A novel mechanism of action of ketoconazole: inhibition of the NorA efflux pump system and biofilm formation in multidrug-resistant Staphylococcus aureus
Received 10 January 2019
Accepted for publication 11 March 2019
Published 14 June 2019 Volume 2019:12 Pages 1703—1718
Checked for plagiarism Yes
Review by Single-blind
Peer reviewer comments 2
Editor who approved publication: Professor Suresh Antony
Rehab M Abd El-Baky,1,2 Tim Sandle,3 James John,4 Gamal El-Din AA Abuo-Rahma,5 Helal F Hetta6,7
1Department 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, USA
Background: 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<0.01) and decreased the efflux of EtBr. Furthermore, a time-kill assay revealed that combinations of levofloxacin with ketoconazole or carbonyl cyanide m-chlorophenylhydrazone showed no growth for the tested strains after 24 h in comparison to the effect of levofloxacin alone. Docking studies and the ability of ketoconazole to diminish the relative expression of NorA gene in comparison to control (untreated strains) confirmed its action as an efflux pump inhibitor.
Conclusion: The findings showed that the antifungal ketoconazole has no antibacterial activity but can potentiate the activity of the fluroquinolones against MDR S. aureus via inhibiting efflux pump and biofilm formation in vitro.
Keywords: ketoconazole, efflux pump, biofilm, Nor genes
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