Introduction Bacterial cytotoxic proteins, such as vaginolysin (VLY) produced by Gardnerella vaginalis, are thought to be virulence factors that in vitro alter cell integrity and local immunity. VLY may play a significant role in bacterial vaginosis (BV), therefore we assessed whether G. vaginalis dominant vaginal microbiota and immune markers were associated with higher concentrations of VLY in reproductive-age women.

Methods Forty women self-collected mid-vaginal swabs in a cross-sectional study. Microbial communities were characterised by 16S rRNA gene amplicon sequence analysis. VLY (ng/ml) was detected by ELISA and normalised by a cube root transformation. Absolute bacterial abundance of G. vaginalis (log₁₀ transformed) was estimated by multiplying its relative abundance by the sample total bacterial burden estimated by qPCR. Pro-inflammatory immune markers were quantified by Luminex and categorised above and below the median. Multivariate linear regression models evaluated factors associated with VLY abundance and controlled for confounders, including smoking and history of vaginal douching.

Results Vaginal microbiota clustered into 3 community state types (CSTs); 2 dominated by Lactobacillus spp. (Lactibacillus iners (CST-III) or L. crispatus (CST-I)), and one lacking Lactobacillus spp. and characterised by BV-associated bacteria including G. vaginalis and Atopobium vaginae (CST-IV). In the multivariate analysis, CST-IV, G. vaginalis bacterial load, a high Nugent Gram stain score, and a more basic vaginal pH (all p<0.03) were positively associated with increasing concentrations of VLY. TNF-α, TGF-β and IL-8 were inversely associated with VLY but only TNF-α remained significant in multivariate analysis (p=0.01).

Conclusion This study confirms that vaginal microbiota lacking lactobacilli, as well as other clinical indicators of BV, were associated with higher concentrations of VLY in vivo. Inflammatory markers were inversely associated with VLY. Because VLY may alter the vaginal microbiota and local inflammation, the role of VLY in BV warrants further evaluation.