Variations in lactobacilli extracellular metabolome.

<p>Box plots represent the concentration (mM) of extracellular metabolites which vary significantly among the diverse <i>Lactobacillus</i> species considered. Metabolites were quantified in cell free supernatants by ¹H-NMR. Lines within the boxes indicate the median values of the metabolite concentration and each box represents the interquartile range (25–75th percentile). The bottom and top bars indicate the 10th and 90th percentiles, respectively. Boxes were colored in grey to highlight the <i>Lactobacillus</i> species groupings that show significantly different concentration of the corresponding metabolite (P<0.05, Bonferroni-adjusted).</p

Phylogenetic tree based on lactobacilli 16S rRNA sequences.

<p>The Neighbor-Joining method was used to infer evolutionary history. The evolutionary distances were computed using the Maximum Likelihood method based on Tamura-Nei model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172483#pone.0172483.ref032" target="_blank">32</a>]. The tree is drawn to scale, with branch lengths measured in number of substitutions per site. The bootstrap values inferred from 1000 replicates is shown next to the branches. The analysis involved 40 nucleotides sequences. All positions containing gaps and missing data were eliminated. The tree was obtained by using MEGA 6 software.</p

Cluster analysis of MALDI-TOF MS spectra obtained from the <i>Lactobacillus</i> strains included in the study.

<p>In the MSP dendrogram, relative distance between isolates is displayed as arbitrary units. Zero indicates complete similarity and 1,000 indicates maximum dissimilarity.</p

Novel approaches for the taxonomic and metabolic characterization of lactobacilli: Integration of 16S rRNA gene sequencing with MALDI-TOF MS and ¹H-NMR

<div><p>Lactobacilli represent a wide range of bacterial species with several implications for the human host. They play a crucial role in maintaining the ecological equilibrium of different biological niches and are essential for fermented food production and probiotic formulation. Despite the consensus about the ‘health-promoting’ significance of <i>Lactobacillus</i> genus, its genotypic and phenotypic characterization still poses several difficulties. The aim of this study was to assess the integration of different approaches, genotypic (16S rRNA gene sequencing), proteomic (MALDI-TOF MS) and metabolomic (¹H-NMR), for the taxonomic and metabolic characterization of <i>Lactobacillus</i> species. For this purpose we analyzed 40 strains of various origin (intestinal, vaginal, food, probiotics), belonging to different species. The high discriminatory power of MALDI-TOF for species identification was underlined by the excellent agreement with the genotypic analysis. Indeed, MALDI-TOF allowed to correctly identify 39 out of 40 <i>Lactobacillus</i> strains at the species level, with an overall concordance of 97.5%. In the perspective to simplify the MALDI TOF sample preparation, especially for routine practice, we demonstrated the perfect agreement of the colony-picking from agar plates with the protein extraction protocol. ¹H-NMR analysis, applied to both culture supernatants and bacterial lysates, identified a panel of metabolites whose variations in concentration were associated with the taxonomy, but also revealed a high intra-species variability that did not allow a species-level identification. Therefore, despite not suitable for mere taxonomic purposes, metabolomics can be useful to correlate particular biological activities with taxonomy and to understand the mechanisms related to the antimicrobial effect shown by some <i>Lactobacillus</i> species.</p></div

List of <i>Lactobacillus</i> strains included in the present study, genotypic identification, 16S rDNA accession numbers, MALDI TOF MS identification and source.

<p>List of <i>Lactobacillus</i> strains included in the present study, genotypic identification, 16S rDNA accession numbers, MALDI TOF MS identification and source.</p

Variations in lactobacilli intracellular metabolome.

<p>Box plots represent the concentration (mM) of intracellular metabolites which vary significantly among the diverse <i>Lactobacillus</i> species considered. Metabolites were quantified in cellular lysates by ¹H-NMR. Lines within the boxes indicate the median values of the metabolite concentration and each box represents the interquartile range (25–75th percentile). The bottom and top bars indicate the 10th and 90th percentiles, respectively. Boxes were colored in grey to highlight the <i>Lactobacillus</i> species groupings that show significantly different concentration of the corresponding metabolite (P<0.05, Bonferroni-adjusted).</p

Isolation of Vaginal Lactobacilli and Characterization of Anti-<i>Candida</i> Activity

<div><p>Healthy vaginal microbiota is dominated by <i>Lactobacillus</i> spp., which form a critical line of defence against pathogens, including <i>Candida</i> spp. The present study aims to identify vaginal lactobacilli exerting <i>in vitro</i> activity against <i>Candida</i> spp. and to characterize their antifungal mechanisms of action. <i>Lactobacillus</i> strains were isolated from vaginal swabs of healthy premenopausal women. The isolates were taxonomically identified to species level (<i>L</i>. <i>crispatus</i> B1-BC8, <i>L</i>. <i>gasseri</i> BC9-BC14 and <i>L</i>. <i>vaginalis</i> BC15-BC17) by sequencing the 16S rRNA genes. All strains produced hydrogen peroxide and lactate. Fungistatic and fungicidal activities against <i>C</i>. <i>albicans</i>, <i>C</i>. <i>glabrata</i>, <i>C</i>. <i>krusei</i>, <i>C</i>. <i>tropicalis</i>, <i>C</i>. <i>parapsilosis</i> and <i>C</i>. <i>lusitaniae</i> were evaluated by broth micro-dilution method. The broadest spectrum of activity was observed for <i>L</i>. <i>crispatus</i> BC1, BC4, BC5 and <i>L</i>. <i>vaginalis</i> BC15, demonstrating fungicidal activity against all isolates of <i>C</i>. <i>albicans</i> and <i>C</i>. <i>lusitaniae</i>. Metabolic profiles of lactobacilli supernatants were studied by ¹H-NMR analysis. Metabolome was found to be correlated with both taxonomy and activity score. Exclusion, competition and displacement experiments were carried out to investigate the interference exerted by lactobacilli toward the yeast adhesion to HeLa cells. Most <i>Lactobacillus</i> strains significantly reduced <i>C</i>. <i>albicans</i> adhesion through all mechanisms. In particular, <i>L</i>. <i>crispatus</i> BC2, <i>L</i>. <i>gasseri</i> BC10 and <i>L</i>. <i>gasseri</i> BC11 appeared to be the most active strains in reducing pathogen adhesion, as their effects were mediated by both cells and supernatants. Inhibition of histone deacetylases was hypothesised to support the antifungal activity of vaginal lactobacilli. Our results are prerequisites for the development of new therapeutic agents based on probiotics for prophylaxis and adjuvant therapy of <i>Candida</i> infection.</p></div

Interference of vaginal lactobacilli with <i>C</i>. <i>albicans</i> adhesion to HeLa cells.

<p>Exclusion (A), competition (B) and displacement (C) experiments were performed. The results were expresses as percentages of adherent yeasts per HeLa cell and compared with adhesion without lactobacilli (control value). The control value was taken as 100% of adhesion (black bars). White and grey bars show the adhesion of <i>C</i>. <i>albicans</i> in presence of <i>Lactobacillus</i> cells and supernatants, respectively. Statistical significance was determined at <i>P</i> < 0.05*, <i>P</i> < 0.01** and <i>P</i> < 0.001***. Error bars represent SEM.</p

Adhesion of vaginal lactobacilli to HeLa cells.

<p>The results were expressed as average number of adherent bacteria per cell. Error bars represent SEM.</p

Correlation between metabolome of lactobacilli and fungistatic/fungicidal activity towards <i>C</i>. <i>albicans</i> and <i>C</i>. non-<i>albicans</i>.

<p>(A) Biplot of a PCA performed on the total metabolites identified by ¹H-NMR in <i>Lactobacillus</i> cell free supernatants. Expl. Var, explained variance. (B) Box plots representing the distribution of <i>Lactobacillus</i> species in relation to the metabolome. Lines within the boxes indicate the median values of the samples groups corresponding to <i>L</i>. <i>crispatus</i>, <i>L</i>. <i>gasseri</i> and <i>L</i>. <i>vaginalis</i> species. (C) Box plots representing the distribution of fungistatic/fungicidal activity scores towards <i>C</i>. <i>albicans</i> and <i>C</i>. non-<i>albicans</i> in relation to the metabolome. Lines within the boxes indicate the median values of the samples groups corresponding to the different activity scores (0–4 for <i>C</i>. <i>albicans</i>; 0–5 for <i>C</i>. non-<i>albicans</i>). Each box represents the interquartile range (25–75th percentile). The bottom and top bars indicate the 10th and 90th percentiles, respectively. Outlier values are indicated (BC10 and BC15).</p