Evaluation of Modulatory Activities of Lactobacillus crispatus Strains in the Context of the Vaginal Microbiota

Abstract

It has been widely reported that members of the genus Lactobacillus dominate the vaginal microbiota, which is represented by the most prevalent species Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners. L. crispatus is furthermore considered an important microbial biomarker due to its professed beneficial implications on vaginal health. In order to identify molecular mechanisms responsible for health-promoting activities that are believed to be elicited by L. crispatus, we performed in silico investigations of the intraspecies biodiversity of vaginal microbiomes followed by in vitro experiments involving various L. crispatus strains along with other vaginal Lactobacillus species mentioned above. Specifically, we assessed their antibacterial activities against a variety of pathogenic microorganisms that are associated with vaginal infections. Moreover, coculture experiments of L. crispatus strains showing the most antibacterial activity against different pathogens revealed distinct ecological fitness and competitive properties with regard to other microbial colonizers. Interestingly, we observed that even phylogenetically closely related L. crispatus strains possess unique features in terms of their antimicrobial activities and associated competitive abilities, which suggests that they exert marked competition and evolutionary pressure within their specific environmental niche. IMPORTANCE The human vaginal microbiota includes all microorganisms that colonize the vaginal tract. In this context, a vaginal microbiota dominated by Lactobacillus and specifically by Lactobacillus crispatus is considered a hallmark of health. The role of L. crispatus in maintaining host health is linked to its modulatory activity toward other members of the vaginal ecosystem and toward the host. In this study, in vitro experiments followed by genetic analyses of the mechanisms used by L. crispatus in colonizing the vaginal ecological niche, particularly in the production of different antimicrobial compounds, were evaluated, highlighting some intriguing novel aspects concerning the genetic variability of this species. Our results indicate that this species has adapted to its niche and may still undergo adaptation to enhance its competitiveness for niche colonization.

Keywords: Lactobacillus crispatus; antibacterial activities; vaginal microbiota.

FIG 1

Antibacterial activity of Lactobacillus strains against different pathogens. (a) A phylogenetic tree of 17 Lactobacillus crispatus strains, including eight isolated from humans, eight from chicken, and one from Equus caballus, and its association with the diameter of the inhibition halos obtained for each Lactobacillus species grown with various (opportunistic) pathogens. (b) A phylogenetic tree of 17 Lactobacillus crispatus strains, including eight isolated from humans, eight from chicken, and one from Equus caballus, and its association with antimicrobial activity of Lactobacillus CFS toward Candida albicans ATCC 32032, tested following EUCAST guidelines. OD_530 nm values at 24 h and 48 h were normalized for positive controls, and results are expressed as inhibition (%) of Candida growth. Excursion represents the difference between the inhibition (%) between 48 h and 24 h.

FIG 2

Evaluation of antimicrobial compounds in L. crispatus strains. (a) A graphical count of bacteriocin-associated loci identified in L. crispatus and represented as a bar plot. (b) pH decrease (due to lactic acid production) during fermentation in simulated vaginal fluid (SVF). The variation of pH is reported as a function of time.

FIG 3

Phylogenetic tree of L. crispatus and related inhibition data. (a and c) A phylogenetic tree of 17 Lactobacillus crispatus strains, eight isolated from human, eight from chicken, and one from Equus caballus, is related to bacteriocin distribution with relative gene count (a) and to inhibition halo, expressed in millimeters, of nine different species used as a control test (c). (b) Statistical analysis relative to impact of bacteriocin LCBs 3, 4, and 5 versus the nine species tested.

FIG 4

Evaluation of Lactobacillus load in coculture experiments. Quantitative PCR evaluation of the relative number of Lactobacillus and Gardnerella strains in coculture experiments. The graph highlights the average abundance observed through qPCR at 6 h, 10 h, 24 h, and 30 h. (a) Coculture results of two different experiments in which PRL2021 or LB57 L. crispatus strains were grown together with L. gasseri V105C. (b) Relative load of L. crispatus strains (PRL2021 or LB57) with L. iners LMG 14328. (c) Relative number of L. crispatus strains (PRL2021 or LB57) with L. jensenii V94G. (d) qPCR evaluation between L. crispatus strains (PRL2021 or LB57) and Gardnerella vaginalis LMG 7832. (e) Relative load of all microorganisms used in the cocultivation experiments when cocultivated together.