Lactic acid bacteria are considered an inexhaustible source of bioactive compounds; indeed, products from their metabolism are known to have immunomodulatory and anti-inflammatory activity. Recently, we demonstrated that Cell-Free Supernatants (CFS) obtained from Lactobacillus (L.) acidophilus, Lactiplantibacillus (L.) plantarum, Lacticaseibacillus (L.) rhamnosus, and Limosilactobacillus (L.) reuteri can impair Candida pathogenic potential in an in vitro model of epithelial vaginal infection. This effect could be ascribed to a direct effect of living lactic acid bacteria on Candida virulence and to the production of metabolites that are able to impair fungal virulence. In the present work, stemming from these data, we deepened our knowledge of CFS from these four lactic acid bacteria by performing a metabolomic analysis to better characterize their composition. By using an untargeted metabolomic approach, we detected consistent differences in the metabolites produced by these four different lactic acid bacteria. Interestingly, L. rhamnosus and L. acidophilus showed the most peculiar metabolic profiles. Specifically, after a hierarchical clustering analysis, L. rhamnosus and L. acidophilus showed specific areas of significantly overexpressed metabolites that strongly differed from the same areas in other lactic acid bacteria. From the overexpressed compounds in these areas, inosine from L. rhamnosus returned with the best identification profile. This molecule has been described as having antioxidant, anti-inflammatory, anti-infective, and neuroprotective properties. The biological significance of its overproduction by L. rhamnosus might be important in its probiotic and/or postbiotic activity.

Candida albicans (C. albicans) can behave as a commensal yeast colonizing the vaginal mucosa, and in this condition is tolerated by the epithelium. When the epithelial tolerance breaks down, due to C. albicans overgrowth and hyphae formation, the generated inflammatory response and cell damage lead to vulvovaginal candidiasis (VVC) symptoms. Here, we focused on the induction of mitochondrial reactive oxygen species (mtROS) in vaginal epithelial cells after C. albicans infection and the involvement of fungal burden, morphogenesis and candidalysin (CL) production in such induction. Bioluminescent (BLI) C. albicans, C. albicans PCA-2 and C. albicans 529L strains were employed in an in vitro infection model including reconstituted vaginal epithelium cells (RVE), produced starting from A-431 cell line. The production of mtROS was kinetically measured by using MitoSOX^™ Red probe. The potency of C. albicans to induced cell damage to RVE and C. albicans proliferation have also been evaluated. C. albicans induces a rapid mtROS release from vaginal epithelial cells, in parallel with an increase of the fungal load and hyphal formation. Under the same experimental conditions, the 529L C. albicans strain, known to be defective in CL production, induced a minor mtROS release showing the key role of CL in causing epithelial mithocondrial activation. C. albicans PCA-2, unable to form hyphae, induced comparable but slower mtROS production as compared to BLI C. albicans yeasts. By reducing mtROS through a ROS scavenger, an increased fungal burden was observed during RVE infection but not in fungal cultures grown on abiotic surface. Collectively, we conclude that CL, more than fungal load and hyphae formation, seems to play a key role in the rapid activation of mtROS by epithelial cells and in the induction of cell-damage and that mtROS are key elements in the vaginal epithelial cells response to C. albicans.

In recent years, the search for new compounds with antibacterial activity has drastically increased due to the spread of antibiotic-resistant microorganisms. In this study, we analyzed Cell-Free Supernatant (CFS) from Bacillus siamensis, assessing its potential antimicrobial activity against some of the main pathogenic microorganisms of human interest. To achieve this goal, we exploited the natural antagonism of skin-colonizing bacteria and their ability to produce compounds with antimicrobial activity. Biochemical and molecular methods were used to identify 247 strains isolated from the skin. Among these, we found that CFS from a strain of Bacillus siamensis (that we named CPAY1) showed significant antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, and Candida spp. In this study, we gathered information on CFS’s antimicrobial activity and on its sensitivity to chemical–physical parameters. Time–kill studies were performed; anti-biofilm activity, antibiotic resistance, and plasmid presence were also investigated. The antimicrobial compounds included in the CFS showed resistance to the proteolytic enzymes and were heat stable. The production of antimicrobial compounds started after 4 h of culture (20 AU/mL). CPAY1 CFS showed antimicrobial activity after 7 h of bacteria co-culture. The anti-biofilm activity of the CPAY1 CFS against all the tested strains was also remarkable. B. siamensis CPAY1 did not reveal the presence of a plasmid and showed susceptibility to all the antibiotics tested.

Vaginal ecosystem is a unique environment where, in physiological conditions, lactobacilli dominate. However, pathogenic microbial species responsible for vaginitis and vaginosis can also harbor vaginal microbiota. To extend our previously published data, we analyzed here both the anti-Candida and anti-inflammatory properties of the vaginal gel formulation, Respecta^® Balance Gel (RBG), commercialized as an adjuvant to treat vaginitis and vaginosis. We evaluated its activity by an in vitro model where a monolayer of A-431 vaginal epithelial cells was infected by Candida albicans in the presence of RBG or the placebo formulation (pRBG). Specifically, we tested the RBG capacity to counteract C. albicans virulence factors and their anti-inflammatory properties. Our results show that, unlike the placebo, RBG reduces C. albicans adhesion, its capacity to form hyphae and C. albicans-induced vaginal cell damage. Interestingly, both RBG and pRBG reduce LPS-induced IL-8 secretion (with RBG being the most effective), demonstrating that also the placebo retains anti-inflammatory properties. From our experimental approach, we highlighted the possible role of farnesol on such effects, but we would like to point out that lactic acid, polydextrose and glycogen too must be relevant in the actual application. In summary, our results show that RBG impairs C. albicans virulence and is able to reduce the inflammation in the vaginal environment, ultimately allowing the establishment of a balanced vaginal ecosystem.

Probiotics are living microbes that provide benefits to the host. The growing data on health promotion, following probiotics administration, increased interest among researchers and pharmaceutical companies. Infections of the lower genital tract in females, caused by a wide range of pathogens, represent one of the main areas for the use of probiotics and postbiotics. Vulvovaginal candidiasis (VVC) affects 75% of women of reproductive age at least once during their lifetime, with 5–8% developing the recurrent form (RVVC). The disease is triggered by the overgrowth of Candida on the vaginal mucosa. Here, in order to establish its probiotic potential in the context of VVC, we evaluated the anti-fungal effects of the spore-producing Bacillus coagulans LMG S-24828 against C. albicans and C. parapsilosis as well as its beneficial effects in counteracting Candida vaginal infection in vitro. Our results show that both live B. coagulans and its Cell-Free Supernatant (CFS) exerted antifungal activity against both fungi. Moreover, live B. coagulans reduced hyphal formation, inhibited C. albicans adhesion to vaginal epithelial cells, showed co-aggregation capacity, and exerted a protective effect on vaginal epithelial cells infected with C. albicans. These data suggest that B. coagulans LMG S-24828 may provide benefits in the context of Candida vaginal infections.

Thermo-tolerance is a virulence factor responsible for the emergence of new fungal pathogens, including Candidozyma auris (formerly classified as Candida auris, C. auris). It has been shown that in C. auris the thermo-tolerance, as well as other virulence traits, such as the ability to aggregate, to form pseudo-hyphae, or to produce melanin are strain-specific features. Here, we investigated the impact of different temperatures (25 °C, 37 °C and 42 °C) on the phenotypic and virulence profile of C. auris strain CDC B11903. The results show a positive correlation between the resistance to antifungals and increasing temperature from 25 °C to 37 °C, while no differences were observed between 37 °C and 42 °C, except for Anidulafungin. Furthermore, C. auris growth was impaired at 25 °C as compared to 37 °C and 42 °C. Except for the haemolytic activity, which increased with rising temperatures, phospholipase, lipase and biofilm production were found at all tested temperatures. Moreover, the ability to produce melanin was observed only at 37 °C and 42 °C. The capacity to grow as pseudo-hyphae or in clusters and to adhere to both biotic and abiotic surfaces were observed at all the temperatures tested, with increased propensity of C. auris to adhere to abiotic surfaces with rising temperatures. The results underline the thermo-tolerance of C. auris strain B11903 and its increased virulence profile at human body temperature both in physiological (37 °C) and febrile state (42 °C).