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Background To determine the prevalence of intestinal S. aureus colonization of patients at a large teaching hospital and determine the molecular characteristics of the identified strains. The second objective of this research was to determine risk factors associated with S. aureus intestinal colonization. Methods A cross-sectional study of 781 specimens from inpatients and outpatients at the University of Iowa Hospitals and Clinics Clinical Microbiology Laboratory was conducted. S. aureus was identified using traditional culture methodologies. Methicillin-resistance was determined via PCR of the mecA gene. PVL PCR, spa typing, and antimicrobial sensitivity testing were also done. A nested case-control study was done on a subset of patients with all colonized patients defined as cases and non-colonized controls. Medical record abstractions were done to identify risk factors for intestinal colonization in the nested study. Results Out of 625 patients included in the final study, 58 were positive for S. aureus (9.3%). One isolate was positive for the PVL gene. A high number of isolates were resistant to multiple antibiotics including oxacillin (43.1%), erythromycin (51.7%), and levofloxacin (41.4%). All isolates were susceptible to vancomycin, daptomycin, linezolid, and quinupristin-dalfopristin. In the nested study, having a disease or condition of the gastrointestinal tract significantly increased the odds of intestinal colonization (OR: 1.96, 95% CI: 1.04–3.7; aOR: 13.9, 95% CI: 1.67–115.7). No other variables were significantly associated with increased odds of colonization. Conclusions S. aureus was identified from the stool of patients at the University of Iowa Hospitals and Clinics, with a large number of those isolates being resistant to antibiotics and may serve a reservoir for subsequent infections as well as asymptomatic transmission.