Investigating Bacterial Vaginosis Pathogenesis Using Peptide Nucleic Acid-Fluorescence in situ Hybridization with a Focus on the Roles of Gardnerella species, Prevotella bivia, and Fannyhessea vaginae

Background Bacterial vaginosis (BV) is a vaginal dysbiosis characterized by polymicrobial communities of BV-associated bacteria (BVAB) adhered to the vaginal epithelium. Despite decades of research, its etiology remains unknown. We aimed to investigate BV biofilm formation over time among women who developed incident BV (iBV) using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH), focusing on three key BVAB (Gardnerella species, Prevotella bivia, and Fannyhessea vaginae). Methods Heterosexual, non-pregnant women ages 18-45 with optimal vaginal microbiota were enrolled to self-collect twice-daily vaginal specimens for 60 days. iBV was defined as a Nugent score of 7-10 on ≥4 consecutive specimens. For women who developed iBV (cases), Gardnerella spp., P. bivia, and F. vaginae were visualized and quantified by PNA-FISH for up to 14 days prior to iBV, the day of iBV, and 3 days post-iBV. Cases were matched to women maintaining optimal vaginal microbiota (controls) based on age, race, and contraceptive method. Control specimens were matched to case specimens by day of menses. Results Among 135 women enrolled, 18 developed iBV and were matched to 18 controls. Pooled median Gardnerella spp. counts significantly increased starting 5 days before iBV, while pooled median F. vaginae counts significantly increased on the day of iBV diagnosis. In contrast, pooled median P. bivia counts were not significantly different between groups. Conclusion These data suggest that Gardnerella spp. are early colonizers of the BV biofilm while F. vaginae is a secondary colonizer. P. bivia was not found to be significantly different between iBV case and control specimens.