Lactobacillus Biofilms Influence Anti- Candida Activity

Abstract

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.

Keywords: Candida; Lactobacillus; biofilm; health benefits; metabolome; vaginal microbiota.

FIGURE 1

Biofilm formation by Lactobacillus strains, evaluated by crystal violet staining (average ± st. dev.). *p < 0.05 selected species vs. other Lactobacillus species.

FIGURE 2

Fungistatic activity of Lactobacillus bf-CFS and pk-CFS toward clinical Candida isolates, tested following EUCAST guidelines (pH 7). OD 530 nm values were normalized for positive controls and data were expressed as inhibition (%) of Candida growth. Fungistatic activity color scale was reported (10% activity variation).

FIGURE 3

Fungistatic activity of L. crispatus BC1, L. crispatus BC6, L. gasseri BC11, L. gasseri BC12, L. vaginalis BC17, and L. plantarum BC19 bf-CFS and pk-CFS toward clinical Candida isolates, tested in simulated vaginal fluid, pH 4.2. OD 530 nm values were normalized for positive controls and data were expressed as inhibition (%) of Candida growth. Fungistatic activity color scale was reported.

FIGURE 4

t-SNE cluster analysis on CFS fungistatic activity dataset. Three groups were identified, corresponding to low, medium, and high activity. Samples derived from biofilm and planktonic cultures were signed in black and red, respectively.

FIGURE 5

Box plot of Lactobacillus CFS metabolite concentrations. Concentrations were calculated as differences from MRS broth (mmol/L). Lines within the boxes indicate the median values of the samples. Each box represents the interquartile range (25–75th percentile). The extremes of the bars indicate the 10th and 90th percentiles, respectively. Outlier values are indicated (+). Glucose median and acetate interquartile range are not represented because they were out of the range considered.

FIGURE 6

(A) Biplot of a PCA performed on Lactobacillus bf-CFS and pk-CFS metabolome and anti-Candida activity data. (B) Boxplot of CFS distribution on PC1 based on anti-Candida activity score. Lines within the boxes indicate the median values of the samples. Each box represents the interquartile range (25–75th percentile). The extremes of the bars indicate the 10th and 90th percentiles, respectively.