The Molecular Microbiology of Bacterial Vaginosis

Fred Hutchinson Cancer Research Center, Seattle WA

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Abstract

DESCRIPTION (provided by applicant): Bacterial vaginosis (BV) is a condition that affects millions of women and is linked to several serious health conditions, including preterm labor, cervical intraepithelial neoplasia, and HIV infection. About half of women with BV complain of a malodorous vaginal discharge, and half are asymptomatic. The cause of BV is not known, though current evidence suggests that women with BV undergo a change in the bacterial flora of the vagina. No single cultivated bacterium has been definitively determined to cause BV. Advanced methods in molecular biology have recently been used to study environmental and human ecosystems, allowing investigators to detect and identify microbes without cultivation. These studies reveal many novel, cultivation-resistant bacteria, and expand our understanding of the microbial diversity in these niches. We propose to apply the same molecular methods to the microbial ecosystem of the human vagina. The Specific Aims are to: 1. Create a census of the bacteria that inhabit the normal vagina. Vaginal fluid samples from 4 women without BV will be obtained and subjected to broad range PCR to directly amplify bacterial 16S rDNA without cultivation. The PCR products will be cloned into E. coli, and the clones screened by performing PCR on the inserts. Inserts of the correct size will be analyzed with PCR RFLP analysis, and unique inserts will be sequenced. Phylogenetic analysis of these 16S rDNA sequences will allow us to identify bacteria, or to infer evolutionary relationships for novel bacteria; 2. Create a census of the bacteria that inhabit the vagina of women with BV. Vaginal fluid samples from 4 women with BV will be subjected to the analysis outlined in aim 1; 3. Identify bacteria or bacterial communities that may be the cause of BV. Bacteria that are only associated with BV in our initial cohort will be selected for further study. Specific PCR assays will be developed and validated for each candidate pathogen. In future studies, vaginal fluid samples from a larger cohort of women will be assayed to determine if a candidate pathogen is specifically associated with BV.