Genetic structure and antimicrobial resistance of foodborne Escherichia coli in Australia

235 Pages·2017·2.96 MB·English

by  Belinda Vangchhia

Genetic structure and antimicrobial
resistance of foodborne Escherichia
coli in Australia

Belinda Lallawmkimi Vangchhia

A thesis submitted for the Degree of Doctor of Philosophy

Evolution and Ecology
Research School of Biology
The Australian National University
July, 2017
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DECLARATION
I declare that the work contained in this thesis titled “Genetic structure and antibiotic
resistance of foodborne Escherichia coli in Australia” is the outcome of my original
research. To the best of my knowledge, this thesis contains results that are solely from
this study except where due references have been provided. None of the work has been
submitted for obtaining a degree to any other University.

Belinda Lallawmkimi Vangchhia
July, 2017

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ACKNOWLEDGEMENTS
My sincerest gratitude goes to my Supervisor and guru, David Gordon for his excellent
supervision and patience. I could not have possibly asked for better guidance and
mentorship! Right from basic lab skills to data analysis and even debugging
Bioinformatics programs, you have taught me a great big deal. For all of these and much
more, I am truly grateful.
A massive thanks to the professionals from whom I have learned and gained so much,
Michaela Blyton, Claire O’ Brien, and my Supervisory panel members, Dr Peter Collignon
and Dr Karina Kennedy. A big gratitude to Michaela for her massive help with the
statistical analyses. My labmates, Sam Burn, Angel, Heli, Buddhie, Ojas, Judith, Alex,
Delia, the friendship, research tips and lab skills I learned from you all is innumerable.
Thank you!
My beautiful family, especially Dad, Mum and Jonathan, for their endless prayers and
mental support. Nancy, Mapuia, Maama, Domy, you all are my constant motivators! So
much gratitude to my precious nieces Mamteii and Bawihteii, for always being my ray
of sunshine and happy pills with their enthusiasm and unceasing positive energy.
Ray, our endless Skype calls without paying heed to our time difference kept me sane
for 3 years, for that I am indebted. My sisters in Christ, Gisse and Ila, thank you for your
inspirational faith and prayers. Juanito, your patience and encouragement got me
through my final months in Canberra, muchas gracias. And not to forget my Graduate
House and ANU Union family, Maria, Jyotsna, Bo, Savitri, Yasmin, Sarita, Mayank, Henri,
Kimlong, Samunda, Deth, Evelyn, Kumar, Jenny who never gave me the chance to feel
homesick. The ever vibrant conversations, tears and laughter shared were priceless and
made this journey so worthwhile! You all are my blessings.
Above all, praise to the living God who guided me throughout this venture, I know for
certain that I would not be where I am today if not for His mercy and grace. I dedicate
this work for His glory alone.
“I can do all thing through Christ who strengthens me”.

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ABSTRACT
Poultry meat is thought to harbour the most “human-like” E. coli strains, capable of
causing both intestinal and extra-intestinal infections in humans. Furthermore, poultry
meat is also known to harbour multi-drug resistant E. coli strains. In this study, extensive
sampling of retail poultry meat products sold in major supermarkets and independent
butchers in Canberra was undertaken. Phenotyping and genotyping of the E. coli isolated
from the meat samples was then performed to better understand the evolution and
diversity of these strains and their significance to human health. Using a whole genome
sequence based approach, the strains were then compared to clinical and commensal
human E. coli isolates to determine if there is transmission between the two hosts.
A highly diverse population of E. coli in poultry meat was identified in this study.
Although the exact contamination route is hard to determine, our findings suggest that
poultry meat is frequently contaminated, not only with E. coli isolates from poultry birds
but also significantly from other sources, possibly during processing chains and
transport. In addition, the genetic structure comparison of human and poultry meat E.
coli strains indicate that there is cross-transmission between the two hosts, but at low
levels. Therefore, poultry meat serves as a low risk transmission route for zoonotic E.
coli strains that can cause extra-intestinal infections. Our findings also suggest that the
risk of exposure to intestinal or diarrheal pathogenic E. coli strains through poultry meat
consumption and handling is even lower than exposure to extra-intestinal pathogenic E.
coli (ExPEC) strains.
Furthermore, poultry meat in Canberra region does not serve as a highly significant
carrier of antimicrobial resistant E. coli unlike several other countries. Resistance was
commonly observed to antibiotic classes approved for use in poultry industries (like
ampicillin and tetracycline), but was low for critically important antibiotics to humans
which are banned in the industry (like fluoroquinolone and 3rd generation
cephalosporins). In addition, no carbapenem and colistin resistance was observed,
which has recently been identified in poultry meat products in many countries including
China, United Kingdom, Denmark. Poultry meat in Canberra is therefore more
frequently contaminated with E. coli strains from the birds rather than from other
contamination sources.
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TABLE OF CONTENTS

Chapter 1: General Introduction
1.1 Escherichia coli……………………………………………………………….…………………….………………11
1.2 Genetic diversity and evolution of E. coli………………………………….……………………………12
1.3 Ecological structure of E. coli……………………………………………………..…………………………13
1.4 Classification of E. coli…………………………………………………………………………………………..14
1.4.1 Phylogenetic groups (Phylogroups)…………………………………..……..….…………15
1.4.2 Serotypes or serogroups……………...…….…………..………………….………………….16
1.4.3 Multi-locus Sequence Typing (MLST)………………………………………..….…………17
1.4.4 Other molecular characterization techniques of E. coli….……………..………...17
1.4.5 Whole genome sequencing………………….…………………..….………………………..18
1.5 Poultry farming: From farm to consumption…………………………………………………………19
1.5.1 The poultry industry………………………………………….……………………………………20
1.5.2 Poultry meat and E. coli………………………………………………….…………….………..21
1.5.3 Antimicrobial use in poultry industry, Australia…………….……………………….22
1.5.4 Antimicrobial resistance…………………………….…………………………………………..24
1.6 Extra-intestinal pathogenic E. coli (ExPEC) and avian pathogenic E. coli (APEC)…..….27
1.7 Poultry meat as reservoir of ExPEC and APEC…………………………………………………….…..30
1.8 Intestinal pathogenic E. coli (IPEC) and poultry meat……………………………………………..31
1.9a Research Aims…………………………………………………………………………………………………….33
1.9b References………………………………………………………………………………………………………….34

Chapter 2: Factors associated with the presence, genetic diversity, and antimicrobial
sensitivity of Escherichia coli in poultry samples collected from Canberra,
Australia
2.1 Abstract………………………………………………………………………………………………………….……57
2.2 Introduction………………………………………………………………………………………………..……….58
2.3 Materials and Methods……………………………………………………………………………….……….59
2.3.1 Sampling………………………………………………………………………………………….…….59
2.3.2 Isolation of E. coli…………………………………………………………………………………..59
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2.3.3 Molecular characterization…………………………………………………………………..61
2.3.4 Antimicrobial susceptibility testing……………………………………………………….62
2.3.5 Statistical analyses………………………………………………………………………………..62
2.3.5.1 Presence of E. coli and number of REP-types in a sample……….62
2.3.5.2 Distribution of phylogenetic groups (phylogroups)…………………63
2.3.5.3 Antimicrobial resistance…………………………………………………………63
2.4 Results…………………………………………………………………………………………………………………64
2.4.1 Presence of E. coli…………………………………………………………………………………64
2.4.2 Number of REP-types……………………………………………………………………………66
2.4.3 Phylogenetic group (phylogroup) distribution………………………………………68
2.4.4 Antimicrobial resistance…………………………………………………………………….…72
2.5 Discussion…………….…………………………………………………………………………………………….78
2.6 References………………………………………………………………………………………………………….83
2.7 Supplemental Materials……………………………………………………………………………………..87

Chapter 3: Genetic structure of Escherichia coli in poultry meat from Canberra,
Australia
3.1 Abstract……………………………………………………………………………………………………………100
3.2 Introduction…………………………………………………………………………………………………..…101
3.3 Materials and Methods…………………………………………………………………………………….103
3.3.1 Strains………………………………………………………………………………………………..103
3.3.2 Whole genome sequencing………………………………………………………………..103
3.3.3 In silico typing…………………………………………………………………………………….103
3.3.4 Phylogenetic tree………..……………………………………………………………………..104
3.4 Results………………………………………………………………………………………………………………104
3.4.1 MLST…………………………………………………………………………………………………..104
3.4.2 Serotype diversity……………………………………………………………………………...108
3.4.3 Virulence factors (VFs) distribution…………………………………………………….109
3.4.4 Resistance profile……………………………………………………………………………….112
3.4.5 Incompatibility (Inc) groups distribution…………………………………………….116
3.5 Discussion…………………………………………………………………………………………………………117
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3.6 References……………………………………………………………………………………………………….125
3.7 Supplemental Materials……………………………………………………………………………………133

Chapter 4: Comparison of Escherichia coli in poultry meat and humans with focus on
ST117, ST95, ST131 and ST69
4.1 Abstract……………………………………………………………………………………………………………154
4.2 Introduction……………………………………………………………………………………………………..154
4.3 Materials and Methods…………………………………………………………………………………….157
4.3.1 Sampling…………………………………………………………………………………………….157
4.3.2 Isolation of E. coli……………………………………………………………………………….157
4.3.3 Strain characterization……………………………………………………………………….158
4.3.4 Whole genome sequencing………………………………………………………………..159
4.3.5 In silico MLST and Serotyping……………………………………………………………..159
4.3.6 Phylogenetic tree and variable gene content inference……………………..159
4.3.7 Plasmid content………………………………………………………………………………….160
4.4 Results………………………………………………………………………………………………………………161
4.4.1 ST117…………………………………………………………………………………………………161
4.4.2 ST95…………………………………………………………………………………………………..165
4.4.3 ST131…………………………………………………………………………………………………168
4.4.4 ST69…………………………………………………………………………………………………..170
4.5 Discussion…………………………………………………………………………………………………………172
4.6 Conclusion………………………………………………………………………………………………………..175
4.7 References………………………………………………………………………………………………………..176
4.8 Supplemental Materials…………………………………………………………………………………...183

Chapter 5: General Discussion
5.1 Overview of E. coli in poultry meat…………………………………………………………………..189
5.2 Risks associated with poultry meat E. coli……….……………………………………………....191
5.3 Commercial poultry versus backyard poultry……………………………………………………196
5.4 Conclusion……………………………………………………………………………………………………….198
5.5 Future directions……………………………………………………………………………………………..199
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5.6 References………………………………………………………………………………………………………200

Appendix: Publication
Phylogenetic Diversity, Antimicrobial Susceptibility and Virulence Characteristics of
Phylogroup F Escherichia coli in Australia……………………………………………………….….…209

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Chapter 1

General Introduction

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