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

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

Fungistatic/fungicidal activity of <i>Lactobacillus</i> strains against <i>Candida</i> isolates.

<p>First symbol, fungistatic activity; second symbol, fungicidal activity.</p><p>Fungistatic/fungicidal activity of <i>Lactobacillus</i> strains against <i>Candida</i> isolates.</p

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

Fungistatic and fungicidal activity scores of lactobacilli towards <i>C</i>. <i>albicans</i> and <i>C</i>. non-<i>albicans</i> isolates.

<p>Fungistatic and fungicidal activity scores of lactobacilli towards <i>C</i>. <i>albicans</i> and <i>C</i>. non-<i>albicans</i> isolates.</p

Characterization of the vaginal lactobacilli: taxonomy, pH of cultural supernatants and production of antimicrobial compounds.

<p>*nd: not determined</p><p>Characterization of the vaginal lactobacilli: taxonomy, pH of cultural supernatants and production of antimicrobial compounds.</p

Substituted 3-(5-Imidazo[2,1-<i>b</i>]thiazolylmethylene)-2-indolinones and Analogues: Synthesis, Cytotoxic Activity, and Study of the Mechanism of Action

The synthesis of substituted 3-(5-imidazo­[2,1-<i>b</i>]­thiazolylmethylene)-2-indolinones and analogues is reported. Their cytotoxic activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. The action of selected compounds was examined for potential inhibition of tubulin assembly in comparison with the potent colchicine site agent combretastatin A-4. The most potent compounds also strongly and selectively inhibited the phosphorylation of the oncoprotein kinase Akt in cancer cells. The effect of the most interesting compounds was examined on the growth of HT-29 colon cancer cells. These compounds caused the cells to arrest in the G2/M phase of the cell cycle, as would be expected for inhibitors of tubulin assembly