Overexpression of MexAB-OprM efflux pump in carbapenem-resistant Pseudomonas aeruginosa
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ORIGINAL PAPER
Overexpression of MexAB‑OprM efflux pump in carbapenem‑resistant Pseudomonas aeruginosa Ya‑ping Pan1 · Yuan‑hong Xu1 · Zhong‑xin Wang1 · Ya‑ping Fang2 · Ji‑lu Shen1
Received: 13 December 2015 / Revised: 12 March 2016 / Accepted: 28 March 2016 © Springer-Verlag Berlin Heidelberg 2016
Abstract 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 highlevel 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 carbapenemresistant 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 Communicated by Erko Stackebrandt. Electronic supplementary material The online version of this article (doi:10.1007/s00203-016-1215-7) contains supplementary material, which is available to authorized users. * Ji‑lu Shen [email protected] 1
Department of Laboratory Medicine, The First Affiliated Hospital of Anhui Medical University, Anhui Medical University, Hefei, Anhui, China
2
Department of Laboratory Medicine, The Second Affiliated Hospital of Anhui Medical University, Anhui Medical University, Hefei, Anhui, China
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 EDTAdisk 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. Keywords Carbapenem-resistant · Efflux pump · MexAB-OprM · Pseudomonas aeruginosa
Introduction Pseudo
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