Vaginal Lactobacilli Supernatants Protect from Herpes Simplex Virus Type 1 Infection in Cell Culture Models.

A healthy vaginal microbiota hosts Lactobacillus as the most predominant genus. Lactobacilli play a role in human health through the production of diverse antimicrobial substances that can act against human pathogens or modulate the immune system. Previous reports highlighted the ability of vaginal lactobacilli to counteract viruses causing STIs, e.g., HIV-1 and HSV-2. In this report, we analyze the activity of supernatants of vaginal lactobacilli against HSV-1 infection, which is becoming increasingly relevant as a STI. We show that the supernatants of two vaginal Lactobacillus species (i.e., L. crispatus and L. gasseri) were active at neutralizing HSV-1 infection in two different cell lines of human and simian origin. Specifically, we demonstrate that L. crispatus strains are the most effective in antiviral activity, as evidenced by the comparison with a vaginal pathogen taken as reference. The effect was specific and not attributable to the generic toxicity of the supernatants to the cells. Our results pave the way for the development of probiotics to limit the impact of HSV-1 infection on women's health.

Exopolysaccharides from vaginal lactobacilli modulate microbial biofilms.

BACKGROUND: Exopolysaccharides (EPS) secreted by beneficial lactobacilli exert a plethora of positive activities, but little is known about their effects on biofilms of opportunistic vaginal pathogens and especially on biofilms of lactobacilli themselves. Here, the EPS produced by six vaginal lactobacilli, belonging to Lactobacillus crispatus (BC1, BC4, BC5) and Lactobacillus gasseri (BC9, BC12, BC14) species were isolated from cultural supernatants and lyophilized. RESULTS: Lactobacillus EPS were chemically characterized in terms of monosaccharide composition by liquid chromatography (LC) analysis coupled to UV and mass spectrometry (MS) detection. Moreover, the ability of EPS (0.1, 0.5, 1 mg/mL) to stimulate the biofilm formation of lactobacilli and to inhibit the formation of pathogens' biofilms was evaluated by crystal violet (CV) staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Isolated EPS (yields 133-426 mg/L) were heteropolysaccharides mainly composed of D-mannose (40-52%) and D-glucose (11-30%). For the first time we demonstrated that Lactobacillus EPS were able to stimulate in a dose-dependent manner (p < 0.05) the formation of biofilms of ten strains belonging to L. crispatus, L. gasseri and Limosilactobacillus vaginalis species, in terms of cell viability (84-282% increase at 1 mg/mL) and especially biofilm biomass (40-195% increase at 1 mg/mL), quantified with MTT assay and CV staining, respectively. EPS released from L. crispatus and L. gasseri were found to better stimulate the biofilms of the same producer species rather than that of other species, including producing strains themselves and other strains. Conversely, the biofilm formation of bacterial (Escherichia coli, Staphylococcus spp., Enterococcus spp. and Streptococcus agalactiae) and fungal (Candida spp.) pathogens was inhibited. The anti-biofilm activity was dose-dependent and was more marked for L. gasseri-derived EPS (inhibition up to 86%, 70%, and 58% at 1 mg/mL, 0.5 mg/mL, and 0.1 mg/mL, respectively), whilst L. crispatus-derived EPS resulted overall less efficient (inhibition up to 58% at 1 mg/mL and 40% at 0.5 mg/mL) (p < 0.05). CONCLUSIONS: Lactobacilli-derived EPS favour the biofilm formation of lactobacilli preventing, at the same time, that of opportunistic pathogens. These results support the possible employment of EPS as postbiotics in medicine as a therapeutic/preventive strategy to counteract vaginal infections.

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.

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.

Drug-in-cyclodextrin-in-polymeric nanoparticles: A promising strategy for rifampicin administration.

The aim of this work was to develop novel chitosan (CH) based nanoparticles (NPs) for rifampicin (RIF) delivery. RIF, a lipophilic molecule, was incorporated inside NPs as a complex with an anionic cyclodextrin, sulphobutyl-ether-ß-cyclodextrin (SBE-ß-CD). NPs were then prepared through the ionic gelation method by exploiting the interaction between CH and SBE-ß-CD-RIF complex (CH/SBE-ß-CD-RIF NPs), possibly in the presence of other crosslinkers, like carboxymethylcellulose (CH/SBE-ß-CD-RIF/CMC NPs) and pentasodium tripolyphosphate (CH/SBE-ß-CD-RIF/TPP NPs). NPs were then characterized for their size, ζ-potential, morphology, yield, drug loading, stability, mucoadhesion, in vitro drug release and antimicrobial activity. Results demonstrated that the functional properties of loaded NPs, like their size, ζ-potential, and stability, varied on the basis of the CH/crosslinker weight ratio. Interestingly, all the developed NPs had a round shape and were characterized by high yield values and mucoadhesive properties. Among them, NPs based on CH/SBE-ß-CD-RIF and CH/SBE-ß-CD-RIF/CMC have gained high drug loading, provided a sustained release of RIF and showed the best antimicrobial activity. Thus, both types of NPs may be considered as promising nanocarriers for the release of RIF.

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.