Enterococcal bacteremia in mice is prevented by oral administration of probiotic Bacillus spores

Abstract

Whether and how probiotics promote human health is a controversial issue. Their claimed benefit for counteracting gastrointestinal infection is linked predominantly to reducing pathogen abundance within the intestinal microbiota. Less understood mechanistically is the reported value that probiotics could have in reducing systemic infections. Enterococcus faecalis is an opportunistic pathogen that causes systemic infection after translocation through the intestinal epithelium, particularly in hospitalized and immune-depleted patients receiving antibiotic therapy. In this study, we used an E. faecalis mouse infection model with wild-type and isogenic mutant strains deficient in genes of the E. faecalis Fsr (fecal streptococci regulator) quorum-sensing system. We show that E. faecalis translocation from the mouse gut into the blood is mediated by the Fsr quorum-sensing system through production of the protease GelE, which compromises intestinal epithelium integrity. Furthermore, we demonstrate that orally administered probiotic Bacillus subtilis spores blocked E. faecalis translocation from the gut to the bloodstream and subsequent systemic infection in mice by inhibiting Fsr activity. These findings demonstrate that a key aspect of Enterococcus pathogenesis is controlled by quorum sensing, which can be targeted with probiotic Bacillus spores.

Fig. 1.

The E. faecalis Fsr quorum-sensing system does not impact intestinal colonization. A, Colonization without pretreatment. E. faecalis [wild-type (WT) or isogenic ΔfsrB mutant] was administered (2 × 10⁹ CFU in 200 μl each) by oral gavage, and CFU in the entire small or large intestines were determined after sacrifice 3 days afterwards. n=5/group. B, C, Colonization with pretreatment. Mice received CY for 3 days (−6 to −4), then antibiotics for 3 days (−3 to −1), after which E. faecalis [wild-type (WT) or isogenic ΔfsrB mutant] was administered on one (B) or 2 (C) consecutive days (2 × 10⁹ CFU in 200 μl each) by oral gavage, and CFU in the entire small or large intestines were determined after sacrifice 3 days afterwards (B, n=5/group, C, n=9/group,). A-C, All error bars show the geometric mean and geometric SD. No statistically significant differences were found in any WT-ΔfsrB comparison by unpaired two-tailed t (A,B) or Mann-Whitney tests (C). Parametric (t) or non-parametric (Mann-Whitney) tests were used dependent on the results of normality and log normality analyses of all data (see methods).

Fig. 2.

In-vivo translocation of E. faecalis through the gut epithelium is due to quorum-sensing control of gelatinase. A, Experimental setup. Mice were pre-treated with cyclophosphamide (CY) (days −6 to −4) and an antibiotic mix (days −3 to −1) and received E. faecalis wild-type (WT) or the indicated isogenic mutants by oral gavage (2 × 10⁹ CFU in 200 μl) the next day. 24 h later, mice were sacrificed, the blood was collected by cardiac puncture and CFU were determined by plating. B, CFU in the blood. n=5. Data under the detection limit (LOD, dashed line) were set to 0 and are depicted below the x axis. Statistical analysis is by Poisson regression versus WT. C, Permeability of the gut epithelium. Permeability was determined by administration of FITC-dextran 4 h before sacrifice. n=5. Statistical analysis is by 1-way ANOVA with Dunnett’s post-test versus values obtained with the WT. D, Leukocyte numbers in the blood (WBC, total white blood cells; Neu, neutrophils; Lymph, lymphocytes; Mono, monocytes). n=5. Statistical analysis is by 2-way ANOVA with post-tests versus values obtained with the WT within each leukocyte group. B, Error bars show the geometric mean and geometric SD. C,D, Error bars show the mean ± SD.

Fig. 3.

The E. faecalis Fsr quorum-sensing is essential for translocation through the intestinal epithelium and subsequent systemic infection. A, Mice received CY for 3 days (−6 to −4), then antibiotics for 3 days (−3 to −1), after which E. faecalis (WT, ΔfsrB, or ΔgelE isogenic deletion strains) were administered on two consecutive days (2 × 10⁹ CFU in 200 μl each) by oral gavage. On day 1 or 3 the indicated parameters were determined in two different experiments (n=9–10/group). B, CFU in blood and organs. Data under the detection limit (LOD, dashed line) were set to 0 and are depicted below the x axis. Statistical analysis is by Poisson regression versus WT. C, Permeability of the gut epithelium. Permeability was determined by administration of FITC-dextran 4 h before sacrifice. Statistical analysis is by Kruskal-Wallis and Dunn’s multiple comparisons tests. D, Histological examination of intestinal epithelia (jejunum). Areas encircled with white dashed lines mark examples of areas with pronounced immune cell infiltration. Green arrows, crypts; yellow arrows, villi. B, Error bars show the geometric mean and geometric SD. C, Error bars show the mean ± SD.

Fig. 4.

The impact of the E. faecalis Fsr quorum-sensing system on experimental systemic infection of intestinal origin is mainly due to translocation effects. A, Direct impact on systemic infection. Mice received 2 × 10⁸ CFU E. faecalis wild-type (WT) or the indicated isogenic mutant bacteria by intravenous injection, without any CY or antibiotic pre-treatment. Three days post infection cardiac puncture was performed, and the blood, liver and kidneys were collected. There were no bacteria found in the blood. n=4–5. Statistical analysis is by two-tailed unpaired Mann-Whitney test. B, Co-colonization model. Mice received CY for 3 days (−6 to −4), then antibiotics for 3 days (−3 to −1), after which E. faecalis WT and ΔfsrB bacteria were administered in equal amounts (1 × 10⁹ CFU in 200 μl each) twice (on two consecutive days) by oral gavage and compared to separate infection with 2 × 10⁹ WT or ΔfsrB bacteria (also given twice on two consecutive days). On day 3 the indicated parameters were determined (n=5/group). C, Histological examination of the intestinal epithelium (jejunum). Areas encircled with white dashed lines mark examples of areas with pronounced immune cell infiltration. Green arrows, crypts; yellow arrows, villi. D, CFU in the blood and intestines. Statistical analysis is by Mann-Whitney tests (intestines) or Poisson regression (organs, blood). Data under the detection limit (LOD, dashed line) were set to 0 and are depicted below the x axis. A,D, Error bars show the geometric mean and geometric SD.

Fig. 5.

B. subtilis fengycin and surfactin lipopeptides inhibit E. faecalis Fsr quorum-sensing. A, Growth, proteolytic activity, fsr and gelE/sprE gene expression during growth of the indicated E. faecalis strains. B, Suppression of proteolytic activity by culture filtrates of the indicated strains. C,D Suppression of proteolytic activity by purified fengycin (C) or surfactin (D). E,F Suppression of fsrBDC (E) or gelE/sprE (F) expression by purified fengycin or surfactin (10 μM). G, Growth and proteolytic activity during growth of E. faecalis OG1RF under influence of the Fsr-autoinducing peptide GBAP. Statistical analysis is by unpaired two-tailed t-tests. H,I Reversion of the fengycin- (H) or surfactin- (I) caused suppression effect on E. faecalis OG1RF proteolytic activity by GBAP. B-F,H,I, Statistical analysis is by 1-way ANOVA with Dunnett’s post-tests versus the respective control groups. A-I, n=3. All error bars show the mean ± SD, except for growth curves, where error bars show the geometric mean and geometric SD. B-I, *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Proteolytic activity or gene expression was determined in cultures grown for 3 h.

Fig. 6.

Oral administration of B. subtilis spores decreases E. faecalis epithelial translocation and eliminates E. faecalis bacteremia by a quorum-quenching mechanism. A, Experimental setup. Mice were pre-treated with CY (days −6 to −4) and an antibiotic mix (days −3 to −1) and received B. subtilis spores (of wild-type or fengycin/surfactin lipopeptide-deficient Δsfp mutant) by oral gavage on the two subsequent days (2 × 10⁷ CFU in 200 μl each), and E. faecalis OG1RF, OG1RF ΔfsrB, or TX0104 (2 × 10⁹ CFU in 200 μl) the next day. 24 h later, mice were sacrificed, the blood was collected by cardiac puncture and CFU were determined by plating. Epithelial permeability was determined by administration of FITC-dextran 4 h before sacrifice. n=5/group. B, CFU in the small and large intestine and the blood. Data under the detection limit were set to 0 and are depicted on/below the x axis. Statistical analysis is by 1-way ANOVA or Kruskal-Wallis test dependent on the result of normality and log-normality tests of the data to be compared (intestines) or Poisson regression (blood). No comparison in the intestine data was statistically significant. n=4–5. C, Epithelial permeability. Permeability was determined by administration of FITC-dextran 4 h before sacrifice. Statistical analysis is by 1-way ANOVA and Tukey’s post-tests (OG1RFΔfsrB, TX0104) or Kruskal-Wallis test with Dunn’s multiple comparisons test (OG1RF). n=5/group. D, Leukocyte numbers. WBC, total white blood cells; Neu, neutrophils; Lymph, lymphocytes; Mono, monocytes. n=4–5. B, Error bars show the geometric mean and geometric SD. C,D, Error bars show the mean ± SD.

Fig. 7.

Oral administration of B. subtilis spores decreases systemic E. faecalis infection of intestinal origin via quorum-quenching. A, Experimental setup. Mice were treated with CY for 3 days (−6 to −4), then with antibiotics for 3 days (−3 to −1), and received B. subtilis spores (of wild-type or fengycin/surfactin lipopeptide-deficient Δsfp mutant) by oral gavage on the two subsequent days (2 × 10⁷ CFU in 200 μl each), and E. faecalis OG1RF on the next two days (2 × 10⁹ CFU in 200 μl each). CFU in the small and large intestine, organs, and the blood were determined after sacrifice on day 1 or 3 in two different experiments (n=8/group, n=10/group, respectively). Epithelial permeability was determined by administration of FITC-dextran 4 h before sacrifice. Histological analysis of the intestinal epithelium (jejunum) was performed. B, CFU in the intestines, organs (liver, spleen) and the blood. Non-plotted data were under the detection limit (20, dashed line) and set to 0. Statistical analysis is by Poisson regression. C, Epithelial permeability. Statistical analysis is by Friedman and Dunn’s multiple comparisons tests. D, Leukocyte numbers. WBC, total white blood cells; Neu, neutrophils; Lymph, lymphocytes; Mono, monocytes. E, Histology. Areas encircled with white dashed lines mark examples of areas with pronounced immune cell infiltration. Green arrows, crypts; yellow arrows, villi. B, Error bars show the geometric mean and geometric SD. C,D, Error bars show the mean ± SD.

Fig. 8.

Model of Fsr quorum-sensing and B. subtilis probiotic-mediated reduction of E. faecalis bacteremia. The Fsr system comprises the fsrA, fsrB, fsrD, and fsrC gene operon. FsrD is secreted and post-translationally modified by FsrB, to the mature GBAP peptide, which stimulates the FsrC-FsrA two-component system by binding to the FsrC transmembrane histidine kinase receptor. The activated FsrA response regulator then increases transcription of the fsrABDC locus (quorum-sensing feedback loop) in addition to that of the two target genes, gelE (gelatinase) and sprE (serine protease), located immediately downstream of the fsrABDC operon. As shown in this study, orally administered B. subtilis spores germinate in the gut and produce fengycin/surfactin lipopeptides that inhibit GBAP-mediated induction of the Fsr quorum-sensing system of E. faecalis. As a result, gelatinase production-mediated destruction of the gut epithelium is avoided and E. faecalis cannot transgress into the bloodstream, abolishing E. faecalis systemic infection.