An exploration of ecological concepts in the context of antimicrobial resistance and the use of phytochemical compounds within the ruminant gut microbiome by Natalie Cécile Knox A thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science University of Manitoba Copyright © 2013 by Natalie Cécile Knox ii ACKNOWLEDGMENTS I would like to express my sincere gratitude to the late Dr. Denis Krause for serving as my advisor and Dr. Rick Holley for serving as my co-advisor. To Dr. Krause, I will always be indebted to you for being a great mentor and teacher. You will be greatly missed. In addition, I would like to thank Dr. Holley for your kind advice and guidance throughout my program. Special thank you to Dr. Kim Ominski for your kind support and advice as you have, in my mind, acted as another co-advisor throughout my degree. Special thanks to my committee members Drs. Karin Wittenberg, George Zhanel, and Tim McAllister for their time and insightful contributions along the way. I would also like to thank Dr. Gary Crow for his sound statistical advice throughout my research. And thank you to the Canadian Dairy Commission for the financial support throughout my graduate degree. The animal trial would not have been possible without the help of Terri Garner, Janice Haines, Chad Malfait, Darcy Catellier and staff at the Glenlea Research Centre, many thanks to all of you. And of course a huge thank you to Jenelle Hamblin and Kristen Bouchard-Teasdale for their friendships and patience while teaching me what agriculture was all about. A special thank you also goes out to Ehsan Khafipoor, Sanjiv Bhandari, Amit Setia, Juan Hernandez, Stephanie Cheng, and Roman Kotlowski for teaching me all the fundamentals of molecular biology and statistical methods. To Victoria Tkachuk and Ainsley Little, thank you for always being there to help me with lab work but most of all, thank you for your friendship! Many thanks to all the graduate students, faculty, and staff of the Department of Animal Science for your support and friendships along the way, you’ve made the last 5 years memorable. Finally, I would like to express my utmost gratitude to my mom and dad for their endless iii support and encouragement. No words can express how grateful I am for everything you have done for me. Thank you to my brothers, Jude and Serge for always being there for me whenever I needed them. To my new family, Fran, Ted, Angie, Dustin, Pam, and Marc, thank you for all your patience and support throughout what seemed to you as a never ending degree. And lastly, a special thank you to my husband Greg for his love and encouragement throughout this journey! iv DEDICATION I dedicate this thesis to my parents. You are the reason I’ve made it this far! Thank you. v ABSTRACT Secondary plant metabolites have recently been gaining interest in livestock production systems following the ban of in-feed antibiotics within the European Union. The rise in antimicrobial resistance found in pathogenic and non-pathogenic bacteria has lead to increased interest in the research community regarding the use of phythochemicals as an alternative to antibiotics. The purpose of this research was to evaluate the impact of including phytochemicals in a livestock production system. Specifically, a high tannin-containing forage, sainfoin (Onobrychis viciifolia), was evaluated in vitro for its antimicrobial effect on Escherichia coli. We determined that phytochemicals alone are not as inhibitory as synthetic antibiotics. Thus, the use of combination therapy to deter the development of antimicrobial resistance was evaluated. A myriad of plant compounds were screened for their synergistic interactions with ciprofloxacin. Geraniol, an essential oil, was identified to possess good antimicrobial activity and synergistic interactions with ciprofloxacin. Therefore the effect of long term exposure to both ciprofloxacin and geraniol were examined. Results demonstrated that once an antimicrobial concentration threshold was reached, resistance to ciprofloxacin increased markedly in the presence of both geraniol and ciprofloxacin. Finally, an in vivo trial was conducted in which forty steers were fed sainfoin or alfalfa over a 9-week period to evaluate its ability to reduce E. coli shedding and its impact on gut microbiota in the context of popular theoretical ecology concepts. Results from the in vivo study indicate that sainfoin was able to promote a slight decrease in generic E. coli shedding which could be maintained throughout the trial. Using high-throughput sequencing, the effect of sainfoin on the microbial ecosystem of the ruminant gut was evaluated. Sainfoin induced a significant shift in the microbial community structure of the rumen and to a lesser vi extent in the hindgut. Using ecology theories, a hypothesis was formulated regarding the mechanisms that mediate the development of tolerance and the fundamental ecological processes controlling microbial population shifts. Understanding how the gut ecosystem functions and predicting its behaviour in the presence of various fluctuating environmental conditions will enable more efficient manipulation of the rumen and promote best management practices in livestock production. vii FOREWARD This thesis was prepared following a manuscript format. The thesis is comprised of five manuscripts each corresponding to a chapter. Manuscript I and II have been published in the Canadian Journal of Plant Science and the Journal of Applied and Environmental Microbiology, respectively. Manuscript III, IV, and V will be submitted to the Journal of Applied and Environmental Microbiology. All manuscripts are formatted in accordance with the guidelines of the Canadian Journal of Plant Science. viii TABLE OF CONTENTS ACKNOWLEDGEMENTS..............................................................................................ii DEDICATION...................................................................................................................iv ABSTRACT........................................................................................................................v FOREWARD....................................................................................................................vii TABLE OF CONTENTS................................................................................................viii LIST OF TABLES............................................................................................................xii LIST OF FIGURES.........................................................................................................xiv LIST OF COPYRIGHTED MATERIAL FOR WHICH PERMISSION WAS OBTAINED.....................................................................................................................xvii LIST OF ABBREVIATIONS.........................................................................................xix CHAPTER ONE: GENERAL INTRODUCTION..........................................................1 CHAPTER TWO: LITERATURE REVIEW.................................................................3 2.1 Microbial ecosystem of the ruminant gut............................................................3 2.2 How to study microbial ecosystems?..................................................................7 2.3 Next-generation sequencing technology.............................................................8 2.4 Introduction to antimicrobial resistance..............................................................9 2.5 Microbial mechanisms of antimicrobial resistance.............................................9 2.6 Agriculture and antimicrobial resistance...........................................................12 2.7 Combination therapy and methodologies to assess antimicrobial synergism, additivity, and antagonism..................................................................................................16 2.8 Phytochemicals..................................................................................................26 ix 2.9 Tannins..............................................................................................................33 2.10 Ecology theories................................................................................................54 2.11 Research hypothesis and objectives...............................................................60 CHAPTER THREE: MANUSCRIPT I.........................................................................61 Condensed tannin concentrations found in vegetative and mature forage legumes grown in western Canada….....................................................................................................61 3.1 Abstract..................................................................................................................62 3.2 Introduction............................................................................................................63 3.3 Material and methods.............................................................................................64 3.4 Results and discussion............................................................................................68 CHAPTER BRIDGE TO CHAPTER FOUR................................................................80 CHAPTER FOUR: MANUSCRIPT II...........................................................................81 Potential to reduce Escherichia