Antimicrobial properties of Lactobacillus cell-free supernatants against multidrug-resistant urogenital pathogens

Abstract

The healthy vaginal microbiota is dominated by Lactobacillus spp., which provide an important critical line of defense against pathogens, as well as giving beneficial effects to the host. We characterized L. gasseri 1A-TV, L. fermentum 18A-TV, and L. crispatus 35A-TV, from the vaginal microbiota of healthy premenopausal women, for their potential probiotic activities. The antimicrobial effects of the 3 strains and their combination against clinical urogenital bacteria were evaluated together with the activities of their metabolites produced by cell-free supernatants (CFSs). Their beneficial properties in terms of ability to interfere with vaginal pathogens (co-aggregation, adhesion to HeLa cells, biofilm formation) and antimicrobial activity mediated by CFSs were assessed against multidrug urogenital pathogens (S. agalactiae, E. coli, KPC-producing K. pneumoniae, S. aureus, E. faecium VRE, E. faecalis, P. aeruginosa, P. mirabilis, P. vulgaris, C. albicans, C. glabrata). The Lactobacilli tested exhibited an extraordinary ability to interfere and co-aggregate with urogenital pathogens, except for Candida spp., as well as to adhere to HeLa cells and to produce biofilm in the Lactobacillus combination. Lactobacillus CFSs and their combination revealed a strong bactericidal effect on the multidrug resistant indicator strains tested, except for E. faecium and E. faecalis. The antimicrobial activity was maintained after heat treatment but decreased after enzymatic treatment. All Lactobacilli showed lactic dehydrogenase activity and production of D- and L-lactic acid isomers on Lactobacillus CFSs, while only 1A-TV and 35A-TV released hydrogen peroxide and carried helveticin J and acidocin A bacteriocins. These results suggest that they can be employed as a new vaginal probiotic formulation and bio-therapeutic preparation against urogenital infections. Further, in vivo studies are needed to evaluate human health benefits in clinical situations.

Keywords: Lactobacilli; MDR-urogenital infection; antimicrobial activity; supernatants; vaginal probiotics.

FIGURE 1

Co‐aggregation ability of Lactobacilli after 5 h incubation at room temperature in PBS (pH 7.4). Results are presented as the average of at least three independent experiments, and the error bars correspond to standard deviations. Statistical significance was evaluated by ANOVA with Fisher’s least significant difference (LSD) (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

FIGURE 2

Bacterial adhesion to HeLa cell layer. (a) Adhesion indexes (ADI; the number of bacteria/100 HeLa cells). (b) Cell layers observed after Giemsa staining using light microscopy: (1) L. gasseri 1A‐TV; (2) L. fermentum 18A‐TV; (3) L. crispatus 35A‐TV; (4) Adhesion to HeLa cell monolayer as a negative control

FIGURE 3

In vitro antimicrobial activity of cell‐free supernatants (CFSs) on indicator strains by time‐killing curves analysis. The gray dotted line indicates a 3‐log₁₀ decrease in the number of CFU/ml versus the number at the baseline (bactericidal effect), while the blue dotted line indicates a general control growth

FIGURE A1

In vitro antimicrobial activity of cell‐free supernatants (CFSs) on each single indicator strain by time‐killing curve analysis. *The black dotted line indicates a growth control of each indicator strain, the solid black line with circles indicates the CFS of L. gasseri 1A‐TV, while the line with rectangles indicates the CFS of L. fermentum 18A‐TV, the line with triangles indicates the CFS of L. crispatus 35 A‐TV, and the line with triangles facing down indicates the CFS Lactobacilli mix

FIGURE A2

Effects of pH on CFS antimicrobial activity on indicator strain growth. Comparison of growth curves at 6 and 24h (OD = 600 nm for bacterial strains and OD = 530 for Candida spp.) of control indicator strains with respect to the growth of untreated Lactobacilli CFS, CFS adjusted to pH 5.5, 6.5, and 7.5. The results are expressed as mean ± standard deviations of values obtained from triplicate experiments