Investigation Of Distribution And Effects Of Gene Expression Of Four Efflux Pumps In Pseudomonas Aeruginosa With Multi-drug Resistance

Objective:To investigate the effects of overexpression of effiux pump MexAB-OprM,MexXY-OprM and expression of MexCD-OprJ,MexEF-OprN in multidrug resistant Pseudomonas aeruginosa,the distribution of gene expression and mutantion of regulation gene in our hospital.Methods:Collected non-duplicated clinical isolates of 58 Pseudomonas aeruginosa strain,in which 40 multiple drug resis tant strains,non-multiple drug-resistant strains 18 strains in our hospital in 2008,one wild strain,and one quality control strains.To preliminarily genetic phenotype screen with Efflux pumps inhibitor(phenylalanine-L -βnaphthylamide,Phe-Arg-β-naph thylamide,PAβN)and K-B paper dispersion method with four kinds of antibiotics.using,RT-PCR qualitative,Real time-PCR semi-quantitative to analysis of gene expression,to study the distribution of effiux pump gene;To analyse the role of efflux pump gene expression in multi-drug resistance of Pa with control study.PCR amplification of regulation gene and sequencing and to analysis mutation of its regulation gene.Results:With screening of gene phenotype the positive rate were MexAB-OprM 45.0%(18/40),MexCD-OprJ 30.0%(12/40),MexEF-OprN 42.5%(17/40),MexXY-OprM 12.5%(5/40) respectively in multi-drug resistant group;To amplifacte mexA,C,E,X gene represent each effiux pump system with RT-PCR,showed the expression of mexA and mexX positive rate were 100%(58/58) and 22.5%(9/40), semi-quantitative analysis of overexpression strains(PA mexA,X 2^CT/rpsL的2^-CT /Wild PA mexA,X 2^-C/TrpsL的2^-CT＞1) positive rate was 55.0%(22/40) and 22.5%(9/40) respectively with Real time-PCR,RT-PCR mexC positive rate was 22.5%(9/40),mexE positive rate was 45.0%(18/40).The percent agreement between gene amplification and efflux pump phenotype showed that MexAB-OprM was 100%(18/18),MexCD-OprJ was 55.6%(5/9),MexEF-OprN 64.7%(11/17),MexXY-OprM 40.0% (2/5);However,overexpression of both mexA and mexX with Real-timePCR showed positive rate were 0.0%(0/18),while mexC and mexE with RT-PCR expression rate were 11.1%(2/18),38.9%(7/18) in susceptible grup respectively.between group of multi-drug resistant and non-multi-drug resistant group,the difference level of mexA and mexX expression was significant(P＜0.00 1,P=0.045),while mexC and mexE expression had no significant(P=0.306,P=0.778).Compare the sequence of regulation gene of MexR,NfxB,MexZ to which in GeneBank found that overexpression of mexA strain Pa20 carried gene mutations in mexR and had aminoacid substitution,164 GTG→GAG, 126Vai→Glu,Pa40 had no gene mutations,the overexpression of MexXY-OprM strain Pa34 had gene mutation and amino acid substitution, 164 GCG→GAG,55 Ala→Glu.expression of mexC strain Pa2,Pa40 both had gene mutation but no aminoacid substitution.Conclusions:1)Most of multi-drug resistant Pa existed the effiux mechanism and mostly due to MexAB-OprM and MexXY-OprM overexpression in our hospital.2) K-B paper dispersion method combined phenylalanine-naphthyl amide arginine-β(PAβN) can be used as efflux pump MexAB-OprM gene phenotype creening.3) Efflux pump MexAB-OprM and MexXY-OprM overexpression existed its regulation gene mutation.