A novel mechanism of action of ketoconazole: inhibition of the NorA efflux pump system and biofilm formation in multidrug-resistant Staphylococcus aureus Rehab M Abd El-Baky,1,2 Tim Sandle,3 James John,4 Gamal El-Din AA Abuo-Rahma,5 Helal F Hetta6,71Department of Microbiology and Immunology, Faculty of Pharmacy, Minia University, Minia 61519, Egypt; 2Microbiology and Immunology Department, Faculty of Pharmacy, Deraya University, Minia 11566, Egypt; 3School of Health Sciences, Division of Pharmacy & Optometry, University of Manchester, Manchester, UK; 4Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Chennai, India; 5Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, Minia 61519, Egypt; 6Department of Medical Microbiology & Immunology, Faculty of Medicine, Assiut University, Assiut, Egypt; 7Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USABackground: The rapid emergence of antimicrobial resistance among Gram-positive organisms, especially staphylococci, has become a serious clinical challenge. Efflux machinery and biofilm formation are considered two of the main causes of antimicrobial resistance and therapy failure.Aim: Our study aims to evaluate the antibiofilm and efflux pump inhibitory activity of the antifungal ketoconazole against multidrug-resistant (MDR) Staphylococcus aureus.Methods: Ketoconazole was tested for its effect on the following: minimum inhibitory concentrations (MICs) of ciprofloxacin, norfloxacin, levofloxacin, and ethidium bromide (EtBr) by the broth microdilution method, the efflux of EtBr by NorA-positive MDR S. aureus, and the relative expression of NorA, NorB, and NorC efflux pump genes. Docking studies of ketoconazole were performed using 1PW4 (glycerol-3-phosphate transporter from Escherichia coli which was the representative structure from the major facilitator superfamily).Results: Ketoconazole significantly decreased the MICs of levofloxacin, ciprofloxacin, norfloxacin, and EtBr (a substrate for efflux pump) by 8 to 1024-fold (P