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.

In this study, we sought to find novel bacterial and metabolic hallmarks for bacterial vaginosis (BV). We studied the vaginal microbiome and metabolome of vaginal fluids from BV-affected patients (n = 43) and healthy controls (n = 37) by means of an integrated approach based on quantitative polymerase chain reaction (qPCR) and proton nuclear magnetic resonance ((1)H-NMR). The correlations between the clinical condition and vaginal bacterial communities were investigated by principal component analysis (PCA). To define the metabolomics signatures of BV, 100 discriminant analysis by projection on latent structure (PLS-DA) models were calculated. Bacterial signatures distinguishing the health condition and BV were identified by qPCR. Lactobacillus crispatus strongly featured the healthy vagina, while increased concentrations of Prevotella, Atopobium and Mycoplasma hominis specifically marked the infection. (1)H-NMR analysis has led to the identification and quantification of 17 previously unreported molecules. BV was associated with changes in the concentration of metabolites belonging to the families of amines, organic acids, short chain fatty acids, amino acids, nitrogenous bases and monosaccharides. In particular, maltose, kynurenine and NAD(+) primarily characterised the healthy status, while nicotinate, malonate and acetate were the best metabolic hallmarks of BV. This study helps to better understand the role of the vaginal microbiota and metabolome in the development of BV infection. We propose a molecular approach for the diagnosis of BV based on quantitative detection in the vaginal fluids of Atopobium, Prevotella and M. hominis, and nicotinate, malonate and acetate by combining qPCR and (1)H-NMR.

Coronary artery disease is the most common cause of cardiac failure in the Western world, and to date there is no alternative to bypass surgery for severe coronary atherosclerosis. We report that c-kitpositive cardiac progenitor cells (CPCs) activated with insulin-like growth factor 1 and hepatocyte growth factor before their injection in proximity of the site of occlusion of the left coronary artery in rats, engrafted within the host myocardium forming temporary niches. Subsequently, CPCs divided and differentiated into endothelial cells and smooth muscle cells and, to a lesser extent, into cardiomyocytes. The acquisition of vascular lineages appeared to be mediated by the up-regulation of hypoxia-inducible factor 1␣, which promoted the synthesis and secretion of stromal-derived factor 1 from hypoxic coronary vessels. Stromal-derived factor 1 was critical in the conversion of CPCs to the vascular fate. CPCs formed conductive and intermediate-sized coronary arteries together with resistance arterioles and capillaries. The new vessels were connected with the primary coronary circulation, and this increase in vascularization more than doubled myocardial blood flow in the infarcted myocardium. This beneficial effect, together with myocardial regeneration attenuated postinfarction dilated myopathy, reduced infarct size and improved function. In conclusion, locally delivered activated CPCs generate de novo coronary vasculature and may be implemented clinically for restoration of blood supply to the ischemic myocardium.coronary blood flow ͉ infarct size ͉ myocardial regeneration ͉ stem cells ͉ vasculogenesis

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 vaginal microbiota, dominated by Lactobacillus spp., plays a key role in preventing HIV-1 transmission. Here, we investigate whether the anti-HIV effect of lactobacilli is mediated by extracellular vesicles (EVs) released by these bacteria. Human cervico-vaginal and tonsillar tissues ex vivo, and cell lines were infected with HIV-1 and treated with EVs released by lactobacilli isolated from vaginas of healthy women. EVs released by L. crispatus BC3 and L. gasseri BC12 protect tissues ex vivo and isolated cells from HIV-1 infection. This protection is associated with a decrease of viral attachment to target cells and viral entry due to diminished exposure of Env that mediates virus-cell interactions. Inhibition of HIV-1 infection is associated with the presence in EVs of several proteins and metabolites. Our findings demonstrate that the protective effect of Lactobacillus against HIV-1 is, in part, mediated by EVs released by these symbiotic bacteria. If confirmed in vivo, this finding may lead to new strategies to prevent male-to-female sexual HIV-1 transmission.

The vaginal microbiota plays a crucial role in maintaining the health and functioning of the female genital tract, preventing the colonization of urogenital pathogens and sexually transmitted infections. In this study, we characterized the vaginal bacterial communities and the metabolome associated to Chlamydia trachomatis infection (CT: 20 women), compared to healthy condition (H: 22 women) and bacterial vaginosis (BV: 19 women). A microarray-based tool (VaginArray), implemented with a real-time PCR for Gardnerella vaginalis, was used to determine the vaginal bacterial composition, whereas the metabolic profiles were assessed by a proton-based nuclear magnetic resonance (1H-NMR) spectroscopy. CT infection was characterized by bacterial and metabolic signatures similar to healthy condition, even though higher amounts of Lactobacillus iners, as well as depletion of some amino acids, biogenic amines, and succinate marked CT infection. Moreover, the frequency of Lactobacillus crispatus was higher in asymptomatic CT-positive patients than in women with CT-correlated symptoms. We also confirmed the marked differences in the microbiome and metabolome between healthy and BV-affected women. In conclusion, we highlighted microbial and metabolic peculiarities of the vaginal ecosystem in the case of CT infection, even though further studies are needed to understand if the observed alterations precede the infection onset or if the pathogen itself perturbs the vaginal environment.

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 min). 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.