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Toxin E. Coli Serogroups on Fresh Produce Development of a Phage Cocktail to Control the Top 7 Shiga -toxin E. coli Serogroups on Fresh Produce by Yiran Ding A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirement of the degree of MASTER OF SCIENCE Department of Food and Nutritional Sciences University of Manitoba Winnipeg Copyright © 2018 by Yiran Ding I I hereby declare that I am the sole author of this thesis. I authorize the University of Manitoba to lend this thesis to other institutions or individuals for the purpose of scholarly research. Yiran Ding I further authorize the University of Manitoba to reproduce this thesis by photocopying or by other means in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. Yiran Ding II ACKNOWLEDGMENTS First of all, I want to thank my parents, Luosi Ding and Xu Li, for their encouragements and supports in every way I can think about throughout my life. I would never have been able to achieve anything without their love and care. I also would like to thank my grandparents for believing in me and encouraging me to go through the difficult times. I owe my sincerest gratitude to my relatives back home, for staying in touch and warming my heart in the coldest winter. I am extremely thankful to my Advisor, Dr. Narvaez, for her generous, knowledge, patience, encouragement, and inspiration for me to explore deeper into the topics. She provided me many chances to learn and triggered my passion for food and microbiology. I want to thank her for answering my silly questions all the time and reminding me there is always room for improvement. Thank you, Dr. Narvaez, for holding the light and showing the right direction on my way to food science. I would also like to thank my co-advisor, Dr. McAllister, my committee members, Dr. Holley, Dr. Nadon, and Dr. Niu for their suggestions and willingness to help me. I also want to thank Dr. Rodas of Health Canada and the Lethbridge Research Center respectively for their training and assistance throughout the project. I want to express my appreciation to Xuan Zhang, for being not just a colleague but also my friend who helped me both inside and outside the laboratory. My way to the goal was more interesting with Xuan walking by my side. And I want to say thank you to Matthew Wells, for his kindness and patient when he helped me to improve my writing skill. I would also like to thank many of the Food Science staff; Carola, Jennifer, Pat, Denise, and Yang for your kind assistance in many ways. III Last but not least, this project was made possible by funding from Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, ON, Canada. IV DEDICATION To my parents, Luosi and Li V Organization of the thesis This thesis is divided into five chapters and includes one manuscript, which is located in chapter three. The first chapter gives a brief introduction about using bacteriophages as a biocontrol method against 7 STEC serogroups. General objectives are summarized at the end of the chapter. The second chapter is the literature review including the general background information of shiga-toxigenic E. coli including: history, categories, symptoms, virulence factors, transmission, epidemiology, and major outbreaks caused by STEC. The current pre- and postharvest chemical and physical control methods in food, and fresh produce is also covered. Phage molecular information and current applications are also included in this chapter. The third chapter is the methods, results, and discussion of the experimental part of this thesis including the phage lytic activity assay, spot-inoculation tests on lettuce and sprouts, and the scale-up validation study. Chapter 4 is the appendix study of where a STEC phage cocktail was combined with the commercial phage cocktail SalmofreshTM and tested for its effectiveness against STEC. In addition, the monitoring of emergence of bacterial resistance against phage was also reported in this chapter. Chapter 5 states the overall conclusion arising from the thesis. VI Table of Contents Acknowledgements ...................................................................................................................... III Dedication ..................................................................................................................................... V Organization of thesis ................................................................................................................. VI List of Tables .............................................................................................................................. XI List of Figures ........................................................................................................................... XIII Abstract ................................................................................................................................... XVI Chapter 1 1. General Introduction .................................................................................................................. 1 1.1. Organic produce and food safety. ........................................................................................... 2 1.2. Objectives ............................................................................................................................... 4 Chapter 2 2. Literature Review ....................................................................................................................... 5 2.1. E. coli History ......................................................................................................................... 5 2.2. Classification ........................................................................................................................... 6 2.2.1. Shiga-toxigenic E. coli (STEC) ........................................................................................... 8 2.2.2. STEC O157: H7 reservoirs and resistance to environmental hurdles ................................. 9 2.2.3. STEC Non-O157: H7 reservoirs and resistance to environmental hurdles ...................... 10 2.3. STEC detection methods ....................................................................................................... 11 2.4. STEC virulence factors ......................................................................................................... 12 2.5. Symptoms of STEC infection ............................................................................................... 15 2.6. Transmission of STEC infections ............................................................................... 16 2.6.1. Contaminated food and drinks ........................................................................................... 16 2.6.2. Person-to-person transmission ........................................................................................... 18 2.6.3. Animal Contact .................................................................................................................. 18 2.7. Epidemiology of STEC in human infection .......................................................................... 19 2.7.1. Outbreaks related to produce commodities ........................................................................ 23 2.8. Current antimicrobial interventions on fresh produce to reduce STEC ................................ 26 VII 2.8.1. Government regulations on food safety ............................................................................. 26 2.8.2. Chemical interventions ...................................................................................................... 27 2.8.3. Non-chemical disinfection methods .................................................................................. 32 2.9. Phages ................................................................................................................................... 35 2.9.1. Phage taxonomic classification .......................................................................................... 35 2.9.2. Lytic and lysogenic pathway ............................................................................................. 38 2.9.2.1. Lytic pathway .................................................................................................................. 38 2.9.2.2. Lysogenic pathway ......................................................................................................... 39 2.9.3. Advantages using phage technology as a biocontrol method ........................................... 39 2.9.4. Limitations ......................................................................................................................... 40 2.9.5. Lysis from without ............................................................................................................. 41 2.9.6. Bacterial resistance and phage evasion tactics ................................................................... 42 2.9.7. Previous phage interventions in food ................................................................................. 46 2.9.8. Conclusion ......................................................................................................................... 50 Chapter 3 3. In vitro and in vivo phage cocktail effectiveness against the top 7 STEC. .............................. 51 3.1. Introduction ..........................................................................................................................
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