BIFIDOBACTERIUM BREVE BC104: ORALLY ADMINISTERED VAGINAL PROBIOTIC FOR PREVENTING GENITAL AND URINARY TRACT INFECTIONS

Purpose: The first aim of this study was to investigate the probiotic potential of Bifidobacterium breve BC104, isolated from a vaginal swab1, by assessing its antimicrobial activity against bacteria and fungi responsible for urogenital diseases (Candida spp., C. trachomatis, E. coli, E. faecalis, E. faecium, S. faecium, E. hirae, S. aureus and P. mirabilis) and against gastrointestinal pathogens (E. coli enterotoxigentic, S. typhimurium and Y. enterocolitica). To provide functional benefits to the gut and urogenital tract, oral probiotics must survive passage through the acidic stomach and colonize colon. Thus, tolerance to bile salts and low pH, as well as adhesion to Caco-2 and HeLa cells, were investigated. The second aim was to develop an oral gastro-resistant and mucoadhesive formulation able to favour bacterial survival during storage and following administration and bacterial intestinal and vaginal colonization. Methods: Antifungal activity was tested against Candida spp. by broth micro-dilution method. The anti-Chlamydia activity was measured as reduction in Chlamydia infectivity to HeLa cells treated with BC104. The antibacterial activity against the other pathogens was tested by using agar spot test2. The capability of Bifidobacterium to adhere to Caco-2 and HeLa cells was assessed by microscopy after Giemsa staining. To mimic gastric and intestinal conditions, Bifidobacterium cells were suspended in simulated gastric juice (pH 2) for three hours and then in simulated intestinal fluid (pH 7) for other three hours. Aliquots were taken every hours to estimate viability over time3. The oral formulation was obtained by spray-drying followed by prilling. Firstly, the overnight culture of BC104 was suspended in prebiotic media, containing an encapsulating agent (reconstituted skim milk 10%) and prebiotic (inulin 5%, oligofructose 5% and ascorbic acid 2.5%)4. Spray-drying process was performed at constant inlet temperature of 120°C. Secondly, gastro-resistant mucoadhesive beads were obtained by dripping and cooling of molten lauric acid (75%, p/p) containing CMC (10%, p/p) and spray-dried microcapsules (15%, p/p). Viability after the manufacturing procedure was evaluated as well as BC104 release from the beads. Results: B. breve BC104 exhibited a strong fungistatic activity against C. albicans and was able to reduce Chlamydia infectivity to HeLa cells. Moreover, BC104 exerted a good antibacterial activity towards all other pathogens examined, in particular against Escherichia coli, Staphylococcus aureus and Yersinia enterocolitica. BC104 showed a good ability to adhere to Caco-2 but not to HeLa cells, accordingly to the fact that bifidobacteria are a minor component of vaginal ecosystem, while they are essential in maintaining gastrointestinal homeostasis. Even after three hours at pH 2 and further exposure to the shift of pH under intestinal condition, BC104 maintained elevated cell concentration (> 105 CFU/mL), showing an intrinsic ability to resist to gastric acidity. Microcapsules containing B. breve showed high survival after spray drying (1010 CFU/g) and after 4 weeks of storage at 4°C. BC104 maintained also a good viability after incorporation inside beads (1.7*107 CFU/g). Conclusions: The results show that B. breve BC104 possess a good antimicrobial activity and can be deliver in oral gastro-resistant formulation. 1 Parolin, C., Marangoni, A., Laghi, L., Foschi, C., Ñahui Palomino, R.A., Calonghi, N., Cevenini, R., Vitali, B. Isolation of Vaginal Lactobacilli and Characterization of Anti-Candida Activity. PLoS One 2015, 10(6), e0131220. 2 Schillinge,r U., Lücke, F.K. Antibacterial activity of Lactobacillus sake isolated from meat. Appl Environ Microbiol 1989, 55(8), 1901-6. 3 Fernández, M.F., Boris, S., Barbés, C. Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract. J Appl Microbiol 2003, 94(3), 449-55. 4 Fritzen-Freire, C.B., Prudencio, E.S., Amboni, R.D.M.C., Pinto, S.S., Negrao-Murakami, A.N., Murakami, F. S. Microencapsulation of bifidobacteria by spray drying in the presence of prebiotics. Food Research Internationa 2011, 45, 306-312.