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Bacterial vaginosis (BV) is a highly prevalent condition associated with adverse health outcomes. Gram stain analysis of vaginal fluid is the standard for confirming the diagnosis of BV, wherein abundances of key bacterial morphotypes are assessed. These Lactobacillus,  Gardnerella ,  Bacteroides , and  Mobiluncus  morphotypes were originally linked to particular bacterial species through cultivation studies, but no studies have systematically investigated associations between uncultivated bacteria detected by molecular methods and Gram stain findings. In this study, 16S-rRNA PCR/pyrosequencing was used to examine associations between vaginal bacteria and bacterial morphotypes in 220 women with and without BV. Species-specific quantitative PCR (qPCR) and fluorescence in Situ hybridization (FISH) methods were used to document concentrations of two bacteria with curved rod morphologies:  Mobiluncus  and the fastidious BV-associated bacterium-1 (BVAB1). Rank abundance of vaginal bacteria in samples with evidence of curved gram-negative rods showed that BVAB1 was dominant (26.1%), while  Mobiluncus  was rare (0.2% of sequence reads). BVAB1 sequence reads were associated with  Mobiluncus  morphotypes (p<0.001). Among women with curved rods, mean concentration of BVAB1 DNA was 2 log units greater than  Mobiluncus  (p<0.001) using species-specific quantitative PCR. FISH analyses revealed that mean number of BVAB1 cells was 2 log units greater than  Mobiluncus  cells in women with highest Nugent score (p<0.001).  Prevotella  and  Porphyromonas  spp. were significantly associated with the “Bacteroides morphotype,” whereas  Bacteroides  species were rare. Gram-negative rods designated  Mobiluncus  morphotypes on Gram stain are more likely BVAB1. These findings provide a clearer picture of the bacteria associated with morphotypes on vaginal Gram stain.

More Than Meets the Eye: Associations of Vaginal Bacteria with Gram Stain Morphotypes Using Molecular Phylogenetic Analysis