Analysis of adherence, biofilm formation and cytotoxicity suggests a greater virulence potential of Gardnerella vaginalis relative to other bacterial-vaginosis-associated anaerobes

Abstract

Worldwide, bacterial vaginosis (BV) is the most common vaginal disorder in women of childbearing age. BV is characterized by a dramatic shift in the vaginal microflora, involving a relative decrease in lactobacilli, and a proliferation of anaerobes. In most cases of BV, the predominant bacterial species found is Gardnerella vaginalis. However, pure cultures of G. vaginalis do not always result in BV, and asymptomatic women are sometimes colonized with low numbers of G. vaginalis. Thus, there is controversy about whether G. vaginalis is an opportunistic pathogen and the causative agent of many cases of BV, or whether BV is a polymicrobial condition caused by the collective effects of an altered microbial flora. Recent studies of the biofilm-forming potential and cytotoxic activity of G. vaginalis have renewed interest in the virulence potential of this organism. In an effort to tease apart the aetiology of this disorder, we utilized in vitro assays to compare three virulence properties of G. vaginalis relative to other BV-associated anaerobes. We designed a viable assay to analyse bacterial adherence to vaginal epithelial cells, we compared biofilm-producing capacities, and we assessed cytotoxic activity. Of the BV-associated anaerobes tested, only G. vaginalis demonstrated all three virulence properties combined. This study suggests that G. vaginalis is more virulent than other BV-associated anaerobes, and that many of the bacterial species frequently isolated from BV may be relatively avirulent opportunists that colonize the vagina after G. vaginalis has initiated an infection.

Fig. 1.

Adherence of G. vaginalis BV-associated anaerobes to vaginal epithelial cells. Bacteria were grown anaerobically in sBHIG at 37 °C for 24 h. Bacteria (green) cultures were standardized to ensure equal numbers, and added to vaginal epithelial cells (red). After incubation for 15 min, non-adherent bacteria were removed by washing. Qualitative estimates of adherence were ascertained by confocal microscopy. P. bivia strain 29303 (not shown) did not exhibit adherence.

Fig. 2.

Biofilm formation of G. vaginalis strains and various BV-associated anaerobes. (a) Bacteria were grown anaerobically in sBHIG at 37 °C for 24 h. Non-adherent cells were removed from the wells, and the adherent bacteria were stained with safranin. OD₅₉₅ readings were determined to ensure similar growth of each species. Experiments were repeated three times, and produced similar results each time; representative images are shown. (b) Quantitative assessment of the biofilm-forming capacities were made by dissolving the safranin stain in 33 % acetic acid and measuring OD₅₆₂. Eight technical replicates and 3 biological replicates (n=24) were performed on each strain and the absorbance readings were averaged. Error bars represent standard deviations. P. bivia strain BVD is not shown but similar to P. bivia strain 29303, it did not exhibit biofilm formation.

Fig. 3.

Cytotoxic changes of vaginal epithelial cell monolayers challenged with G. vaginalis strains and various BV-associated anaerobes. Bacteria were grown anaerobically in sBHIG at 37 °C for 24 h, and cultures were standardized to ensure equal numbers. Bacteria were added to vaginal epithelial cells, and incubated for 4 h. Light microscopy images were taken after incubation for 4 h. P. bivia strain BVD (not shown) did not exhibit cytotoxicity, and produced results that were similar to those of P. bivia strain 29303.