Antimicrobial and co-aggregative properties of vaginal lactobacilli for their inclusion as active pharmaceutical ingredients for pharmabiotic products.

PRISCILLA DE GREGORIO; MARIA SILVINA, JUAREZ TOMAS; MARIA ELENA FÁTIMA NADER

Rosario

Congreso; 2da Reunión Internacional de Ciencias Farmacéuticas RICIFA,; 2012

UNR, UNC

Introduction Urogenital tract infections (UGTI) in women generate high morbidity and recurrence rates, probably due to failures of the antimicrobial treatments that are not able to restore the lactobacilli dominated vaginal microbiome. Pharmabiotics are promising alternatives to prevent or treat UGTI, and are defined as “live or dead microorganisms as well as microbial constituents and metabolites which can beneficially interact with the host”(1). In pharmaceutical biotechnology, microbial cells and/or their metabolites are API of the pharmabiotics. Our research group isolated and selected human vaginal lactobacilli, which could be included as API in pharmabiotic products to re-establish the balance of the urogenital tract(2). The aim of this work was to evaluate the antimicrobial and co-aggregative properties of selected lactobacilli on pathogens causing UGTI in “in vitro” assays.   Materials and methods Vaginal lactobacilli (Lactobacillus gasseri CRL1263, L. gasseri CRL1509, Lactobacillus reuteri CRL1324, Lactobacillus salivarius CRL1328 and Lactobacillus rhamnosus CRL1332) and urogenital pathogens (Streptococcus agalactiae NH16, NH17, NH18 and GB96, Staphylococcus aureus and Candida albicans F1 and F8) were grown in LAPTg broth at 37°C. The effect of their microbial supernatants was evaluated with the agar plate diffusion method, using lactobacilli and pathogens, both as indicator and/or as producer of antimicrobial substances. Associative cultures of L. gasseri CRL1263 or CRL1509 and S. agalactiae NH17 were performed in LAPTg broth at 37°C. The inocula were 106 and 107 colony forming units (CFU)/ml for lactobacilli and 105 CFU/ml for S. agalactiae. Bacterial growth was determined by the plate dilution method using MRS (pH 5.5) for lactobacilli and Todd Hewitt for streptococci. The antimicrobial lactic acid and hydrogen peroxide levels produced during the microbial growth were determined by HPLC and the peroxidase chromogenic methods, respectively. Co-aggregation between lactobacilli and pathogens was assayed monitoring the modifications of the optical density at 600nm of bacterial suspensions in PBS buffer. Data were analyzed using ANOVA-general linear model.   Results L. gasseri CRL1509 and L. reuteri CRL1324 slightly inhibited the growth of S. agalactiae NH17 and/or GB96. L. reuteri CRL1324 was the only strain that resulted inhibited by the S. agalactiae NH16 pathogen. In the associative cultures of lactobacilli and S. agalactiae, pathogen growth was significantly inhibited faster (from 4 and 8 h of incubation) at higher Lactobacillus inoculum, following the increase of lactic acid and H2O2 levels. The lowest number of viable pathogen (reduction of at least 5 log units) was reached from 12 h co-culture. The highest co-aggregation values were recorded for C. albicans or S. agalactiae strains in presence of L. gasseri CRL1509 or L. gasseri CRL 1263, respectively.   Conclusions Inhibitory and co-aggregative abilities of candidate API lactobacilli would promote the elimination of pathogens. The Lactobacillus strains studied are excellent candidates to be included in a pharmabiotic product, contributing thus to its functionality in relation to the protection of the urogenital tract health   References 1) Shanahan F. Therapeutic implications of manipulating and mining the microbiota. JPhysiol.2009;587 (17):4175-9. 2) Juárez Tomás MS, Saralegui Duhart CI, De Gregorio PR, Vera Pingitore E, Nader-Macías ME. Urogenital pathogen inhibition and compatibility between vaginal Lactobacillus strains to be considered as probiotic candidates. Eur JObstet Gynecol Reprod Biol.2011;159(2):399-406.