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Background & objectives:The emergence and rapid spread of carbapenem resistance mediated by metallo-beta-lactamase (MBL) in Pseudomonas aeruginosa is of major concern due to limited therapeutic options. This study was aimed at detecting the presence of MBL and its association with integrons in imipenem-resistant P. aeruginosa isolates and to determine their genetic relatedness.Methods:A total of 213 P. aeruginosa isolates were collected from two tertiary care centres and tested against anti-pseudomonal antibiotics by antimicrobial susceptibility testing, followed by the detection of MBL production by combined disk method. Minimum inhibitory concentration (MIC) of meropenem was determined by E-test. Multiplex polymerase chain reaction (PCR) was performed for the detection of blaSPM, blaIMP, blaVIM, blaNDM, blaGIM and blaSIM. PCR was carried out to characterize the variable region of class 1 integron. Transcongujation assay was carried out for the confirmation of plasmid-mediated resistance. Enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR was performed for determining the genetic relatedness among P. aeruginosa isolates.Results:Of the 213 P. aeruginosa isolates, 22 (10%) were found to be carbapenem resistant and these were from pus 18 (82%), urine 2 (9%), sputum 1 (5%) and tracheal wash 1 (5%). Among 22 isolates, 18 (81.8%) were found to be MBL producers by phenotypic method and MIC range of meropenem was 8 to >32 µg/ml. PCR amplification showed that 20 (91%) isolates carried any one of the MBL genes tested: blaVIM and blaNDM in seven (32%) and six (27%) isolates, respectively; blaVIM and blaNDM in three (14%); blaIMP and blaNDM in two (9%); blaVIM and blaIMP in one (5%) isolate. The blaVIM, blaIMP and blaNDM were found to co-exist in one isolate. None of the isolates were positive for blaSPM, blaSIM and blaGIM. All 22 isolates carried class I integron. Of the 20 MBL-positive isolates, transconjugants were obtained for 15 isolates. ERIC-PCR analysis showed all isolates to be clonally independent.Interpretation & conclusions:Our results showed 10.3 per cent of carbapenem resistance among P. aeruginosa isolates, and the coexistence of MBL-encoding genes among P. aeruginosa mediated by class I integron.

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Background: Intrinsic resistance in Pseudomonas aeruginosa is largely due to overexpression of efflux pumps, among which members of the Resistance Nodulation cell Division family (RND) viz., MexAB-oprM, MexXY-OprM, MexCD-OprJ & MexEF-OprN are predominant. MDR infections are difficult to treat with antibiotics and hence combinations of antibiotics with efflux pump inhibitors (EPIs) have been proposed. Studies on efflux mediated resistance among MDR are lacking. Therefore the present study is aimed to determine the effect of efflux pump inhibitor and overexpression of efflux pumps in clinical isolates of P. aeruginosa.Methods & Materials: A total of 213 P. aeruginosa clinical isolates were collected and antimicrobial susceptibility testing (AST) was done by standard techniques. Forty MDR isolates were chosen and included for the present study. EPI test was carried out by determining minimum inhibitory concentration (MIC) of antibiotics with and without EPI (Phe-Arg ␤-naphthylamide) at a concentration of 50 g/ml. Reporter antibiotics used were carbenicillin(MexAB-OprM), erythromycin(MexCD-OprJ), norfloxacin(MexEF-OprN) and gentamicin(MexXY-OprM). In the case of 21 isolates, expression of efflux pump encoding genes (mexA, mexC, mexE, mexX) was determined by quantitative Real Time PCR with rpoD gene as endogenous control. Wild-type PAO1, PAO mexR::Gm, PAONB, PAO↑EF, PAO mexZ::Gm were used as reference strains.Results: EPI test showed 4-64 fold MIC reduction in 23/40(58%) isolates for carbenicillin and erythromycin and in 32/40(80%) isolates for norfloxacin. No MIC reduction was observed in 32 (80%) isolates for gentamicin whereas one isolate showed 64-fold reduction. Quantitative analysis revealed that on comparison with PAO1 strain, expression of mexA and mexE increased by 3-10 and 2-3 fold respectively in all isolates. mexC expression ranged from 5-24 fold increase in 14(67%) isolates, 73-294 fold and 630-fold in 4 and 1 isolate respectively. One isolate each showed 2 and 17 fold difference in mexX expression whereas no expression was observed for the remaining isolates.Conclusion: The present study documents the prevalence of efflux pump mediated resistance among MDR isolates of P. aeruginosa. All MDR isolates over-expressed at least one of the efflux pumps. Among the RND family genes studied, mexC showed a wide range as well as high levels of expression indicating its role in multidrug resistance in P. aeruginosa.http://dx.