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Whole Genome Sequencing Analysis of Antimicrobial Resistant Escherichia Coli : from Food to Human

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Whole Genome Sequencing Analysis of Antimicrobial Resistant Escherichia Coli : from Food to Human This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Whole genome sequencing analysis of antimicrobial resistant Escherichia coli : from food to human Guo, Siyao 2020 Guo, S. (2020). Whole genome sequencing analysis of antimicrobial resistant Escherichia coli : from food to human. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/145480 https://doi.org/10.32657/10356/145480 This work is licensed under a Creative Commons Attribution‑NonCommercial 4.0 International License (CC BY‑NC 4.0). Downloaded on 04 Oct 2021 19:05:03 SGT WHOLE GENOME SEQUENCING ANALYSIS OF ANTIMICROBIAL RESISTANT Escherichia coli: FROM FOOD TO HUMAN GUO SIYAO SCHOOL OF CHEMICAL AND BIOMEDICAL ENGINEERING 2020 WHOLE GENOME SEQUENCING ANALYSIS OF ANTIMICROBIAL RESISTANT Escherichia coli: FROM FOOD TO HUMAN GUO SIYAO 202 222 00020 20022 2 Statement of Originality I hereby certify that the work embodied in this thesis is the result of original research, is free of plagiarised materials, and has not been submitted for a higher degree to any other University or Institution. 22 April 2020 . Date Guo Siyao 3 Supervisor Declaration Statement I have reviewed the content and presentation style of this thesis and declare it is free of plagiarism and of sufficient grammatical clarity to be examined. To the best of my knowledge, the research and writing are those of the candidate except as acknowledged in the Author Attribution Statement. I confirm that the investigations were conducted in accord with the ethics policies and integrity standards of Nanyang Technological University and that the research data are presented honestly and without prejudice. 3 April 2020 . Date Joergen Schlundt 4 Authorship Attribution Statement This thesis contains material from 2 papers published in the following peer- reviewed journals in which I am listed as an author. Chapter 3 is published as Guo, S., Tay, M.Y., Aung, K.T., Seow, K.L., Ng, L.C., Purbojati, R.W., Drautz- Moses, D.I., Schuster, S.C. and Schlundt, J., 2019. Phenotypic and genotypic characterization of antimicrobial resistant Escherichia coli isolated from ready- to-eat food in Singapore using disk diffusion, broth microdilution and whole genome sequencing methods. Food control, 99, pp.89-97. and Guo, S., Tay, M.Y., Thu, A.K., Seow, K.L.G., Zhong, Y., Ng, L.C. and Schlundt, J., 2019. Conjugative IncX1 Plasmid Harboring Colistin Resistance Gene mcr- 5.1 in Escherichia coli Isolated from Chicken Rice Retailed in Singapore. Antimicrobial agents and chemotherapy, 63(11). The summary of work in Chapter 1 included the abstract of the above papers The contributions of the co-authors are as follows: • Prof Joergen Schlundt provided the initial project direction and edited the manuscript drafts. • I prepared the manuscript drafts. The manuscript was revised by Dr Moon and Mr. Aung K.T. • I co-designed the study with Prof Joergen Schundt, Dr Moon and Mr. Aung K.T and performed most of the laboratory work at the School of Chemical and Biomedical Engineering and Environmental Health Institute. I also analyzed the data. • All DNA extraction, including sample preparation and quality control, was conducted by Kelyn and Zhong Yang. • Ng, L.C., Purbojati, R.W., Drautz-Moses, D.I., Schuster, S.C. provide suggestions to experiments design and data analysis, and they also revised the manuscript. 5 22 April 2020 . Date Guo Siyao 6 Acknowledgements First of all, I would like to express my sincere gratitude to my supervisor Prof. Joergen Schlundt for the continuous support of my Ph. D study and research. His patience, humor and activated attitude towards research and life inspired me a lot during my Ph. D career. He is always encouraging us to think critically, broaden our eyes and make a contribution to the society and world. He is always good at comforting me when I feel nervous or anxious. He is not only a supervisor for my Ph. D but also a spiritual advisor in my life. My sincere thanks also goes to Dr. Moon Tay, Mr. Aung Kyaw Thu, who provided guidance at the initial stage of my research. Their insightful comments and encouragement benefit my research from various perspectives. I also thank my fellow lab mates and my colleagues Mrs Zhong Yang, Kelyn Seow and others for the stimulating discussions and unconditional help during experiments. Also I thank my friends who accompanied me in the last four years. My thesis was finished in a special period --- the whole world is fighting against the pandemic COVID-19. I would like to express my special thankfulness to those people who are in the frontline of stopping the virus and saving lives. Last but not the least, I would like to thank my family: my parents and my sister for supporting me spiritually throughout writing this thesis and my life in general. 7 Table of Contents STATEMENT OF ORIGINALITY .....................................................................................3 SUPERVISOR DECLARATION STATEMENT ...................................................................4 AUTHORSHIP ATTRIBUTION STATEMENT ...................................................................5 ACKNOWLEDGEMENTS ...............................................................................................7 TABLE OF CONTENTS ..................................................................................................8 SUMMARY OF WORK ................................................................................................ 11 PUBLICATIONS .......................................................................................................... 17 LIST OF TABLES ......................................................................................................... 18 LIST OF FIGURES ....................................................................................................... 19 ABBREVIATIONS ....................................................................................................... 21 CHAPTER 1 INTRODUCTION ...................................................................................... 23 1.1 RESEARCH BACKGROUND ...................................................................................... 23 CHAPTER 2 LITERATURE REVIEW .............................................................................. 27 2.1 ANTIMICROBIAL RESISTANCE.................................................................................. 27 2.1.1 Antimicrobial agents ............................................................................... 27 2.1.2 The mechanism of antimicrobial resistance ............................................. 33 2.1.3 Global Antimicrobial Resistance Surveillance System ............................... 34 2.2 ESBL-PRODUCING BACTERIA ................................................................................. 40 2.2.1 Beta-lactam antimicrobials...................................................................... 40 2.2.2 Emergence of ESBL .................................................................................. 42 2.2.3 Prevalence of ESBL in food and food-producing animals (worldwide and in Singapore) ....................................................................................................... 44 2.3 COLISTIN AND COLISTIN RESISTANCE ........................................................................ 48 2.4 AMR IN FOOD CHAIN AND ITS TRANSMISSION TO HUMAN ............................................ 52 2.5 AMR GENES AND HORIZONTAL GENE TRANSFER ....................................................... 58 2.6 AMR CONTROL FROM ONE HEALTH PERSPECTIVE ...................................................... 61 2.7 ENTEROBACTERIACEAE AND E. COLI ........................................................................ 64 2.8 NEXT GENERATION SEQUENCING ............................................................................ 66 8 CHAPTER 3 PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF ANTIMICROBIAL RESISTANT E. COLI IN READY-TO-EAT FOOD IN SINGAPORE ..................................... 78 3.1 INTRODUCTION .................................................................................................. 78 3.2 MATERIAL AND METHODS .................................................................................... 80 3.2.1. Bacterial isolates .................................................................................... 80 3.2.2. Phenotypic antimicrobial resistance characterization ............................. 80 3.2.3. Extended-Spectrum Beta-Lactemase confirmatory testing ...................... 82 3.2.4. DNA extraction and whole genome sequencing ...................................... 83 3.2.5. Analysis of whole genome sequencing data ............................................ 84 3.2.6 Evaluation of WGS for AMR prediction .................................................... 85 3.2.7 Conjugation experiment and stability testing of plasmid .......................... 86 3.3 RESULTS ........................................................................................................... 86 3.3.1. Phenotypic characterization analysis of antimicrobial resistance ............ 86 3.3.2. Genotypic characterization analysis of antimicrobial resistance .............. 91 3.3.3. Comparison of phenotypic and genotypic AMR data ............................... 93 3.3.4. Location analysis of ESBL gene, and colistin and quinolone resistance gene .......................................................................................................................
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