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.

Limosilactobacillus vaginalis Exerts Bifidogenic Effects: A Novel Postbiotic Strategy for Infant Prebiotic Supplementation.

Infant microbiota shaping strictly influences newborns' well-being and long-term health, and babies born by cesarean-section and formula-fed generally show low microbial gut diversity and are more prone to develop various disorders. The supplementation with beneficial microbes of vaginal origin or derivatives (postbiotics, including heat-inactivated cells) represents a valid strategy to drive the correct gut microbiota shaping. Here, we explored for the first time the bifidogenic activity of a heat-killed vaginal strain (Limosilactobacillus vaginalis BC17), in addition to the assessment of its safety. L. vaginalis BC17 whole genome was sequenced by Nanopore technology and highlighted the absence of antibiotic resistance genes and virulence factors, indicating the strain safety profile for human health. MIC values confirmed that L. vaginalis BC17 is susceptible to widely employed antibiotics. Heat-killed BC17 cells significantly enhanced the planktonic growth of Bifidobacterium spp. For the first time, stimulating effects were observed also toward biofilm formation of bifidobacteria and their pre-formed biofilms. Conversely, heat-killed BC17 cells exerted antibacterial and anti-biofilms activities against Gram-positive and Gram-negative pathogens. Lyophilized heat-killed BC17 cells were formulated in a sunflower oil suspension (1010 heat-killed cell/g) intended for infant oral intake. This possessed optimal technological (i.e., re-dispersibility and stability) and functional properties (i.e., bifidogenic activity) that were maintained even after pre-digestion in acidic conditions.

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.

Prebiotic Activity of Vaginal Lactobacilli on Bifidobacteria: from Concept to Formulation.

The gut of babies born vaginally is rapidly colonized by Bifidobacterium spp. after birth, while in infants born by cesarean section (C-section), the presence of bifidobacteria drops dramatically, increasing the risk of developing gastrointestinal disorders. Considering that newborns naturally come into contact with maternal lactobacilli as they pass through the birth canal, the aim of this work is to exploit for the first time the bifidogenic activity exerted by the cell-free supernatants (CFSs) from lactobacilli of vaginal origin, belonging to the species Lactobacillus crispatus, Lactobacillus gasseri, Limosilactobacillus vaginalis, and Lactiplantibacillus plantarum. CFSs were recovered after 7 h, 13 h, and 24 h of fermentation and assessed for the ability to stimulate the planktonic growth and biofilms of Bifidobacterium strains belonging to species widely represented in the gut tract. A bifidogenic effect was observed for all CFSs; such activity was maximal for CFSs recovered in exponential phase and was strongly dependent on the species of lactobacilli. Importantly, no stimulating effects on an intestinal Escherichia coli strain were observed. CFSs from L. vaginalis BC17 showed the best bifidogenic profile since they increased bifidobacterial planktonic growth by up to 432% and biofilm formation by up to 289%. The CFS at 7 h from BC17 was successfully formulated with a hyaluronic acid-based hydrogel aimed at preventing and treating breast sores in lactating women and exerting bifidogenic activity in infants born mainly by C-section. IMPORTANCE Bifidobacteria in the gut tract of infants play crucial roles in the prevention of gastrointestinal diseases and the maturation of the immune system. Consequently, strategies to trigger a bifidogenic shift in the infant gut are highly desirable. Evidences suggest that the presence of a maternal vaginal microbiota dominated by health-promoting lactobacilli and the development of a bifidobacterium-enriched gut microbiota in newborns are interconnected. In this context, we found out that the cell-free supernatants from lactobacilli of vaginal origin were able to effectively stimulate the proliferation of Bifidobacterium spp. grown in free-floating and biofilm forms. The cell-free supernatant from Limosilactobacillus vaginalis BC17 showed excellent bifidogenic behavior, which was preserved even after its incorporation into a nipple formulation for lactating women. Lactobacilli derivatives, such as cell-free supernatants, have gained increasing interest by virtue of their safer profile than that of living cells and can be proposed as an ecosustainable approach to favor gut colonization of infants by bifidobacteria.

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.

Development and Characterization of Azithromycin-Loaded Microemulsions: A Promising Tool for the Treatment of Bacterial Skin Infections.

In recent years, the treatment of bacterial skin infections has been considered a major healthcare issue due to the growing emergence of antibiotic-resistant strains of Staphylococcus aureus. The incorporation of antibiotics in appropriate nanosystems could represent a promising strategy, able to overcome several drawbacks of the topical treatment of infections, including poor drug retention within the skin. The present work aims to develop microemulsions containing azithromycin (AZT), a broad-spectrum macrolide antibiotic. Firstly, AZT solubility in various oils, surfactants and co-surfactants was assessed to select the main components. Subsequently, microemulsions composed of vitamin E acetate, Labrasol® and Transcutol® P were prepared and characterized for their pH, viscosity, droplet size, zeta potential and ability to release the drug and to promote its retention inside porcine skin. Antimicrobial activity against S. aureus methicillin-resistant strains (MRSA) and the biocompatibility of microemulsions were evaluated. Microemulsions showed an acceptable pH and were characterized by different droplet sizes and viscosities depending on their composition. Interestingly, they provided a prolonged release of AZT and promoted its accumulation inside the skin. Finally, microemulsions retained AZT efficacy on MRSA and were not cytotoxic. Hence, the developed AZT-loaded microemulsions could be considered as useful nanocarriers for the treatment of antibiotic-resistant infections of the skin.

Human Breast Milk: A Source of Potential Probiotic Candidates.

This study focuses on the isolation of lactobacilli/bifidobacteria from human breast milk and their first characterization, in the perspective to find new probiotic candidates to be included in food products. More specifically, breast-milk-isolated strains demonstrated a very good aptitude to adhere to intestinal cells, in comparison with L. rhamnosus GG strain, taken as reference. The same behavior has been found for hydrophobicity/auto-aggregation properties. A remarkable antagonistic activity was detected for these isolates not only against spoilage and pathogenic species of food interest, but also against the principal etiological agents of intestinal infections. Indeed, isolated strains impaired spoilage and pathogenic species growth, as well as biofilm formation by gut pathogens. In addition, breast milk strains were characterized for their antibiotic susceptibility, displaying species-specific and strain-specific susceptibility patterns. Finally, to assess their technological potential, the fermentation kinetics and viability of breast milk strains in pasteurized milk were investigated, also including the study of the volatile molecule profiles. In this regard, all the strains pointed out the release of aroma compounds frequently associated with the sensory quality of several dairy products such as acetic acid, diacetyl, acetoin, acetaldehyde. Data here reported point up the high potential of breast-milk-isolated strains as probiotics.

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.

Influence of Lactobacillus Biosurfactants on Skin Permeation of Hydrocortisone.

One of the most widely used strategies to improve drug diffusion through the skin is the use of permeation enhancers. The aim of this work was to investigate the effect of two biosurfactants (BS), produced by Lactobacillus crispatus BC1 and Lactobacillus gasseri BC9, on the skin permeation profile of hydrocortisone (HC, model drug). HC aqueous solubility and in vitro diffusion studies through porcine skin were performed in the presence of BC1-BS and BC9-BS at concentrations below and above critical micellar concentrations (CMC). Moreover, skin hydration tests and differential scanning calorimetry (DSC) analysis were performed to further investigate BS interaction with the outermost layer of the skin. Both BS increased HC solubility, especially at concentrations above their CMC. At concentrations below the CMC, drug permeation through the skin was improved, as the result of a dual effect: a) the formation of a superficial lipophilic environment, as confirmed by the reduction in skin hydration and b) the interaction between BS and the stratum corneum (SC), as demonstrated by the DSC curves. From the obtained data, it appears that BC1-BS and BC9-BS could represent new promising green excipients for drug permeation enhancement through the skin.

Anti-Candida Activity of Hyaluronic Acid Combined with Lactobacillus crispatus Lyophilised Supernatant: A New Antifungal Strategy.

Vulvovaginal candidiasis (VVC) and recurrencies are common in reproductive-aged women. The emergence of Candida strains resistant to conventional antimycotic drugs prompted the search for alternative therapies. Hyaluronic acid (HA), a uniform and linear glycosaminoglycan, has been proposed as an anti-Candida agent. Vaginal lactobacilli and their derivatives, including cell free culture supernatants (CFS), represent potential strategies for the treatment of Candida infections. In the present paper, the anti-Candida potential of HA and lyophilised CFS (LCFS), obtained from the vaginal strain Lactobacillus crispatus BC5, was investigated. HA and LCFS proved to be active towards a panel of clinical Candida isolates belonging to different species in a dose dependent manner and their association maintained the antifungal activity. Notably, also Candida species generally resistant to conventional antifungals resulted sensitive. A vaginal matrix based on microcrystalline cellulose and containing effective doses of both agents was developed and characterised. This vaginal formulation showed mucoadhesive ability and almost abrogated Candida albicans growth. In conclusion, HA and LCFS from L. crispatus BC5 are thus good candidates to design a new therapeutic strategy to counteract VVC, and the proposed vaginal matrix represents a promising prototype.

Insight into phenotypic and genotypic differences between vaginal Lactobacillus crispatus BC5 and Lactobacillus gasseri BC12 to unravel nutritional and stress factors influencing their metabolic activity.

The vaginal microbiota, normally characterized by lactobacilli presence, is crucial for vaginal health. Members belonging to L. crispatus and L. gasseri species exert crucial protective functions against pathogens, although a total comprehension of factors that influence their dominance in healthy women is still lacking. Here we investigated the complete genome sequence and comprehensive phenotypic profile of L. crispatus strain BC5 and L. gasseri strain BC12, two vaginal strains featured by anti-bacterial and anti-viral activities. Phenotype microarray (PM) results revealed an improved capacity of BC5 to utilize different carbon sources as compared to BC12, although some specific carbon sources that can be associated to the human diet were only metabolized by BC12, i.e. uridine, amygdalin, tagatose. Additionally, the two strains were mostly distinct in the capacity to utilize the nitrogen sources under analysis. On the other hand, BC12 showed tolerance/resistance towards twice the number of stressors (i.e. antibiotics, toxic metals etc.) with respect to BC5. The divergent phenotypes observed in PM were supported by the identification in either BC5 or BC12 of specific genetic determinants that were found to be part of the core genome of each species. The PM results in combination with comparative genome data provide insights into the possible environmental factors and genetic traits supporting the predominance of either L. crispatus BC5 or L. gasseri BC12 in the vaginal niche, giving also indications for metabolic predictions at the species level.

Lactobacillus crispatus BC1 Biosurfactant Counteracts the Infectivity of Chlamydia trachomatis Elementary Bodies.

Lactobacilli-derived biosurfactants (BS) have shown promising effects as antimicrobial molecules. Since Lactobacillus crispatus plays a crucial role in maintaining vaginal eubiosis, BS from this species could represent novel therapeutic agents to counteract sexually transmitted pathogens, such as Chlamydia trachomatis (CT). The aim of the present study was to assess the inhibitory effects of a BS produced by the vaginal strain L. crispatus BC1 on the infectivity of CT elementary bodies (EBs). For concentrations ranging between 1 and 0.5 mg/mL at 60-min contact time, L. crispatus BC1 BS displayed a highly significant anti-CT activity, with about 50% reduction of EB infectivity towards HeLa cells. To identify the components responsible for chlamydial inhibition, a panel of selected fatty acids, including those present in BS lipopeptidic structure, was tested against CT EBs. Pentadecanoic acid, myristic acid, ß-hydroxy-myristic acid, and ß-hydroxy-palmitic acid were able to significantly reduce EBs infectivity up to 5-0.5 µg/mL, concentrations that resulted to be non-toxic for HeLa cells. These data can contribute to the understanding of the biological role of lactobacilli in the vaginal niche, as well as to promote the application of their produced BS as an innovative and antibiotic-sparing anti-chlamydial strategy.

Probiotic and Metabolic Characterization of Vaginal Lactobacilli for a Potential Use in Functional Foods.

The main aim of this work was to verify the metabolic and functional aptitude of 15 vaginal strains belonging to Lactobacillus crispatus, Lactobacillus gasseri, and Limosilactobacillus vaginalis (previously Lactobacillus vaginalis), already characterized for their technological and antimicrobial properties. In order to evaluate the metabolic profile of these vaginal strains, a phenotype microarray analysis was performed on them. Functional parameters such as hydrophobicity, auto-aggregation, deconjugation of bile salts, adhesion to an intestinal cell line (Caco-2), and a simulated digestion process were evaluated for these strains. A good number of these strains showed hydrophobicity values higher than 70%. Regarding the auto-aggregation assay, the most promising strains were L. crispatus BC9 and BC1, L. gasseri BC10 and BC14, and L. vaginalis BC17. Moreover, L. crispatus BC4, BC6, BC7, and BC8 were characterized by strong bile salts hydrolase activity (BHS). In addition, L. crispatus BC8 and L. vaginalis BC17 were characterized by a medium ability to adhere to Caco-2 cells. Data related to digestion process showed a strong ability of vaginal lactobacilli to withstand this stress. In conclusion, the data collected show the metabolic versatility and several exploitable functional properties of the investigated vaginal lactobacilli.

Lactobacillus crispatus BC1 Biosurfactant Delivered by Hyalurosomes: An Advanced Strategy to Counteract Candida Biofilm.

The emergence of resistance to antifungal drugs has made the treatment of vulvovaginal candidiasis (VVC) very challenging. Among natural substances, biosurfactants (BS) produced by Lactobacillus have gained increasing interest in counteracting Candida infections for their proven anti-adhesive properties and safety profile. In the present study, liposomes (LP-BS) or liposomes coated with hyaluronic acid (HY-LP-BS) were prepared in the presence of the BS isolated from the vaginal strain Lactobacillus crispatus BC1 and characterized in terms of size, ζ potential, stability and mucoadhesion. The anti-biofilm activity of free BS, LP-BS and HY-LP-BS was investigated against different Candida albicans and non-albicans strains (C. glabrata, C. lusitaniae, C. tropicalis, C. krusei and C. parapsilosis), clinically isolated from patients affected by VVC. The inhibition of biofilm formation and the dispersal of pre-formed biofilm were evaluated. The obtained phospholipid vesicles showed suitable size for vaginal application and good stability over the storage period. HY-LP-BS exhibited good mucoadhesive properties and the best anti-biofilm profile, both in preventing or limiting the surface colonization by a broad spectrum of Candida species. In conclusion, the formulation of a novel antifungal agent derived from the vaginal microbiota into mucoadhesive nanocarriers appears to be a promising biotherapeutic strategy to counteract vulvovaginal candidiasis.

Lactobacilli as Anti-biofilm Strategy in Oral Infectious Diseases: A Mini-Review.

The spread of biofilm-related diseases in developed countries has led to increased mortality rates and high health care costs. A biofilm is a community of microorganisms that is irreversibly attached to a surface, behaving very differently from planktonic cells and providing resistance to antimicrobials and immune response. Oral diseases are an excellent example of infection associated with the formation of highly pathogenic biofilms. It is generally accepted that, when the oral homeostasis is broken, the overgrowth of pathogens is facilitated. Among them, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are the main etiological agents of periodontitis, while Streptococcus mutans is strongly associated with the onset of dental caries. Other microorganisms, such as the fungus Candida albicans, may also be present and contribute to the severity of infections. Since the common antibiotic therapies usually fail to completely eradicate biofilm-related oral diseases, alternative approaches are highly required. In this regard, the topical administration of probiotics has recently gained interest in treating oral diseases. Thus, the present mini-review focuses on the possibility of using Lactobacillus spp. as probiotics to counteract biofilm-mediated oral infections. Many evidence highlight that Lactobacillus living cells can impede the biofilm formation and eradicate mature biofilms of different oral pathogens, by acting through different mechanisms. Even more interestingly, lactobacilli derivatives, namely postbiotics (soluble secreted products) and paraprobiotics (cell structural components) are able to trigger anti-biofilm effects too, suggesting that they can represent a novel and safer alternative to the use of viable cells in the management of biofilm-related oral diseases.

Evaluation of the fate of Lactobacillus crispatus BC4, carried in Squacquerone cheese, throughout the simulator of the human intestinal microbial ecosystem (SHIME).

Lactobacillus crispatus strain BC4, isolated from the human healthy vaginal environment and characterised by a strong antimicrobial activity against urogenital pathogens and foodborne microorganisms, was employed as a probiotic culture in the cheesemaking of the soft cheese Squacquerone. Such cheese is intended as a "gender food", that could be used as a hedonistic dietary strategy to reduce the incidence of woman vaginal dysbiosis and infections, given the evidence that a probiotic strain able to survive to the entire digestive process once ingested, can pass from intestine to vagina. This work was aimed to evaluate the resistance of L. crispatus BC4, carried in Squacquerone cheese, to different challenges of the human gastrointestinal tract, including the colon stage. The digestion process was tested using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). The viability and metabolic activity of L. crispatus BC4 during the colon simulation were monitored by qPCR and gas chromatography, respectively, also in the presence of a complex microbiota. The results showed that L. crispatus BC4 survival was not affected by the gastric condition, while it was significantly affected by bile salts and pancreatic juice in small intestine conditions, where it decreased of approx. 0.6 log (colony-forming units) CFU/g. Differently, during colon simulation L. crispatus BC4 was able to grow in sterile colon conditions and to maintain viability in the presence of a complex microbiota. Moreover, during colon simulation, L. crispatus BC4 was metabolically active as demonstrated by the higher production of short chain fatty acids (SCFA) and lactate. In the presence of a complex gut microbiota, a decrease of lactate was observed, due to its conversion into propionate (anti-cholesterol activity) and butyrate (anti-inflammatory activity) by cross-feeding. However, no differences in propionate and butyrate production could be observed between control cheese and cheese containing L. crispatus BC4. Despite this may appear as a negative outcome, it must be taken into account that, in this setup, only a single dose of the cheese was tested and the outcome of the colonization and impact of the gut microbiota might be different when daily repeated doses are tested.

Metabolic profiling of Candida clinical isolates of different species and infection sources.

Candida species are the most common cause of opportunistic fungal infections. Rapid identification and novel approaches for the characterization of these fungi are of great interest to improve the diagnosis and the knowledge about their pathogenic properties. This study aimed to characterize clinical isolates of Candida spp. by proteomics (MALDI-TOF MS) and metabolomics (1H-NMR), and to correlate their metabolic profiles with Candida species, source of infection and different virulence associated parameters. In particular, 49 Candida strains from different sources (blood, n = 15; vagina, n = 18; respiratory tract, n = 16), belonging mainly to C. albicans complex (61%), C. glabrata (20%) and C. parapsilosis (12%) species were used. Several extracellular and intracellular metabolites showed significantly different concentrations among isolates recovered from different sources of infection, as well as among different Candida species. These metabolites were mainly related to the glycolysis or gluconeogenesis, tricarboxylic acid cycle, nucleic acid synthesis and amino acid and lipid metabolism. Moreover, we found specific metabolic fingerprints associated with the ability to form biofilm, the antifungal resistance (i.e. caspofungin and fluconazole) and the production of secreted aspartyl proteinase. In conclusion, 1H-NMR-based metabolomics can be useful to deepen Candida spp. virulence and pathogenicity properties.

Biosurfactant from vaginal Lactobacillus crispatus BC1 as a promising agent to interfere with Candida adhesion.

BACKGROUND: Lactobacillus spp. dominating the vaginal microbiota of healthy women contribute to the prevention of urogenital and sexually transmitted infections. Their protective role in the vagina can be mediated by Lactobacillus cells themselves, metabolites or bacterial components, able to interfere with pathogen adhesion and infectivity. Vulvovaginal candidiasis (VVC) is a common genital infection, caused by the overgrowth of opportunistic Candida spp. including C. albicans, C. glabrata, C. krusei and C. tropicalis. Azole antifungal drugs are not always efficient in resolving VVC and preventing recurrent infections, thus alternative anti-Candida agents based on vaginal probiotics have gained more importance. The present work aims to chemically characterize the biosurfactant (BS) isolated from a vaginal Lactobacillus crispatus strain, L. crispatus BC1, and to investigate its safety and antiadhesive/antimicrobial activity against Candida spp., employing in vitro and in vivo assays. RESULTS: BS isolated from vaginal L. crispatus BC1 was characterised as non-homogeneous lipopeptide molecules with a critical micellar concentration value of 2 mg/mL, and good emulsification and mucoadhesive properties. At 1.25 mg/mL, the BS was not cytotoxic and reduced Candida strains' ability to adhere to human cervical epithelial cells, mainly by exclusion mechanism. Moreover, intravaginal (i.va.) inoculation of BS in a murine experimental model was safe and did not perturb vaginal cytology, histology and cultivable vaginal microbiota. In the case of i.va. challenge of mice with C. albicans, BS was able to reduce leukocyte influx. CONCLUSIONS: These results indicate that BS from vaginal L. crispatus BC1 is able to interfere with Candida adhesion in vitro and in vivo, and suggest its potential as a preventive agent to reduce mucosal damage occasioned by Candida during VVC.