Analysis Of Antibiotics Resistance Among Pseudomonas Aeruginosa And Correlated Study On Efflux Pump MexAB-OprM In Carbapenem-resistant Pseudomonas Aeruginosa

Objective：1. To investigate the distribution and resistance to13clinical drugs of clinicalP.aeruginosa.In order to provide guidance for the reasonable clinical use ofantibiotics and the basis for clinicians during anti-infective therapy and hospitalinfection control.2. To learn the role of efflux pump MexAB-OprM in carbapenem-resistantP.aeruginosa.3. To study the mutations of the regulatory genes mexR,nalC and nalD in effluxpump MexAB-OprM phenotype positive strains.Methods：The sensitivity of459clinical isolates of P.aeruginosa to13antibiotics weretested by disk diffusion method;the minimum inhibitory concentrations(MIC)ofimipenem and meropenem with or without pump inhibitor MC207110for75carbapenem-resistant P.aeruginosa strains were determined by agar dilutionmethod.PCR method was used to amplify the regulated genes of active efflux pumpMexAB-OprM,such as mexR,nalC and nalD in the strains overexpression the effluxpump.At the same time using the BLAST method analyed the PCR products withDNA sequencing.Real-time PCR method was used to study the level of mRNAexpressing of mexA in active efflux pump MexAB-OprM and housekeeping generpoD. Result：The antimicrobial susceptibility of459P.aeruginosa strains reveled that theresistance rate of P.aeruginosa to aztreonam(82.3%)was higher than otherantimicrobial agents,while the resistance rate of P.aeruginosa to amikacin(15.2%)wasthe lowest.About23.4%and22.6%of P.aeruginosa were resistant to imipenem andmeropenem respectively.Among of75P.aeruginosa strains,there were total13strainsbelong to efflux pump positive phenotype,the positive rate was17.3%(13/75).Amongof13positive phenotype,10strains were overexpressed the efflux pump ofmexAB-oprM.The regulatory genes mexR,nalC and nalD were expressing positive inthe10strains.Nine isolates had a Gly71â†'Glu change in NalC,of which eight isolateshad a Ser209â†'Arg change in NalC at the same time.Only one strain had aThr158â†'Ile change in NalD.Eight strains had mutations in MexR.The highestrelative expression of mexA in PA4,PA5and PA2was10.26times,8.83times and6.12times respectively.Conciusions：1.In this study,most clinical P.aeruginosa were recovered fromsputum(64.7%),and17.4%of P.aeruginosa strains were recoverd from respiratorymedicine department which is higher than other departments.2.In this study,clinical isolated P.aeruginosa had a lowest resistant rate toamikacin,followed by cefoperazone/sulbactam,cefepime,meropenem andimipenem.So clinicians could choose one or two drugs for clinical empiric therapy.3.Overexpression efflux pump MexAB-OprM strains are related to the regulatorygenes mutations.4.High expression of efflux pump MexAB-OprM plays an important role incarbapenems P.aeruginosa.5.The regulatory gene mexR of efflux pump MexAB-OprM had been found two new mutations,Leu54â†'Pro and Pro37â†'Arg,which were probably the reasons ofPA2,PA4and PA5strains overexpression.