Role of vaginal lactobacilli in counteracting Chlamydia trachomatis infectivity in an in vitro model

Introduction: Lactobacilli play a fundamental role in maintaining the ecological equilibrium of the vaginal niche, preventing the overgrowth of endogenous microorganisms and impeding the colonisation of pathogens. Although many studies have focused on the mechanisms displayed by lactobacilli in counteracting several urogenital pathogens, a few data are available on the interaction between lactobacilli and Chlamydia trachomatis (CT). The aim of this study was therefore to assess the in vitro activity of different vaginal Lactobacillus strains against CT infectivity, investigating two different fractions of bacteria (cell pellets and cell-free supernatants), by three different mechanisms of action (competition, exclusion and displacement). Methods: A total of 17 Lactobacillus strains, isolated from vaginal swabs of healthy premenopausal women and belonging to L. crispatus (BC1; BC3-BC8), L. gasseri (BC9-BC14), L. vaginalis (BC16-BC17) and L. plantarum (BC18-19) species, were included in the study. The capacity of lactobacilli cell pellets (CP) and cell-free supernatants (CFS) to interfere with CT adhesion and entry in HeLa epithelial cells was evaluated, by means of competition, exclusion and displacement mechanisms. In particular, lactobacilli fractions corresponding to 5×107 colony forming units (CFU) were incubated with 5×103 CT elementary bodies (EBs) of strain GO/86, (serotype D), following different timelines. CT infection was evaluated by counting chlamydia inclusion forming units (IFUs) by direct immunofluorescence. Moreover, on the basis of CT infectivity interference results, 5 lactobacilli were selected for dose-effect assays and the same experiments were repeated, using CP or CFS fractions with 5×106 and 5×105 lactobacilli cells, in order to verify if a dose-dependent activity was present. Finally, the capacity of Lactobacillus strains to adhere to HeLa cells was assessed as well: results were read at light-microscopy and HeLa cells were scored for the presence and number of lactobacilli attached. Results: We found that lactobacilli cell pellets were the most active fraction in counteracting CT infectivity, particularly by means of an exclusion strategy, and that L. crispatus was the most effective species, even though a strain-specific activity was detected. Moreover, the anti-chlamydial activity was not correlated with the level of lactobacilli adhesion on epithelial cells and it was significantly maintained with low numbers of lactobacilli, although in presence of a dose-response effect. Conclusion: We identified specific vaginal Lactobacillus strains (L. crispatus BC4, L. crispatus BC5, L. crispatus BC7, L. gasseri BC14 and L. plantarum BC19) able to interfere with CT EBs adhesion and entry in epithelial cells and we were able to shed light on the mechanisms displayed by lactobacilli in counteracting CT infectivity. A major potential application lies on the use of these Lactobacillus strains as probiotics for the prophylaxis and/or adjuvant therapy of CT infections.