Study On The Mechanism Of Pseudomonas Aeruginosa Resistant To Imipenem

Obiective:To investigate the drug resistance of imipenem-resistant Pseudomonas aeruginosa（IRPA）isolated from inpatients and to detect the expression of metalloenzyme,extended-spectrumβ-lactamase and AmpC enzyme genes and the distribution of integrons,and explore the relationship between the deletion of membrane protein op D₂ and the expression of Mex AB-Opr M,Mex XY-Opr M,Mex CD-Opr J and MexEF-OprN in active efflux system and bacterial resistance.These studies will provide the basis for rational use of antibiotics and effective control of drug-resistant strains through systematically studying the mechanism of PA resistant to imipenem.Methods:34 isolates of IRPA isolated from clinical specimens were collected during Apr 2015 to Jul 2016 in our hospital.The bacterial identification and antimicrobial susceptibility were tested by Vitek-2 Compact and Phoneix-100 bacterial identification and susceptibility system.The expression of carbapenemase genes（IMP,VIM,SIM,GIM,SPM,OXA-2 and OXA-10）,extended spectrumβ-lactamase genes（TEM,VEB,SHV,PER）,and Amp C enzyme genes（PDC,DHA）was detected by PCR method.The integrase genes including int Il,int I2 and int I3 were detected by PCR,and the integrase variable region was amplified from the integrase positive strain.The resistance gene cassette carried by the integron variable region was sequenced and analyzed.Fluorescence quantitative PCR was used to detect the deletion of oprD₂ gene in IRPA and the overexpression of Mex AB-OprM,Mex XY-OprM,MexCD-OprJ and MexEF-Opr N in the efflux system of PA.Additionally,the regulatory genes including MexR,nalC,nalD,MexZ,nfx B,MexT₁ and MexT₂ were also amplified by PCR and sequencing Analysis.Results:IRPA isolated from sputum samples possessed the highest percentage that accounts for 70.6%,followed by urine samples（14.7%）,wound secretions（5.9%）,throat swabs（5.9%）and blood samples（2.9%）.According to the distribution of departments,brain surgery had largest proportion that accounted for 58.8%,followed by the ICU,accounting for 14.7%.The rates of 34 strains resistant to meropenem,cefepime,and aztreonam were 85.3%,88.2%and 91.2%,respectively.Whereas,the resistant rate to amikacin was the lowest（2.9%）.Of 34 IRPA,there were 27 strains（79.4%）with TEM-1gene,while all of 34 strains carried PDC-73 genes（100.0%）.But,the resistant genes including IMP,VIM,SIM,GIM,SPM,VEB,SHV,PER,OXA-2,OXA-10 and DHA weren’t found by PCR.23 strains（67.6%）were detected by PCR,while class II or III integron wasn’t found.4 strains carring with I integron（17.4%）showed I integron variable regions by PCR and sequencing,which shared 1 strain（750 bp）and 3 strains（2500 bp）,respectively.The variable region with 750 bp carried drug resistance gene cassette dfrA17 that was confirmed by sequencing.Of 34 IRPA,11 strains（32.4%）showed the loss of oprD₂ genes,and 17 strains（50%）exhibited overexpression of active efflux systems,including 8（23.5%）in MexA,9（26.5%）in MexA,11（32.4%）in MexC,and10（29.4%）in MexE.Conclusion:The production of TEM-1 extended-spectrumβ-lactamase,the loss of oprD₂,the overexpression of the active efflux pump,and the transport of the mobile elements are the main causes of PA resistant to multidrugand resistant gene dispersal in the present hospital.