Distribution and effects of gene expression of four efflux pumps in Pseudomonas aeruginosa with multi-drug resistance in Hunan province

Abstract

Objective To investigate the effects and distribution of 4 efflux pumps and correlated mutation of regulatory gene in multi-drug resistance Pseudomonas aeruginosa (Pa) in Hunan province. Methods Forty non-duplicated clinical strains of multiple-drug-resistant Pseudomonas aeruginosa were collected in Hunan in 2008, then the phenotype was screened with efflux pumps inhibitor phenylalanine-L-β-naphthylamide and 4 antimicrobial susceptibility test discs. Genes of the efflux pump membrane fusion protein were amplified by PCR, and correlated efflux pump regulatory genes were amplified and sequenced to analyze the role of efflux pump gene expression in multi-drug resistance compared to that of 18 strains with non-multi-drug resistance. Re-suits The positivity rates of MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM were 45.0% (18/40), 30.0% (12/40), 42.5% (17/40) and 12.5% (5/40) respectively with phenotype screening in multi-drug resistance group. The positivity rates of mexA, mexC, mexE and mexX were 100% (58/58), 22.5% (9/40), 45.0% (18/40) and 22.5% (9/40) with RT-PCR. The overexpressed positivity rates of mexA and mexX were 55.0% (22/40) and 22.5% (9/40) respectively by semi-quantitative analysis with real-time PCR. However, no over-expression of mexA and mexX in non-multi-drug resistance group with real-time PCR. The positive expression rates of mexC and mexE with RT-PCR were 11.1% (2/18), 38.9% (7/18) in non-multi-drug resistance group. The difference of overexpression of mexA and mexX was significant(P<0.001, P=0.045) between two groups. The strain Pa20 with mexA overexpressian displayed gene mutations in mexR(164GTC→GAG) and amino acid substitution(126Val→Glu), the strain Pa34 with overexpression of mexX had 164GCG→GAG,55Ala→GIu. Conclusion Most of Pa with multi-drug resistance contained the resistant mechanism of efflux pumps and most-ly due to the overexpression of MexAB-OprM and MexXY-OprM in Hunan. There are mostly regulatory genes mutation in the strains with overexpression of MexAB-OprM and MexXY-OprM. Key words: Pseudomonas aeruginosa; Multi-drug resistance; Efflux pump; Efflux pumps inhibitor