Nutrient Availability and Biofilm Polysaccharide Shape the Bacillaene-Dependent Antagonism of Bacillus subtilis against Salmonella Typhimurium

Abstract

Salmonella enterica is one of the most common foodborne pathogens and, due to the spread of antibiotic resistance, new antimicrobial strategies are urgently needed to control it. In this study, we explored the probiotic potential of Bacillus subtilis PS-216 and elucidated the mechanisms that underlie the interactions between this soil isolate and the model pathogenic strain S. Typhimurium SL1344. The results reveal that B. subtilis PS-216 inhibits the growth and biofilm formation of S. Typhimurium through the production of the pks cluster-dependent polyketide bacillaene. The presence of S. Typhimurium enhanced the activity of the P_pksC promoter that controls bacillaene production, suggesting that B. subtilis senses and responds to Salmonella. The level of Salmonella inhibition, overall P_pksC activity, and P_pksC induction by Salmonella were all higher in nutrient-rich conditions than in nutrient-depleted conditions. Although eliminating the extracellular polysaccharide production of B. subtilis via deletion of the epsA-O operon had no significant effect on inhibitory activity against Salmonella in nutrient-rich conditions, this deletion mutant showed an enhanced antagonism against Salmonella in nutrient-depleted conditions, revealing an intricate relationship between exopolysaccharide production, nutrient availability, and bacillaene synthesis. Overall, this work provides evidence on the regulatory role of nutrient availability, sensing of the competitor, and EpsA-O polysaccharide in the social outcome of bacillaene-dependent competition between B. subtilis and S. Typhimurium. IMPORTANCE Probiotic bacteria represent an alternative for controlling foodborne disease caused by Salmonella enterica, which constitutes a serious concern during food production due to its antibiotic resistance and resilience to environmental stress. Bacillus subtilis is gaining popularity as a probiotic, but its behavior in biofilms with pathogens such as Salmonella remains to be elucidated. Here, we show that the antagonism of B. subtilis is mediated by the polyketide bacillaene and that the production of bacillaene is a highly dynamic trait which depends on environmental factors such as nutrient availability and the presence of competitors. Moreover, the production of extracellular polysaccharides by B. subtilis further alters the influence of these factors. Hence, this work highlights the inhibitory effect of B. subtilis, which is condition-dependent, and the importance of evaluating probiotic strains under conditions relevant to the intended use.

Keywords: Bacillus subtilis; Salmonella enterica; biofilm; enteric pathogen; microbial competition; nutrients; polysaccharides; probiotics.

FIG 1

The influence of nutrients on cell counts of Bacillus subtilis BM1097 and Salmonella Typhimurium SL1344 GFP in monocultures and cocultures. Bacterial cells were grown in tryptic soy broth (TSB), 1/5 TSB, and 1/20 TSB media and CFU/mL values were determined after 24 h of static incubation at 37°C. Data sets represent means and standard deviation of three biological replicates. Statistically significant results (P < 0.05) were determined using Student’s t test and are indicated with an asterisk (*).

FIG 2

Spatial distribution of B. subtilis BM1097 (red) and S. Typhimurium SL1344 GFP (green) and thickness of S. Typhimurium SL1344 GFP biofilm in different growth media. (A to C) 3D view of B. subtilis wild-type (WT) strain BM1097 and S. Typhimurium SL1344 GFP monocultures and cocultures in TSB (A), 1/5 TSB (B), and 1/20 TSB medium (C) after 24 h of static incubation at 37°C. Only orthogonal views of B. subtilis and S. Typhimurium cocultures in three different media are shown. The middle image in the orthogonal view represents the first slice (319 μm × 319 μm), which was measured from the bottom of the well. Side and top images in the orthogonal view represent the y to z and x to z stacks, respectively, with z-stack 50 μm in size. (D) Thickness of S. Typhimurium SL1344 GFP biofilm in monocultures and cocultures in TSB, 1/5 TSB, and 1/20 TSB media after 24 h of static incubation at 37°C. (E) Adhesion of S. Typhimurium SL1344 GFP on polystyrene surface after 24 h of static incubation in monoculture and coculture with B. subtilis WT strain BM1097 at 37°C in TSB medium. Data sets represent means and standard deviation of three biological replicates. A Student’s t test was used to determine statistically significant differences (P < 0.05) to monoculture, which are marked with an asterisk (*).

FIG 3

The role of bacillaene in competition with Salmonella. (A) Effect of different B. subtilis strains on S. Typhimurium SL1344 GFP growth in TSB medium after 24 h of static incubation at 37°C. (B) Adhesion of S. Typhimurium SL1344 GFP on polystyrene surface after 24 h of static cocultivation with the B. subtilis PS-216 Δpks mutant (BM1957) at 37°C in TSB medium. Data sets represent mean values with the standard deviation of three biological replicates. A Student’s t test was used to determine statistically significant differences (P < 0.05) to monoculture, which are marked with an asterisk (*). (C) Transcriptional activity of P_pksC-yfp promoter of the B. subtilis BM1884 strain in monoculture and coculture with S. Typhimurium SL1344. Measurements of P_pksC-yfp promoter activities were performed in TSB and 1/20 TSB media every half-hour (for clarity, only data points measured every hour are shown). Results are presented as relative units, as described in the Methods section, using a constitutively expressed P₄₃-mKate2 promoter as a proxy for B. subtilis biomass.

FIG 4

The influence of biofilm matrix deficiency on bacillaene-mediated antagonism against S. Typhimurium. (A) 3D view of monoculture and cocultures of B. subtilis PS-216 ΔepsA-O mutant (BM1310) (red) and S. Typhimurium SL1344 GFP (green) and orthogonal view of coculture in TSB medium after 24 h of static incubation at 37°C. In the orthogonal view, the middle image shows the first slice (319 μm × 319 μm) measured from the bottom of the well. In the orthogonal view, side and top images represent the y to z and x to z stacks, respectively, with z-stack 50 μm in size. A similar spatial distribution was observed in 1/20 TSB medium (data not shown). (B) Growth inhibition of S. Typhimurium SL1344 GFP in coculture with the B. subtilis PS-216 WT strain (BM1097), B. subtilis PS-216 ΔepsA-O mutant (BM1310), and B. subtilis PS-216 ΔepsA-O Δpks double mutant (BM1906). Measurements of CFU/mL were performed after 24 h of coincubation in static conditions in TSB and 1/20 TSB media at 37°C. Data sets represent mean values with the standard deviation of three biological replicates. A Student’s t test was used to determine statistically significant differences (*, P < 0.05) to the B. subtilis PS-216 ΔepsA-O mutant (BM1310). (C) Transcriptional activity of the P_pksC-yfp promoter of the B. subtilis BM1884 and BM1901 strains. Measurements of the P_pksC-yfp promoter activity were performed in TSB and 1/20 TSB media every half-hour (for clarity, only data points measured every hour are shown). Results are presented as relative units, as described in Methods, and using the constitutively expressed P₄₃-mKate2 promoter as a proxy for B. subtilis biomass.