Access Type

Open Access Dissertation

Date of Award

January 2020

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Immunology and Microbiology

First Advisor

Kevin R. Theis

Abstract

Next-generation sequencing technologies, especially 16S rRNA gene and metagenomic sequencing have allowed investigations of low microbial biomass tissues of the human body. While these sequencing methodologies have provided large amounts of reliable data for higher microbial biomass sites, such as the mouth, intestine, and vagina, tissues of low microbial biomass sites are subject to specific caveats that were not appropriately considered in early investigations of these sites. Low microbial biomass sites of particular interest have included those of the reproductive and urinary systems. Utilization of DNA sequencing methodologies have allowed researchers to challenge existing paradigms of sterility around these sites that were historically considered sterile, including but not limited to the placenta, the endometrium, and the bladder. While a thorough and complete understanding of the microbial signals in urogenital compartments is necessary for the best patient care and treatment, premature conclusions that redefine historical paradigms can have harmful consequences on patient health, especially for pregnant women with whom microorganisms have been associated with multiple adverse pregnancy outcomes.

In this dissertation, I present a lack of evidence for a placental microbiota in humans using multiple modes of microbiological inquiry. Through culture, quantitative real-time PCR (qPCR), 16S rRNA gene sequencing, and metagenomics we found no evidence of bacterial signals beyond those also present in background technical controls. This work with human subjects was subsequently complemented by work in mice, in which we investigated the bacterial signals in the murine placenta and fetus, as well as multiple murine tissue control sites; we again found no consistent evidence of a placental microbiota or in utero colonization through multiple microbiological methodologies. Conversely, investigations of the urine of pregnant women revealed evidence of a low abundance bladder microbiota. We found bacterial signals that clearly exceeded those of technical controls, suggesting that a shift in sterility paradigm for the upper urinary tract may be warranted. Specifically, through bacterial culture, qPCR, and 16S rRNA gene sequencing we found evidence of a bladder microbiota in pregnant women that showed strong variation among individuals and consisted of Ureaplasma urealyticum and Gram-positive anaerobic cocci. A more thorough understanding of the bladder microbiota in pregnant women across gestation will allow healthcare professionals to address urinary and bladder symptoms in a way that alleviates or prevents pregnancy complications.

This body of work provides strategies for the thorough investigation of low microbial biomass sites and demonstrates the high degree of evidence necessary to overturn classic paradigms of sterility in perinatal medicine and host biology in general.

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