Enterotoxicity of a nonribosomal peptide causes antibiotic-associated colitis

Antibiotic therapy disrupts the human intestinal microbiota. In some patients rapid overgrowth of the enteric bacterium Klebsiella oxytoca results in antibiotic-associated hemorrhagic colitis (AAHC). We isolated and identified a toxin produced by K. oxytoca as the pyrrolobenzodiazepine tilivalline and demonstrated its causative action in the pathogenesis of colitis in an animal model. Tilivalline induced apoptosis in cultured human cells in vitro and disrupted epithelial barrier function, consistent with the mucosal damage associated with colitis observed in human AAHC and the corresponding animal model. Our findings reveal the presence of pyrrolobenzodiazepines in the intestinal microbiota and provide a mechanism for colitis caused by a resident pathobiont. The data link pyrrolobenzodiazepines to human disease and identify tilivalline as a target for diagnosis and neutralizing strategies in prevention and treatment of colitis.The human digestive tract houses several hundred microbial species (1). This complex community, known collectively as the intestinal microbiota, is important for human digestive physiology, immune function, and protection from pathogens (2). In healthy humans homeostasis between the microbiota and the host is maintained. Perturbed homeostasis, a state known as dysbiosis, is defined by shifts in bacterial abundances, their altered local distribution, and modified metabolic activities. Infections or nutritional or therapeutic stressors can induce dysbiosis and alter the integrity of the mucosal barrier. The pathogenesis of many disorders has been associated with dysbiotic changes in the intestinal microbiota, including metabolic diseases, cancer, allergies, and afflictions of the bowel, liver, and lung (3–6). The underlying mechanisms are poorly understood; however, recent work is beginning to show that microbial metabolites are important mediators of complex interactions between the enteric microbiota and the host. Thus, bacterial metabolites produced in a dysbiotic intestinal environment together with host factors may be important determinants in the pathogenesis of disease (7, 8).Many drug therapies, and especially antibiotics, disrupt the human intestinal microbiota. Up to 30% of patients taking antibiotics experience diarrhea (9). With the exception of infection with toxin-producing Clostridium difficile, the underlying causes for the vast majority of diarrhea or colitis cases after antibiotic therapy are not known. A link to antibiotic-driven changes in microbial abundances and overgrowth of certain bacterial species is observed; however, the pathophysiological mechanisms remain to be determined (10). The expanding species are often not pathogens (11, 12) and in the majority of cases no single organism seems to explain either the presence or absence of disease.In contrast to this general view, antibiotic-associated hemorrhagic colitis (AAHC) is a disease associated with antibiotic-driven enterobacterial overgrowth, which is dominated by a single organism, Klebsiella oxytoca. This bacterium is a resident of the gut in 2–10% of healthy individuals (13–15). In AAHC, a brief therapy with penicillins triggers dysbiosis with sudden onset of bloody diarrhea and abdominal cramps (14). K. oxytoca constitutively expresses resistance to amino- and carboxypenicillins (16). Antibiotic clearance of a niche in the colon facilitates overgrowth of K. oxytoca (10⁷ cfu/g feces during acute phases of AAHC compared with 10² cfu/g feces in healthy subjects) (15). Our earlier work showed that the dysbiotic population shift dominated by K. oxytoca causes colitis (14), thereby identifying the resident organism as a pathobiont (17).Here we aimed to characterize the pathogenicity of K. oxytoca in colitis. We identified the nonribosomal peptide tilivalline from K. oxytoca and demonstrate its causative action in an animal model of AAHC. Unlike most known enterotoxins, tilivalline is not a protein but a pyrrolobenzodiazepine metabolite not previously linked with the intestinal microbiota and human disease. Our findings define a mechanism whereby antibiotic-driven enterobacterial overgrowth and concomitant production of the small molecule enterotoxin ultimately leads to colitis.