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Objective: To characterize the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) transmission and infections in a level IIIC neonatal intensive care unit (NICU) and identify barriers to MRSA control., Setting and Design: Retrospective cohort study in a university-affiliated NICU with an MRSA control program including weekly nares cultures of all neonates and admission nares cultures for neonates transferred from other hospitals or admitted from home., Methods: Medical records were reviewed to identify neonates with NICU-acquired MRSA colonization or infection between April 2007 and December 2011. Compliance with hand hygiene and an MRSA decolonization protocol were monitored. Relatedness of MRSA strains were assessed using pulsed-field gel electrophoresis (PFGE)., Results: Of 3,536 neonates, 74 (2.0%) had a culture grow MRSA, including 62 neonates with NICU-acquired MRSA. Nineteen of 74 neonates (26%) had an MRSA infection, including 8 who became infected before they were identified as MRSA colonized, and 11 of 66 colonized neonates (17%) developed a subsequent infection. Of the 37 neonates that underwent decolonization, 6 (16%) developed a subsequent infection, and 7 of 14 (50%) that remained in the NICU for 21 days or more became recolonized with MRSA. Using PFGE, there were 14 different strain types identified, with USA300 being the most common (31%)., Conclusions: Current strategies to prevent infections-including active identification and decolonization of MRSA-colonized neonates-are inadequate because infants develop infections before being identified as colonized or after attempted decolonization. Future prevention efforts would benefit from improving detection of MRSA colonization, optimizing decolonization regimens, and identifying and interrupting reservoirs of transmission.