Aims: To assess the ability of vaginal lactobacilli to form biofilm under different culture conditions and to determine the relationship between their growth and the capability of biofilm formation by selected strains. Methods and Results: Fifteen Lactobacillus strains from human vagina were tested for biofilm formation by crystal violet staining. Only Lactobacillus rhamnosus Centro de Referencia para Lactobacilos Culture Collection (CRL) 1332, Lact. reuteri CRL 1324 and Lact. delbrueckii CRL 1510 were able to grow and form biofilm in culture media without Tween 80. However, Lact. gasseri CRL 1263 (a non-biofilm-forming strain) did not grow in these media. Scanning electron microscopy showed that Lact. rhamnosus CRL 1332 and Lact. reuteri CRL 1324 formed a highly structured biofilm, but only Lact. reuteri CRL 1324 showed a high amount of extracellular material in medium without Tween. Conclusions: Biofilm formation was significantly influenced by the strain, culture medium, inoculum concentration, microbial growth and chemical nature of the support used for the assay. Significance and Impacts of the Study: The results allow the selection of biofilm-forming vaginal Lactobacillus strains and the conditions and factors that affect this phenomenon.

Maternal GBS colonization is one of the most important risk factors for developing disease in newborns. Lactobacillus reuteri CRL1324 could be considered as a new biological agent to reduce infections caused by this micro-organism.

The aim of this work was to evaluate the effects of beneficial human vaginal lactobacilli (Lb) on urogenital pathogens through in vitro and in vivo experiments. Co-aggregative and antimicrobial properties between five vaginal Lb strains and urogenital pathogens or potential pathogens (Streptococcus agalactiae, Staphylococcus aureus and Candida albicans strains) were assayed. Also, associative cultures of Lb strains and S. agalactiae were performed and bacterial growth, pH, lactic acid and hydrogen peroxide (H 2 O 2 ) were determined at different times. Based on the results obtained, the in vivo studies were assayed in mice with Lactobacillus gasseri CRL 1509 or Lactobacillus salivarius CRL 1328 inoculated intravaginally (i.v.) and then challenged i.v. with S. agalactiae. Results were analysed by ANOVA (repeated measures and general linear models). Most of the Lb strains increased the percentage of aggregation of S. agalactiae strains. Only one strain (Lactobacillus reuteri CRL 1324) positively affected the aggregation of S. aureus and none increased the aggregation of C. albicans. The inhibition of the growth of S. agalactiae strains by production of organic acids by lactobacilli was evidenced. The Lb-S. agalactiae co-cultures showed a significant inhibition of the pathogen after 4 h and 8 h of incubation. Parallel increases in lactic acid and H 2 O 2 levels were observed. However, in the experimental murine model, no significant differences were obtained in the number of streptococci recovered from the vaginal tract of control mice and those inoculated with Lb. In conclusion, vaginal Lb exhibited in vitro coaggregative and antimicrobial effects on S. agalactiae strains, suggesting that they could be promising candidates for protection against S. agalactiae challenge. However, as these effects were not evidenced in the murine model used, further animal studies under different experimental conditions should be conducted to evaluate the preventive effect of Lb against challenge with S. agalactiae.

This is the first study on auto-aggregation, adhesion and biofilm formation of vaginal Lactobacillus strains by phenotypic and genetic assays.

Adhesion and biofilm formation are strain properties that reportedly contribute to the permanence of lactobacilli in the human vagina. The kinetics of biofilm formation and the chemical nature of the biofilm matrix formed by Lactobacillus reuteri CRL (Centro de Referencia para Lactobacilos Culture Collection) 1324 and Lactobacillus rhamnosus CRL 1332, vaginal beneficial strains, were evaluated in this work. Crystal violet-stained microplate assay and techniques of epifluorescence, electron and confocal microscopy were applied. The highest density and complexity of biofilms of both vaginal lactobacilli were observed at 72 h of incubation. Protease, proteinase K, α-chymotrypsin and trypsin treatments efficiently detached L. reuteri CRL 1324 biofilm that was also partially affected by α-amylase. However, L. rhamnosus CRL 1332 biofilm was slightly affected by protease, proteinase K and α-amylase. Confocal microscopy revealed greater amount of polysaccharides in L. rhamnosus CRL 1332 biofilm matrix than in L. reuteri CRL 1324 biofilm matrix. The results indicate that proteins are one of the main components of the L. reuteri CRL 1324 biofilm, while the biofilm matrix of L. rhamnosus CRL 1332 is composed of carbohydrates and proteins. The results obtained support the knowledge, understanding and characterization of two biofilm-forming vaginal Lactobacillus strains.