Functional and safety characterization of beneficial vaginal lactic acid bacteria for the design of vaginal hygiene products.

AIMS: The aim was to complement the evaluation of functional and safety properties of beneficial vaginal lactic acid bacteria to select the most adequate strains as potential probiotics for their inclusion in a vaginal probiotic formula. METHODS AND RESULTS: Twenty-four beneficial vaginal lactic acid bacteria strains previously isolated from healthy women were characterized by applying phenotypical and genetic techniques. The biofilm formation and exopolysaccharide production by the different strains showed a strong influence of the growth medium on the expression of these properties. The evaluated strains evidenced different antibiotic susceptibility patterns by phenotypic tests, while the resistance genes were not always correlated with the phenotypic profiles. None of the evaluated strains showed lecithinase, gelatinase, or ß-hemolytic activity. Based on the results obtained, Lactobacillus gasseri CRL 1320 and Lacticaseibacillus rhamnosus CRL 1332 were selected to advance in the design of the products. CONCLUSIONS: A wide variability in the properties expressed by beneficial vaginal lactic acid bacteria strains were shown, evidencing a strain-specific and independent behaviour of the species/metabolic group where classified. The selection criteria applied provided a valuable tool for the selection of the best vaginal lactobacilli strains for their inclusion in formulas for vaginal application. SIGNIFICANCE AND IMPACT OF THE STUDY: Since probiotics are an important tool for improving human health, the designed formula are an excellent alternative to reconstitute the vaginal microbiota and prevent/treat urogenital tract infections in women. The results obtained contribute to deepen the characterization of homologous beneficial vaginal lactic acid bacteria and advance in the design of vaginal probiotic products.

Screening of autochthonous vaginal beneficial lactobacilli strains by their growth at high temperatures for technological applications.

The pharmaceutical industry shows an emerging interest in formulas that contain live and beneficial microorganisms, also known as probiotics or pharmabiotics, which in many cases, are host-specific. The resistance to higher temperature is an essential feature of these microorganisms when working on the design of products for vaginal formula. In order to obtain a high number of viable cells and a prolonged shelf life in the designed product, it is required to apply technological procedures using high temperatures or abrupt changes of them, which result in conditions that are different from the optimal growth temperature and can affect the metabolic capabilities of the bacteria when administered to the host in order to reestablish the ecological mucosa. The aim of this work was to evaluate the behavior of 30 different species and strains of autochthonous beneficial vaginal lactobacilli (BVL) when exposed to high temperatures, determine their survival capabilities and analyze their pre-adaptation to those temperatures, in order that they still maintain their viability after technological processes and further conservation. BVL were exhibited to temperatures higher than optimal, with the purpose of evaluating their growth kinetics and parameters. Later, they were exposed to higher temperatures, and then, returned to their optimal, to determine if they were able to grow again. The strains that showed higher resistance were selected, and their viability and beneficial properties studied further. The growth kinetics of strains exposed to higher temperatures showed different patterns, which provided evidence that the thermal adaptation is strain-dependent and is not related to any particular species and/or metabolic group in which the strains were taxonomically classified. The pre-adaptive step allowed the growth of some of the strains, preserving their viability and probiotic properties after the high temperatures were applied. The results shows that BVL can be exposed to high temperatures used in different technological processes that are applied for pharmabiotic formulations, such as spray dried or vacuum rotary evaporation, and/or during the conservation period. The results obtained indicate that some specific BVL strains resist high temperatures and grow afterwards at optimal conditions.

Survival of Beneficial Vaginal Lactobacilli (BVL) to Different Gastrointestinal Tract Conditions.

BACKGROUND: Lactobacilli are the dominant bacteria in the healthy vaginal tract, preventing the income of pathogenic microorganisms, either sexually or not transmitted. Probiotics are used to restore the vaginal microbiome by local administration. However, the ascendant colonization is proposed as a way to restore the vaginal balance, and to exert some complementary effects on the host, situation that requires that probiotic strains resist the gastrointestinal tract passage. OBJECTIVE: To determine which probiotic vaginal strains were able to resist different gastrointestinal factors (pH, bile salts, and enzymes) to advance in the design of oral formulas. METHODS: Different protocols were applied to evaluate the growth of 24 beneficial vaginal lactic bacteria (BVL) strains at low pH and high bile salts (individually evaluated) and in combined protocols. The viability of the strains in simulated gastrointestinal tract conditions was studied to select the most resistant strains. RESULTS: A low number of BVL was able to grow at low pH. Most of the strains did not survive at high bile salts concentration. The passage through pH first and bile salts later showed that only three strains were able to survive. In the simulated intestinal conditions, only Lactobacillus gasseri CRL1290, L. jensenii CRL1313, and L. jensenii CRL1349 decrease one or two logarithmic growth units (UFC/ml) at the end of the assay, maintaining their beneficial properties. CONCLUSION: The behavior of BVL in the conditions assayed is not related to specific strain or metabolic group, because the resistance is strain-specific. The results highlight the importance of the screening performed in a way to select the most adequate strains to be included in the oral designed formula for the restoration of the vaginal tract microbiome.

Anti-Candida activity of beneficial vaginal lactobacilli in in vitro assays and in a murine experimental model.

Vulvovaginal candidiasis (VVC) is one of the most frequent infections affecting women worldwide. Healthy vaginal microbiota is dominated by lactobacilli, which form a strong defense line against pathogens. In this work, in vitro antimicrobial properties of thirty vaginal Lactobacillus strains were evaluated against eleven Candida vaginal clinical isolates, employing three different methods. Also, the effect of intravaginal (i.va.) administrations (preventive, therapeutic and preventive-therapeutic) of L. reuteri CRL1324 or L. rhamnosus CRL1332 strains against the i.va. challenge with Candida albicans C2 (C.a.) was evaluated in a murine experimental model. From the results of agar overlay and liquid medium assays the selected lactobacilli strains have shown to inhibit the growth of at least one Candida strain. The inhibition was mainly due to the effect of organic acids. Anti-Candida activity was not evidenced in the agar plate diffusion method. In the experimental murine model, only preventive-therapeutic administration of both lactobacilli was able to significantly reduce viable C.a. numbers recovered in vaginal washes and the leukocyte influx induced by the fungi. In conclusion, lactobacilli exhibited in vitro and in vivo antimicrobial effects on Candida, suggesting that they could be promising candidates for protection against VVC.

Contraception: Influence on Vaginal Microbiota and Identification of Vaginal Lactobacilli Using MALDI-TOF MS and 16S rDNA Sequencing.

BACKGROUND: The vaginal microbiome is influenced by a wide variety of factors, including contraceptive methods. AIM: To evaluate the effect of contraceptive methods on vaginal microbiota and to compare MALDI-TOF MS and 16S rDNA sequencing for lactobacilli identification. PATIENTS AND METHODS: One hundred and one (101) women consulting for birth control were included in a prospective study. Their vaginal content was sampled and analyzed once before they started using the contraceptive method of their choice, and twice after the initiation of contraception, at three months (94/101 women attended) and at six months (89/101 women attended). The relative frequencies of yeasts and trichomonas were analyzed. MALDI-TOF MS and 16S rDNA sequence analysis were applied for the identification of lactobacilli in their vaginal microbiota. The following contraceptive methods were assessed: Combined Oral Contraceptive Pill (COCP), Condom (CON) and The Rhythm Method (RHYT). McNemar's statistical test was applied. RESULTS: A statistically significant association between COCP and normal microbiota was observed after three months (p< 0.01) and after six months (p< 0.0001), when the vaginal microbiota was modified. At six months, inflammatory reaction was detected in 3/7 women in the CON group, while 6/7 patients using RHYT showed the same state. Yeast colonization increased with the COCP. Identification of lactobacilli by MALDI-TOF MS analysis compared to 16S rDNA sequencing yielded 92.9% concordant results. Lactobacillus gasseri and L. crispatus were the predominant species. CONCLUSION: The pattern of vaginal states was significantly modified. Hormone administration apparently corrected the alterations and retained a normal vaginal state. MALDI-TOF MS has the potential of being an accurate tool for the identification of vaginal lactobacilli species L. murinus was for the first time isolated from the vagina.