
p
h
a
g
e
0

5
0
0
0

1
0
0
0
0

2
5
0
0
0

3
0
0
0
0

3
5
0
0
0

4
0
0
0
0

4
2
6
0
0

4
7
7
2

2

C
l
a
s
s

1

i
n
t
e
g
r
o
n
C
l
a
s
s

1

i
n
t
e
g
r
o
n
D
R
-
R
D
R
-
L
M
u
l
t
i

d
r
u
g

r
e
s
i
s
t
a
n
c
e

r
e
g
i
o
n

24

Until now there is no evidence that virulence Iactors are present in SGI1 but
similarities between SGI1 and SPI are noted
11
. Both harbor large segments oI DNA Ilanked by
small direct repeats which have diIIerent GC contents (41) compared to the chromosomal
DNA (51-53) oI S. Typhimurium. and both harbor cryptic and Iunctional genes encoding
mobility Iactors.

1.4.2. Resistance to antimicrobial agents

How do bacteria in general or salmonellae in particular 'learn to deIeat antibiotics? A selective
pressure is imposed on bacterial populations by the use oI antimicrobial agents and antibiotic
resistance is selected Ior. This selective pressure may come Irom diIIerent sources (Fig 3)
135
.
Bacteria can become resistant to antibiotics by mutation oI existing genes or by acquisition oI
resistance genes Irom other bacteria. Molecular biology helps us to understand what the various
resistance mechanisms are. although we still have to learn many oI the details. Antibiotic
resistance oI bacteria can be divided into seven basic groups depending on the mechanisms
involved: (i) the presence oI an enzyme that inactivates the antibiotic; (ii) the presence oI an
alternative enzyme Ior the enzyme that is inhibited by the antibiotic; (iii) mutation in the target
oI the antibiotic. which reduces binding oI the antibiotic to the target; (iv) modiIication oI the
target oI the antibiotic. which reduces binding oI the antibiotic to the target; (v) reduced uptake
oI the antibiotic; (vi) active eIIlux oI the antibiotic and (vii) overexpression oI the target oI the
antibiotic
37
. Since antimicrobial resistance is a process oI bacterial evolution. mechanisms oI
resistance which have not been detected in salmonellae but in other bacteria sometimes will also
be mentioned below.

Antibiotic use
for growth
promotion,
prophyIaxis,
and therapy
Antibiotic
use for
prophyIaxis,
and therapy
Food
animaIs
Non-food
animals
Surface water
Feces
Slurry Waste water Culture plants
Food
Animal
feed
Animal products
HospitaIised
patients
Humans in
community
Feces
Hospital
admission
Antibiotic use
for therapy and
prophyIaxis
Antibiotic use
for therapy and
prophyIaxis
Direct contact
Direct contact
Selective pressures
Main reservoirs
Antibiotic use
for growth
promotion,
prophyIaxis,
and therapy
Antibiotic
use for
prophyIaxis,
and therapy
Food
animaIs
Non-food
animals
Surface water
Feces
Food
Animal
feed
Animal products
HospitaIised
patients
Humans in
community
Feces
Hospital
admission
Antibiotic use
for therapy and
prophyIaxis
Antibiotic use
for therapy and
prophyIaxis
Direct contact
Direct contact
Antibiotic use
for growth
promotion,
prophyIaxis,
and therapy
Antibiotic
use for
prophyIaxis,
and therapy
Food
animaIs
Food
animaIs
Non-food
animals
Non-food
animals
Surface water
Feces
Food
Animal
feed
Animal products
HospitaIised
patients
HospitaIised
patients
Humans in
community
Humans in
community
Feces
Hospital
admission
Antibiotic use
for therapy and
prophyIaxis
Antibiotic use
for therapy and
prophyIaxis
Direct contact
Direct contact
Selective pressures
Main reservoirs

Fig 3. Ecological relationship between antibiotic selective pressure and antibiotic
resistance in bacteria Irom diIIerent reservoirs and route oI transmission. (adapted Irom
135
)

Chapter 1
25
Resistance to betalactams

-lactam antibiotics act on penicillin binding proteins (PBPs) which are involved in cell wall
synthesis. In Salmonella isolates. resistance to -lactams is oIten due to the production oI -
lactamases. which destroy the lactam ring. In contrast. mutations in PBPs resulting in reduced
aIIinity Ior -lactam antibiotics are commonly observed in streptococci. Haemophilus spp.. and
Neisseria spp.
94
. The acquisition oI an alternative penicillin binding protein is another
mechanism by which resistance to -lactam antibiotics can be obtained. e.g. the PBP2a oI
staphylococci is encoded by the mecA gene which is located on the chromosome. The
acquisition oI a mecA gene by staphylococci leads to methicillin-resistant Staphvlococcus
aureus (MRSA). Recently. MRSA strains have been reported to be able to excise the
'staphylococcal cassette chromosome mec (SCCmec)- a mobile genetic element that carries
the mecA gene. This contributes to the horizontal transIer oI antimicrobial resistance oI MRSA
strains
61
. Resistance to beta-lactams is less Irequently caused by a reduced uptake oI the
antibiotic due to changes in the cell wall or active eIIlux. Several classiIication systems Ior -
lactamases have been published
37
. One classiIication scheme is based on their nucleotide
sequence and distinguishes classes A through D. Class A. C. and D enzymes have serine at their
active site. while class B enzymes have Iour zinc atoms in their active site. They are also
diIIerent in terms oI their preIerred substrates
15
.

Resistance to extended-spectrum cephalosporins (ESCs. such as ceIotaxime.
ceIotriaxone. and ceItazidime) are oI special concern since these antimicrobials are commonly
used to treat children with serious inIections or patients with inIections with MDR strains. Till
2004. ESC-resistant salmonellae had been reported in 43 countries
3. 6. 19. 53. 72. 76. 83. 136
.

Resistance to ESC can be caused by the extended-spectrum -lactamases (ESBL) which
belong to the class A mentioned above (Table 5). A commonly used 'working deIinition is that
the ESBLs are -lactamases capable oI conIerring bacterial resistance to penicillins. Iirst-.
second-. and third-generation cephalosporins. and aztreonam (but not to cephamycins and
carbapenems) by hydrolysis oI these antibiotics and that they are inhibited by -lactamase
inhibitors. such as clavulanic acid
93
.

An increased Minimal Inhibitory Concentration (MIC) oI ceItazidime. ceIotaxime.
ceItriaxone or aztreonam is considered as an indication Ior the presence oI ESBLs. True ESBLs
are inhibited by clavulanic acid. ConIirmatory tests Ior ESBL activity are based on the synergy
between clavulanic acid and cephalosporin(s) Ior which the isolate was initially Iound to be
resistant. Several methods can be used to test Ior ESBL activity: (i) the double disk test In this
test both ceItazidime. ceItriaxone and amoxicillin/clavulanic acid disks are used. The test is
cheap and reliable
35
. but the optimal separation oI the two discs varies Irom strain to strain and
some ESBL producers may easily be missed
74
. The ESBL production is inIerred Irom the Iact
that the inhibition zone oI either cephalosporin is expanded by clavulanic acid. (ii)
Combination disc methods In these methods the inhibition zones oI cephalosporin discs are

26
compared to those oI the same cephalosporin plus clavulanate. According to the supplier. either
the diIIerence in zone diameter or the ratio oI diameters is compared. Increases oI > 5 mm
or > 50 in the presence oI clavulanate implies ESBL production
74
. (iii) Etest strips have a
cephalosporin gradient at one end and a cephalosporin plus clavulanate gradient at the other.
ESBL production is inIerred iI the MIC ratio Ior cephalosporin alone: cephalosporin
clavulanate is > 8. These tests are more expensive. Although they are considered accurate and
precise
74
. a large number oI the Etests can not be interpreted when the reading oI one oI the
antibiotics is out oI the range on the test strip (when the isolates are completely resistant to
cephalosporins) and no ratio can be determined
35
.

Table 5. Extended-Spectrum Cephalosporin resistance in non-typhoid Salmonella serovars.
-lactamases Serotypes Country Origin ReI.
1EM- and SHJ-tvpe Extended Spectrum Beta-Lactamases (ESBL)
TEM-3 Kedougou
Typhimurium
France.
Morocco
H
H
83

83

TEM-4 Mbandaka Tunisia H
TEM-20 Paratyphi B.Java The Netherlands
53

TEM-25 Mbandaka France H
83

TEM-27 Othmarschen Spain H
83

TEM-52 Enteritidis
Saintpaul. Agona. Stanley
Blockley. Paratyphi B. Virchow
Thompson. London
Typhimurium
Korea. The Netherlands
Korea
The Netherlands
The Netherlands
The Netherlands
Hungary. England and Wales
H. A
H
A
H
A
H
53

83

53

53

53

83

TEM-63 Isangi The Netherlands H
53

SHV-2 Wien
Corvallis
Vichow
Tunisia
Bulgaria
The Netherlands
H
H
A
83

76

53

SHV-2a Mbandaka
Typhimurium
Tunisia
Poland. Canada. Tunisia
H
H
83

83

SHV-5 SenItenberg
Typhimurium
India
Romania. Austria
H
H
83

83

SHV-12 Enteritidis
35:c:1.2
Concord
Italy
Senegal
The Netherlands
H
H. A
H
83

83

53

Aon-1EM-, non-SHJ ESBL tvpes
CTX-M-1 Albany. Anatum. Tyhimurium.
Choleraesuis. Enteritidis. Wien
InIantis. Isangi. Vichow. Saintpaul.
Kentucky. Mbandaka. SenItenberg
na
na
na
na
na
na
na
na
3

3

3

3

CTX-M-2 Typhimurium
InIantis. Agona
Enteritidis. Oranienburg
Virchow
Anatum
Argentina. England and Wales
Argentina
Argentina
Argentina
The Netherlands
England and Wales
H
H
H
A
H
H
83

83

83

83

53

83

Chapter 1
27
Cont. Table 5
-lactamases Serotypes Country Origin ReI.
CTX-M-3

Typhimurium
Mbandaka.
Oranienburg. Wien
Anatum
Poland.
The Netherlands
Poland
Tunisia. Taiwan
H
H
H
H
83

53

83

83

CTX-M-4 Typhimurium Russia H
83

CTX-M-9 Vichow Spain H
83

CTX-M-14 London
Enteritidis
Panama
Korea
Hongkong
Taiwan
H
H
H
136

19

72

CTX-M-28 Isangi The Netherlands H
53

CTX-type Typhimurium Greece. Hungary. Belarus H
83

OXA type Saintpaul. Enteritidis Korea na
83

PER-1 Typhimurium Turkey H
83

KPC-2 Cubana USA H
83

AmpC -lactamases
CMY-2 SenItenberg.
Typhimurium.
Newport.
Ohio. Bredeney.
Agona. Give. Heidelberg.
Motevideo.
Mikawasima. DuesseldorI
Derby. Wagenia
Worthington
Algeria. Taiwan
Canada.USA. Taiwan
USA. France
Canada
USA
USA
Spain
Taiwan
England and Wales
H
H. A
H. A. F
F
H
H
H
H
H
72

83

83

83

83

83

83

72

6

CMY-2 like Typhimurium Taiwan H
72

CMY-4 Wien
SenItenberg
Tunisia
England and Wales
H
H
83

6

CMY-7 Typhimurium Australia H
83

CMY-type Enteritidis
Typhimurium. Heidelberg
Italy
Romania
H
H
83

83

DHA-1 Enteritidis
SenItenberg
Saudi Arabia
England and Wales
H
H
83

6

ACC-1 Livingstone. Mbandaka
Bareilly. Braenderup. InIantis
Tunisia
The Netherlands
H
A
83

53

H. humans; A. animals; F. Iood. na. not available (data available Irom 1988 to 2005).

In 1985. approximately 20 diIIerent -lactamases had been distinguished based on
their overall electric charge. Isoelectric Iocusing (IEF) was used to examine -lactamases on
thin polyacrylamide gels employing ampholines (multicharged compounds that Iorm a pH
gradient). As a consequence. -lactamases are diIIerentiated on the basis oI their isoelectric
points (pI. the pH at which the protein bears no net charge and is electrophoretically
immobile)
94
. Today. more than 500 diIIerent -lactamases have been characterized and it is
more than likely that additional enzymes will continue to be identiIied. ThereIore. IEF is no
longer suIIicient Ior diIIerentiation oI -lactamases
94
. AmpliIication oI the gene by PCR
Iollowed by nucleotide sequencing can be used to determine a speciIic Iamily oI ESBLs
compared to their progenitors. In laboratories where systematic sequencing oI the target genes
is not available. restriction Iragment length polymorphism (RFLP) analysis oI a relevant

28
Iragment oI the gene generated by PCR (RFLP-PCR) or PCR-single-strand conIormation
polymorphism (PCR-SSCP) can indicate the presence oI a TEM or SHV type ESBL
37
.

The most widely occurring ESBLs are derived by point mutation Irom the TEM
(named aIter Temoneira. a Greek patient) or SHV (reIerring to sulIhydryl variable)
penicillinases Ior which the genes are located on plasmids
37. 93
. The vast maiority oI TEM and
SHV-type penicillinases are reIerred to as ceItazidimases. based on the Iact that a higher level
oI resistance to ceItazidime than to ceIotaxime is obtained
3
. CTX-M type -lactamases have a
very potent hydrolytic activity against ceIotaxime. Organisms producing CTX-M type
-lactamases have MICs Ior ceIotaxime in the Iully resistant range (~64 g/ml). whereas MICs
Ior ceItazidime are usually in the apparently susceptible range (2-8 g/ml). Consequently. the
ceIotaximases (which show a higher level oI resistance to ceIotaxime than to ceItazidime) are
mostly CTX-M-type enzymes
3
. The OXA-type -lactamases are so named aIter their oxacillin-
hydrolysing ability. The OXA-type ESBL is however rarely encountered in Salmonella. The
-lactamases belonging to one oI the Iamilies mentioned above diIIer in only a Iew amino acids
compared to their ancestors. but these changes remarkably inIluence the spectrum oI activity oI
these enzymes
37
. For example. TEM-52. the ESBL commonly Iound in third-generation
cephalosporin-resistant Salmonella isolates. is diIIerent Irom the parent TEM-1 (causing
resistance against ampicillin at a greater rate than against carbenicillin. oxacillin and
cephalothin) at amino acids Glu104Lys. Met182Thr and Gly238Ser
112
. The total number
oI ESBLs now characterized exceeds 200. Details on the nomenclature oI ESBLs can be Iound
on the website hosted by Jacoby and Bush (http://www.lahey.org/study)
93
.

Resistance to ESC can also be caused by plasmid-mediated AmpC -lactamases. Like
ESBLs. plasmid-mediated AmpC -lactamases have a broad substrate spectrum that includes
penicillins. cephalosporins (apart Irom zwitterionic cephalosporins) and monobactams. In
contrast to ESBLs. AmpC -lactamases even hydrolyze cephamycins. They are inhibited by
EDTA but not by clavulanic acid
93
. In many genera AmpC -lactamases (class C) are
chromosomally located. but an increasing number oI plasmid-mediated AmpC -lactamases is
being reported. AmpC -lactamases are usually inducible via a system that involves the ampD.
ampG. ampR genes encoding -lactam resistance and intermediates in the peptidoglycan
recycling.

The disk test used to detect production oI plasmid-mediated AmpC -lactamases in
isolates that lack a chromosomally encoded AmpC -lactamase was decribed by Black et al
(2005). This test is based on the use oI Tris EDTA to permeabilize a bacterial cell and release
-lactamases into the environment
9
(see Chapter 7). Perez-Perez et al. described a multiplex
PCR to detect and diIIerentiate between the diIIerent types and Iamilies oI plasmid-mediated
AmpC -lactamases based on the sequences oI 29 plasmid-mediated ampC genes
95
. Direct
sequence analysis will also allow determination oI the type oI a plasmid-mediated AmpC -
lactamases.

Chapter 1
29
Plasmid-mediated class C -lactamases have been named with the inconsistency
typical oI -lactamase nomenclature aIter to the resistance to cephamycins (CMY). cefoxitin
(FOX). and monobactam (MOX) or latamoxeI (LAT) that they induce. aIter the type oI -
lactamase. such as AmpC type (ACT) or Ambler class C (ACC). aIter the site oI discovery.
such as the Miriam Hospital in Providence. I.R. (MIR-1) or Dhahran hospital in Saudi Arabia
(DHA). and aIter the name oI the patient who provided the original sample. Bilal (BIL-1). More
recently. -lactamases which are able to hydrolyze carbapenems have been described. They
belong to the Ambler class B. MBL (metallo -lactamase) or class A -lactamases. An example
oI such a -lactamases is KPC (Klebsiella pneumonia carbapenemase). Reanalysis oI several
plasmid-mediated AmpC genes revealed that the amino acid sequences oI CMY-2. BIL-1. and
LAT-2 are identical. as were LAT-1 and LAT-4 and LAT-3 and CMY-6
96
. CMY-2. the TEM-
and SHV- types as well as the CTX-M-1 group are most prevalent among Salmonella isolates
Irom human and animal origin whereas DHA and ACC-types have recently emerged.

In many bacteria such as Enterobacter cloacae. Citrobacter freundii. and
Pseudomonas aeruginosa. overproduction oI the intrinsic chromosomal AmpC -lactamases
provides resistance to both oxyimino- and-7--methoxy cephalosporines and monobactams
96
.
Unlike most other clinically signiIicant Enterobacteriaceae. Salmonella is considered to be
ampC minus due to the absence oI a structural ampC gene. ThereIore. AmpC -lactamases in
Salmonella are essentially produced Irom acquired genes. These genes encode potent -
lactamases known as the plasmid-mediated. imported. transmissible. Ioreign. or mobile AmpC
-lactamases
83
.

Resistance to aminoglycosides

Aminoglycosides bind to the bacterial ribosome and interIere with protein synthesis. Resistance
to these antimicrobials is widespread and can be caused by modiIying enzymes. changes in cell
permeability by eIIlux systems and changes in the cellular target by rRNA mutations
37. 114
. High
level aminoglycoside resistance most oIten results Irom acquisition oI genes that encode
modiIying enzymes and these genes are oIten plasmid-borne
114
. More than 50 aminoglycoside-
modiIying enzymes have already been described. Most oI the genes encoding them are
associated with Gram-negative bacteria
37
. Depending on the type oI modiIication they bring
about. these enzymes are classiIied as aminoglycoside acetyltransIerases (aac). which use
acetyl Co-A to modiIy speciIic amino groups on the aminoglycoside. aminoglycoside
adenylyltransIerases (aad) (also named aminoglycoside nucleotidyltransIerases. ant). which
use ATP to adenylylate speciIic hydroxyl groups. and aminoglycoside phosphotransIerases
(aph). which use ATP to phosphorylate speciIic hydroxyl groups
37. 114
. The gene names Iollow
this nomenclature. but a Iurther subclassiIication is made when diIIerent genes encode enzymes
that have the same substrate speciIicity
37
. The aac genes. which mediate resistance to
gentamicin. are located on plasmids in S. Typhimurium and on the chromosomal DNA oI
S. HaiIa. S. Newport and S. Kentucky. In the latter two the acc genes are part oI the SGI1
variants present in these serovars. OI the more than 20 aad (also called ant) genes described in

30
various serovars oI Salmonella. the aadA genes mediate resistance to streptomycin and
spectinomycin whereas the aadB genes conIer resistance to gentamicin. kanamycin and
tobramycin. The aph genes responsible Ior resistance to diIIerent aminoglycosides have been
detected in diIIerent serovars oI Salmonella (Choleraesuis. Typhimurium. Typhi. Enteritidis)
81
.
Recently. the gene armA coding Ior a 16S rRNA methylase which conIers high-level resistance
to all clinically available aminoglycosides except streptomycin by chemically modiIying the
cellular target site has been described in S. Enteritidis
81
.

Genotyping methods such as DNA hybridization or the use oI 5 sets oI primers in a
single PCR reaction have been employed to detect the presence oI as many as 4 aminoglycoside
modiIying genes. Three multiplex PCR assays covering a total oI 7 aminoglycoside resistance
genes can also be used to detect aminoglycoside modiIying genes
37
. Genes encoding
aminoglycoside modiIying enzymes are oIten gene cassettes associated with integrons.
Nucleotide sequencing oI the gene cassettes will allow determination oI the type oI
aminoglycoside modiIying enzymes present in particular cases. Due to at least two alternatively
used diIIerent nomenclatures Ior genes encoding the same aminoglycosides in databases. gene
designation may be conIusing. Identical genes have occasionally received diIIerent
designations. while structurally diIIerent genes have been given the same designation.
ThereIore a thorough revision oI the current nomenclature is urgently needed
81
.

Resistance to fluoroquinolones

Fluoroquinolone antibiotics exert their antibacterial eIIect by inhibition oI certain bacterial
topoisomerase enzymes. namely DNA gyrase (bacterial topoisomerase II) and topoisomerase
IV. These essential bacterial enzymes alter the topology oI dsDNA within cells. In most
bacteria. the chromosome exists as a single circle oI dsDNA which is maintained in a highly
negatively supercoiled conIormation. This energetically activated Iorm is required Ior cellular
processes like replication and transcription. Deoxyribonucleic acid gyrase and topoisomerase
IV are heterotetrameric proteins composed oI two subunits. designated A and B. In Salmonella.
the genes encoding the A and the B subunits are reIerred to as gvrA and gvrB (DNA gyrase) or
parC and parE (DNA topoisomerase IV)
112
. DNA gyrase is the only enzyme responsible Ior the
negative supercoiling oI chromosomal DNA. and accomplishes this by nicking the DNA
strands. supercoiling the DNA. and resealing the nick
14
. DNA topoisomerase IV is
predominantly responsible Ior separation oI daughter DNA strands during cell division. In
Gram-negative bacteria. DNA gyrase is the primary target and DNA topoisomerase IV is the
secondary target Ior quinolones. whereas in some Gram-positive organisms DNA
topoisomerase IV is the primary target
112
. Fluoroquinolones bind to DNA and this complex then
binds to DNA gyrase. This results in the DNA gyrase being 'poisoned and permanently
attached to the bacterial chromosome via a covalent bond. ThereIore. Iluoroquinolones stabilize
the breaks in DNA and inhibit DNA synthesis resulting in an induction oI the bacterial SOS
response. and bacteria die rapidly
14
.

Chapter 1
31
Alteration oI the target enzymes appears to be the most important Iactor in the
acquisition oI resistance to quinolones. The maiority oI the mutations described to date have
been Iound in the N-terminus oI the GyrA and GyrB proteins in a region called the Quinolone
Resistance-Determining Region (QRDR). located between amino acids Ala67-Gln106 in gvrA
and Asp426-Lys447 in gvrB
57
. The Irequency oI gvrB mutations. compared to those in gvrA. is
relatively low in most species. The two amino acids Ser83 and Asp87 in GyrA. are the most
commonly substituted amino acid residues in resistant isolates. In almost all instances. amino
acid substitutions within the QRDR region involve the replacement oI a hydroxyl group with a
bulky hydrophobic residue. This suggests that mutations in gvrA induce changes in the
conIormation oI the binding site and/or changes in the charge oI the protein that may be
important Ior quinolone-DNA gyrase interaction
37
. While all mutations in the gvrA gene oI
E. coli have been within the QRDR. in salmonellae a number oI strains have been identiIied
with a mutation at codon Ala119 which is located outside the originally deIined QRDR
57
.
Topoisomerase IV mutations have been characterized in several bacteria. including Salmonella
isolates. However. mutations in parC and parE oI salmonellae occur not as Irequent as in
E. coli and it has been suggested that they do not play an important role in quinolone resistance
in Salmonella. It is also possible that they are only required to achieve high-level resistance
57
.

It is important to note that in Campvlobacter species a high level oI Iluoroquinolone
resistance can be reached by a single point mutation in gvrA in the presence oI a constitutively
expressed multidrug eIIlux pump (CmeABC). In contrast. the acquisition oI Iluoroquinolone
resistance in Salmonella and also in other Gram-negative bacteria requires the stepwise
accumulation oI gvrA mutations and overexpression oI eIIlux pumps
138
.

Since mutations in the topoisomerase genes cannot completely account Ior the
resistance phenotype oI strains. which harbor identical mutations but have a diIIerent MIC Ior
ciproIloxacin. an additional mechanism Ior resistance has been suggested. A decreased
Iluoroquinolone uptake might also be the result oI a decrease in bacterial permeability or
overexpression oI eIIlux pumps
57
. A decrease oI quinolone permeability as a result oI a
decreased expression oI the outer membrane porin OmpF has been demonstrated
14
in Gram-
negative bacteria but has not been proven in Gram-positive bacteria
37
. Quinolone resistance
resulting Irom the multiple antimicrobial resistance (MAR) phenotype is oIten associated with
cross-resistance to many other structurally unrelated antimicrobials such as beta-lactams.
puromycin. tetracyclines. chloramphenicol. and resistance to oxidative stress agents and
desinIectants. Less is known about the Mar phenotype in salmonellae. The role oI a reduced
OmpF expression in the Mar locus oI salmonellae is also not clear
57
. Quinolone resistance can
be mediated by an eIIlux system. It has been suggested that the acrAB eIIlux pump may be the
primary mechanism oI ciproIloxacin resistance in S. Typhimurium isolates
45
. Overexpression oI
MarA and AcrB in S. Choleraesuis also induces the Mar phenotype. Active eIIlux oI
antimicrobials and multiple mutations in gvrA and parC contribute to high level ciproIloxacin
resistance in this serovar
22. 57
.


32
Resistance to quinolones can be also caused by the presence oI a gene responsible Ior
plasmid-mediated quinolone resistance (PMQR). qnr
120
. The Qnr Iamily includes resistance
determinants. like QnrA. QnrB and QnrS
87
. The proteins provide resistance to quinolones like
nalidixic acid but not to Iluoroquinolones. according to break points oI the NCCLS. The qnrA
gene is associated with a sul1-type class 1 integron (see integron section) iust downstream oI a
conserved region element (CR1. 3`-CS-qacE and sulI) and immediately upstream Irom a
second qacE1 and sul1 gene. and Ilanked at one side by OrI513. which was previously
identiIied in In6 and In7
75
. The qnr gene has been detected with a low Irequency in Shigella.
Klebsiella. Salmonella and E. coli. In addition to carrying qnr. the 56-kb plasmid (pMG252)
originating Irom Klebsiella pneumoniae also contained other determinants conIerring resistance
to third-generation cephalosporins. chloramphenicol. kanamycin. gentamicin. streptomycin.
tobramycin. sulIaIurazole. trimethoprim and mercuric chloride. This plasmid has a broad host
range and aIter its transIer Irom E. coli by coniugation. quinolone resistance was expressed in
Pseudomonas aeruginosa. Citrobacter freundii. and S. Typhimurium. In a recent report. a
human S. Enteritidis isolate cultured in Hongkong carried a qnr gene which was linked to
bla
CTX-M-14
19
. A human Enterobacter cloacae strain Irom a Dutch outbreak contained qnrA1
associated with the bla
CTX-M-9
extended-spectrum -lactamase
92
. Although at present qnr-
positive strains occur relatively seldom. widespread horizontal transIer oI qnr between strains
and co-selection oI quinolone resistance upon exposure to other antimicrobials may happen in
the Iuture iI proper use oI antimicrobials in both human and veterinary medicine is not
implemented
57
. Several variants oI qnr-containing plasmids associated with class 1 integrons
Irom diIIerent species oI Enterobacteriaceae are depicted in Fig 4.

Various methods to detect point-mutations such as sequence-speciIic oligonucleotide
probe hybridization. radioisotopic or nonradioisotopic single-strand conIormation
polymorphism (SSCP). mismatch ampliIication mutation assay (MAMA) PCR. combining
allele-speciIic PCR and RFLP (AS-PCR-RFLP) and/or sequencing oI the target genes have
been reported
37
. Real-time PCR can also be used to assess the mutations in gvrA in
S. Typhimurium isolates
131
. Giraud et al.. developed the AS-PCR-RFLP. a simple and rapid
assay. Ior screening point mutations responsible Ior all amino acid changes observed in the
S. Typhimurium gvrA gene at codons 81. 83 and 87
44
. The nucleotide substitutions can then be
determined by sequencing the amplicon oI the target gene (see Chapter 3 and 6). Mutations
detected in the DNA topoisomerase genes oI Salmonella isolates are shown in Table 6.

Salmonella isolates with a MIC oI > 2mg/L Ior ciproIloxacin also showed a resistant
phenotype to cyclohexane. Cyclohexane resistance in Salmonella can be induced via a Mar-
dependent pathway and is associated with an active eIIlux pump. ThereIore. the cyclohexane
resistance assay
5
can be used as a screening method Ior these resistance mechanisms in
Salmonella isolates
73
. II it is suspected that changes in permeability are the cause or part oI the
cause oI quinolone resistance in Gram-negative bacteria. outer membrane proteins can be
extracted. electrophorized. visualized and compared with known controls to detect eventual
changes
14
.
Chapter 1
33

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34
Table 6. Mutations detected in the DNA gvrase and topoisomerase IJ genes oI salmonellae
57
.
gvrA gvrB parC parE
Codon Sub. Codon Sub. Codon Sub. Codon Sub.
Ala67 Pro Tyr420 Cys Tyr57 Ser Glu453 Gly
Asp72 Gly Arg437 Leu Thr66 Ile Ser458 Pro
Val73 Ile Ser464 Tyr/Phe Gly78 Asp His461 Tyr
Gly81 Cys/Ser/His/Asp Ser80 Arg/Ile Ala498 Thr
Asp82 Gly/Asn Glu84 Lys/Gly Val521 Gly
Ser83 Tyr/Phe/Ala
Asp87 Asn/Gly/Tyr/Lys
Leu98 Val
Ala119 Ser/Glu/Val
Ala131 Gly
Glu139 Ala
Asp144 Asp (silent mutation)
Sub.. substitution.

Resistance to tetracyclines
Tetracyclines probably penetrate bacterial cells by passive diIIusion. Tetracycline acts by
binding to the 30S ribosomal subunit. resulting in the inhibition oI protein synthesis. To date.
more than 30 diIIerent tetracycline resistance (Tet) and three oxytetracycline resistance (Otr)
determinants have been characterized
105
. Most oI the resistance genes code Ior proteins
involved in one oI the two most important mechanisms oI tetracycline resistance. namely eIIlux
pumps or ribosomal protection. Enzymatic inactivation oI the antibiotic (encoded by the tetX
gene) is not a clinical important mechanism
112
.

EIIlux pumps are mediated by energy-dependent antiporter systems and exchange a
proton Ior a tetracycline-cation complex. Most eIIlux pump proteins (except TetB in Gram-
negative bacteria) conIer resistance to tetracycline but not to minocycline or glycycycline.
Tetracycline eIIlux proteins have amino acid sequences and a protein structure similar to other
eIIlux proteins involved i.e. in multiresistance to chroramphenicol. quinolones. and quarternary
ammonium ions. Multidrug eIIlux pumps transport their substrates straight out oI the cell into
the surrounding medium
20
.

EIIlux determinants in Gram-negative bacteria (TetA-E. TetG-H) share a common
genetic organization. which is diIIerent Irom that in Gram-positive bacteria. The Gram-negative
eIIlux genes are widely distributed and normally located on large plasmids. most oI which are
coniugative. These plasmids oIten carry other antibiotic resistance genes. heavy metal
resistance genes and/or virulence Iactors
20
. Thus selection Ior any oI these Iactors selects Ior the
plasmid and contributes to the dramatic increase in the number multiple-drug resistant bacteria.
Tetracycline resistance can result Irom the production oI a protein that interacts with ribosomes.
These proteins protect the ribosome Irom the action oI the antibiotic and conIer a broad
spectrum oI resistance to tetracyclines (tetracycline. doxycycline and minocycline)
20
.
Tetracycline resistance determinants are listed in Table 7. The number oI tet genes has reached
the end oI the Roman alphabet. and numbers are being assigned. tet(30) is the beginning oI this
number designation.
Chapter 1
35
Table 7. Tetracycline-resistance determinants
20. 104. 105. 112
.
Name Location Mechanism Distribution
TetA P EIIlux Gram-negative
TetB P. C(rare) EIIlux Gram-negative
TetC P EIIlux Gram-negative
TetD P EIIlux Gram-negative
TetE P EIIlux Gram-negative
TetG P EIIlux Gram-negative
TetH P EIIlux Gram-negative
Tet I - EIIlux (likely) -
TetJ - EIIlux -
TetK P. C EIIlux Gram-positive
TetL P. C(rare) EIIlux Gram-positive
TetM C. P(rare) Ribosomal Gram-positive. Gram-negative
TetO P. C Ribosomal Gram-positive. Gram-negative
TetP P Ribosomal Gram-positive
TetQ C Ribosomal Gram-positive. Gram-negative
TetS P Ribosomal Gram-positive
TetX P Enzymatic Gram-negative
TetU - unknown Staphylococcus
TetV - EIIlux -
TetT - Ribosomal -
TetY - EIIlux -
TetZ - EIIlux -
TetW - Ribosomal Gram-positive
Tet30 - EIIlux Gram-negative
Tet31 - EIIlux Gram-negative
OtrA P Ribosomal Gram-positive
OtrB P EIIlux -
OtrC P unknown -
P. plasmid; C. chromosome; -. not available

Salmonella isolates are oIten Iound to carry tet(A)- tet(B)- tet(C)- tet(D)- tet(E) which
are associated with non-coniugative plasmids
81. 112
. Recently. tet(G) and tet(L) eIIlux genes have
also been described in Salmonella
11. 105
. In integrons. the tet genes have not yet been Iound but
they are oIten associated with plasmids and transposons. This can partly explain the spread oI
tet genes among a wider bacterial range
105
. In several Salmonella serovars (Typhimurium.
Agona. Albany. Paratyphi B. Newport. Derby. Cerro. DusseldorI and Emek). the tet(G) gene
has been Iound in the resistance gene hotspot oI Salmonella Genomic Island 1 and its variants
12.
29. 30. 39. 68
.

DNA-DNA hybridization with the structural genes as probes is still the standard
method used to distinguish between tet genes
69
. A new gene is identiIied by its inability to
hybridize under stringent conditions with probes Ior any oI the known tet genes; this criterion
indicates less than 80 sequence identity. II this is demonstrated Ior a new gene. a letter or a
number designation is given in accordance with the nomenclature standards proposed by
Levy
69. 105
. PCR assays have also been developed to detect tet(K) and tet(L) or tet(M) and
tet(Q)
37
.

36
Resistance to sulfonamides and trimethoprim

SulIonamides are analogs oI p-aminobenzoic acid. They competitively inhibit the
dihydropteroate synthetase (DHPS). Trimethoprim is an analog oI dihydroIolic acid. It interacts
with dihydroIolate reductase (DHFR). These two enzymes are part oI the Iolate biosynthetic
pathway required Ior thymine production and bacterial cell growth. The Iirst sulIonamide
compounds were used in 1932. whereas trimethoprim was Iirst used in 1962
60
. Since 1968. the
combination oI the two drugs (trimethoprim and sulIamethoxazole) has been used because oI its
low cost and the Iact that it reduces selection Ior antibiotic resistance to either drug
104
.

Nevertheless. resistance to sulIonamide and trimethoprim is now widespread.
Chromosomally encoded sulIonamide resistance has been described. Alterations in DHPS could
lead to a low aIIinity Ior sulIonamides
112
. In Salmonella. sul1. sul2 and sul3 coding Ior diIIerent
types oI DHPS have been identiIied. The sul1 gene will be discussed in relation to integrons.
The sul2 gene is oIten linked with the streptomycin resistance genes strA-strB Iound on
plasmids in various Salmonella serovars. In Salmonella. the sul3 gene has been detected on
large multi-resistance plasmids
81
.

Resistance to trimethoprim can be intrinsic or acquired. Intrinsic resistance is due to
permeability barriers. Iolate auxotrophy (Lactobacillus spp. and Enterococcus spp. i.e. are able
to use preIormed exogenous Iolates and exhibit a reduced susceptibility to sulIonamides and
trimethoprim) or DHFR enzymes with low aIIinity Ior the drug. Chromosomal acquired
resistance to trimethoprim may be due to: overproduction oI the DHFR; mutations in the DHFR
structural gene folA or mutations that inactivate thymidylate synthetase. an enzyme that
converts deoxyuridylate to thymidylate
112
. High-level resistance to trimethoprim in
Enterobacteriaceae is almost always caused by the acquisition oI DNA that encodes a
trimethoprim-resistant DHFR with an alternative active site
37. 104
. Plasmid-borne. transIerable or
cassette-associated DHFRs are present in Gram-negative bacteria. So Iar. more than 30 diIIerent
trimethoprim resistance mediating dihydrofolate reductase (dfr) genes have been identiIied.
They are subdivided into two maior classes (1 and 2). (nowadays reIerred to as A and B or dfrA
and dfrB) on the basis oI their structure. ThereIore. dihydroIolate reductase genes can have
diIIerent designations. For example. class A type 1 dihydroIolate reductase still can be Iound in
the GenBank database as dhfrI. dfrI or dfrA1 (GenBank accession number AF382145.
AJ419168 and DQ23813. respectively). While dfrB genes have not yet been identiIied in
Salmonella. 13 oI 20 diIIerent known dfrA genes. mostly cassette-borne genes located in
integrons. have been identiIied by sequence determination in various Salmonella enterica
serovars
36. 60. 81
. The cassette-borne TMP resistance genes (dfrA1. dfrA5. dfrA7. dfrA12. dfrA14.
dfrB1. dfrB2. dfrB3) seem to be more widespread than those that are not cassette-borne (dfrA3.
dfrA8. dfrA9. and dfrA10)
60
.

Chapter 1
37
Resistance to chloramphenicol

Chloramphenicol binds to the 50S ribosomal subunit and inhibits the peptidyltransIerase step in
protein synthesis. Resistance to chloramphenicol is generally due to inactivation oI the
antibiotic by an enzyme. chloramphenicol acetyltransIerase (CAT). which adds an acetyl group
to hydroxyl groups oI chloramphenicol
109
. CATs are able to inactivate chloramphenicol as well
as thiamphenicol and azidamphenicol. The cat genes oI Gram-negative and Gram-positive
bacteria show little homology. and variants oI diIIerent enzymes have been described. Type A
and type B CATs diIIer Irom each other in their structure on the basis oI their amino acid
sequences
113
. The catA1 gene has been Iound in serovars Typhimurium. Agona. Derby. Dublin.
Newport. Virchow and Pullorum. The catA2 gene has been detected on a multiresistance
plasmid in S. Choleraesuis. S. Enteritidis. S. Typhimurium and S. Derby. The catB2. catB3 and
catB8 genes are mainly gene cassettes oI class 1 integrons on plasmids in S. Typhimurium and
S. Enteritidis
81. 113
.

In Gram-negative bacteria. resistance to chloramphenicol can also be due to
chromosomal mutations that result in a decreased outer membrane permeability. Expression oI
the ClmA protein (encoded by the clmA gene) appears to result in the reduced production oI the
outer membrane porins OmpA and OmpC and in a decreased uptake oI chloramphenicol. The
clmA genes have been identiIied in plasmid-borne class 1 integrons oI S. Typhimurium. S.
Muenchen. and S. Agona
81
. The floR gene (also called clmA-like gene) encoding cross
resistance to chloramphenicol and florIenicol via an eIIlux pump was Iound in the resistance
gene cluster oI Salmonella Genomic Island 1
11
. A transposable element is assumed to be
involved in the spread oI floR since the floR genes detected on plasmids in E. coli and K.
pneumoniae are Ilanked by transposase-like reading Irames
113
. The absence oI the floR gene in
some SGI1 variants (SGI1-B. -C. -D. -G) implies the excision oI this gene Irom these genomic
islands. Hybridization or PCR assays using degenerated primers have been used to detect cat
genes. However. the study oI chloramphenicol resistance on a molecular level has received little
attention so Iar. probably because chloramphenicol is seldom used Ior the treatment oI severe
inIections
37
. More than one chloramphenicol resistance gene in the same Salmonella isolates
has also been reported i.e. catAfloR in S. Typhimurium var Copenhagen; catA2cmlA in S.
Choleraesuis or catA1catA2 in S. Derby or S. Typhimurium
81
.

1.4.3. Spread of antibiotic resistance

Bacteria can gain resistance to antibiotics. but at a cost. Resistance genes or gene mutations
may increase the time needed Ior DNA replication and may drain energy that could otherwise
be used Ior growth and reproduction. In the presence oI antibiotics. resistant bacteria have an
advantage in growth compared to susceptible bacteria. This is an acceptable price to pay. But in
the absence oI antibiotics. resistant bacteria have to meet a Iitness cost. Fitness is a measure oI
how well a bacterial strain can inIect a host. persist and proliIerate. and get transmitted to a new
host. Fitness cost is deIined as a reduced growth and persistence oI resistant bacteria in the host
and environment or an increased clearance Irom the inIected host and a reduced transmission

38
between hosts. and is typically measured via laboratory experiments in culture media or animal
models
139
. For instance. a single mutation in DNA gyrase in Salmonella is enough to cause
nalidixic acid resistance but not suIIicient to cause high-level Iluoroquinolone resistance
57
.
However. this strain with a single mutation may become Iitter iI it gains other mutations. so-
called compensating mutations. An easier and quicker way Ior a bacterium to become resistant
to an antibiotic is to acquire a resistance gene or genes Irom other bacteria although this route
also entails a biological Iitness cost. Bacteria can acquire new genes by transduction.
transIormation or coniugation (Fig 5)
70
. In bacteriophage transduction. the bacteria need the
right phage receptor on their surIace. a trait that is usually restricted to a closely related group oI
bacteria. In natural transIormation. Iragments oI DNA are taken up Irom the external
environment by a specialized set oI proteins. In both cases. the DNA must integrate into the
genome by homologous recombination. Thus. bacteria must be close enough to each other
genetically Ior homologous recombination to be possible. These types oI transIer tend to occur
mainly between members oI the same species or between members oI very closely related
species. Coniugation has no such limitation. This process is the direct cell-to-cell transIer oI
DNA through a protein complex that connects the membranes oI two bacteria
109
. Coniugation is
considered the most common mode oI acquiring resistance genes. The spread oI resistance
genes. especially between members oI diIIerent species. is a very serious threat. There are two
types oI coniugative elements: coniugative plasmids and coniugative transposons
109
. ThereIore.
coniugative plasmids. transposons and integrons represent important genetic elements
contributing to the spread oI antimicrobial resistance.

Fig 5. TransIer oI resistance genes. (a) coniugation. (b) transduction. (c)
transIormation. (kindly provided by Leverstein-van Hall
67
)

Chapter 1
39
Dissemination of resistance genes via conjugative plasmids

Not all plasmids are capable oI selI-transIer or are coniugative plasmids. Plasmids that transIer
themselves by coniugation must carry a number oI genes encoding proteins needed Ior the
coniugation process (tra genes). Thus. selI-transmissible plasmids are usually at least 25 kb in
size. It also important to note that some plasmids which can not transIer themselves can still be
mobilizable by selI-transmissible plasmids. These are called mobilizable plasmids and can be
much smaller than selI-transmissible plasmids since they need only one or two genes (mob
genes) Ior transIer Irom one bacterial cell to another
109
.

Although most Salmonella enterica serovars do not possess plasmids. the serovars
which are Irequently associated with inIections in humans and Iarm animals like serovars
Enteritidis. Typhimurium. Dublin. Choleraesuis. Gallinarum. Pullorum and Abortus-ovis oIten
carry plasmids oI 2 kb to more than 200 kb in size. Plasmids can be classiIied into 3 groups
with regard to their Iunctions: the serovar speciIic virulence plasmids. oIten 50-100 kb in size;
the high molecular weight plasmids related to antibiotic resistance; and the low molecular
weight plasmids with a size lower than 20 kb with mostly unknown Iunctions
108
. Guerra et al.
described an IncFII plasmid oI about 140 kb carrying virulence determinants (spvA. spvB. spvC
and rck genes encoding Salmonella plasmid virulence proteins and resistance to complement
killing protein. respectively) and antibiotic resistance genes (oxa1/aadA1. qacE. sul1. catA1
and tetB genes) in a Salmonella Typhimurium strain
49
. Chu et al. also reported that some S.
Choleraesuis isolates harbour virulence plasmids oI 136 kb or 140 kb containing sul1 and
bla
TEM-1
genes
21
. Recently. Daly et al.
26
reported the characterization oI two multidrug resistance
gene containing regions with a high degree oI structural similarity. Iound on a 12kb S.
Enteritidis IncI plasmid and in the 10 kb chromosomal resistance gene region oI
S. Typhimurium DT 193 (Fig 6). Both regions contained common elements oI class 1 integrons
and a strB-strA-sul2-repC-repA region derived Irom the RSF1010 plasmid (EMBL accession
no. M28829) Ilanked by two inverted-IS26 elements in the Iormer (S. Enteritidis) and by the
integron and an inverted IS element in the latter (S. Typhimurium). The tnpM gene. known to
be part oI the transposase machinery oI the Tn21-like transposons is present on the IncI plasmid
but absent in the DT193 resistance island. A Iurther diIIerence between both sequences is their
opposite orientation with respect to the integrons and the Iact that the integron located on the
S. Enteritidis plasmid is devoid oI its 3`CS. This suggests that the development oI multidrug
resistance occurred via the acquisition oI both conserved and highly diIIerent genetic traits
through independent rounds oI insertion and recombination.

Spread of antibiotic resistance via transposons

Transposons. 'easily mobilizable genetic elements. range in size Irom a Iew kb to more than
150 kb
112
. Transposons are Ilanked by DNA segments known as insertion sequences. which
encode the enzyme that catalyzes transposition (transposase) and provide the ends recognized
by the transposase when it cuts and pastes the DNA during an insertion event
109
. Transposons
can integrate either in plasmids or in the bacterial chromosome. Numerous transposons have

40
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Chapter 1
41
been described and many oI them carry one or more antibiotic resistance determinants. Since
transposons may be present in multiple copies. this enhances the eIIectiveness in expression oI
the resistance they encode. Transposons. Ior instance. Tn5. Tn7.Tn10 and Tn21 have been
Iound in Gram-negative bacteria and Tn554. Tn916. Tn4001 in Gram-positive bacteria
112
.

It is important to distinguish between gene cassettes. integrons and transposons.
Radstrom et al proposed that integrons are transposons or at least transposon derivatives
99
. But
while tranposons are capable oI moving themselves. integrons are not. Recchia and Hall stated
that though gene cassettes are mobile elements they are not transposons. Their conclusion was
derived Irom the Iact that gene cassettes are distinct Irom transposons in three important Iacts.
First. gene cassettes do not include any genes encoding proteins that catalyse their movement.
The mobility oI cassettes is mediated by the integrase. Second. gene cassettes are not Ilanked by
inverted repeats. nor are they Ilanked by a duplication oI the target sequences. Third. the
mechanism used Ior mobilization oI the gene cassette involves conservative site speciIic
recombination
102
. In short. resistance gene cassettes can be integrated into integrons; integrons
can be associated with transposons; transposons enhance the movement oI resistance genes.

Spread of multiresistance via SCI1- an integrative mobilisable element

In all Salmonella enterica serovars. SGI1 is Ilanked by imperIect 18 bp direct repeats at the leIt
and right iunctions in the Salmonella chromosome. which suggests that the origin oI this
sequence may be Irom the donor Irom which SGI1 was obtained by horizontal transIer
29
. In
1999. a study by Schmieger and Schicklmaier
111
showed that the chromosomally-located
resistance gene cluster oI S. Typhimurium can be eIIectively transduced to another strain by the
P22-like phage ES18 and by the phage PDT17. Doublet et al.
31
reported that SGI1 is an
integrative mobilizable element. SGI1 appeared to be transmissible to E. coli and non-SGI1
containing Salmonella enterica recipients only in the presence oI additional coniugative
Iunctions. The Iunctions were provided by the coniugative IncC plasmid R55 in coniugation
experiments in vitro. These data suggest that SGI1 is mobile and that the MDR region is a
hotspot Ior the integration and loss oI genes. These two properties make SGI1 a well equipped
tool Ior the adaptation oI bacterial cells to new antibiotic challenges and Ior the spread oI
resistance genes to other Salmonella and probably other species
39
.

1.5. Previous studies on antimicrobial resistance in non-typhoid Salmonella in Vietnam
and The Netherlands

From The Netherlands. several publications on antimicrobial resistance in non-typhoid
Salmonella Irom human and animal origin have appeared
123. 124
. In these manuscripts mainly
levels and patterns oI resistance among Salmonella serovars isolated Irom diIIerent host species
during the period 1984-2001 are discussed. Resistance to tetracycline. ampicillin.
chloramphenicol. and trimethoprim-sulIamethoxazole was Irequently observed and this
resistance increased during this period in the serovar Typhimurium. Since 2002. monitoring oI
antimicrobial resistance and antibiotic usage in animals is conducted in the proiect

42
'Antimicrobial Resistance Research in Animals Iunded by the Ministry oI Agriculture. Nature
and Food Quality
78
. The annual reports are available online via the website www.vwa.nl or
www.cidc-lelystad.nl. Trends with respect to the development oI phenotypic resistance to
antibiotics in Salmonella. Campvlobacter and E. coli isolated Irom Iood animals and humans
are discussed in these reports.

However. knowledge on the genetic mechanisms behind the development oI resistance
among salmonellae is limited. In two recent investigations van Duiikeren et al. investigated
integrons in a small number oI gentamicin- and cotrimoxazole-resistant Salmonella isolates
Irom horses
125
and Hasman et al. studied the diversity oI ESBL genes in Salmonella enterica
isolated Irom animals. Iood products and human patients
53
. The genetic characteristics oI Dutch
MDR Salmonella Irom humans and animals are not well known. Likewise it is not well studied
whether mobile genetic elements contribute to the development oI new MDR strains.

No longitudinal study on non-typhoid Salmonella has been conducted in Vietnam yet.
Several small studies in hospitals or institutes are available but these local resistance data were
not always communicated to physicians and veterinarians. Furthermore. practitioners did not
realise the importance oI these data or ignored them due to the availability oI broad-spectrum
antibiotics on the market. Only data on antimicrobial resistance in Salmonella Typhi Irom
humans in Vietnam have been published
65. 97
.

2. THESIS OUTLINE

2.1. The aim of this thesis was to compare antimicrobial resistance oI non-typhoid Salmonella
isolates obtained Irom human and animal origin in Vietnam (a developing country) and in The
Netherlands (a developed country) with Iocus on the genetic basis oI this resistance. Stepwise.
the obiectives oI the study described in this thesis were:
- to determine the level and pattern oI antimicrobial resistance among Salmonella serovars in
the two countries.
- to address the role oI integrons in antimicrobial resistance oI salmonellae.
- to determine whether resistant Salmonella isolates oI the same serovars are clonal or distinct
strains.
- to understand whether horizontal transIer oI resistance determinants between Salmonella and
other Enterobacteriaceae species is possible.
- to examine iI there is any diIIerence in terms oI in vitro pathogenicity between the two
multi-drug resistant phage types oI S. Typhimurium pt 90 (dominant in Vietnam) and pt 506
(dominant in The Netherlands).

2.2. The structure of this thesis

Chapter 1 provides the readers with a review on the current situation with respect to resistance
oI non-typhoid Salmonella to diIIerent antimicrobial agents and the various mechanisms by
Chapter 1
43
which antibiotic resistance is obtained or by which antibiotic resistance determinants can be
spread.

One oI the Iirst requirements Ior a study on antimicrobial resistance in Salmonella is
obtaining inIormation on the distribution oI Salmonella serovars Irom human and animal origin
on a national or regional level. Certain serovars are usually dominant in a certain animal
species. although a shiIt in the dominant serovar can be observed over time. Antimicrobial use
is diIIerent in human medicine and in veterinary medicine and diIIers Irom country to country.
As a result. one may speculate about a diIIerent outcome oI the development oI resistance to
antimicrobials among Salmonella serovars. In Vietnam. there is very limited data regarding the
distribution oI Salmonella serovars Irom humans and animals. ThereIore. Chapter 2 is Iocused
on the discussion oI this topic.

The main obiectives oI this thesis are presented in Chapter 3. Chapter 4. Chapter 5
and Chapter 6 in which integron-borne resistance is investigated using Salmonella isolates oI
various serovars. Irom diIIerent sources. In these chapters. the molecular biology oI
antimicrobial resistance is studied and particular attention is given to integron-associated gene
cassettes and the multi-drug resistance region oI Salmonella Genomic Islands. Chapter 4 was
designed to understand whether Salmonella isolates Irom diIIerent reservoirs shared the same
resistance phenotypes and genotypes. The implication oI the results regarding typing methods
described in Chapter 5 is helpIul Ior an epidemiological investigation oI S. Dublin. In
Chapter 6 the potential oI transmission oI resistance by a direct exchange oI resistance
determinants between diIIerent bacterial species (S. Typhimurium isolates and E. coli K12) is
determined. Non-integron borne Iluoroquinolone resistance is addressed partly in Chapter 3
and Chapter 6. Chapter 7 describes research into the genetic basis oI resistance to third
generation cephalosporins in Salmonella and closely related species. Coniugal transIer oI
resistance Iactors is demonstrated in vitro between clinical isolates oI Enterobacteriaceae. One
oI the surprising Iindings in the study documented in chapter 2 was the predominance oI S.
Typhimurium phage type 90 among Vietnamese S. Typhimurium isolates. This led to
experiments described in Chapter 8 which address the question whether there is a diIIerence in
terms oI in vitro pathogenicity between the two multi-drug resistant phage types oI S.
Typhimurium pt 90 and pt 506 (also known as DT 104).

Chapter 9 is a general discussion. The research described in this thesis supports the
contention that there is no limitation in antibiotic resistance among non-typhoid Salmonella
with regard to the diIIerent serovars. the source oI isolates. the geographic region Irom which
the isolates were obtained. the kinds oI antimicrobial agents against which resistance is
observed and the resistance mechanisms involved. The overall message Irom the studies is that
constant national and international programs on prudent use oI antimicrobial agents in human
and veterinary medicine as well as international surveillance on antimicrobial resistance are
needed. Recommendations in that respect are also proposed.


44
REFERENCES

1. Ahmed. A.M.. H. Nakano. and T. Shimamoto. 2005. Molecular characterization oI integrons in non-typhoid
Salmonella serovars isolated in Japan: description oI an unusual class 2 integron. J. Antimicrob. Chemother.
55:371-374.
2. Antunes. P.. J. Machado. J.C. Sousa. and L. Peixe. 2004. Dissemination amongst humans and Iood products
oI animal origin oI a Salmonella Typhimurium clone expressing an integron-borne OXA-30 beta-lactamase.
J. Antimicrob. Chemother. 54:429-434.
3. Arlet. G.. T.J. Barrett. P. Butaye. A. Cloeckaert. M.R. Mulvey. and D.G. White. 2006. Salmonella resistant
to extended-spectrum cephalosporins: prevalence and epidemiology. Microbes InIect. 8:1945-1954.
4. Asai. T.. M. Itagaki. Y. Shiroki. M. Yamada. M. Tokoro. A. Koiima. K. Ishihara. H. Esaki. Y. Tamura. and
T. Takahashi. 2006. Antimicrobial resistance types and genes in Salmonella enterica InIantis isolates Irom
retail raw chicken meat and broiler chickens on Iarms. J. Food Prot. 69:214-216.
5. Asako. H.. K. Kobayashi. and R. Aono. 1999. Organic solvent tolerance oI Escherichia coli is independent
oI OmpF levels in the membrane. Appl. Environ. Microbiol. 65:294-296.
6. Batchelor. M.. K.L. Hopkins. E.J. ThrelIall. F.A. CliIton-Hadley. A.D. Stallwood. R.H. Davies. and E.
Liebana. 2005. Characterization oI AmpC-mediated resistance in clinical Salmonella isolates recovered Irom
humans during the period 1992 to 2003 in England and Wales. J. Clin. Microbiol. 43:2261-2265.
7. Bax. R.. R. Bywater. G. Cornaglia. H. Goossens. P. Hunter. V. Isham. V. Jarlier. R. Jones. I. Phillips. D.
Sahm. S. Senn. M. Struelens. D. Taylor. and A. White. 2001. Surveillance oI antimicrobial resistance--what.
how and whither? Clin. Microbiol. InIect. 7:316-325.
8. Bennett. P.M. 1999. Integrons and gene cassettes: a genetic construction kit Ior bacteria. J Antimicrob
Chemother 43:1-4.
9. Black. J.A.. E.S. Moland. and K.S. Thomson. 2005. AmpC disk test Ior detection oI plasmid-mediated
AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal AmpC beta-lactamases. J. Clin.
Microbiol. 43:3110-3113.
10. Bouallegue-Godet. O.. Y. Ben Salem. L. Fabre. M. Demartin. P.A. Grimont. R. Mzoughi. and F.X. Weill.
2005. Nosocomial outbreak caused by Salmonella enterica serotype Livingstone producing CTX-M-27
extended-spectrum beta-lactamase in a neonatal unit in Sousse. Tunisia. J. Clin. Microbiol. 43:1037-1044.
11. Boyd. D.. G.A. Peters. A. Cloeckaert. K.S. Boumedine. E. Chaslus-Dancla. H. Imberechts. and M.R.
Mulvey. 2001. Complete nucleotide sequence oI a 43-kilobase genomic island associated with the multidrug
resistance region oI Salmonella enterica serovar Typhimurium DT104 and its identiIication in phage type
DT120 and serovar Agona. J. Bacteriol. 183:5725-5732.
12. Boyd. D.. A. Cloeckaert. E. Chaslus-Dancla. and M.R. Mulvey. 2002. Characterization oI variant
Salmonella Genomic Island 1 multidrug resistance regions Irom serovars Typhimurium DT104 and Agona.
Antimicrob. Agents Chemother. 46:1714-1722.
13. Boyd. D.A.. G.A. Peters. L. Ng. and M.R. Mulvey. 2000. Partial characterization oI a genomic island
associated with the multidrug resistance region oI Salmonella enterica Typhimurium DT104. FEMS
Microbiol. Lett. 189:285-291.
14. Brown. J.C.. and S.G.B. Amyes. 1998. Quinolone resistance. In WoodIord. N. & Johnson. A. P. Methods in
molecular medicine. New Jersey: Humana Press Inc.. 617-639.
15. Bush. K.. G.A. Jacoby. and A.A. Medeiros. 1995. A Iunctional classiIication scheme Ior beta-lactamases and
its correlation with molecular structure. Antimicrob. Agents Chemother. 39:1211-1233.
16. Cao. Y.. and F. Hayes. 1999. A newly identiIied. essential catalytic residue in a critical secondary structure
element in the integrase Iamily oI site-speciIic recombinases is conserved in a similar element in eucaryotic
type IB topoisomerases. J Mol Biol 289:517-527.
17. Chalker. J.. S. Ratanawiiitrasin. N.T. Chuc. M. Petzold. and G. Tomson. 2005. EIIectiveness oI a multi-
component intervention on dispensing practices at private pharmacies in Vietnam and Thailand--a
randomized controlled trial. Soc. Sci. Med. 60:131-141.
Chapter 1
45
18. Chen. S.. S. Zhao. D.G. White. C.M. Schroeder. R. Lu. H. Yang. P.F. McDermott. S. Ayers. and J. Meng.
2004. Characterization oI multiple-antimicrobial-resistant Salmonella serovars isolated Irom retail meats.
Appl. Environ. Microbiol. 70:1-7.
19. Cheung. T.K.. Y.W. Chu. M.Y. Chu. C.H. Ma. R.W. Yung. and K.M. Kam. 2005. Plasmid-mediated
resistance to ciproIloxacin and ceIotaxime in clinical isolates oI Salmonella enterica serotype Enteritidis in
Hong Kong. J. Antimicrob. Chemother. 56:586-589.
20. Chopra. I.. and M. Roberts. 2001. Tetracycline antibiotics: mode oI action. applications. molecular biology.
and epidemiology oI bacterial resistance. Microbiol. Mol. Biol. Rev. 65:232-260.
21. Chu. C.. C.H. Chiu. W.Y. Wu. C.H. Chu. T.P. Liu. and J.T. Ou. 2001. Large drug resistance virulence
plasmids oI clinical isolates oI Salmonella enterica serovar Choleraesuis. Antimicrob. Agents Chemother.
45:2299-2303.
22. Chu. C.. L.H. Su. C.H. Chu. S. Baucheron. A. Cloeckaert. and C.H. Chiu. 2005. Resistance to
Iluoroquinolones linked to gvrA and parC mutations and overexpression oI acrAB eIIlux pump in
Salmonella enterica serotype Choleraesuis. Microb. Drug Resist. 11:248-253.
23. Collis. C.M.. and R.M. Hall. 1995. Expression oI antibiotic resistance genes in the integrated cassettes oI
integrons. Antimicrob. Agents Chemother. 39:155-162.
24. Daly. M.. and S. Fanning. 2000. Characterization and chromosomal mapping oI antimicrobial resistance
genes in Salmonella enterica serotype Typhimurium. Appl. Environ. Microbiol. 66:4842-4848.
25. Daly. M.. and S. Fanning. 2004. Integron analysis and genetic mapping oI antimicrobial resistance genes in
Salmonella enterica serotype Typhimurium. Methods Mol. Biol. 268:15-32.
26. Daly. M.. L. Villa. C. Pezzella. S. Fanning. and A. Carattoli. 2005. Comparison oI multidrug resistance gene
regions between two geographically unrelated Salmonella serotypes. J. Antimicrob. Chemother. 55:558-561.
27. Davies. J.. and V. Webb. 2004. Antibiotic resistance in bacteria. In Schaechter. M. & Lederberg. J. The desk
encyclopedia oI microbiology. London. UK: Elsevier Academic Press. 25-46.
28. Di Conza. J.. J.A. Ayala. P. Power. M. Mollerach. and G. Gutkind. 2002. Novel class 1 integron (InS21)
carrying bla
CTX-M-2
in Salmonella enterica serovar InIantis. Antimicrob. Agents Chemother. 46:2257-2261.
29. Doublet. B.. R. Lailler. D. Meunier. A. Brisabois. D. Boyd. M.R. Mulvey. E. Chaslus-Dancla. and A.
Cloeckaert. 2003. Variant Salmonella Genomic Island 1 antibiotic resistance gene cluster in Salmonella
enterica serovar Albany. Emerg. InIect. Dis. 9:585-591.
30. Doublet. B.. P. Butaye. H. Imberechts. D. Boyd. M.R. Mulvey. E. Chaslus-Dancla. and A. Cloeckaert. 2004.
Salmonella Genomic Island 1 multidrug resistance gene clusters in Salmonella enterica serovar Agona
isolated in Belgium in 1992 to 2002. Antimicrob. Agents Chemother. 48:2510-2517.
31. Doublet. B.. D. Boyd. M.R. Mulvey. and A. Cloeckaert. 2005. The Salmonella Genomic Island 1 is an
integrative mobilizable element. Mol. Microbiol. 55:1911-1924.
32. Ebner. P.. K. Garner. and A. Mathew. 2004. Class 1 integrons in various Salmonella enterica serovars
isolated Irom animals and identiIication oI genomic island SGI1 in Salmonella enterica var. Meleagridis. J.
Antimicrob. Chemother. 53:1004-1009.
33. Edrington. T.S.. C.L. Schultz. K.M. BischoII. T.R. Callaway. M.L. Looper. K.J. Genovese. Y.S. Jung. J.L.
McReynolds. R.C. Anderson. and D.J. Nisbet. 2004. Antimicrobial resistance and serotype prevalence oI
Salmonella isolated Irom dairy cattle in the southwestern United States. Microb. Drug Resist 10:51-56.
34. Fierer. J.. and M. Swancutt. 2000. Non-typhoid Salmonella: a review. Curr. Clin. Top InIect. Dis. 20:134-
157.
35. Floriin. A.. S. Niissen. F.J. Schmitz. J. VerhoeI. and A.C. Fluit. 2002. Comparison oI E test and double disk
diIIusion test Ior detection oI extended spectrum beta-lactamases. Eur J Clin Microbiol InIect Dis 21:241-
243.
36. Fluit. A.C.. and F.J. Schmitz. 1999. Class 1 integrons. gene cassettes. mobility. and epidemiology. Eur. J.
Clin. Microbiol. InIect. Dis. 18:761-770.
37. Fluit. A.C.. M.R. Visser. and F.J. Schmitz. 2001. Molecular detection oI antimicrobial resistance. Clin.
Microbiol. Rev. 14:836-871.
38. Fluit. A.C.. and F.J. Schmitz. 2004. Resistance integrons and super-integrons. Clin. Microbiol. InIect.
10:272-288.

46
39. Fluit. A.C. 2005. Towards more virulent and antibiotic-resistant Salmonella? FEMS Immunol. Med.
Microbiol. 43:1-11.
40. Fluit. A.C.. J.T. van der Bruggen. F.M. Aarestrup. J. VerhoeI. and W.T.M. Jansen. 2006. Priorities Ior
antibiotic resistance surveillance in Europe. Clin. Microbiol. InIect. 12:410-417.
41. Gassama-Sow. A.. A. Aidara-Kane. N. Raked. F. Denis. and M.C. Ploy. 2004. Integrons in Salmonella
Keurmassar. Senegal. Emerg. InIect. Dis. 10:1339-1341.
42. Gebreyes. W.A.. S. Thakur. P.R. Davies. J.A. Funk. and C. Altier. 2004. Trends in antimicrobial resistance.
phage types and integrons among Salmonella serotypes Irom pigs. 1997-2000. J. Antimicrob. Chemother.
53:997-1003.
43. Gebreyes. W.A.. and S. Thakur. 2005. Multidrug-resistant Salmonella enterica serovar Muenchen Irom pigs
and humans and potential interserovar transIer oI antimicrobial resistance. Antimicrob. Agents Chemother.
49:503-511.
44. Giraud. E.. A. Brisabois. J.L. Martel. and E. Chaslus-Dancla. 1999. Comparative studies oI mutations in
animal isolates and experimental in vitro- and in vivo-selected mutants oI Salmonella spp. suggest a
counterselection oI highly Iluoroquinolone-resistant strains in the Iield. Antimicrob. Agents Chemother.
43:2131-2137.
45. Giraud. E.. A. Cloeckaert. D. KerboeuI. and E. Chaslus-Dancla. 2000. Evidence Ior active eIIlux as the
primary mechanism oI resistance to ciproIloxacin in Salmonella enterica serovar Typhimurium. Antimicrob.
Agents Chemother. 44:1223-1228.
46. Goossens. H.. M. Ferech. R. vander Stichele. M. Elseviers. and Ior the ESAC Proiect Group. 2005.
Outpatient antibiotic use in Europe and association with resistance: a cross-national database study. Lancet
365:579-586.
47. Guardabassi. L.. S. Schwarz. and D.H. Lloyd. 2004. Pet animals as reservoirs oI antimicrobial-resistant
bacteria. J. Antimicrob. Chemother. 54:321-332.
48. Guerra. B.. S.M. Soto. J.M. Arguelles. and M.C. Mendoza. 2001. Multidrug resistance is mediated by large
plasmids carrying a class 1 integron in the emergent Salmonella enterica serotype |4.5.12:i:-|. Antimicrob.
49. Guerra. B.. S. Soto. R. Helmuth. and M.C. Mendoza. 2002. Characterization oI a selI-transIerable plasmid
Irom Salmonella enterica serotype Typhimurium clinical isolates carrying two integron-borne gene cassettes
together with virulence and drug resistance genes. Antimicrob. Agents Chemother. 46:2977-2981.
50. Hall. R.M.. and C.M. Collis. 1995. Mobile gene cassettes and integrons: capture and spread oI class 1
integrons among Salmonella serotypes. Mol. Microbiol. 15:593-600.
51. Hall. R.M.. and C.M. Collis. 1998. Antibiotic resistance in gram-negative bacteria: the role oI gene cassettes
and integrons. Drug Resist Updat 1:109-119.
52. Hall. R.M.. C.M. Collis. M.J. Kim. S.R. Partridge. G.D. Recchia. and H.W. Stokes. 1999. Mobile gene
cassettes and integrons in evolution. Ann N Y Acad Sci 870:68-80.
53. Hasman. H.. D. Mevius. K. Veldman. I. Olesen. and F.M. Aarestrup. 2005. beta-Lactamases among
extended-spectrum beta-lactamase (ESBL)-resistant Salmonella Irom poultry. poultry products and human
patients in The Netherlands. J. Antimicrob. Chemother. 56:115-121.
54. Helmuth. R. 2000. Antibiotic resistance in Salmonella. In Wray. C. & Wray. A. Salmonella in domestic
animals. WallinIord. UK: CABI publishing. 89-106.
55. Henning. K.J.. and K. Sepkowitz. 1999. Antibiotic use policy. In Amstrong. D. & Cohen. S. InIectious
diseases. Mosby. 1-8.
56. Hohmann. E.L. 2001. Nontyphoidal salmonellosis. Clin. InIect. Dis. 32:263-269.
57. Hopkins. K.L.. R.H. Davies. and E.J. ThrelIall. 2005. Mechanisms oI quinolone resistance in Escherichia
coli and Salmonella. recent developments. Int. J. Antimicrob. Agents 25:358-373.
58. Huang. T.M.. Y.F. Chang. and C.F. Chang. 2004. Detection oI mutations in the gvrA gene and class I
integron Irom quinolone-resistant Salmonella enterica serovar Choleraesuis isolates in Taiwan. Vet.
Microbiol. 100:247-254.
59. Humphrey. T. 2000. Public health aspects oI Salmonella inIections. In Wray. C. & Wray. A. Salmonella in
domestic animals. New York. USA: CABI Publishing. 245-263.
Chapter 1
47
60. Huovinen. P.. L. Sundstrom. G. Swedberg. and O. Skold. 1995. Trimethoprim and sulIonamide resistance.
61. Jansen. W.T.. M.M. Beitsma. C.J. Koeman. W.J. van Wamel. J. VerhoeI. and A.C. Fluit. 2006. Novel
mobile variants oI Staphylococcal Cassette Chromosome mec in Staphvlococcus aureus. Antimicrob. Agents
Chemother. 50:2072-2078.
62. Kosek. M.. C. Bern. and R.L. Guerrant. 2003. The global burden oI diarrhoeal disease. as estimated Irom
studies published between 1992 and 2000. Bull. World Health Organ. 81:197-204.
63. Kuyvenhoven. M.M.. F.A.M. van Balen. and T.J.M. Verheii. 2003. Outpatient antibiotic prescriptions Irom
1992 to 2001 in The Netherlands. J. Antimicrob. Chemother. 52:675-678.
64. Kwon. H.J.. T.E. Kim. S.H. Cho. J.G. Seol. B.J. Kim. J.W. Hyun. K.Y. Park. S.J. Kim. and H.S. Yoo. 2002.
Distribution and characterization oI class 1 integrons in Salmonella enterica serotype Gallinarum biotype
Gallinarum. Vet. Microbiol. 89:303-309.
65. Le. T.A.. M. Leiay-Collin. P.A. Grimont. T.L. Hoang. T.V. Nguyen. F. Grimont. and M.R. Scavizzi. 2004.
Endemic. epidemic clone oI Salmonella enterica serovar Typhi harboring a single multidrug-resistant
plasmid in Vietnam between 1995 and 2002. J Clin Microbiol 42:3094-3099.
66. Lee. W.C.. M.J. Lee. J.S. Kim. and S.Y. Park. 2001. Foodborne illness outbreaks in Korea and Japan studied
retrospectively. J. Food Prot. 64:899-902.
67. Leverstein-van Hall. M.A. 2002. The growing problem oI drug resistant bacteria: the role oI horizontal gene
transIer (PhD Thesis). Utrecht University. 191.
68. Levings. R.S.. D. LightIoot. S.R. Partridge. R.M. Hall. and S.P. Diordievic. 2005. The Genomic Island
SGI1. containing the multiple antibiotic resistance region oI Salmonella enterica serovar Typhimurium
DT104 or variants oI it. is widely distributed in other S. enterica serovars. J. Bacteriol. 187:4401-4409.
69. Levy. S.B.. L.M. McMurry. V. Burdett. P. Courvalin. W. Hillen. M.C. Roberts. and D.E. Taylor. 1989.
Nomenclature Ior tetracycline resistance determinants. Antimicrob. Agents Chemother. 33:1373-1374.
70. Levy. S.B. 1998. The challenge oI antibiotic resistance. Sci Am 278:46-53.
71. Li. Q.. J.A. Skyberg. M.K. Fakhr. J.S. Sherwood. L.K. Nolan. and C.M. Logue. 2006. Antimicrobial
susceptibility and characterization oI Salmonella isolates Irom processed bison carcasses. Appl. Environ.
Microbiol. 72:3046-3049.
72. Li. W.C.. F.Y. Huang. C.P. Liu. L.C. Weng. N.Y. Wang. N.C. Chiu. and C.S. Chiang. 2005. CeItriaxone
resistance oI nontyphoidal Salmonella enterica isolates in Northern Taiwan attributable to production oI
CTX-M-14 and CMY-2 beta-lactamases. J. Clin. Microbiol. 43:3237-3243.
73. Liebana. E.. C. Clouting. C.A. Cassar. L.P. Randall. R.A. Walker. E.J. ThrelIall. F.A. CliIton-Hadley. A.M.
Ridley. and R.H. Davies. 2002. Comparison oI gvrA mutations. cyclohexane resistance. and the presence oI
class I integrons in Salmonella enterica Irom Iarm animals in England and Wales. J. Clin. Microbiol.
40:1481-1486.
74. Livermore. D.M.. and N. WoodIord. 2004. Laboratory detection and reporting oI bacteria with extended-
spectrum beta-lactamases. Health Protection Agency-Colindale. London. 1-14.
75. Mammeri. H.. M. Van De Loo. L. Poirel. L. Martinez-Martinez. and P. Nordmann. 2005. Emergence oI
plasmid-mediated quinolone resistance in Escherichia coli in Europe. Antimicrob. Agents Chemother.
49:71-76.
76. Markovska. R.. K. Rachkova. I. Schneider. E. Keuleyan. and A. BauernIeind. 2005. Multiresistant SHV-2-
producing Salmonella enterica Serotype Corvallis in Bulgaria. J. Chemother. 17:568-569.
77. Mead. P.S.. L. Slutsker. V. Dietz. L.F. McCaig. J.S. Bresee. C. Shapiro. P.M. GriIIin. and R.V. Tauxe. 1999.
Food-related illness and death in the United States. Emerg. InIect. Dis. 5:607-625.
78. Mevius. D.J.. and W. van Pelt. MARAN-2004- Monitoring oI antimicrobial resistance and antibiotic usage
in animals in The Netherlands in 2004. http://www.cidc-lelystad.wur.nl/NR/rdonlyres/7F79ACE6-0FD2-
41AB-81B2-BB17FA89603C/11382/MARAN2004web1.pdI (10 August 2006. date last accessed).
79. Michael. G.B.. M. Cardoso. and S. Schwarz. 2005. IdentiIication oI an aadA2 gene cassette Irom Salmonella
enterica subsp. enterica serovar Derby. J Vet Med B InIect Dis Vet Public Health 52:456-459.

48
80. Michael. G.B.. M. Cardoso. and S. Schwarz. 2005. Class 1 integron-associated gene cassettes in Salmonella
enterica subsp. enterica serovar Agona isolated Irom pig carcasses in Brazil. J. Antimicrob. Chemother.
55:776-779.
81. Michael. G.B.. P. Butaye. A. Cloeckaert. and S. Schwarz. 2006. Genes and mutations conIerring
antimicrobial resistance in Salmonella: an update. Microbes InIect. 8:1898-1914.
82. Mikasova. E.. H. Drahovska. T. Szemes. T. Kuchta. R. Karpiskova. M. Sasik. and J. Turna. 2005.
Characterization oI Salmonella enterica serovar Typhimurium strains oI veterinary origin by molecular
typing methods. Vet. Microbiol. 109:113-120.
83. Miriagou. V.. P.T. Tassios. N.J. Legakis. and L.S. Tzouvelekis. 2004. Expanded-spectrum cephalosporin
resistance in non-typhoid Salmonella. Int. J. Antimicrob. Agents 23:547-555.
84. Mulvey. M.R.. D.A. Boyd. L. Baker. O. Mykytczuk. E.M. Reis. M.D. Asensi. D.P. Rodrigues. and L.K. Ng.
2004. Characterization oI a Salmonella enterica serovar Agona strain harbouring a class 1 integron
containing novel OXA-type beta-lactamase (bla
OXA-53
) and 6'-N-aminoglycoside acetyltransIerase genes
|aac(6')-I30|. J. Antimicrob. Chemother. 54:354-359.
85. NETHMAP. 2004. Verbrugh. H. A. & Neeling. d. A. J. Delivery oI antibiotics by the commuinity
pharmacies vesus general practitioners with their own pharmacy. 19.
86. Nogrady. N.. I. Gado. A. Toth. and J. Paszti. 2005. Antibiotic resistance and class 1 integron patterns oI non-
typhoidal human Salmonella serotypes isolated in Hungary in 2002 and 2003. Int. J. Antimicrob. Agents
26:126-132.
87. Nordmann. P.. and L. Poirel. 2005. Emergence oI plasmid-mediated resistance to quinolones in
Enterobacteriaceae. J. Antimicrob. Chemother. 56:463-469.
88. Nunes-Duby. S.E.. H.J. Kwon. R.S. Tirumalai. T. Ellenberger. and A. Landy. 1998. Similarities and
diIIerences among 105 members oI the Int Iamily oI site-speciIic recombinases. Nucleic Acids Res. 26:391-
406.
89. Okumura. J.. S. Wakai. and T. Umenai. 2002. Drug utilisation and selI-medication in rural communities in
Vietnam. Soc. Sci. Med. 54:1875-1886.
90. O'Mahony. R.. M. Saugy. N. Leonard. D. Drudy. B. Bradshaw. J. Egan. P. Whyte. M. O'Mahony. P. Wall.
and S. Fanning. 2005. Antimicrobial resistance in isolates oI Salmonella spp. Irom pigs and the
characterization oI an S. InIantis gene cassette. Foodborne Pathog. Dis. 2:274-281.
91. O'Ryan. M.. V. Prado. and L.K. Pickering. 2005. A millennium update on pediatric diarrheal illness in the
developing world. Semin. Pediatr. InIect. Dis. 16:125-136.
92. Paauw. A.. A.C. Fluit. J. VerhoeI. and M.A. Leverstein-van Hall. 2006. Enterobacter cloacae outbreak and
emergence oI quinolone resistance gene in Dutch hospital. Emerg. InIect. Dis. 12:807-812.
93. Paterson. D.L.. and R.A. Bonomo. 2005. Extended-spectrum beta-lactamases: a clinical update. Clin.
94. Payne. D.J.. and C.J. Thomson. 1998. Molecular approachs Ior the detection and identiIication oI beta-
lactamases. In WoodIord. N. & Johnson. A. P. Molecular bacteriology: protocols and clinical applications.
Totowa. NJ: Humanna Press Inc..
95. Perez-Perez. F.J.. and N.D. Hanson. 2002. Detection oI plasmid-mediated AmpC beta-lactamase genes in
clinical isolates by using multiplex PCR. J. Clin. Microbiol. 40:2153-2162.
96. Philippon. A.. G. Arlet. and G.A. Jacoby. 2002. Plasmid-determined AmpC-type beta-lactamases.
97. Phung le. V.. H. Ryo. and T. Nomura. 2002. SpeciIic gvrA mutation at codon 83 in nalidixic acid-resistant
Salmonella enterica serovar Typhi strains isolated Irom Vietnamese patients. Antimicrob. Agents
Chemother. 46:2052-2053.
98. Quinn. P.J.. M.E. Carter. B. Markey. and G.R. Carter. 1994. Clinical veterinary microbiology. Mosby-
Yearbook Europe Limited. England.
99. Radstrom. P.. O. Skold. G. Swedberg. J. Flensburg. P.H. Roy. and L. Sundstrom. 1994. Transposon Tn5090
oI plasmid R751. which carries an integron. is related to Tn7. Mu. and the retroelements. J. Bacteriol.
176:3257-3268.
Chapter 1
49
100. Rankin. S.C.. H. Aceto. J. Cassidy. J. Holt. S. Young. B. Love. D. Tewari. D.S. Munro. and C.E. Benson.
2002. Molecular characterization oI cephalosporin-resistant Salmonella enterica serotype Newport isolates
Irom animals in Pennsylvania. J. Clin. Microbiol. 40:4679-4684.
101. Recchia. G.D.. and R.M. Hall. 1995. Plasmid evolution by acquisition oI mobile gene cassettes: plasmid
pIE723 contains the aadB gene cassette precisely inserted at a secondary site in the incQ plasmid RSF1010.
Mol. Microbiol. 15:179-187.
102. Recchia. G.D.. and R.M. Hall. 1995. Gene cassettes: a new class oI mobile element. Microbiology
141:3015-3027.
103. Recchia. G.D.. and R.M. Hall. 1997. Origins oI the mobile gene cassettes Iound in integrons. Trends
Microbiol. 5:389-394.
104. Roberts. M.C. 1998. Resistance to tetracyclines. macrolides. trimethoprim and sulIonamides. In WoodIord.
N. & Johnson. A. P. Methods in molecular medicine. New Jersey: Humana Press Inc.. 641-663.
105. Roberts. M.C. 2005. Update on acquired tetracycline resistance genes. FEMS Microbiol. Lett. 245:195-203.
106. Rodriguez. I.. M.R. Rodicio. M.C. Mendoza. and M. Cruz Martin. 2006. Large coniugative plasmids Irom
clinical strains oI Salmonella enterica serovar Virchow contain a class 2 integron in addition to class 1
integrons and several non-integron-associated drug resistance determinants. Antimicrob. Agents Chemother.
50:1603-1607.
107. Ruiz. J.. L. Capitano. L. Nunez. D. Castro. J.M. Sierra. M. Hatha. J.J. Borrego. and J. Vila. 1999.
Mechanisms oI resistance to ampicillin. chloramphenicol and quinolones in multiresistant Salmonella
Typhimurium strains isolated Irom Iish. J. Antimicrob. Chemother. 43:699-702.
108. Rychlik. I.. D. Gregorova. and H. Hradecka. 2006. Distribution and Iunction oI plasmids in Salmonella
enterica. Vet. Microbiol. 112:1-10.
109. Salyers. A.A.. and D.D. Whitt. 2002. Bacterial pathogenesis: a molecular approach. 2nd. ASM Press.
Washington DC. 168-183.
110. Schlundt. J.. H. ToyoIuku. J. Jansen. and S.A. Herbst. 2004. Emerging Iood-borne zoonoses. Rev. Sci. Tech.
23:513-533.
111. Schmieger. H.. and P. Schicklmaier. 1999. Transduction oI multiple drug resistance oI Salmonella enterica
serovar Typhimurium DT104. FEMS Microbiol. Lett. 170:251-256.
112. Schmitz. F.J.. and A.C. Fluit. 1999. Mechanisms oI resistance. In Amstrong. D. & Cohen. S. InIectious
diseases. Mosby. 1-14.
113. Schwarz. S.. C. Kehrenberg. B. Doublet. and A. Cloeckaert. 2004. Molecular basis oI bacterial resistance to
chloramphenicol and IlorIenicol. FEMS Microbiol. Rev. 28:519-542.
114. Shaw. K.J.. F.J. Sabatelli. L. Naples. P. Mann. R.S. Hare. and G.H. Miller. 1998. The application oI
molecular techniques Ior the study oI aminoglycoside resistance. In WoodIord. N. & Johnson. A. P. Methods
in molecular medicine. New Jersey: Humana Press Inc.. 463.
115. Shea. K.M. 2003. Antibiotic resistance: what is the impact oI agriculture uses oI antibiotics on children
health? Pediatrics 112:253-258.
116. Soto. S.M.. M.A. Gonzalez-Hevia. and M.C. Mendoza. 2003. Antimicrobial resistance in clinical isolates oI
Salmonella enterica serotype Enteritidis: relationships between mutations conIerring quinolone resistance.
integrons. plasmids and genetic types. J. Antimicrob. Chemother. 51:1287-1291.
117. Su. L.H.. C.H. Chiu. C. Chu. and J.T. Ou. 2004. Antimicrobial resistance in nontyphoid Salmonella
serotypes: a global challenge. Clin. InIect. Dis. 39:546-551.
118. Tankson. J.D.. P.J. Fedorka-Cray. C.R. Jackson. and M. Headrick. 2006. Genetic relatedness oI a rarely
isolated Salmonella: Salmonella enterica serotype Niakhar Irom NARMS animal isolates. J. Antimicrob.
Chemother. 57:190-198.
119. The World Bank. http://www.worldbank.org/data/quickreIerence/quickreI.html (11 August 2006. date last
accessed).
120. Tran. J.H.. and G.A. Jacoby. 2002. Mechanism oI plasmid-mediated quinolone resistance. Proc. Natl. Acad.
Sci. U S A 99:5638-5642.
121. Uzzau. S.. D.J. Brown. T. Wallis. S. Rubino. G. Leori. S. Bernard. J. Casadesus. D.J. Platt. and J.E. Olsen.
2000. Host adapted serotypes oI Salmonella enterica. Epidemiol. InIect. 125:229-255.

50
122. Vaillant. V.. H. de Valk. E. Baron. T. Ancelle. P. Colin. M.C. Delmas. B. DuIour. R. Pouillot. Y. Le Strat. P.
Weinbreck. E. Jougla. and J.C. Desenclos. 2005. Foodborne inIections in France. Foodborne Pathog. Dis.
2:221-232.
123. van Duiikeren. E.. W.J.B. Wannet. M.E.O.C. Heck. W. van Pelt. M.M. Sloet van Oldruitenborgh-
Oosterbaan. J.A.H. Smit. and D.J. Houwers. 2002. Sero types. phage types and antibiotic susceptibilities oI
Salmonella strains isolated Irom horses in The Netherlands Irom 1993 to 2000. Vet. Microbiol. 86:203-212.
124. van Duiikeren. E.. W.J.B. Wannet. D.J. Houwers. and W. van Pelt. 2003. Antimicrobial susceptibilities oI
Salmonella strains isolated Irom humans. cattle. pigs. and chickens in The Netherlands Irom 1984 to 2001. J.
Clin. Microbiol. 41:3574-3578.
125. van Duiikeren. E.. A.T.A. Box. P. Schellen. D.J. Houwers. and A.C. Fluit. 2005. Class 1 integrons in
Enterobacteriaceae isolated Irom clinical inIections oI horses and dogs in The Netherlands. Microb. Drug
Resist. 11:383-386.
126. Van Duong. D.. C.W. Binns. and T. Van Le. 1997. Availability oI antibiotics as over-the-counter drugs in
pharmacies: a threat to public health in Vietnam. Trop. Med. Int. Health 2:1133-1139.
127. van Duynhoven. Y.T.H.P.. C.M. de Jager. L.M. Kortbeek. H. Vennema. M.P.G. Koopmans. F. van Leusden.
W.H.M. van der Poel. and M.J.M. van den Broek. 2005. A one-year intensiIied study oI outbreaks oI
gastroenteritis in The Netherlands. Epidemiol. InIect. 133:9-21.
128. van Pelt. W.. M.A. de Wit. W.J. Wannet. E.J. Ligtvoet. M.A. Widdowson. and Y.T. van Duynhoven. 2003.
Laboratory surveillance oI bacterial gastroenteric pathogens in The Netherlands. 1991-2001. Epidemiol.
InIect. 130:431-441.
129. Villa. L.. C. Mammina. V. Miriagou. L.S. Tzouvelekis. P.T. Tassios. A. Nastasi. and A. Carattoli. 2002.
Multidrug and broad-spectrum cephalosporin resistance among Salmonella enterica serotype Enteritidis
clinical isolates in southern Italy. J. Clin. Microbiol. 40:2662-2665.
130. Villa. L.. and A. Carattoli. 2005. Integrons and transposons on the Salmonella enterica serovar
Typhimurium virulence plasmid. Antimicrob. Agents Chemother. 49:1194-1197.
131. Walker. R.A.. N. Saunders. A.J. Lawson. E.A. Lindsay. M. Dassama. L.R. Ward. M.J. Woodward. R.H.
Davies. E. Liebana. and E.J. ThrelIall. 2001. Use oI a LightCycler gvrA mutation assay Ior rapid
identiIication oI mutations conIerring decreased susceptibility to ciproIloxacin in multiresistant Salmonella
enterica serotype Typhimurium DT104 isolates. J. Clin. Microbiol. 39:1443-1448.
132. Wang. M.. J.H. Tran. G.A. Jacoby. Y. Zhang. F. Wang. and D.C. Hooper. 2003. Plasmid-mediated
quinolone resistance in clinical isolates oI Escherichia coli Irom Shanghai. China. Antimicrob. Agents
Chemother. 47:2242-2248.
133. Weill. F.X.. F. Guesnier. V. Guibert. M. Timinouni. M. Demartin. L. Polomack. and P.A.D. Grimont. 2006.
Multidrug resistance in Salmonella enterica serotype Typhimurium Irom humans in France (1993 to 2003).
J. Clin. Microbiol. 44:700-708.
134. Willemsen. I.. A. Groenhuiizen. D. Bogaers. A. Stuurman. P. Keulen. and J. Kluytmans. 2006. Determinants
oI inapprooriate (IA) use oI antibiotics identiIied in prevalence survey. Ned. Tiidschr. Geneeskd. 14:S52.
135. Witte. W. 1998. Medical consequences oI antibiotic use in agriculture. Science 279:996-997.
136. Yong. D.. Y. Lim. W. Song. Y.S. Choi. D.Y. Park. H. Lee. J.H. Yum. K. Lee. J.M. Kim. and Y. Chong.
2005. Plasmid-mediated. inducible AmpC beta-lactamase (DHA-1)-producing Enterobacteriaceae at a
Korean hospital: wide dissemination in Klebsiella pneumoniae and Klebsiella oxvtoca and emergence in
Proteus mirabilis. Diagn. Microbiol. InIect. Dis. 53:65-70.
137. Zhang. H.. L. Shi. L. Li. S. Guo. X. Zhang. S. Yamasaki. S. Miyoshi. and S. Shinoda. 2004. IdentiIication
and characterization oI class 1 integron resistance gene cassettes among Salmonella strains isolated Irom
healthy humans in China. Microbiol. Immunol. 48:639-645.
138. Zhang. Q.. J. Lin. and S. Pereira. 2003. Fluoroquinolone-resistant Campvlobacter in animal reservoirs:
dynamics oI development. resistance mechanisms and ecological Iitness. Anim. Health Res. Rev. 4:63-71.
139. Zhang. Q.. O. Sahin. P.F. McDermott. and S. Payot. 2006. Fitness oI antimicrobial-resistant Campvlobacter
and Salmonella. Microbes InIect. 8:1972-1978.
140. Zhao. S.. A.R. Datta. S. Ayers. S. Friedman. R.D. Walker. and D.G. White. 2003. Antimicrobial-resistant
Salmonella serovars isolated Irom imported Ioods. Int. J. Food. Microbiol. 84:87-92.
Chapter 1
51
141. Zhao. S.. P.J. Fedorka-Cray. S. Friedman. P.F. McDermott. R.D. Walker. S. Qaiyumi. S.L. Foley. S.K.
Hubert. S. Ayers. L. English. D.A. Dargatz. B. Salamone. and D.G. White. 2005. Characterization oI
Salmonella Typhimurium oI animal origin obtained Irom the National Antimicrobial Resistance Monitoring
System. Foodborne Pathog. Dis. 2:169-181.
142. Zhao. S.. P.F. McDermott. S. Friedman. S. Qaiyumi. J. Abbott. C. Kiessling. S. Ayers. R. Singh. S. Hubert.
J. SoIos. and D.G. White. 2006. Characterization oI antimicrobial-resistant Salmonella isolated Irom
imported Ioods. J. Food Prot. 69:500-507.

52

DIstrIbutInn nf Sa|mone||a enterlca
scrnvars frnm humans, !Ivcstnck and
mcat In VIctnam and thc dnmInancc nf
Sa|mone||a TvhImurIum Phagc Tvc 90

An T. T. Vo
, LngeIine van Duijkeien
, Ad C. IIuil
Max L. O.
C. Heck
, Anjo Veiliuggen
, Hennv M. L. Maas
Win Caaslia

1
Dcpar|ncn| cf |nnunc|cgu and |nfcc|icus Discascs. |acu||u cf Vc|crinaru
Mcdicinc. U|rccn| Unitcrsi|u
2
|ij|nan-lin||cr Ccn|cr. Unitcrsi|u Mcdica| Ccn|cr U|rccn|
3
Na|icna| |ns|i|u|c cf Puo|ic Hca||n and |nc |ntircnncn|. 8i||nctcn. Tnc
Nc|ncr|ands
4
|acu||u cf Anina| Scicncc and Vc|crinaru Mcdicinc. Ncng |an Unitcrsi|u.
Vic|nan

Veleiinaiv MiciolioIogv 113: 153-158. 2OO6

Distribution oI Salmonella enterica serovars in Vietnam
54
ABSTRACT

Epidemiologically unrelated non-typhoid Salmonella isolates Irom humans (n56) and animal
origin (n241. Irom Iaeces. carcasses and meat) in Vietnam were investigated. Salmonella
Typhimurium. S. Anatum. S. Weltevreden. S. Emek. and S. Rissen were the most prevalent
serovars. S. Typhimurium phage type 90 was predominant among S. Typhimurium isolates. The
serotype and phage type distribution oI the Salmonella isolates was diIIerent Irom that in
Europe and America. Many sero- and phage types Iound in humans were also Iound in cattle.
pigs. and poultry suggesting that Iood producing animals are an important source oI human
non-typhoid Salmonella inIection in Vietnam.

Keywords: Salmonella. human. animal. meat. serotype. phagetype 90. Vietnam.
Chapter 2
55
INTRODUCTION

Non-typhoid Salmonella inIections in humans continue to be a maior problem. in terms oI both
morbidity and economic costs
10
. The maiority oI the 2500 Salmonella serovars are capable oI
causing inIections in humans. Most human Salmonella outbreaks are associated with the
consumption oI contaminated products Irom animal origin
15
although non-Ioodborne
Salmonella inIection in humans may be transmitted during contact with animals. contaminated
water. or the environment.

The widespread distribution oI Iood is a global challenge in Salmonella control. With
increasing travel and global trade. outbreaks involving widely scattered cases are occurring
more Irequently. Contaminated Iood produced in one country may cause illness in another.
demonstrating the importance oI national control programmes. Besides sero-typing. phage
typing has played a central role in epidemiological studies in Salmonella Typhimurium and
Salmonella Enteritidis
1. 13
.

The aim oI this study was to examine the distribution oI serovars and phage types
among Salmonella strains isolated Irom humans. cattle. pigs and poultry in Vietnam in order to
contribute to the understanding oI the epidemiology oI Salmonella.

MATERIALS AND METHODS

Bacterial isolates
A total oI 297 epidemiologically unrelated isolates were included in this study. The isolates
were Irom humans (n56). cattle (n63). pigs (n111). and poultry (n67). The 56 clinical
human isolates oI unrelated patients with diarrhoea and Iever were obtained Irom Iive
provincial hospitals and two Pasteur Institutes in Vietnam.

Sampling
The animal isolates were collected Irom pigs. cattle. chickens and ducks (Table 1) in 13
provinces oI South Vietnam during the year 2004. Faecal samples Irom healthy animals were
taken at the slaughterhouses (78) and Irom healthy or sick animals on Iarms (12). The
animals sampled came Irom diIIerent Ilocks or herds. For poultry. only samples Irom healthy
animals were collected due to the risk oI avian Ilu inIection during the study period. Rectal
Iaecal samples were taken Irom pigs and cattle. Faecal samples Irom poultry were taken Irom
the distal part oI the intestinal tract in the slaughterhouses or Irom pooled samples Irom 5 to 10
animals on the Iarms. About 25 g oI Iaeces was collected Irom each animal (except Ior poultry
on the Iarms) and placed in a sterile sampling bag. kept in an ice-box at 4
0
C and transported to
the diagnostic laboratories within 24h. For carcass and meat samples. a carcass-swabbing
method using cotton surgical gauzes (10 cm 20 cm 5 mm) was applied to the whole
carcasses oI poultry. pork or beeI in the slaughterhouses. No carcass samples were taken Irom
animals Irom which rectal Iaecal samples were collected. Approximately 25 g oI minced meat
56
or cut meat was sampled in supermarkets. markets or restaurants and transported to the
laboratory in the same conditions described Ior Iaecal samples. Five to 20 samples were taken at
each animal Iarm. slaughterhouse. market or restaurant. Samples were analysed at Nong Lam
University laboratories and two laboratories oI the Provincial Departments oI Animal Health oI
Vietnam.

isolation
The bacterial culture procedure was carried out as described in standard ISO-6579
6
. BrieIly.
Iaecal or meat samples were mixed with BuIIer Peptone Water (BPW) at a 1:9 ratio. using a
stomacher Ior 1 min and incubated at 37
0
C Ior 18-24 h. Then. one ml and 0.1 ml oI the culture
was transIerred to 10 ml oI Mller-KauIImann Tetrathionate Broth (MKTT) and 10 ml oI
Rappaport Vassiliadis Soya Broth (RVS). respectively. and incubated at 42
0
C Ior 24 48 h. The
culture was then streaked onto Xylose Lysine Decarboxylase Agar (XLD) and Brilliant Green
Agar (BGA). Colonies suspected oI being Salmonella were transIerred to Nutrient Agar plates
and were biochemically characterized using Triple Sugar Iron Agar (TSI). Lysine
Decarboxylase Broth (LDC). Urea Agar. and Tryptone Broth. All media (except Lysine
Decarboxylase Broth supplied by Merck) were purchased Irom Oxoid (UK). II more than one
sample Irom a slaughterhouse. Iarm. market or supermarket was Salmonella positive. only one
isolate was randomly chosen and included in this study.

Sero typing and phage typing
Salmonella isolates were serotyped by slide and microtitre agglutination Ior O and H antigen
(the antisera were purchased Irom Statens Serum Institut. Denmark) according to the latest
version oI the KauIImann and White scheme
9
at the Diagnostic Labotarory Ior InIectious
Diseases and Perinatal Screening oI the Dutch National Institute oI Public Health and the
Environment (RIVM).

Bacteriophage typing was used Ior S. Typhimurium and S. Enteritidis. The pattern oI
lysis produced by inIection with typing phages was recorded and designated in accordance with
the standard schemes. UT indicates untypeable. which means that a tested isolate was not lysed
by any typing phages. RDNC indicates that the isolate reacted with some oI the typing phages
but did not conIorm to any recognized phage type in the typing scheme. S. Typhimurium phage
types were determined using the method described by Anderson et al.
1
and the interpretive
Table 1. Origins oI the investigated Salmonella isolates.
No. No. No. investigated isolates
Samples taken positive () Irom Iaeces
Irom
carcasses
Irom meat Total
Pigs 534 264 (49.4) 77 (14
*
) 23 11 111
Cattle 390 107 (27.4) 37 (16
*
) 16 10 63
Chicken 257 99 (38.5) 18 35 11 64
Ducks 34 7 (20.5) 3 0 0 3
* isolates Irom animals with diarrhoea
Chapter 2
57
guidelines supplied by the Public Health Laboratory Service (PHLS) in Colindale. United
Kingdom. In addition. the Dutch phage typing system was also used
5
. Salmonella Enteritidis
isolates were phage typed using the English phage typing system as described by Ward et al.
13
.

RESULTS

The percentage oI Salmonella-positive samples was 20.5. 27.4. 38.5 and 49.4 Ior duck. cattle.
chicken and pig samples. respectively (Table 1). We Iound 38 serovars among the 297
Salmonella isolates originating Irom humans and animals. The 10 most common serovars
represented approximately 79 oI the collection (Table 2). The predominant serovars
(S. Typhimurium. S. Anatum. S. Weltevreden. S. Emek. and S. Rissen) accounted Ior about 59
oI the isolates. The distribution oI Salmonella serovars among the diIIerent sources is shown in
Table 2. S. Typhimurium (37.5) was the most common serovar among the 56 Salmonella
isolates Irom humans. Iollowed by S. Enteritidis (12.5) and S. Weltevreden (7.1). In cattle.
serovar Anatum. Weltevreden and Lexington were predominant and represented 23.8. 17.5
and 15.8. oI the 63 bovine isolates. respectively. Among the 111 porcine isolates. S. Anatum
(26.1) and S. Typhimurium (20.7) were the most common serovars. Iollowed by
S. Weltevreden (15.3). S. Derby (11.7) and S. Rissen (11.7). S. Emek (38.8) and
S. Blockley (20.9) were the most prevalent serovars among 67 isolates originating Irom
poultry.

Table 2. Distribution oI the most common Salmonella serovars isolated in Vietnam Irom
humans. cattle. pigs and poultry.
Salmonella serovars Humans Cattle (*) Pigs (*) Poultry Total ()
Typhimurium 21 3 23 (3) - 47 (15.8)
Anatum 1 15 (7) 29 (4) 1 46 (15.5)
Weltevreden 4 11 (2) 17 (3) 2 34 (11.4)
Emek 2 26 28 (9.4)
Rissen 5 13 1 19 (6.4)
Derby 3 (1) 13 (1) 1 17 (5.7)
Blockley 1 14 15 (5.1)
S. (I).4. 5. 12: b.- 2 3 4 (2) 1 10 (3.4)
Lexington 10 (1) 10 (3.4)
Hadar 2 6 8 (2.7)
Newport 1 1 6 0 8 (2.7)
London - 1 2 4 7 (2.4)
Enteritidis 7 7 (2.4)
Albany 1 3 4 (1.3)
Panama 1 2 3 (1.0)
Rubislaw 3 (3) 3 (1.0)
Kedougou 2 2 (0.7)
Schwarzengrund 2 2 (0.7)
Tallahassee 2 2 (0.7)
Others 11 8 (2) 2 (1) 4 25 (8.3)
Total 56 63 111 67 297
* isolates Irom animals with diarrhoea
58
The distribution oI Salmonella
Typhimurium phage types is shown in
Table 3. OI the 47 S. Typhimurium
isolates. the Dutch phage type (pt) 90 was
predominant and accounted Ior 57.5 oI
all S. Typhimurium isolates Irom humans.
pigs and cattle. These 27 isolates could
not be typed with the English phage
typing system (see Table 3).
S. Typhimurium RDNC accounted Ior
nearly 20 oI the phage types oI this
serovar (by the Dutch system). The Dutch
phage type 506 (corresponding with
DT 104 in the English phage typing
system) was Iound at a low rate (4.3).
Among the S. Enteritidis isolates. two
phage types were Iound. pt 1 (n1) and
pt 7 (n2). Four isolates did not react with
any oI the phages used.

DISCUSSION

Surveillance oI Salmonella serovars and
phage-types Irom human and animal
sources is relevant Ior detecting national and global outbreaks. Ior identiIying the source oI an
inIection and Ior implementing prevention and control measures since the distribution oI
Salmonella serovars may diIIer between countries. To our knowledge. this is the Iirst study
comparing the serotypes and phage types oI human Salmonella isolates to those oI Iarm animals
in Vietnam. We observed that S. Typhimurium. S. Anatum. S. Weltevreden. and S. Emek were
the most common serovars in South Vietnam. S. Typhimurium was the most prevalent serovar
in isolates oI human origin. Pigs can be considered an important reservoir oI S. Typhimurium
since this serovar was Irequently isolated Irom pig Iaeces and pork. S. Anatum and
S. Weltevreden were isolated Irom humans. cattle. pigs and chickens. A similar Iinding was
reported Irom Thailand
3
. While S. Typhimurium and S. Dublin are known to be predominant
among isolates Irom cattle in Europe. Australia and America
12. 15
we Iound S. Anatum.
S. Weltevreden and S. Lexington to be the most prevalent bovine Salmonella serovars in
Vietnam. Among poultry isolates. S. Emek and S. Blockley predominated. S. Enteritidis was not
cultured Irom poultry. although S. Enteritidis was oIten isolated Irom humans in Vietnam. In
contrast. S. Enteritidis is Irequently isolated Irom poultry in many European countries and in the
USA and this animal species is an important source Ior human S. Enteritidis inIections in these
countries
12. 15
. Our Iindings are in accordance with those oI Tran et al.
11
who conducted an
investigation oI Salmonella spp in pigs. chickens and ducks in Mekong delta provinces. They
Iound only one S. Enteritidis isolate among 80 animal isolates. Thus. human S. Enteritidis
Table 3. Distribution oI Salmonella enterica
Typhimurium phage types in Vietnam.
No. oI
isolates
Source
English
phagetype
Dutch
phagetype
9 human UT
a
90
2 cattle UT 90
16 pig UT 90
1 cattle UT 510
1 pig UT 508
2 pig UT 507
1 human UT RDNC
b
1 human UT UT
1 pig UT UT
1 human 104 506
1 pig 104 506
1 human U302 507
2 human U302 RDNC
1 pig U302 RDNC
1 human 195 507
1 human 20 RDNC
2 human 12 RDNC
2 human RDNC RDNC
1 pig RDNC RDNC
a
UT untypeable
b
RDNC reaction does not conIorm to any recognized
phage types.
Chapter 2
59
inIections in Vietnam probably originate Irom other sources. Another explanation Ior the low
prevalence oI S. Enteritidis in the present study might be that 95 oI the poultry samples were
taken Irom broilers due to the restricted access oI poultry Iarms during the bird Ilu outbreak.
ThereIore only a small number oI laying hens were sampled. The percentage oI Salmonella
positive samples in our study is much higher than the 7.1 reported by Tran et al. One possible
explanation is the diIIerence in the amount oI sample used Ior culturing in their study. which
was 1g. whereas in our study 25g was sampled. Overall. the data Irom the present study indicate
that the distribution oI Salmonella serovars in Vietnam was similar to that oI other South-East
Asian countries
3. 14
but diIIerent Irom that oI European countries and the USA
8. 12. 15
. One
possible reason Ior this is the diIIerences in animal husbandry between the continents.
Determination oI the distribution oI Salmonella serovars is important in the epidemiology oI
salmonellosis. It provides evidence oI possible sources oI Iood borne inIection in a climate oI
increasing international travel and trade in Iood products oI animal origin and may lead to
improved prevention and control measures.

The Dutch pt 90 was the most common phage type among the S. Typhimurium isolates
in Vietnam and was Iound among bovine. porcine and human S. Typhimurium isolates. The
dominance oI this phage type is considerably diIIerent Irom that in European countries and
America where DT 104 (corresponding to pt 506 in the Dutch phage typing system) has been
common among S. Typhimurium isolates. The Dutch phage typing system perIormed better in
typing our Vietnamese S. Typhimurium isolates than the English phage typing system
(Table 3). Seventy-Iour percent oI the S. Typhimurium isolates could be phage-typed with the
Dutch typing system compared to 21 in the English typing system. It is important to note that
although the English phage typing method was able to type most oI the S. Typhimurium isolates
Irom European countries and the United States oI America
4. 8. 12
. this system could not type 77
(10/13) oI Turkish S. Typhimurium isolates
2
and 12.2 (27/221) oI Japanese S. Typhimurium
isolates
7
.

In conclusion. the distribution oI Salmonella serovars and phage types in Vietnam is
considerably diIIerent Irom that in Europe and America. The same serotypes and phage types
were Iound in humans and Iood animals with the exception oI S. Enteritidis. which would
suggest that Iarm animals are an important source oI human non-typhoid Salmonella inIection
in Vietnam.

ACKNOWLEDGMENTS

This study was Iunded by a grant oI the Vietnamese government to Proiect 322 oI the Ministry
oI Education and Training. We appreciate the help oI Associate ProI. Nguyen Ngoc Tuan. Dr.
Nguyen Nhu Pho. Le Huu Ngoc. Van Thien Bao. Huynh Van Diem. Nguyen Kim Hoang. and
physicians. veterinarians and technicians who assisted in the sampling procedures and isolation
oI Salmonella. We thank Dr. Nancy Bleumink and Carolien Flemming oI Utrecht University.
The Netherlands Ior assistance and technical guidance. We are grateIul to Linda Ward and the
60
staII oI the Public Health Laboratory Service (PHLS) in Colindale. United Kingdom Ior
providing the S. Typhimurium phages.

REFERENCES
1. Anderson. E.S.. L.R. Ward. M.J. Saxe. and J.D. de Sa. 1977. Bacteriophage-typing designations oI Salmonela
typhimurium. J. Hyg. (Lond) 78:297-300.
2. Ang-Kucuker. M.. V. Tolun. R. Helmuth. W. Rabsch. O. Buyukbaba-Boral. D. Torumkuney-Akbulut. S.
Susever. and O. Ang. 2000. Phage types. antibiotic susceptibilities and plasmid proIiles oI Salmonella
Typhimurium and Salmonella Enteritidis strains isolated in Istanbul. Turkey. Clin. Microbiol. InIect. 6:593-
599.
3. Bangtrakulnonth. A.. S. Pornreongwong. C. Pulsrikarn. P. Sawanpanyalert. R.S. Hendriksen. D.M.A. Lo Fo
Wong. and F.M. Aarestrup. 2004. Salmonella serovars Irom humans and other sources in Thailand. 1993-2002.
Emerg. InIect. Dis. 10:131-136.
53:997-1003.
5. Guinee. P.A.. W.J. van Leeuwen. and D. Pruys. 1974. Phage typing oI S. Typhimurium in The Netherlands. 1.
The phage typing system. Zentralbl. Bakteriol. 226:194-200.
6. International Organization Ior Standardization. 1993. ISO 6579- Microbiology- General guidance on methods
Ior detection oI Salmonella. 3rd. International Organization Ior Standardization. Geneva. Switzerland.
7. Izumiya. H.. J. Teraiima. S. Matsushita. K. Tamura. and H. Watanabe. 2001. Characterization oI multidrug-
resistant Salmonella enterica serovar Typhimurium isolated in Japan. J. Clin. Microbiol. 39:2700-2703.
8. Pasquali. F.. A. de Cesare. A. Ricci. C. Kehrenberg. S. Schwarz. and G. ManIreda. 2004. Phage types.
ribotypes and tetracycline resistance genes oI Salmonella enterica subsp. enterica serovar Typhimurium strains
isolated Irom diIIerence origins in Italy. Vet. Microbiol. 103:71-76.
9. PopoII. M.Y. 2001. Antigenic Iormulas oI the Salmonella serovars. 8th. WHO Collaborating Center Ior
ReIerence and Research on Salmonella. Paris. France.
10. Tirado. C.. and K. Schmidt. 2001. WHO surveillance programme Ior control oI Ioodborne inIections and
intoxications: preliminary results and trends across greater Europe. World Health Organization. J. InIect. 43:80-
84.
11. Tran. T.P.. T.L.K. Ly. T.T. Nguyen. M. Akiba. N. Ogasawara. D. Shinoda. T.A. Okatani. and H. Hayashidani.
2004. Prevalence oI Salmonella spp. in pigs. chickens and ducks in the Mekong Delta. Vietnam. J. Vet. Med.
Sci. 66:1011-1014.
12. van Duiikeren. E.. W.J.B. Wannet. D.J. Houwers. and W. van Pelt. 2002. Serotype and phage type distribution
oI Salmonella strains isolated Irom humans. cattle. pigs. and chickens in The Netherlands Irom 1984 to 2001. J.
13. Ward. L.R.. J.D. de Sa. and B. Rowe. 1987. A phage-typing scheme Ior Salmonella Enteritidis. Epidemiol.
InIect. 99:291-294.
14. WHO Global Salm-Surv. Top 15 Salmonella serotype list Irom each country.
http://thor.dIvI.dk/pls/portal/GSS.COUNTRYDATASETREP.show (23 March 2005. date last accessed).
15. Wray. C.. and A. Wray. 2000. Salmonella in domestic animals. CABI Publishing. New York. USA.

AntImIcrnbIa! rcsIstancc, c!ass 1 Intcgrnns
and a nnvc! varIant nf GcnnmIc Is!and 1
In Isn!atcs frnm VIctnam
An T. T. Vo
1,3
1
, Ad C. IIuil
2
,

Win
Caaslia
1

1
Dcpar|ncn| cf |nnunc|cgu and |nfcc|icus Discascs. |acu||u cf Vc|crinaru
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AnlinicioliaI Agenls and Chenolheiapv. nn !Inc 2 Ar. 2OO7

Drug resistance in non-typhoid Salmonella in Vietnam
62
ABSTRACT

The antimicrobial resistance pattern oI 297 epidemiologically unrelated non-typhoid Salmonella
isolates obtained Irom humans and animals in Vietnam was investigated by using a disk
diIIusion assay. The characteristics oI class 1 integrons. Salmonella Genomic Islands and the
horizontal transIer oI resistance determinants were investigated. Resistance to gentamicin.
kanamycin. chloramphenicol. streptomycin. trimethoprim. ampicillin. nalidixic acid.
sulIonamides. and tetracycline was Iound in 13 to 50 oI the isolates. Forty percent oI the
isolates were resistant to more than one antimicrobial agent. Nine distinct integron types were
detected in 28 oI the isolates belonging to 11 Salmonella serovars. Integrons were Iound Ior
the Iirst time in serovars Kedougou and Tallahassee. The integrons contained the aadA1.
aadA2. aadA5. bla
PSE-1
. bla
OXA-30.
dfrA1. dfrA12. dfrA17. and sat genes. as well as unknown
open reading Irames. Most integrons were associated with coniugative plasmids. which could
transIer their antimicrobial resistance determinants to Escherichia coli or Salmonella
Enteritidis. or with Salmonella Genomic Island 1 or its variants. The resistance gene cluster in
serovar Emek identiIied by PCR mapping and nucleotide sequencing contained a new SGI1
variant called SGI1-N. High-level resistance to Iluoroquinolones was Iound in 3 multiresistant
S. Typhimurium isolates and was associated with mutations in the gvrA gene leading to the
amino acid changes Ser83Phe and Asp87Asn.

Key words: antimicrobial resistance. Salmonella. integron. genomic island. Vietnam. humans.
animals.

Chapter 3
63
INTRODUCTION

Non-typhoid Salmonella inIection is one oI the main zoonotic diseases in developed
11. 13
and
developing countries
1. 44
. The ease with which people can travel between distant countries and
the exchange oI Iood between countries by global trade has contributed to the spread oI Iood-
borne diseases. Multidrug-resistant (MDR) Salmonella isolates are a direct threat to human
health when this multidrug resistance interIeres with treatment or an indirect threat when
resistance can be transIerred to other human pathogens
12
. ThereIore. antimicrobial susceptibility
monitoring is important Ior the detection oI resistant clinical isolates and Ior the surveillance oI
antimicrobial resistance.

A strong relationship between MDR Salmonella strains and the presence oI integrons
has been proved
29. 38
. Class 1 integrons are the most common integron type present in clinical
isolates oI the Iamily Enterobacteriaceae. Class 1 integrons. along with transIerable elements
like coniugative plasmids or transposons. play an important role in the carriage and
dissemination oI antimicrobial resistance genes due to their ability to incorporate or excise one
or more resistance gene cassettes
14. 21
. Recently. antibiotic resistance gene clusters in class 1
integrons located on the chromosomal Salmonella Genomic Island 1 (SGI1) have been
demonstrated in S. Typhimurium DT104
4
. The SGI1-associated MDR region consists oI a
complex integron carrying the aadA2. bla
PSE.
floR. tetR. and tet(G) genes. In several Salmonella
serovars. including strains oI S. Typhimurium DT104. a number oI SGI1 variants (SGI1-A to J)
have been detected
3. 7. 9. 31. 32
. SGI1 is transmissible. but only in the presence oI a helper plasmid.
This mobility oI SGI1 by coniugative mobilization may contribute to the spread oI antibiotic
resistance genes between diIIerent S. enterica serovars and between Salmonella and other
bacterial pathogens
8
.

The aims oI this study were to investigate (i) the antimicrobial resistance oI
Vietnamese Salmonella isolates collected Irom humans. livestock and meat (ii) the prevalence
and characteristics oI class 1 integrons in these isolates and (iii) the resistance gene clusters
present in SGI1.


Isolates
A total oI 297 epidemiologically unrelated isolates Irom Vietnam were investigated. The
isolates originated Irom humans (n56). cattle (n63). pigs (n111) and poultry (n 67). All
animal isolates were collected during the year 2004. The animal isolates were cultured Irom
Iaeces. carcasses and meat. Faecal samples Irom healthy animals were taken at slaughterhouses
(78) and Irom healthy or sick animals on Iarms (12) as previously described
42
. The animal
samples came Irom diIIerent Ilocks or herds. II more than one sample Irom a slaughterhouse.
Iarm. market or supermarket was Salmonella positive. only one isolates was randomly chosen
and included in this study. The 56 clinical human isolates oI unrelated patients with diarrhoea
and Iever were obtained Irom Iive provincial hospitals and two Pasteur Institutes in Vietnam.
64
These isolates have been isolated during the year 2004. The methods used Ior the isolation and
identiIication oI the isolates have been described
42
. The isolates included in the present study
belonged to 38 serovars oI Salmonella. S. Typhimurium. S. Anatum. S. Weltevreden. S. Emek
and S. Rissen were the most prevalent serovars. S. Typhimurium phage type 90 (in the Dutch
phage typing system). which has no recognized phage type in the English phage typing system.
was predominant among the S. Typhimurium isolates
19
.

Antimicrobial susceptibility testing
The antimicrobial susceptibility oI the isolates was determined according to the guidelines oI
the Clinical and Laboratory Standards Institute (CLSI). Iormerly NCCLS
34
. Agar diIIusion
assays were perIormed on Muller-Hinton agar and with disks containing 15 diIIerent
antimicrobial agents (Oxoid. UK). The antimicrobials tested (Table 1) were ampicillin
10 g (A). amoxicillin/clavulanic acid 30/15 g (Ac). ceIalothin 30 g (Ce). ceItazidime 30 g
(CI). chloramphenicol 30 g (C). ciproIloxacin 5 g (Ci). colistin 10 g (Co). gentamicin 10 g
(G). kanamycin 30 g (K). nalidixic acid 30 g (Na). norIloxacin 10 g (No). streptomycin
10 g (S). tetracycline 30 g (T). trimethoprim 5 g (Tp). and sulIonamides 300 g (Su). The
interpretive categories susceptible. intermediate or resistant were used according to the CLSI
guidelines
5
except Ior colistin where the zone criteria oI 11 (resistant) and 14mm
(susceptible) were used
17
. Escherichia coli ATCC 25922 and E. coli ATCC 35218 were used as
quality control organisms.

Detection of class 1 integrons
All isolates were tested Ior the presence oI class 1 integrons. The presence oI integrons was
determined by PCR ampliIication oI the class 1 integrase speciIic intI1 gene
28
. Template DNA
was obtained by the whole cell boiled lysate method as described elsewhere
30
. Integron gene
cassettes were detected by PCR using the 5`-CS and 3`-CS primer set as described previously
30
.
CS-PCR products were run on 0.7 agarose gels Ior at least 3 hours at 100 V and visualized
under UV-light aIter ethidium bromide staining. Since the 3`-CS Iragment oI class 1 integrons
is not always as conserved as its name indicates
20
. an integrase-positive isolate does not always
yield an amplicon in the CS-PCR. When this was the case. an integrase-inverted PCR was used
to characterise the gene cassettes. BrieIly. one g oI genomic DNA oI the isolate was cleaved
with the restriction endonuclease SphI. The Iragments were ligated and subiected to PCR using
int-OUT and CS-F primers. Since the smallest size Ior a gene cassette inserted into an integron
is about 400 bp only Iragments larger than 500 bp generated Irom the inverted PCR were
sequenced.

Characterization of integrons
CS-PCR products with the same size were puriIied using the QiaQuick PCR puriIication kit
(Qiagen. Germany) and analysed by restriction Iragment length polymorphism (RFLP). The
amplicons were digested with at least two diIIerent restriction endonucleases and considered
identical iI they showed the same RFLP patterns. The restriction endonucleases used were
HpaII. HincII. BclI. NciI. and EcoRI.
Chapter 3
65

Nucleotide sequencing of gene cassettes
One representative oI each RFLP type was randomly chosen Ior nucleotide sequencing. For
isolates with a unique integron. puriIied CS-PCR products were cloned in the pGEM-T easy
Vector (Promega. USA). Colonies carrying the inserted Iragment were picked Irom Luria
Bertani plates containing ampicillin (100 g/ml). 40 l (100 mM) IPTG and 40 l (2) X-Gal.
The inserted Iragments were obtained by PCR. using T7 and Sp6 primers under the same
conditions as Ior the CS-PCR. The ampliIication products were puriIied and sequenced. The T7
and Sp6 primers were used Ior sequencing both ends oI the diIIerent amplicons under study. In
addition. Ior the 2000 bp amplicon obtained by CS-PCR. an internal primer was used to
continue the sequencing until the resistance genes in each amplicon were identiIied. For isolates
carrying two integrons which only diIIered about 50 bp in size. CS-PCR products were also
cloned in the pGEM-T easy Vector. The two plasmids with diIIerent inserts were selected based
on restriction enzyme (EcoRI or HpaII) analysis and used Ior sequencing. Dideoxy sequencing
was perIormed on an ABI 3730 Sequencer. DNA sequences were analysed with the Clone
Manager Suite and by consulting the GenBank database via the BLAST network service. The
nucleotide sequences oI the gene cassettes have been deposited in the GenBank database under
the accession numbers shown in Table 2.

Bacterial conjugation
A coniugation experiment was perIormed as described
27
to determine whether the integrons oI
the Salmonella isolates could transIer their resistance determinants to another Salmonella
serovar or to another bacterial species (E. coli). RiIampicin-resistant (RiI
R
) and
sulIamethoxazole-susceptible (Sul
S
) E. coli K12 was used as recipient. All 83 integron carrying
Salmonella isolates were used as donors. In addition. a plasmid-Iree susceptible S. Enteritidis
isolate. which was made resistant to riIampicin. was also used as recipient. In this second
coniugation experiment. only Salmonella isolates which could transIer their resistance
determinants to E. coli in the Iirst coniugation experiment were used as donors. Both donor
(int-positive sulIamethoxazole-resistant Salmonella isolates) and acceptor bacteria were
cultured in LB broth until an OD
660
0.5-0.6 was reached. The mating process in which donor
(sulIamethoxazole-resistant isolates) and recipient were present in a 1:9 ratio (v/v) was
perIormed in LB broth. Incubation took place overnight in a water-bath at 37
0
C.
Transconiugants were selected by plating 50 l oI the mating culture on MacConkey (Oxoid.
UK) agar plates containing both riIampicin (50 g/ml) and sulIamethoxazole (512 g/ml).
Colonies were selected based on their resistance to both antimicrobials and puriIied by
subculture on MacConkey agar containing antibiotics and then nutrient agar (NA. Oxoid. UK)
without antibiotics. The transconiugants were tested Ior their biochemical characteristics. using
the API 20E system (bioMerieux. France) and the Salmonella transconiugants were serotyped
using antisera (Staten Serum Institute. Denmark) against antigens oI S. Enteritidis. The
transconiugants were tested as described above Ior their susceptibility patterns and the presence
oI class 1 integrons.

66
Detection of Genomic Island 1 and its variants
Integron-positive isolates (n12) Irom various Salmonella serovars were selected Ior analysis oI
the presence oI SGI1 on the basis oI their integron proIiles and antibiotic resistance patterns.
First. the isolates were examined Ior the presence oI the leIt and right iunction oI SGI1 by PCR.
Then the order oI the antibiotic resistance gene cluster was tested as described
7
. Template DNA
was prepared using the High Pure PCR Preparation Kit (Roche. Germany). The primers used
Ior ampliIication oI the leIt and right iunctions oI SGI1 and the linking sequences in the
antibiotic resistance gene cluster were previously described
7. 31
. The PCRs were carried out in a
total volume oI 25 l volumes containing 2.5 l oI 10 PCR buIIer (HT Biotechnology.
England). 0.5 l 10 deoxynucleotide triphosphate mix (2mM each). 50 pmol oI each primer.
1.25 U Taq DNA polymerase. and 1 l oI template DNA. To ampliIy Iragments larger than
3.5 kb. Taq Plus polymerase was used instead oI Taq DNA polymerase (HT Biotechnology.
England). Thermal cycling conditions consisted oI a hot start cycle oI 94
0
C Ior 3 min. Iollowed
by 35 cycles with: 1 min at 94
0
C. 1 min at 50 to 65
0
C (depending on the primers). 1 to 5 min at
72
0
C (depending on the expected amplicon size) and a Iinal step at 72
0
C Ior 10 min. The
expected sizes oI the PCR products were based on nucleotide sequences present in GenBank
under accession number AF261825. S. Typhimurium N216 carrying SGI1 and S. Albany N107
containing SGI1-F
41
were included as positive controls. Fragments oI S. Emek V14 generated
by PCR mapping (Figure 1) were either directly sequenced or cloned into the pGEM-easy
vector beIore sequencing to conIirm the gene identity and the linkage between the genes.

Determination of the circular extrachromosomal form of SGI1
From isolates harbouring SGI1 or its variant types. 8 representative isolates carrying SGI1 or
one oI its variants were randomly chosen and examined Ior the presence oI the circular
extrachromosomal Iorm oI SGI1 by PCR (SGIc-PCR). The PCR was perIormed as described
8

using primers oriented towards the leIt and right chromosomal SGI1 iunctions and plasmid
DNA extracted with the Qiagen plasmid midi kit (Qiagen. Germany) as template DNA. The
obtained PCR product was subsequently sequenced.

Fluoroquinolone resistance
Three Iluoroquinolone (norIloxacin/ciproIloxacin)-resistant Salmonella Typhimurium isolates
(V57. V58 and V60) were Iurther studied with respect to the resistance mechanism involved. A
PCR as described by Paauw et al
36
was used in order to investigate whether the class 1 integron-
associated- qnrA gene was present. A qnr-carrying Enterobacter cloacae strain
36
was used as
positive control. Since a mutation in the target enzyme Ior Iluoroquinolones. GyrA. is regularly
Iound in salmonellae
15. 18. 22. 33
. the 3 Iluoroquinolone-resistant isolates were subiected to allele-
speciIic PCR and RFLP analysis (AS-PCR-RFLP) as described
18
to detect mutations related to
quinolone resistance in codons 81. 83 and 87 oI the gvrA gene. The gvrA mutations were
conIirmed by nucleotide sequencing oI the products generated by PCR using GyrA-F and
GyrA-R as primers
18
.
Chapter 3
67

RESULTS

Phenotypical resistance. One hundred-and-ten (37) Vietnamese Salmonella isolates were
Iully susceptible to all 15 antimicrobials tested (Table 1). No ceItazidime-resistant isolate was
Iound. Nearly two thirds oI the collection (187 isolates) showed resistance to at least one
antimicrobial agent. More than 40 (n125) oI the isolates belonging to 17 serovars were
multidrug-resistant (resistant against 2 antimicrobials) (Table 1 and Table 3).

Table 1. Number oI resistant Salmonella isolates belonging to diIIerent serovars isolated Irom
humans. cattle. pigs and poultry in Vietnam by an agar diIIusion method
*
.

Sources/ serovars A Ac Ce CI S G K C Na No Ci T Su Tp Co
MDR
isolates
N ()
Human (56) 24

2 2

23 14 11 13 21 2 2 30 28 18
28 (50)
Typhimurium (21) 14 2 1 14 11 10 6 11 2 2 15 15 12
Enteritidis (7) 4 4 4 4
Emek (2) 2 2 2 2
Others (26) 6 1 5 3 1 5 8 11 7 4
Cattle (63) 18 2 1

6 2 2 3 10 1 1 19 7 4

12 (19)
Anatum (15) 12 1 2 1 9 14 2 2
Typhimurium (3) 3 1 3 2 2 2 1 1 1 3 3 2
Others (45) 3 1 1 2 2
Pig (111) 35

25 20 21 8 6 69 26 24
34
(30.6)
Anatum (29) 13 3 2 4 24 2 1
Typhimurium (23) 18 18 18 18 2 18 18 18
Derby (13) 2 2 2 2 1 2 13 2 2
Others (46) 2 2 1 3 14 4 3
Poultry (67) 9 2 3

23 2 14 34 54 30 40 35 1
51
(76.1)
Emek (26) 1 1 1 2 28 26 1 28 28
Blockley (14) 14 14 1 18 14 1 1
Albany (3) 3 1 3 3 2 3 3 1
Others (24) 5 1 2 8 2 7 13 8 3
TOTAL (297) 86 6 6 77 38 48 58 91 3 3 148 101 81 1 125 (42)
resistant
``
29 2.0 2 0 26 13 16 20 31 1 1.0 50 34 27 0.3

intermediate 1 8 2 0 19 0 0.3 5 8 0 0.6 9 0 0 0

susceptible 70 90 96 100 55 87 83 75 61 99 98.4 41 66 73 99.7

Abbreviations used: N. number oI the isolates tested A. ampicillin (10 g); Ac. amoxicillin /clavulanic acid (30 g);
Ce. cephalothin (30 g); CI. ceItazidime (30 g); S. streptomycin (10 g); G. gentamicin (10 g); K. kanamycin
(30 g); C. chloramphenicol (30 g). Na. nalidixic acid (30 g); No. norIloxacin (10 g); Ci. ciproIloxacin (5 g); T.
tetracycline (30 g); Su. sulIonamides (300 g); Tp. trimethoprim (5 g). Co. colistin (10 g); MDR. multidrug-
resistant.
*
The number oI isolates resistant to a particular antimicrobial agent is given below each antimicrobial.
**
The percentage oI the total number oI isolates resistant. intermediate resistant or susceptible Ior a particular
antimicrobial is indicated in the last three rows below each antimicrobial.

68
Resistance to six or more antimicrobials was Iound in 51 isolates (17). Resistance to
streptomycin. ampicillin. sulIonamides. trimethoprim. and tetracycline and was Iound in 26 to
50 oI the isolates. In addition. resistance to gentamicin was Iound among human (14) and
porcine (20) salmonellae. especially S. Typhimurium isolates. OI the poultry isolates. 80
were resistant against nalidixic acid. Three norIloxacin-resistant S. Typhimurium isolates were
Iound and all were isolates Irom humans (n3).

Integrons and gene cassettes. The prevalence oI class 1 integrons was high (28). Nine
diIIerent proIiles oI class 1 integrons (Table 2 and 3) were detected in 83 isolates belonging to
11 serovars including serovars Kedougou and Tallahassee. which have not been reported to
carry class 1 integrons beIore. The gene cassettes Iound in these integrons included the aadA1.
aadA2 and aadA5 genes encoding resistance to streptomycin and spectinomycin. the bla
PSE-1

and bla
OXA-30
genes conIerring resistance to -lactams. the dfrA1. dfrA12 and dfrA17 genes
encoding resistance against trimethoprim. the sat gene mediating streptothricin resistance and
open reading Irames encoding proteins oI an unknown Iunction.

Table 2. Characterization oI class 1 integrons oI Salmonella isolates Irom human and animal
origin in Vietnam.
IP
a Size in bp
(isolate ID)
RE
b
1 Fragments (bp) RE 2 Fragments (bp) Gene cassette
Accession
number
I 1010 (V237) EcoRI 561; 449 HpaII 411; 246; 138; 73; 61; 57; 24 aadA2 DQ238100

1197 HincII 703; 351; 143 HpaII 826; 371 blaPSE-1 DQ238099
1242 (V84) dfrA1.orfC DQ238102 II
1198

blaPSE-1 DQ238101
V 1010 (V171) EcoRI 561; 449 HpaII 411; 246; 138; 73; 61; 57; 24 aadA2 DQ238098
X -
XI 1242 (V14) HincII 656; 490; 96 HpaII 762; 480 dfrA1. orfC DQ238097
XII 1914 (V80) HincII 1303; 611 HpaII 538; 464; 246; 196; 138; 116;
73; 57; 24
aadA2. orfF. dfrA12 DQ238105
1700 (V57) dfrA17. aadA5
1914 aadA2. orfF. dfrA12
XIII
2010 blaOXA-30. aadA1 DQ861642
1914 (V58) aadA2. orfF. dfrA12 XIV
2010 blaOXA-30. aadA1
XV 627 (V48) BclI 480; 147 NciI 351; 151; 125 sat (partial) DQ284538

a
integron proIile nomenclature Iollowed that Irom a previous study
41
. ProIiles (XI-XV) are designated in this study.
b
RE: restriction endonuclease
- no product obtained in CS-PCR or inverted PCR

Phenotypic resistance to a certain antimicrobial drug was observed in all isolates
carrying the corresponding gene cassettes. The transIer oI the integrons and the antimicrobial
resistance determinants (AAcGKSTSuTp. CSTSuTp. and CSSu) to E. coli was possible Irom
17 oI the 83 integron-positive isolates oI serovar Typhimurium. Anatum and Agona.
respectively (Table 3). Ten S. Typhimurium isolates Irom 17 isolates tested could transIer their
integrons and resistance determinants to S. Enteritidis. This was demonstrated by the Iact that
Chapter 3
69
the E. coli and S. Enteriditis transconiugants were int-positive and obtained the phenotypic
resistance patterns oI the donors.

Genomic Island 1. In serovar Typhimurium. Derby. Albany. and Tallahassee.
SGI1 and the variants SGI1-C and SGI1-F were Iound (Table 3). The three S. Emek isolates
with the phenotypic resistance pattern CNaSSuTp. CNaTpSu. and AAcCeCNaTTpSu (V14.
V28 and V116. respectively) were positive in the PCRs Ior SGI1-J
31
but negative in the PCR
using the DB-T1/MDR-B primers which suggests a new variant oI SGI1 without a transposase
in the IS6100 element as described in SGI1-J
31
(Figure 1). In detail. these S. Emek strains
yielded positive PCR results Ior the leIt iunction oI SGI1. For the right iunction oI SGI1. iI a
primer in the sequence oI the vidY gene was used as reverse primer. the PCR results were
positive but negative iI a primer in the sequence oI the int2 gene was used. The dfrA1. orfC.
sul1. floR. tet. orf1. orf2 genes were present. PCR products oI the expected sizes were obtained
Irom PCRs using primers to ampliIy the Iragments between the genes as indicated in Figure 1.
S. Emek isolate V14 was also used to Iurther investigate the Iragments generated by PCR using
nucleotide sequencing. Sequencing data revealed that this isolate had a similar gene cluster as
described Ior SGI1-J
31
but it does not contain IS6100. The sequence oI a Iragment oI
approximately 750 bp obtained by PCR using the sul-OUT/ S044 primer showed homology
with a gene encoding the sul1delta Iusion protein oI the S. Typhimurium carrying SGI1
4
and oI
Acinetobacter baumannii
16
. We propose the name SGI1-N Ior the variant Iound in the S. Emek
V14 isolate.

Circular form of SGI1. To study the possible excision oI SGI1 Irom the genome and its
presence as a circular Iorm. a PCR speciIic Ior the circular Iorm oI SGI1 was used. The SGI1 in
3 S. Typhimurium isolates proved to be present in its circular Iorm. Nucleotide sequencing
showed that the Iragment oI ca. 430 bp obtained by SGIc-PCR Irom a SGI1 carrying
S. Typhimurium isolate (V54) was identical to the sequence oI the S004 gene. the right end oI
SGI1 up to the insertion site oI the cryptic retronphage
8
in the sequence deposited in the
GenBank under accession number AF261825.2. No PCR product indicating the presence oI the
circular Iorm oI SGI1 was obtained with DNA Irom other serovars.

Fluoroquinolone resistance. No qnrA gene was Iound in the three norIloxacin/ciproIloxacin-
resistant Salmonella Typhimurium isolates (V57. V58 and V60). However. AS-PCR-RFLP
revealed that all 3 isolates had double point mutations in their gvrA gene (Table 5) at Ser-83 and
Asp-87. Nucleotide sequencing oI the Iragments. spanning the 'Quinolone Resistance
Determining Region (QRDR) showed a substitution in the codon TCC (Ser) at position to 83
TTC (Phe) and in the codon GAC (Asp) at position 87 to AAC (Asn).

70
Table 3. Antimicrobial resistance characteristics oI MDR Salmonella isolates Irom human and
animal origin in Vietnam.
Multidrug-resistance patterns Serovars (animal /human isolates)

IP types
Coniugation
Ec SE
SGI 1
Type Excision
SSu Derby (2/0) V(2)
a

- nt SGI1-C nt
ATNa Anatum (13/0) - nt nt nt nt
CSSu Agona (1/0) X (1) (1) - - nt
STSu London (3/0) - nt nt nt nt
ACTNa Anatum (1/0) - nt nt nt nt
ASSuGSt Kedougou (0/1) XV (1) - nt - nt
ASTSu Enteritidis (0/4) - nt nt nt nt
CSuTpNa Emek (23/1) XI (24) - nt SGI1-N -
CTSuTp Panama (1/0) - nt nt nt nt
STKNa Blockley (13/0) - nt nt nt nt
STSuNa Tm pt 507 (0/1). Hadar (0/1) - nt nt nt nt
ACSuTpNa Albany (1/0) II (1) - nt SGI1-F -
ACTSSu Tm pt 506 (0/1) I (1) - nt SGI1
ACTSuTp Panama (1/1) X (2) - nt - nt
ATSuTpNa Anatum (1/0) X (1) - nt - nt
CSSuTpNa Emek (0/1) XI (1) - nt SGI1-N -
CSuTpGNa Emek (2/0) XI (2) - nt - nt
CSTKNa Blockley (0/1) - nt nt nt
CSTSuTp Anatum (1/0) XII (1) (1) nt - nt
ACSTSuG Kedougou (0/1) X (1) - nt - nt
ACSTSuNa Tm RDNC (0/1). Tm pt 506 (1/0) I (2) - nt SGI1
ASTSuGNa Tm RDNC (0/1) I (1) - nt SGI1
ASTSuTpG Tm 90 (4/0) XII (4) (1) (1) - nt
ASTSuTpNa Schwarzengrund (1/0) XII (1) - nt - nt
ACSTSuTpNa Albany (1/1) II (2) - nt SGI1-F -
ATSuTpAcCeNa Schwarzengrund (1/0) XII (1) - nt - nt
ACSTSuTpNa Tallahassee (2/0) II (2) - nt SGI1-F -
CSTSuTpKNa Blockley (1/0) - nt nt nt nt
ASTSuTpGK
Tm pt 90 (8/6). Tm pt 507 (2/0).
Tm RDNC (1/0). Tm pt 510 (1/0)
XII (18) (10) (6) - nt
ASTSuTpGNa S.enterica (I) 4. (5) 12:1: - (0/1) XII (1) - nt - nt
ASTSuTpGNa Tm UT (1/0) XII (1) (1) (1) - nt
ACSTSuTpCeNa Anatum (1/0) V (1) - nt - nt
ACTSuTpAcCeNa Emek (1/0) XI (1) - nt SGI1-N -
ACSTSuTpCoNa Albany (1/0) II (1) - nt SGI1-F -
ASTSuTpGKNa Tm 90 (1/1) XII (2) (1) (1) - nt
ACSTSuTpGK Derby (1/0) XII (1) - nt - nt
ACSTSuTpGK Tm 90 (2/0) XII (2) (1) (1) - nt
ACSTSuTpGNa Tm 90 (1/0) - nt nt nt nt
ASTSuTpKGNa Tm 90 (1/0). Tm RDNC (1/0) - nt nt nt nt
ACSTSuTpAcGKNaNoCi Tm 507 (0/1). Tm UT (0/1) XIV (2) (1) - - nt
ACSTSuTpAcCeGKNaNoCi Tm UT (0/1) XIII (1) - nt - nt

Abbreviations used: A. ampicillin; C. chloramphenicol; S. streptomycin; St. streptothricin; T. tetracycline; Su.
sulIonamides; Tp. trimethoprim. Ac. amoxicillin; Ce. cephalothin; G. gentamicin; K. kanamycin; Na. nalidixic acid;
No. norIloxacin; Ci. ciproIloxacin; Co. colistin.
Tm. Typhimurium; RDNC. reaction does not conIorm to any recognized phage types; UT. untypeable phage;
Ec. E. coli as the recipient; SE. S. Enteritidis as the recipient; QRDR. quinolone resistance determining region.
- not Iound; nt. not tested.
a
number in brackets: number oI isolate(s).
Chapter 3
71
DISCUSSION

To date very little data have been published on antimicrobial resistance among non-typhoidal
Salmonella serovars Irom Vietnam
25
. A phenotypic resistance study is the Iirst step oI such an
antimicrobial resistance investigation. The data Irom the present study indicated a high rate oI
antimicrobial resistance among Vietnamese Salmonella isolates. More than halI oI the isolates
showed resistance to at least one antibiotic. The resistance percentages to chloramphenicol.
streptomycin. ampicillin. sulIonamides. and tetracycline Iound in the present study were
comparable to those Iound in other countries
10. 11. 40
and can thereIore be considered a
worldwide problem. The high rate oI resistance oI the Vietnamese isolates against
aminoglycosides (gentamicin and kanamycin) and trimethoprim diIIers Irom the low rate oI
resistance against these antimicrobials among salmonellae isolated in 10 European countries
40
.
An explanation Ior this observation may be the increasing and inappropriate use oI antibiotics
during the last ten years in Vietnam especially in the intensive animal husbandry in which
antibiotics are being used on a large scale Ior prophylaxis. as growth enhancer. and Ior therapy.
In 2002. gentamicin and trimethoprim. Ior example. were used Irequently in animal husbandry
in Vietnam
6
.

The prevalence oI integrons Iound in Salmonella varies Irom country to country and
depends on the origin oI the isolates. II both human and animal Salmonella isolates are
included. 28. 20. and 16 oI the Vietnamese. English. and Dutch non-typhoid salmonellae
isolates. respectively. were Iound to carry class 1 integrons as demonstrated in this study and in
the literature
38. 41
. Among the 9 proIiles oI class 1 integrons Iound. gene cassettes encoding
resistance to aminoglycosides (aadA1. aadA2. and aadA5). -lactams (bla
PSE-1.
bla
OXA-30
) and
trimethoprim (dfrA1. dfrA12. and dfrA17) were Irequently detected. The data oI the genotypic
and phenotypic resistance assays in the present study indicated that apparently there is a
relationship between the use oI these antimicrobials in the last decades in human medicine and
in the veterinary sector in Vietnam. In addition. the sat gene encoding resistance to
streptothricin was detected.

An important observation in the current study was the high prevalence oI class 1
integrons. especially in S. Typhimurium pt 90. In this study an integron oI about 1.95 kb with
the aadA2. orfF and dfrA12 genes was the predominant integron proIile detected in
S. Typhimurium pt 90. S. Schwarzengrund. S. Anatum and S. Derby isolates. This type oI
class 1 integron has also been detected in S. Cholerasuis in Taiwan
24
. and S. Gallinarum in
Korea
26
. in S. Schwarzengrund Irom catIish and squid roll imported Irom Thailand and Taiwan.
respectively. to the United States
47
. During the same period. in European countries and the
United States. human and animal S. Typhimurium strains (especially DT104) with the two
integrons oI the aadA2 and bla
PSE-1
genes were the most prevalent type
35. 43. 46
. Thus. diIIerent
types oI integrons can be dominant in diIIerent geographic regions. Also in this study.
S. Typhimurium pt 90 isolates carried integrons and antibiotic resistance determinants against 7
to 8 diIIerent antimicrobials that could be transIerred to S. Enteritidis and to E. coli.
S. Typhimurium pt 90 is the most common phage type in Vietnam
42
. This suggests that
72
S. Typhimurium pt 90 may play an important role in the spread oI class 1 integrons and
antimicrobial resistance determinants among Enterobacteriaceae in this country. Remarkably.
this is the Iirst time that 3 integrons (with amplicons oI 1.7 kb. 1.95 kb and 2.0 kb) were
detected in a single isolate (V57). This isolate was cultured Irom a serious case oI human
salmonellosis in Ho Chi Minh City. The isolate is classiIied as Salmonella Typhimurium U320
in the English phage typing system. The isolate was resistant to 13 antimicrobials including the
Iluoroquinolones. The spread oI such a Salmonella strains is hazardous and should be
controlled.

Resistance to nalidixic acid (35) and decreased susceptibility to Iluoroquinolones
(15) oI the isolates in the present study were even higher than in other Asian countries
11. 25
.
Resistance to relatively new antimicrobials like norIloxacin and ciproIloxacin was Iound only
among the invasive human Salmonella isolates. This is oI particular concern because
ciproIloxacin is the drug oI choice Ior the treatment oI human Salmonella inIections. Mutations
leading to substitutions at amino acid 83 and 87 oI the QRDR may be in part responsible Ior the
high level oI resistance to Iluoroquinolones (MIC norIloxacin 32-64 g/ ml) among the 3
MDR resistant Salmonella Typhimurium isolates. These mutations lead to the substitution oI
Ser Ior Phe and Asp Ior Asn. at positions 83 and 87. respectively. This is the Iirst report on
mutations in two codons in gvrA oI Vietnamese salmonellae. Similar mutations have been
Iound in S. Cholerasuis isolated Irom pigs in Taiwan
24
. It is important to note that the
acquisition oI Iluoroquinolone resistance in Salmonella requires the stepwise accumulation oI
gvrA mutations or the overexpression oI eIIlux pumps
45
. A single mutation in gvrA oI
Salmonella can be suIIicient to cause high-level resistance to nalidixic acid but additional
mutations are required to attain high-levels oI resistance to Iluoroquinolones
39
. Mutations in
two codons are rarely Iound among Iield isolates oI Salmonella while mutations at either Ser83
and or Asp87 are very commonly observed
23
. Resistance to antimicrobials in human Salmonella
isolates can be the result oI antibiotic misuse in human medicine: in Vietnam patients can easily
buy antimicrobial drugs in any pharmacy without a prescription and stop treatment at any time.
In addition. abuse oI antibiotics in veterinary practice may have an important inIluence on
selection oI Iluoroquinolone-resistant Salmonella isolates.

Unlike plasmid-mediated resistance. which may disappear in the absence oI selective
pressure. chromosomally mediated resistance is oIten maintained. Many MDR Salmonella
isolates in this study contained SGI1 or one oI its variants. This study is the Iirst to document
the presence oI class 1 integrons in serovar Tallahassee and Kedougou. as well as oI SGI1-C in
serovar Tallahassee. Thus worldwide. class 1 integrons and SGI1 are more and more recognized
as signiIicant determinants oI multiple drug resistance in an increasing number oI Salmonella
serovars. S. Emek is one oI the dominant serovars Iound in poultry in Vietnam
42
. Strikingly. a
new SGI1 variant in a S. Emek isolate is deIined. This genomic island is comprised oI a
resistance gene cluster dfrA1-sul1-floR-tet(G)-sul1 although no phenotypic resistance to
tetracycline and chloramIenicol was Iound in the S. Emek isolate. The antibiotic resistance gene
cluster oI SGI1 is constituted oI a complex class 1 integron that belongs to the In4 group
3
. The
multidrug resistance region is deIined as the Iragment between the tnpR and IS6100 sequences
Chapter 3
73
in SGI1 and its variants
2
. In the In4 group oI integrons. a 3`-CS (conserved segment) including
a copy oI IS6100 but not oI the transposition gene has been detected
37
. Our Iinding oI the novel
variant oI SGI1 (SGI1-N) contributes to the hypothesis that the transIer oI SGI1 as well as
deletion and insertion events
3
. are responsible Ior the spread and the diversity oI SGI1 among
Salmonella serovars
3. 7. 31
. In the present study. SGI1 was detected as circular extrachromosomal
DNA in S. Typhimurium DT104 isolates but not in other SGI1 carrying serovars. This suggests
that S. Typhimurium DT104 may play a key role in the spread oI SGI1 among Salmonella
serovars because the extrachromosomal circular intermediate oI SGI1 can be transIerred in the
presence oI a helper plasmid providing the mating apparatus as described previously
8
.

Fig. 1. Map oI a novel variant SGI1-N Iound in S. Emek (V14) in the present study. The map oI
SGI1-J (Levings et al.. 2005) is given Ior comparison. Fragments in bold arrows were
conIirmed by nucleotide sequencing. The gray shades indicate resistance genes.

In summary. high rates oI multidrug resistance and oI the presence oI integrons Iound
among the Salmonella isolates in this study suggests that legislation to enIorce a more prudent
use oI antibiotics in both human and veterinary medicine should be implemented by the
authorities in Vietnam. The association oI antimicrobial resistance determinants with
transIerable elements may promote the rapid dissemination oI antibiotic resistance among
Enterobacteriaceae. The diversity oI transIerable and novel multiresistance determinants
observed in Salmonella serovars indicates that international co-operation is needed in order to
limit the emergence and the spread oI MDR Salmonella isolates. especially in the context oI
increased international travel and trade in Iood products oI animal origin.

ACKNOWLEDGMENTS

This study was supported by a grant Irom the Vietnamese government in the Proiect 322. We
thank Hans Mittenburg oI AST Farma B.V. Ior kindly providing clavulanic acid. and
AnneMarie Verel oI Eiikman-Winkler Institute Ior technical assistance with MIC
determination.

500 bp 500 bp

SGI1-N thdf int intI1 dfrA1 orfC qac sulI floR tetR tetC orf1 orf2 groEL/intI1 qac sul1 S044 vidY
SGI1-J thdf int intI1 dfrA1 orfC qac sulI floR tetR tetC orf1 orf2 groEL/intI1 qac sul1 orf 5 orf 1 S044 vidY

74
REFERENCES
1. Bangtrakulnonth. A.. S. Pornreongwong. C. Pulsrikarn. P. Sawanpanyalert. R.S. Hendriksen. D.M.A. Lo Fo
Wong. and F.M. Aarestrup. 2004. Salmonella serovars Irom humans and other sources in Thailand. 1993-
2002. Emerg. InIect. Dis. 10:131-136.
2. Boyd. D.. G.A. Peters. A. Cloeckaert. K.S. Boumedine. E. Chaslus-Dancla. H. Imberechts. and M.R.
Mulvey. 2001. Complete nucleotide sequence oI a 43-kilobase genomic island associated with the multidrug
resistance region oI Salmonella enterica serovar Typhimurium DT104 and its identiIication in phage type
DT120 and serovar Agona. J. Bacteriol. 183:5725-5732.
3. Boyd. D.. A. Cloeckaert. E. Chaslus-Dancla. and M.R. Mulvey. 2002. Characterization oI variant
Salmonella Genomic Island 1 multidrug resistance regions Irom serovars Typhimurium DT104 and Agona.
4. Boyd. D.A.. G.A. Peters. L. Ng. and M.R. Mulvey. 2000. Partial characterization oI a genomic island
associated with the multidrug resistance region oI Salmonella enterica Typhimurium DT104. FEMS
5. CLSI. M100 S14. PerIormance standards Ior antimicrobial susceptibility testing; Iourteenth inIormational
supplement.). Place. 2004.
6. Dinh. T.T.. N.T. Nguyen. T.T.A. Vo. T.H. Le. B.L. Vo. and T.N. Khuong. 2003. Initial survey on the use oI
antibiotics in Iarms and antibiotic residues in pork and chicken meat in Binh Duong Province. J. Vet. Sci.
Tech. 10:50-58. (in Vietnamese).
9. Ebner. P.. K. Garner. and A. Mathew. 2004. Class 1 integrons in various Salmonella enterica serovars
isolated Irom animals and identiIication oI genomic island SGI1 in Salmonella enterica var. Meleagridis. J.
Antimicrob. Chemother. 53:1004-1009.
11. Esaki. H.. A. Morioka. K. Ishihara. A. Koiima. S. Shiroki. Y. Tamura. and T. Takahashi. 2004.
Antimicrobial susceptibility oI Salmonella isolated Irom cattle. swine and poultry (2001-2002): report Irom
the Japanese Veterinary Antimicrobial Resistance Monitoring Program. J. Antimicrob. Chemother. 53:266-
270.
12. FAO. OIE. and WHO. Joint Iirst FAO/OIE/WHO expert workshop on non-human antimicrobial usage and
antimicrobial resistance: scientiIic assessment. http://www.who.int/IoodsaIety/publications/micro/en/amr.pdI
(25 February 2005. date last accessed).
13. Fisher. I.S.T.. and E.J. ThrelIall. 2005. The Enter-net and Salm-gene databases oI Ioodborne bacterial
pathogens that cause human inIections in Europe and beyond: an international collaboration in surveillance
and the development oI intervention strategies. Epidemiol. InIect. 133:1-7.
15. Fluit. A.C.. M.R. Visser. and F.J. Schmitz. 2001. Molecular detection oI antimicrobial resistance. Clin.
16. Fournier. P.E.. D. Vallenet. V. Barbe. S. Audic. H. Ogata. L. Poirel. H. Richet. C. Robert. S. Mangenot. C.
Abergel. P. Nordmann. J. Weissenbach. D. Raoult. and J.M. Claverie. 2006. Comparative genomics oI
multidrug resistance in Acinetobacter baumannii. PLoS Genet 2:e7.
17. Gales. A.C.. A.O. Reis. and R.N. Jones. 2001. Contemporary assessment oI antimicrobial susceptibility
testing methods Ior polymyxin B and colistin: review oI available interpretative criteria and quality control
guidelines. J. Clin. Microbiol. 39:183-190.
18. Giraud. E.. A. Brisabois. J.L. Martel. and E. Chaslus-Dancla. 1999. Comparative studies oI mutations in
animal isolates and experimental in vitro- and in vivo-selected mutants oI Salmonella spp. suggest a
Chapter 3
75
counterselection oI highly Iluoroquinolone-resistant strains in the Iield. Antimicrob. Agents Chemother.
43:2131-2137.
19. Guinee. P.A.. W.J. van Leeuwen. and D. Pruys. 1974. Phage typing oI S. Typhimurium in The Netherlands.
1. The phage typing system. Zentralbl. Bakteriol. 226:194-200.
20. Hall. R.M.. H.J. Brown. D.E. Brookes. and H.W. Stokes. 1994. Integrons Iound in diIIerent locations have
identical 5' ends but variable 3' ends. J. Bacteriol. 176:6286-6294.
21. Hall. R.M.. and C.M. Collis. 1995. Mobile gene cassettes and integrons: capture and spread oI class 1
integrons among Salmonella serotypes. Mol. Microbiol. 15:593-600.
22. Hooper. D.C. 1999. Mechanisms oI Iluoroquinolone resistance. Drug Resist. Updat. 2:38-55.
23. Hopkins. K.L.. R.H. Davies. and E.J. ThrelIall. 2005. Mechanisms oI quinolone resistance in Escherichia
coli and Salmonella: recent developments. Int. J. Antimicrob. Agents 25:358-373.
24. Huang. T.M.. Y.F. Chang. and C.F. Chang. 2004. Detection oI mutations in the gvrA gene and class I
integron Irom quinolone-resistant Salmonella enterica serovar Choleraesuis isolates in Taiwan. Vet.
Microbiol. 100:247-254.
25. Isenbarger. D.W.. C.W. Hoge. A. Sriian. C. Pitarangsi. N. Vithayasai. L. Bodhidatta. K.W. Hickey. and P.D.
Cam. 2002. Comparative antibiotic resistance oI diarrheal pathogens Irom Vietnam and Thailand. 1996-
1999. Emerg. InIect. Dis. 8:175-180.
26. Kwon. H.J.. T.E. Kim. S.H. Cho. J.G. Seol. B.J. Kim. J.W. Hyun. K.Y. Park. S.J. Kim. and H.S. Yoo. 2002.
Distribution and characterization oI class 1 integrons in Salmonella enterica serotype Gallinarum biotype
Gallinarum. Vet. Microbiol. 89:303-309.
27. Leverstein-van Hall. M.A.. A.T.A. Box. H.E.M. Blok. A. Paauw. A.C. Fluit. and J. VerhoeI. 2002. Evidence
oI extensive interspecies transIer oI integron-mediated antimicrobial resistance genes among multidrug-
resistant Enterobacteriaceae in a clinical setting. J. InIect. Dis. 186:49-56.
28. Leverstein-Van Hall. M.A.. A. Paauw. A.T.A. Box. H.E.M. Blok. J. VerhoeI. and A.C. Fluit. 2002. Presence
oI integron-associated resistance in the community is widespread and contributes to multidrug resistance in
the hospital. J. Clin. Microbiol. 40:3038-3040.
29. Leverstein-van Hall. M.A.. H.E.M. Blok. A.R.T. Donders. A. Paauw. A.C. Fluit. and J. VerhoeI. 2003.
Multidrug resistance among Enterobacteriaceae is strongly associated with the presence oI integrons and is
independent oI species or isolate origin. J. InIect. Dis. 187:251-259.
30. Levesque. C.. L. Piche. C. Larose. and P.H. Roy. 1995. PCR mapping oI integrons reveals several novel
combinations oI resistance genes. Antimicrob. Agents Chemother. 39:185-191.
31. Levings. R.S.. D. LightIoot. S.R. Partridge. R.M. Hall. and S.P. Diordievic. 2005. The Genomic Island
SGI1. containing the multiple antibiotic resistance region oI Salmonella enterica serovar Typhimurium
DT104 or variants oI it. is widely distributed in other S. enterica serovars. J. Bacteriol. 187:4401-4409.
32. Meunier. D.. D. Boyd. M.R. Mulvey. S. Baucheron. C. Mammina. A. Nastasi. E. Chaslus-Dancla. and A.
Cloeckaert. 2002. Salmonella enterica serotype Typhimurium DT-104 antibiotic resistance genomic island I
in serotype Paratyphi B. Emerg. InIect. Dis. 8:430-433.
33. Miko. A.. K. Pries. A. Schroeter. and R. Helmuth. 2005. Molecular mechanisms oI resistance in multidrug-
resistant serovars oI Salmonella enterica isolated Irom Ioods in Germany. J. Antimicrob. Chemother.
56:1025-1033.
34. National Committee Ior Clinical Laboratory Standards. 2001. PerIormance standards Ior antimicrobial disk
and dilution susceptibility tests. M31-A2- Approved standard. 2nd. NCCLS. Wayne. PA. USA.
35. O'Mahony. R.. M. Saugy. N. Leonard. D. Drudy. B. Bradshaw. J. Egan. P. Whyte. M. O'Mahony. P. Wall.
and S. Fanning. 2005. Antimicrobial resistance in isolates oI Salmonella spp. Irom pigs and the
characterization oI an S. InIantis gene cassette. Foodborne Pathog. Dis. 2:274-281.
37. Partridge. S.R.. G.D. Recchia. H.W. Stokes. and R.M. Hall. 2001. Family oI class 1 integrons related to In4
Irom Tn1696. Antimicrob. Agents Chemother. 45:3014-3020.
76
38. Randall. L.P.. S.W. Cooles. M.K. Osborn. L.J.V. Piddock. and M.J. Woodward. 2004. Antibiotic resistance
genes. integrons and multiple antibiotic resistance in thirty-Iive serotypes oI Salmonella enterica isolated
Irom humans and animals in the UK. J. Antimicrob. Chemother. 53:208-216.
39. Ruiz. J.. D. Castro. P. Goni. J.A. Santamaria. J.J. Borrego. and J. Vila. 1997. Analysis oI the mechanism oI
quinolone resistance in nalidixic acid-resistant clinical isolates oI Salmonella serotype Typhimurium. J.
Med. Microbiol. 46:623-628.
40. ThrelIall. E.J.. I.S.T. Fisher. C. Berghold. P. Gerner-Smidt. H. Tschape. M. Cormican. I. Luzzi. F.
Schnieder. W. Wannet. J. Machado. and G. Edwards. 2003. Antimicrobial drug resistance in isolates oI
Salmonella enterica Irom cases oI salmonellosis in humans in Europe in 2000: results oI international multi-
centre surveillance. Euro. Surveill. 8:41-45.
41. Vo. A.T.T.. E. van Duiikeren. A.C. Fluit. and W. Gaastra. 2006. Antibiotic resistance. integrons and
Genomic Island SGI1 among non-typhoid Salmonella serovars in The Netherlands. Int. J. Antimicrob.
Agents 28:172-179.
42. Vo. A.T.T.. E. van Duiikeren. A.C. Fluit. M.E.O.C. Heck. A. Verbruggen. H.M.E. Maas. and W. Gaastra.
2006. Distribution oI Salmonella enterica serovars Irom humans. livestock and meat in Vietnam and the
dominance oI Salmonella Typhimurium phage type 90. Vet. Microbiol. 113:153-158.
Multidrug resistance in Salmonella enterica serotype Typhimurium Irom humans in France (1993 to 2003).
44. Yang. S.J.. K.Y. Park. S.H. Kim. K.M. No. T.E. Besser. H.S. Yoo. B.K. Lee. and Y.H. Park. 2002.
Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated Irom animals
in Korea: comparison oI phenotypic and genotypic resistance characterization. Vet. Microbiol. 86:295-301.
45. Zhang. Q.. J. Lin. and S. Pereira. 2003. Fluoroquinolone-resistant Campvlobacter in animal reservoirs:
dynamics oI development. resistance mechanisms and ecological Iitness. Anim. Health Res. Rev. 4:63-71.
46. Zhao. S.. P.J. Fedorka-Cray. S. Friedman. P.F. McDermott. R.D. Walker. S. Qaiyumi. S.L. Foley. S.K.
Hubert. S. Ayers. L. English. D.A. Dargatz. B. Salamone. and D.G. White. 2005. Characterization oI
Salmonella Typhimurium oI animal origin obtained Irom the National Antimicrobial Resistance Monitoring
System. Foodborne Pathog. Dis. 2:169-181.
47. Zhao. S.. P.F. McDermott. S. Friedman. S. Qaiyumi. J. Abbott. C. Kiessling. S. Ayers. R. Singh. S. Hubert.
J. SoIos. and D.G. White. 2006. Characterization oI antimicrobial-resistant Salmonella isolated Irom
imported Ioods. J. Food Prot. 69:500-507.

AntIbIntIc rcsIstancc, Intcgrnns and
GcnnmIc Is!and amnng
nnn-tvhnIda! 5crnvars
In Thc Ncthcr!ands

An T. T. Vo
1,4
1
, Ad C. IIuil
2
,

Win I. .
Wannel
3
, Anjo Veiliuggen
3
, Hennv M. L. Maas
3
,

Win
Caaslia
1

1
Dcpar|ncn| cf |nnunc|cgu and |nfcc|icus Discascs.|acu||u cf
Vc|crinaru Mcdicinc. U|rccn| Unitcrsi|u
2
3
Na|icna| |ns|i|u|c cf Puo|ic Hca||n and |nc |ntircnncn|.
8i||nctcn. Tnc Nc|ncr|ands
4
|acu||u cf Anina| Scicncc and Vc|crinaru Mcdicinc. Ncng |an
Unitcrsi|u. Vic|nan

InleinalionaI IouinaI of AnlinicioliaI Agenls 172-179. 2OO6

Antibiotic resistance. integrons and SGI1 in Salmonella in The Netherlands
78
ABSTRACT

The obiective oI the present study was to investigate the antimicrobial resistance patterns.
characteristics oI integrons and their gene cassettes and the presence oI Salmonella Genomic
Island 1 in non-typhoid Salmonella isolates Irom human and animal origin. Epidemiologically
unrelated Dutch non-typhoid Salmonella strains (n237) originating Irom Iood producing
animals and human cases oI salmonellosis were tested Ior their susceptibility to 15
antimicrobial agents. Resistance to 14 oI these antimicrobials. including the third-generation
cephalosporins was detected. Resistance to sulphonamides. ampicillin. tetracycline.
streptomycin. trimethoprim and nalidixic acid was common (10 or more oI the strains were
resistant). Resistance against 3 or more antimicrobials was observed in 57 isolates. The same
237 strains were studied Ior the prevalence oI class 1 integrons. their gene cassettes and the
presence oI Salmonella Genomic Island 1 (SGI1). Thirty-six (15.2) isolates carried class 1
integrons. These integrons had ten distinct proIiles based on the size oI the integron and
restriction Iragment length polymorphism analysis. Integrons were detected Ior the Iirst time in
serovars Indiana and SenItenberg. Multidrug-resistance was strongly associated with the
presence oI class 1 integrons in which the aadA2. aadA1. bla
PSE-1
. dfrA1. dfrA5. dfrA14 or sat
genes were present as determined by nucleotide sequence determination. The presence oI gene
cassettes or combinations oI gene cassettes not Iound beIore in integrons in Salmonella was
observed. Salmonella Genomic Island 1 or its variants (SGI-B. -C and -F) were present in 16
isolates that belonged either to serovar Typhimurium. Derby or Albany. Regardless oI whether
the isolate was oI human or animal origin the same resistance phenotype. integron proIile and
SGI1 structure could be observed.

Keywords: antimicrobial resistance. integrons. Salmonella. Salmonella Genomic Island 1.

Chapter 4
79
INTRODUCTION

Salmonellosis is a maior zoonotic disease in humans which caused 68 oI the outbreaks oI
Iood-borne diseases reported in Europe between 1993- 1998. In The Netherlands. 50.000 cases
oI salmonellosis are reported each year
34
. The maiority oI the 2500 Salmonella serovars can
cause Iood borne salmonellosis in humans
21
.

Antimicrobial resistance in Salmonella spp. is a maior health problem in human and
veterinary medicine worldwide
15
. Many antimicrobial resistance genes are associated with
genetic elements called integrons
24
which can be located on transposons and plasmids. but also
on the chromosome. They are able to integrate and express genes coding Ior antibiotic
resistance
20. 32
. Class 1 integrons. the most common integron type
20
. have been detected in many
countries in diIIerent Salmonella serovars
16
may be located on the so-called Salmonella
Genomic Island 1 (SGI1)
3. 10. 13. 26. 27
. SGI1 is an integrative 43 kb chromosomal element
12
on
which antibiotic resistance genes are clustered. Ilanked by 2 class 1 integrons
1. 2. 5
. Strains
containing SGI1 are usually resistant to ampicillin (and amoxicillin). streptomycin (and
spectinomycin). chloramphenicol (and IlorIenicol). sulphonamides and tetracycline. Strains
carrying SGI1 variants (SGI1-A to SGI1-J) with diIIerent antibiotic resistance proIiles have also
been Iound in several Salmonella serovars
3. 10. 11. 26
. It is important to study the spread oI
antibiotic resistance to understand the relationship between antibiotic resistance genes. class 1
integrons and the Salmonella Genomic Island 1 because integrons and transIerable elements are
responsible Ior today`s spread oI resistance genes in the bacterial population and increase the
overall resistance gene pool.

The aim oI the present study was to determine (1) the antimicrobial resistance proIiles
oI salmonellae isolated Irom humans. cattle. pigs. chickens and Iood products in The
Netherlands (2) the rate and molecular characterization oI class 1 integrons in these strains and
(3) the genetic basis oI SGI1.


Bacteria
The 237 epidemiologically unrelated Salmonella isolates in this study were derived Irom the
collection oI 3265 isolates obtained in 2004 by the Dutch National Institute oI Public Health
and the Environment (RIVM) and the Veterinary Microbiological Diagnostic Center (VMDC)
oI Utrecht University. All isolates were conIirmed to be Salmonella based on colony
morphology and biochemical tests
14
. The isolates in this study were chosen to represent
diIIerent serotypes as well as diIIerent origins oI isolation i.e.. Irom humans (n114). cattle
(n44). pigs (n24). chickens (n47). and Iood products (n8. Irom meat or eggs). All human
isolates were Irom clinical cases. OI the animal isolates. 73 were Irom clinical cases (mainly
cattle and pigs) and 17 Irom healthy animals (mostly chickens). The isolates were serotyped
according to the latest version oI the KauIImann-White scheme using slide and microtiterplate
80
agglutination
30
. Serovar Enteritidis isolates were phage-typed as described by Ward
35
. Serovar
Typhimurium isolates were phage-typed using the Dutch Phage typing system
19
.

Antimicrobial susceptibility
Antimicrobial susceptibility was tested by the agar diIIusion technique using commercial disks
(Oxoid. UK) according to the guidelines oI the Clinical and Laboratory Standards Institute
(CLSI. Iormerly the National Committee Ior Clinical Laboratory Standards - NCCLS)
29
. The
antimicrobials tested were ampicillin 10 g (A). amoxicillin/clavulanic acid 30 g (Ac).
ceIalothin 30 g (Cp). ceItazidime 30 g (CeI). chloramphenicol 30 g (C). ciproIloxacin 5 g
(Ci). colistin 10 g (Co). gentamicin 10 g (G). kanamycin 30 g (K). nalidixic acid 30 g
(Na). norIloxacin 10 g (No). streptomycin 10 g (S). tetracycline 30 g (T). trimethoprim 5 g
(Tp). and sulphonamides 300 g (Su). Escherichia coli 25922 was used as control organism.

Detection of class 1 integrons
Template DNA oI all 237 isolates was prepared by the boiled lysate procedure
25
. Integrons were
detected by PCR ampliIication oI the class 1 integrase speciIic int1 gene
23
. Subsequently. the
size oI any inserted gene cassette oI the integrase-positive isolates was determined by PCR
using primers Ior the conserved segment regions (CS-PCR)
25
. Since the 3`segment oI class 1
integron is not always conserved. some integrase-positive isolates yielded no product in the CS-
PCR. In this case. the gene cassette oI the isolate was characterized by an inverted PCR Ior the
integrase gene. BrieIly. one g oI genomic DNA was cleaved with the restriction endonuclease
SphI. The DNA obtained in this way were ligated and subiected to PCR using the int-OUT and
CS-F primers (Table 1). PCR products were resolved by electrophoresis at 100V Ior 3 h on
0.7 agarose gels containing ethidium bromide and visualized under ultraviolet light. Only
amplicons oI about 1 kb were sequenced.

Gene cassette characterization
CS-PCR amplicons oI the same size were restricted with two restriction endonucleases and
were considered identical iI they had the same RFLP pattern aIter digestion with both enzymes
(Table 2). One sample oI each representative RFLP type was randomly chosen Ior sequencing.
In the case oI a unique integron. puriIied CS-PCR products were cloned in the pGEM-T easy
Vector (Promega. Madison. WI. USA). Colonies containing plasmids with the inserted
Iragment were picked Irom Luria Bertani plates containing ampicillin (100 g/ml). 40 l IPTG
(100 mM per plate) and 40 l X-Gal (2 per plate). The target Iragments were obtained by
PCR. using T7 and Sp6 primers under the same conditions as described Ior the CS-PCR. The
ampliIication products were puriIied using the Qia Gel Extraction kit (Qiagen. Hilden.
Germany) and the nucleotide sequence was determined. For sequencing oI the diIIerent
amplicons Irom both ends the T7 and Sp6 primers were used. For the CS-amplicon oI 2000 bp.
an internal primer was synthesized (based on the sequence obtained) and used to continue
sequencing until the resistance genes inserted in the amplicon were identiIied. For isolates
containing two integrons which only diIIered 50 bp in size. CS-PCR products were cloned in
Chapter 4
81
Primer Gene Fragment
ampliIication
PCR
product
Sequence (5`-3`)

ReI
int1-F int1 int1 242 TCT CGG GTA ACA TCA AGG
24
int1-R int1 AGG AGA TCC GAA GAC CTC
24
int-OUT int1 AAG TGG TTC GCA TCC TCG This study
CS-F CS Cassette (s)
a
GGC ATC CAA GCA GCA AG
23
CS-R CS Cassette (s) AAG CAG ACT TGA CCT GA
23
U7-L12 thdf LeIt iunction 500 ACA CCT TGA GCA GGG CAA G
10
LJ-R1 int1 AGT TCT AAA GGT TCG TAG TCG
10
104-RJ S044 Right iunction 515 TGA CGA GCT GAA GCG AAT TG
10
C9-L2 int2 AGC AAG TGT GCG TAA TTT GG
10
104-D vidY 500 ACC AGG GCA AAA CTA CAC AG
10
dIrA1-F dfrA1 drfA1..orfC 1057 CCA GCA GCA AGC GCG TTA CG
10
orIC-R orfC TCT CGA ATC AAG CAG GAA CC
10
cml01 floR floR 494 TTT GGW CCG CTM TCR GAC
10
cml15 floR SGA GAA RAA GAC GAA GAA G
10
int1 intI1 intI.. aadA2 1135 GCT CTC GGG TAA CAT CAA GG
10
aad aadA2 GAC CTA CCA AGG CAA CGC TA
10
sulTER sul1delta sulI.. floR 942 AAG GAT TTC CTG ACC CTG
10
F3 floR AAA GGA GCC ATC AGC AGC AG
10
F4 floR floR..tetR 598 TTC CTC ACC TTC ATC CTA CC
10
F6 tetR TTG GAA CAG ACG GCA TGG
10
tetR tetR tetR..tetA 1559 GCC GTC CCG ATA AGA GAG CA
10
tetA tetA GAA GTT GCG AAT GGT CTG CG
10
int2 groEL int1..pse1 1338 TTC TGG TCT TCG TTG ATG CC
10
pse1 pse-1 CAT CAT TTC GCT CTG CCA TT
10
pse-L pse-1 pse1..S044 4400 AAT GGC AAT CAG CGC TTC CC
10
MDR-B S044 GAA TCC GAC AGC CAA CGT TCC
10
TEM-F blaTEM blaTEM ATG AGT ATT CAA CAT TTC CGT GTC G This study
TEM -R blaTEM ACC AAT GCT TAA TCA GTG AGG CA This study
TEM-S1 blaTEM For sequencing ACA ACG ATC GGA GGA CCG This study
TEM-S2 blaTEM For sequencing GCG GTT AGC TCC TTC GGT This study
SHV-F blaSHV blaSHV GTA TTG AAT TCA TGC GTT ATA TTC GCC TGT GTA
4
SHV-R blaSHV CAG AAT TCG GCT AGC GTT GCC AGT GCT CGA
4
CTX-F blaCTX-M blaCTX-M 538 CGA TGT GCA GTA CCA GTAA A. Paauw
b

CTX-R blaCTX-M ATA TCG TTG GTG GTG CC A. Paauw
b

the pGEM-T easy Vector (Promega. Madison. WI. USA). Plasmids with diIIerent inserts were
selected on the basis oI restriction enzyme analysis using EcoRI or HpaII.
Table 1. Primers used Ior PCRs.
a
size depending on the gene cassette(s) inserted
b
A. Paauw. personal communication (2006)

PuriIied plasmids were then used Ior DNA sequence determination. Dideoxy sequencing was
perIormed on an ABI 3730 Sequencer (Foster. CA. USA). DNA sequences were analysed with
the Clone Manager Suit and by consulting the GenBank database oI the National Center Ior
Biotechnology InIormation via the BLAST network service. The nucleotide sequences oI the
gene cassettes have been deposited in the GenBank database under the accession numbers in
Table 2.

Genomic Island 1 mapping
Integron-positive isolates were investigated Ior the presence oI SGI1. The isolates were Iirst
examined by PCR Ior the presence oI the leIt and right iunction oI SGI1. Next the presence oI
sequences Irom the antibiotic resistance gene cluster was determined by PCR using primers
(Table 1) described previously
10
and in this study. Template DNA was prepared with the High
Pure PCR Preparation Kit (Roche. Mannheim. Germany). PCR was perIormed in a total volume
82
oI 25 l containing 2.5 l oI 10 PCR buIIer (HT Biotechnology. Cambridge. England).
0.5 l 10 deoxynucleotide triphosphate mix (2 mM each). 50 pmol oI each primer. 1.25 U Taq
DNA polymerase. and 1 l oI template DNA. To ampliIy Iragments larger than 3.5 kb. Taq
Plus polymerase was used instead oI Taq DNA polymerase (HT Biotechnology. Cambridge.
England). Thermal cycling conditions consisted oI a hot start cycle at 94
0
C Ior 3 min. Iollowed
by 35 cycles oI 1 min at 94
0
C. 1 min at 50
0
C to 65
0
C (depending on the primers used). 1 to
5 min at 72
0
C (depending on the expected amplicon size) and a Iinal step oI 72
0
C Ior 10 min.
Annealing temperatures Ior primers used in the SGI1 mapping were 50
0
C except Ior int1/aad.
tetR/tetA (54
0
C). sulTER/F3 and pse-L/MDR-B (60
0
C).

Beta-lactamase production
CeItazidime-resistant isolates were investigated Ior extended-spectrum -lactamase (ESBL)
production. The isolates were also tested Ior the TEM. SHV and CTX-M type -lactamases by
PCR ampliIication using primers listed in Table 1
4
. The expected product was sequenced and
analysed according to to Lahey`s scheme
22
. E. coli 09A488. Klebsiella pneumoniae 09A018
and Enterobacter cloacae 03773 which were previously shown to harbor TEM. SHV and CTX-
M type -lactamase. respectively. were used as positive control.

Statistical analysis
All statistical analyses were perIormed in MicrosoIt Excel 2000 using Chi square tests.
DiIIerences were considered signiIicant at p0.05.

RESULTS

In the present study. 128 (54) isolates were susceptible to all 15 antibiotics tested (Figure 1).
Sixty-nine (29) salmonellae were resistant to 2 antimicrobials. Antimicrobial resistance was
not solely associated with a particular Salmonella serovar. Resistant isolates belonged to 25
diIIerent serovars including Typhimurium (35/51). Dublin (10/37). Enteritidis (13/41). Vichow
(8/8). Hadar. Paratyphi B (5/5). Derby (6/7). Newport (3/6). Mbankada (3/7). Brandenburg
(2/6). SenItenberg (2/7). Stanley (2/3). Livingstone (1/2). Agona (1/3). Anatum (1/3). Panama
(1/4). InIantis (1/5). Albany. Blockley. Braenderup. Corvallis. HaiIa. Glostrup. Indiana. London
(1/1). However. resistance to antimicrobials was high (nearly 70) in serovar Typhimurium.
S. Typhimurium pt 506 (DT 104) isolates were resistant to 2 to 7 antimicrobials. Resistance to
sulphonamides. ampicillin. tetracycline. streptomycin. trimethoprim. nalidixic acid.
chlorampenicol. cephalothin. amoxicillin/ clavulanic acid. ceItazidime. kanamycin.
ciproIloxacin. norIloxacin and gentamicin was Iound in 29. 21. 17. 17. 13. 12. 9. 4. 2. 1. 0.8.
0.8. 0.4 and 0.4 oI the isolates. respectively.

Most isolates in the present study were susceptible to the third-generation
cephalosporins. However. 3 (1.3 ) ceItazidime-resistant isolates (2 S. Typhimurium Irom
human and cattle and one S. Braenderup Irom chicken) were observed. No ESBL producing
strain was Iound.
Chapter 4
83
0 0
3
9 9 10
4
1
128
40
9
12
6
3 2 1
0
20
40
60
80
100
120
140
0 1 2 3 4 5 6 7
Number of antimicrobials
N
u
m
b
e
r

o
f

i
s
o
l
a
t
e
s

r
e
s
i
s
t
a
n
t

t
o
int+
int-

Fig 1. The relationship between class 1 integrons and
multidrug resistance (Chi square test. p0.001)

Resistance to nalidixic acid was
common in serovar Hadar (100)
and serovar Virchow (87.5).
Three isolates showing resistance to
Iluoroquinolones (norIloxacin. n1;
ciproIloxacin. n2) and 3 isolates
with decreased susceptibility
(reduction in the diameter oI the
inhibition zone) to these
antimicrobials (norIloxacin. n2;
ciproIloxacin. n1) were present.
Multidrug-resistant salmonellae
were obtained more Irequently
Irom animal origin (86. 50.
30. and 23 oI Iood. pig. cattle.
and chicken isolates; respectively)
than Irom humans (19). There was a strong relationship between multidrug-resistance and the
presence oI class 1 integrons (p0.001) (Fig. 1).

Class 1 integrons were detected in 36 (15) Salmonella isolates oI 11 diIIerent
serovars. Integrons were Iound predominantly in S. Typhimurium DT 104 (n13; 100).
However. class 1 integrons were also identiIied in other phage types oI S. Typhimurium and
other serovars (Table 3). Data analysis showed that there is a strong relationship between the
presence oI class 1 integrons and resistance to sulphonamide. chloramphenicol. ampicillin.
tetracycline. streptomycin. or trimethoprim (p0.001 in order oI decreasing strength oI
correlation) while resistance to other antimicrobials tested was integron-independent. Ten class
1 integron proIiles (IPs) could be deIined based on the number and size oI the amplicons
obtained (Table 2. Table 3).

Table 2. Characterization oI class 1 integrons oI Salmonella isolates in The Netherlands.
IP
Size in bp
(isolate ID) *RE 1
Fragments
(approx. bp) RE 2
Fragments
(approx. bp) Gene cassette
Accession
number
I 1000 (N216) EcoRI 550; 450 HpaII 400; 250; 150; 50 aadA2 DQ133165
1200 HincII 700; 350; 150 HpaII 800; 400 blaPSE-1 DQ133161
II 1250 (N107) dfrA1 DQ123842
1200 blaPSE-1 DQ133163
III 700 (N111) dfrA5 DQ133160
IV 1000 (N328) BclI 700; 300 HpaII 580; 240; 120; 60 aadA1 DQ133159
V 1000 (N80) EcoRI 550; 450 HpaII 400; 250; 150; 50 aadA2 DQ133165
VI 1200 (N209) HincII 700; 350; 150 HpaII 800; 400 blaPSE-1 DQ133162
VII 1500 (N133) HincII 1000; 500 HpaII 700; 500; 200; 100 aadA1. dfrA1 DQ133164
VIII 2000 (N279) BclI 700; 650; 450 HpaII 700; 500; 300; 200; 100 aadA2. sat DQ133166
IX 1200 (N246) dfrA14. orfC DQ228133
X - (N178)

* RE: restriction endonuclease
- no product Iound Irom CS-PCR or inverted PCR
84

Four isolates (oI serovar S. InIantis. S. Livingstone. and S. Mbandaka and
S. SenItenberg) were integron positive but yielded no product in the CS-PCR. IP-IX was Iound
in S. SenItenberg using inverted PCR. IP-X was observed in 3 isolates (S. InIantis.
S. Livingstone. and S. Mbandaka) where no products in either CS-PCR or inverted PCR were
obtained. Gene cassettes encoding resistance to streptomycin (aadA1). streptomycin and
spectinomycin (aadA2). streptothricin (sat). beta-lactam (bla
PSE-1
). trimethoprim (dfrA1. dfrA5
and dfrA14) were observed. Complete copies oI SGI1 or one oI its variants were Iound in 16
(44 ) oI the class 1 integron-positive isolates (Table 3 and Fig. 2).

Table 3. Resistance patterns in class 1 integron-positive Salmonella enterica isolates. their
integrons and SGI1 types.
ID
Resistance proIile
(intermediate)
Serovar
(source oI isolate
3
)
Amplicon size (bp) IP
4
SGI types
N216 ACeICSTSuTp(Ac) Typhimurium DT104
1
(H) 1000;1200 I SGI1
N235 ACSTSuTp(Ac) Typhimurium DT104 (P) 1000;1200 I SGI1
N246 ACSTSuTp(Ac) SenItenberg (C) 1200
*
IX -
N193 ACNaTSuTp(AcS) Derby (H) 1000;1200 I SGI1
N140 ACpNaTSuTp(AcS) Virchow (C) 1500 VII -
N94 ACSTSu(Ac) Typhimurium DT104 (P) 1000;1200 I SGI1
N176 ACSTSu(Ac) Typhimurium DT104 (H) 1000;1200 I SGI1
N188 ACSTSu Typhimurium DT104 (H) 1000;1200 I SGI1
N326 ACSTSu(Ac) Non-DT104
2
(B) 1000 ;1200 I SGI1
N104 ACNaSSu(T) Typhimurium DT104 (F) 1000;1200 I SGI1
N167 AKTSuTp(Cp) Non-DT104 (F) 2000 VIII -
N203 ASTSuTp Indiana (C) 1500 VII -
N107 ACNaSuTp(AcST) Albany (C) 1200; 1250 II SGI1-F
N108 ACSSu(AcT) Typhimurium DT104 (H) 1000;1200 I SGI1
N320 ACSSu(AcT) Typhimurium DT104 (B) 1000;1200 I SGI1
N194 ACSSu(AcTp) Non-DT104 (H) 1000;1200 I SGI1
N209 AAcNaSu Typhimurium DT104 (H) 1200 VI SGI1-B
N327 CNoSuTp Typhimurium DT104 (B) 1000 V -
N279 ASTSu(AcCp) Non-DT104 (F) 2000 VIII -
N133 ATSuTp(S) Virchow (H) 1500 VII -
N149 ATSuTp InIantis (C) - X -
N111 NaTSuTp HaiIa (H) 700 III -
N295 CSSu Dublin (B) 1000 IV -
N199 ASuTp Mbandaka (C) - X -
N277 TSuTp (S) Non-DT104 (P) 1500 VII -
N80 SSu DT104 Typhimurium (P) 1000 V SGI1-C
N74 SSu(Ci) Derby (P) 2000 VIII -
N178 SuTp(S) Livingstone (H) - X -

1
DT 104: pt 506 in Dutch phage typing system * amplicon oI invertedint PCRs
2
Non-DT 104: S. Typhimurium other phage types than pt 506 - not Iound
3
H. human; P. pig; C. chicken; B. bovine
4
class 1 integron proIiles

Chapter 4
85
Fig. 2. Map oI SGI1 highlighting targets and Iragment sizes in PCRs used Ior mapping SGI1
antibiotic resistance gene clusters. (a) SGI1 Iound in S. Typhimurium (N216); (b) SGI1 Iound
in S. Derby (N193); (c) SGI1-F Iound in S. Albany (N107); (d) SGI1-C Iound in S.
Typhimurium (N80); (e) SGI1-B Iound in S. Typhimurium (N209). The gray shades indicate
resistance genes. (modiIied Irom reI. 10).

SGI1 was Iound in 12 S. Typhimurium isolates and one S. Derby isolate. SGI1-F was
present in a single S. Albany isolate. There was a strong relationship between the presence oI
two class 1 integrons and the presence oI SGI1 (p0.001). Two other isolates oI
S. Typhimurium carrying only a single integron contained an incomplete SGI1. known as
SGI-B (strain N209) or SGI1-C (strain N80). Both yielded the expected products Ior the leIt
(thdf and int genes) and right iunction (S044 and int2 genes) oI these SGI1s. Isolate N80 also
yielded a single 1200 bp product in the ampliIication oI the Iragment between the int1 and
aadA2 genes. Isolate N209 yielded no products in the int2- pseL PCR. but a 900 bp product was

500bp 900 bp 4500bp 515 bp

(e) thdf int intI1 pse1 qacE1 sulI S044 int2 vidY
(a) thdf int intI1 aadA2 qacE1 sulI floR tetR tetG groEL/int pse1 qacE1 S044 int2 vidY

494 bp 4400 bp 515 bp

500 bp 1135 bp 942 bp 598 bp 1559 bp 1338 bp

(d) thdf int intI1 aadA2 qacE1 sulI S044 int2 vidY

500 bp 1135 bp 515 bp

(c) thdf int intI1 dfrA1 orf qacE1 sulI floR tetR tetG groEL/intI1 pse1 qacE1 S044 vidY

1057 bp 494 bp 4400 bp 500 bp

3000 bp
500 bp 942 bp 598 bp 1559 bp 1338 bp

(b) thdf int intI1 aadA2 qacE1 sulI floR tetR tetG groEL/intI1 pse1 qacE1 S044 vidY

494 bp 4400 bp 500 bp

500 bp 1135 bp 942 bp 598 bp 1559 bp 1338 bp

86
generated by ampliIication with the int1 and pseL primers. No products were obtained Irom
other combined PCRs as shown in Figure 2. except Ior the ampliIication oI the 4.5 kb Iragment
Irom pse-1 to S044 Ior isolate N209. Thus. the Salmonella Genomic Island in strains N209 and
N80 (Table 2) were classiIied as SGI1-B and -C. respectively.

DISCUSSION

The main Iinding in this study was the widespread presence oI multidrug resistance among
salmonellae Irom human and animal origin and the diversity oI class 1 integrons and SGI1 in
diIIerent Salmonella serovars including serovars in which class 1 integons have not been Iound
beIore. Class 1 integrons have been detected in diIIerent Salmonella serovars including
Typhimurium. Virchow. Hadar. Dublin. Derby. HaiIa. InIantis. Albany. and Mbandaka
beIore
18. 31. 36
. however. to our knowledge. not in S. SenItenberg and S. Indiana. This Iinding
suggests the spread oI integrons. which are located on a mobile genetic element among the
many serovars oI Salmonella. In contrast to a study Irom Scotland
6
. no class 1 integrons were
Iound among serovar Enteritidis in this study. In The Netherlands. antimicrobial resistance in
S. Enteritidis isolates is still low (3)
28. 33
. whereas in Scotland S. Enteritidis isolates were
multi-resistant and carried integrons
6
. In general. our data suggest that acquisition oI integrons
is not limited to speciIic Salmonella serovars but may occur in any serovar.

AmpliIication oI gene cassettes by the CS-PCR in integrase-positive isolates is
sometimes negative because the primers do not always anneal to the 3`-conserved segment
25
.
This was also observed in the present study. An inverted-integrase PCR proved to be an
eIIective method Ior the study (at least part oI) the gene cassettes present therein. Most
integron-positive serovars carried gene cassettes that were described earlier
8. 9. 18. 31. 37
. However.
neither the dfrA5 gene cassette in S. HaiIa nor the combination oI the gene cassettes aadA2 and
sat in Salmonella Typhimurium or the combination oI dfrA14 and an unknown gene in S.
SenItenberg have been reported beIore. The dfrA5 gene has been Iound in S. Wien (accession
number AY827837). The aadA2 and sat genes have been described on a plasmid oI a highly
invasive and resistant S. Cholerasuis strain
7
. The gene dfrA14 has only been observed in E. coli
previously (accession number AJ884725) but not in Salmonella. So. one may speculate that
integrons or the gene cassettes they contain have been exchanged not only among Salmonella
serovars but also among Enterobacteriaceae. Two class 1 integrons with sizes oI approximately
1000 bp and 1200 bp were Iound in almost all S. Typhimurium DT104 isolates in accordance
with the Iindings oI Randall et al.
31
. In the present study. S. Typhimurium DT 104 isolates not
only had a pentadrug resistance phenotype (ACSSuT). a tetradrug resistance phenotype
(CSSuT) or a SSu resistance phenotype as reported beIore
17. 18. 31. 36
. but also other multiple
resistance phenotypes (Table 3). These phenotypes are characteristic Ior SGI1 and its variants.

SGI1 is an important determinant oI multi-drug resistance. SGI1 or variants with
diIIerent gene cassettes located in two integrons have been Iound in many serovars oI
Salmonella enterica
26
. The high rate with which SGI1 is present in S. Typhimurium DT 104
isolates in the present and previous studies
10. 26. 27
suggests that the SGI1 variant with the Iive
Chapter 4
87
antibiotic resistance genes aadA2. sul1. floR. tet. and pse. is the oldest and probably the Iirst to
arise. Its variants in other Salmonella serovars
26
probably arose as the result oI recombination
events. Deletion is a possible explanation Ior the structure oI SGI1-B and SGI1-C in the two
S. Typhimurium isolates which contain only one integron Ilanked by the leIt and right iunction
oI SGI1. The Iact that PCR products Ior isolate N209 could only be obtained with int1- pseL
primers. but not with int2-pseL primers suggests another location oI the bla
PSE 1
gene at the leIt
iunction oI SGI1 due to a single crossover event at intI1
3
. The genes Iound adiacent to the leIt
iunction oI SGI1 such as the int and xis genes could have an important role in the excision and
integration oI integron-located gene cassettes
2
. In this study the expression oI the tetracycline
resistance gene (tetG) varied Irom Iull resistance among SGI1 carrying S. Typhimurium isolates
to intermediate susceptibility in a SGI1-F positive S. Albany isolate.

In summary. multidrug-resistance was strongly associated with the presence oI class 1
integrons in salmonellae. We Iound gene cassettes or combinations oI gene cassettes not
reported previously in integrons in Salmonella. Class 1 integrons were detected Ior the Iirst time
in serovars Indiana and SenItenberg. SGI1 and its variants (SGI1-B. -C and -F) were Iound in
the serovars Typhimurium. Derby and Albany. Similar resistance patterns. integron types and
SGI1 structures were Iound regardless the source oI the isolate (human or animal).

ACKNOWLEGEMENTS

This study was Iunded by a grant oI Vietnamese Government to An T.T. Vo. We thank Max
Heck. Frans Bensink oI RIVM; Marcel de Zoete oI I&I Ior technical help and Armand Paauw
oI Eiikman-Winkler Institute Ior providing control strains.

REFERENCES
1. Arcangioli. M.A.. S. Leroy-Setrin. J.L. Martel. and E. Chaslus-Dancla. 1999. A new chloramphenicol and
IlorIenicol resistance gene Ilanked by two integron structures in Salmonella Typhimurium DT104. FEMS
2. Boyd. D.. G.A. Peters. A. Cloeckaert. K.S. Boumedine. E. Chaslus-Dancla. H. Imberechts. and M.R. Mulvey.
2001. Complete nucleotide sequence oI a 43-kilobase genomic island associated with the multidrug resistance
region oI Salmonella enterica serovar Typhimurium DT104 and its identiIication in phage type DT120 and
serovar Agona. J. Bacteriol. 183:5725-5732.
3. Boyd. D.. A. Cloeckaert. E. Chaslus-Dancla. and M.R. Mulvey. 2002. Characterization oI variant Salmonella
Genomic Island 1 multidrug resistance regions Irom serovars Typhimurium DT104 and Agona. Antimicrob.
4. BradIord. P.A. 1999. Automated thermal cycling is superior to traditional methods Ior nucleotide sequencing oI
bla(SHV) genes. Antimicrob. Agents Chemother. 43:2960-2963.
5. Briggs. C.E.. and P.M. Fratamico. 1999. Molecular characterization oI an antibiotic resistance gene cluster oI
Salmonella Typhimurium DT104. Antimicrob. Agents. Chemother. 43:846-849.
6. Brown. A.W.. S.C. Rankin. and D.J. Platt. 2000. Detection and characterisation oI integrons in Salmonella
enterica serotype Enteritidis. FEMS Microbiol. Lett. 191:145-149.
7. Chiu. C.H.. P. Tang. C. Chu. S. Hu. Q. Bao. J. Yu. Y.Y. Chou. H.S. Wang. and Y.S. Lee. 2005. The genome
sequence oI Salmonella enterica serovar Choleraesuis. a highly invasive and resistant zoonotic pathogen.
Nucleic Acids Res. 33:1690-1698.
88
8. Daly. M.. and S. Fanning. 2000. Characterization and chromosomal mapping oI antimicrobial resistance genes
in Salmonella enterica serotype Typhimurium. Appl. Environ. Microbiol. 66:4842-4848.
9. del Cerro. A.. S.M. Soto. and M.C. Mendoza. 2003. Virulence and antimicrobial- resistance gene proIiles
determined by PCR-based procedure Ior Salmonella isolated Irom samples oI animal origin. Food Microbiol.
20:431-438.
Salmonella Genomic Island 1 multidrug resistance gene clusters in Salmonella enterica serovar Agona isolated
in Belgium in 1992 to 2002. Antimicrob. Agents Chemother. 48:2510-2517.
13. Ebner. P.. K. Garner. and A. Mathew. 2004. Class 1 integrons in various Salmonella enterica serovars isolated
Irom animals and identiIication oI genomic island SGI1 in Salmonella enterica var. Meleagridis. J. Antimicrob.
Chemother. 53:1004-1009.
14. Edwards. P.R.. and W.H. Ewing. 1986. Edwards and Ewing's identiIication oI Enterobacteriaceae. 4th.
Elseviers Science Publishing Co.Inc.. New York.
15. FAO. OIE. and WHO. Joint Iirst FAO/OIE/WHO expert workshop on non-human antimicrobial usage and
antimicrobial resistance: scientiIic assessment. http://www.who.int/IoodsaIety/publications/micro/en/amr.pdI
(25 February 2005. date last accessed).
16. Fluit. A.C. 2005. Towards more virulent and antibiotic-resistant Salmonella? FEMS Immunol. Med. Microbiol.
43:1-11.
17. Gebreyes. W.A.. and C. Altier. 2002. Molecular characterization oI multidrug-resistant Salmonella enterica
subsp. enterica serovar Typhimurium isolates Irom swine. J. Antimicrob. Chemother. 40:2813-2822.
18. Guerra. B.. S. Soto. S. Cal. and M.C. Mendoza. 2000. Antimicrobial resistance and spread oI class 1 integrons
among Salmonella serotypes. Antimicrob. Agents Chemother. 44:2166-2169.
20. Hall. R.M.. and H.W. Stokes. 1993. Integrons: novel DNA elements which capture genes by site-speciIic
recombination. Genetica 90:115-132.
21. Humphrey. T. 2000. Public health aspects oI Salmonella inIections. In Wray. C. & Wray. A. (ed). Salmonella
in domestic animals. New York. USA: CABI Publishing. pp. 245-263.
22. Lahey Clinic. Amino acid sequences Ior TEM. SHV. and OXA extended-spectrum and inhibitor resistant beta-
lactamases. http://www.lahey.org/Studies/ (31 Jan. date last accessed).
23. Leverstein-Van Hall. M.A.. A. Paauw. A.T.A. Box. H.E.M. Blok. J. VerhoeI. and A.C. Fluit. 2002. Presence oI
integron-associated resistance in the community is widespread and contributes to multidrug resistance in the
hospital. J. Clin. Microbiol. 40:3038-3040.
26. Levings. R.S.. D. LightIoot. S.R. Partridge. R.M. Hall. and S.P. Diordievic. 2005. The Genomic Island SGI1.
containing the multiple antibiotic resistance region oI Salmonella enterica serovar Typhimurium DT104 or
variants oI it. is widely distributed in other S. enterica serovars. J. Bacteriol. 187:4401-4409.
27. Meunier. D.. D. Boyd. M.R. Mulvey. S. Baucheron. C. Mammina. A. Nastasi. E. Chaslus-Dancla. and A.
Cloeckaert. 2002. Salmonella enterica serotype Typhimurium DT-104 antibiotic resistance genomic island I in
serotype Paratyphi B. Emerg. InIect. Dis. 8:430-433.
Chapter 4
89
28. Mevius. D.J.. and W. van Pelt. MARAN-2004- Monitoring oI antimicrobial resistance and antibiotic usage in
animals in The Netherlands in 2004. http://www.cidc-lelystad.wur.nl/NR/rdonlyres/7F79ACE6-0FD2-41AB-
81B2-BB17FA89603C/11382/MARAN2004web1.pdI (10 August 2006. date last accessed).
29. National Committee Ior Clinical Laboratory Standards. 2001. PerIormance standards Ior antimicrobial disk and
dilution susceptibility tests. M31-A2- Approved standard. 2nd. NCCLS. Wayne. PA. USA.
genes. integrons and multiple antibiotic resistance in thirty-Iive serotypes oI Salmonella enterica isolated Irom
humans and animals in the UK. J. Antimicrob. Chemother. 53:208-216.
32. Stokes. H.W.. and R.M. Hall. 1989. A novel Iamily oI potentially mobile DNA elements encoding site-speciIic
gene-integration Iunctions: integrons. Mol. Microbiol. 3:1669-1683.
33. van Duiikeren. E.. W.J.B. Wannet. D.J. Houwers. and W. van Pelt. 2003. Antimicrobial susceptibilities oI
Salmonella strains isolated Irom humans. cattle. pigs. and chickens in The Netherlands Irom 1984 to 2001. J.
34. van Pelt. W.. M.A. de Wit. W.J. Wannet. E.J. Ligtvoet. M.A. Widdowson. and Y.T. van Duynhoven. 2003.
Laboratory surveillance oI bacterial gastroenteric pathogens in The Netherlands. 1991-2001. Epidemiol. InIect.
130:431-441.
35. Ward. L.R.. J.D. de Sa. and B. Rowe. 1987. A phage-typing scheme Ior Salmonella Enteritidis. Epidemiol.
InIect. 99:291-294.
36. White. D.G.. S. Zhao. P.F. McDermott. S. Ayers. S. Friedman. J. Sherwood. M. Breider-Foley. and L.K. Nolan.
2003. Characterization oI integron mediated antimicrobial resistance in Salmonella isolated Irom diseased
swine. Can. J. Vet. Res. 67:39-47.
37. Zhao. S.. A.R. Datta. S. Ayers. S. Friedman. R.D. Walker. and D.G. White. 2003. Antimicrobial-resistant
Salmonella serovars isolated Irom imported Ioods. Int. J. Food. Microbiol. 84:87-92.
90

C!ass 1 Intcgrnns In Dutch Sa|mone||a
enterlca scrnvar Dub!In Isn!atcs frnm
c!InIca! cascs nf bnvInc sa!mnnc!!nsIs

An T. T. Vo
1,4
1
, Ad C. IIuil
2
,

Max L. O. C. Heck
3
,
Anjo Veiliuggen
3
, Kin van dei ZvaIuv
3
,

Win Caaslia
1

1
Dcpar|ncn| cf |nnunc|cgu and |nfcc|icus Discascs.|acu||u cf Vc|crinaru
2
3
Na|icna| |ns|i|u|c cf Puo|ic Hca||n and |nc |ntircnncn|. 8i||nctcn. Tnc
Nc|ncr|ands
4
Vic|nan

Veleiinaiv MiciolioIogv : 192-2OO. 2OO6

Class 1 integrons and the genetic diversity oI the Dutch bovine Salmonella Dublin isolates
92
ABSTRACT

FiIty nine Salmonella enterica serovar Dublin (Salmonella Dublin) isolates Irom clinical cases
oI bovine salmonellosis between 1993 and 2004 were tested Ior their susceptibility to 15
antimicrobial agents and the presence oI class 1 integrons. Integrons were Iurther analyzed by
conserved segment PCR-RFLP. DNA sequencing was used to identiIy the inserted gene
cassette. Twelve (20.3 ) isolates were multidrug-resistant. A combination oI resistance against
chloramphenicol. streptomycin and sulphonamides was the most common phenotype observed.
Multidrug-resistance (MDR) was Iound to be strongly associated with the presence oI
integrons. since a class 1 integron with the aadA1 gene cassette encoding resistance to
streptomycin and spectinomycin was Iound in all 12 multidrug-resistant isolates. The presence
oI the aadA1 gene in Salmonella Dublin has not been reported beIore. None oI the integron
carrying Salmonella Dublin isolates could transIer its antimicrobial resistance to E. coli K12 by
coniugation. Analysis oI plasmid proIiles and pulsed Iield gel electrophoresis (PFGE) patterns
showed at least some clonality among the Salmonella Dublin isolates. but 11 diIIerent types
could be distinguished based on both XbaI and BlnI-PFGE patterns. Thus. the Dutch Salmonella
Dublin strains were closely related but not clonal.

Keywords: Salmonella. integron. genotyping. antimicrobial resistance. cattle. Salmonella
Dublin
Chapter 5
93
INTRODUCTION

In Europe. Salmonella enterica subspecies enterica Dublin (Salmonella Dublin) is one oI the
most prevalent Salmonella serovars isolated Irom cattle
32
. In The Netherlands. Salmonella
Dublin accounted Ior 53 oI the Salmonella isolates Irom cattle obtained between 1993 and
2000
30
. Bovine Salmonella Dublin inIections may result in subclinical excretion. latent
carriership. or in clinical disease causing enteritis. septicaemia. meningitis. abortion.
osteomyelitis. arthritis. terminal dry gangrene. depressed milk yield. or pneumonia
22. 32
.
Although Salmonella Dublin is adapted to cattle
22. 32
. it may occasionally inIect other animal
species including humans. In humans. these inIections can result in severe invasive disease
3
and
are oIten associated with the consumption oI contaminated dairy products
12. 29
. In 1979. a milk-
borne Salmonella Dublin outbreak aIIected at least 700 persons in Scotland
25
. In 1995. a similar
cheese-borne outbreak occurred in Switzerland and in France
29
. Recently. a Dutch patient with
sepsis and an inIected aneurysm caused by Salmonella Dublin died as a result oI rupture oI the
aorta
12
.

The emergence oI multi-drug resistance (MDR) among Salmonella enterica has been
reported worldwide
7. 10. 26
. Resistance genes are oIten carried on integrons. Integrons are genetic
elements that. although normally immobile themselves. may contain gene cassettes which can
be transIerred to other integrons or other sites in the bacterial genome. Class 1 integrons. the
maior integrons Iound in Enterobacteriaceae. consist oI a 5`-conserved segment with an
integrase gene (int). an adiacent attI site in which the gene cassettes are integrated and a
3`-conserved segment which includes a qacE and a sulI gene
8
. Gene cassettes and integrons
have clearly played an important role with respect to acquired antibiotic resistance in
Salmonella spp.
24
. However. to date there is little inIormation on integrons in S. Dublin
5. 17
. The
purpose oI this study was to determine the antimicrobial susceptibility oI a collection oI 59
Dutch Salmonella Dublin isolates. to speciIy the prevalence and the characteristics oI class 1
integrons in these isolates. and to investigate their epidemiological relationship.


Bacterial isolates
FiIty-nine Salmonella Dublin isolates were obtained Irom the Veterinary Microbiological
Diagnostic Center (VMDC) oI Utrecht University and the Dutch National Institute oI Public
Health and the Environment (RIVM). The isolates were collected between 1993 and 2004 Irom
clinical cases oI bovine salmonellosis. The isolates are epidemiogically unrelated and isolated
Irom the samples originating Irom diIIerent Iarms and diIIerent geographical regions in The
Netherlands. The isolates were cultured Irom Iaeces (n21). milk (n5). intestine (n5). lung
(n4). abomasa (Iourth stomach) (n4). lymph node (n3). liver (n2). synovia (n2). blood
(n2). kidney (n2). spleen (n2). brain (n2). and other organs (n5). The age oI the animal
Irom which the isolates were obtained ranged Irom 1 month to 8 years. Most oI them were dairy
cattle oI Holstein Friesian breed or Braunvieh Brown. Epidemiological inIormation on the 12
integron-positive isolates is listed in Table 1. All isolates were identiIied as Salmonella spp.
94
based on their colony morphology on selective media. and biochemical testing
6
. Serovar
identiIication was perIormed using microtitre and slide agglutination methods according to the
latest version oI the KauIImann and White scheme
21
at the Diagnostic Laboratory Ior InIectious
Diseases and Perinatal Screening oI the Dutch National Institute oI Public Health and the
Environment (RIVM). The Dutch S. Dublin isolates are indicated with the preIix N in the
Iigures.

All isolates were tested Ior their susceptibility to 15 antimicrobial agents by the Kirby Bauer
disk diIIusion assay on Mller-Hinton agar (Oxoid. UK) according to a standard procedure oI
the Clinical and Laboratory Standards Institute (CLSI. Iormerly the National Committee Ior
Clinical Laboratory Standards NCCLS)
19
. The antimicrobials tested were: ampicillin (AMP;
10 g). amoxicillin/clavulanic acid (AMC; 30 g). cephalothin (CEF; 30 g). ceItazidime
(CAZ; 30 g). chloramphenicol (CHL; 30 g). ciproIloxacin (CIP; 5 g). colistin (COL;
10 g). gentamicin (GEN; 10 g). kanamycin (KAN; 30 g). nalidixic acid (NAL; 30 g).
norIloxacin (NOR; 10 g). streptomycin (STR; 10 g). tetracycline (TET; 30 g). trimethoprim
(TMP; 5 g). and sulphonamides (SUL; 300 g). In addition. the integron-carrying Salmonella
Dublin isolates were tested against spectinomycin (SPE; 100 g) to conIirm the agreement
between the phenotypic resistance and genotypic resistance observed. These isolates were also
tested against IlorIenicol (FLO; 30 g) in order to Iigure out any relationship between their
susceptibility to chloramphenicol and IlorIenicol. The antibiotic disks were purchased Irom
Oxoid (UK) except Ior IlorIenicol (BBL. USA). Isolates were scored as susceptible.
intermediate resistant or resistant according to inhibition zone diameters recommended Ior
Enterobacteriaceae by the CLSI except Ior colistin where the zone criteria oI 11 (resistant)
and 14 mm (susceptible) were used
9
. Escherichia coli ATCC 25922 was used Ior quality
control.

Detection of class 1 integrons by PCR
Total DNA was prepared by the boiled lysate method
16
. Alkaline lysis
13
was used Ior the
analysis oI chromosomal and plasmid DNA.
Table 1. The PFGE proIiles. antimicrobial susceptibility patterns oI multidrug-resistant class 1
integron-carrying S. Dublin isolates Irom cattle in The Netherlands
PFGE proIiles R-types
ID no.

Year
Sources
Geographic
regions
XbaI BlnI
N018
N054
N056
N064
N065
N295
N302
N305
N308
N311
N323
N328
2001
1993
1995
1995
1996
2004
2004
2004
2004
2004
2003
2003
Feces (calI)
Blood (dairy cow)
Feces ( dairy cow)
Synovia (calI)
Synovia (calI)
Spleen (dairy cow)
Lung (dairy cow)
Abomasum (dairy cow)
Intestines (dairy cow)
Feces (dairy cow)
Intestines (dairy cow)
Brain (dairy cow)
Leeuwarden
Barsingerhorn
Zeewolde
Abcoude
Nederhorst
Lutiegast
Griipskerk
Baaium
Deventer
Ameland
Oudega
Niieberkoop
I
IIb
IIc
IIc
IIc
IIa
IIa
IIa
IIc
IIc
IIc
IIc
III
IV
VI
I
I
I
I
I
I
I
I
V
CHL.STR. SPE. SUL
CHL.STR. SPE. SUL
CHL. SPE. SUL
CHL.STR. SPE. SUL
CHL.STR. SPE. SUL
CHL.STR. SPE. SUL
CHL.STR. SPE. SUL
CHL.STR. SPE. SUL
CHL.STR. SPE. SUL
CHL.STR. SPE. SUL
CHL.STR. SPE. SUL
CHL.STR. SPE. SUL

Chapter 5
95
All isolates were tested Ior the presence oI class 1 integrons by PCR as described
15
.
The size oI any inserted gene cassette(s) in the integron-positive (int-positive) isolates was
determined by CS-PCR according to the method oI Levesque et al.
16
. The int-positive control
isolate was Salmonella Typhimurium (V9908674)
31
.

Characterization of integrons by restriction fragment length polymorphism (RFLP)
In order to determine whether diIIerent isolates yielding CS-PCR ampliIication products oI a
similar size carried identical integrons. the products were puriIied using the QiaQuick PCR
puriIication kit (Qiagen. Germany) and digested with the restriction endonuclease BclI and
HpaII (Invitrogen. USA) according to the manuIacturers` instructions. Integrons oI the same
size were considered diIIerent iI they had distinct RFLP patterns (at least one band diIIerence).

Cloning and sequencing
When int-positive Salmonella Dublin isolates had identical RFLP patterns. the CS-PCR product
Irom one isolate. which was randomly chosen. was sequenced. The CS-PCR product was
puriIied and cloned in the pGEM-T easyVector (Promega. USA). Colonies carrying the target
Iragment were picked by blue-white screening Irom Luria-Bertani plates containing ampicillin
100 g/ml. 40 l 100 mM IPTG per plate and 40 l 2 X-Gal per plate. Plasmid DNA Irom
white colonies was checked Ior the presence oI the target Iragment by puriIication with a
QIAprep Spin Miniprep kit (Qiagen. Germany) and digestion with the restriction endonuclease
EcoRI (10 ng/l) at 37
0
C Ior 2 hours.

The CS-Iragment was cycle sequenced using SP6 and T7 primers (Promega. USA).
Dideoxy sequencing was perIormed on the ABI Prism 310 Sequencer. Sequences obtained were
compared to those in the GenBank database using the SeqMan II program (DNAStar Inc..
USA).

Conjugation experiments
In order to determine whether antimicrobial resistance determinants in Salmonella Dublin
isolates could be transIerred to other species. a coniugation experiment was perIormed in which
a riIampicin-resistant (RiI
R
) and sulIamethoxazole-susceptible (Sul
S
) E. coli K12 isolate was
used as a recipient
14
. Both donor (integron carrying sulIamethoxazole-resistant S. Dublin
isolates) and acceptor bacteria were cultured in Luria Bertani (LB) broth until an OD
660
0.5-
0.6 was reached. The mating process in which donor and recipient were present in an 1:9 ratio
(v/v) was perIormed in LB broth. Incubation took place overnight in a water-bath at 37
0
C.
Transconiugants were selected on MacConkey (Oxoid. UK) agar plates containing both
riIampicin (50 g/ml) and sulIamethoxazole (512 g/ml). Red colonies were selected and
puriIied by subculture on MacConkey agar and nutrient agar (NA. Oxoid. UK) without
antibiotics. Biochemical characteristics. susceptibility patterns. and the presence oI class 1
integrons oI the transconiugants were tested as described above. The donor control strain was a
Salmonella Typhimurium isolate obtained Irom a human clinical case in Vietnam. which can
transIer its resistance determinants to E. coli K12.
96
Plasmid profiles
Plasmid proIiling was used in order to investigate the genetic diversity oI our Salmonella
Dublin isolates. The plasmid analysis was perIormed as previously described by Kado and
Liu
13
. with some modiIications. BrieIly. 1.5 ml oI an overnight culture in BHI broth was
centriIuged at 13000 rpm Ior 5 min. The pellet was resuspended in 50 l TE (50 mM Tris.
1 mM EDTA. (pH 8.0). Cells were lysed by mixing with 100 l (50 mM Tris. 3 sodium
dodecyl sulphate (SDS. pH 12.6) and incubated in a water bath Ior 30 min at 56
0
C. Then.
100 l oI phenol:chloroIorm:isoamyl alcohol (25:24:1. v/v/v) was added. The suspension was
mixed vigorously until it was homogeneously milk-white. AIter centriIugation at 13000 rpm Ior
15 min. 40 l oI the upper aqueous phase was careIully transIerred to a clean EppendorI tube.
Twenty-Iive l oI this plasmid extraction sample was analyzed on a 0.7 agarose gel. AIter
electrophoresis Ior 3 h. the gel was stained with ethidium bromide (0.5 g/ml) Ior 30 min. DNA
bands were visualized under UV transillumination. The reIerence isolate was Salmonella
Typhimurium phage type 13 containing Iive plasmids oI diIIerent sizes (180 kbp. 82 kbp.
39 kbp. 5.5 kbp and 4.4 kbp).

Southern blot hybridization
The location oI the integrons in 12 integron-carrying Salmonella Dublin isolates was assessed
by Southern blot hybridization using two probes. The integrase probe was generated by PCR
using int-F-DIG and int-R speciIic primers. A probe generated by PCR using the primers
(5`-ATGACCAGCCACACTGGAAC-3` and 5`-TTAGCCGGTGCTTCTTCTGC-3`) designed
based on the 16S ribosomal RNA gene oI Salmonella Dublin (accession number AF22789) was
used as a control. The plasmid and chromosomal DNA in the gel were transIerred to a nylon
membrane by capillary action. Iixed by UV irradiation (Stratalinker. USA). and analysed by
Southern blot analysis
18
using a digoxigenin labelled probe. A DIG labelling and detection kit
(Roche. Germany) was used to label the probe and to detect hybridized targets according to the
manuIacturer`s instructions. Between the hybridizations the membrane was stripped as
described
1
.

Pulsed Field Gel Electrophoresis
The twelve int-positive isolates and 10 randomly chosen int-negative isolates were investigated
using Pulsed Field Gel Electrophoresis (PFGE). BrieIly. a pellet Irom one ml oI overnight
bacterial culture (in trypticase soy broth) was obtained by centriIugation at 13000 rpm Ior
10 min. The pellet was resuspended in 300 l TEN buIIer (10 mM Tris/HCl. 100 mM EDTA.
150 mM NaCl. pH8.0) and diluted to an OD
600
oI 2 in 100 l (6 mM Tris/HCl. 100 mM
EDTA. 1 M NaCl. pH8) using a Universal Photometer. Next 100 l melted 1.2 agarose
containing 1 SDS (BioRad. USA). held at a temperature oI 50
0
C. was added to a microtube
containing 100 l cell suspension (OD
600
2). The mixture was quickly and careIully dispensed
into a sample mold (BioRad. USA). AIter solidiIication. the plug was transIerred to a tube
containing 200 l (5 mM Tris/ HCl. 100 mM EDTA. 1M NaCl. 1 N-lauroyl sarcosyl. 0.5
natrium desoxycholate. (pH8.0). 1mg/ml lysozyme) and incubated at 37
0
C overnight. Then.
the plugs were incubated overnight in 400 l (50 mM Tris/HCl. 50 mM EDTA. 1 N- lauroyl
Chapter 5
97
sarcosyl. 1mg/ml proteinase K) at 55
0
C. AIter deproteinisation. the plugs were washed 5 times
at room temperature with 10 ml oI 1.0 Tris EDTA Ior 15-30 min per wash with moderate
shaking. The agarose plug embedded bacterial DNA was digested with 40 U oI XbaI or BlnI
according to the manuIacturer`s instructions (Roche. Switzerland) at 37
0
C Ior 4 h. The plugs
were then placed in a 1 Seakem Gold PFGE agarose gel (BioRad. USA) and restriction
Iragments were separated by electrophoresis in 0.5 Tris borate EDTA (TBE) buIIer with
recirculation at 14
0
C using a CHEF DR III system (BioRad. USA). Pulse times were set up
Irom 2 to 64 sec during a 20 h run at 6 V/cm. The gel was stained with ethidium bromide
(0.5 g/ml) Ior 30 min. DNA bands were visualized under UV transillumination. The reIerence
isolate was PulseNet Salmonella Braenderup (H9812). The PFGE patterns oI our bovine
Salmonella Dublin isolates aIter digestion with XbaI were compared to each other and to those
oI the human Salmonella Dublin isolates (without the preIix N in their ID in Fig. 2) Irom other
European countries available Irom a database oI the Salm-gene proiect. a European
collaboration Ior DNA Iingerprinting oI Iood-related salmonellosis
28
(the data were downloaded
Irom the network oI the Public Health Laboratory Service. Laboratory oI Enteric Pathogens.
London. United Kingdom to that oI the National Institute oI Public Health and Environment
RIVM. The Netherlands) using Bionumerics soItware V.4.00 (Applied Maths. Belgium). Types
were deIined as diIIerent when their PFGE patterns had at least one band diIIerence.

RESULTS

Disk diIIusion testing oI the 59 Salmonella Dublin isolates revealed that 44 isolates (74.6)
were susceptible to all 15 antimicrobial agents tested. All isolates were susceptible to
ampicillin. amoxicillin with clavulanic acid. cephalothin. ceItazidime. ciproIloxacin.
norIloxacin. colistin. gentamicin. kanamycin. tetracycline and trimethoprim. Resistance was
Iound against streptomycin. chloramphenicol. sulphonamides and nalidixic acid. Resistance
patterns oI resistant Salmonella Dublin isolates are shown in Table 1. The Salmonella Dublin
isolates susceptible to all antimicrobials and the isolates that were resistant to only one
antimicrobial were all int-negative. All 12 multidrug-resistant isolates were int-positive and
contained a CS-PCR amplicon oI approximately 1000 bp. The RFLP patterns oI these
amplicons were identical. BclI cleaves the amplicon in 2 Iragments oI 700 and 300 bp and
HpaII cleaves the amplicon into 4 Iragments oI 580. 240. 130 and 50 bp. Sequencing oI the
1000 bp CS-PCR product showed that it contained the aadA1 gene (Genbank accession no.
AJ746361). The coniugation experiment showed that none oI the multidrug-resistant
Salmonella Dublin isolates could transIer its antimicrobial resistance to the E. coli K12
recipient. Southern blot hybridization showed that the integrons oI the 12 Salmonella Dublin
isolates were located on the chromosome since the integrase probes hybridized with the same
target DNA as the 16S ribosomal RNA probes oI Salmonella Dublin (data not shown).

Two plasmid proIiles were Iound among the 59 Salmonella Dublin isolates (Fig.1).
Isolates with proIile II carried only one plasmid oI 72 kb. This was the most common proIile
among the Salmonella Dublin isolates (n57; 96.6) and was Iound in both int-positive and
98

1 2 3

180 kb
82 kb
39 kb

chromosomal DNA

5.7 kb

4.4 kb

Fig 1. Plasmid proIiles oI Salmonella Dublin strains
Lane 1: S . Typhimurium pt 13 (reIerence strain)
Lane 2: Plasmid proIile I: 72 kb. 3.3 kb (isolates
N18 and N54)
Lane 3: Plasmid proIile II: 72 kb (other remaining
int-positive isolates and int-negative isolates)

int-negative isolates. Two isolates
with proIile I contained a second
plasmid oI 3.3 kb in addition to the
plasmid oI 72 kb (N18 and N54. both
int-positive isolates).

XbaI-PFGE analysis revealed that
Salmonella Dublin isolates carrying
class 1 integrons had slightly
diIIerent patterns Irom the int-
negative isolates although they were
still highly homologous (~90)
(Fig. 2). The twelve int-positive
Salmonella Dublin isolates could be
divided into 2 types. OI these two.
XbaI-type I included N18 which
diIIered Irom all other isolates.
Isolate N18 possessed one band oI
about 900 kb in size instead oI two
bands oI about 400 kb and 480 kb in
size (Fig. 2). XbaI-type II included
isolates which could be divided into 7 subtypes (isolates in a subtype have 100 homology).
Subtype IIa included N305. N302. N295 and isolate number 012149 Irom Denmark (data oI this
isolate are Irom the Salm-gene proiect)
28
. Subtype IIb included N54 and isolate number 012139
Irom Denmark. Subtype IIc contained N56. N65. N64. N323. N308. N311. N328. isolate
number 012145 Irom Denmark and 009514 Irom Scotland. The int-negative Salmonella Dublin
isolates were distributed among subtypes IId and IIe (Fig. 2). The Salmonella Dublin isolates
within the clusters could be easily distinguished by BlnI-PFGE analysis. Class 1 int-positive
isolates belonged to BlnI-types I (n8). III (n1). IV (n1). V (n1) and VI (n1). Int-negative
S. Dublin isolates belonged to BlnI-types II. VII. VIII and IX. A combination oI the two PFGE
proIiles allowed clear diIIerentiation oI 11 Salmonella Dublin strains (Fig. 2).

DISCUSSION

The most important Iinding in this study was the high rate oI class 1 integrons in
epidemiologically unrelated Dutch Salmonella Dublin isolates. These Iindings are in contrast
with a study by Randall et al
23
who Iound no class 1 integrons among 30 Salmonella Dublin
isolates originating Irom the United Kingdom. In another study investigating 100 Salmonella
Dublin isolated Irom humans and animals in the United Kingdom only Iive multidrug-resistant
isolates were Iound and oI these. only three harboured class 1 integrons
17
. ThereIore the rate oI
class 1 integrons was much higher in the present study (20.3) than in the study oI Liebana et
al. (3)
17
or in the study by Randall et al. (0)
23
. One possible explanation Ior these
diIIerences might be that here only bovine strains were investigated whereas Liebana et al.
17

Chapter 5
99
also included human isolates in their study. Another explanation might be diIIerences in animal
husbandry practices or antimicrobial usage in cattle Iarming between The Netherlands and the
United Kingdom. Salmonella Dublin isolates carrying integrons should be controlled as
regional and international transports oI animals are becoming more and more common and may
lead to the global spread oI multidrug-resistant Salmonella Dublin strains.

Fig. 2. Dendogram demonstrating the genetic relationship oI S. Dublin including 12 class 1
integron positive isolates (dot). 10 class 1 integron negative isolates (star) and isolates Irom
Denmark (DKSSI) and Scotland (GZSSRL) aIter digestion with XbaI or with BlnI. The
combined types were also indicated. The analysis oI the bands generated was perIormed using
the Dice coeIIicient and un-weighted pair group method with arithmetic averages. The numerals
at the top oI the Iingerprint indicate molecular weights in Kb.

Another Iinding oI this study was that phenotypical resistance to two or more
antimicrobials was strongly associated with the presence oI class 1 integrons in Salmonella
Dublin. In accordance with the results oI other studies on Salmonella
2
. isolates susceptible to all
antimicrobials did not contain class 1 integrons. Many oI the Dutch Salmonella Dublin isolates
showed phenotypical resistance against streptomycin. chloramphenicol and sulphonamides.
These isolates carried integrons with only the gene cassette aadA1 encoding Ior streptomycin
resistance. To our knowledge. this is the Iirst report documenting the presence oI aadA1 in
Salmonella Dublin although it has previously been reported in S. Typhimurium
11
and other
Enterobacteriaceae
14
. This indicates that antibiotic resistance genes associated with class 1
Dice (Opt:1.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [10.0%-93.0%]
PFGE-XbaI
1
0
0
9
5
9
0
PFGE-XbaI
5
0
.0
0
1
0
0
.0
0
2
0
0
.0
0
3
0
0
.0
0
4
0
0
.0
0
6
0
0
.0
0
1
0
0
0
1
5
0
0
Kb
PFGE-BInI
005702
N03
N251
N30
N33
N16
N26
N31
N317
N34
N59
006564
009514
012145
N308
N311
N323
N328
N56
N64
N65
012139
N54
012149
N295
N302
N305
006563
N18
DKSS
DKSS
GZSSRL
DKSS
DKSS
DKSS
DKSS
e
e
e
e
e
d
d
d
d
d
d
d
c
c
c
c
c
c
c
c
c
b
b
a
a
a
a
a
V
V
ID isolates
XbaI
Types
BlnI
Types
Combined
Types
100
integrons may be exchanged between Salmonella serovars and between Enterobacteriaceae.
Further research is necessary to conIirm this hyphothesis.

Sequencing oI the CS-product showed that no gene encoding chloramphenicol
resistance was located on the class 1 integrons oI the bovine S. Dublin strains in this study. In
The Netherlands. chloramphenicol is not longer used in cattle since it was Iorbidden in 1994 but
chloramphenicol resistance appears to be stably maintained. This might be explained by the Iact
that IlorIenicol is being used to treat bovine respiratory inIections in the Netherlands since
1995. Acquired resistance to IlorIenicol may develop and show cross-resistance to
chloramphenicol. However. all Salmonella Dublin isolates in the present study were susceptible
to IlorIenicol. Thus. other possible explanations Ior the maintained resistance against
chloramphenicol are the use oI chloramphenicol in the past or the co-selection oI
chloramphenicol resistance through the use oI other antimicrobials.

The epidemiological relatedness oI Salmonella Dublin was studied using plasmid
analysis and PFGE. All isolates in the present study had at least one plasmid oI 72 kb. This
plasmid is known to be a serotype-speciIic plasmid (SSP) oI Salmonella Dublin and contributes
to the virulence oI the organism in the mouse model
27
. The Iact that it was Iound in all our
isolates indicates a high level oI stability oI the SSP. Until now. plasmid proIile I (the 3.3 and
the 72 kb plasmids) has only been reported Irom Germany and the UK
4. 17
. while plasmid
proIile II (only the 72 kb plasmid) seems to have a worldwide distribution among Salmonella
Dublin
4
. The coniugation experiment using the mating procedure described in the present study
showed that none oI the MDR Salmonella Dublin isolates could transIer its antimicrobial
resistance to the E. coli K12 recipient. In a previous study. Salmonella Dublin isolates were
observed to contain Iewer transmissible R plasmids than S. Typhimurium
3
. This is in
accordance with our Iinding that the integrons in Salmonella Dublin isolates are located on the
chromosome. The integration oI resistance determinants into the bacterial chromosome may
contribute to the enhanced persistence oI phenotypic resistance even in the absence oI
antibiotic.

In addition to plasmid analysis. PFGE was used to investigate the genetic relationship
between the Salmonella Dublin isolates. Salmonella Dublin isolates were shown to have a
typical PFGE pattern. characteristic Ior this serovar. iI its genomic DNA was digested with the
restriction enzyme XbaI. AIter digestion with XbaI. the maiority oI the Dutch bovine
Salmonella Dublin isolates appeared to be very closely related to each other and to human
S. Dublin isolates Irom other European countries. In one study conducted in England. Wales
and Ireland. some bovine Salmonella Dublin isolates were reported to be diIIerent. when XbaI
was used
17
. Another interesting Ieature oI the present study was that the PFGE proIiles in which
the genomic DNA was cut with the restriction enzyme BlnI instead oI XbaI were even more
discriminative (7 BlnI types compared with 2 XbaI types). The PFGE proIiles produced aIter
digestion with BlnI resulted in a better discrimination oI the int-positive and int-negative
isolates. This indicates that the Dutch bovine Salmonella Dublin isolates are actually diIIerent
strains and not similar as indicated by the XbaI proIiles alone. In contrast with the Iinding oI
Chapter 5
101
Browning et al
4
. our Salmonella Dublin isolates were not clonal. Thus. BlnI can be used in
PFGE genotyping oI Salmonella Dublin isolates in national and international surveillance.
especially when isolates seem to belong to the same genomic lineages
17. 20
.

In conclusion. the combination oI the presence oI antimicrobial resistance genes and
the high prevalence oI class 1 integrons among Salmonella Dublin isolates may lead to the
geographical spread oI antibiotic resistance since the international transport oI live animals and
animal products is growing. PFGE patterns using the restriction enzyme BlnI demonstrated
more genetic diversity among Salmonella Dublin isolates than when the restriction enzyme
XbaI was used. ThereIore. it is advised to use BlnI in addition to XbaI Ior PFGE analysis oI
Salmonella Dublin.

ACKNOWLEDGMENTS

We thank Dr. Wim Wannet. Henk Brunings. and Henny Maas Irom the LIS. RIVM. Bilthoven.
and Mariike Keestra and the staII oI the VMDC. Utrecht University Ior assistance and technical
guidance.

REFERENCES
1. Ausubel. F.M.. R. Brent. R.E. Kingston. D.D. Moore. J.G. Seidman. J.A. Smith. and K. Struhl. 2005.
Current protocols in molecular biology. John Wiley and Sons. Inc.. USA.
2. Baggesen. D.L.. D. Sandvang. and F.M. Aarestrup. 2000. Characterization oI Salmonella enterica serovar
Typhimurium DT104 isolated Irom Denmark and comparison with isolates Irom Europe and the United
States. J. Clin. Microbiol. 38:1581-1586.
3. Brackelsberg. C.A.. L.K. Nolan. and J. Brown. 1997. Characterization oI Salmonella Dublin and Salmonella
Typhimurium (Copenhagen) isolates Irom cattle. Vet. Res. Commun. 21:409-420.
4. Browning. L.M.. C. Wray. and D.J. Platt. 1995. Diversity and molecular variation among plasmids in
Salmonella enterica serotype Dublin based on restriction enzyme Iragmentation pattern analysis. Epidemiol.
InIect. 114:237-248.
6. Edwards. P.R.. and W.H. Ewing. 1986. Edwards and Ewing's identiIication oI Enterobacteriaceae. 4th.
Elseviers Science Publishing Co.Inc.. New York.
7. Esaki. H.. A. Morioka. A. Koiima. K. Ishihara. T. Asai. Y. Tamura. H. Izumiya. J. Teraiima. H. Watanabe.
and T. Takahashi. 2004. Epidemiological characterization oI Salmonella Typhimurium DT104 prevalent
among Iood-producing animals in the Japanese veterinary antimicrobial resistance monitoring program
(1999-2001). Microbiol. Immunol. 48:553-556.
9. Gales. A.C.. A.O. Reis. and R.N. Jones. 2001. Contemporary assessment oI antimicrobial susceptibility
testing methods Ior polymyxin B and colistin: review oI available interpretative criteria and quality control
guidelines. J. Clin. Microbiol. 39:183-190.
53:997-1003.
11. Guerra. B.. S. Soto. S. Cal. and M.C. Mendoza. 2000. Antimicrobial resistance and spread oI class 1
integrons among Salmonella serotypes. Antimicrob. Agents Chemother. 44:2166-2169.
102
12. Jacobs. P.P.. A.M. van Elsacker-Niele. and I.J. Visser. 2002. A man with a Salmonella Dublin-inIected
aneurysm oI the abdominal aorta. Ned. Tiidschr. Geneeskd. 146:520-524.
13. Kado. C.I.. and S.T. Liu. 1981. Rapid procedure Ior detection and isolation oI large and small plasmids. J.
Bacteriol. 145:1365-1373.
14. Leverstein-van Hall. M.A.. A.T.A. Box. H.E.M. Blok. A. Paauw. A.C. Fluit. and J. VerhoeI. 2002. Evidence
oI extensive interspecies transIer oI integron-mediated antimicrobial resistance genes among multidrug-
resistant Enterobacteriaceae in a clinical setting. J. InIect. Dis. 186:49-56.
15. Leverstein-Van Hall. M.A.. A. Paauw. A.T.A. Box. H.E.M. Blok. J. VerhoeI. and A.C. Fluit. 2002. Presence
oI integron-associated resistance in the community is widespread and contributes to multidrug resistance in
the hospital. J. Clin. Microbiol. 40:3038-3040.
17. Liebana. E.. L. Garcia-Migura. C. Clouting. C.A. Cassar. F.A. CliIton-Hadley. E.A. Lindsay. E.J. ThrelIall.
S.A. Chappell. and R.H. Davies. 2002. Investigation oI the genetic diversity among isolates oI Salmonella
enterica serovar Dublin Irom animals and humans Irom England. Wales and Ireland. J. Appl. Microbiol.
93:732-744.
18. Maniatis. T.. E.F. Fritsch. and J. Sambrook. 1982. Molecular cloning: a laboratory manual Cold Spring
Harbor Laboratory. Cold Spring Harbor.
20. Olsen. J.E.. and M. Skov. 1994. Genomic lineage oI Salmonella enterica serovar Dublin. Vet. Microbiol.
40:271-282.
22. Quinn. P.J.. M.E. Carter. B. Markey. and G.R. Carter. 1994. Clinical veterinary microbiology. Mosby-
Yearbook Europe Limited. England.
genes. integrons and multiple antibiotic resistance in thirty-Iive serotypes oI Salmonella enterica isolated
Irom humans and animals in the UK. J. Antimicrob. Chemother. 53:208-216.
24. Recchia. G.D.. and R.M. Hall. 1997. Origins oI the mobile gene cassettes Iound in integrons. Trends
25. Small. R.G.. and J.C. Sharp. 1979. A milk-borne outbreak due to Salmonella Dublin. J. Hyg. (Lond). 82:95-
100.
26. Soto. S.M.. M.A. Gonzalez-Hevia. and M.C. Mendoza. 2003. Antimicrobial resistance in clinical isolates oI
Salmonella enterica serotype Enteritidis: relationships between mutations conIerring quinolone resistance.
integrons. plasmids and genetic types. J. Antimicrob. Chemother. 51:1287-1291.
27. Terakado. N.. T. Sekizaki. K. Hashimoto. and S. Naitoh. 1983. Correlation between the presence oI a IiIty-
megadalton plasmid in Salmonella Dublin and virulence Ior mice. InIect. Immun. 41:443-444.
28. The European Commission. Strengthening international Salmonella surveillance through strain typing and
diIIerentiation (Salm-gene). http://www.salmgene.net (Accessed 19 April 2005. date last accessed).
29. Vaillant. V.. S. Haeghebaert. J.C. Desenclos. P. Bouvet. F. Grimont. P.A. Grimont. and A.P. Burnens. 1996.
Outbreak oI Salmonella Dublin inIection in France. November - December 1995. Euro. Surveill. 1:9-10.
30. van Duiikeren. E.. W.J.B. Wannet. D.J. Houwers. and W. van Pelt. 2002. Serotype and phage type
distribution oI Salmonella strains isolated Irom humans. cattle. pigs. and chickens in The Netherlands Irom
1984 to 2001. J. Clin. Microbiol. 40:3980-3985.
31. van Duiikeren. E.V.. A.T. Box. P. Schellen. D.J. Houwers. and A.C. Fluit. 2005. Class 1 integrons in
Resist. 11:383-386.
32. Wray. C.. and R.H. Davies. 2000. Salmonella inIection in cattle. In Wray. C. & Wray. A. Salmonella in
domestic animals. WallinIord. UK: CABI publishing. 169-190.

A nnvc! gcnnmIc Is!and 1
and rarc Intcgrnn tvcs In
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, Ad C. IIuil
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IouinaI of AnlinicioliaI Chenolheiapv. :594-599. 2OO7

Class 1 integrons in equine Salmonella Typhimurium
104
ABSTRACT

Objectives: To investigate the genotypic resistance oI integron-carrying S. Typhimurium
isolates Irom horses and their genetic relationship.
Methods: Sixty-one Salmonella isolates were screened Ior the presence oI class 1 integrons by
PCR. The gene cassettes oI integron-positive isolates were detected by PCR. restriction
Iragment length polymorphism typing and sequencing. The potential Ior the transIer oI
resistance determinants was investigated by coniugation experiments. The presence oI
Salmonella Genomic Island 1 (SGI1) or its variants was studied by PCR and nucleotide
sequencing. PFGE was used to genotype the isolates.
Results: Eight distinct XbaI-PFGE proIiles and seven integron types were observed among 26
integron-carrying S. Typhimurium isolates. The gene cassettes detected were dfrA1. dfrA7.
dfrA14. aadA1. aadA2. aadB. and bla
PSE
. A rare type oI integron Iound in nine isolates carried
the dfrA14 and aadA1 gene cassettes. Twelve S. Typhimurium DT104 isolates contained
Salmonella Genomic Island 1 or one oI its variants (SGI1. SGI1-B and SGI1-C). A novel
variant oI SGI1 designated SGI1-M was identiIied in one isolate in which the aadA2 gene oI
SGI1 was replaced by the aadB gene. TransIer oI integrons and antimicrobial resistance
determinants to E. coli K12 via coniugation was possible with nine isolates. Resistance to
Iluoroquinolones in nine isolates was caused by mutations in the gvrA gene leading to the
amino acid changes Ser83Ala and Asp87Asn.
Conclusions: The integron-positive clinical S. Typhimurium isolates Irom horses belong to
distinct strains. The data demonstrate the capability oI S. Typhimurium to acquire additional
antibiotic resistance determinants and underline the need Ior the prudent use oI antimicrobials.

Key words: Salmonella spp.. genotyping. SGI1. multidrug resistance. coniugation.
Chapter 6
105
INTRODUCTION

Over the last decade. a large increase in multidrug-resistant (MDR) Salmonella enterica isolates
has been documented
27
. In humans. Salmonella Typhimurium (S. Typhimurium) is a maior
aetiologic agent oI Iood-borne salmonellosis. In The Netherlands. S. Typhimurium is the
predominant serovar causing salmonellosis in horses and this serovar was more oIten resistant
to antimicrobial agents when compared to other Salmonella serovars
28
. MDR Salmonella
isolates Irom horses can be transIerred to humans by direct contact or indirectly through the
Iood chain
8
.

Multidrug resistance is strongly linked to the presence oI class 1 integrons
18
. Integrons
are genetic elements that recognize and capture mobile gene cassettes (oIten encoding antibiotic
resistance) by site-speciIic recombination
14
. Three classes oI integrons have been described.
Class 1 integrons are the most common integrons Iound in clinical Salmonella isolates
10
.
Salmonella Genomic Island 1 (SGI1) is a 43-kilobase (kb) genomic island. which contains a
complex integron
23
. Variants oI SGI1 (A-L) have been Iound in several Salmonella serovars
including S. Typhimurium
1. 5. 20
. SGI1 can be transIerred to other bacteria like Escherichia coli
in the presence oI a helper plasmid
6
.

Van Duiikeren et al.
30
described that a particular type oI
integron. which was identiIied by restriction Iragment length polymorphism (1.600 bp. Type
XIII). was exclusively detected in equine S. Typhimurium isolates. This observation prompted
the current investigation.

In the present study we investigated (i) the genetic relationship oI 26 clinical equine
integron-carrying MDR S. Typhimurium isolates. (ii) the characteristics oI the antimicrobial
resistance determinants among the isolates. and (iii) the potential to transIer these antibiotic
resistance determinants to other bacterial species.


Bacterial isolates
During the period 1993 to 2005. a total oI 406 clinical equine Salmonella isolates was collected
by the Veterinary Microbiological Diagnostic Center (VMDC) oI Utrecht University. The
Netherlands. The isolates were identiIied as Salmonella by biochemical testing and Iurther
characterized by serotyping. Based on the susceptibility testing data which were recorded by the
VMDC. 61 Salmonella group B isolates Irom this collection were selected. based on their
resistance to at least one antimicrobial drug. All isolates were cultured Irom diIIerent horses
that belonged to diIIerent owners living throughout the country. The 61 isolates were screened
Ior the presence oI class 1 integrons using PCR ampliIication oI the class 1 integrase gene
17
.
The 26 integron-carrying S. Typhimurium isolates were phage-typed using the Dutch phage
typing system
13
. Phage type 506 in the Dutch phage typing system corresponds to phagetype
DT104 in the English phage typing system
29
. The 26 isolates (Table 1) including 8 isolates
described in a previous study
30
were tested Ior their antimicrobial susceptibility by the disk
106
diIIusion assay using Neo-sensitab disks (Rosco. Denmark) based on the procedure
recommended by the Dutch Committee on Guidelines Ior Susceptibility Testing CRG
3
. The
antimicrobials tested were ampicillin (30 g). amoxicillin/clavulanic acid (30/15 g). ceIalexin
(30 g). ceItioIur (30 g). Ilumequine (30 g). enroIloxacin (10 g). streptomycin (100 g).
gentamicin (40 g). kanamycin (100 g). chloramphenicol (60 g). tetracycline (80 g). and
trimethoprim/sulIamethoxazole (5.2/240 g). In addition. the isolate H37 was tested Ior its
susceptibility to tobramycin (40 g) since its integron contained the aadB gene encoding
resistance to aminoglycosides.

Gene cassette characterization
The gene cassettes inserted in the integrons oI the isolates were determined by PCR with
primers Ior the conserved segment regions (CS-PCR)
19
. CS-PCR amplicons oI the same size
were subiected to restriction Iragment length polymorphism (RFLP) typing and were
considered identical iI they had the same RFLP pattern aIter digestion with at least two
enzymes (Table 1). The RFLP patterns obtained were compared with those Irom a previous
study
31
. II these data were not available. a representative oI each RFLP type was randomly
chosen Ior nucleotide sequencing on an ABI 3730 Sequencer. The obtained nucleotide
sequences have been deposited in GenBank (accession numbers DQ388123. DQ388124.
DQ388125 and DQ388126).

Detection of Salmonella Genomic Island 1 and its variants
All 26 isolates were examined Ior the presence oI the leIt and right iunction oI SGI1. The
presence oI sequences Irom the antibiotic resistance gene cluster was determined by PCR as
described
4. 7. 20
. Three S. Typhimurium isolates carrying SGI1. SGI1-B and SGI1-C.
respectively
31
. were included as controls. To conIirm the gene identity and the linkage between
genes. the products generated by PCR mapping (Fig. 1) were either sequenced or cloned using
DNA oI S. Typhimurium isolate H37 as template.

The two transconiugants obtained Irom the coniugation oI isolate H16 and H18 and E. coli were
examined Ior the insertion oI SGI1or SGI1-C into their chromosome. PCR assays were
perIormed as described previously
6
. II SGI1 or SGI1-C can be transIerred Irom Salmonella
H16 or H18. respectively. and inserted into the E. coli genome. we would expect that SGI1 will
be Ilanked by the thdF gene and the tnaL gene oI the E. coli transconiugants.
6

Detection of gvrA mutations by allele-specific (AS)-PCR-RFLP assay and nucleotide
sequencing
Nine enroIloxacin-resistant S. Typhimurium isolates were tested by AS-PCR and RFLP
analysis
11
to detect mutations in codons 81. 83 and 87 oI the gvrA gene. When more than one
isolate had the same RFLP pattern. one representative Iragment was chosen Ior nucleotide
sequencing. The enroIloxacin-resistant isolates were also tested Ior the presence oI the qnrA1
gene by PCR
24
.
Chapter 6
107
F
i
g
.

1
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108
Bacterial conjugation and plasmid analysis
A coniugation experiment was perIormed to determine whether the integrons and resistance
determinants oI the 26 S. Typhimurium isolates could be transIerred to E. coli. A riIampicin-
resistant (RiI
R
) and sulIamethoxazole-susceptible (Sul
S
) E. coli K12 strain was used as the
recipient as described
32
.

The transconiugants were tested Ior their biochemical characteristics by
the API 20E system (bioMerieux. Marcy-l'Etoile. France) and their susceptibility patterns and
integrons were determined as described above. In addition. plasmid analysis was perIormed
using the phenol-chloroIorm extraction procedure
16
Ior both the Salmonella donors and the
E. coli transconiugants. The reIerence strain was a S. Typhimurium phage type 13 strain
containing Iive plasmids ranging in size between 4.4 and 180 kb
32
.

Pulsed Field Gel Electrophoresis
To determine the genetic relationship among the 26 integron-carrying S. Typhimurium isolates.
PFGE analysis was perIormed as described
32
. The reIerence isolates were PulseNet
S. Braenderup and S. SenItenberg. PFGE proIiles were deIined as diIIerent when their PFGE
patterns had at least one band diIIerence.

Southern blot hybridization
To determine whether integrons were located at the same position oI the S. Typhimurium
genome Ior all isolates. Southern blot hybridization was perIormed by the capillary blot
procedure using the nine enroIloxacin resistant isolates. A luminescent DIG labelling and
detection kit (Roche. Mannheim. Germany) was used according to the manuIacturer`s
instructions.

RESULTS

Twenty-six (43 ) S. Typhimurium isolates carried at least one integron. The phage types.
resistance phenotypes. integron types. characteristics oI inserted gene cassettes. SGI1 types.
XbaI-PFGE proIiles. and the results oI the coniugation experiments are summarized in Table 1.
The integron-carrying isolates belonged to 8 diIIerent phage types. had nine diIIerent
phenotypic resistance proIiles and were resistant against one to nine antimicrobial agents. Eight
distinct PFGE proIiles were deIined (Table 1 and Fig 2). Seven integron types were Iound. Nine
S. Typhimurium isolates oI diIIerent phage types with resistance pattern ACSSuTEFGK and
integron type XVI. were grouped in PFGE proIile I or II. The 13 S. Typhimurium DT 104
isolates were grouped in PFGE proIiles III or IV. Ten oI these isolates had the resistance
phenotype ACSSuT and contained a type I integron. The two isolates which were non-typeable
by phages and carried a type VII integron were classiIied into PFGE proIile V or VI. The two
isolates oI pt 204 and RDNC. respectively. both with integron type XVIII. were classiIied into
proIile VII or VIII.

Chapter 6
109
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110
Fig 2. XbaI-PFGE proIiles (I-VIII) oI integron-carrying
S. Typhimurium isolates Irom horses in The
Netherlands. M marker strain. S. Braenderup.
I II III IV V VI VII VIII M

1135 kbp
452.7 kbp
244.4 kbp
138.9 kbp
33.3 kbp

The cassettes present in the integrons carried the aadA1. aadA2 and aadB genes
encoding resistance to aminoglycosides; the dfrA1. dfrA7 and dfrA14 genes conIerring
resistance to trimethoprim. and the bla
PSE-1
gene encoding resistance to ampicillin. Nine
S. Typhimurium isolates carried an integron with the dfrA14-aadA1 gene cassettes. Southern
blot hybridization with an integrase-speciIic probe showed that the integrons in eight oI nine
isolates tested hybridized to similar sized Iragments suggesting that these integrons have a
similar position on the chromosome.

Ten S. Typhimurium DT 104 isolates contained SGI1. one isolate carried SGI1-B and
one isolate contained SGI1-C (Table 1). Based on the nucleotide sequencing results. the
antibiotic resistance cluster in isolate H37 (Fig. 1) is part oI a new SGI1 variant Ior which we
propose the name SGI1-M. The aadA2 gene encoding resistance against spectinomycin and
streptomycin oI SGI1 and other variants (SGI1-A. -C. -D. -E. -I) was replaced in this variant by
the aadB gene encoding kanamycin. gentamicin and tobramycin resistance. The isolate H37
indeed showed phenotypic resistance to kanamycin. gentamicin and tobramycin.

The nine enroIloxacin-resistant S. Typhimurium isolates had mutations in the two
codons Ior amino acids at positions 83 and 87 oI the gvrA gene as shown by AS-PCR-RFLP.
Nucleotide sequence analysis conIirmed that mutations were present at codon 83 and 87 where
Chapter 6
111
nucleotides TCC and GAC were replaced by GCC and AAC. respectively. leading to amino
acid substitutions Ser83Ala and Asp87Asn. No qnrA1-carrying isolate was Iound.

Integrase ampliIication using genomic DNA oI the transconiugants indicated that nine
S. Typhimurium isolates belonging to diIIerent phage types could transIer integrons to E. coli
(Table 1). The antibiotic resistance phenotype oI the transconiugants and the sizes oI the
plasmids detected are shown in Table 1. It should be noted that a large 90-kb plasmid was Iound
in halI oI the transconiugants. However. no plasmid was detected in some transconiugants
although they had a resistance phenotype (ACSSuTRiI) similar to that oI the donor.

The isolates H16 and H18 which carried SGI1 and SGI1-C. were tested Ior their ability
to transIer SGI1 to E. coli. The transconiugants had the resistance patterns ASSuRiI
(transconiugant Irom H16 and E. coli) and ASuRiI (transconiugant Irom H18 and E. coli)
(Table 1). Integrons were detected in these transconiugants.

DISCUSSION

This study describes the antibiotic resistance phenotypes and resistance genes oI 26 integron-
carrying MDR S. Typhimurium isolates Irom horses. the ability oI these isolates to transIer their
antimicrobial resistance determinants to E. coli and their genetic relationship. These data
indicate that equine S. Typhimurium isolates may be potential risk Iactors Ior both animal and
human health because they can easily spread their resistance determinants and because oI the
close contact between horses and humans.

An interesting Iinding in our study was that isolates oI diIIerent phage types can have
the same PFGE proIile. carry the same integron type and show a similar resistance phenotype.
Vice versa. isolates oI the same phage type can have diIIerent PFGE proIiles. contain distinct
integrons in various genomic islands (SGI1. SGI1-C or SGI1-B) and have diIIerent resistance
phenotypes. The combination oI phage typing. PFGE analysis and the analysis oI the integrons
indicated that the equine integron-carrying S. Typhimurium isolates are not clonal but belong to
a number oI diIIerent strains. These data and the great potential oI horizontal transIer indicate
that the multidrug resistance is due to acquired resistance rather than to the spreading a single
clone.

Apart Irom resistance to sulphonamides. resistance to ampicillin. chloramphenicol.
streptomycin and tetracycline was commonly observed regardless oI the phage types oI the
isolates. Phenotypic resistance to these antimicrobials in the S. Typhimurium DT104 isolates
may be caused by the presence oI the resistance genes (aadA2. bla
PSE-1
.

floR. tet(G) and sul1)
associated with SGI1. In isolates oI phage types other than DT 104. integron-associated
resistance genes (aadA1. dfrA1. dfrA7. dfrA14) were responsible Ior part oI the resistance
phenotype detected. In these non-DT 104 isolates resistance to gentamicin. kanamycin and
enroIloxacin was Irequently observed. but integron-associated gene cassettes encoding these
resistances were not Iound. The trimethoprim resistance gene cassettes (dfrA1. dfrA7 and
112
dfrA14) were Irequently detected in integrons in the present study. This is in accordance with a
previous report on high percentages oI phenotypic resistance to sulphonamides and
trimethoprim in equine salmonellae in The Netherlands
28
. It seems that resistance to
trimethoprim and sulphonamides is due to the Irequent use oI these antimicrobials Ior the
treatment oI horses. In The Netherlands. trimethoprim/sulphonamide combinations are the Iirst
choice in the treatment oI equine salmonellosis. All horses in the present study were clinically
ill and were probably treated with trimethoprim/sulphonamides or other antimicrobials beIore
the samples Ior culturing were taken. However. the exact data on the usage oI antimicrobials in
the horses were not available.

An important Iinding was that the nine MDR S. Typhimurium isolates carrying a rare
integron type with the dfrA14 and aadA1 gene cassettes. belong to distinct strains because
diIIerent phage types and two distinct PFGE patterns were observed. Four oI these isolates were
able to transIer their integron and the resistance determinants encoding Ior ampicillin.
chloramphenicol and tetracycline resistance to E. coli. This clearly indicates the potential oI
these strains Ior gene transIer to other members oI the Enterobacteriaceae. These nine isolates
also showed resistance to Ilumequine and enroIloxacin. This resistance is caused by mutations
leading to the amino acid changes Ser83Ala and Asp87Asn in GyrA. In Salmonella. a
single mutation in gvrA can be suIIicient to cause high-level resistance to nalidixic acid but
additional mutations are required to attain high-level resistance to Iluoroquinolones
26
. In
previous studies. single amino acid changes at Ser83Phe. and Asp87Asn/Gly were most
commonly observed
2. 12. 21. 25
. The mutation at both codons mentioned above were previously
detected in in vitro experiments
11. 22
and they were also described in 6 S. Typhimurium isolates
obtained Irom humans and cattle in Germany.
15
The presence oI enroIloxacin-resistant
Salmonella isolates in Dutch horses is unexpected because quinolones are not licensed Ior the
use in horses in The Netherlands. A possible explanation is that these isolates originate Irom
other animal species or humans.

A 90 kb plasmid can be transIerred Irom Salmonella Typhimurium to E. coli.
including the antimicrobial resistance genes that are present on them. In most transconiugants at
least one plasmid was present. but in some cases. no plasmid could be observed although the
transconiugants had obtained a resistance phenotype similar to that oI the donor. A possible
explanation Ior this phenomenon is the presence oI a low-copy plasmid. which could not be
detected with the procedure used. Another explanation may be that the resistance determinants
were present on a coniugative transposon and may be integrated into the chromosome oI the
recipients
9
.

Another interesting Ieature oI the present study is the presence oI a coniugative
plasmid-associated integron and a chromosomally located integron in the same S. Typhimurium
DT104 strain. Evidence Ior the presence oI a coniugative plasmid-associated integron includes
the presence oI integrons in the E. coli transconiugants obtained aIter mating between
S. Typhimurium H16 or H18 and E. coli K12; and the phenotypic resistance to ampicillin.
streptomycin. sulphonamide and riIampicin observed in the transconiugants. Salmonella
Chapter 6
113
isolates H16 and H18 contained SGI1 and SGI1-C. However. these genomic islands appear not
to be transIerred because neither resistance to chloramphenicol or tetracycline nor structures oI
SGI1 in the E. coli chromosome were detected in the transconiugants.

A novel variant oI SGI1 was Iound in a S. Typhimurium DT 104 isolate. In this isolate
the aadA2 gene present in the Iirst integron oI SGI1 or its variants (SGI1-A. -C. -D. E. I)
4. 5. 20
is
replaced by the aadB gene. The presence oI this new type oI SGI1 with the resistance gene
cluster aadB-sul1-floR-tet(G)-bla
PSE-1
coincided with the resistance to aminoglycosides.
sulphonamides. tetracycline. chloramphenicol/IlorIenicol and ampicillin observed in this
isolate. It is proposed to name this variant SGI1-M. The resistance to streptomycin and
trimethoprim is probably not encoded by gene cassettes integrated in integrons in this isolate.

ACKNOWLEDGEMENTS

This work was supported by a grant Irom the Vietnamese Government. We thank Anio
Verbruggen. Henny Maas. and Max Heck oI the Dutch National Institute oI Public Health and
the Environment and Marc Wsten oI the Department oI InIectious Diseases and Immunology.
Faculty oI Veterinary Medicine. Utrecht University Ior their help.

REFERENCES
2. Chu. C.. L.H. Su. C.H. Chu. S. Baucheron. A. Cloeckaert. and C.H. Chiu. 2005. Resistance to Iluoroquinolones
linked to gvrA and parC mutations and overexpression oI acrAB eIIlux pump in Salmonella enterica serotype
Choleraesuis. Microb. Drug Resist. 11:248-253.
3. Commissie Richtliinen Gevoeligheidsbepalingen (CRG). 2000. Interpretatie van gevoeligheidsonderzoek en
gevoeligheidscriteria voor antibacteriele middelen in Nederland. Ned. Tiidschr. Med. Microbiol 8:79-81.
7. Ebner. P.. K. Garner. and A. Mathew. 2004. Class 1 integrons in various Salmonella enterica serovars isolated
Irom animals and identiIication oI genomic island SGI1 in Salmonella enterica var. Meleagridis. J. Antimicrob.
Chemother. 53:1004-1009.
8. Espie. E.. H. De Valk. V. Vaillant. N. Quelqueieu. F. Le Querrec. and F.X. Weill. 2005. An outbreak oI
multidrug-resistant Salmonella enterica serotype Newport inIections linked to the consumption oI imported
horse meat in France. Epidemiol. InIect. 133:373-376.
9. Fluit. A.C.. and F.J. Schmitz. 2004. Resistance integrons and super-integrons. Clin. Microbiol. InIect. 10:272-
288.
10. Fluit. A.C. 2005. Towards more virulent and antibiotic-resistant Salmonella? FEMS Immunol. Med. Microbiol.
43:1-11.
114
11. Giraud. E.. A. Brisabois. J.L. Martel. and E. Chaslus-Dancla. 1999. Comparative studies oI mutations in animal
isolates and experimental in vitro- and in vivo-selected mutants oI Salmonella spp. suggest a counterselection
oI highly Iluoroquinolone-resistant strains in the Iield. Antimicrob. Agents Chemother. 43:2131-2137.
12. Griggs. D.J.. K. Gensberg. and L.J. Piddock. 1996. Mutations in gvrA gene oI quinolone-resistant Salmonella
serotypes isolated Irom humans and animals. Antimicrob. Agents Chemother. 40:1009-1013.
15. Heisig. P.. B. Kratz. E. Halle. Y. Graser. M. Altwegg. W. Rabsch. and J.P. Faber. 1995. IdentiIication oI DNA
gvraseA mutations in ciproIloxacin-resistant isolates oI Salmonella Tvphimurium Irom men and cattle in
Germany. Microb. Drug Resist. 1:211-218.
Bacteriol. 145:1365-1373.
17. Leverstein-Van Hall. M.A.. A. Paauw. A.T.A. Box. H.E.M. Blok. J. VerhoeI. and A.C. Fluit. 2002. Presence oI
integron-associated resistance in the community is widespread and contributes to multidrug resistance in the
hospital. J. Clin. Microbiol. 40:3038-3040.
21. Liebana. E.. C. Clouting. C.A. Cassar. L.P. Randall. R.A. Walker. E.J. ThrelIall. F.A. CliIton-Hadley. A.M.
Ridley. and R.H. Davies. 2002. Comparison oI gvrA mutations. cyclohexane resistance. and the presence oI
class I integrons in Salmonella enterica Irom Iarm animals in England and Wales. J. Clin. Microbiol. 40:1481-
1486.
22. Miko. A.. K. Pries. A. Schroeter. and R. Helmuth. 2005. Molecular mechanisms oI resistance in multidrug-
resistant serovars oI Salmonella enterica isolated Irom Ioods in Germany. J Antimicrob Chemother 56:1025-
1033.
23. Mulvey. M.R.. D.A. Boyd. A.B. Olson. B. Doublet. and A. Cloeckaert. 2006. The genetics oI Salmonella
Genomic Island 1. Microbes InIect. 8:1915-1922.
25. Piddock. L.J.. V. Ricci. I. McLaren. and D.J. Griggs. 1998. Role oI mutation in the gvrA and parC genes oI
nalidixic-acid-resistant Salmonella serotypes isolated Irom animals in the United Kingdom. J. Antimicrob.
Chemother. 41:635-641.
26. Ruiz. J.. D. Castro. P. Goni. J.A. Santamaria. J.J. Borrego. and J. Vila. 1997. Analysis oI the mechanism oI
quinolone resistance in nalidixic acid-resistant clinical isolates oI Salmonella serotype Typhimurium. J. Med.
27. Su. L.H.. C.H. Chiu. C. Chu. and J.T. Ou. 2004. Antimicrobial resistance in nontyphoid Salmonella serotypes:
a global challenge. Clin. InIect. Dis. 39:546-551.
28. van Duiikeren. E.. W.J.B. Wannet. M.E.O.C. Heck. W. van Pelt. M.M. Sloet van Oldruitenborgh-Oosterbaan.
J.A.H. Smit. and D.J. Houwers. 2002. Sero types. phage types and antibiotic susceptibilities oI Salmonella
strains isolated Irom horses in The Netherlands Irom 1993 to 2000. Vet. Microbiol. 86:203-212.
Chapter 6
115
30. van Duiikeren. E.. A.T.A. Box. P. Schellen. D.J. Houwers. and A.C. Fluit. 2005. Class 1 integrons in
Resist. 11:383-386.
31. Vo. A.T.T.. E. van Duiikeren. A.C. Fluit. and W. Gaastra. 2006. Antibiotic resistance. integrons and Genomic
Island SGI1 among non-typhoid Salmonella serovars in The Netherlands. Int. J. Antimicrob. Agents 28:172-
179.
32. Vo. A.T.T.. E. van Duiikeren. A.C. Fluit. M.E. Heck. A. Verbruggen. K. van der Zwaluw. and W. Gaastra.
2006. Class 1 integrons in Dutch Salmonella enterica serovar Dublin isolates Irom clinical cases oI bovine
salmonellosis. Vet. Microbiol. 117:192-200.

116

CharactcrIstIcs nf cxtcndcd-scctrum
ccha!nsnrIn-rcsIstant EntcrnbactcrIaccac
Isn!atcs frnm anIma!s
An T. T. Vo
1,3
1
, Ad C. IIuil
2
,

Win
Caaslia
1

1
Dcpar|ncn| cf |nnunc|cgu and |nfcc|icus Discascs. |acu||u cf
Vc|crinaru Mcdicinc. U|rccn| Unitcrsi|u
2
3
|acu||u cf Anina| Scicncc and Vc|crinaru Mcdicinc. Ncng |an
Unitcrsi|u. Vic|nan

Iail of lhis chaplei is accepled foi pulIicalion in Veleiinaiv MiciolioIogv

Extended-spectrum cephalosporin resistant Enterobacteriaceae isolates Irom animals
118
Abstract
Eight cetioIur/ceItazidime-resistant isolates (Iour Escherichia coli. three Klebsiella pneumoniae
and one Salmonella Braenderup) Irom horses and chicken were studied Ior the presence oI
extended-spectrum -lactamases (ESBL). AmpC -lactamases and class 1 integrons. The aim
was to contribute to the knowledge on this type oI isolates Irom animals. which is still limited.
The potential Ior horizontal transIer oI resistance genes among these clinical isolates was also
studied. Resistance Ior up to 11 antimicrobials was Iound Ior six isolates. A remarkable Iinding
was that ESBL- and integron encoded resistance genes were present in six equine isolates (even
Irom one month old Ioals). AmpC-type genes were Iound in one equine isolate and one chicken
isolate. Nucleotide sequence analysis revealed that the isolates carried the bla
CTX-M-1
. bla
CMY-2

and bla
ACC-1
genes besides the bla
TEM-1
and bla
SHV-1
genes
.
This is the Iirst report describing the
in vitro coniugal transIer oI the bla
ACC-1
and bla
CTX-M-1
genes between clinical
Enterobacteriaceae isolates (Irom Salmonella to Salmonella and Irom E. coli to Salmonella.
respectively). Gene cassettes encoding resistance to aminoglycosides (aadA1. aadA2 and
aadA5). and trimethoprim (dfrA1. dfrA12 and dfrA17) were Iound on the integrons present in
the isolates. The cassette arrays oI the dfrA1-aadA1 and dfrA17-aadA5 genes in the two
integrons oI a single E. coli isolate have not yet been described beIore.

Key words: Extended spectrum -lactamases. Enterobacteriaceae. animal. antibiotic
resistance.

Chapter 7
119
INTRODUCTION
The introduction oI the third-generation cephalosporins (TGC) in the early 1980s improved
clinical practice. TGC. are oIten used in a clinical setting. especially Ior the treatment oI
children. They are eIIective against most -lactamase-producing organisms. UnIortunately.
soon aIter their introduction resistance to expanded-spectrum cephalosporins (ESC) was
detected
5
. In veterinary medicine. TGC like ceItioIur. ceIquinome and ceIaperazone are used
Ior the treatment oI serious inIections in animals. ESC resistance has been studied in detail in
Enterobacteriaceae isolated Irom humans and to a lesser extent in isolates Irom Iood-producing
animals
19. 21
. Data on ESC-resistant Enterobacteriaceae Irom animals is limited
15
.

Commonly. TGC resistance in Enterobacteriaceae relates to the production oI
extended-spectrum -lactamases (ESBL. class A oI Ambler`s scheme)
1
or AmpC -lactamases
(class C)
21
. ESBLs conIer resistance to the penicillins. Iirst-. second-. and third-generation
cephalosporins and aztreonam (but not to cephamycins or carbapenems) and are usually
inhibited by -lactamase inhibitors like clavulanic acid. AmpC -lactamases have an even
broader resistance spectrum (even to cephamycins) and are not blocked by commercially
available -lactamase inhibitors
21
.

In addition. ESC-resistant strains oIten are also resistant to Iluoroquinolones
18. 22
. or
contain integrons encoding resistance to sulphonamide. aminoglycosides and
chloramphenicol
18. 26
. which even Iurther narrows the options Ior treatment. Integrons are gene
capture systems that contain genetic determinants Ior recognition and incorporation oI mobile
gene cassettes
9
. Among three distinct classes oI integrons described in clinical
Enterobacteriaceae isolates. class 1 integrons are the most signiIicant. II resistance determinants
are located on selI-transmissible plasmids. the chance that resistance spreads to other bacteria is
highly increased.

In the present work the antimicrobial resistance characteristics oI seven TGC-resistant
Escherichia coli. Klebsiella pneumoniae isolates Irom horses and a Salmonella Braenderup
isolates Irom a chicken were determined. Our Iocus was on genes responsible Ior resistance to
TGC and resistance genes associated with class 1 integrons. Subsequently. the potential oI in
vitro selI-transIer oI the resistance elements between the TGC-resistant isolates and diIIerent
multidrug-resistant (MDR) clinical Salmonella isolates was investigated.


Bacterial isolates
In the present study. the criterion Ior isolate selection was resistance to ampicillin and ceItioIur
or ceItazidime because ceItioIur was used to test susceptibility oI bacteria to third-generation
cephalosporins in the Veterinary Microbiology Diagnostic Center (VMDC) oI Utrecht
University. The isolates were Irom the collection oI 33 ceItioIur-resistant bacteria oI the
Veterinary Microbiology Diagnostic Center (VMDC) oI Utrecht University in 2004. Seven

120
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.

Chapter 7
121
epidemiologically unrelated Enterobacteriaceae isolates were selected (Iour equine Escherichia
coli and three equine Klebsiella pneumonia isolates E1-E4 and K1-K3). A ceItazidime-resistant
Salmonella Braenderup isolate (S1) Irom a chicken. which was described as isolate N153 in a
previous study
23
was also included. The isolates were cultured Irom diIIerent horses coming
Irom diIIerent regions in The Netherlands (Table 1). Species and serovars were identiIied by
conventional methods at the VMDC and at the RIVM (the Dutch National Institute oI Public
Health and the Environment). The Salmonella isolates. used in the coniugation experiments (see
below) were Irom a Salmonella collection obtained Irom humans and animals in Vietnam
25
.
Only norIloxacin- or kanamycin resistant and ceItazidime susceptible isolates were selected
(Table 2). A clinical E. coli isolate with a conIirmed FOX -lactamase was used as positive
control in the AmpC -lactamase genes determination.

Susceptibility oI the isolates to antimicrobials was tested by a disk diIIusion assay using Iso-
sensitest agar (Oxoid. UK) and Neo-sensitab discs (Rosco. Denmark) based on the procedure
recommended by the Dutch Committee on Guidelines Ior Susceptibility Testing CRG
7
. The
antimicrobials used were ampicillin (30 g). amoxicillin/ clavulanic acid (30/15 g). cephalexin
(30 g). ceItazidime (30 g). ceItioIur (30 g). ceIotaxime (30 g). streptomycin (100 g).
gentamicin (40 g). kanamycin (100 g). tetracycline (80 g). norIloxacin (10 g).
chloramphenicol (60 g). sulphonamides (240 g). and trimethoprim (5.2 g). To determine the
appropriate concentration oI antibiotics Ior the coniugation experiments. a microbroth dilution
method was used Ior the donors and the recipients with the antimicrobials kanamycin.
norIloxacin. and ceItazidime (Sigma. Germany) according to the guidelines oI the Clinical and
Laboratory Standards Institute (CLSI. Iormerly the National Committee Ior Clinical Laboratory
Standards - NCCLS)
16
. E. coli ATCC 25922 and E. coli ATCC 35218 were used as reIerence
strains.

Extended Spectrum Beta-Lactamases (ESBL) investigation
Double Disk 1est
The eight isolates were tested Ior their phenotypic resistance to ESBL. The test is based on the
synergy between clavulanate and the indicator cephalosporins as described
8. 11
. BrieIly. two
disks containing ceIotaxime (30 g) and ceItazidime (30 g) respectively were placed at
20-30 mm Irom a disk containing amoxicillin/clavulanic acid (20 g/10 g) on a Mueller-
Hinton agar (MHA) plate inoculated with the test isolate. When aIter overnight incubation the
zone oI either cephalosporin was expanded by clavulanate. ESBL production was indicated.

Cenetic characterization of ESBL genes
The same isolates were analysed Ior the presence oI TEM. SHV. OXA and CTX-M type ESBLs
by PCR using the primers described in the previous studies
4. 10. 17. 18
. Template DNA was
prepared by the whole bacteria cell boiled lysate method
13
. PCR products oI the expected size
were extracted Irom agarose gel using the Qiaquick Gel Extraction kit (Qiagen. Germany). The
puriIied PCR products were sequenced on an ABI 3730 sequencer (Foster. USA). Nucleotide
122
sequences were analyzed using Clone Manager Suite (version 8). Mutations were determined
by consulting sequence data in the GenBank database and on website
http://www.lahey.org/study. E. coli 09A488. K. pneumoniae 09A018 and Enterobacter cloacae
03773 which harbour TEM. SHV and CTX-M-9 type -lactamase. respectively. were included
as positive controls.

AmpC-type -Lactamases investigation
AmpC Disk 1est
The isolates were also investigated Ior the presence oI AmpC-type -lactamases. In this test
Tris-EDTA is used to permeabilise the bacterial cell wall which releases the -lactamases into
the external environment
3
. BrieIly. ceIoxitin disks (30 g) were placed on the surIace oI a MHA
plate inoculated with a lawn oI ceIoxitin-susceptible E. coli ATCC 25922. Several colonies oI
each test organism were applied on Iilter paper disks soaked in Tris-EDTA (20 l oI a 1:1
mixture oI saline and 100 Tris-EDTA. 1 M Tris/HCl. 0.1 M EDTA. (pH 8.0)). and placed on
the same MHA plate almost touching the ceIoxitin disk. AIter overnight incubation. an
indentation or a Ilattening oI the zone oI inhibition (indicating enzymatic inactivation oI
ceIoxitin) was considered an indication Ior AmpC production.

Cenetic characterization of plasmid-mediated AmpC -lactamase genes
The presence oI the AmpC -lactamase genes Irequently Iound in Enterobacteriaceae was
tested by multiplex PCR using the MOX. CIT. DHA. ACC. EBC and FOX primers as
described
20
. The nucleotide sequence oI the amplicons was determined to conIirm the identity
oI the -lactamase gene in question. The procedures Ior puriIication oI PCR products.
sequencing. and sequence analysis were the same as used Ior the ESBL genes. Sequences were
analysed by searching the GenBank database. E. coli pNU2936. pMG247 (a giIt Irom Dr. G.A.
Jacoby) carrying MOX-1 and DHA-1 -lactamase. respectively. were used as positive controls.

Class 1 integron detection and characterization of gene cassettes
The detection oI class 1 integrons and the genetic characterization oI inserted gene cassettes
were perIormed as described
23
. BrieIly. a PCR using primers targeting the class 1 integrase
speciIic int1 gene (int-PCR) was perIormed Ior all eight ESC-resistant isolates. The gene
cassettes oI the integron-carrying isolates were then ampliIied by conserved-segment PCR
(CS-PCR). The amplicons obtained Irom the CS-PCR were puriIied using the QiaQuick PCR
puriIication kit (Qiagen. Germany) and were compared by restriction Iragment length
polymorphism (RFLP) analysis with two diIIerent restriction endonucleases (HpaII and NciI).
Representative amplicons Irom each RFLP type were randomly chosen Ior nucleotide
sequencing. For isolates which had a unique integron. puriIied (Gel Extraction kit. Qiagen.
Germany) CS-PCR products were sequenced directly using the CS primers. For the isolate
carrying 2 integrons (which only diIIer about 50 bp in size). the puriIied CS-PCR products were
cloned into a pGEM-T easy Vector. The Iragment inserted into the vector was ampliIied using
T7 and Sp6 primers. The amplicons obtained were puriIied Irom agarose gel and sequenced
Chapter 7
123
using T7 and Sp6 primers. The obtained nucleotide sequences were analyzed with the Clone
Manager Suite by consulting the GenBank database via the BLAST network service.

Nucleotide accession numbers
The sequences oI the ESBL genes detected in this study. the bla
CTX-M-1
Irom E. coli isolate E1.
the bla
CMY-2
Irom E. coli isolate E2. and the bla
CTX-M-1
Irom K. pneumoniae isolate K2. are listed
in the GenBank database under accession numbers DQ663489. DQ663490. and DQ663591.
respectively. The sequences oI the 1.65 and 1.7 kb-amplicons containing the dfrA1-aadA1 and
dfr17- aadA5 genes Irom E. coli isolate E3 have been assigned the GenBank accession numbers
DQ663487 and DQ663488.

Conjugation assay
A coniugation experiment was perIormed to study whether ESC resistance genes can be
transIerred between diIIerent Enterobacteriaceae and whether ESC-resistant isolates can play a
role in the horizontal transIer oI antibiotic resistance. To Iind suitable acceptor bacteria Ior the
mating experiments. nine MDR Salmonella isolates were selected as mentioned in Materials
and Methods. The scheme oI the coniugation experiments is shown in Table 2. Coniugation was
carried out using the broth mating method
24
except that the MacConkey agar was supplemented
with a combination oI ceItazidime (8 g/ml) and kanamycin (256 g/ml) or a combination oI
ceItazidime (8 g/ml) and norIloxacin (32 g/ml) to select Ior transconiugants. The
transconiugants were identiIied as Salmonella in the API 20E system (bioMerieux. France) and
by slide agglutination with antisera speciIic Ior the O antigens oI Salmonella (Staten Serum
Instute. Denmark). Transconiugants were also assayed Ior their susceptibility to 15
antimicrobial agents and the antibiograms were compared with those oI the donors and
recipients. The presence oI the ESC resistance genes in the transconiugants was demonstrated
by PCR.

Plasmid Profiling and Southern Hybridization
Transconiugants. donors and recipients were subiected to plasmid analysis using a modiIied
alkaline lysis method
12
to determine which plasmid carrying ESC genes had been transIerred.
Subsequently. Southern blot hybridization
14
with bla
ACC.
bla
CTX-M
or bla
CMY
probes was
perIormed to detect the target genes. 1.5 ml oI an overnight culture in Brain Heart InIusion
(BHI) broth was centriIuged at 14.000 rpm Ior 10 min. The pellet was resuspended in 50 l TE
(50 mM Tris. 1 mM EDTA. (pH 8.0). Cells were lysed by mixing them with 450 l
(50 mM Tris. 3 sodium dodecyl sulphate (pH 12.6)) and incubated in a water bath Ior 60 min
at 56
0
C. Next. 500 l oI a mixture oI phenol:chloroIorm:isoamyl alcohol (25:24:1. v/v/v) was
added. The suspension was mixed vigorously until it was homogeneously milk-white. AIter
centriIugation at 13000 rpm Ior 30 min at 4
0
C. 300 l oI the upper aqueous phase was careIully
transIerred to a clean EppendorI tube. FiIty l oI this plasmid extraction sample was analyzed
on a 0.7 agarose gel in 40 mM Tris acetate. 2 mM sodium EDTA. (pH 7.9). The pH was
adiusted with acetic acid. AIter electrophoresis Ior 6 h. the gel was stained with ethidium
bromide (0.5 g/ml) Ior 30 min. DNA bands were visualized under UV transillumination.
124
S. Typhimurium phage type 13 containing Iive plasmids oI diIIerent sizes (180 kbp. 82 kbp.
39 kbp. 5.5 kbp and 4.4 kbp) was used as a standard Ior the determination oI plasmid sizes.
Chromosomal and plasmid DNA was transIerred to a N
Hybond membrane by capillary action.

air dried and Iixed by cross-linking under UV light. The digoxigenin labelled probes (bla
ACC
.
bla
CTX-M
and bla
CMY
) were generated by PCR using the primers (CIT-F/CIT-R. CTX-F/CTX-R
and ACC-F/ACC-R. Table 3) and the PCR Dig Probe Synthesis kit (Roche. Germany). For each
probe. a separate hybridization was perIormed. as described
14
. Hybridization took place at 60
0
C
Ior the bla
ACC
and bla
CMY
probes and at 55
0
C Ior the bla
CTX-M

probe. The presence oI any
hybridized targets was detected using the alkaline phosphate-coniugated antibody DNA
detection kit (Roche. Germany) and NTB/BCIB (bromo-4-chloro-3`-indolyphosphate p-
toluidine salt/ nitro-blue tetrazolium chloride). Between hybridizations the membrane was
stripped as described by the manuIacturer.

RESULTS
Resistance phenotype. All seven equine ceItioIur-resistant E. coli. K. pneumoniae isolates and
a ceItazidime-resistant S. Braedenrup isolate originating Irom a chicken were multidrug
resistant. The phenotypic resistance patterns oI the isolates are documented in Table 1. Seven
isolates were positive in the phenotypic tests Ior ESC: six isolates in the double disk test Ior
ESBL conIirmation and one isolate in the AmpC disk test (Table 1).

-lactamase genes. ESBL genes were detected in Iive oI the six isolates positive in the double
disk test (Table 1). The presence oI TEM-. SHV- and CTX-M type ESBL genes was Iound by
PCR and conIirmed by nucleotide sequencing. The CTX-M-1 gene. was detected in two E. coli
isolates (E1 and E3) and three K. pneumoniae isolates (K1. K2. and K3). One E. coli isolate
(E4) was considered an ESBL producer with an unidentiIied mechanism since this isolate was
positive in the double disk test but neither TEM type-. SHV-type-. CTX-M type-. nor OXA-
type- ESBL genes could be detected.

Two isolate tested positive in the AmpC disk test. one was PCR positive Ior the LAT
and CMY group oI -lactamases and the other produced a product with ACC primers.
Nucleotide sequencing Iollowed by a BLAST search conIirmed the identity oI the gene
encoding the AmpC-type -lactamase. The bla
CMY-2
gene was Iound in the E. coli isolate (E2).
The bla
ACC-1
gene was identiIied in the S. Braenderup isolate (S1). In addition to the genes
encoding Ior ESC-resistance. TEM-1 and SHV-1 -lactamase genes were present in six out oI
eight isolates. In two K. pneumoniae isolates. three genes encoding -lactamases (TEM-1.
SHV-1. and CTX-M-1) were detected (Table 1).

Class 1 integron and resistance gene cassettes. Six integron-carrying isolates were identiIied
by int-PCR. The results are summarized in Table 1. Three types oI class 1 integrons were
Iound. Gene cassettes encoding resistance to aminoglycosides (aadA1. aadA2 and aadA5) or to
trimethoprim (dfrA1. dfrA12 and dfrA17) were present in these integrons. Remarkably. the
presence oI the two class 1 integrons (amplicons oI 1650 and 1700 bp with dfrA1-aadA1 and
Chapter 7
125
dfrA17-aadA5. respectively) was detected in an E. coli isolate (E3) cultured Irom a 1 month-old
Ioal.

Table 3. Primers used in this study.
Primer Target(s)
PCR
product
size
Sequence (5`-3`. as synthesized)

ReI.
TEM-F bla
TEM
929
ATG AGT ATT CAA CAT TTC CGT
GTC G
This study
TEM -R ACC AAT GCT TAA TCA GTG AGG CA
This study
TEM-S1 For sequencing ACA ACG ATC GGA GGA CCG
This study
TEM-S2 For sequencing GCG GTT AGC TCC TTC GGT
This study
SHV-F bla
SHV
900
GTA TTG AAT TCA TGC GTT ATA TTC
GCC TGT GTA
4

SHV-R
CAG AAT TCG GCT AGC GTT GCC
AGT GCT CGA
4

SHV-FW For sequencing TATCGGCCCTCACTCAAGGA
This study
SHV-RV For sequencing GGGTTAGCGTTGCCAGTGCT
This study
CTX-F bla
CTX-M
538 CGA TGT GCA GTA CCA GTAA
18
CTX-R ATA TCG TTG GTG GTG CC
18

CTX-FW For sequencing CTTCCAGAATAAGGAATCCCA
This study
CTX-RV For sequencing GTTTCCGCTATTACAAACCGT
This study
OXA1-F bla
OXA1-group
820
ATGAAAAACACAATACATATCAACT
TCGC
17

OXA1-R GTGTGTTTAGAATGGTGATCGCATT
17

OXA2-F bla
OXA2-group
602 ACGATAGTTGTGGCAGACGAAC
10

OXA2-R ATYCTCTTTGGCGTATCRATATTC
10

FOX-F bla
FOX -1 to -5b
190 AACATGGGGTATCAGGGAGATG
20

FOX-R CAAAGCGCGTAACCGGATTGG
20

EBC-F bla
MIR-1. ACT-1
302 TCGGTAAAGCCGATGTTGCGG
20

EBC-R CTTCCACTGCGGCTGCCAGTT
20

ACC-F bla
ACC
346 AACAGCCTCAGCAGCCGGTTA
20

ACC-R TTCGCCGCAATCATCCCTAGC
20

DHA-F bla
DHA-1. -2
405 AACTTTCACAGGTGTGCTGGGT
20

DHA-R CCGTACGCATACTGGCTTTGC
20

CIT-F bla
LAT-1 to -4. CMY-2 to -7. BIL-1
462 TGGCCAGAACTGACAGGCAAA
20

CIT-R TTTCTCCTGAACGTGGCTGGC
20

MOX-F bla
MOX -1. 2. CMY-1. -8 to -11
520 GCTGCTCAAGGAGCACAGGAT
20

MOX-R CACATTGACATAGGTGTGGTGC
20

CMY-FW For sequencing AACACACTGATTGCGTCTGAC
20

CMY-RV For sequencing CTGGGCCTCATCGTCAGTTA
20

Horizontal transfer of integrons and resistance determinants. Resistance determinants could
be exchanged between Salmonella isolates and E. coli isolates but not between Salmonella and
K. pneumoniae. In the 30 coniugations perIormed. 9 transconiugants were obtained (Table 2).
(i) TGC-resistant E. coli as a donor; Salmonella Typhimurium as a recipient: E. coli isolate
E1 could transIer its bla
CTX-M-1
gene to S. Typhimurium S57. S58 and S60 since these genes
were detected by PCR in the donor (E1) and the 3 transconiugants (S57E1. S58E1. S60E1)
but not in the recipients (S57. S58. S60). Phenotypic resistance to ceItazidime was conIirmed in
the 3 transconiugants. Plasmid proIiles oI the donor. the recipients and the transconiugants were
similar (data not shown) (ii) Salmonella Typhimurium as a donor; TGC-resistant E. coli as
a recipient: No diIIerence was observed between the plasmid proIile oI the E. coli recipient
(E2) and those oI the E. coli transconiugants (E2S15. E2S305. E2S309) (data not shown).
However. E. coli isolate E2 which carries the bla
CMY-2
gene obtained a kanamycin resistance
126
determinant Irom the donors (S15. S305 and S309) since the transconiugants (E2S15.
E2S305. E2S309) were phenotypically resistant to both ceItazidime and kanamycin. (iii)
TGC-resistant Salmonella Braenderup as a donor; Salmonella Typhimurium as a
recipient: S. Braenderup isolate S1 had two large plasmids oI approximately 80 kb and 30 kb.
The 30 kb plasmid on which the bla
ACC-1
gene was located could be horizontally transIerred
since the bla
ACC-1
probe hybridized with the plasmid Irom the S. Braenderup donor (S1) and the
S. Typhimurium transconiugants (S57S1. S58S1. and S60S1) (data not shown).

DISCUSSION

The presence oI ESBL and AmpC type genes encoding resistance to extended-spectrum
cephalosporins among Enterobacteriaceae isolates Irom a chicken and horses (including horses
oI 1 month old) is an alarming Iinding. In The Netherlands. extended-spectrum cephalosporins
(ceItioIur. ceIquinome) are prescription-only medicines approved Ior the treatment oI
inIections oI the respiratory tract and pododermatitis interdigitalis in cattle and pigs
6
. but they
were not licensed Ior treatment oI horses during the study period. However. ceItioIur is used
oII-label Ior the treatment oI equine inIections.

There is no evidence that extended-spectrum cephalosporins have been used in poultry
production in The Netherlands. ThereIore the origin oI the ceItazidime resistance in the S.
Braenderup isolate Irom a chicken is not clear. Several bla
ACC-1
carrying Salmonella isolates
have also been Iound in a study by Hasman et al. in Dutch chicken
10
. It is important to note that
one oI their isolates was a bla
ACC-1
containing S. Braenderup isolate collected Irom broiler in
2001. The S. Braenderup isolate Irom our study was cultured 3 years later than the one oI
Hasman et al. but they have the same ESC resistance gene. One may speculate about the
persistence or transmission oI a bla
ACC-1
-carrying S. Braenderup strains in chickens in The
Netherlands although we do not have any data regarding the epidemiological relationship
between these two isolates. Another possibility Ior the emergence oI bla
ACC-1
among Salmonella
serovars might be the result oI horizontal transIer by coniugative plasmids. This study is the
Iirst to prove that the bla
ACC-1
gene was successIully transIerred via a coniugative plasmid Irom
one Salmonella isolate (S. Braederup) to other clinical isolates oI a diIIerent Salmonella serovar
(S. Typhimurium) and expressed the resistance phenotype in the recipient.

To the best oI our knowledge. the presence oI the cassette arrays in two class 1
integrons (one which yields a CS-amplicon oI 1700 bp containing the dfrA17-aadA5 genes and
one yielding a CS-amplicon oI 1650 bp containing the dfrA1-aadA1 genes) in a single E. coli
isolate has not been described beIore. Interestingly. the gene cassettes oI these two integrons
contain distinct genes (dfrA1-aadA1 and dfrA17-aadA5) but encode resistance against the same
antibiotics: the dfrA1 and dfrA17 genes both against trimethoprim; the aadA1 and aadA5 genes
both against streptomycin and spectimomycin. Besides the genes encoding resistance to
extended-spectrum cephalosporins. almost all (six out oI seven) isolates carried one or two
other genes encoding -lactam resistance. two to Iour genes encoding resistance to
aminoglycosides or trimethoprim and apparently also other resistance determinants were
Chapter 7
127
present. Five isolates were resistant to up to 11 antimicrobials including Iluoroquinolones. Such
multidrug resistance in pathogenic bacteria severely limits therapeutic options. This is a serious
concern Ior veterinary medicine but also Ior human medicine since direct transIer oI ESC-
resistant isolates Irom animals to humans has been documented
2
.

Coniugal experiments to transIer antimicrobial resistance are usually perIormed with
reIerence strains (E. coli K12) or strains adapted Ior coniugation
10
. In the present study. in vitro
coniugation was perIormed between clinical isolates. The data indicate the ease oI the spread oI
multiple resistance determinants among Enterobacteriaceae. especially E. coli and Salmonella.
Horse and chicken are an underreported source oI TGC-resistant Enterobacteriaceae in The
Netherlands.

ACKNOWLEGEMENTS

This work was Iunded by the Vietnamese Government (Proiect 322).

REFERENCES
1. Ambler. R.P.. A.F. Coulson. J.M. Frere. J.M. Ghuysen. B. Joris. M. Forsman. R.C. Levesque. G. Tiraby. and
S.G. Waley. 1991. A standard numbering scheme Ior the class A beta-lactamases. Biochem J 276:269-270.
2. Arlet. G.. T.J. Barrett. P. Butaye. A. Cloeckaert. M.R. Mulvey. and D.G. White. 2006. Salmonella resistant
to extended-spectrum cephalosporins: prevalence and epidemiology. Microbes InIect. 8:1945-1954.
3. Black. J.A.. E.S. Moland. and K.S. Thomson. 2005. AmpC disk test Ior detection oI plasmid-mediated
AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal AmpC beta-lactamases. J. Clin.
Microbiol. 43:3110-3113.
4. BradIord. P.A. 1999. Automated thermal cycling is superior to traditional methods Ior nucleotide sequencing
oI bla
SHV
genes. Antimicrob. Agents Chemother. 43:2960-2963.
5. BradIord. P.A. 2001. Extended-spectrum beta-lactamases in the 21st century: characterization.
epidemiology. and detection oI this important resistance threat. Clin. Microbiol. Rev. 14:933-951.
6. Bureau Diergeneesmiddelen. 2005. Overzicht van alle geregistreerde diergeneesmiddelen. Den Haag. 176.
7. Commissie Richtliinen Gevoeligheidsbepalingen (CRG). 2000. Interpretatie van gevoeligheidsonderzoek en
gevoeligheidscriteria voor antibacteriele middelen in Nederland. Ned. Tiidschr. Med. Microbiol. 8:79-81.
8. Emery. C.L.. and L.A. Weymouth. 1997. Detection and clinical signiIicance oI extended-spectrum beta-
lactamases in a tertiary-care medical center. J. Clin. Microbiol. 35:2061-2067.
10. Hasman. H.. D. Mevius. K. Veldman. I. Olesen. and F.M. Aarestrup. 2005. beta-Lactamases among
extended-spectrum beta-lactamase (ESBL)-resistant Salmonella Irom poultry. poultry products and human
patients in The Netherlands. J. Antimicrob. Chemother. 56:115-121.
11. Jarlier. V.. M.H. Nicolas. G. Fournier. and A. Philippon. 1988. Extended broad-spectrum beta-lactamases
conIerring transIerable resistance to newer beta-lactam agents in Enterobacteriaceae: hospital prevalence and
susceptibility patterns. Rev. InIect. Dis. 10:867-878.
Bacteriol. 145:1365-1373.
14. Maniatis. T.. E.F. Fritsch. and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring
Harbor Laboratory. Cold Spring Harbor.
128
15. Miriagou. V.. P.T. Tassios. N.J. Legakis. and L.S. Tzouvelekis. 2004. Expanded-spectrum cephalosporin
resistance in non-typhoid Salmonella. Int. J. Antimicrob. Agents 23:547-555.
17. Olesen. I.. H. Hasman. and F.M. Aarestrup. 2004. Prevalence oI beta-lactamases among ampicillin-resistant
Escherichia coli and Salmonella isolated Irom Iood animals in Denmark. Microb. Drug Resist. 10:334-340.
19. Paterson. D.L.. and R.A. Bonomo. 2005. Extended-spectrum beta-lactamases: a clinical update. Clin.
20. Perez-Perez. F.J.. and N.D. Hanson. 2002. Detection oI plasmid-mediated AmpC beta-lactamase genes in
clinical isolates by using multiplex PCR. J. Clin. Microbiol. 40:2153-2162.
21. Philippon. A.. G. Arlet. and G.A. Jacoby. 2002. Plasmid-determined AmpC-type beta-lactamases.
22. Tolun. V.. O. Kucukbasmaci. D. Torumkuney-Akbulut. C. Catal. M. Ang-Kucuker. and O. Ang. 2004.
Relationship between ciproIloxacin resistance and extended-spectrum beta-lactamase production in
Escherichia coli and Klebsiella pneumoniae strains. Clin. Microbiol. InIect. 10:72-75.
23. Vo. A.T.T.. E. van Duiikeren. A.C. Fluit. and W. Gaastra. 2006. Antibiotic resistance. integrons and
Genomic Island SGI1 among non-typhoid Salmonella serovars in The Netherlands. Int. J. Antimicrob.
Agents 28:172-179.
24. Vo. A.T.T.. E. van Duiikeren. A.C. Fluit. M.E. Heck. A. Verbruggen. K. van der Zwaluw. and W. Gaastra.
2006. Class 1 integrons in Dutch Salmonella enterica serovar Dublin isolates Irom clinical cases oI bovine
salmonellosis. Vet. Microbiol. 117:192-200.
26. Wang. M.. J.H. Tran. G.A. Jacoby. Y. Zhang. F. Wang. and D.C. Hooper. 2003. Plasmid-mediated
quinolone resistance in clinical isolates oI Escherichia coli Irom Shanghai. China. Antimicrob. Agents
Chemother. 47:2242-2248.

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Conpaialive InnunoIogv, MiciolioIogv & Infeclious Diseases : 11-18. 2OO7

Comparison oI in vitro pathogenicity oI two Salmonella Typhimurium phagetypes
130
ABSTRACT

The in vitro pathogenicity oI Salmonella enterica serovar Typhimurium phage type (pt) 90 and
pt 506 (also known as DT 104) isolates Irom human and porcine origin was studied in adhesion
and invasion assays to the human cell line Caco-2 and the porcine cell line IPI-2. Interleukin-8
(IL-8) production by these two cell lines in response to stimulation by the two Salmonella
phage types was also measured. Generally. Salmonella Typhimurium pt 506 and pt 90 adhered
to and invaded Caco-2 cells and IPI-2 cells equally well. The release oI IL-8 by Caco-2 cells or
by IPI-2 cells was similar. independent oI the Salmonella phage type used Ior stimulation oI the
cells. These data suggest that S. Typhimurium pt 90 has a similar ability to cause Salmonella
inIections as S. Typhimurium DT 104.

Key words: Salmonella Typhimurium. phage type. adhesion. invasion. IL-8. epithelial cell.
Chapter 8
131
INTRODUCTION

Salmonella Typhimurium is one oI the most common causes oI salmonellosis worldwide
26
. The
prevalence oI the predominant phage types oI S. Typhimurium diIIers between diIIerent
regions
2. 3. 23. 27
. Salmonella Typhimurim phage type (DeIinitive Type or DT) 104. which
corresponds to S. Typhimurium phage type (pt) 506 in the Dutch phage typing system
23
. is the
predominant S. Typhimurium phage type in many European countries and the United States
3
. In
Vietnam. however. S. Typhimurium Dutch pt 90 (which has no recognized phage type in the
English typing system). is the most prevalent phage type oI S. Typhimurium
25
.

Despite their close genetic relationship. diIIerences in epidemiology and virulence oI
diIIerent Salmonella serovars and phage types are common. Salmonella lipopolysacharides
(LPS). a surIace structure. serves as the attachment site Ior bacteriophages
9. 18
. The sensitivity
and ability oI the bacteriophages to distinguish diIIerent closely related Salmonella strains is the
basis Ior phage typing systems
1. 6
. The surIace structures are also oIten virulence Iactors and as
such targets oI the innate and adaptive host deIence.

Upon inIection by salmonellae. enterocytes do not act as passive victims oI inIection
but signal inIection to the immune system. The recognition oI the Salmonella Ilagellin protein
by Toll-like receptor 5 (TLR-5) oI host cells leads to the release oI inIlammatory mediators like
cytokines and chemokines (such as IL-8) Irom diIIerent types oI host cells
8
. IL-8 secretion leads
to the attraction and activation oI polymorphonuclear leukocytes causing acute inIlammation
7
.

In vitro studies can reveal important principles oI pathogenesis
19
. Adhesion and
invasion in host cells can be regarded as exponents oI the pathogenesis oI S. Typhimurium
14
. In
the present study. adhesion and invasion experiments using two intestinal epithelial cell lines
were perIormed in order to compare the pathogenicity oI S. Typhimurium pt 506 and
S. Typhimurium pt 90 Irom human and porcine origin. In addition. the IL-8 production by these
two cell lines in response to stimulation by isolates oI the two S. Typhimurium phage types
were measured since the magnitude oI the cytokine response. which IL-8 used as a marker.
reIlects the intensity oI the host-pathogen interaction.


Bacteria
The Salmonella Typhimurium isolates were Irom two collections oI non-typhoid salmonellae
cultured Irom humans and animals in The Netherlands and in Vietnam as described in previous
studies
24. 25
. Serotyping and phage typing was perIormed to classiIy the isolates. Four
S. Typhimurium pt 506 isolates Irom humans (N176. N216) and pigs (N94. N235) in The
Netherlands and Iour S. Typhimurium pt 90 isolates Irom humans (V15. V22) and pigs (V226.
V291) in Vietnam were randomly chosen Irom the mentioned collections and included in this
study (Table 1). A wild-type Salmonella Enteritidis 706 strain was used as a positive control in
all assays using the procedure described elsewhere
22
. For the adhesion and invasion
132
experiments. an overnight culture oI bacteria was diluted 1/100 in Luria Bertani (LB) broth.
grown Ior 2 h. collected by centriIugation (1.500 g Ior 15 min) and resuspended in plain
Dulbecco`s ModiIied Eagle Medium (DMEM) (Invitrogen) to a concentration oI approximately
10
8
bacteria/ ml.

Cell cultures
The human colon adenocarcinoma cell line (Caco-2. ATCC CRC 11268) that is known to
exhibit enterocyte-like diIIerentiation patterns. was used
16
. At late conIluency Caco-2 cells
display the structural and Iunctional diIIerentiation characteristics oI small intestinal
enterocytes
11
. Caco-2 cells were cultured in high glucose (4.5 g/l) Dulbecco`s ModiIied Eagle
Medium (DMEM) supplemented with 1 (v/v) non-essential amino acids (Flow Laboratories.
The Netherlands). 10 mM NaHCO
3
. 25 mM HEPES. 4 mM glutamine (Flow Laboratories). and
20 (v/v) Ietal calI serum (Cambrex. Belgium) in a humidiIied atmosphere oI 5 CO
2
at 37
0
C.
The swine miniature male ileum cell line (IPI-2. ECACC 93100622) immortalized by
transIection with an SV40 plasmid which yields morphologically heterogeneous colonies oI
typical porcine epithelial cells
15
was also used. IPI-2 cells were cultured in the same medium
but supplemented with 10 Ietal calI serum and 0.024 IU/ml insulin. Cell medium was changed
three times a week. Caco-2 cells and IPI-2 cells were seeded in 12-well plates (Greiner) and
cultured Ior 19 days and 5 days at 40.000 cells/cm
2
and 50.000 cells/cm
2
. respectively. IPI-2
cells were used at conIluency.

Adhesion assay
The adhesion and invasion assays were perIormed essentially as described
11. 16
. BrieIly.
epithelium cells were washed twice with plain DMEM and incubated Ior at least 1 hour in this
medium prior to addition oI the bacterial suspension. AIter incubating the cells and the bacteria
Ior one hour
12
. the bacterial suspension was removed to exclude the unattached bacteria. The
monolayers oI epithelial cells were washed 3 times with DMEM. and 1 ml Triton X-100 in PBS
was added Ior 5 min at room temperature to release the bacteria Irom the cells. The number oI
adherent bacteria was estimated by plating serial dilutions. All experiments were perIormed in
triplicate.

Invasion assay
AIter an incubation oI one hour as described in the adhesion assay. the bacterial suspension was
removed. Cells were washed once with DMEM. One ml oI DMEM containing colistin
(300 g/ml) was added and incubated Ior 2 hours (37
0
C. 5 CO
2
) to kill all extracellular
bacteria. Next. the cell culture supernatant was collected and stored at 20
0
C Ior IL-8
determination. The cells were washed 3 times with DMEM and lysed in 1 Triton X-100 in
PBS. The number oI intracellular bacteria was determined by plate counting. All tests were
perIormed in triplicate.

IL-8 determination by sandwich ELISA
IL-8 concentrations were determined using the human IL-8 Cytosets
TM
antibody pair kit
containing matched. pretitred and Iully optimized capture and detection antibodies. recombinant
Chapter 8
133
standards and streptavidin-horseradish peroxidase (Biosource Europe S.A.. Belgium). The assay
was perIormed according to the manuIacturer`s speciIications. Porcine and human IL-8 have
80 homology (GenBank accession no. BAC06611 and NP000575. respectively) which
explains the cross reactivity oI human ELISA with porcine IL-8. ThereIore. the equivalence
unit to human IL-8 was indicated Ior porcine IL-8.

Statistical analysis
As all experiments were perIormed in triplicate. the mean values and the standard deviation
were calculated and compared using the independent-samples t test Ior groups oI isolates or
one-way ANOVA Ior individual isolates. DiIIerences were considered signiIicant at p 0.05.
SPSS 12.0.1 Ior Windows was used.

RESULTS

The results oI the adhesion and invasion experiments are shown in Table 1. Generally. no
signiIicant diIIerences (p~0.05) in adherence and invasion to Caco-2 cells and IPI-2 cells by the
two phage types oI S. Typhimurium was observed. Large diIIerences were however observed
between individual isolates (p0.05) oI the same phage type (e.g. invasion oI Caco-2 cells by
V15 compared to V22) and this result was reproducible. since the experiments were perIormed
in triplicate and the results oI the repeated testing Ior a certain strain were similar as can be seen
Irom the standard deviations.

Exposure oI the Caco-2 cells or IPI-2 cells to 200-500 bacteria/cell Ior 1 h induced
IL-8 production signiIicantly higher than that in the unstimulated cells (Table 1) (p0.05). No
signiIicant diIIerence was observed between the IL-8 levels released by Caco-2 cells or IPI-2
cells (p~0.05) in stimulation oI S. Typhimurium phage type 506 compared to that oI
S. Typhimurium pt 90 isolates.

DISCUSSION

S. Typhimurium DT 104 initially emerged in cattle in England and Wales. Subsequently. the
strain has been isolated Irom many animal species including poultry. sheep. pigs and horses and
has spread to other European countries and the United States. S. Typhimurium DT 104 has
caused human Salmonella outbreaks worldwide. In the present study the S. Typhimurium pt 506
(DT 104) and S. Typhimurium pt 90 isolates used exhibited the same level oI adhesion to and
invasion oI Caco-2 cells and IPI-2 cells. This suggests that the ability oI these two phage types
to cause gastroenteritis is similar since the capacity to invade epithelial cells enables Salmonella
to colonize and cross the epithelial barriers and starts the process oI inIlammatory diarrhoea
14
.

Initial adherence helps to bring Salmonella in close contact with the host cells. Better
adhesion generally means a higher chance oI invasion but a bacterial strain with a high capacity
to adhere will not always invade eukaryotic cells to a higher extent. For instance isolate V15
attached Caco-2 cells better than isolate V22 (21.6 and 2 bacteria/ cell. respectively) but lower
134
number oI isolate V15 invaded Caco-2 cells compared to isolate V22 (3.5 and 11.2 bacteria/100
cells. respectively).

The next step in the interaction between salmonellae and host cells involves the
secretion oI virulence Iactors by type III secretion systems (TTSS)
5
. The TTS apparatus present
in all Salmonella enterica strains. resembles a needle-like structure and iniects Salmonella
Ilagellin protein into eukaryotic cells. The interaction between Ilagellin proteins and TLR-5
lead to IL-8 production by the eukaryotic cells
8
. It is known that Caco-2 cells express TLR-5
21
.
Recently. a porcine ieiunal epithelial cell line (IPEC-J2) has been deIined to express TLR-5
4
.
IL-8 production by Caco-2 cells was Iound aIter exposure to puriIied Salmonella Ilagellin but
not detected in the absence oI puriIied Ilagellin
21
. Induction oI an IL-8 release by epithelial cells
Table 1. Comparison oI the adhesion. invasion capacity oI Salmonella Typhimurium isolates oI
phage type 506 and phage type 90 to Caco-2 and IPI-2 cells and Salmonella induced IL-8
production by Caco-2 and IPI-2 cells.
Cell line Strain ID Phage type Adhesion
1
Invasion
2
IL-8
3
N216 pt 506 25.8 6.3 13.6 3.5
425.3 32.2
N176 pt 506 22 0.6 2.6 1.4
323.4 39.1
N235 pt 506 3.6 0.5 4.5 0.8

501.0 39.4
N94 pt 506 2.5 0.4 1 1.0
375.4 41.9
Mean Ior the group oI pt 506 11.4 10.9 5.44 5.3 406.3 75.8
V15 pt 90 21.6 17.2 3.5 0.7
329.0 48.2
V22 pt 90 2 0.3 11.2 1.0
388.6 40.3
V291 pt 90 2.2 0.5 8.6 4.6
376.5 40.3
V226 pt 90 1.7 0.1 7 5.4
274.8 29.8
Mean Ior the group oI pt 90 9 13.8 7.67 4.2 345.8 57.5
Caco-2
S. Enteritidis (control) 1.6 1.3 3.5 0.6
654 98
N216 pt 506 7 3.4 1.3 0.7
391.1 34.2
N176 pt 506 11 5.1 2.8 2.9
563.5 12.4
N235 pt 506 7.7 2.6 17.2 3.0

475.0 32.9
N94 pt 506 1.5 0.4 3.2 0.1
425.3 32.2
Mean Ior the group oI pt 506 6.88 4.6

6.09 6.9 466.7 71.9
V15 pt 90 1.8 0.1 2.2 1.3
312.0 68.2
V22 pt 90 2.8 0.3 23 9.7
531.9 40.1
V291 pt 90 4.6 1.2 17 12
406.1 22.5
V226 pt 90 1.8 0.1 4.3 2.6
506.4 152
Mean Ior the group oI pt 90 2.73 1.3 11.64 11.3 439.4 117
IPI-2
S. Enteritidis (control) 2.8 1.5 3.5 1.5
400 120
1
mean SD oI the number oI bacteria attached per cell Irom triplicate wells.
2
mean SD oI the number oI bacteria that invaded 100 cells Irom triplicate wells.
3

mean SD oI IL-8 (pg/ml) secretion by the eukaryote cells aIter 1 h exposure to S. Typhimurium isolates. measured
Irom triplicate wells.
Unstimulated Caco-2 and IPI-2 cells released 12.9 0.14 and 54.8 0.07 pg/ml IL-8. respectively.

Chapter 8
135
is dependent on the invasive capacity oI the Salmonella strain
13. 19. 20
. In the present study. a
better invasion oI salmonellae leading to a higher IL-8 production by human and porcine cell
lines was observed in almost all oI Salmonella isolates except the isolate N176 to IPI-2 cell.
IL-8 promotes neutrophil transmigration
17
into the intestinal lumen. leading to enterocyte
iniury. Data oI the present study show that high IL-8 levels were released by Caco-2 or IPI-2
cells in stimulation oI the S. Typhimurium isolates regardless diIIerences oI the two phage
types. ThereIore. in addition to comparative levels oI invasion to the cell lines oI
S. Typhimurium pt 506 and S. Typhimurium pt 90. the isolates oI these two phage types also
stimulated the cell lines to produce a high amount oI IL-8. One may speculate that a strong
inIlammatory reaction may occur in the host. InIections caused by S. Typhimurium pt 506 (or
DT 104) are well documented
10
. The present study suggests that S. Typhimurium pt 90 has a
similar ability to cause Salmonella inIections.

In conclusion. S. Typhimurium phage type 506 and S. Typhimurium phage type 90
adhered to and invaded human and porcine epithelial cells equally well. Both phage types
induced a similar IL-8 response. ThereIore the diIIerences in the surIace structure mediated the
diIIerences oI the two phage types oI S. Typhimurium may not inIluence in vitro pathogenicity
oI S. Typhimurium phage type 506 and S. Typhimurium phage type 90 regarding adhesion.
invasion Caco-2 and IPI-2 cell lines and stimulation these cell lines to produce IL-8.

REFERENCES
1. Anderson. E.S.. L.R. Ward. M.J. Saxe. and J.D. de Sa. 1977. Bacteriophage-typing designations oI Salmonela
typhimurium. J. Hyg. (Lond) 78:297-300.
599.
3. Baggesen. D.L.. D. Sandvang. and F.M. Aarestrup. 2000. Characterization oI Salmonella enterica serovar
Typhimurium DT104 isolated Irom Denmark and comparison with isolates Irom Europe and the United States.
4. Burkey. T.E. Expression oI Toll-like receptors in porcine immune cells and tissues.
http://hdl.handle.net/2097/153 (16 July 2006. date last accessed).
5. Ehrbar. K.. and W.D. Hardt. 2005. Bacteriophage-encoded type III eIIectors in Salmonella enterica subspecies
1 serovar Typhimurium. InIect. Genet. Evol. 5:1-9.
7. Harada. A.. N. Sekido. T. Akahoshi. T. Wada. N. Mukaida. and K. Matsushima. 1994. Essential involvement oI
interleukin-8 (IL-8) in acute inIlammation. J. Leukoc. Biol. 56:559-564.
8. Hayashi. F.. K.D. Smith. A. Ozinsky. T.R. Hawn. E.C. Yi. D.R. Goodlett. J.K. Eng. S. Akira. D.M. Underhill.
and A. Aderem. 2001. The innate immune response to bacterial Ilagellin is mediated by Toll-like receptor 5.
Nature 410:1099-1103.
9. Heller. K.J. 1992. Molecular interaction between bacteriophage and the gram-negative cell envelope. Arch.
Microbiol. 158:235-248.
10. Helms. M.. S. Ethelberg. and K. Molbak. 2005. International Salmonella Typhimurium DT104 inIections.
1992-2001. Emerg. InIect. Dis. 11:859-867.
136
11. Hendriks. H.. A. van Asten. J. Koninkx. W. Kok. B. van der Zeiist. and J. van Diik. 1996. Interactions between
Salmonella Enteritidis and the enterocyte-like human carcinoma cell line Caco-2. In Bardocz. S.. Nekrep. F. V.
& Pusztai. A. EIIects oI antinutrients on the nutritional value oI legume diets. Luxemburg: OIIice OIIicial
Publications European Communities. 137-139.
12. Hendriks. H.. A. van Asten. J. Koninkx. B. van der Zeiist. and J. van Diik. 1996. Interactions between
Salmonella enteritidis and the enterocyte-like human colon carcinoma cell line Caco-2. In Bardocz. S.. Nekrep.
F. & Pusztai. A. EIIects oI antinutrients on the nutritional value oI legume diets. Luxembourg: Cost 98. OIIice
Ior OIIicial Publications oI the European Communities.
13. Hobbie. S.. L.M. Chen. R.J. Davis. and J.E. Galan. 1997. Involvement oI mitogen-activated protein kinase
pathways in the nuclear responses and cytokine production induced by Salmonella Typhimurium in cultured
intestinal epithelial cells. J. Immunol. 159:5550-5559.
14. Jones. G.W.. L.A. Richardson. and J.L. vanden Bosch. 1980. Phases in the interaction between bacteria and
animal cells. In Berkeley. R. C. W.. Luynch. J. M.. Melling. J.. Rutter. P. R. & Vincent. B. Microbial adhesion
to surIaces. London: Ellis Horwood Limited. 211-219.
15. KaeIIer. B.. E. Bottreau. P. Velge. and P. Pardon. 1993. Epithelioid and Iibroblastic cell lines derived Irom the
ileum oI an adult histocompatible miniature boar (d/d haplotype) and immortalized by SV40 plasmid. Eur. J.
Cell Biol. 62:152-162.
16. Kusters. J.G.. G.A. Mulders-Kremers. C.E. van Doornik. and B.A. van der Zeiist. 1993. EIIects oI multiplicity
oI inIection. bacterial protein synthesis. and growth phase on adhesion to and invasion oI human cell lines by
Salmonella Typhimurium. InIect. Immun. 61:5013-5020.
17. McCormick. B.A.. S.P. Colgan. C. Delp-Archer. S.I. Miller. and J.L. Madara. 1993. Salmonella Typhimurium
attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils. J.
Cell Biol. 123:895-907.
18. Salgado. C.J.. M. Zayas. and R. VillaIane. 2004. Homology between two diIIerent Salmonella phages:
Salmonella enterica serovar Tvphimurium phage P22 and Salmonella enterica serovar Anatum var. 15
phageepsilon34. Virus Genes 29:87-98.
19. Scherer. C.A.. and S.I. Miller. 2001. Molecular pathogenesis oI salmonellae. In Groisman. E. A. Principles oI
bacterial pathogenesis. Academic press. 247-333.
20. Schierack. P.. M. NordhoII. M. Pollmann. K.D. Weyrauch. S. Amasheh. U. Lodemann. J. Jores. B. Tachu. S.
Kleta. A. Blikslager. K. Tedin. and L.H. Wieler. 2006. Characterization oI a porcine intestinal epithelial cell
line Ior in vitro studies oI microbial pathogenesis in swine. Histochem. Cell Biol. 125:293-305.
21. van Asten. F. 2005. The moving story on the Ilagella oI Salmonella serotype Enteritidis (PhD thesis). Utrecht.
The Netherlands.
22. van Asten. F.J.. H.G. Hendriks. J.F. Koninkx. B.A. Van der Zeiist. and W. Gaastra. 2000. Inactivation oI the
Ilagellin gene oI Salmonella enterica serotype Enteritidis strongly reduces invasion into diIIerentiated Caco-2
cells. FEMS Microbiol. Lett. 185:175-179.
24. Vo. A.T.T.. E. van Duiikeren. A.C. Fluit. and W. Gaastra. 2006. Antibiotic resistance. integrons and Genomic
Island SGI1 among non-typhoid Salmonella serovars in The Netherlands. Int. J. Antimicrob. Agents 28:172-
179.
26. WHO Global Salm-Surv. Top 15 Salmonella serotype list Irom each country.
http://thor.dIvI.dk/pls/portal/GSS.COUNTRYDATASETREP.show (23 March 2005. date last accessed).
Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated Irom animals in
Korea: comparison oI phenotypic and genotypic resistance characterization. Vet. Microbiol. 86:295-301.

5ummarIzIng dIscussInn

Summarizing discussion
138
Surveillance studies on antimicrobial resistance at a national level are necessary Ior a correct
understanding oI the current situation in a country and Ior the implementation oI measures to
control a Iurther transmission oI antibiotic resistance. All evidence available today indicates
that the acquisition and transmission oI antibiotic resistance genes in bacterial pathogens has
taken place since the late 1940s. when the use oI antibiotics Ior treatment oI inIectious diseases
was introduced. The best support Ior this notion are the studies by Hughes and Datta
4
who
examined the 'Murray Collection. a collection oI Gram-negative pathogens obtained Irom
clinical cases in the pre-antibiotic era. No evidence oI resistance to antibiotics in current use
was shown in these strains. So. the use oI antibiotics to cure bacterial inIectious diseases at the
same time induces resistance to these antibiotics. With this concept in mind the studies on the
antibiotic resistance among non-typhoid Salmonella isolates. originating Irom humans and
animals Irom Vietnam and The Netherlands described in this thesis were initiated. DiIIerences
in the use oI antibiotics in human and veterinary medicine between the two countries probably
resulted in a diIIerent situation with respect to antimicrobial resistance in Salmonella. Integrons
were chosen as a marker throughout the experiments because they are well-deIined genetic
determinants with a gene capture system involved in resistance to various agents and thereIore
associated with the development oI multidrug resistance and integrons are oIten located on
mobile genetic elements. The availability oI extrinsic resistance genes (or gene cassettes) is
required to generate novel integron variants.

Differences in the distribution of Salmonella serovars and phage types in Vietnam
compared to that in Europe and The United States.
The data presented in Chapter 2 show that the Irequency with which particular Salmonella
serovars occur in Vietnam is diIIerent Irom that in Europe and the United States. regardless oI
the origin oI the isolates (animal or human). Similar data have been obtained in other Asian
countries. but no explanation Ior this phenomenon is currently available. Among human isolates
Irom Vietnam S. Typhimurium is the most common serovar. Iollowed by S. Enteritidis and S.
Weltevreden. In cattle. serovars Anatum. Weltevreden and Lexington are predominant. Among
the porcine isolates. S. Anatum. S. Typhimurium. S. Weltevreden. S. Derby and S. Rissen are
the most common serovars. S. Emek and S. Blockley are the most prevalent serovars among the
isolates originating Irom poultry. S. Enteritidis is Irequently isolated Irom poultry in many
European countries and in the United States and this animal species is an important source Ior
human S. Enteritidis inIections in these countries. In contrast. human S. Enteritidis inIections in
Vietnam probably originate Irom other sources since no S. Enteritidis isolates were detected
among isolates Irom animals. Our Iindings are in accordance with those in another study in
Vietnam where only one S. Enteritidis isolate has been Iound among 80 animal isolates.
Another explanation Ior the low Irequency with which S. Enteritidis is observed in our study
might be that 95 oI the poultry samples were taken Irom broilers due to the restricted access
to poultry Iarms during the bird Ilu outbreak at the time the Vietnamese isolate collection was
carried out. ThereIore only a small number oI laying hens could be sampled. In Europe.
Australia and the United States. S. Typhimurium and S. Dublin are known to be predominant
among isolates Irom cattle. This was not observed in Vietnam. as is likely the case in Thailand.
Chapter 9
139
However. data Ior the distribution oI Salmonella serovars in speciIic species oI animals are not
available Irom this country.

Overall. it can be said that the data obtained in the present study indicate that the
distribution oI Salmonella serovars in Vietnam is similar to that in other South-East Asian
countries but diIIerent Irom that oI European countries and the United States. These data are
important in the epidemiology oI salmonellosis. The exact reason Ior these diIIerent
distributions is not yet known. but the data may provide evidence Ior possible sources oI Iood-
borne inIections that are diIIerent in diIIerent geographical regions in spite oI the increasing
international travel and trade in animals and Iood products oI animal origin.

In Vietnam. S. Typhimurium pt 90 (according to the Dutch phage typing system).
which has no recognized phage type in the English typing system. is the most common phage
type present among S. Typhimurium isolates. The incidence oI this phage type is diIIerent Irom
that in European countries and the United States where S. Typhimurium DT 104 (corresponding
to pt 506 in the Dutch phage typing system) is the most common phage type observed among S.
Typhimurium isolates. It is important to note that the English phage typing system is suitable
Ior typing most Salmonella isolates Irom European countries and the United States but not Ior
typing Salmonella isolates Irom Asian countries as discussed in this chapter.

Although phage typing is very helpIul in understanding the population structure oI
Salmonella it is insuIIicient to answer the question whether MDR strains present in a certain
region or during a particular outbreak belong to a single clone or are distinct strains. Typing by
Pulsed Field Gel Electrophoresis can be a good candidate Ior this purpose as shown in the
Salm-gene proiect (a European collaboration Ior DNA Iingerprinting oI Iood-related
salmonellosis) where the electronic database is available Ior participating institutes to make
comparisons between diIIerent strains Irom diIIerent countries (see Chapter 3).

The in vitro pathogenesis of two MDR S. Typhimurium phage types.
Phage typing demonstrates the presence or absence oI certain receptors Ior the surIace adhesion
oI these phages but restriction/modiIication systems or the presence oI lysogenic phages can
also inIluence the Iact whether or not a phage can grow on a particular strain. However. the
presence or absence oI certain receptors is the most important Iactor and the surIace structure oI
South-East Asian S. Typhimurium isolates probably is diIIerent Irom that oI S. Typhimurium
isolates Irom other parts oI the world. This might inIluence the pathogenicity oI Salmonella
strains. In Chapter 8. the results show that S. Typhimurium pt 506 and pt 90. the dominant
phage types in Europe and Vietnam. respectively. adhered to and invaded a human epithelial
cell line (Caco-2) and a swine epithelial cell line (IPI-2) equally well. The release oI IL-8 by
Caco-2 cells or IPI-2 cells was comparable. independent oI the Salmonella phage type used Ior
stimulation oI the cells. These data suggest that S. Typhimurium pt 90 may have a similar
ability to cause inIection as S. Typhimurium DT104. This partly answers the question raised
above. whether or not diIIerent surIace structures (diIIerent phage types) are oI inIluence on the
in vitro pathogenicity oI S. Typhimurium.
140

Comparison of phenotypic resistance and the characteristics of class 1 integrons of
Vietnamese and Dutch salmonellae.
Generally. resistance to sulphonamide. ampicillin. tetracycline. streptomycin. trimethoprim and
nalidixic acid was observed among the isolates Irom both countries. Similar results have
actually been observed worldwide. MDR resistant isolates Irom both human and non-human
origin have been isolated in both countries. However. the percentage oI resistant isolates
(especially to gentamicin). and oI MDR is higher among Salmonella isolates Irom Vietnam
(Chapter 3) than Irom The Netherlands (except Ior ceItazidime) (Chapter 4). The prevalence oI
integrons in Vietnamese salmonellae (28) is signiIicantly higher than in Dutch salmonellae
(15.2). An explanation Ior this observation might be the increasing and inappropriate use oI
antibiotics during the last ten years in Vietnam. especially in the intensive animal husbandry in
which antibiotics are being used on a large scale as prophylaxis. growth enhancer and Ior
therapy. In 2002. gentamicin and trimethoprim. Ior example. were used Irequently in animal
husbandry in Vietnam.

It is important Ior Dutch authorities to realize that although the percentage oI MDR
Salmonella isolates is lower in The Netherlands than in Vietnam. this Iigure is still high (one
Iourth oI the Dutch salmonellae were resistant to 3 or more antimicrobials). Nevertheless. it can
be said that within Europe. the Scandinavian countries and The Netherlands have a relatively
limited problem with antibiotic resistance. This did not occur by accident. but is the result oI an
active policy oI prevention oI transmission oI pathogens in and outside hospitals. The use oI
antibiotics diIIers also strongly between countries in the EU. In Belgium and France general
practitioners on average prescribe three to Iive times more antibiotics than their colleagues in
The Netherlands
13
. What causes this? Are Belgians or Frenchmen suIIering Irom inIectious
diseases more oIten? This explanation is not very likely. The explanation has to be Iound
elsewhere. namely with those that prescribe or sell antibiotics and with those that consume them
as patients. as well as in the diIIerent ways in which health care is organized in diIIerent
countries. In addition. there are cultural diIIerences which are the basis Ior the diIIerences in
use oI antibiotics. as we have mentioned above Ior Vietnam and The Netherlands.

Several novel types oI integrons or combinations oI gene cassettes and genomic islands
in various Salmonella serovars were detected (Chapter 3 and Chapter 4). Integrons Iound in
isolates Irom humans. pigs. cattle. chicken and horses in the two countries harbored gene
cassettes responsible Ior resistance against aminoglycosides (aadA1. aadA2. aadA5. and aadB).
trimethoprim (dfrA1. dfrAA5. dfrA7. dfrA12. dfrA14. and dfrA17). and beta-lactams (bla
PSE-1

and bla
OXA-30
). These Iindings can be explained by the use oI these antimicrobials during the
last decades in the two countries. Since the sul1 gene. which encodes resistance to
sulphonamides is a core part oI most class 1 integrons. co-selection oI resistance might be
enhanced by the selective pressure imposed by the use oI this antimicrobial drug. OIten
integron-carrying salmonellae are also resistant to other antimicrobials (such as tetracycline.
chloramphenicol. gentamicin. kanamycin and quinolones. see also Chapter 6) than the
antimicrobials Ior which the gene cassettes encode resistance. ThereIore. screening Ior
Chapter 9
141
integron-carrying strains can be part oI the classiIication oI MDR strains in laboratories.
Knowledge oI the mechanism oI the development and transmission oI resistance may govern
therapeutic choices and pave the way Ior laboratory reports oI susceptibility test results to
clinicians and even inIluence the development oI new antimicrobials.

The genetic basis of antimicrobial resistance in specific serovars of S. Dublin from cattle
and S. Typhimurium from horses.
In Chapter 5. Dutch S. Dublin was chosen as a research subiect Ior study oI antibiotic resistance
transmission because it is one oI the most prevalent serovars in cattle in The Netherlands but
not in Vietnam. ThereIore it was expected that the prudent use oI antibiotics in The Netherlands
would show in the resistance proIile oI these isolates. Resistance against streptomycin.
chloramphenicol. sulphonamides and nalidixic acid was observed. The rate with which class 1
integrons (20.3) were Iound in epidemiologically unrelated S. Dublin isolates in this study
was higher than in a study by Liebana et al
11
(3) or in the one by Randall et al
12
(0) in the
United Kingdom. The presence oI integrons in the chromosome oI S. Dublin containing the
aadA1 gene described in this Chapter has not been reported beIore. Analysis oI plasmids and
genomic Iingerprinting by Pulsed Field Gel Electrophoresis (PFGE) showed that the Dutch S.
Dublin strains were closely related but not clonal in nature.

In Chapter 6. a novel variant oI Salmonella Genomic Island 1. SGI1-M Iound in a
Dutch equine S. Typhimurium DT104 isolate is also described. In this case. the aadA2 gene
located in the antibiotic resistance cluster oI Salmonella Genomic Island 1 (SGI1. see
Introduction) is replaced by the aadB gene in the SGI1-M carrying isolate. This change leads to
diIIerences in the phenotypic resistance: SGI1-carrying isolates are resistant Ior Iive antibiotics
(ampicillin. chloramphenicol. tetracycline. streptomycin and sulphonamide) whereas the SGI1-
M-carrying isolate is resistant against at least 7 antibiotics (ampicillin. chloramphenicol.
tetracycline. gentamicin. kanamycin. tobramycin and sulphonamide). A rare integron type with
the dfrA14 and aadA1 genes was detected in 9 non-DT104 S. Typhimurium isolates. These 9
isolates were also resistant to Iluoroquinolones due to mutations in the gvrA gene leading to
substitutions at codons encoding the amino acids Ser83Ala and Asp87Asn. The presence
oI enroIloxacin-resistant Salmonella isolates in Dutch horses is unexpected because quinolones
(like acid nalidixic. Ilumequine and Iluoroquinolones) are not registered Ior use in horses in The
Netherlands. Thus. the expectations with respect to antibiotic resistance in these isolates
expressed above were not met; on the contrary the resistance was higher than observed in
studies Irom some surrounding countries. What might be the explanation Ior these Iindings?
The presence oI the resistance observed in these strains may be caused by oII-label use oI
Iluoroquinolones in equine medication in The Netherlands. One may also speculate about the
spread oI such Salmonella isolates via animal transport between The Netherlands and Germany
where similar resistant strains have been Iound in both humans and cattle. The alternative
hypothesis that quinolone-resistant salmonellae in horses originate Irom other Iood producing
animal species that received quinolones as prophylaxis and treatment or Irom humans that
received Iluoroquinolones as therapy in The Netherlands is also possible.

142
In contrast to several studies Irom which it was concluded that multidrug resistant
S. Typhimurium DT104 has been clonally transmitted between European countries and the
United States. this study showed that the Dutch equine S. Typhimurium isolates (including the
DT104) belonged to distinct strains. Together with the great potential oI horizontal transIer oI
resistance determinants between these S. Typhimurium strains. it is likely that multiresistance to
antimicrobials among the equine S. Typhimurium strains is due rather to acquired resistance
than to the transmission oI a single clonal strain. The data in this study indicate that equine S.
Typhimurium isolates may be potential risk Iactors Ior both animal and human health because
they can easily spread resistance determinants and because oI the close contact between horses
and humans.

In the Dutch veterinary sector. extended spectrum cephalosporins (ceItioIur.
ceIquinome) are prescription-only medicines approved Ior the treatment oI diseases such as
inIections oI the respiratory tract. and oI pododermatitis interdigitalis in cattle and pigs. Until
May 2006. these antimicrobial agents were not allowed Ior use in chickens nor in horses. The
presence oI ESBL and AmpC type genes encoding resistance to extended spectrum
cephalosporins among Enterobacteriaceae isolates Irom horses and a chicken (including a horse
oI 1 month old) is a remarkable Iinding (Chapter 7). It means that the transmission oI these
resistant isolates could occur between animal species or between animals and humans. Improper
use oI antibiotics in veterinary and human medicine in The Netherlands might also contribute. It
is also worthwhile to note that there are two classes oI antibiotics (quinolones and third-
generation cephalosporins) that have a higher propensity Ior development oI resistance.

Horizontal exchange of resistance determinants between Salmonella serovars or between
Salmonella and E. coli by in vitro conjugation.
The potential oI in vitro horizontal transIer oI integrons and resistance determinants by
coniugation between Salmonella isolates oI diIIerent serovars (Chapter 3) or between
Salmonella and a reIerence E. coli K12 strain (Chapter 3 and Chapter 6) or between clinical
isolates oI Salmonella and E. coli (Chapter 7) was clearly demonstrated. Data in this chapter
also show that the bla
ACC-1
gene responsible Ior resistance to ceItazidime oI a Dutch S.
Braenderup isolate could be transIerred by coniugation. So. Salmonella isolates that were
successIul in horizontal genetic exchange belonged to diIIerent serovars and were isolated in
both countries. The results shown in this thesis indicate that clinical Salmonella isolates can
play a role as donor or recipient in the horizontal exchange oI antibiotic resistance determinants
between diIIerent serovars or bacterial species. The role as a reservoir oI multiple drug
resistance oI Salmonella should be highlighted.

CONCLUDING REMARKS

- Similarities and diIIerences in the distribution oI Salmonella serovars. phage types oI
S. Typhimurium. integron types and SGI1 observed in Salmonella isolates Irom Vietnam and
The Netherlands are described in this thesis. The data indicate that the precise epidemiological
determination oI a Salmonella strain during an outbreak oI salmonellosis or when
Chapter 9
143
imported/exported-Ioods are examined is warranted. Only then a correct decision in the control
on emergence oI resistant strains can be made. The data in this thesis will contribute to the
knowledge oI MDR salmonellae. They show that although MDR salmonellae is present
worldwide their distribution seems diIIerent in various geographic locations. For instance. the
MDR Salmonella phage type oI S. Typhimurium observed with the highest Irequency is
diIIerent between diIIerent countries: S. Typhimurium DT104 is the most dominant serovar in
most European countries
15
and North America
7. 16
; whereas non-DT104 S. Typhimurium is the
most dominant serovar in Asian countries
1. 17.

Chapter 2
. and in Australia a diIIerent serovar
(Heidelberg) is most common
5
. The distribution oI antibiotic resistance encoding genomic
islands in Salmonella serovars in Australia and Asia
10. Chapter 3
seems diIIerent Irom that in
Europe and Canada
2. 6. 14
. Likewise. the mutations in the gvrA gene leading to substitutions in
the amino acids Ser83Ala and Asp87Asn are Iound in Dutch
Chapter 6
and German S.
Typhimurium strains
8
. whereas Ser83Phe and Asp87Asn substitutions are Iound in
Vietnamese S. Typhimurium
Chapter 3
and Taiwanese S. Cholerasuis isolates
9
.

- Resistance to antimicrobials in salmonellae was high (up to 50 or more oI the isolates tested
were resistant to at least one antibiotic)
5. 12
. Higher levels oI resistance in Salmonella were
observed in countries where antibiotics are used Ireely (e.g. Vietnam
Chapter3
) or in high amounts
(e.g. France)
3
compared to countries where the use oI antibiotics is controlled and lower
amounts are used (e.g. The Netherlands. see also above
Chapter 4
). The same holds Ior the
prevalence oI class 1 integrons. There is no evidence that the Dutch Salmonella isolates were
imported into The Netherlands neither were the Vietnamese Salmonella isolates imported into
Vietnam. ThereIore. the presence oI antibiotic resistant Salmonella strains as well as integron-
carrying Salmonella strains in Vietnam and The Netherlands is mainly a direct result oI how
antibiotics are used in both countries. A way to reduce resistance to antimicrobials in bacteria
including Salmonella is that legislation requiring a more prudent use oI antibiotics in both
human and veterinary medicine should be implemented by local and national authorities.
especially in Vietnam.

- Detection oI new types oI integrons. novel variants oI SGI1. and the potential oI transIer oI
antibiotic resistance determinants by coniugation in salmonellae Irom The Netherlands and
Vietnam contributes to the explanation oI the Ilexibility oI Salmonella to deal with antibiotics
and the widespread occurrence oI these antibiotic resistance determinants among not only
Salmonella serovars but also among Enterobacteriaceae. Firstly. integrons with distinct genes
but conIerring the same resistance phenotype were Iound to be present in a single isolate
Chapter 3
.
One antibiotic resistance gene may be lost during evolution/ replication; others will remain and
help bacterial cells to retain its resistance. Secondly. deletion. exchange or/ and insertion oI
genes Irom/ in SGI1 are probably responsible Ior the spread and the diversity oI SGI1 among
MDR Salmonella strains (SGI1-N
Chapter 3
. SGI1-M
Chapter 6
). A single isolate can contain integrons
located on coniugative plasmids or on the chromosome. The presence oI resistance
determinants on mobile elements
Chapter 3. Chapter 6
contributes to the transmission oI the resistance
they encode. As a result. the transmission oI Salmonella strains. resistant to 10-13
antimicrobials
Chapter 3. Chapter 6
is not only possible but also a reality. It is obvious that this
144
compromises therapy Ior both humans and animals and that this should be monitored and
controlled in both countries. At the same time. the integration oI resistance determinants in the
chromosome
Chapter 5
also can enhance the persistence oI resistance even in the absence oI
antimicrobials. ThereIore. it is important to realize that reduction or discontinuation oI
antimicrobial use alone will not always result in a decrease in the Irequency by which
antibiotic-resistant isolates are recovered. at least not on the short term
18
. Resistant Salmonella
strains may still retain their 'Iitness in the absence oI antibiotics. Thus. it is impossible to get
rid oI antibiotic resistance when it arises. However. we can control or reduce antibiotic
resistance iI more eIIorts will be made by authorities. scientists. veterinarians. physicians and
the general public in both countries.

RESEARCH/ ACTIONS IN FUTURE

To deal with the problem oI antibiotic resistance we should Iirst have to determine the extend oI
the problem. This thesis contributes to this by describing the antibiotic resistance types and
levels in Salmonella in both The Netherlands and Vietnam. In addition. the mechanisms
involved in resistance were studied as well as transmission routes. Nevertheless. additional
studies are required Ior other countries and species. However. even without complete
knowledge oI the extend oI the problem it is clear that action should be taken. These actions
include general measures like education and enIorcement oI prudent use oI antibiotics. but also
direct interventions like hygienic measures when the extend oI the problem is known. For
Salmonella and possibly other species the Iocus should not only be on antibiotic resistance.
because Salmonella is an important pathogen. A strong physical association oI both virulence
and antibiotic resistance genes is possible in Salmonella. As mentioned in Chapter 1. the
Salmonella Pathogenicity Island and the Salmonella Genomic Island both contain Iunctional
genes encoding mobile elements. In addition. the Iormation oI cointegrates oI resistance and
virulence plasmids is possible in Salmonella. Selection Ior one oI them will automatically lead
to selection Ior the other. The relation between virulent Iactors and antibiotic resistance
determinants in strains oI S. Typhimurium at molecular level will oIIer a very interesting topic
Ior Iurther research.

REFERENCES
599.
3. Cailhol. J.. R. Lailler. P. Bouvet. S. La Vieille. F. Gauchard. P. Sanders. and A. Brisabois. 2006. Trends in
antimicrobial resistance phenotypes in non-typhoid Salmonellae Irom human and poultry origins in France.
Epidemiol. InIect. 134:171-178.
4. Davies. J.. and V. Webb. 2004. Antibiotic resistance in bacteria. In Schaechter. M. & Lederberg. J. The desk
encyclopedia oI microbiology. London. UK: Elsevier Academic Press. 25-46.
Chapter 9
145
5. Davos. D.. and H. Hocking. Antimicrobial resistance in Salmonella spp oI human and non-human origin in
Australia 2006. International symposium Salmonella and Salmonellosis. Colin. P. & Clement. G. Saint-Malo.
France. 145-148
7. Gebreyes. W.A.. and C. Altier. 2002. Molecular characterization oI multidrug-resistant Salmonella enterica
subsp. enterica serovar Typhimurium isolates Irom swine. J. Antimicrob. Chemother. 40:2813-2822.
8. Heisig. P.. B. Kratz. E. Halle. Y. Graser. M. Altwegg. W. Rabsch. and J.P. Faber. 1995. IdentiIication oI DNA
gvrase A mutations in ciproIloxacin-resistant isolates oI Salmonella Tvphimurium Irom men and cattle in
Germany. Microb. Drug Resist. 1:211-218.
9. Huang. T.M.. Y.F. Chang. and C.F. Chang. 2004. Detection oI mutations in the gvrA gene and class I integron
Irom quinolone-resistant Salmonella enterica serovar Choleraesuis isolates in Taiwan. Vet. Microbiol.
100:247-254.
11. Liebana. E.. L. Garcia-Migura. C. Clouting. C.A. Cassar. F.A. CliIton-Hadley. E.A. Lindsay. E.J. ThrelIall.
S.A. Chappell. and R.H. Davies. 2002. Investigation oI the genetic diversity among isolates oI Salmonella
enterica serovar Dublin Irom animals and humans Irom England. Wales and Ireland. J. Appl. Microbiol.
93:732-744.
genes. integrons and multiple antibiotic resistance in thirty-Iive serotypes oI Salmonella enterica isolated Irom
humans and animals in the UK. J. Antimicrob. Chemother. 53:208-216.
13. Verburgh. H.E. 2003. Resistentie: cultuurverschillen en beleid. In Antibiotica en resistentie. Cahiers Bio-
wetenschappen en Maatschappii. 44-49.
14. Weill. F.X.. L. Fabre. B. Grandry. P.A. Grimont. and I. Casin. 2005. Multiple-antibiotic resistance in
Salmonella enterica serotype Paratyphi B isolates collected in France between 2000 and 2003 is due mainly to
strains harboring Salmonella genomic islands 1. 1-B. and 1-C. Antimicrob. Agents Chemother. 49:2793-2801.
Multidrug resistance in Salmonella enterica serotype Typhimurium Irom humans in France (1993 to 2003). J.
16. White. D.G.. S. Zhao. P.F. McDermott. S. Ayers. S. Friedman. J. Sherwood. M. Breider-Foley. and L.K. Nolan.
2003. Characterization oI integron mediated antimicrobial resistance in Salmonella isolated Irom diseased
swine. Can. J. Vet. Res. 67:39-47.
Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated Irom animals in
Korea: comparison oI phenotypic and genotypic resistance characterization. Vet. Microbiol. 86:295-301.
18. Zhang. Q.. O. Sahin. P.F. McDermott. and S. Payot. 2006. Fitness oI antimicrobial-resistant Campvlobacter
and Salmonella. Microbes InIect. 8:1972-1978.

146

Ncdcr!andsc samcnvattIng vnnr dc !cck


Werden kort na hun introductie begin iaren veertig van de vorige eeuw. deze middelen ter
bestriiding van inIectieziekten veroorzaakt door bacterien nog met argwaan bezien. niet kort
daarna hadden antibiotica de status van wondermiddelen. Tientallen iaren heeIt de mensheid in
de veronderstelling geleeId dat inIectieziekten geen bedreiging meer vormden voor de
gezondheid. Dit optimisme was op zeker moment zelIs zo groot dat antibiotica ook werden
gebruikt bii virale inIecties. terwiil virussen helemaal niet gevoelig ziin voor antibiotica. Dit
verkeerde gebruik oI zo men wil misbruik van antibiotica vond ook plaats binnen de
veehouderii. waar vele tonnen antibiotica aan het voer van niet zieke dieren werden toegevoegd
omdat ze daar zo lekker van groeiden.

Ook bii het gebruik van antibiotica bleek echter een wet uit de natuurkunde. 'actie is
reactie opgeld te doen. Bacterien reageerden op het Ieit dat zii werden aangevallen door het
veranderen van hun erIeliike eigenschappen (mutatie) en door het opnemen van gedeelten van
de erIeliike eigenschappen van organismen (bacterien en schimmels) die zelI in staat ziin tot het
maken van bepaalde antibiotica. Als gevolg van deze reactie konden korte tiid na de introductie
van antibiotica al de eerste bacterien gesoleerd worden die ongevoelig (immuun) geworden
waren tegen bepaalde antibiotica. De bacterien hadden evenwel nog meer verrassingen in petto.
zii bleken zeer Ianatieke verzamelaars van de stukies erIeliik materiaal die hen immuun maken
voor antibiotica en zoals een postzegelverzamelaar ziin postzegels in een album stopt. zo
stopten bacterien hun verzameling ook in een album (een integron). Op deze manier ziin de
beste verzamelaars ongevoelig geworden voor wel meer dan tien verschillende soorten
antibiotica en ziin er nu zelIs bacterien die niet meer gevoelig ziin voor wat voor antibioticum
dan ook en waartegen dus geen behandeling meer mogeliik is. Daar waar 'Samen werken.
samen leven het motto is van de regeringsverklaring. zou samenwerken om samen te overleven
het motto kunnen ziin van bacterien. Zii brengen dit in praktiik doordat zii het album met hun
integron verzameling doorgeven van de ene bacteriesoort naar de andere. Hiermee kan in een
klap een bacterie die gevoelig en goed behandelbaar met antibiotica was. ongevoelig voor wel
dertien verschillende antibiotica worden en daardoor een inIectie met zo`n bacterie
onbehandelbaar.

In dit proeIschriIt is onderzoek beschreven naar de gevoeligheid voor antibiotica van
de bacterie Salmonella. die bii mens en dier darminIecties kan veroorzaken. Omdat (verkeerd)
gebruik van antibiotica van invloed is op de ontwikkeling van ongevoeligheid voor antibiotica
is bii dit onderzoek een vergeliiking gemaakt tussen Salmonella bacterien aIkomstig van de
mens. het varken. het rund en de kip in Vietnam en Nederland. Daar waar in Nederland
antibiotica uitsluitend op recept verkriigbaar ziin is het in Vietnam mogeliik deze in de vriie
verkoop te betrekken. Dit leidt tot aanzienliike verschillen in de manier waarop met antibiotica
wordt omgegaan in beide landen. Voorbeelden daarvan ziin het niet nemen van een kuur met de
iuiste dosis. het niet voldoende lang nemen van een kuur. het niet slechts nemen van een kuur
bii bacteriele inIecties en het nog steeds gebruiken van antibiotica als groeibevorderaar in
Vietnam. Ongevoeligheid voor antibiotica van bacterien aIkomstig uit Vietnam
bleek veelvuldig voor te komen. In Nederland. waar al iarenlang de ontwikkeling van
Nederlandse samenvatting voor de leek

ongevoeligheid van tegen antibiotica wordt bestudeerd en biigehouden was het

probleem nauweliiks minder. In sommige diersoorten werd zelIs ongevoeligheid waargenomen
voor antibiotica waarvan het gebruik bii die diersoort niet is toegestaan. In dit onderzoek ziin
zowel in Vietnam als in Nederland ook bacterien aangetroIIen waarvan de
verzameling in hun integron album uniek is en nog nergens ter wereld eerder is beschreven.
Aan het eind van het proeIschriIt wordt ingegaan op de maatregelen die zouden kunnen worden
genomen om het tii van de antibioticum ongevoeligheid te keren. De noodzaak van
voortdurende inventarisatie van de toestand op plaatseliik. landeliik. regionaal en mondiaal
niveau wordt beklemtoond. even als de gezamenliike verantwoordeliikheid die overheid en
burgers hebben wat betreIt het onoordeelkundig gebruik van antibiotica.

Tm tt !un n

D khang khang sinh cua Salmonella
152
Non-typhoid Salmonella la mt trong nhung vi khun gy tiu chay cp tinh thuong
gp nht o cac nuoc phat trin va dang phat trin. Chung la nguyn nhn gy tu vong cho
khoang 2 triu nguoi mi nm. Non-typhoid Salmonella la thut ngu am chi cac Salmonella
thuc bt cu serovar nao cua Salmonella enterica ma khng phai Salmonella enterica serovar
Typhi (gy bnh thuong han) hoc Salmonella enterica Paratyphi (gy bnh pho thuong han).
Da s cac truong hop vim rut tiu chay do non-typhoid Salmonella khng cn phai can thip
bng khang sinh. Tuy nhin. trong nhung truong hop nhim trung gy nguy him dn tinh
mang. hoc nhim trung toan thn o nguoi gia. tre em. nguoi bi suy giam min dich thi liu
phap khang sinh la cn thit. Vi th. tinh trang cac chung vi khun non-typhoid Salmonella d
khang khang sinh gia tng ngay cang nhiu khp noi trn th gioi dang la mt mi lo ngai co
tinh toan cu. Su tn tai cua cac chung Salmonella khang khang sinh khng chi lam gioi han
kha nng diu tri bnh do chung gy nn cho nguoi va thu. chung con co th la noi tich tru cac
ngun gene khang khang sinh d tu dy truyn gene khang thuc cho cac vi khun khac.
Cac nghin cuu giam sat v d khang khang sinh o cp d quc gia la thuc su cn thit
d chung ta co duoc nhung kin thuc dung dn v hin trang cua vn d khang thuc tai quc
gia do. Tu do. nhung bin phap kim soat chng su lan tran cua d khang khang sinh co th
duoc tng cuong. Vic su dung khang sinh trong phong tri bnh tu nhung nm 1940 d dng
thoi tao ra mt ap luc chon loc. tu do su d khang cua vi khun voi khang sinh s xut hin o
mt muc d nht dinh. Su d khang do thu nhn gene khang thuc co vai tro quan trong trong
su lan tran tinh trang khang khang sinh cua cac chung vi khun. Nm 1995. Hall va cng su d
m ta cac yu t di truyn co kha nng thu nhn gene (gene capture system) cua nhiu loai vi
khun. goi la integron (Chuong 1). Integron co kha nng nhn bit. bt giu mt hoc nhiu
gene cassette khang thuc. Cac gene duoc goi la cac cassette vi chi khi chen vao integron chung
moi co th biu hin duoc tinh trang d khang. O trang thai tu do. gene cassette la nhung gene
khng hoan chinh (thiu promotor cho qua trinh giai m di truyn). Do integron co kha nng thu
giu 1 hay nhiu gene cassette. cac chung vi khun co mang integron thuong la da d khang (d
khang voi nhiu khang sinh).
Lun an nay m ta va thao lun cac nghin cuu v d khang khang sinh cua cac chung
vi khun non-typhoid Salmonella phn lp tu nguoi. thit gia suc gia cm va thu nui o Vit nam
va Ha lan voi gia thuyt rng su khac bit trong vic su dung khang sinh trong nhn y va thu y
o hai quc gia gop phn vao su khac bit v tinh trang d khang cua Salmonella. Cac integron
lop 1 (class 1 integron) duoc chon nhu mt di tuong nghin cuu chinh o cac thi nghim vi
chung khng chi lin quan dn da d khang cua cac chung vi khun thuc Enterobacteriaceae
ma chung con thuong hin din trong cac yu t di truyn co kha nng di chuyn gop phn vao
su lan tran d khang khang sinh. Dao chua gene khang thuc nm trn nhim sc th cua
Salmonella (Salmonella Genomic Island 1. SGI1) cung duoc nghin cuu vi chung thuong mang
nhiu integron va cac gene khang khang sinh nm ngoai integron. D khang voi Iluoroquinolon.
voi cephlosporin th h thu 3 cung duoc nghin cuu vi dy la nhung khang sinh ph rng chu
cht trong diu tri nhim trung do cac nhiu loai vi khun gy bnh trong nhn y va thu y khoa.
Cac chung vi khun Salmonella duoc dinh danh bng cac qui trinh tiu chun. Chung
duoc phn loai bng serotyping va phagetyping (hai hinh thuc ph bin trong phn loai
Salmonella) voi h thng phn loai cua Anh quc va Ha lan. Su khac bit v phn b cua cac
serovar va phage type cua cac chung Salmonella phn lp tu Vit nam va Ha lan duoc trinh bay
Tom tt lun an

153
o Chuong 2. Nhung du liu nay s gop phn trong vic danh gia v dich t hoc cua cac chung
Salmonella phn lp tu dich hoc thuc phm xut nhp khu. Chi khi co nhung danh gia dung
dn v ngun gc cua cac chung vi khun. vic ngn chn su ly lan moi co hiu qua. Du liu
va thao lun trong chuong nay cung cho thy nhu cu v mt phuong phap dinh danh va phn
loai tt hon cn duoc ap dung (nu co th) vi h thng phn loai bng phage cua Anh quc d
khng th phn loai hu ht cac chung vi khun Salmonella Typhimurium cua cac nuoc chu A.
Ct DNA b gene vi khun nay bng cac enzyme gioi han va thuc hin din di trn gel voi xung
din truong dao (Pulsed Field Gel Electrophoresis. PFGE) co th la mt chon lua trong tuong
lai. nht la khi cac du liu din tu co th duoc kt ni va so sanh lin quc gia. PFGE kt hop
voi k thut dinh danh plasmid (plasmid proIiling). d xac dinh rng cac chung S. Dublin phn
lp tu bo (Chuong 5) va S. Typhimuirum tu ngua (Chuong 6) tu 10 nm qua o Ha lan la
nhung chung co lin h v di truyn nhung khng thuc cung 1 dong (clone).
Kiu hinh d khang cua cac chung Salmonella duoc xac dinh bng khang sinh d hoc
phuong phap vi pha long. Kt qua so sanh v kiu hinh d khang va dc dim cua cac class 1
integron trong cac chung Salmonella phn lp tu Vit nam (297 chung) va Ha lan (237 chung)
trong nhin cuu ct ngang (nm 2004) duoc trinh bay o Chuong 3 va Chuong 4. Du liu cho
thy rng d khang cua Salmonella voi sulphonamide. ampicillin. tetracycline. streptomycin.
trimethoprim va acid nalidixic la ph bin o ca 2 nuoc cung nhu trn th gioi. Tuy nhin. ti l
d khang (dc bit voi gentamicin). da d khang cao hon trong cac chung Salmonella phn lp
tu Vit nam so voi cac chung phn lp tu Ha lan (ngoai tru voi ceItazidime). Dc tinh cua
integron va gen khang thuc duoc nghin cuu dua trn k thut nn tang tu PCR (PCR-based
techiques). kim tra su da hinh bng phn ct DNA voi cac enzyme (Restriction Fragment
Length Polymorphism. RFLP). va doc trinh tu gene (sequencing). Co mi lin quan cht giua
da d khang va su hin din cua integron trong cac chung vi khun cua nghin cuu nay. Tn
sut phat hin integron cung cao o cac chung cua Vit nam (28) hon la o cac chung cua Ha
lan (15.2). Trong nghin cuu nay. khng co chung cu nao cho thy cac chung Salmonella cua
Vit nam hay chung Salmonella cua Ha lan la tu nuoc ngoai dua vao. Do do. su hin din cua
cac chung Salmonella khang thuc hay Salmonella co mang integron chu yu la kt qua truc
tip cua cach su dung khang sinh trong nhn y va chn nui thu y o tai chinh 2 nuoc nay
(Chuong 1). O Vit nam. khang sinh chua duoc kim soat cht ch. Khang sinh duoc bay ban
cng khai va vic mua khang sinh khng cn toa thuc cua bac si la rt ph bin. Thm vao do.
nhn thuc cua nguoi tiu dung trong vic su dung khang sinh con rt han ch nn vic ngung
thuc giua liu trinh diu tri. dung thuc duoi liu. rut ngn liu trinh xay ra ca trong nhn y va
thu y. Vic chn nui tp trung voi qui m lon va v sinh chn nui chua tt cung lam gia tng
nhu cu su dung khang sinh phong bnh. kich thich tng truong. Cac chinh sach va lut l lin
quan dn quan li. phn phi. va su dung khang sinh o Vit nam cn duoc gioi chuc co trach
nhim xem xet va tng cuong quan ly mt cach hiu qua hon. Vn d d khang khang sinh o Ha
lan tuong di gioi han so voi cac nuoc khac o chu u do nhung chinh sach tich cuc trong vic
t chuc chm soc y t. k toa. mua. ban va su dung khang sinh; trong phong tranh su lan truyn
mm bnh va vi khun khang thuc. Tuy nhin cac nha chuc trach Ha lan cung cn luu y rng
tuy ti l cac chung Salmonella da d khang cua Ha lan thp hon cua Vit nam. con s mt phn
tu cac chung Salmonella phn lp tu nuoc nay d khang voi 3 khang sinh tro ln la khng nho.
D khang khang sinh cua Salmonella
154
Mt s class 1 integron moi. hoc su phi hop cua cac gene cassette trong cac
integron. hoc su hin din cua cac integron tai SGI1 chua tung duoc cng b d duoc phat
hin trong cac chung Salmonella thuc nhiu serovar tu Vit nam va Ha lan (Chuong 3 dn
Chuong 6). Cac integron nay co mang cac gene lin quan dn d khang voi aminoglycoside
(aadA1. aadA2. aadA5 m hoa su d khang voi streptomycin va spectinomycin; aadB m hoa
su d khang voi gentamicin. kanamycin. tobramycin). trimethoprim (dfrA1. dfrA5. dfrA7.
dfrA12. dfrA14 va dfrA17). beta-lactams (bla
PSE-1
va bla
OXA-30
). Diu nay co th tu vic su dung
cac khang sinh nu trn qua nhung thp nin vua qua o ca Ha lan va Vit nam. Vi sul1 gene.
gene m hoa su d khang sulIonamide. la mt phn co ban cua hu ht cac class 1 integron. su
chon loc dng thoi (co-selection) tinh trang d khang nhiu khang sinh s co th xay ra khi ap
loc chon loc voi mt hay nhiu khang sinh trn xut hin. Hon th nua. cac chung Salmonella
co mang integron thuong d khang voi nhiu khang sinh khac nhu tetracycline.
chloramphenicol (nht la trong cac chung nay co mang dao chua gene da d khang SGI1).
gentamicin. kanamycin va ca Iluoroquinolone. D khang voi enroIloxacin cua Salmonella (phn
lp tu nguoi o Vit nam. Chuong 3; hoc tu ngua o Ha lan. Chuong 6) la do 2 dt bin dim o
gvrA gene dn toi su thay di amino acid o vi tri 83 va 87 trong vung quyt dinh su d khang
quinolone (Quinolone Resistance Determining Region).
Mc du d khang khang sinh o non-typhoid Salmonella hin din khp noi trn th
gioi. lun an nay cung phn tich cac du liu va cho thy duong nhu su phn b cua cac chung
Salmonella da d khang chim uu th o tung khu vuc la khac nhau (Chuong 9). Nghin cuu
cung khng dinh su linh hoat cua vi khun trong vn d di pho voi khang sinh. Nhiu gene lin
quan dn cung mt tinh trang d khang co th tn tai trong 1 chung vi khun. Nu 1 gene mt di
trong qua trinh nhn di hoc tin hoa. cac gene con lai vn tn tai giup vi khun giu nguyn
kha nng d khang khang sinh. Ngoai ra. su loai bo. thm vao hoc trao di gene khang thuc
hoc integron co th la co ch ma nhiu bin th (variant) SGI1 ra doi d phu hop voi mi
truong sng cua vi khun voi nhung ap luc chon loc khac nhau. Hon th nua. voi cac yu t
khang thuc nm trn nhim sc th vi khun s co kha nng bao tn cac tinh trang d khang
khang sinh trong mt thoi gian dai. Qua thi nghim tip hop phi lm sang (in vitro coniugation)
cac Salmonella phn lp tu Vit nam va Ha lan th hin rng chung co mang cac gene khang
thuc (da d khang lin kt voi integron. Chuong 3; khang cephalosporim th h 3. Chuong 7)
nm trn plasmid tip hop. d dang trao cho vi khun khac (Salmonella thuc serovar khac hoc
E. coli) d lam gia tng kha nng d khang voi khang sinh cua qun th vi khun. R rang la.
chung ta khng th loai bo hoan toan su d khang cua vi khun di voi khang sinh mt khi
chung xut hin. Tuy nhin. chung ta co th kim soat va gioi han vn d nay nu cac nha chuc
trach. nha khoa hoc. nguoi cng tac trong nganh y t va thu y cung nhu cng dng dn cu o
tung dia phuong. quc gia. khu vuc va quc t co nhiu n luc hon nua cho vn d nay
(Chuong 1).
Vi Salmonella la mt loai vi khun gy bnh. nghin cuu d v dc luc cua cac chung
Salmonella da d khang co th la mt trong nhung d tai cho cac nghin cuu trong tuong lai.
Chuong 8 trinh bay nhung thi nghim cho thy kha nng gy bnh (muc d bam dinh. xm
nhim va kich thich san sinh IL-8 trn t bao nguoi va heo) tuong duong cua 2 dong
S. Typhimurium da d khang phn lp tu Vit nam (pt 90) va tu Ha lan (pt 50 hay DT 104).

Acknnw!cdgcmcnts

Li cam ta
First of all I would like to thank Aong Lam Universitv of Ho Chi Minh Citv and Jietnamese
Covernment (Project 322 of 1he Ministrv of Education and 1raining) who granted this studv
and gave me the chance to understand The Netherlands and some other European countries. I
also thank the Department of Infectious Diseases and Immunologv. Utrecht Univeisitv for the
kind support during the time I was doing this research.

I deeplv thank Prof. Dr. W. Gaastra for being a good leader of the proiect and for treating me
nicelv and softlv. I respect vou verv much. Wim, in both science and human life. I would like to
express mv deep gratitude to Dr. E. van Duiikeren for understanding mv efforts and alwavs
being there in the most difficult times of mv PhD life. I learned from vou. Engeline, not onlv
knowledge of Salmonella. antimicrobial resistance. and academic English but also the wav of
organizing a proiect and having confidence in science. Please send mv regards to vour Sint and
Piet for presenting me a poem. green tree and chocolate in the last winters. I feel a great
admiration for Dr. A.C. Fluit. Thank vou. Ad. for vour hundreds of scientific emails that vou
replied or sent me. You contributed remarkablv to orient the studv. to help me set up the
experiments and to improve the manuscripts. To mv opinion vou are a good example of a
genuine scientist and a highlv responsible teacher.

Im indebted to manv Jietnamese people who have given me assistance or encouragement.
especiallv during the time of sample collecting and Salmonella isolation. Thev are the teachers.
Prof. Dr. N.N. 1un. Dr. N.N. Pho, Prof. P.S. 1in. Prof. Dr. T.T. Dn, and Dr. D.D. Dng,
mv colleague Agoc, the veterinarians. co Ahung, anh 1hut. chi Chu. chi 1rinh. anh Hiu.
anh Long. chi Phi. anh 1ruong. anh 1n. chi 1uvt. Long. Ha, and 1rang, the phvsicians.
Dr. N. Jinh. chi 1ht. chi Hong. and co Mai, mv uncle Bnh, chu 1hng. mv students.
especiallv Bao and Dim.

Im verv grateful to Prof. Dr. 1os van Putten for his acceptance of mv PhD proposal and for his
kind encouragement to do this research. A special thanks goes to Marcel for being
understanding and helpful. I thank Marijke. Lieke, Aancv. Aiwat. Stephanie. Marc. Remon.
Aina. Liana. Andrie. Marille, Merel, Ceert and Linda in the Infectionbiologv Division for the
good things thev gave me and valuable scientific experience thev shared with me through all
these vears. I thank Dirk. 1aap. Carolien. Ankv, 1ohan and the staff of the JMDC, Corrie and
people of ITC, Anton. Rob of I&I, Ali and Sandor of J&J, R. Pauling. Hellen. Adja. and
Rosita of BIC for their high responsibilitv and their warm help.

Deep appreciation I wish to express to mv Dutch colleagues Wim. Max. Anjo. Hennv. Kim.
Henk and Frans of the RIJM. Henno. Peter and Fons of the Pathologv Department who either
contributed to the studv as co-authors or generouslv helped me in Salmonella serotvping. phage
tvping. genotvping and cell investigation, to Maurine. Anne Marie, Risma and Armand of the
Eiikman-Winkler for technical guidance and providing the materials, to Prof. Stefan Schwarz
of the Institut fr Tierzucht (Germanv). to Hans Mittengurg of AST Farma B.J (The

Netherlands). and to Linda Ward and the staff of the Public Health Laboratorv Service (UK)
for their kind help from a distance.

I would also like to sav thank vou to mv best Dutch friend. Lea. Ill remember vour zuurkool.
Dutch lessons. Dutch stories and a heater which saved me in the winters. I am grateful to Svlvia
who kindlv helped me in expressing mv feelings about the beautv of the lotus and tulip. I am
thankful to the veterinarians. Dick, Maarten, Marieke and Astrid who gave me a warm
welcome and useful knowledge during the field trips to animal farms or to animal clinics.

Dear co Hoa. I bet vou will not forget the experiences in The Netherlands we shared such as
'verkeerde trein` to Houten. 'missing boat` in Giethoorn. 'high adventure`in the Euromast
or the'Wizard-Roche invention kit` in the lab. Co Hoa. Dai. Minh. Anh, chi 1huv, Hung and
Lan (Jietnamese). Ludmila (Ukranian). Aivada (Thai). Li (Taiwanese). and Bvoung (Korean).
mv heartfelt thanks goes to vou all for the helpful advice and assistance that vou have given me.
I also thank colleagues. friends and students whose names I did not mention but who have
encouraged me in different wavs.

Con gui tam long bit on vo han toi B Me. Nghi luc song va phuong phap lam vic ma Bo d
dav con. giup con vuot qua nhng kho khn cua khoa nghin cuu nav. Ca dao. tuc ng ma Me
truvn giup con duoc vu thuong bao boc noi nav. Con cng gui loi cam on toi Ba M. nguoi
d ht long voi cac con. cac chau. d la cho dua d tui con phan dau cho mot tuong lai tot dep
hon. To mv dear brother. mv sisters-in-law and brothers-in-law. especiallv. Aam. Xuvn. 1huv
and H. I owe vou for what vou have done for mv daughter and mv familv during the time anh
Bao and I were studving abroad.

Dear anh Bao. I am reallv moved bv vour love for me and our daughter. You are mv darling.
mv husband and mv closest friend who shares with me happiness and miserv wherever we are. I
appreciated so much vour support for this studv and vour taking care of little Khanh when I
was not at home. Dear puppv Minh Khanh. mom thanks vou for vour reading poems and fairv
tales. singing songs. drawing postcards and telling stories that warmed me up in the cold
weekends in the office. I am motivated bv vour sweet voice to finish this studv. I love both of vou
so much.

J Thi Tra An

.Du di xa that la xa
Chng dau vui duoc nhu nha cua em

CURRICULUM VITAE

V Thi Tra An (A.T.T. Vo) was born on the 10
th
oI February 1974 in Ha Nam. Vietnam.
1979 - 1989: received primary education in Phu Cuong. Binh Duong Province. Vietnam.
1989 - 1991: Iollowed secondary education at the Vo Minh Duc High School. Binh
Duong Province. Vietnam.
1991 - 1996: passed the national examination and studied the undergraduate program in
Veterinary Medicine at Nong Lam University (Iormerly Agriculture and
Forestry University). Ho Chi Minh City. Vietnam.
25 Jul 1996: obtained the Doctor oI Veterinary Medicine diploma with a thesis on 'Use
oI Lactobacillus acidophilus as a probiotic Ior diarrhea prevention in
piglets.
Jan 1997: was employed as an assistant lecturer in the Department oI Veterinary
Pharmacology and Internal Medicine. Faculty oI Animal Husbandry and
Veterinary Medicine. Nong Lam University.
Jan 1999: became a iunior lecturer. teaching in the Iield oI veterinary pharmacology
Ior undergraduate students in the Department oI Veterinary Pharmacology
and Internal Medicine. Faculty oI Animal Husbandry and Veterinary
Medicine. Nong Lam University.
2000 - 2002: studied Ior Master oI Science in Veterinary Medicine at Nong Lam
University and worked as a lecturer in the Faculty oI Animal Husbandry
and Veterinary Medicine. Nong Lam University.
31 Mar 2002: was given the degree oI Master oI Science in Veterinary Medicine with a
thesis entitled 'Antibiotic use in chicken husbandry and antibiotic residues
in broiler meat in Ho Chi Minh City.
May 2003: passed the national examination and got a scholarship Irom the Vietnamese
Government (Proiect 322 oI the Ministry oI Education and Training) Ior a
PhD study.
Jun 2003: obtained a grant Irom the Government oI Thailand Ior a 'Faculty and
Student Exchange Program in Animal Science at Maeio University.
Chiangmai. Thailand.
Nov 2003-2007: perIormed PhD research on 'Antibiotic resistance in Salmonella at the
Department oI InIectious Diseases and Immunology. Faculty oI Veterinary
Medicine. Utrecht University. The Netherlands.
Sep 2007- work as a staII member oI the Faculty oI Animal Husbandry and Veterinary
Medicine. Nong Lam University. Vietnam.

LI5T OF PUBLICATION5

Vo Thi Tra An. Nguyen Ngoc Tuan. Nguyen Nhu Pho. 2002. Antibiotic use and antibiotic
residues in chicken meat in Ho Chi Minh City. Journal of Jeterinarv Science and Techniques.
9. 53-57. (in Vietnamese).
Vo Thi Tra An. Nguyen Ngoc Tuan. Nguyen Nhu Pho. 2002. Survey on chicken production in
Ho Chi Minh City. Journal of Animal Husbandrv. 2. 11-13. (in Vietnamese).
Dinh Thien Thuan. Nguyen Ngoc Tuan. Vo Thi Tra An. Le Thanh Hien. Vo Ba Lam. Khuong
Thi Ninh. 2003. Initial survey on the use oI antibiotics on Iarms and their residues in pork and
chicken meat in Binh Duong Province. Journal of Jeterinarv Science and Techniques. 10. 50-
58. (in Vietnamese).
Vo Thi Tra An. 2005. Some molecular techniques Ior studying antimicrobial resistance in
Salmonella. Journal of Science and Techniques in Agriculture and Forestrv. 2: 249-255. (in
Vietnamese).
Vo Thi Tra An. Nguyen Ngoc Tuan. Le Huu Ngoc. 2006. Prevalence oI Salmonella in Ieces
and carcasses oI cattle. pigs and chickens in some provinces oI South Vietnam. Journal of
Jeterinarv Science and Techniques. 13: 37-42. (in Vietnamese).
An T.T. Vo. Engeline van Duiikeren. Ad C. Fluit. Max E.O.C. Heck. Anio Verbruggen. Henny
M.E. Maas. Wim Gaastra. 2006. Distribution oI Salmonella enterica serovars Irom humans.
livestock and meat in Vietnam and the dominance oI Salmonella Typhimurium phage type 90.
Jeterinarv Microbiologv.113:153-158.
A.T.T. Vo. E. van Duiikeren. A.C. Fluit. W. Wannet. A. Verbruggen. H.M.E. Maas. W.
Gaastra. 2006. Phenotypic and genotypic characterization oI antimicrobial resistance among
Dutch Salmonella isolates. Dutch Magazine for Medical Microbiologv. 14:S75.
E.J.A. Veldhuizen. H. Hendriks. S. Kallhove. A. Vo. W. Gaastra. P. Tooten. H.P. Haagsman.
2006. Salmonella Typhimurium causes upregulation oI porcine beta-deIencins in a porcine
intestinal cell line. Dutch Magazine for Medical Microbiologv. 14:S32.
An T.T. Vo. Engeline van Duiikeren. Ad C. Fluit. Wim Gaastra. 2006. High potential on
transIer oI antimicrobial resistance determinants among integron-carrying Salmonella
Typhimurium strains Irom horses in The Netherlands. European Jeterinarv Conference
Amsterdam. Abstract Vooriaarsdagen. 91.
An T.T. Vo. Engeline van Duiikeren.

Ad C. Fluit.

Max E.O.C. Heck. Anio Verbruggen. Henny
M.E. Maas.

Wim Gaastra. 2006. Antimicrobial resistance. class 1 integrons and Salmonella
Genomic Island 1 among Salmonella enterica serovars in Vietnam. In Proceedings of
International Svmposium Salmonella and Salmonellosis. Saint-Malo. France. 10-12 May 2006.
161-164.

An T.T. Vo. Engeline van Duiikeren. Ad C. Fluit

. Max E.O.C. Heck. Anio Verbruggen. Kim
van der Zwaluw.

Wim Gaastra. 2006. Class 1 integrons and the genetic diversity oI Salmonella
enterica serovar Dublin. In Proceedings of International Svmposium Salmonella and
Salmonellosis. Saint-Malo. France. 10-12 May 2006. 235-236.
An T.T. Vo. Engeline van Duiikeren. Ad C. Fluit. Max E.O.C. Heck. Anio Verbruggen . Kim
van der Zwaluw.

Wim Gaastra. 2006. Class 1 integrons in Dutch Salmonella enterica serovar
Dublin isolates Irom clinical cases oI bovine salmonellosis. Jeterinarv Microbiologv. 117:192-
200.
An T.T. Vo. Engeline van Duiikeren. Ad C. Fluit. Wim Gaastra. 2006. Antibiotic Resistance.
integrons and Genomic Island SGI1 among non-typhoid Salmonella serovars in The
Netherlands. International Journal of Antimicrobial Agents. 28:172-179.
An T.T. Vo. Engeline van Duiikeren. Ad C. Fluit. Wim Gaastra. 2006. Characterization oI
resistance genes associated with class 1 integrons in non-typhoid Salmonella. In Proceedings of
Workshop of Biotechnologv in Agriculture. Ho Chi Minh City. Vietnam.
An T.T. Vo. Engeline van Duiikeren. Ad C. Fluit. Henno G.C.J.M. Hendriks. Peter C.J. Tooten.
Wim Gaastra. 2007. Comparison oI the in vitro pathogenicity oI two Salmonella Typhimurium
phage types. Comparative Immunologv. Microbiologv & Infectious Diseases. 30:11-18.
An T.T. Vo. Engeline van Duiikeren. Ad C. Fluit. Wim Gaastra. 2007. A novel Salmonella
Genomic Island 1 and rare integron types in Salmonella Typhimurium isolates Irom horses in
The Netherlands. Journal of Antimicrobial Chemotherapv. 59:594-599.
An T.T. Vo. Engeline van Duiikeren. Ad C. Fluit. Wim Gaastra. 2007. Antimicrobial resistance.
class 1 integrons and a novel variant oI Genomic Island 1 in Salmonella isolates Irom Vietnam.
Antimicrobial Agents and Chemotherapv. On line 2 Apr 2007.
An T.T. Vo. Engeline van Duiikeren. Ad C. Fluit. Wim Gaastra. 2007. Characteristics oI
extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates
Irom horses. Jeterinarv Microbiologv. Accepted.

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Antibiotic resistance in

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