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Background: Surgical antimicrobial prophylaxis (SAP) is a leading indication for antibiotic use in Australian hospitals with established high rates of inappropriate prescribing. Optimal administration of SAP for patients at high risk of methicillin-resistant Staphylococcus aureus (MRSA) infections presents additional complexities. A greater understanding of barriers to optimal SAP in this cohort is required to inform targeted antimicrobial stewardship strategies, optimize SAP, and reduce the rate of surgical site infections (SSIs). Methods: A multiple-choice questionnaire appraising knowledge and barriers to optimal SAP was electronically distributed to key stakeholders. Data from the questionnaire were collated and analyzed using Survey Monkey® (Momentive Inc., San Mateo, CA) data analysis tools. Results: Eighty-three persons provided full or partial responses to the questionnaire. There were 19% of respondents who considered MRSA colonization status of patients to be only "somewhat important" when selecting appropriate SAP. Additionally, 62% of responses did not correctly identify the appropriate SAP regimen for patients who are colonized with MRSA. Several barriers to optimal SAP were identified including poor understanding of SAP guidelines, lack of timely identification of patients confirmed to be colonized with MRSA, inaccurate documentation of antibiotic and surgical start times, and limitations of the current operating room management software program. Conclusions: The high level of engagement from most key stakeholders demonstrates accountability and an overall desire to improve SAP. Barriers identified in this audit should be considered by facilities wishing to optimize compliance with SAP guidelines and consequently reduce SSIs, in particular for patients who are at high risk of MRSA infections. [ABSTRACT FROM AUTHOR]