More Easily Cultivated Than Identified: Classical Isolation with Molecular Identification of Vaginal Bacteria Targeted Locus (Loci). human vaginal metagenome

Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria that have been identified with molecular methods and associated with BV. It is unclear if this is due to the inability to adequately propagate these bacteria, or to correctly identify them in culture. Vaginal fluid from 15 women was plated on six different media using classical cultivation approaches. Individual isolates were identified by 16S rRNA gene sequencing, and compared with validly published species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene PCR and pyrosequencing. Pyrosequencing reads and bacterial isolates from the same set of samples were compared to evaluate cultivation efficacy. We isolated and identified 101 distinct bacterial strains spanning six phyla. Bacteria isolated include: 1) novel strains with <98% 16S rRNA sequence identity to published species (Dialister sp. Type 2, Eggerthella sp. Type 1), 2) phylogenetically diverse bacteria (Prevotella spp.), 3) bacteria previously isolated from other body sites but not the vagina (Atopobium deltae, Fenollaria massiliensis), and 4) bacteria formerly isolated from the vagina (Dialister micraerophilus, Veillonella montpellierensis). Pyrosequencing showed that novel strains Peptoniphilaceae bacterium DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV. Genomes of 31 strains are being sequenced by the Human Microbiome Project. We isolated a diverse set of novel and clinically significant anaerobes from the vagina using conventional approaches with systematic molecular identification. Several previously “uncultivated” bacteria from the human vagina are amenable to cultivation using conventional methods.