Antibiotics are often used in the clinic to treat bacterial infections, but the effects of these drugs on microbiota composition and on intestinal immunity are poorly understood. Citrobacter rodentium was used as a model enteric pathogen to investigate the effect of microbial perturbation on intestinal barriers and susceptibility to colitis. Streptomycin and metronidazole were used to induce alterations in the composition of the microbiota prior to infection with C. rodentium. Metronidazole pretreatment increased susceptibility to C. rodentium-induced colitis over that of untreated and streptomycin-pretreated mice, 6 days postinfection. Both antibiotic treatments altered microbial composition, without affecting total numbers, but metronidazole treatment resulted in a more dramatic change, including a reduced population of Porphyromonadaceae and increased numbers of lactobacilli. Disruption of the microbiota with metronidazole, but not streptomycin treatment, resulted in an increased inflammatory tone of the intestine characterized by increased bacterial stimulation of the epithelium, altered goblet cell function, and thinning of the inner mucus layer, suggesting a weakened mucosal barrier. This reduction in mucus thickness correlates with increased attachment of C. rodentium to the intestinal epithelium, contributing to the exacerbated severity of C. rodentium-induced colitis in metronidazole-pretreated mice. These results suggest that antibiotic perturbation of the microbiota can disrupt intestinal homeostasis and the integrity of intestinal defenses, which protect against invading pathogens and intestinal inflammation.The intestinal microbiota can be thought of as an organ system, essential for nutrient acquisition, metabolism of indigestible compounds, defense against colonization by pathogens, and the development of intestinal architecture and the immune system (40,43). Compositional changes in the intestinal microbiota can lead to severe dysregulation of the physiological and immunological intestinal homeostasis, with serious adverse consequences for the host (43). A well-known case of this is antibiotic treatment, and previous studies have shown that antibiotic treatment can predispose the host to enteric infections (48). A recent investigation by Sekirov et al. showed that various doses of antibiotic treatments predispose mice to increased colonization by Salmonella enterica serovar Typhimurium and intestinal pathology (48). Additionally, Brandl et al. showed that administration of a broad-spectrum combination of metronidazole (Met), neomycin, and vancomycin promotes infection by vancomycin-resistant enterococci (9). Further, there are significant differences in microbiota compositions of inflammatory bowel disease (IBD) patients and healthy individuals, further implicating microbial factors in the initiation and perpetuation of colitis (17,19,32,46). It is unknown if such changes precede and contribute to the onset of IBD or are simply a result of IBD. Moreover,