ABSTRACT: Bacterial vaginosis (BV), the most common vaginal infection among women worldwide, is characterized by an imbalance in the vaginal microbiota with a reduction of protective Lactobacillus species and overgrowth of facultative and strictly anaerobic bacteria. Although the development of a polymicrobial biofilm on the vaginal epithelium is a hallmark of BV, interactions between key BV-associated bacteria (BVAB) [i.e. Gardnerella vaginalis, Fannyhessea vaginae, and Prevotella bivia] present in the biofilm remain largely unexplored. In this study, we aimed to analyze the transcriptome of triple-species biofilms growing in a medium simulating vaginal tract secretions (mGTS) compared to a rich medium, New York City III (NYCIII). As such, triple-species biofilms formed by G. vaginalis, F. vaginae, and P. bivia were grown in either NYCIII or mGTS in anaerobic conditions. After that, RNA was extracted and cDNA libraries were constructed and sequenced. We first characterized the biofilm composition in both media by qPCR and observed that G. vaginalis was the most prevalent species in both conditions. RNA-sequencing data analysis revealed a total of 964, 596, and 656 differentially expressed genes in G. vaginalis, F. vaginae, and P. bivia, respectively, when comparing the transcriptome of biofilms in mGTS versus NYCIII. We identified 927 upregulated and 37 downregulated genes for G. vaginalis, 492 upregulated and 104 downregulated for F. vaginae, and 166 upregulated and 490 downregulated for P. bivia. Gene ontology enrichment showed that metabolic and biosynthetic processes were common within upregulated genes for G. vaginalis, while downregulated genes were associated with transmembrane transporters. For F. vaginae, transmembrane transporters were identified among upregulated genes, while downregulated genes were related to metabolism and cellular process, in which several KEGG pathways linked to metabolism were recognized. For P. bivia, upregulated genes were associated with transmembrane processes while downregulated genes were associated with metabolic processes; KEGG pathways were also identified. This work highlighted the adaptation of 3 key BVAB growing on a medium that simulates in vivo conditions and will contribute to a better understanding of bacterial interactions that are important in the pathogenesis of incident BV.
