Gardnerella species exhibit synergy in their ability to displace Lactobacillus crispatus adhered to HeLa cells.

Background: Bacterial vaginosis (BV) is the most common vaginal infection in reproductive-age women. It is associated with adverse pregnancy complications, such as preterm birth and low birth weight, in addition to an increased risk of acquisition of HIV and sexually transmitted infections. BV is characterized by a vaginal dysbiosis, involving loss of protective Lactobacillus species (including L. crispatus) and overgrowth of facultative and strict anaerobic bacteria, with Gardnerella species playing a predominant role. However, despite extensive research on BV pathogenesis, its etiology remains unclear, and the sequence of events leading to the displacement of lactobacilli by anaerobic bacteria in women has not yet been fully elucidated. Until 2019, all bacteria belonging to the Gardnerella genus were considered part of the species G. vaginalis. However, it is now recognized that different Gardnerella species exist, each with varying virulence potentials. Recent data have shown that multiple subgroups of Gardnerella spp. are frequently detected simultaneously in the vaginal microbiota of women with BV. With this in mind, we aimed to test the hypothesis that different combinations of known Gardnerella species, isolated from the vaginal microbiota of women with BV, have an enhanced ability to compete against Lactobacillus crispatus, pre-adhered to HeLa cells, thereby facilitating the early stages of BV development. Methods: Adhesion assays of dual combinations of Gardnerella spp. (G. vaginalis, G. leopoldii, G. swidsinskii, G. piotii) were performed on an in vitro model of HeLa cells, covered with and without L. crispatus. Quantification of the species used in our assays was subsequently performed using quantitative polymerase chain reaction (qPCR). Results: Our results revealed synergy between different Gardnerella spp., demonstrating their ability to overcome the presumed protective effect of L. crispatus, thereby creating favorable conditions for the development of a polymicrobial biofilm characteristic of BV. The combination of G. vaginalis and G. leopoldii showed the greatest synergistic effect on initial adhesion to HeLa cells while the combination of G. leopoldii and G. swidsinskii had the greatest ability to reduce L. crispatus colonization. Conclusions: Although this in vitro study does not unequivocally prove that BV is initiated by the disruption of normal vaginal microbiota by Gardnerella spp., it strongly supports this possibility, contributing to a better understanding of BV etiology.