Vaginal Lactobacillus Impair Candida Dimorphic Switching and Biofilm Formation

Lactobacillus spp. generally dominate the vaginal microbiota and prevent pathogen adhesion and overgrowth, including Candida spp., by various mechanisms. Although Candida spp. can be commensal, in certain conditions they can become pathogenic, causing vulvovaginal candidiasis. The insurgence of candidiasis is related to the expression of Candida virulence factors, including morphologic switching and biofilm formation. Germ tubes, pseudohyphae, and hyphae promote Candida tissue invasion, biofilms increase persistence and are often resistant to antifungals and host immune response. Here, we explored the inhibitory activity of vaginal Lactobacillus strains belonging to Lactobacillus crispatus, Lactobacillus gasseri, Limosilactobacillus vaginalis, and Lactiplantibacillus plantarum species towards Candida virulence factors. With the aim to investigate the interrelation between mode of growth and functionality, supernatants were collected from lactobacilli planktonic cultures and, for the first time, from adherent ones, and were evaluated towards Candida dimorphic switching and biofilm. Candida biofilms were analyzed by multiple methodologies, i.e., crystal violet staining, MTT assay, and confocal microscopy. Lactobacillus supernatants reduce Candida switching and biofilm formation. Importantly, L. crispatus supernatants showed the best profile of virulence suppression, especially when grown in adherence. These results highlight the role of such species as a hallmark of vaginal eubiosis and prompt its employment in new probiotics for women's health

Lactobacilli extracellular vesicles: potential postbiotics to support the vaginal microbiota homeostasis

Background: Lactobacillus species dominate the vaginal microflora performing a first-line defense against vaginal infections. Extracellular vesicles (EVs) released by lactobacilli are considered mediators of their beneficial effects affecting cellular communication, homeostasis, microbial balance, and host immune system pathways. Up to now, very little is known about the role played by Lactobacillus EVs in the vaginal microenvironment, and mechanisms of action remain poorly understood. Results: Here, we hypothesized that EVs can mediate lactobacilli beneficial effects to the host by modulating the vaginal microbiota colonization. We recovered and characterized EVs produced by two vaginal strains, namely Lactobacillus crispatus BC5 and Lactobacillus gasseri BC12. EVs were isolated by ultracentrifugation and physically characterized by Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS). EVs protein and nucleic acids (DNA and RNA) content was also evaluated. We explored the role of EVs on bacterial adhesion and colonization, using a cervical cell line (HeLa) as an in vitro model. Specifically, we evaluated the effect of EVs on the adhesion of both vaginal beneficial lactobacilli and opportunistic pathogens (i.e., Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, and Enterococcus faecalis). We demonstrated that EVs from L. crispatus BC5 and L. gasseri BC12 significantly enhanced the cellular adhesion of all tested lactobacilli, reaching the maximum stimulation effect on strains belonging to L. crispatus species (335% and 269% of average adhesion, respectively). At the same time, EVs reduced the adhesion of all tested pathogens, being EVs from L. gasseri BC12 the most efficient. Conclusions: Our observations suggest for the first time that EVs released by symbiotic Lactobacillus strains favor healthy vaginal homeostasis by supporting the colonization of beneficial species and preventing pathogens attachment. This study reinforces the concept of EVs as valid postbiotics and opens the perspective of developing postbiotics from vaginal strains to maintain microbiota homeostasis and promote women’s health

Lactobacillus Biofilms Influence Anti-Candida Activity

Lactobacilli are the dominant members of the healthy human vaginal microbiota and represent the first defense line from pathogen infection, including vulvovaginal candidiasis. Biofilm is the predominant microbial growth form in nature, and the formation of biofilms inside the human body has important implications in health and disease. In particular, the formation of biofilm by members of the human resident microbiota is desirable, as it can improve microbial persistence and influence functionality. In the present study, we investigated the capability of 16 vaginal Lactobacillus strains (belonging to Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus vaginalis, and Lactobacillus plantarum species) to form biofilms, and we correlated their mode of growth to anti-Candida activity. L. plantarum strains were the best biofilm producers, and high variability was registered in the level of biofilm formation among L. crispatus and L. gasseri strains. Culture supernatants derived from Lactobacillus biofilm and planktonic growth were tested toward a panel of Candida clinical isolates (Candida albicans, Candida glabrata, Candida lusitaniae, Candida tropicalis, Candida krusei, and Candida parapsilosis) and their metabolome assessed by 1H-NMR. L. crispatus and L. plantarum strains exhibited the best fungistatic profile, and biofilms enhanced their anti-Candida activity; on the contrary, L. gasseri strains were more effective when grown in a planktonic mode. Biofilm/planktonic mode of growth also affects Lactobacillus metabolism, mainly influencing nitrogen and amino acid pathways, and anti-Candida activity is instead strictly related to carbohydrate metabolism. The present study underlined the strict interdependence between microbial mode of growth, metabolism, and functional properties. Biofilm formation by members of the healthy human microbiota represents a crucial issue in the field of microbial physiology and host–microbiota interactions, beyond supporting the development of new antimycotic strategies based on probiotics grown in adherence.Fil: Parolin, Carola. Universidad de Bologna; ItaliaFil: Croatti, Vanessa. Universidad de Bologna; ItaliaFil: Laghi, Luca. Universidad de Bologna; ItaliaFil: Giordani, Barbara. Universidad de Bologna; ItaliaFil: Tondi, Maria Rosaria. Universidad de Bologna; ItaliaFil: de Gregorio, Priscilla Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Foschi, Claudio. Universidad de Bologna; ItaliaFil: Vitali, Beatrice. Universidad de Bologna; Itali

THE HEALTH-PROMOTING EFFECT OF EXTRACELLULAR VESICLES RELEASED BY VAGINAL LACTOBACILLI

Lactobacilli (LB) species dominate the vaginal microflora performing a first line defence against infections (1). Lactobacilli metabolites mediate the health-promoting effect acting as antimicrobials or are used for quorum-sensing and cross-talk communication (2). In this sense, extracellular vesicles (EVs) from LB demonstrate an antimicrobial activity (3,4), modulate host’s nervous and immune system (5) and induce apoptosis in cancerous liver cells (6). Notably, EVs derived from vaginal LB isolates reduce HIV-1 infection (7). Here, we investigate the health-promoting activity of EVs produced by two strains of LB isolated from human vagina (L. gasseri BC12 and L. crispatus BC5) in terms of anti-adhesive effect on pathogens and pro-adhesive effect on LB. EVs were recovered from LB cultures by ultracentrifuge cycles and yield (1010 EVs/mL), size (90-150 nm diameter) and protein content were characterized. Adhesion assays, performed on HeLa cells monolayer, demonstrated that LB-EVs were able to reduce at least 25% the adhesion of all pathogens tested: Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Streptococcus agalactiae. Moreover, LB-EVs stimulated more than 26% the adhesion of all LB strains, including the same producer strains and two other vaginal isolates belonging to the same species (L. gasseri BC9, BC13 and L. crispatus BC1, BC3). L. gasseri BC12 EVs demonstrated the best health-promoting profile. These results highlighted that the barrier effect exerted by lactobacilli can be mediated by EVs released in the vaginal environment. This opens to the hypothesis of using postbiotics from human vaginal bacteria as a new strategy to prevent/treat human infections