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Biosynthesis and degradation of H2O2 by vaginal lactobacilli.

ABSTRACT: Hydrogen peroxide production by vaginal lactobacilli represents one of the most important defense mechanisms against vaginal colonization by undesirable microorganisms. To quantify the ability of a collection of 45 vaginal Lactobacillus strains to generate H(2)O(2), we first compared three published colorimetric methods. It was found that the use of DA-64 as a substrate rendered the highest sensitivity, while tetramethyl-benzidine (TMB) maintained its linearity from nanomolar to millimolar H(2)O(2) concentrations. Generation of H(2)O(2) was found to be especially common and strong for L. jensenii strains, while it was variable among L. crispatus and L. gasseri strains. Biosynthesis of H(2)O(2) only occurred upon agitation of the cultures, but the H(2)O(2)-producing machinery was already present in them before aeration started. Calcium, magnesium, manganese, and zinc ions did not affect H(2)O(2) production, while Cu(2+) inhibited the growth of Lactobacillus jensenii CECT 4306, which was chosen as a model strain. Cultures with Fe(3+), hemin, and hemoglobin did not accumulate H(2)O(2). Fe(3+) activated an extracellular peroxidase that destroyed the H(2)O(2) being produced by the cultures. This protected the lactobacilli against its antimicrobial effect. The production of the enzyme appears to be constitutive, the Fe(3+) ions being a necessary cofactor of the reaction.

SUBMITTER: Martin R

PROVIDER: S-EPMC2805228 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Biosynthesis and degradation of H2O2 by vaginal lactobacilli.

Martín Rebeca R Suárez Juan E JE

Applied and environmental microbiology 20091130 2

Hydrogen peroxide production by vaginal lactobacilli represents one of the most important defense mechanisms against vaginal colonization by undesirable microorganisms. To quantify the ability of a collection of 45 vaginal Lactobacillus strains to generate H(2)O(2), we first compared three published colorimetric methods. It was found that the use of DA-64 as a substrate rendered the highest sensitivity, while tetramethyl-benzidine (TMB) maintained its linearity from nanomolar to millimolar H(2)O ...[more]

PMID: 19948869

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Project description:BackgroundExopolysaccharides (EPS) secreted by beneficial lactobacilli exert a plethora of positive activities, but little is known about their effects on biofilms of opportunistic vaginal pathogens and especially on biofilms of lactobacilli themselves. Here, the EPS produced by six vaginal lactobacilli, belonging to Lactobacillus crispatus (BC1, BC4, BC5) and Lactobacillus gasseri (BC9, BC12, BC14) species were isolated from cultural supernatants and lyophilized.ResultsLactobacillus EPS were chemically characterized in terms of monosaccharide composition by liquid chromatography (LC) analysis coupled to UV and mass spectrometry (MS) detection. Moreover, the ability of EPS (0.1, 0.5, 1 mg/mL) to stimulate the biofilm formation of lactobacilli and to inhibit the formation of pathogens' biofilms was evaluated by crystal violet (CV) staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Isolated EPS (yields 133-426 mg/L) were heteropolysaccharides mainly composed of D-mannose (40-52%) and D-glucose (11-30%). For the first time we demonstrated that Lactobacillus EPS were able to stimulate in a dose-dependent manner (p < 0.05) the formation of biofilms of ten strains belonging to L. crispatus, L. gasseri and Limosilactobacillus vaginalis species, in terms of cell viability (84-282% increase at 1 mg/mL) and especially biofilm biomass (40-195% increase at 1 mg/mL), quantified with MTT assay and CV staining, respectively. EPS released from L. crispatus and L. gasseri were found to better stimulate the biofilms of the same producer species rather than that of other species, including producing strains themselves and other strains. Conversely, the biofilm formation of bacterial (Escherichia coli, Staphylococcus spp., Enterococcus spp. and Streptococcus agalactiae) and fungal (Candida spp.) pathogens was inhibited. The anti-biofilm activity was dose-dependent and was more marked for L. gasseri-derived EPS (inhibition up to 86%, 70%, and 58% at 1 mg/mL, 0.5 mg/mL, and 0.1 mg/mL, respectively), whilst L. crispatus-derived EPS resulted overall less efficient (inhibition up to 58% at 1 mg/mL and 40% at 0.5 mg/mL) (p < 0.05).ConclusionsLactobacilli-derived EPS favour the biofilm formation of lactobacilli preventing, at the same time, that of opportunistic pathogens. These results support the possible employment of EPS as postbiotics in medicine as a therapeutic/preventive strategy to counteract vaginal infections.

Project description:The human vaginal microbiome is dominated by bacteria from the genus Lactobacillus, which create an acidic environment thought to protect women against sexually transmitted pathogens and opportunistic infections. Strikingly, lactobacilli dominance appears to be unique to humans; while the relative abundance of lactobacilli in the human vagina is typically >70%, in other mammals lactobacilli rarely comprise more than 1% of vaginal microbiota. Several hypotheses have been proposed to explain humans' unique vaginal microbiota, including humans' distinct reproductive physiology, high risk of STDs, and high risk of microbial complications linked to pregnancy and birth. Here, we test these hypotheses using comparative data on vaginal pH and the relative abundance of lactobacilli in 26 mammalian species and 50 studies (N = 21 mammals for pH and 14 mammals for lactobacilli relative abundance). We found that non-human mammals, like humans, exhibit the lowest vaginal pH during the period of highest estrogen. However, the vaginal pH of non-human mammals is never as low as is typical for humans (median vaginal pH in humans = 4.5; range of pH across all 21 non-human mammals = 5.4-7.8). Contrary to disease and obstetric risk hypotheses, we found no significant relationship between vaginal pH or lactobacilli relative abundance and multiple metrics of STD or birth injury risk (P-values ranged from 0.13 to 0.99). Given the lack of evidence for these hypotheses, we discuss two alternative explanations: the common function hypothesis and a novel hypothesis related to the diet of agricultural humans. Specifically, with regard to diet we propose that high levels of starch in human diets have led to increased levels of glycogen in the vaginal tract, which, in turn, promotes the proliferation of lactobacilli. If true, human diet may have paved the way for a novel, protective microbiome in human vaginal tracts. Overall, our results highlight the need for continuing research on non-human vaginal microbial communities and the importance of investigating both the physiological mechanisms and the broad evolutionary processes underlying human lactobacilli dominance.

Project description:BACKGROUND: Bacterial vaginosis (BV), which is highly prevalent in the African population, is one of the most common vaginal syndromes affecting women in their reproductive age placing them at increased risk for sexually transmitted diseases including infection by human immunodeficiency virus-1. The vaginal microbiota of a healthy woman is often dominated by the species belonging to the genus Lactobacillus namely L. crispatus, L. gasseri, L. jensenii and L. iners, which have been extensively studied in European populations, albeit less so in South African women. In this study, we have therefore identified the vaginal Lactobacillus species in a group of 40 African women from Soweto, a township on the outskirts of Johannesburg, South Africa. METHODS: Identification was done by cultivating the lactobacilli on Rogosa agar, de Man-Rogosa-Sharpe (MRS) and Blood agar plates with 5% horse blood followed by sequencing of the 16S ribosomal DNA. BV was diagnosed on the basis of Nugent scores. Since some of the previous studies have shown that the lack of vaginal hydrogen peroxide (H2O2) producing lactobacilli is associated with bacterial vaginosis, the Lactobacillus isolates were also characterised for their production of H2O2. RESULTS: Cultivable Lactobacillus species were identified in 19 out of 21 women without BV, in three out of five women with intermediate microbiota and in eight out of 14 women with BV. We observed that L. crispatus, L. iners, L. jensenii, L. gasseri and L. vaginalis were the predominant species. The presence of L. crispatus was associated with normal vaginal microbiota (P = 0.024). High level of H2O2 producing lactobacilli were more often isolated from women with normal microbiota than from the women with BV, although not to a statistically significant degree (P = 0.064). CONCLUSION: The vaginal Lactobacillus species isolated from the cohort of South African women are similar to those identified in European populations. In accordance with the other published studies, L. crispatus is related to a normal vaginal microbiota. Hydrogen peroxide production was not significantly associated to the BV status which could be attributed to the limited number of samples or to other antimicrobial factors that might be involved.

Project description:BackgroundLactobacilli are gatekeepers of vaginal ecosystem impeding growth of pathogenic microbes and their diversity varies across populations worldwide. The present study investigated diversity of human vaginal microbiota among females of Northeast India, who are distinct in dietary habits, lifestyle, and genomic composition from rest of India.ResultsAltogether, 154 bacterial isolates were obtained from vaginal swab samples of 40 pregnant and 29 non-pregnant females. The samples were sequenced for 16 s rRNA gene and analysed for identification using a dual approach of homology search and maximum likelihood based clustering. Molecular identification based on 16S rRNA gene sequence confirmed the isolates belonging to 31 species. Lactobacilli constituted 37.7% of the bacterial isolates with 10 species and other Lactic Acid Bacteria (39.61%) represented another 10 species, some of which are opportunistic pathogens. The remaining of the communities are mostly dominated by species of Staphylococcus (14.28%) and rarely by Propionibacterium avidum (3.90%), Bacillus subtilis, Escherchia coli, Janthinobacterium lividum, and Kocuria kristinae (each 0.64%). Interestingly Lactobacillus mucosae and Enterococcus faecalis, which are globally uncommon vaginal microbes is found dominant among women of Northeast India. This tentatively reflects adaptability of particular Lactobacillus species, in distinct population, to better compete for receptors and nutrients in vaginal epithelium than other species. Further, intrageneric 16S rRNA gene exchange was observed among Enterococcus, Staphylococcus, and two species of Lactobacillus, and deep intraspecies divergence among L. mucosae, which pinpointed possibility of emergence of new strains with evolved functionality. Lactobacilli percentage decreased from young pregnant to aged non-pregnant women with maximum colonization in trimester II.ConclusionThe study highlighted importance of assessment of vaginal microbiota, Lactobacillus in particular, across different population to gain more insight on female health.

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Biosynthesis and degradation of H2O2 by vaginal lactobacilli.

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Biosynthesis and degradation of H2O2 by vaginal lactobacilli.

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