Do nomadic lactobacilli fit as potential vaginal probiotics? The answer lies in a successful selective multi-step and scoring approach

Abstract

Background: The goal of this study was to create a multi-strain probiotic gel that would foster a lactobacilli-dominated vaginal microbiota in pregnant women and ensure appropriate eubiosis for the newborn. Nomadic lactobacilli (95 strains), mostly isolated from food sources, were preliminarily screened for functional traits before being characterized for their capability to inhibit the two vaginal pathogens Streptococcus agalactiae and Candida albicans, which may lead to adverse pregnancy-related outcomes. Eight best-performing strains were chosen and furtherly investigated for their ability to produce biofilm. Lastly, the two selected potential probiotic candidates were analyzed in vitro for their ability to reduce the inflammation caused by C. albicans infection on the reconstituted human vaginal epithelium (HVE).

Results: Lactiplantibacillus plantarum produced both isomers of lactic acid, while Lacticaseibacillus paracasei produced only L-isomer. The production of hydrogen peroxide was strain-dependent, with the highest concentrations found within Lact. paracasei strains. The auto-aggregation capacity and hydrophobicity traits were species-independent. S. agalactiae 88II3 was strongly inhibited both at pH 7.0 and 4.0, whereas the inhibition of C. albicans UNIBZ54 was less frequent. Overall, L. plantarum strains had the highest pathogen inhibition and functional scoring. L. plantarum C5 and POM1, which were selected as potential probiotic candidates also based on their ability to form biofilms, were able to counteract the inflammation process caused by C. albicans infection in the HVE model.

Conclusions: Our multi-step and cumulative scoring-based approach was proven successful in mining and highlighting the probiotic potential of two nomadic lactobacilli strains (L. plantarum C5 and POM1), being applicable to preserve and improve human vaginal health.

Keywords: Nomadic lactobacilli; Pathogens inhibition screening; Probiotics; Vaginal ecosystem; Vaginal microbiota.

Fig. 1

Functional assays results. Functional assays profiling, categorized by strain, of total lactic acid (A), lactic acid isomers (B), peroxide production (C), and auto-aggregation and hydrophobicity (D) characterization of nomadic lactobacilli strains after 18–24 h at 37 °C of incubation in MRS. Principal component analysis of data gathered during the first screening (E). Dunn test significant statistical differences after FDR correction between groups indicated as follows: * (P < 0.05), ** (P < 0.005), *** (P < 0.0005), **** (P < 0.00005)

Fig. 2

Growth inhibition parameters of Candida albicans and Streptococcus agalactiae from cell free supernatants kept at original acidic pH (pH ca. 4) or adjusted to neutral pH (pH 7) for all the tested strains and divided by species, compared to the control condition (CRT) of growth of C. albicans and S. agalactiae 

Fig. 3

Three dimensional images from biofilms observed under the confocal laser scanning microscopy. Biofilm produced by L. plantarum C5 (a), L. plantarum POM1 (b), L. plantarum 1LS16 (c), L. plantarum D3.15 (d), L. plantarum OE1 (e), L. plantarum D6.6 (f), L. plantarum E3.8 (g), Lact. paracasei 41j (h). Metabolically active cells (green fluorescence) and biofilm exopolysaccharides in the extracellular polymeric matrix (red fluorescence) are shown in the same panel. Bars represent 50 μm; units of the x, y, and z axes are μm. Images are representatives of three biological replicates analyzed in triplicate

Fig. 4

Expression of E-cadherin and Interleukin 8 (mRNA Relative Quantification) in the Human Vaginal Epithelium. Expression rates were calculated as the relative quantification (RQ) data. The data represent mean ± standard deviation. [a–c Means within the same treatment with different letters are significantly different (P < 0.05)]