CHARACTERIZATION AND MOLECULAR MECHANISMS OF ANTIMICROBIAL RESISTANCE IN ...

197 Pages·2004·2.53 MB·English

ABSTRACT
Title of Dissertation: CHARACTERIZATION AND MOLECULAR
MECHANISMS OF ANTIMICROBIAL RESISTANCE
IN FOODBORNE PATHOGENS
Sheng Chen, Doctor of Philosophy, 2004
Dissertation directed by: Associate Professor Jianghong Meng
Department of Nutrition and Food Science
The emergence of antimicrobial resistance bacteria in both the medical and
agricultural fields has become a serious problem worldwide. The use of antimicrobial
agents in animals forprophylaxis and growth promotion favors the selection of
antimicrobial resistance in bacteria. These resistant bacteria may subsequently
transmit to humans through food chain or human-animal interactions. The objectives
of this study were to characterize and determine the molecular mechanisms of
antimicrobial resistance in Salmonella and Escherichia coli. E. coli isolates recovered
from diseased piglets and chicken in China were characterized for the antimicrobial
resistancephenotype and the presenceof mutation in their gyrA, gyrB, parC and parE
genes. Results indicated that multiple-antimicrobial-resistant E. coli, including
fluoroquinolone-resistant variants, are commonly present among diseased swine and
chickens in China. DNA sequencing revealed that double gyrA mutations coupled
with parC mutation conferred high-level resistance to fluoroquinolones. In addition,
Salmonella isolates recovered from retail meats in the United States and China were
characterized for antimicrobial resistance genotypes and the horizontal transfer of
resistance determinants. The antimicrobial resistance genotypes of Salmonella were
consistent to their phenotypes. Genes conferring antimicrobial resistance in
Salmonella are often carried on integrons and plasmids, and could be transmitted
through conjugation. To rapidly screen for the antimicrobial-resistant and virulence
genes from bacteria, a DNA microarray was developed to analyze the antimicrobial
resistance and virulence genes from Salmonella and E. coli. Results indicated that
microarrray was an effective method to rapidly screen antimicrobial resistance and
virulence genes in Salmonella and E coli. Finally, molecular mechanisms of
fluoroquinolone resistance, including over-expression of efflux pumps and target gene
mutations, were characterized among laboratory-induced and field-acquired
fluoroquinolone-resistant Salmonella. The efflux pumps which were overexpressed in
resistant strains were deleted and mutated gyrA and parC genes were replaced to
determine the contribution of efflux pump and target gene mutations in
fluoroquinolone-resistant Salmonella. When Salmonella are exposed to
fluoroquinolone, certain efflux pumps are overexpressed in tandem with particular
mutations in topoisomerase genes (gyrA and parC). Based on deletion mutagenesis
studies, it appears that the most relevant genes with regards to the selection of
fluoroquinolone resistance phenotypes among Salmonella are the AcrAB-tolC efflux
pump and the gyrA portion of DNA gyrase gene.
CHARACTERIZATION AND MOLECULAR MECHANISMS
OF
ANTIMICROBIAL RESISTANCE IN FOODBORNE PATHOGENS
By
Sheng Chen
Dissertation submitted to the Faculty of Graduate School of the
University of Maryland, College Park in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
2004
Advisory Committee:
Professor Jianghong Meng, Chair/Advisor
Professor Thomas W. Castonguay
Professor George A. Bean
Dr. Shaohua Zhao
Dr. Patrick A. McDermott
© Copyright by
Sheng Chen
2004
To my father who left me before my finishing the Ph.D. degree, to my mother and to
my selfless wife and my lovely son
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ACKNOWLEDGEMENT
I would like to express my sincere appreciation to my advisor, Dr. Jianghong
Meng, for his support, guidance, and professional expertise that brought me to where I
am today. Dr. Jianghong Meng gave me a freedom to develop my own ideas, yet was
always willing to answer questions and make sure my project was going in the right
direction. I greatly appreciate his patience and understanding given me by my advisor
over the past four years. I also thank members of my dissertation committee, Dr. Shaohua
Zhao, Dr. Patrick A. McDermott, Dr. Thomas W. Castonguay Dr. George A. Bean, and
Dr. Bernadene Magnuson for helpful discussion and advice during the course of this
research.
I am grateful to the assistance I received from the always helpful staff, in
particular Brain Bonner, Vera Mccoy, Shirley G. Pressley, and Rose Santellano-Milem. I
also thank my fellow lab members, Beilei Ge, Lucy Zhao, Web Giruad, Shenghui Cui,
Yifan Zhang, Jie Zheng and Emily Yhe for their friendship and assistance with various
laboratory tasks, and particularly I want to thank former fellow Dr. Carl Schroeder for his
critical review of all my manuscripts and my thesis
I owe special thanks to my parents for their unfailing support, and constant
concern. I would particular like to remember my kind father, who has devoted his entire
life to me since the moment I was born. He has always been an inspiration for me and I
know he would be very proud to see this dissertation. I also thank my parents-in-law for
their constant support and encouragement.
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Finally, I would like to thank my wife, Shaoli Lin, for her enormous love,
unlimited support, patience, and self scarifice that made my career possible. Special
thanks to my son, Jeric Chen, for his endurance of the time I wasn’t able to spend with
him and for the sunshine personality he’s always shown me with.
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TABLE OF CONTENTS
List of Tables ………………………………………………………………....……….vii

List of Figures ……………………………………………………………..……..…..viii
List of Abbrevations …………………………………………………………………...ix
CHAPTER 1 Introduction...............................................................................................1
ORIGINS AND CONSEQUENCE OF ANTIMICROBIAL RESISTANCE IN FOOD BORNE PATHOGENS
.........................................................................................................................................1
Antimicrobial use in animal husbandry.......................................................................2
The association between the use of antimicrobials in food animals and the occurrence
of antimicrobial-resistant bacteria................................................................................3
Potential mechanisms of increased disease in humans from antimicrobial resistance
in food animals.............................................................................................................5
ANTIMICROBIAL RESISTANCE IN SALMONELLA AND COMMENSAL BACTERIA...................7
Antimicrobial resistance in Salmonella.......................................................................8
Antimicrobial resistance in commensal bacteria.......................................................10
MECHANISMS OF ANTIMICROBIAL ACTION AND RESISTANCE...........................................13
(cid:1)-Lactams...................................................................................................................13
Aminoglycosides........................................................................................................14
Phenicols....................................................................................................................15
Sulfonamides and Trimethoprim................................................................................15
Tetracycline................................................................................................................16
Quinolones/ Fluoroquinolones...................................................................................17
MECHANISMS OF FLUOROQUINOLONE RESISTANCE IN E. COLI AND SALMONELLA..........18
Quinolones and fluoroquinolones..............................................................................18
Interaction of fluoroquinolones with bacterial gyrase and topoisomerase IV...........20
Quinolone resistance due to altered gyrase and topoisomerase IV............................21
Fluoroquinolone resistance and efflux pumps...........................................................22
BRIEF OVERVIEW OF THIS DISSERTATION.........................................................................27
REFERENCE....................................................................................................................29
CHAPTER 2 Characterization of Multiple-Antimicrobial-ResistantE.coli Isolated
form Swine and Chicken in China................................................................................41
ABSTRACT......................................................................................................................41
INTRODUCTION...............................................................................................................42
MATERIALS AND METHODS............................................................................................44
RESULTS.........................................................................................................................48
DISCUSSION....................................................................................................................55
REFERENCE....................................................................................................................60
CHAPTER 3 Characterization of Multiple-Antimicrobial-Resistant Salmonella
Serovars Isolated from Retail Meats.............................................................................64
ABSTRACT......................................................................................................................64
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INTRODUCTION...............................................................................................................65
MATERIALS AND METHODS............................................................................................67
RESULTS.........................................................................................................................76
DISCUSSION....................................................................................................................81
REFERENCE....................................................................................................................86
CHAPTER 4 Identification of Virulence and Antimicrobial-Resistant Genes in
Salmonella and Escherichia coli Using Microarray Analysis......................................90
ABSTRACT......................................................................................................................90
INTRODUCTION...............................................................................................................91
MATERIALS AND METHODS.............................................................................................92
RESULTS AND DISCUSSION............................................................................................101
REFERENCE...................................................................................................................110
CHAPTER 5 The Role of Target Gene Mutation and Efflux Pump in
Fluoroquinolone-Resistant Salmonella........................................................................115
ABSTRACT.....................................................................................................................115
INTRODUCTION..............................................................................................................116
MATERIALS AND METHODS............................................................................................119
RESULTS.......................................................................................................................132
DISCUSSION..................................................................................................................145
REFERENCE..................................................................................................................152
CHAPTER 6 Summary................................................................................................157
CHAPTER 7 Reference...............................................................................................161
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LIST OF TABLES
Table 2-1. Antimicrobial resistance phenotypes of E. coli isolates from diseased
chickens and piglets........................................................................................45
Table 2-2. Primer sequences used to amplify class 1 integrons and the quinolone
resistance-determining regions (QRDR) of gyrA, gyrB, parC and parE......49
Table 2- 3. Serogroups and virulence genes of E. coli isolates from diseased chicken and
swine..............................................................................................................50
Table 2-4. Amino acid substitutions in DNA gyrase and topoisomerase IV and
corresponding fluoroquinolone resistance profiles among E. coli isolated from......52
Table 2-5. Integrons and their gene cassettes in swine and avian E. coli.....................54
Table 3-1. Antimicrobial resistance of Salmonella isolates from retail meats in the
United States and China.................................................................................69
Tble3-2. Sequences of PCR oligonucleotide primers used for the identification of
antimicrobial resistance genes in Salmonella isolates...................................71
Table 3-3. Antimicrobial resistance and resistance gene profiles...................................78
Table 3-4. Antimicrobial susceptibility profiles of donors, recipients, and
transconjugants in the conjugation experimentsa...........................................82
Table 4-1. Bacterial strains, serotypes, antimicrobial resistance profile and their gene
hybridizations to microarray..........................................................................93
Table 4-2. Microarray design, gene location, gene functions and the primer sequence of
antimicrobial-resistant and virulence genes...................................................97
Table 5-1. Known and predicted Salmonella efflux pumps and regulatory genes.......121
Table 5-4. Antimicrobial susceptibility of deletion and replacement mutants of
Salmonella S21............................................................................................139
Table 5-5. Antimicrobial susceptibility of deletion and replacement mutants of
Salmonella field strains................................................................................141
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