Development of carbapenem resistance is common in Pseudomonas aeruginosa. This resistance, which is due mainly to alteration of the OprD porin, the specific uptake pathway of carbapenems, may also result from acquisition of foreign genes encoding Ambler class A, class B, or class D B-lactamases. These enzymes able to hydrolyze carbapenems at various degrees. Thus, detection of carbapenemase producers in clinical laboratories is of utmost importance for the determination of appropriate therapeutic schemes and the implementation of infection control measures. In order to improve the detection of carbapenemase producers, various inhibitor-based tests and enzymatic assays (e.g., NP-Carba) have been proposed as a first screening step prior to the use of confirmatory molecular techniques. However, because most of imipenem-non susceptible strains are just OprD-deficient mutants, these phenotypic or enzymatic tests usually yield low rates of positivity.A new test is developed in order to screen for OprD-deficient mutants thus discriminating carbapenemase producingP. Aeruginosa strains from non-producers. This test combines imipenem and cloxacillin, a strong inhibitor of intrinsic cephalosporinase AmpC. It is based on the observation that imipenem resistance resulting from OprD deficiency requires constitutive and/or carbapenem-induced overproduction of AmpC. Therefore, inhibition of AmpC by cloxacillin is expected to restore partial or complete sensitivity to imipenem in OprD-deficient strains but not in carbapenemase positive strains. The aim of this study was to evaluate the performance of a simple, inexpensive detection method applicable in medical laboratories that used combined disk testing of Imipenem and cloxacillin, in order to discriminate carbapenemase producing P. aeruginosastrains from non-producers(OprD-deficient mutants).Fifty clinical isolates of imipenem resistant P. aeruginosa, which were well characterized by MHT for carbapenamse production and by real time PCR for (bla_IMP) gene and (bla_KPC) gene from previous studies, were used. Combined test testing (CDT) using a carbapenem disk (imipenem) supplemented with various loads of cloxacillin was assessed. Out of 50 P.aeruginosa isolates,15 (30%)isolates were positive by Imipenem-Cloxacillin CDT at conc. 4000.This CDT detected OprD-deficient mutants which were non carbapenamse producer strains. In comparison to (bla_IMP) gene positivity and (bla_KPC) gene positivity, and MHT results, sensitivity and specificity of imipenem-cloxacillin at conc. 4000ug were 93.33% and 75% respectively. In conclusion, the Imipenem-Cloxacillin at conc. 4000 test is a simple and inexpensive presumptive method that can be added to the standard antibiogram for routine screening of OprD-deficient mutants thus discriminating carbapenemase producing P. aeruginosa strains from non-producers.

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