bResistance to the innate defenses of the intestine is crucial for the survival and carriage of Staphylococcus aureus, a common colonizer of the human gut. Bile salts produced by the liver and secreted into the intestines are one such group of molecules with potent antimicrobial activity. The mechanisms by which S. aureus is able to resist such defenses in order to colonize and survive in the human gut are unknown. Here we show that mnhF confers resistance to bile salts, which can be abrogated by efflux pump inhibitors. MnhF mediates the efflux of radiolabeled cholic acid both in S. aureus and when heterologously expressed in Escherichia coli, rendering them resistant. Deletion of mnhF attenuated the survival of S. aureus in an anaerobic three-stage continuous-culture model of the human colon (gut model), which represents different anatomical areas of the large intestine. Staphylococcus aureus is a ubiquitous and highly adaptable human pathogen responsible for a significant global burden of morbidity and mortality. The bacterium lives as a commensal in the nares of 20 to 25% of the population at any one time (1, 2). While nasal colonization is a well-established risk factor for most types of S. aureus infections, several recent studies have suggested that colonization of the intestine, which occurs in ca. 20% of individuals and which by and large has been overlooked, could have important clinical implications (3). Patients with S. aureus intestinal colonization can serve as an important source of transmission, as they often contaminate the adjacent environment (4). Similarly, such patients display an increased frequency of skin colonization (5). A study of intensive care and liver transplant units showed that patients with both rectal and naris colonization by methicillin-resistant S. aureus (MRSA) had a significantly higher risk of disease (40%) than did patients with nasal colonization alone (18%) (6). Furthermore, a study of hospitalized patients in the United States reported cocolonization by S. aureus and vancomycin-resistant enterococci in Ͼ50% of the individuals studied (7). Thus, it is likely that intestinal colonization by S. aureus provides the pathogen a potential opportunity to acquire new antibiotic resistance genes.While the clinical implications of intestinal colonization by S. aureus are still relatively ill defined, it is assumed that carriage is a risk for intestinal infection; S. aureus can induce pseudomembranous colitis that is histologically distinct from that caused by Clostridium difficile (8). Multiple studies have demonstrated frequent intestinal colonization in infants, particularly those who were breast fed, and that there is a positive correlation with the development of allergies (9-13). While a role for S. aureus intestinal carriage in the development of systemic S. aureus disease has not been established, colonization of the intestinal lumen of mice can lead to the pathogen crossing the intestinal epithelial barrier and subsequently spreading to the mesenteric lymph nodes (14, 15)...