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Characterizing the Human Gut Microbiome Through Culture

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Characterizing the Human Gut Microbiome Through Culture CHARACTERIZING THE HUMAN GUT MICROBIOME THROUGH CULTURE CHARACTERIZING THE DIVERSITY AND COMPLEXITY OF THE HUMAN GUT MICROBIOME THROUGH THE COMBINATION OF CULTURE AND CULTURE-INDEPENDENT METHODS By JENNIFER T. LAU, B.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy McMaster University © Copyright by Jennifer T. Lau Ph.D. Thesis – J. Lau; McMaster University – Biochemistry and Biomedical Sciences McMaster University DOCTOR OF PHILOSOPHY (2017) Hamilton, Ontario (Biochemistry and Biomedical Sciences) TITLE: Characterizing the diversity and complexity of the human gut microbiome through the combination of culture and culture-independent methods AUTHOR: Jennifer T. Lau, B.Sc (McMaster University) SUPERVISOR: Dr. Michael G. Surette NUMBER OF PAGES: xv, 210 ii Ph.D. Thesis – J. Lau; McMaster University – Biochemistry and Biomedical Sciences Lay Abstract Bacteria that inhabit the human intestine are important for health, and are involved in several diseases; therefore, it is critical to determine the roles of specific bacteria. I describe a method that results in the growth and recovery of most bacteria in stool, which allows them to be studied in detail. The differences, both in behaviour and in DNA sequences, found within two different bacterial groups were characterized, and extensive variability was observed between closely related bacteria. I studied which bacteria and their functions might be important in Irritable Bowel Syndrome (IBS) by using our method for growing stool bacteria combined with sequencing of all DNA in the stool, but could not find strong support for specific bacteria causing IBS symptoms. This work shows how the ability to grow and isolate bacteria, combined with studying their DNA, allows for better understanding of their functions in the human intestine. iii Ph.D. Thesis – J. Lau; McMaster University – Biochemistry and Biomedical Sciences Abstract The human gut microbiome is the collection of all organisms and their genetic content that inhabit the gastrointestinal tract. An overwhelming number of studies have associated the gut microbiota with health and disease, but with little consensus on which specific bacterial groups are important for causing or maintaining either state. A majority of microbiome studies only identify associations between the gut microbiome and health status, and determining causation requires the isolation and growth of bacterial isolates for further experiments. The goal of this thesis is to demonstrate that the combination of culture-based and culture-independent methods describes greater complexity and diversity in the human gut microbiota than observed by either approach alone. In the first study, a method of culture-enriched molecular profiling could capture the majority of bacterial groups found in fecal samples. Additionally, when compared to culture- independent 16S rRNA gene sequencing, culture detected more bacterial taxa. This method was applied to the targeted culture of the commensal Lachnospiraceae family. The second study explored the diversity in the isolated Lachnospiraceae strains, and compared the genetic diversity of the strains to reference genomes, revealing functional and genetic heterogeneity within the bacterial family. The third study characterized the intra-species phenotypic and genetic diversity in Escherichia coli. E. coli diversity was extensive between individuals, but also within-individuals, in both the phenotypes and genetic profiles. Lastly, a method of culture-enriched metagenomics was applied to a murine IBS microbiota transfer model to identify bacterial members of the microbiota and their functional pathways that may be responsible for the development of gastrointestinal iv Ph.D. Thesis – J. Lau; McMaster University – Biochemistry and Biomedical Sciences and behavioural IBS phenotypes, although no bacterial groups could be conclusively associated with symptoms. Together, the work described demonstrates that culture and culture-independent methods are complementary, and provides more resolution into the structure and diversity of the human gut microbiome than either approach in isolation. v Ph.D. Thesis – J. Lau; McMaster University – Biochemistry and Biomedical Sciences ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Mike Surette, for the opportunity to work in your lab for the past six years. You have created the greatest environment to explore my scientific curiosities, to learn and grow as a scientist, and you have put together a group of the most fantastic people that I have had the pleasure to work alongside – many of whom will remain life-long friends. I could not have asked for a more supportive mentor; thank you for your endless encouragement, the excitement and passion you bring to science, your intelligence that I am in constant awe of, for calming me down when I feel anything but, and most of all, for making me a better scientist. Thank you to my committee members, Dr. Premysl Bercik and Dr. Brian Coombes, for challenging me during committee meetings and giving me a new perspective to look at things, for your interest in my projects and for supporting my career developments. I would also like to thank Lisa Kush, who has always gone above and beyond whenever I needed help. The experience of grad school would not have been the same without the members of the Surette lab, both current and former, that I have had the pleasure to work with and learn from. Thank you to all for your help and support over the years, for the lunchtime company, for everything you guys have taught me, and for sharing your really amazing science with me. I am so happy to be able to consider many of you my friends. Thank you to Dr. Fiona Whelan, for without you, this PhD would not be what it was. Thanks for always being there when I’m feeling overwhelmed, for sharing the latest lab news, for being someone to bounce ideas off, and for being the voice of encouragement vi Ph.D. Thesis – J. Lau; McMaster University – Biochemistry and Biomedical Sciences whenever it was needed. You’ll always be my favourite person to finish a crossword with. Also thank you to the many people that I have collaborated with over the past several years, who have provided me with the chance to broaden my horizons; afterall, collaborations are what drive science. Thank you to my parents, and Justin, for giving me the opportunity to pursue my love of science, even if you didn’t always understand it. I would not have been able to do it without your support. Thanks to Sophie for your daily texts, which are always entertaining, and a reminder that there’s more to life outside the grad school bubble. Thank you to my friends, both back home and at McMaster (and now also all over!), for being there through all these years, and for making the time fly by. I’m so proud of how far we’ve come, and so inspired by all of you. And of course, thank you to Ryan Buensuceso. I wouldn’t have finished this PhD without you, your support, and your encouragement. Thanks for believing that I can do anything; I sure am glad I sat behind you in safety training that first week. vii Ph.D. Thesis – J. Lau; McMaster University – Biochemistry and Biomedical Sciences TABLE OF CONTENTS LAY ABSTRACT ............................................................................................................ iii ABSTRACT ...................................................................................................................... iv ACKNOWLEDGEMENTS ............................................................................................ vi TABLE OF CONTENTS .............................................................................................. viii LIST OF FIGURES ......................................................................................................... xi LIST OF TABLES ......................................................................................................... xiii LIST OF ABBREVIATIONS ....................................................................................... xiv CHAPTER ONE – INTRODUCTION ............................................................................ 1 The human microbiome ........................................................................................... 2 The human gut microbiome .................................................................................... 3 Functions of the gut microbiome ............................................................................. 4 The gut microbiota in disease .................................................................................. 9 The gut microbiota in gastrointestinal diseases and disorders ............................ 9 Inflammatory Bowel Disease ................................................................................. 9 Irritable Bowel Syndrome .................................................................................... 11 Colorectal cancer ................................................................................................. 13 The gut microbiota in metabolic disorders .......................................................... 14 Obesity ................................................................................................................ 14 Type 2 diabetes ................................................................................................... 15 Type 1 diabetes ..................................................................................................
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