JAC Advance Access published online on September 6, 2002
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkf147
© 2002 by The British Society for Antimicrobial Chemotherapy
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In Brief
1 Department of Microbiology, University of Manitoba, Winnipeg
MB R3T 2N2, Canada
* Corresponding author. E-mail: eworobe{at}ms.umanitoba.ca.
Received 15 November 2001
; 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.
Fluoroquinolone resistance of Serratia
marcescens: involvement of a proton gradient-dependent efflux
pump
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