Bactericidal Effects of the Probiotic L. rhamnosus HN001 against Group B Streptococcus

Background:

Group B Streptococcus (GBS) infection is one of the leading bacterial infections causing illness and death in newborns in the United States. Screening of pregnant women for GBS colonization and subsequent treatment is the standard of care but carries the risks of antibiotic side effects, such as C. difficile infection. An alternative approach to prevent or treat GBS vaginal colonization could be the use of probiotics that have antagonistic effects against GBS.

Objective:

The objective of this study was to assess the bactericidal effects of the probiotic L. rhamnosus HN001 against GBS and to evaluate the ability of the probiotic to adhere to epithelial monolayers.   

Methods:

GBS (three clinical isolates, GBS 0191, 0192 and 0193 and an ATCC strain, GBS 12386) were co-cultured with the probiotic in varying proportions in Columbia Broth. GBS were enumerated on blood agar after 24 and 48 h and pH measurements were taken at 48 h. To evaluate the ability of the probiotic to adhere to monolayers of tissue culture cell lines, Madin-Darby canine kidney (MDCK) and Vero cells were inoculated with L. rhamnosus HN001 at 100, 10 or 1 multiplicity of infection (moi) and incubated for 1 h. Monolayers were washed 3 times with PBS and lysed with 0.1% Triton-X. The adherent bacteria were plated on MRS agar at appropriate dilutions and were counted after 48 h.

Results:

L. rhamnosus HN001 has bactericidal activity against all GBS tested. Co-culturing one of the clinical isolates (GBS 0193) (1 x 10³ CFU) with L. rhamnosus HN001 (2 x 10⁸) resulted in an extremely significant (P=0) decrease in the GBS counts both at 24 h and 48 h. The decrease in the number of GBS was associated with a decrease in pH. Counts of the ATCC GBS strain also decreased significantly (P = 0.028 and 0.016 at 24 and 48h, respectively). At higher initial GBS inocula (1.8 x 10⁵) and lower L. rhamnosus inocula (1.7 x 10⁷), a 2 log decrease in GBS 0193 after 24 h, a 3 log decrease in GBS 0192 after 48 h and a log and a 2 log decrease in GBS ATCC12386 and GBS 0191, respectively, at both 24 h and 48 h were achieved (Fig. 1). These decreases in GBS counts were not associated with a decrease in pH; suggesting other mechanisms of killing the pathogen than production of organic acids. One of the desirable features of probiotics is their ability to adhere to epithelial cells. L. rhamnosus HN001 adheres to monolayers of MDCK and Vero cells. There was a direct relation between the number of bound bacteria and the number of added bacteria. The highest number of adherent bacteria was observed on Vero cells at 100 moi, with an average recovered 1.98 x 10⁶ bacteria. Interestingly, the highest ratio of adherence for L. rhamnosus HN001 on vero cells, 11.3%, was achieved at 10 moi.

Conclusions:

In this study we showed that L. rhamnosus HN001 inhibits growth of GBS in vitro. The high adherence of the probiotic to monolayers of tissue culture cell lines should be studied further for its potential to reduce invasion by pathogenic bacteria such as GBS.