We characterized the vaginal ecosystem during common infections of the female genital tract, as vulvovaginal candidiasis (VVC, n = 18) and Chlamydia trachomatis infection (CT, n = 20), recruiting healthy (HC, n = 21) and bacterial vaginosis-affected (BV, n = 20) women as references of eubiosis and dysbiosis. The profiles of the vaginal microbiome and metabolome were studied in 79 reproductive-aged women, by means of next generation sequencing and proton based-nuclear magnetic resonance spectroscopy. Lactobacillus genus was profoundly depleted in all the genital infections herein considered, and species-level analysis revealed that healthy vaginal microbiome was dominated by L. crispatus. In the shift from HC to CT, VVC, and BV, L. crispatus was progressively replaced by L. iners. CT infection and VVC, as well as BV condition, were mainly characterised by anaerobe genera, e.g. Gardnerella, Prevotella, Megasphaera, Roseburia and Atopobium. The changes in the bacterial communities occurring during the genital infections resulted in significant alterations in the vaginal metabolites composition, being the decrease of lactate a common marker of all the pathological conditions. In conclusion, according to the taxonomic and metabolomics analysis, we found that each of the four conditions is characterized by a peculiar vaginal microbiome/metabolome fingerprint.

Healthy vaginal microbiota is dominated by Lactobacillus spp., which form a critical line of defence against pathogens, including Candida spp. The present study aims to identify vaginal lactobacilli exerting in vitro activity against Candida spp. and to characterize their antifungal mechanisms of action. Lactobacillus strains were isolated from vaginal swabs of healthy premenopausal women. The isolates were taxonomically identified to species level (L. crispatus B1-BC8, L. gasseri BC9-BC14 and L. vaginalis BC15-BC17) by sequencing the 16S rRNA genes. All strains produced hydrogen peroxide and lactate. Fungistatic and fungicidal activities against C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis and C. lusitaniae were evaluated by broth micro-dilution method. The broadest spectrum of activity was observed for L. crispatus BC1, BC4, BC5 and L. vaginalis BC15, demonstrating fungicidal activity against all isolates of C. albicans and C. lusitaniae. Metabolic profiles of lactobacilli supernatants were studied by ¹H-NMR analysis. Metabolome was found to be correlated with both taxonomy and activity score. Exclusion, competition and displacement experiments were carried out to investigate the interference exerted by lactobacilli toward the yeast adhesion to HeLa cells. Most Lactobacillus strains significantly reduced C. albicans adhesion through all mechanisms. In particular, L. crispatus BC2, L. gasseri BC10 and L. gasseri BC11 appeared to be the most active strains in reducing pathogen adhesion, as their effects were mediated by both cells and supernatants. Inhibition of histone deacetylases was hypothesised to support the antifungal activity of vaginal lactobacilli. Our results are prerequisites for the development of new therapeutic agents based on probiotics for prophylaxis and adjuvant therapy of Candida infection.

Lactobacillus species dominate the vaginal microbiota of healthy reproductive-age women and protect the genitourinary tract from the attack of several infectious agents. Chlamydia trachomatis, a leading cause of sexually transmitted disease worldwide, can induce severe sequelae, i.e. pelvic inflammatory disease, infertility and ectopic pregnancy. In the present study we investigated the interference of Lactobacillus crispatus, L. gasseri and L. vaginalis, known to be dominant species in the vaginal microbiome, with the infection process of C. trachomatis. Lactobacilli exerted a strong inhibitory effect on Chlamydia infectivity mainly through the action of secreted metabolites in a concentration/pH dependent mode. Short contact times were the most effective in the inhibition, suggesting a protective role of lactobacilli in the early steps of Chlamydia infection. The best anti-Chlamydia profile was shown by L. crispatus species. In order to delineate metabolic profiles related to anti-Chlamydia activity, Lactobacillus supernatants were analysed by ¹H-NMR. Production of lactate and acidification of the vaginal environment seemed to be crucial for the activity, in addition to the consumption of the carbonate source represented by glucose. The main conclusion of this study is that high concentrations of L. crispatus inhibit infectivity of C. trachomatis in vitro.

The emergence and spread of antimicrobial resistance in Neisseria gonorrhoeae (GC) underline the need of ’antibiotic-free’ strategies for the control of gonorrhea. The aim of this study was to assess the anti-gonococcal activity of 14 vaginal Lactobacillus strains, belonging to different species (L. crispatus, L. gasseri, L. vaginalis), isolated from healthy pre-menopausal women. In particular, we performed ‘inhibition’ experiments, evaluating the ability of both lactobacilli cells and culture supernatants in reducing GC viability, at two different contact times (7 and 60 minutes). First, we found that the acidic environment, associated to lactobacilli metabolism, is extremely effective in counteracting GC growth, in a pH- and time-dependent manner. Indeed, a complete abolishment of GC viability by lactobacilli supernatants was observed only for pH values < 4.0, even at short contact times. On the contrary, for higher pH values, no 100%-reduction of GC growth was reached at any contact time. Experiments with organic/inorganic acid solutions confirmed the strict correlation between the pH levels and the anti-gonococcal effect. In this context, the presence of lactate seemed to be crucial for the anti-gonococcal activity, especially for pH values in the range 4.4 – 5.3, indicating that the presence of H+ ions is necessary but not sufficient to kill gonococci. Moreover, experiments with buffered supernatants led to exclude a direct role in the GC killing by other bioactive molecules produced by lactobacilli. Second, we noticed that lactobacilli cells are able to reduce GC viability and to co-aggregate with gonococci. In this context, we demonstrated that released-surface components with biosurfactant properties, isolated from ‘highly-aggregating’ lactobacilli, could affect GC viability. The antimicrobial potential of biosurfactants isolated from lactobacilli against pathogens has been largely investigated, but this is the first report about a possible use of these molecules in order to counteract GC infectivity. In conclusion, we identified specific Lactobacillus strains, mainly belonging to L. crispatus species, able to counteract GC viability through multiple mechanisms. These L. crispatus strains could represent a new potential probiotic strategy for the prevention of GC infections in women.