Characterizing species-specific metabolic signatures in vaginal microbiota across planktonic and biofilm states

Bacterial vaginosis (BV) affects about 29 % of U.S. women, with higher rates in some groups and up to 50-69 % recurrence within a year. It increases the risk of STIs, pregnancy complications, and can cause significant discomfort and reduced quality of life. Prior studies mapped vaginal metabolomes, but individual microbial metabolite signatures remain poorly understood. Given that biofilms exhibit distinct metabolic requirements compared to planktonic cultures, analyzing biofilm vs. planktonic culture metabolites with their unique metabolic needs may uncover novel treatment targets. This study provides a comprehensive metabolomic comparison of key vaginal microbes-<i>Lactobacillus crispatus, Gardnerella vaginalis</i>, and <i>Lactobacillus iners</i> under both planktonic and biofilm conditions. Our analysis showed that metabolite production and consumption vary by microbe and growth mode. <i>G. vaginalis</i> formed biofilms in multiple media, with limited shared metabolic pathways between its biofilm types, indicating unique metabolic processes. Despite L. <i>crispatus</i> suspension and biofilm cultures sharing 142 consumed and 104 produced metabolites, the biofilm culture demonstrated a remarkable metabolic shift. Comparing all three species, we observed convergence in nutrient utilization, but divergence in metabolic outputs reflecting growth-specific adaptations, highlighting the importance of considering microbial state in vaginal microbiome studies.