Caratterizzazione di ceppi di Lactobacillus crispatus con potenzialità probiotiche per la salute della donna

Research on human microbiota and its effects on human health and diseases has become a more prominent area of study in recent years, with the purpose of improving the quality of human life or preventing possible potential illnesses. Human microbiota is a collection of microorganisms living in close association with different body districts, such as gastrointestinal tract, skin and vagina, and contributes to their correct functioning by moderating biochemical and physiological processes. Vaginal microbiota plays a central role in women's health. It is essentially composed of bacteria of the Lactobacillus genus, characterized by a low degree of diversity and by a strong dynamic of change in its composition under the influence of different factors, such as age, diet, hormonal fluctuations, and geography. However, the disruption of vaginal ecosystem, known as dysbiosis, is correlated to diverse diseases state, among the others, bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC). VVC is caused by Candida albicans which, instead of being part of the normal vaginal microflora, becomes a robust opportunistic fungal pathogen and overgrows. It affects about 75% of reproductive-aged women at least once during their lives. Standard treatments, such as antibiotics or fluconazole, are not able to spontaneously restore the normal flora and provide long-term protection, often resulting in relapses and recurrences. A variety of probiotics, primarily Lactobacillus spp., have been identified as potential therapeutic agents, with Lactobacillus crispatus in particular shown to attenuate the virulence of C. albicans. However, no European market products containing L. crispatus are currently available in a vaginal preparation. Taking this lack into consideration, the aim of the present thesis project was to characterize new probiotic strains of L. crispatus and to assess their effectiveness in order to further develop a probiotic blend specifically formulated in treating BV and VVC. Firstly, four different strains were isolated based on different phenotypic attributes (IR Biotyper profiles). Subsequently, they were genetically characterized using Polymerase Chain Reaction (PCR) and Pulsed Field Gel Electrophoresis (PFGE), and metabolically, through API 50 CHL and API ZYM analysis. Aside from the 4 newly identified isolates (LCRA, LCRB, LCRD, and LCRE), the strains LCR04 (L. crispatus) and LP01 (L. plantarum) from Probiotical spa collection were also included. After that, effectiveness of selected strains, administered both alive and heat-treated form, was evaluated on intestinal barrier model, by using Caco-2 cells, and on vaginal epithelium model, by using VK2/E6E7 cells. The interest in the effect on intestinal cells is because a potential supplement product for the improvement of the vaginal microbiome can be applied locally to the vaginal area or taken orally and thus act through the intestine-vagina axis or intestine-estrogen axis. In different experimental settings, the strains safety and their probiotic potential to protect vaginal cells after C. albicans infection were evaluated. Results showed that the strains were effective in reducing cytotoxicity and preserving cell vitality; the alive forms were more effective than the heat-treated form in restoring a normal physiological state in the vaginal epithelium when exposed to C. albicans, while the heat-treated forms had an antioxidant effect. Interestingly, phenotypic and metabolic disparities, were revealed by the analysis performed in the first part of this work, were reflected in different outcomes of the strains. This work provides the foundation for the development of a probiotic blend specifically formulated to prevent and treat bacterial vaginosis and vulvovaginal candidiasis.