Lactobacillus Biofilms Influence Anti-Candida Activity.

Lactobacilli are the dominant members of the healthy human vaginal microbiota and represent the first defense line from pathogen infection, including vulvovaginal candidiasis. Biofilm is the predominant microbial growth form in nature, and the formation of biofilms inside the human body has important implications in health and disease. In particular, the formation of biofilm by members of the human resident microbiota is desirable, as it can improve microbial persistence and influence functionality. In the present study, we investigated the capability of 16 vaginal Lactobacillus strains (belonging to Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus vaginalis, and Lactobacillus plantarum species) to form biofilms, and we correlated their mode of growth to anti-Candida activity. L. plantarum strains were the best biofilm producers, and high variability was registered in the level of biofilm formation among L. crispatus and L. gasseri strains. Culture supernatants derived from Lactobacillus biofilm and planktonic growth were tested toward a panel of Candida clinical isolates (Candida albicans, Candida glabrata, Candida lusitaniae, Candida tropicalis, Candida krusei, and Candida parapsilosis) and their metabolome assessed by 1H-NMR. L. crispatus and L. plantarum strains exhibited the best fungistatic profile, and biofilms enhanced their anti-Candida activity; on the contrary, L. gasseri strains were more effective when grown in a planktonic mode. Biofilm/planktonic mode of growth also affects Lactobacillus metabolism, mainly influencing nitrogen and amino acid pathways, and anti-Candida activity is instead strictly related to carbohydrate metabolism. The present study underlined the strict interdependence between microbial mode of growth, metabolism, and functional properties. Biofilm formation by members of the healthy human microbiota represents a crucial issue in the field of microbial physiology and host-microbiota interactions, beyond supporting the development of new antimycotic strategies based on probiotics grown in adherence.

Probiotic lactobacilli in formulas and hygiene products for the health of the urogenital tract.

Lactobacilli are the predominant microorganisms of the healthy human vagina. A novel alternative for the prevention and treatment of female urogenital tract infections (UGTI) is the inclusion of these microorganisms as active pharmaceutical ingredients in probiotic formulas, and more recently in female hygienic products. Probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host." A list of requirements must be considered during the development of probiotic product/formula for the female urogenital tract (UGT). This review aims to resume the requirements, probiotic characteristics, and clinical trial applied to determine the effect of probiotic and potentially probiotic strains on different woman's physiological and pathological conditions, and in preterm birth prevention. A revision of female hygienic products available in the world market is included, together with novel studies applying nanotechnology for Lactobacillus incorporation in hygienic products. Further studies and well-designed clinical trials are urgently required to complement the current knowledge and applications of probiotics in the female UGT. The use of probiotic formulas and products will improve and restore the ecological equilibrium of the UGT microbiome to prevent and treat UGTI in women under different conditions.

Immobilization of vaginal Lactobacillus in polymeric nanofibers for its incorporation in vaginal probiotic products.

Probiotic products require high number of viable and active microorganisms during storage. In this work, the survival of human vaginal Lactobacillus gasseri CRL1320 and Lactobacillus rhamnosus CRL1332 after nanofiber-immobilization by electrospinning with polyvinyl-alcohol, and during storage was evaluated. The optimization of bacterial immobilization and storage conditions using bioprotectors (skim milk-lactose and glycerol) and oxygen-excluding packaging was carried out, compared with lyophilization. After electrospinning, a higher survival rate of L. rhamnosus (93%) compared to L. gasseri (84%) was obtained in nanofibers, with high viable cells (>107 colony-forming unit/g) of the two probiotics in nanofibers stored at -20°C up to 14 days. The storage in oxygen-excluding packaging was an excellent strategy to extend the shelf-life of L. rhamnosus (up to 1.7 × 108 CFU/g) in nanofibers stored at 4°C during 360 days, with no addition of bioprotectives, resulting similar to freeze-dried-cells. L. rhamnosus was successfully incorporated into polymeric hydrophilic nanofibers with a mean diameter of 95 nm. The composite materials were characterized in terms of morphology, and their physicochemical and thermal properties assessed. Nanofiber-immobilized L. rhamnosus cells maintained the inhibition to urogenital pathogens. Thus, polymeric nanofiber-immobilized L. rhamnosus CRL1332 can be included in vaginal probiotic products to prevent or treat urogenital infections.

Biosurfactant from vaginal Lactobacillus crispatus BC1 as a promising agent to interfere with Candida adhesion.

BACKGROUND: Lactobacillus spp. dominating the vaginal microbiota of healthy women contribute to the prevention of urogenital and sexually transmitted infections. Their protective role in the vagina can be mediated by Lactobacillus cells themselves, metabolites or bacterial components, able to interfere with pathogen adhesion and infectivity. Vulvovaginal candidiasis (VVC) is a common genital infection, caused by the overgrowth of opportunistic Candida spp. including C. albicans, C. glabrata, C. krusei and C. tropicalis. Azole antifungal drugs are not always efficient in resolving VVC and preventing recurrent infections, thus alternative anti-Candida agents based on vaginal probiotics have gained more importance. The present work aims to chemically characterize the biosurfactant (BS) isolated from a vaginal Lactobacillus crispatus strain, L. crispatus BC1, and to investigate its safety and antiadhesive/antimicrobial activity against Candida spp., employing in vitro and in vivo assays. RESULTS: BS isolated from vaginal L. crispatus BC1 was characterised as non-homogeneous lipopeptide molecules with a critical micellar concentration value of 2 mg/mL, and good emulsification and mucoadhesive properties. At 1.25 mg/mL, the BS was not cytotoxic and reduced Candida strains' ability to adhere to human cervical epithelial cells, mainly by exclusion mechanism. Moreover, intravaginal (i.va.) inoculation of BS in a murine experimental model was safe and did not perturb vaginal cytology, histology and cultivable vaginal microbiota. In the case of i.va. challenge of mice with C. albicans, BS was able to reduce leukocyte influx. CONCLUSIONS: These results indicate that BS from vaginal L. crispatus BC1 is able to interfere with Candida adhesion in vitro and in vivo, and suggest its potential as a preventive agent to reduce mucosal damage occasioned by Candida during VVC.

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.

Beneficial rhizobacteria immobilized in nanofibers for potential application as soybean seed bioinoculants.

Seed inoculation with plant growth promoting rhizobacteria (PGPR) is an ideal tool to supply the soil with a high density of beneficial microorganisms. However, maintaining viable microorganisms is a major problem during seed treatment and storage. In this work, an evaluation was made of the effect of bacterial immobilization in nanofibers on the stability (viability and maintenance of beneficial properties) of two potential PGPR, Pantoea agglomerans ISIB55 and Burkholderia caribensis ISIB40. Moreover, the impact of soybean seed coating with nanofiber-immobilized rhizobacteria on bacterial survival during seed storage and on germination and plant growth parameters was determined. Bacterial nanoimmobilization and subsequent seed coating with nanofiber-immobilized rhizobacteria were carried out by electrospinning. The results demonstrate that this technique successfully immobilized P. agglomerans ISIB55 and B. caribensis ISIB40 because it did not affect the viability or beneficial properties of either rhizobacteria. Seed coating with nanofiber-immobilized rhizobacteria improved P. agglomerans ISIB55 and B. caribensis ISIB40 survival on seeds stored for 30 days and contributed to the successful colonization of both bacteria on the plant root. Moreover, seed coating with P. agglomerans ISIB55 increased germination, length and dry weight of the root. Furthermore, seed coating with B. caribensis ISIB40 increased leaf number and dry weight of the shoot. Therefore, the technique applied in the present work to coat seeds with nanofiber-immobilized PGPR could be considered a promising eco-friendly approach to improve soybean production using a microbial inoculant.

Immunomodulation of Lactobacillus reuteri CRL1324 on Group B Streptococcus Vaginal Colonization in a Murine Experimental Model.

PROBLEM: Maternal Group B Streptococcus (GBS) colonization is a risk factor for infectious disease in newborns. One promising strategy is the modulation of vaginal defense to increase the host's ability to combat infection. METHOD OF STUDY: The effect of intravaginal (i.va.) Lactobacillus reuteri CRL1324 inoculation on different immune cell populations, cytokines, and immunoglobulin isotypes in a murine model of GBS vaginal colonization was evaluated. RESULTS: Seven i.va. inoculations of L. reuteri CRL1324 previous to GBS challenge showed an immunomodulatory effect on the cells and mediators of innate immunity, decreasing the number of neutrophils induced by the pathogen and increasing the activated macrophage population. Moreover, increases in B lymphocytes and IgA and IgG subclasses were observed in mice inoculated with L. reuteri CRL1324 and then challenged with GBS. CONCLUSION: Lactobacillus reuteri CRL1324 shows a protective effect against GBS colonization that could be mediated by the modulation of the immune response.

Encapsulation and subsequent freeze-drying of Lactobacillus reuteri CRL 1324 for its potential inclusion in vaginal probiotic formulations.

Probiotic formulations must include a high number of viable and active microorganisms. In this work, the survival of human vaginal Lactobacillus reuteri CRL 1324 during encapsulation, lyophilization and storage, and the activity of encapsulated and/or freeze-dried bacterial cells were evaluated. Extrusion-ionic gelation technique was applied to encapsulate L. reuteri CRL 1324, using xanthan and gellan. Encapsulated and free bacterial cells were freeze-dried with or without lactose and skim milk as lyoprotectors. The different systems obtained were stored at room temperature and at 4°C for 150days. The following determinations were performed: L. reuteri CRL 1324 viability, microorganism released from capsules, survival in a medium simulating the vaginal fluid and maintenance of beneficial properties (growth inhibition of opportunistic pathogenic Streptococcus agalactiae NH 17 and biofilm formation). L. reuteri CRL 1324 encapsulation was efficient, allowing the recovery of a high number of entrapped lactobacilli. The survival of encapsulated L. reuteri during lyophilization and storage was significantly higher in the presence of lyoprotectors. At the end of storage, the highest numbers of viable cells were obtained in free or encapsulated cells freeze-dried with lyoprotectors, stored at 4°C. Encapsulated and/or lyophilized L. reuteri cells maintained their viability in simulated vaginal fluid as well as the ability to inhibit S. agalactiae NH 17 growth and to form biofilm. Encapsulated and freeze-dried L. reuteri CRL 1324 can be included in a suitable pharmaceutical form for vaginal application to prevent or treat urogenital infections in women.

In vitro and in vivo effects of beneficial vaginal lactobacilli on pathogens responsible for urogenital tract infections.

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 (H2O2) 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 H2O2 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 co-aggregative 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.

Beneficial lactobacilli: effects on the vaginal tract in a murine experimental model.

Vaginal probiotics containing lactic acid bacteria with activity towards pathogenic microorganisms that cause urogenital tract infections have been proposed as a valid strategy for their prophylaxis and therapy. A murine experimental model was set up to evaluate the colonization capability of beneficial human lactobacilli and their effects on the mouse vaginal mucosa and innate immune cells. Five Lactobacillus strains were intravaginally inoculated into previously estrogenized BALB/c mice. The significance of the effects observed in the vaginal tract was determined by analysis of variance using the general linear model. The numbers of viable vaginal lactobacilli were significantly higher at proestrous-estrous than those at the metaestrous-diestrous phase and decreased markedly on the days after inoculation. Lactobacilli inoculation did not cause cytological or histological modifications of the murine vaginal tract. Moreover, the intravaginal administration of Lactobacillus salivarius CRL (Centro de Referencia para Lactobacilos culture collection) 1328 and Lactobacillus gasseri CRL 1263 did not affect the amounts of granulocytes and macrophages present in vaginal washings. In conclusion, the results demonstrate that vaginal lactobacilli did not produce adverse effects on the murine vaginal tract. Therefore, they could be proposed as safe probiotic candidates to promote a balanced microbiota in the urogenital tract.