Bacillus subtilis-Mediated Protection from Citrobacter rodentium-Associated Enteric Disease RequiresespHand Functional Flagella

Abstract

Commensals limit disease caused by invading pathogens; however, the mechanisms and genes utilized by beneficial microbes to inhibit pathogenesis are poorly understood. The attaching and effacing mouse pathogen Citrobacter rodentium associates intimately with the intestinal epithelium, and infections result in acute colitis. C. rodentium is used to model the human pathogens enterohemorrhagic Escherichia coli and enteropathogenic E. coli. To confirm that Bacillus subtilis, a spore-forming bacterium found in the gut of mammals, could reduce C. rodentium-associated disease, mice received wild-type B. subtilis spores and 24 h later were infected by oral gavage with pathogenic C. rodentium. Disease was assessed by determining the extent of colonic epithelial hyperplasia, goblet cell loss, diarrhea, and pathogen colonization. Mice that received wild-type B. subtilis prior to enteric infection were protected from disease even though C. rodentium colonization was not inhibited. In contrast, espH and hag mutants, defective in exopolysaccharides and flagellum production, respectively, did not protect mice from C. rodentium-associated disease. A motAB mutant also failed to protect mice from disease, suggesting that B. subtilis-mediated protection requires functional flagella. By expanding our current mechanistic knowledge of bacterial protection, we can better utilize beneficial microbes to prevent intestinal disease caused by pathogenic bacteria, ultimately reducing human disease. Our data demonstrate that wildtype B. subtilis reduced disease caused by C. rodentium infection through a mechanism that required espH and functional flagella.

Oral Administration of a Select Mixture of Bacillus Probiotics Affects the Gut Microbiota and Goblet Cell Function following Escherichia coli Challenge in Newly Weaned Pigs of Genotype MUC4 That Are Supposed To Be Enterotoxigenic E. coli F4ab/ac Receptor Negative

“…In addition, administration of the moderate-dose BLS mix increased the number of GCs in the ileum during F4 ϩ ETEC infection. Consistent with our results, B. subtilis pretreatment reduces the severity of Citrobacter rodentium-induced diarrhea in mice, partly through inhibition of GC loss in the colon (47). Furthermore, Bacteroides thetaiotaomicron increases the number of GCs and the expression of mucin genes in gnotobiotic rats, whereas Faecalibacterium prausnitzii diminishes this effect in a process involving acetate and butyrate metabolism (48).…”

Protection from Intestinal Inflammation by Bacterial Exopolysaccharides

“…B. subtilis is a gram positive spore-forming bacterium present in the gastrointestinal tract of both humans and mice (8, 9). Several groups report that select probiotic strains of B. subtilis relieve the symptoms associated with antibiotic-associated diarrhea and irritable bowel syndrome in human patients; however the mechanisms of protection have not been well established (6, 8).…”

Exopolysaccharides: Sweet success with probiotic therapeutics

“…To test this idea, we used biofilm mutants, predicting that these mutants would not prevent C. rodentium-induced disease. Surprisingly, some biofilm mutants were protective and others were not, even though all of the mutants tested were present at levels comparable to wild type [32] . All protective strains were capable of producing EPS as part of their biofilm matrix, leading us to suggest that EPS is the protective molecule of B. subtilis.…”

“…Our lab has studied the beneficial role of the intestinal microbiota for years and recently, we showed that a single dose of B. subtilis prevents enteric inflammatory disease in mice infected with Citrobacter rodentium [32] . C. rodentium is an attaching and effacing murine pathogen that associates intimately with the epithelium to induce colitis in a manner similar to the human pathogen, enteropathogenic E. coli [33,34] .…”