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Access Type

WSU Access

Date of Award

January 2025

Degree Type

Thesis

Degree Name

M.S.

Department

Immunology and Microbiology

First Advisor

Kevin R. Theis

Abstract

Much like every other part of the human body exposed to the environment, the vagina hosts its own ecological community of microbial species, collectively referred to as the microbiome. A dysbiotic vaginal microbiome is characterized by increased microbial diversity and decreased lactobacilli, the most common form being bacterial vaginosis (BV). Defining the pathogenesis of BV, including cause of recurrence and what roles the microbiota play, is important in preventing the complications of recurrent BV. Sneathia species, are Gram-negative, rod-shaped, anaerobic, non-motile bacteria. There are currently two known species of Sneathia, named S. vaginalis and S. sanguinegens, both of which are prominent members of CST IV of the vaginal microbiome, which is prevalent in women with complications such as bacterial vaginosis. Sneathia is perceived as an indicator of a suboptimal vaginal microbiome, given its associations with BV, STIs, spontaneous preterm birth (sPTB), and other severe pregnancy complications. Yet, the mechanisms by which Sneathia spp. are involved in these complications remain unknown, in part due to how little is known about its virulence and pathogenicity given its fastidious nature and thus our inability to use it in experimental models. Elucidating Sneathia’s metabolism and growth requirements is essential for not only success in its isolation, but also for furthering understanding of its pathogenicity.

The widespread majority of existing Sneathia spp. isolates have been obtained from the blood of patients with sepsis or amniotic fluid of patients with intra-amniotic infection. The lack of Sneathia isolation from the vaginal cavity is likely due to its fastidious nature, paired with its tendency to be dominated by faster growing vaginal bacteria in mixed culture. Pure bacterial cultures obtained from vaginal fluid are necessary for the genomic study of Sneathia’s virulence, pathogenicity, and antibiotic susceptibility in order to facilitate the understanding and treatment of associated gynecological and obstetric diseases such as BV and sPTB. The use of the transport solution, X-gluc dye, MUG fluorescence, and the uxuA primer allowed for the isolation of Sneathia species from the vaginal fluid of women with BV. However, there were some samples that were shown to have a high abundance of Sneathia, but isolation was not successful. This includes a higher incidence of S. vaginalis isolation than S. sanguinegens isolation, alluding to a benefit in refining these techniques to expand on the isolation of both species, especially S. sanguinegens.

It has been shown that Sneathia sanguinegens requires whole blood for optimal growth. Uncovering exactly how S. sanguinegens utilizes whole blood and what aspects of it are required is consequential for cultivation efficiency, homogeneity, and further understanding of its pathogenicity. Overall, Hb alone has yielded the highest growth rate of S. sanguinegens, leading to the new hypothesis that it is a critical part of S. sanguiengens growth and metabolism. Given that supplements added to Hb only hindered S. sanguinegens growth, it is likely that whole blood hosts many inhibitory molecules that are not present in Hb, resulting in a lower growth rate. This new hypothesis must be tested further for a deeper understanding of S. sanguinegens metabolism and why Hb is required for growth.

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