TEMPORAL DYNAMICS OF ESCHERICHIA COLI AND THE MICROBIOME by Jonathan Nathan Vernon Martinson A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology MONTANA STATE UNIVERSITY Bozeman, Montana April 2020 ©COPYRIGHT by Jonathan Nathan Vernon Martinson 2020 All Rights Reserved ii DEDICATION I dedicate this work to my partner (Kristin) and to my family: my dad (Goodwin), my mom (Joanne), and my brothers Philip and Vince for their constant support and encouragement. iii ACKNOWLEDGEMENTS Seth Walk is a tremendous scientist and mentor. The environment he created in the laboratory allowed me to grow as a scientist and as a human. I thank Seth for allowing me to develop and drive a new project within the laboratory and providing me with guidance along the way. I’m always surprised by Seth’s depth of knowledge and his ability to reframe problems as opportunities. The members of the Walk lab have made research very enjoyable. Thank you to Susan Broadaway for providing encouragement and a meticulously organized laboratory. I thank Mike Coryell, Brittany Jenkins, Qian Wang, Mark McAlpine, Ben Deuling, Josh Matts, Jinxing Li, and Genevieve Coe, for the conversation and comradery. Thank you to the host of undergraduates that assisted me in data collection – especially, Garrett Peters. Thank you to Nick Pinkham for assisting me with bioinformatics and invaluable coffee breaks. Next, I would like to thank my dissertation committee: Michael Franklin, Blake Wiedenheft, and James Wilking for providing feedback and advice throughout my PhD. Much thanks to the Kopriva and Murdock foundation for providing me with resources to pursue my research. I would like to thank my friends and fellow graduate students within the Microbiology and Immunology department (you know who you are). Finally, I would like to thank the participants that provided their time (and stool) to my project. This work would not be possible without their generosity. iv TABLE OF CONTENTS 1. ESCHERICHIA COLI RESIDENCY IN THE GUT OF HEALTHY HUMAN ADULTS ..............................................................................................................................1 Abstract ............................................................................................................................3 Introduction ......................................................................................................................4 Temporal E. coli Dynamics in the Pre-PCR Era ..............................................................6 Temporal E. coli Dynamics in the PCR Era ..................................................................13 Theoretical Considerations and Mechanisms for Clonal Turnover ...............................21 Nutrient Competition .............................................................................................22 Competition for Physical Space .............................................................................25 Direct Damage and/or Killing of Competitors.......................................................29 Bacteriocins................................................................................................30 Phages ........................................................................................................30 Response (or the Lack of) to Cellular Stress .........................................................35 Conclusions and Future Directions ...............................................................................36 Needed Areas of Research .....................................................................................37 References Cited ............................................................................................................39 2. RETHINKING GUT MICROBIOME RESIDENCY AND THE ENTEROBACTERIACEAE IN HEALTHY HUMAN ADULTS ......................................49 Abstract .........................................................................................................................52 Introduction ...................................................................................................................52 Methods .........................................................................................................................53 Sample Collection and Processing .........................................................................53 Sampling and Characterization of Resident Clones...............................................53 16S rRNA Sequencing and Analysis .....................................................................53 Results ...........................................................................................................................54 Study Participants and Stool Samples....................................................................54 16S rRNA Sequencing Reveals Similar Microbiomes Within Participants Over Time ......................................................................................54 OUT/ASV Residency Varies Significantly ...........................................................55 Culture-Based Evaluation of Enterobacteriaceae in the Healthy Human Adult Gut ..............................................................................................58 Enterobacteriaceae Residency ..............................................................................58 16S rRNA Sequencing Is Not Diagnostic for Enterobacteriaceae or Escherichia/Shigella .....................................................................................58 Discussion .....................................................................................................................60 References Cited ............................................................................................................62 Supplemental Figures and Tables ..................................................................................64 v TABLE OF CONTENTS CONTINUED 3. PHENOTYPIC PREDICTORS OF ESCHERICHIA COLI RESIDENCY IN THE GUT OF HEALTHY HUMAN ADULTS ..........................................................81 Abstract .........................................................................................................................83 Introduction ...................................................................................................................84 Materials and Methods ..................................................................................................86 Direct Competition Assay ......................................................................................87 Cellulose Production ..............................................................................................89 Catalase Activity ....................................................................................................89 Antibiotic Resistance .............................................................................................90 Results ...........................................................................................................................90 Cellulose Production is Not Significantly Different Among Residents and Transients ...................................................................................90 Antibiotic Resistance Among Residents and Transients Does Not Differ ..........................................................................................................91 Direct Competition in the Gut Sometimes Predicts Turnover Events ...................92 Direct Competition Predicts Residency Time........................................................92 Engraftment of Probiotics and Pathogen Colonization Resistance ........................94 Discussion .....................................................................................................................94 References Cited ............................................................................................................97 Figures and Tables .......................................................................................................100 Legends ................................................................................................................100 Figures..................................................................................................................101 Tables ...................................................................................................................106 4. CONCLUSIONS AND FUTURE STUDIES ..............................................................107 Conclusions .................................................................................................................107 Future Directions .........................................................................................................109 References Cited ..........................................................................................................110 CUMULATIVE REFERENCES CITED ........................................................................111 APPENDIX ......................................................................................................................126 vi LIST OF TABLES Table Page 2.1 Summary of samples, sampling days, number of OTUs and residence time according to operationally defined estimates of time ..................55 S2.1 Summary of non-E. coli Enterobacteriaceae characterization and identification by API 20E biochemical profiling ..........................................78