JAC Advance Access originally published online on September 6, 2002
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Journal of Antimicrobial Chemotherapy (2002) 50, 593-596
© 2002 The British Society for Antimicrobial Chemotherapy
Fluoroquinolone resistance of Serratia marcescens: involvement of a proton gradient-dependent efflux pump
Department of Microbiology, University of Manitoba, Winnipeg MB R3T 2N2, Canada
Received 15 November 2001; returned 7 March 2002; revised 16 May 2002; accepted 14 June 2002
Objective: To determine the presence of a proton gradient-dependent efflux of fluoroquinolone drugs in Serratia marcescens.
Methods: Thirteen clinical isolates of S. marcescens were screened for resistance to four fluoroquinolones: ofloxacin, ciprofloxacin, norfloxacin and nalidixic acid by determining MICs. The presence of a proton gradient-dependent efflux mechanism was assessed using ethidium bromide accumulation assays. Drug accumulation studies for norfloxacin, ciprofloxacin and ofloxacin were performed to determine the drug specificity of efflux. Western transfer of cellular proteins, followed by immunodetection using anti-AcrA (Escherichia coli) antibodies were used to demonstrate the presence of a resistance–nodulation–cell division (RND) pump protein. PCR was used to identify a RND pump-encoding gene using primers for two conserved motifs within inner membrane components of RND proteins. A mutant strain of S. marcescens, UOC-67WL, was isolated by culturing the wild-type strain in the presence of ciprofloxacin in T-soy media and was subjected to the same studies as described above for the clinical isolates.
Results: Ethidium bromide accumulation assays confirmed the presence of a proton gradient-dependent efflux mechanism in S. marcescens. One clinical isolate, T-861, and the mutant strain, UOC-67WL, were found to efflux ciprofloxacin and ofloxacin. Western immunoblot results confirmed overexpression of an AcrA-like protein in T-861 and UOC-67WL. Sequencing of the PCR product showed the presence of a mexF-like gene, which is overexpressed in nfxC mutants of Pseudomonas aeruginosa.
Conclusion: This study reports the presence of a proton gradient-dependent efflux mechanism in S. marcescens.
* Corresponding author. Tel: +1-204-474-8473; Fax: +1-204-474-7603; E-mail: eworobe{at}ms.umanitoba.ca
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