Streptococcus salivarius K12 Limits Group B Streptococcus Vaginal Colonization

Abstract

Streptococcus agalactiae (group B streptococcus [GBS]) colonizes the rectovaginal tract in 20% to 30% of women and during pregnancy can be transmitted to the newborn, causing severe invasive disease. Current routine screening and antibiotic prophylaxis have fallen short of complete prevention of GBS transmission, and GBS remains a leading cause of neonatal infection. We have investigated the ability of Streptococcus salivarius, a predominant member of the native human oral microbiota, to control GBS colonization. Comparison of the antibacterial activities of multiple S. salivarius strains by use of a deferred-antagonism test showed that S. salivarius strain K12 exhibited the broadest spectrum of activity against GBS. K12 effectively inhibited all GBS strains tested, including disease-implicated isolates from newborns and colonizing isolates from the vaginal tract of pregnant women. Inhibition was dependent on the presence of megaplasmid pSsal-K12, which encodes the bacteriocins salivaricin A and salivaricin B; however, in coculture experiments, GBS growth was impeded by K12 independently of the megaplasmid. We also demonstrated that K12 adheres to and invades human vaginal epithelial cells at levels comparable to GBS. Inhibitory activity of K12 was examined in vivo using a mouse model of GBS vaginal colonization. Mice colonized with GBS were treated vaginally with K12. K12 administration significantly reduced GBS vaginal colonization in comparison to nontreated controls, and this effect was partially dependent on the K12 megaplasmid. Our results suggest that K12 may have potential as a preventative therapy to control GBS vaginal colonization and thereby prevent its transmission to the neonate during pregnancy.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

S. salivarius inhibits the growth of GBS in vitro. Images are shown of deferred-antagonism tests with S. salivarius K12 (A) or K12ΔpK12 (B) to examine inhibition of seven GBS invasive clinical isolates, i.e., COH1, A909, NEM316, NCTC 10/84, 515, 2603 V/R, and CJB111 (top to bottom), as described in Materials and Methods. Tests were performed at least twice, and representative results are shown. The numbers of viable GBS A909 CFU (C) or S. salivarius CFU (D) are shown after 4 h postinoculation either in single culture or in coculture. Experiments were carried out at least twice independently with three replicates, and the data for one representative experiment are shown. Data were analyzed using two-way repeated-measures ANOVA (C and D) with Bonferroni's multiple-comparisons posttest. ***, P < 0.001; ****, P < 0.0001.

FIG 2

S. salivarius interacts with human vaginal epithelium in vitro. Adherence (A) and invasion (B) of GBS A909, S. salivarius K12, K12ΔpK12, M18, or M18pK12 to human vaginal epithelial cells (hVEC) were examined at an MOI of 1. (C) Gram stain of hVEC infected for 30 min with GBS A909, S. salivarius K12, K12ΔpK12, M18, or M18pK12 at an MOI of 100. Magnification, ×1,000. (D) Protein expression of IL-8 in hVEC supernatants 4 h postinfection with GBS A909, S. salivarius K12, K12ΔpK12, M18, or M18pK12 at an MOI of 10. Experiments were carried out at least twice independently with three replicates, and data for one representative experiment are shown. Data were analyzed using one-way repeated-measures ANOVA (A, B, and D) with Bonferroni's multiple-comparisons posttest. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

FIG 3

S. salivarius colonizes the vaginal tract and limits GBS colonization in vivo. (A) CD1 mice (n = 9 or 10/group) were colonized with 1 × 107 CFU of GBS A909 or S. salivarius K12 and the vaginal lumen was swabbed daily to determine bacterial loads. (B to D) CD1 mice (n = 7 to 20/group) were colonized with 1 × 107 CFU of GBS and on subsequent days were dosed daily with 1 × 108 CFU S. salivarius K12, K12ΔpK12 (C and D only), or PBS, as described in Materials and Methods. (E) Vaginal lavage fluid samples collected from CD1 mice (n = 7 or 8/group) 2 days postcolonization with 1 × 107 CFU of GBS A909 and 1 day posttreatment with 1 × 108 CFU S. salivarius K12, K12ΔpK12, or PBS were subjected to KC ELISA. Lines represent median values for each group. Data were analyzed using the Mann-Whitney test (A and E) or the Kruskal-Wallis test with Dunn's multiple-comparisons posttest for individual days (B to D). *, P < 0.05; **, P < 0.01.