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JAC Advance Access published online on October 13, 2006
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkl404
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received May 25, 2005
Revised August 30, 2006
Accepted September 7, 2006

Brief report

Analysis of the mechanisms of fluoroquinolone resistance in urinary tract pathogens

Hafizah Y. Chenia 1, Balakrishna Pillay 2, and Dorsamy Pillay 3 *

1 Department of Microbiology, School of Microbiology and Biochemistry, University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa; Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa
2 Department of Microbiology, School of Microbiology and Biochemistry, University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa
3 Department of Microbiology, School of Microbiology and Biochemistry, University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa; Department of Biotechnology, Durban University of Technology, PO Box 1334, Durban 4000, South Africa

* To whom correspondence should be addressed.
Dorsamy Pillay, E-mail: gansen{at}dut.ac.za


  Abstract

Objectives: To characterize the mechanisms of fluoroquinolone resistance in urinary tract pathogens exhibiting a multiple antibiotic resistance phenotype as well as a high-level resistance to fluoroquinolones.

Methods: Nineteen Escherichia coli urinary tract infection pathogens exhibiting high-level resistance to fluoroquinolones were characterized in this study. Alterations in outer membrane proteins (OMPs) and lipopolysaccharide (LPS) were analysed by PAGE. Changes to the quinolone resistance-determining regions (QRDRs) of GyrA and ParC were determined by PCR and DNA sequencing. The presence of the qnrA gene was determined by PCR amplification. Ciprofloxacin uptake was determined spectrophotometrically using the quinolone accumulation assay.

Results: OMP analysis showed decreased expression, the absence of certain proteins or the presence of proteins with altered molecular weights when compared with wild-type strains. Most isolates possessed a smooth LPS phenotype. Isolates had double mutations in GyrA codons 83 and 87, in addition to a ParC alteration at Ser-80/Glu-84. Isolates accumulated varying levels of ciprofloxacin, and upon the addition of carbonyl cyanide m-chlorophenylhydrazone, increased accumulation was observed in all instances. E. coli isolates with a rough LPS phenotype appeared to accumulate higher levels of ciprofloxacin compared with those with a smooth LPS phenotype expressing OmpF normally, or even those not possessing OmpF. All E. coli isolates tested demonstrated active efflux of ciprofloxacin. Plasmid-mediated quinolone resistance (presence of the qnrA gene) was observed in 36.8% of isolates.

Conclusions: A combination of target gene alterations, altered OM permeability, presence of the qnrA gene and active efflux appear to act together to produce a high-level, multiresistance phenotype.

Keywords: fluoroquinolone resistance; OMP; LPS; GyrA and ParC alterations; energy-dependent efflux systems.
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