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A randomized controlled study demonstrated that an optimized intraoperative infection control program targeting basic preventive measures can reduce Staphylococcus aureus transmission and surgical site infections. In this study we address potential limitations of operating room heterogeneity of infections and compliance with behavioral interventions following adoption into clinical practice.A post-implementation prospective case-cohort study.Twenty-three operating rooms at a large teaching hospital.A total of 801 surgical patients [425 (53%) women; 350 (44%) ASA 2, age 54.6 ± 15.9 years] were analyzed for the primary and 804 for the secondary outcomes.A multifaceted, evidence-based intraoperative infection control program involving hand hygiene, vascular care, and environmental cleaning improvements was implemented for 23 operating room environments. Bacterial transmission monitoring was used to provide monthly feedback for intervention optimization.S. aureus transmission (primary) and surgical site infection (secondary).The incidence of S. aureus transmission and surgical site infection before (3.5 months) and after (4.5 months) infection control optimization was assessed. Optimization was defined by a sustained reduction in anesthesia work area bacterial reservoir isolate counts. Poisson regression with robust error variances was used to estimate the incidence risk ratio (IRR) of intraoperative S. aureus transmission and surgical site infection for the independent variable of optimization.Optimization was associated with decreased S. aureus transmission [24% before (85/357) to 9% after (42/444), IRR 0.39, 95% CI 0.28 to 0.56, P .001] and surgical site infections [8% before (29/360) and 3% after (15/444) (IRR 0.42, 95% CI 0.23 to 0.77, P = .005; adjusted for American Society of Anesthesiologists' physical status, aIRR 0.45, 95% CI 0.25 to 0.82, P = .009].An optimized intraoperative infection control program targeting improvements in basic preventive measures is an effective and feasible approach for reducing S. aureus transmission and surgical site infection development.