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Isolation of Vaginal Lactobacilli and Characterization of Anti-Candida Activity.

Parolin C, Marangoni A, Laghi L, Foschi C, Ñahui Palomino RA, Calonghi N, Cevenini R, Vitali B - PLoS ONE (2015)

Bottom Line: Most Lactobacillus strains significantly reduced C. albicans adhesion through all mechanisms.In particular, L. crispatus BC2, L. gasseri BC10 and L. gasseri BC11 appeared to be the most active strains in reducing pathogen adhesion, as their effects were mediated by both cells and supernatants.Our results are prerequisites for the development of new therapeutic agents based on probiotics for prophylaxis and adjuvant therapy of Candida infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.

ABSTRACT
Healthy vaginal microbiota is dominated by Lactobacillus spp., which form a critical line of defence against pathogens, including Candida spp. The present study aims to identify vaginal lactobacilli exerting in vitro activity against Candida spp. and to characterize their antifungal mechanisms of action. Lactobacillus strains were isolated from vaginal swabs of healthy premenopausal women. The isolates were taxonomically identified to species level (L. crispatus B1-BC8, L. gasseri BC9-BC14 and L. vaginalis BC15-BC17) by sequencing the 16S rRNA genes. All strains produced hydrogen peroxide and lactate. Fungistatic and fungicidal activities against C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis and C. lusitaniae were evaluated by broth micro-dilution method. The broadest spectrum of activity was observed for L. crispatus BC1, BC4, BC5 and L. vaginalis BC15, demonstrating fungicidal activity against all isolates of C. albicans and C. lusitaniae. 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 Lactobacillus strains significantly reduced C. albicans adhesion through all mechanisms. In particular, L. crispatus BC2, L. gasseri BC10 and L. gasseri 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 Candida infection.

No MeSH data available.


Related in: MedlinePlus

Correlation between metabolome of lactobacilli and fungistatic/fungicidal activity towards C. albicans and C. non-albicans.(A) Biplot of a PCA performed on the total metabolites identified by 1H-NMR in Lactobacillus cell free supernatants. Expl. Var, explained variance. (B) Box plots representing the distribution of Lactobacillus species in relation to the metabolome. Lines within the boxes indicate the median values of the samples groups corresponding to L. crispatus, L. gasseri and L. vaginalis species. (C) Box plots representing the distribution of fungistatic/fungicidal activity scores towards C. albicans and C. non-albicans 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 C. albicans; 0–5 for C. non-albicans). 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).
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pone.0131220.g001: Correlation between metabolome of lactobacilli and fungistatic/fungicidal activity towards C. albicans and C. non-albicans.(A) Biplot of a PCA performed on the total metabolites identified by 1H-NMR in Lactobacillus cell free supernatants. Expl. Var, explained variance. (B) Box plots representing the distribution of Lactobacillus species in relation to the metabolome. Lines within the boxes indicate the median values of the samples groups corresponding to L. crispatus, L. gasseri and L. vaginalis species. (C) Box plots representing the distribution of fungistatic/fungicidal activity scores towards C. albicans and C. non-albicans 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 C. albicans; 0–5 for C. non-albicans). 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).

Mentions: We identified 40 molecules mainly belonging to the families of aminoacids, organic acids monosaccharides, ketones and alcohols (S3 Table). A Principal Component Analysis (PCA) was performed on entire set of metabolites identified (Fig 1). In the biplot describing the distribution of Lactobacillus strains in relation to the pool of metabolites, PC1 and PC2 accounted for the 45.4% of the whole variance of the investigated samples (Fig 1A). This multivariate analysis showed two interesting correlations: (i) metabolome versus taxonomy (PC1, expl. var 28%) and (ii) metabolome versus fungistatic/fungicidal activity (PC2, expl. var 17.4%). These correlations were best visualized by means of box blots representing the distribution of Lactobacillus species (Fig 1B) and fungistatic/fungicidal activity scores (Fig 1C) in relation to the metabolome. Metabolic profiles varied to a greater extent according to the taxonomy. In particular, metabolome of L. vaginalis significantly differed from those of L. crispatus and L. gasseri (P < 0.05). The highest metabolic heterogeneity was observed within L. crispatus, as demonstrated by the width of the corresponding boxplot. Even fungistatic and fungicidal activities of the vaginal lactobacilli were related to their metabolome. Strains with different activity scores were clearly separated in the vertical direction: the most active strains occupied the lower positions while the less active strains were placed in the higher areas of the two-dimensional space represented by the biplot. A linear correlation was observed between median metabolic variance on PC2 and antifungal activity scores against both C. albicans (fungistatic, R2 = 0.86, P = 0.046; fungicidal, R2 = 0.83, P = 0.02) and C. non-albicans isolates (fungistatic, R2 = 0.99, P = 0.003). The correlation coefficient related to the fungicidal activity against C. non-albicans was not calculated due to the presence of only two activity scores (0 and 1).


Isolation of Vaginal Lactobacilli and Characterization of Anti-Candida Activity.

Parolin C, Marangoni A, Laghi L, Foschi C, Ñahui Palomino RA, Calonghi N, Cevenini R, Vitali B - PLoS ONE (2015)

Correlation between metabolome of lactobacilli and fungistatic/fungicidal activity towards C. albicans and C. non-albicans.(A) Biplot of a PCA performed on the total metabolites identified by 1H-NMR in Lactobacillus cell free supernatants. Expl. Var, explained variance. (B) Box plots representing the distribution of Lactobacillus species in relation to the metabolome. Lines within the boxes indicate the median values of the samples groups corresponding to L. crispatus, L. gasseri and L. vaginalis species. (C) Box plots representing the distribution of fungistatic/fungicidal activity scores towards C. albicans and C. non-albicans 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 C. albicans; 0–5 for C. non-albicans). 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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4476673&req=5

pone.0131220.g001: Correlation between metabolome of lactobacilli and fungistatic/fungicidal activity towards C. albicans and C. non-albicans.(A) Biplot of a PCA performed on the total metabolites identified by 1H-NMR in Lactobacillus cell free supernatants. Expl. Var, explained variance. (B) Box plots representing the distribution of Lactobacillus species in relation to the metabolome. Lines within the boxes indicate the median values of the samples groups corresponding to L. crispatus, L. gasseri and L. vaginalis species. (C) Box plots representing the distribution of fungistatic/fungicidal activity scores towards C. albicans and C. non-albicans 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 C. albicans; 0–5 for C. non-albicans). 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).
Mentions: We identified 40 molecules mainly belonging to the families of aminoacids, organic acids monosaccharides, ketones and alcohols (S3 Table). A Principal Component Analysis (PCA) was performed on entire set of metabolites identified (Fig 1). In the biplot describing the distribution of Lactobacillus strains in relation to the pool of metabolites, PC1 and PC2 accounted for the 45.4% of the whole variance of the investigated samples (Fig 1A). This multivariate analysis showed two interesting correlations: (i) metabolome versus taxonomy (PC1, expl. var 28%) and (ii) metabolome versus fungistatic/fungicidal activity (PC2, expl. var 17.4%). These correlations were best visualized by means of box blots representing the distribution of Lactobacillus species (Fig 1B) and fungistatic/fungicidal activity scores (Fig 1C) in relation to the metabolome. Metabolic profiles varied to a greater extent according to the taxonomy. In particular, metabolome of L. vaginalis significantly differed from those of L. crispatus and L. gasseri (P < 0.05). The highest metabolic heterogeneity was observed within L. crispatus, as demonstrated by the width of the corresponding boxplot. Even fungistatic and fungicidal activities of the vaginal lactobacilli were related to their metabolome. Strains with different activity scores were clearly separated in the vertical direction: the most active strains occupied the lower positions while the less active strains were placed in the higher areas of the two-dimensional space represented by the biplot. A linear correlation was observed between median metabolic variance on PC2 and antifungal activity scores against both C. albicans (fungistatic, R2 = 0.86, P = 0.046; fungicidal, R2 = 0.83, P = 0.02) and C. non-albicans isolates (fungistatic, R2 = 0.99, P = 0.003). The correlation coefficient related to the fungicidal activity against C. non-albicans was not calculated due to the presence of only two activity scores (0 and 1).

Bottom Line: Most Lactobacillus strains significantly reduced C. albicans adhesion through all mechanisms.In particular, L. crispatus BC2, L. gasseri BC10 and L. gasseri BC11 appeared to be the most active strains in reducing pathogen adhesion, as their effects were mediated by both cells and supernatants.Our results are prerequisites for the development of new therapeutic agents based on probiotics for prophylaxis and adjuvant therapy of Candida infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.

ABSTRACT
Healthy vaginal microbiota is dominated by Lactobacillus spp., which form a critical line of defence against pathogens, including Candida spp. The present study aims to identify vaginal lactobacilli exerting in vitro activity against Candida spp. and to characterize their antifungal mechanisms of action. Lactobacillus strains were isolated from vaginal swabs of healthy premenopausal women. The isolates were taxonomically identified to species level (L. crispatus B1-BC8, L. gasseri BC9-BC14 and L. vaginalis BC15-BC17) by sequencing the 16S rRNA genes. All strains produced hydrogen peroxide and lactate. Fungistatic and fungicidal activities against C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis and C. lusitaniae were evaluated by broth micro-dilution method. The broadest spectrum of activity was observed for L. crispatus BC1, BC4, BC5 and L. vaginalis BC15, demonstrating fungicidal activity against all isolates of C. albicans and C. lusitaniae. 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 Lactobacillus strains significantly reduced C. albicans adhesion through all mechanisms. In particular, L. crispatus BC2, L. gasseri BC10 and L. gasseri 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 Candida infection.

No MeSH data available.


Related in: MedlinePlus