PREVALENCE OF SHIGA-TOXIN PRODUCING ESCHERICHIA COLI IN ALBERTA BEEF CATTLE AND CHARACTERISTICS OF NON-PATHOGENIC MICROCIN-PRODUCING ESCHERICHIA COLI CHAMPION BATTLING BACTERIAL VIRULENCE SARAH-JO PAQUETTE Master of Science, University of Lethbridge, 2011 A thesis submitted in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY in BIOMOLECULAR SCIENCE Department of Biological Sciences University of Lethbridge LETHBRIDGE, ALBERTA, CANADA © Sarah-Jo Paquette, 2020 PREVALENCE OF SHIGA-TOXIN PRODUCING ESCHERICHIA COLI IN ALBERTA BEEF CATTLE AND CHARACTERISTICS OF NON-PATHOGENIC MICROCIN- PRODUCING ESCHERICHIA COLI CHAMPION BATTLING BACTERIAL VIRULENCE SARAH-JO PAQUETTE Date of Defence: February 26, 2020 Dr. James Thomas Professor Emeritus Ph.D. Dr. Tim Reuter Research Scientist Ph.D. Thesis Co-Supervisors Dr. Kim Stanford Research Scientist Ph.D. Thesis Examination Committee Member Dr. Victor Gannon Research Scientist Ph.D. Thesis Examination Committee Member Dr. Igor Kovalchuk Professor Ph.D. Internal External Examiner Department of Biological Sciences Faculty of Arts and Science Dr. Rebekah DeVinney Associate Professor Ph.D. External Examiner University of Calgary Calgary, Alberta Dr. Brent Selinger Professor Ph.D. Chair, Thesis Examination Committee ABSTRACT Zoonotic pathogens, like Shiga toxin-producing Escherichia coli (STEC) are a food safety and health risk. Among STEC, O157 is considered a major pathogen; however, globally the number of outbreaks with new emerging STEC strains is rising. Here, quantitative data on STEC O157 were gathered for western-Canada beef cattle and compared to emerging STEC. Quantitative data revealed; O178 is 4-times more numerous than O157 and high Shiga-toxin events are often not attributed to O157. Results suggest current surveillance likely misses emerging STEC due to restricted O-serogroup screening. Secondly, an alternate STEC mitigation strategy was investigated as an alternative approach to use of antibiotics which is controversial in human and animal therapeutics. Competition experiments identified a strong E. coli O103F which incapacitated STEC growth by producing a diffusible antimicrobial, most likely a microcin. The antimicrobial revealed tremendous potential for use along the farm-to-fork continuum or in human intervention to mitigate STEC. iii CONTRIBUTIONS OF AUTHORS The work from this thesis resulted in three manuscripts that have been published (Chapter III, IV and V) and one manuscript currently in preparation for publication (Appendix IV). Publication list: Chapter III - Paquette S-J, Stanford K, Thomas J, Reuter T: Quantitative surveillance of shiga toxins 1 and 2, Escherichia coli O178 and O157 in feces of western- Canadian slaughter cattle enumerated by droplet digital PCR with a focus on seasonality and slaughterhouse location. Plos One. 2018; 13(4):e0195880 The experimental design, laboratory work and first draft of the manuscript were performed by myself. I consulted with my supervisors (Dr. James Thomas and Dr. Tim Reuter), and committee member (Dr. Kim Stanford) during the course of the project to ensure the thoroughness of the experimental design or if I encountered any problems requiring troubleshooting. Dr. Stanford provided statistical analysis guidance and reviewed the manuscript. Dr. Thomas provided guidance with experimental design and reviewed the manuscript. Dr. Reuter provided guidance with experimental design, troubleshooting and manuscript revisions. The majority of the work on this manuscript was performed by myself with guidance from my co-authors. Chapter IV - Paquette S-J, Zaheer R, Stanford K, Thomas J, Reuter T: Competition among Escherichia coli strains for space and resources. Veterinary sciences. 2018; 5(4):93 Similarly to the first manuscript, experimental design, laboratory work and manuscript writing were performed by myself with support from my co-authors. I consulted with my supervisors (Dr. Thomas and Dr. Reuter), co-authors (Dr. Zaheer) and committee member (Dr. Stanford) on experimental design, troubleshooting and iv manuscript revisions. Experimental design was constructed mainly by myself with input from my co-authors to ensure the thoroughness of the experiments. The laboratory work was performed primarily by myself with the help of my laboratory colleagues when needed. I drafted the first version of the manuscript and my co-authors provided revisions which I incorporated into the manuscript. Chapter V - Paquette S-J, Reuter T: Properties of an antimicrobial molecule produced by an Escherichia coli champion. Antibiotics. 2019; 9(1):6 The experimental design, laboratory work and manuscript writing were performed by myself. I consulted with my supervisor Dr. Reuter for troubleshooting and manuscript revisions. Appendix IV - Escherichia coli: using physiological cues to turn on antimicrobial molecule production. v ACKNOWLEDGEMENTS Firstly, I would like to thank my co-supervisors, Dr. James Thomas, University of Lethbridge and Dr. Tim Reuter, Alberta Agriculture and Forestry, for your help, guidance and invaluable input during the course of my PhD project. I couldn’t have done this without your mentorship and insight. I will take the lessons and advice learned along the way, remember and apply them going forward in my career. Everything you have done was and is still greatly appreciated. Thank you to my committee members, Dr. Kim Stanford and Dr. Victor Gannon for taking this journey with me, always willing to answer my questions and imparting your valuable knowledge. Your thoughts, comments and insights from the start of the project, the comprehensive and along the way, have kept me on my toes and expanded my critical thinking. Thank you to Shaun Cook, Dr. Rahat Zaheer, Dr. Yongxiang Zhang, Dr. Kingsley Amoako, Dr. Chad Laing, and Dr. Noriko Goji for your invaluable ideas and advice along the way from reviewing manuscripts, ensuring my experimental design was scientifically sound and for being available for my multitude of questions and I know it was a lot of questions. Please know that the expertise you have all imparted will not be forgotten and has contributed to my skills and knowledge, which I will use as I go forward in my scientific career. Similarly, I have to mention the “lab girls”, Susanne Trapp, Yidong Graham and Cheyenne Sargeant for welcoming me into your lab group, providing an extra set of hands when needed, but most importantly trying to comprehend my excited rumblings vi or odd mutterings while I was working. You were irreplaceable sounding boards, asking questions and offering advice, even if my thought process was not always clear and I talked a mile a minute. Thank you to everyone in the Biological Sciences department at the University of Lethbridge, in particular Katrina Mendez, Laurie Pacarynuk and Helena Danyk for all their help training and advice which allowed me to teach the Biology 1010 laboratories to the best of my abilities. Thank you to Dr. Roy Golsteyn for all your help with the comprehensive exam and for answering all my questions along the way. Likewise, I need to mention, Dr. Brent Selinger, for all your help and guidance with the comprehensive exam and for most importantly starting me on my scientific journey with the independent studies during my undergrad and encouraging my quest for knowledge. The biggest thank you to my friends and family for all their support and allowing me to explain what I was doing and nodding along even though I’m sure it must have seemed like another language. Lastly, I have to thank my parents for everything that they have done; allowing me to pursue all my dreams and never letting me give up when the going got tough. vii TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iii CONTRIBUTIONS OF AUTHORS ................................................................................. iv ACKNOWLEDGEMENTS ............................................................................................... vi TABLE OF CONTENTS ................................................................................................. viii LIST OF TABLES ............................................................................................................ xii LIST OF FIGURES ......................................................................................................... xiii LIST OF ABBREVIATIONS ............................................................................................xv CHAPTER I .........................................................................................................................1 1.1 Background: Human Microbiome .....................................................................1 1.2 Escherichia coli .................................................................................................2 1.3 E. coli: Non-pathogen vs Pathogen ...................................................................3 1.4 STEC ..................................................................................................................5 1.5 Reservoir and Transmission of STEC................................................................6 1.6 STEC Serogroups and Emerging STEC O178 ..................................................9 1.7 STEC Virulence Factors ..................................................................................11 1.7.1 Shiga Toxin Genes ............................................................................11