耐亚胺培南铜绿假单胞菌相关耐药基因的检测

Detection of the relevant resistant genes in Imipenem-resistant Pseudomonas aeruginosa

目的对耐亚胺培南(IMP)的铜绿假单胞菌(IRPa)相关耐药基因进行检测。方法 2003年至2009年从临床标本中分离到(P.aeruginosa)共220株,采用三维试验筛选产β-内酰胺酶的铜绿假单胞菌,应用普通PCR和多重PCR分别检测碳青霉烯酶基因和质粒携带的C类头孢菌素酶(AmpC酶)耐药基因,应用荧光定量RT-PCR的方法检测oprD2基因的表达情况。结果共检出43株产β-内酰胺酶的菌株,其中产AmpC酶、超广谱β-内酰胺酶(ESBLs)、金属β-内酰胺酶(MBLs)和未知酶菌株的构成比分别58.14%(25/43)、18.60%(8/43)、4.65%(2/43)和16.28%(7/43)。74株耐亚胺培南的铜绿假单胞菌中,有2株菌携带IMP-9基因,1株菌携带DHA质粒型AmpC酶基因,其他碳青霉烯酶基因检测为阴性。40株菌株oprD2基因表达蛋白量降低,34株oprD2基因表达蛋白量正常。结论 oprD2基因的突变或蛋白表达量降低是IRPa对亚胺培南耐药的主要原因,AmpC酶可水解亚胺培南可能与铜绿假单胞菌对亚胺培南的耐药有一定的关系,而KPC-1酶和MBLs在铜绿假单胞菌对亚胺培南耐药机制中不是主要因素。

Objective To survey the relevant resistant genes in imipenem-resistant Pseudomonas aeruginosa.Method Two hundred and twenty strains of P.aeruginosa were isolated from hospitalized patients′ samples between 2003 and 2009.Three-dimensional test was used to screen P.aeruginosa which produce β-lactamases.PCR and multiplex PCR were performed to detect Carbapenemase genes and plasmid-mediated AmpC β-lactamase genes respectively.The expression of the oprD2 genes in P.aeruginosa were analyzed with Real-time reverse transcriptase PCR.Result 43 strains producing β-lactamases were isolated;the constitution ratio of AmpC β-lactamases,extended spectrum β-lactamases（ESBLs）,metallo-β-lactamases（MBLs） and unknown type of β-lactamases were 58.14%（25/43）,18.60%（8/43）,4.65%（2/43） and 16.28%（7/43）,respectively.Among the 74 strains of Imipenem-resistant P.aeruginosa,two strains carried IMP-9 gene;the plasmid-mediated AmpC β-lactamase gene DHA was identified in one strain.The other carbapenemase gene detection saw negative results.40 strains showed decreasing expression of oprD2 gene while that of other 34 strains showed normal expression rate.Conclusion The diminished production of oprD2 gene is the major cause of imipenem resistence in P.aeruginosa.AmpC β-lactamases can hydrolyze imipenem feebly,which may has some relation with imipenem resistance.