Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens

Abstract

The human intestine, colonized by a dense community of resident microbes, is a frequent target of bacterial pathogens. Undisturbed, this intestinal microbiota provides protection from bacterial infections. Conversely, disruption of the microbiota with oral antibiotics often precedes the emergence of several enteric pathogens. How pathogens capitalize upon the failure of microbiota-afforded protection is largely unknown. Here we show that two antibiotic-associated pathogens, Salmonella enterica serovar Typhimurium (S. typhimurium) and Clostridium difficile, use a common strategy of catabolizing microbiota-liberated mucosal carbohydrates during their expansion within the gut. S. typhimurium accesses fucose and sialic acid within the lumen of the gut in a microbiota-dependent manner, and genetic ablation of the respective catabolic pathways reduces its competitiveness in vivo. Similarly, C. difficile expansion is aided by microbiota-induced elevation of sialic acid levels in vivo. Colonization of gnotobiotic mice with a sialidase-deficient mutant of Bacteroides thetaiotaomicron, a model gut symbiont, reduces free sialic acid levels resulting in C. difficile downregulating its sialic acid catabolic pathway and exhibiting impaired expansion. These effects are reversed by exogenous dietary administration of free sialic acid. Furthermore, antibiotic treatment of conventional mice induces a spike in free sialic acid and mutants of both Salmonella and C. difficile that are unable to catabolize sialic acid exhibit impaired expansion. These data show that antibiotic-induced disruption of the resident microbiota and subsequent alteration in mucosal carbohydrate availability are exploited by these two distantly related enteric pathogens in a similar manner. This insight suggests new therapeutic approaches for preventing diseases caused by antibiotic-associated pathogens.

Figure 1. Bt facilitates S. typhimurium and C. difficile carbohydrate utilization during emergence

a, Schematic of mouse infection experiments. Germ-free, GF; B. thetaiotaomicron, Bt; S. typhimurium, St; C. difficile, Cd. b, S. typhimurium operons displaying significant differences in gene expression levels in vivo in the presence and absence of Bt, 5 days post-infection. Colors indicate the deviation of each gene's signal above (purple) and below (green) its mean expression value across all six in vivo samples and duplicate in vitro growths conducted in minimal medium. c, Induction of S. typhimurium nanE and fucI in cecal contents 1 day post-infection relative to growth in LB broth [n = 9 and 4 for St and St+Bt, respectively]. d, Competitive index of wild-type St/St-ΔnanAΔfucI in Bt-monoassociated (St+Bt) and ex-germ-free (St) mice 1 day post-infection. Horizontal bars indicate the geometric means of CI values, and individual CI values are represented with dots [n = 5/group]. e, Induction of C. difficile nan genes in cecal contents 3 days post-infection relative to growth in minimal medium containing 0.5% glucose [n = 4/group]. f, C. difficile density in feces 1 day post-infection [n = 4/group]. Error bars indicate SEM.

Figure 2. Bt-liberated sialic acid promotes emergence of S. typhimurium and C. difficile

a, Levels of free sialic acid in cecal contents in GF and gnotobiotic mice monoassociated for 10 days [n=3, 3, 5, 5, respectively]. b, Fold change of expression of S. typhimurium nanE in cecal contents 1 day post-infection relative to growth in vitro [n = 9, 4, 5, 5, respectively]. c, Induction of C. difficile nanE expression in cecal contents 3 days post-infection relative to growth in minimal medium containing 0.5% glucose [n = 4/group]. d, C. difficile density in feces 1 day post-infection [n = 5/group]. e, C. difficile density 1 day post-infection in feces of PBS or exogenous free sialic acid (SIA) treated mice. [n = 4–5/group]. f, Induction of C. difficile nanE gene expression 1 day post-infection in feces of PBS or exogenous free sialic acid (SIA) treated mice relative to growth in minimal medium containing 0.5% glucose [n = 5/group]. Error bars indicate SEM.

Figure 3. S. typhimurium and C. difficile utilize mucin-derived monosaccharides resulting from antibiotic treatment of conventional mice

a, Levels of free sialic acid in cecal contents of conventional mice (CONV), antibiotic-treated mice 1 day (Ab D1) and 3 days (Ab D3) post-treatment [n= 8, 9, 3, respectively]. b, Competitive index of wt S. typhimurium versus S. typhimurium mutants in cecal contents (St-ΔnanA) or feces (St-ΔnanAΔfucI) of antibiotic-treated conventional mice. Horizontal bars indicate the geometric means of CI values, and individual CI values are represented with dots [n=5 and 9, respectively]. c, Induction of C. difficile nanA and nanT expression in fecal samples 1 day post-infection of antibiotic-treated conventional mice relative to growth in minimal medium containing 0.5% glucose [n = 4/group]. d, Density of wt C. difficile or a mutant deficient in sialic acid consumption (Cd-nanT⁻) 3 days post-infection in feces of antibiotic-treated conventional mice. [n = 10/group]. Error bars indicate SEM.