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Overexpression of MexAB-OprM efflux pump in carbapenem-resistant Pseudomonas aeruginosa
- Pan, Ya-ping, Xu, Yuan-hong, Wang, Zhong-xin, Fang, Ya-ping, Shen, Ji-lu
- Archives of microbiology 2016 v.198 no.6 pp. 565-571
- Pseudomonas aeruginosa, agar, amino acids, beta-lactamase, gene overexpression, imipenem, meropenem, messenger RNA, minimum inhibitory concentration, multiple drug resistance, outer membrane proteins, phenotype, point mutation, protein structure, quantitative polymerase chain reaction, regulator genes, sequence analysis, transporters
- Efflux pump systems are one of the most important mechanisms conferring multidrug resistance in Pseudomonas aeruginosa. MexAB-OprM efflux pump is one of the largest multi-drug resistant efflux pumps with high-level expression, which is controlled by regulatory genes mexR, nalC, and nalD. This study investigated the role of efflux pump MexAB-OprM in 75 strains of carbapenem-resistant P. aeruginosa and evaluated the influence of point mutation of the regulatory genes. The minimum inhibitory concentrations of imipenem and meropenem, with or without MC207110, an efflux pump inhibitor, were determined by agar dilution method to select the positive strains for an overexpressed active efflux pump. Carba NP test and EDTA-disk synergy test were used for the detection of carbapenemase and metallo-β-lactamases, respectively. The gene mexA, responsible for the fusion protein structure, and the reference gene rpoD of the MexAB-OprM pump were amplified by real-time PCR. The quantity of relative mRNA expression was determined simultaneously. By PCR method, the efflux regulatory genes mexR, nalC, and nalD and outer membrane protein OprD₂ were amplified for the strains showing overexpression of MexAB-OprM and subsequently analyzed by BLAST. Among the 75 P. aeruginosa strains, the prevalence of efflux pump-positive phenotype was 17.3 % (13/75). Carba NP test and EDTA-disk synergy test were all negative in the 13 strains. PCR assay results showed that ten strains overexpressed the MexAB-OprM efflux pump and were all positive for the regulatory genes mexR, nalC, and nalD. Sequence analysis indicated that of the ten isolates, nine had a mutation (Gly → Glu) at 71st amino acid position in NalC, and eight also had a mutation (Ser → Arg) at 209th position in NalC. Only one strain had a mutation (Thr → Ile) at the 158th amino acid position in NalD, whereas eight isolates had mutations in MexR. In conclusion, overexpression of efflux pump MexAB-OprM plays an important role in carbapenem-resistant P. aeruginosa. The mutations of regulatory genes may be a main factor contributing to overexpression of MexAB-OprM.