Background BV is a highly prevalent dysbiotic condition associated with adverse reproductive and health outcomes in women. BV is marked by loss of certain lactobacilli and acquisition of complex communities of anaerobic bacteria. Gram stain is the gold standard for diagnosis, wherein the abundances of four bacterial morphotypes are assessed; Lactobacillus, Gardnerella, Bacteroides and Mobiluncus. Newly described uncultivated bacteria are highly specific for BV, but no studies have investigated the association between these bacteria and Gram stain morphotypes.

Methods We examined the association of bacteria detected by broad-range 16S-rRNA gene PCR/pyrosequencing with bacterial morphotypes detected in Gram stains from 220 women with and without BV. We also used species-specific quantitative PCR and fluorescence in situ hybridization (FISH) methods to document concentrations of two bacteria with curved rod morphologies: Mobiluncus species and the uncultivated BV-associated bacterium-1 (BVAB1).

Results We provide evidence that curved Gram-negative rods designated Mobiluncus morphotypes by Gram stain are more likely BVAB1. Rank abundance plots of vaginal bacteria in women with curved rods (Nugent 9–10) showed that BVAB1 was the dominant bacterium (26%), while relative abundance of Mobiluncus was only 0.2%. BVAB1 sequence reads were also associated with Mobiluncus morphotypes (p = 7.4E-06). Among women with Nugent scores 9–10, the mean concentration of BVAB1 DNA was 2-log units greater than Mobiluncus (p < 0.001). FISH analyses also revealed that among women with Nugent scores of 10, the mean number of BVAB1 cells was greater than Mobiluncus cells (p < 0.001). In addition, we noted that Prevotella and Porphyromonas spp. are significantly associated with the Bacteroides morphotype, whereas Bacteroides species are rare.

Conclusions Gram stain morphotype designations for BV need revision to account for novel vaginal bacteria. These findings have major implications for studies using Gram stains as a proxy to describe the vaginal microbiota.