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Evaluating Historical Paradigms of Sterility in Perinatal Microbiology and Ramifications Orf Pregnancy Outcomes

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Evaluating Historical Paradigms of Sterility in Perinatal Microbiology and Ramifications Orf Pregnancy Outcomes Wayne State University Wayne State University Dissertations January 2020 Evaluating Historical Paradigms Of Sterility In Perinatal Microbiology And Ramifications orF Pregnancy Outcomes Jonathan Greenberg Wayne State University Follow this and additional works at: https://digitalcommons.wayne.edu/oa_dissertations Part of the Microbiology Commons, and the Obstetrics and Gynecology Commons Recommended Citation Greenberg, Jonathan, "Evaluating Historical Paradigms Of Sterility In Perinatal Microbiology And Ramifications orF Pregnancy Outcomes" (2020). Wayne State University Dissertations. 2468. https://digitalcommons.wayne.edu/oa_dissertations/2468 This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. EVALUATING HISTORICAL PARADIGMS OF STERILITY IN PERINATAL MICROBIOLOGY AND RAMIFICATIONS FOR PREGNANCY OUTCOMES by JONATHAN MURRAY GREENBERG DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2020 MAJOR: BIOCHEMISTRY, MICROBIOLOGY, & IMMUNOLOGY Approved by: Advisor Date DEDICATION This dissertation is dedicated to my parents and sister, who have all been immeasurably supportive over my graduate career and more importantly my entire life. I could not have done this without you. ii ACKNOWLEDGMENTS There is no way for me to express fully my sincere gratitude to all the individuals who have had an impact on my life and throughout my time here at Wayne State. None of it would have been possible without Dr. Kevin Theis. Rotating in his lab was one of the best decisions of my life and quite literally got me to where I am. His unwavering support throughout my time as a student and a scientist has meant the world to me, I am so grateful, and his guidance as a scientist and a professional has been fundamental to my development as a researcher. I would also like to thank the members of my committee: Dr. Withey, Dr. Pellett, Dr. Chen, and Dr. Luca who have supported me throughout my research, been patient, and given feedback that has undoubtedly contributed to my success. From the Theis Lab, I must thank Dr. Andrew Winters, who has taught me many laboratory skills and helped sustain me by sharing many home-cooked meals. I also need to thank Madison Ahmad for always being willing to get coffee with me, supplying candy, and being a constant in the lab that I could always count on. I would also like to thank Emma Graffice, a new member of the lab, but one who quickly became invaluable and a supportive friend. I would like to thank Amruth Atyam, and the other undergraduate researchers who have spent time in the lab and allowed me to take on a mentorship role. And of course, I must thank the Perinatal Research Branch for allowing me to be a part of their work. From the BMI department, I am grateful for the enormous support us students receive from April Wolak, Mary Dismuke, Joseph Fiore, and Lynette Ray. Mel Clay, you have contributed to all of our success by aiding us in the routine, but essential tasks that are required to do good science. Thank you to all the departmental faculty members that have played a role in making our experiences as students better through the various committees, and especially Drs. Holland and Thipparthi who were directly involved in my recruitment to Wayne State. I have to thank my friends, new and old, and especially those I have met since moving to Detroit, I could not have been this successful without your support. And to Paul Breen, who has been a great friend and helped keep me accountable during our mutual tenure. And finally, thank you to my family, my parents Jeff and Liz Greenberg. Your love and encouragement have allowed me to come this far, and I cannot begin to fathom the sacrifices you have made in order to support me. iii TABLE OF CONTENTS DEDICATION............................................................................................................................... ii ACKNOWLEDGMENTS ........................................................................................................... iii LIST OF TABLES ....................................................................................................................... ix LIST OF FIGURES ..................................................................................................................... xi CHAPTER 1: INTRODUCTION ................................................................................................ 1 Microbial communities, their diversity, and advancements in their characterization ............ 1 The human microbiome in the context of disease and low microbial biomass tissues ............ 4 Host-microbe interactions and consequences of premature shifts in sterility paradigms ....... 9 CHAPTER 2: DOES THE HUMAN PLACENTA DELIVERED AT TERM HAVE A MICROBIOTA? ......................................................................................................................... 14 I. Abstract .................................................................................................................................. 14 II. Introduction .......................................................................................................................... 15 III. Methods and Materials ........................................................................................................ 21 DNA extraction from placental tissues .................................................................................. 21 Metagenomic sequencing of extracted DNA from placental tissue and background technical control samples ...................................................................................................................... 23 Metagenomic sequence data processing using MG-RAST .................................................... 24 Reanalysis of published placental data after reclassification into ASVs ........................ 25 ASV sequence data processing using DADA2 ....................................................................... 25 Subsampling of 16S rRNA gene sequence data for ASV vs OTU comparisons ..................... 26 iv Decontam program for the removal of likely background DNA contaminants ..................... 26 Rationale for use of isNotContaminant method and thresholds ............................................ 26 Filtering of contaminant ASVs from the primary nested dataset using decontam ................ 29 Filtering of contaminant ASVs from the secondary touchdown dataset using decontam ..... 30 Statistical analyses ................................................................................................................. 31 Figure generation .................................................................................................................. 32 IV. Results ................................................................................................................................. 32 Metagenomic surveys of the bacterial profiles of placental tissues ...................................... 32 Bacterial profiles of placental tissues and technical controls from the primary nested PCR dataset characterized using OTU and ASV approaches ........................................................ 39 Bacterial profiles of placental tissues from the primary nested PCR dataset after removing any ASVs that were identified in controls .............................................................................. 39 Bacterial profiles of placental tissues from the primary nested PCR dataset after filtering out likely background DNA contaminants identified through the program decontam .......... 43 Bacterial profiles of placental tissues and technical controls from the secondary touchdown PCR dataset characterized using OTU and ASV approaches ............................................... 43 Bacterial profiles of placental tissues from the secondary touchdown PCR dataset after removing any ASVs identified in controls .............................................................................. 49 Bacterial profiles of placental tissues from the secondary touchdown PCR dataset after filtering out likely background DNA contaminants identified through the program decontam ............................................................................................................................................... 51 V. Discussion ............................................................................................................................ 55 Metagenomic surveys of a potential placental microbiota .................................................... 56 Classifying 16S rRNA gene sequence data as operational taxonomic units (OTUs) versus as amplicon sequence variants (ASVs) ....................................................................................... 58 CHAPTER 3: DOES THE MOUSE PLACENTA HAVE A MICROBIOTA? CULTURE AND MOLECULAR SURVEYS OF THE MURINE MICROBIOTA ................................. 62 v I. Abstract .................................................................................................................................. 62 II. Introduction .......................................................................................................................... 63 III. Methods and Materials .......................................................................................................
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