Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 2017 The Female Urogynecological Microbiome: Determining Niche Specificity Krystal Thomas-White Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Microbiology Commons Recommended Citation Thomas-White, Krystal, "The Female Urogynecological Microbiome: Determining Niche Specificity" (2017). Dissertations. 2860. https://ecommons.luc.edu/luc_diss/2860 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. Copyright © 2017 Krystal Thomas-White i LOYOLA UNIVERSITY CHICAGO THE FEMALE UROGYNECOLOGICAL MICROBIOME: DETERMINING NICHE SPECIFICTY A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY PROGRAM IN MICROBIOLOGY AND IMMUNOLOGY BY KRYSTAL THOMAS-WHITE CHICAGO, IL AUGUST, 2017 i Copyright by Krystal Thomas-White, 2017 All Rights Reserved ii ACKNOWLEDGMENTS I would like to thank the large team of people that made this thesis possible. I thank my mentor Alan Wolfe, who always gave me the space to explore my ideas and whose door was always open. I would like to thank each of my committee members. My committee Chair, Karen Visick, for making sure I was always on track and helped me through my prelims as well as my thesis work. Linda Brubaker, for being a second mentor to me and showing me how to lead with grace. To Catherine Putonti, for always being happy to advise and help with the bioinformatics. And to Adam Driks, who taught me that understanding complex issues usually means understanding the nuance of the issue. I thank my lab members, both past and present. This work would not have been possible without Meghan Pearce, who laid the ground work, and the teamwork of Evann Hilt and Travis Price. Thank you for always being there to work through problems, pick up the slack, and throughout it all, maintaining the highest standards. In addition, I would like to thank Roberto Limeira for continuing to manage these projects in my absence. Thank you to all the other members of the lab, (Bob Davis, Dave Christiansen, Bozena Zemaitaitis, Linda Hu, AJ Walker, Bruno Lima, Giuseppe Pistone, Danielle Johansen) who made my day to day experience enjoyable. I would like to thank the rest of the Loyola Education and Research Collaborative (LUEREC) who taught me how a talented team of interdisciplinary scientists work together. In iii particularly I would like to thank Susanne Taege, who developed this study with me; Mary Tulke, without whom not a single sample would be processed; and Gina Kuffel, who did all the sequencing and was always there to troubleshoot a problem. In addition, I would like to thank the other urogynecology professors and fellows (Beth Mueller, Cynthia Brincat, Colleen Fitzgerald, Tanaka Dune, and Megan Brady) for their willingness develop projects and to recruit patients. I extend my thanks to Trevor Lawley and the members of his lab at the Wellcome Trust Genome Campus for all their help with genomic sequencing. It was a pleasure to work with Sam Forster and Nitin Kumar, and learn their methods for phylogenetic analysis. Finally, I would like to thank my Family for all their love and support. This could not have been completed without the unfailing encouragement from my parents, Kimber and Matt Hoey, and my husband, Patrick Thomas. I would particularly like to thank Patrick not only for all the understanding, patience, and encouragement, but also for all his technical expertise. iv I dedicate this work to my husband, Patrick. Your encouragement, love, knowledge, and support made this work possible. v TABLE OF CONTENTS ACKNOWLEDGMENTS iii LIST OF TABLES xii LIST OF FIGURES xvi LIST OF ABBREVIATIONS xxii ABSTRACT xxvi CHAPTER I: INTRODUCTION AND LITERATURE REVIEW 1 Historical Perspectives 1 The Human Microbiome Project (HMP) 4 Sequencing Technologies 4 Visualizing the results of the HMP 8 The Bladder is Not Sterile 10 The Female Urinary Microbiota in Relation to Health and Disease 13 Similarities between the FUM and Vaginal Microbiome, and Relationship to Estrogen 15 Difficulties in Studying Niche Interconnectivity 18 Using CRISPR Arrays to Assess Recent Phage Exposure History 23 Summary 24 CHAPTER II: MATERIALS AND METHODS 27 Clinical Study Design (The Estrogen Study) 27 Study Design Overview 27 Patient Recruitment and Clinical Assessment 28 Recruitment 28 Visit 1 (Baseline) 28 Visit 2 (12-week) 29 Inclusion Criteria 30 Exclusion Criteria 30 Clinical Sample Collection 31 Power Calculation and Sample Size 31 Laboratory Assessments 33 vi Sample Processing and Storage 33 Standard Urine Culture (SUC) and Enhanced Urinary Quantitative Culture (EQUC) Procedures 33 Enriched Culture Samples 35 16S rRNA Sequencing 36 Statistical Analysis 36 Microbial Whole Genome Sequence and Analysis 37 Whole Genome Sequencing Library Prep 37 Whole Genome Assembly and Annotation 38 Phylogenetic Analysis 39 Synteny 41 Identifying Genomic Signatures (CDS, Domain, and CRISPR analysis) 41 CDS Analysis 42 Domain Analysis 42 CRISPR Array Analysis 43 CHAPTER III: A GLOBAL COMPARISON OF BLADDER GENOMES TO GUT AND VAGINAL GENOMES 44 Overview of Study Design 44 Phylogenetic Diversity of Cultivated Bladder Isolates 44 Comparison of Functional Markers from the Bladder, Vaginal, and Gut Microbiomes 45 Summary 49 CHAPTER IV: BLADDER AND VAGINAL COMMUNITY STRUCTURE SHIFTS FOLLOWING ESTROGEN TREAMENT 51 Overview 51 Diversity Decreases in the Bladder and Vagina Following Treatment 52 The Biomass Does Not Change in Response to Estrogen 56 The Frequency of Lactobacillus-dominant Profiles Increases by 12-weeks 57 The Bladder and Vaginal Microbiota Profiles are More Similar by 12-weeks 60 Summary 63 CHAPTER V: NICHE SPECIFICITY BETWEEN THE VAGINAL AND BLADDER MICROBIOMES WITHIN AN INDIVIDUAL 65 Overview 65 Bladder and Vaginal Species Accounting within Individuals 65 vii Genetic Analysis of Species Sets 69 Corynebacterium amycolatum 72 Escherichia coli 73 Lactobacillus iners 78 Lactobacillus crispatus 82 Aerococcus urinae 85 Gardnerella vaginalis 88 Streptococcus anginsosus 90 Corynebacterium coyleae 93 A Comparison of Multiple Strains Within an Individual 97 Summary 101 CHAPTER VI: DISCUSSION 102 Summary of data 102 Implications and Discussion 105 Exploring the Implications of Interconnectivity 107 Implications for Infection and Probiotics 112 Future Directions 113 Reference Genome Database 114 Enriched Culture Samples 114 Interpreting Voided Urine 117 Long-term Estrogen Effects 117 APPENDIX A: INTRODUCTION TO THE APPENDECES: ADDITIONAL RESEARCH 119 APPENDIX B: THE FEMALE URINARY MICROBIOME IN URINARY URGENCY INCONTINENCE 121 Overview of Appendix B 122 Abstract 123 Introduction 124 Materials and Methods 125 Results 129 Discussion 138 APPENDIX C: THE FEMALE URINARY MICROBIOME: A COMPARISON OF WOMEN WITH AND WITHOUT URGENCY URINARY INCONTINENCE 144 Overview of Appendix C 145 viii Abstract 145 Introduction 147 Results 149 Discussion 162 Materials and Methods 167 Supplemental Material 175 APPENDIX D: INCONTINENCE MEDICATION RESPONSE RELATES TO THE FEMALE URINARY MICORBIOTA 178 Overview of Appendix D 179 Abstract 180 Introduction 181 Methods 182 Results 188 Discussion 199 Supplemental Material 204 Addendum 209 APPENDIX E: EVALUATION OF THE URINARY MICROBIOME OF WOMEN WITH UNCOMPLICATED STRESS URINARY INCONTINENCE 216 Overview of Appendix E 217 Abstract 218 Introduction 219 Materials and Methods 220 Results 224 Discussion 233 Conclusion 236 Addendum 237 APPENDIX F: THE INTERACTION BETWEEN ENTEROBACTERIACEAE AND CALCIUM OXYLATE DEPOSITS 240 Overview of Appendix F 241 Abstract 241 Introduction 242 Materials and Methods 243 Results 249 Discussion 258 ix Conclusion 263 Supplemental Data 266 Addendum 268 APPENDIX G: GENOMES OF GARDNERELLA STRAINS UNCOVER AN ABUNDANCE OF PROPHAGES WITHIN THE BLADDER MICROBIOME 273 Overview of Appendix G 274 Abstract 274 Background 275 Materials and Methods 277 Results and Discussion 283 Conclusions 295 Supplemental Data 297 APPENDIX H: THE CLINICAL URINE CULTURE: ENHANCED TECHNIQUES IMPROVE DETECTION OF CLINICALLY RELEVANT MICROORGANISMS 302 Overview of Appendix H 303 Abstract 303 Introduction 304 Materials and Methods 305 Results 310 Discussion 319 Supplementary Data 323 APPENDIX I: MICROORGANISMS IDENTIFIED IN THE MATERNAL BLADDER: DISCOVERY OF THE MATERNAL URINARY MICROBIOME 328 Overview of Appendix I 329 Abstract 329 Introduction 330 Methods and Materials 332 Results 335 Discussion 342 Supplementary Material 347 Addendum 349 APPENDIX J: PRESENT BUT UNACCOUNTED FOR: THE UROPATHOGENS IN PRE-SURGICAL URINARY MICROBIOMES 355 x Overview of Appendix J 356 Abstract 356 Introduction 358 Methods 359 Results 363 Discussion 371 APPENDIX K: A NEW SPECIES OF GARDNERELLA, G. URINALIS 375 Overview of Appendix K 376 Results 377 Discussion 379 APPENDIX L: MOTILITY OF URINARY ISOLATES 381 Overview of Appendix L 382 APPENDIX M: R-SCRIPTS 374 APPENDIX N: LISTS OF ISOLATES USED IN THE NICHE COMPARISON STUDIES 388 REFERENCE LIST 404 VITA 437 xi LIST OF TABLES Table 1. Comparison between standard urine culture (SUC) and the expanded quantitative urine culture (EQUC) culture methods 12 Table 2. Culture Conditions for All Estrogen Study Samples 35 Table 3. A List of Isolates that are 100% Identical Between the Bladder and Vagina by DAPC 48 Table 4. Calculating