Access Type
Open Access Embargo
Date of Award
January 2023
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Immunology and Microbiology
First Advisor
Kevin R. Theis
Abstract
The advent of Next Generation Sequencing technologies prompted novel investigations of the microbiome. These revealed both increased diversity of known microbiomes such as those of the gut, oral cavity and vagina, but also bacterial DNA in tissues previously considered sterile such as the blood, liver, brain, and placenta. Thus, previous paradigms of sterility, based primarily on culture data, were called into question. However, interpretation of bacterial DNA signals from low-microbial biomass tissues is complex due to the strong potential for contamination and interference from the surrounding environment. Yet, many new methodologies are being developed to help isolate valid bacterial DNA signals, should they exist. The implications of these low-microbial biomass microbiomes, should they exist, would reshape our understanding of host biology, including reproduction and fetal development. In the meantime, the role of known microbiomes in human disease is also being explored. Specifically, preterm birth, which is the leading cause of neonatal morbidity and mortality worldwide, has been associated with variation in the structure of the vaginal microbiome, although the exact association remains ambiguous. Nevertheless, since ascension from the vagina is the main route for bacteria to gain access to the amniotic cavity and elicit an inflammatory response culminating in a premature labor, the vaginal microbiome is heavily implicated as a direct cause of preterm birth. Herein, we first evaluate the data from publicly available 16S rRNA gene sequencing surveys of the placenta. With a full re-analysis of the data enabling cross-study Amplicon Sequence Variant comparisons, we demonstrate that the bacterial DNA signal from placental samples strongly mirrors that of technical control samples, which capture background DNA contamination, from the same study. Furthermore, no well-defined bacterial patterns exist across all placental studies, except for clear mode of delivery associated contaminants such as Lactobacillus, a principal member of the vaginal microbiome. This re-analysis was followed by validation of a signal enhancement technique for isolating microbial DNA from human host DNA, thereby, in theory, improving the quality of a low-microbial biomass bacterial DNA signal. However, we found that placental tissue from term cesarean deliveries did not yield bacterial DNA signals regardless of signal enhancement and that exposure to placental tissue was lethal for Escherichia coli and Streptococcus agalactiae, two principal bacteria involved in intra-amniotic and placental invasion. Next, we evaluated the association between the vaginal microbiome and birth outcomes through longitudinal vaginal sampling of pregnant women who ultimately experienced term or preterm delivery. Through metagenomic sequencing, it was revealed that the vaginal microbiome was most distinct – demonstrated significantly increased alpha diversity – in cases of early preterm prelabor rupture of membranes and that these increases were present across gestation, even at first sampling. Furthermore, increases in alpha diversity were associated with a shift in microbial function and increased abundance of several notorious bacteria including Sneathia vaginalis, Sneathia sanguinegens, several Prevotella spp., Mobiluncus mulieris, and S. agalactiae. Finally, given that Sneathia spp. are emerging pathogens without well-defined culture protocols and few established cultured representatives, we attempted to improve Sneathia spp. viability during transport, colony detection, DNA detection, and growth in the laboratory. Through these efforts, we were able to validate an appropriate transport medium to maintain viability, highlight Sneathia spp. colonies with X-gluc, develop Sneathia spp. specific primers, and determine Sneathia spp. growth requirements. Collectively, these projects have helped to settle longstanding debates surrounding the existence of microbiomes of the upper female reproductive tract, clarify microbial roles of the vaginal microbiome in adverse pregnancy outcomes, and provide investigative foundations for the exploration of specific bacteria implicated in these adverse pregnancy outcomes, especially spontaneous preterm birth.
Recommended Citation
Panzer, Jonathan, "From Culture To Metagenomics: A Comprehensive Evaluation Of The Female Reproductive Tract Microbiome In Pregnancy" (2023). Wayne State University Dissertations. 3912.
https://digitalcommons.wayne.edu/oa_dissertations/3912