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JAC Advance Access originally published online on February 8, 2007
Journal of Antimicrobial Chemotherapy 2007 59(3):378-386; doi:10.1093/jac/dkl504
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© The Author 2007. 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

A combined phenotypic and genotypic method for the detection of Mex efflux pumps in Pseudomonas aeruginosa

Narcisa Mesaros1, Youri Glupczynski2, Laëtitia Avrain1, Nancy E. Caceres1, Paul M. Tulkens1,* and Françoise Van Bambeke1

1 Unité de Pharmacologie cellulaire et moléculaire, Brussels, Université catholique de Louvain, UCL 7370 avenue E. Mounier 73, B-1200 Bruxelles, Belgium 2 Laboratoire de Microbiologie, Cliniques universitaires UCL de Mont-Godinne, avenue G. Therasse 1, B-5530 Yvoir, Belgium

Received 10 August 2006; returned 29 September 2006; revised 10 November 2006; accepted 14 November 2006

* Corresponding author. Tel: +32-2-7622136 / 7647371; Fax: +32-2-7647373; E-mail: tulkens{at}facm.ucl.ac.be

Background: Mex efflux pumps contribute to multidrug resistance in Pseudomonas aeruginosa. Evidencing their expression in clinical isolates would help in rationalizing antibiotic selection.

Methods: We have developed a combined phenotypic and genotypic approach for the differential diagnosis of resistance mediated by four major transporters (MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM). The methodology was validated using reference strains harbouring only one specific transporter and its applicability evaluated towards seven selected clinical isolates, the resistance mechanisms of which could not be assigned by conventional techniques. Phenotypic detection used MIC measurements with reporter antibiotics [carbenicillin (MexAB-OprM); erythromycin (MexCD-OprJ); norfloxacin and imipenem (MexEF-OprN); gentamicin (MexXY-OprM)] with and without Phe-Arg-ß-naphthylamide. Genotypic detection was made by semi-quantitative reverse transcription PCR (RT-PCR) for mexC and mexE, and by quantitative competitive RT-PCR and real-time PCR for mexA and mexX (correlation between both methods: > 88 % ; overexpression levels ranging between 4.8 and 8.1).

Results: Convergence between phenotypic and genotypic methods was observed in control strains for all pumps. For clinical isolates, convergence was obtained in 6 of 7 strains for MexXY-OprM and MexEF-OprM, and in 5 of 7 for MexAB-OprM and MexCD-OprJ, mostly due to hard to interpret phenotypic data.

Conclusions: The data plead for combining phenotypic and genotypic approaches in the diagnosis of efflux-mediated resistance in P. aeruginosa.

Keywords: multidrug resistance , Phe-Arg-ß-naphthylamide , diagnostic , efflux pump , Pseudomonas aeruginosa


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