A combined phenotypic and genotypic method for the detection of Mex efflux pumps in Pseudomonas aeruginosa.
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-beta-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.
Mesaros
Narcisa
N
Unité de Pharmacologie cellulaire et moléculaire, Brussels, Université catholique de Louvain, UCL 7370 avenue E. Mounier 73, B-1200 Bruxelles, Belgium.
Glupczynski
Youri
Y
Avrain
Laëtitia
L
Caceres
Nancy E
NE
Tulkens
Paul M
PM
Van Bambeke
Françoise
F
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000502:physiology*; D024881:Drug Resistance, Bacterial; D005838:Genotype; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000502:physiology*; D010641:Phenotype; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001425:Bacterial Outer Membrane Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C118598:MexA protein, Pseudomonas aeruginosa; C118597:MexB protein, Pseudomonas aeruginosa; C470189:MexC protein, Pseudomonas aeruginosa; C121980:MexD protein, Pseudomonas aeruginosa; C470190:OprJ protein, Pseudomonas aeruginosa; C402263:OprM protein, Pseudomonas aeruginosa; C105708:OprN protein, Pseudomonas aeruginosa