IRPA对β-内酰胺酶类抗菌药物主要耐药机制研究

Study on main drug resistance mechanism of imipenem-resistant Pseudomonas aeruginosa to β-lactam antibacterial drugs

目的研究该院耐亚胺培南的铜绿假单胞菌β-内酰胺酶的基因分布、膜孔蛋白OprD2的缺失情况及外排泵MexAB-OprM的表达情况。方法收集该院2015年4月至2016年7月由全自动细菌鉴定仪筛选出来的耐亚胺培南的铜绿假单胞菌,采用K-B法进行耐药表型确认,采用聚合酶链反应(PCR)检测耐亚胺培南铜绿假单胞菌金属酶基因IMP、VIM、SIM、GIM、SPM,超广谱β-内酰胺酶基因TEM、VEB、SHV、PER、OXA-2、OXA-10;对扩增阳性产物进行基因测序,采用实时荧光定量PCR检测铜绿假单胞菌膜孔蛋白OprD2的缺失情况及mexA的表达水平。结果该院耐亚胺培南铜绿假单胞菌金属酶基因均未检出;超广谱β-内酰胺酶基因TEM阳性20株(83.3%),其他型未有检出;阳性产物基因测序经比对确认为TEM-1型;膜孔蛋白OprD2缺失6株(25%),外排泵MexAB-OprM过度表达6株(25%)。结论该院耐亚胺培南铜绿假单胞菌对β-内酰胺酶类的耐药机制主要以由超广谱β-内酰胺酶基因TEM-1型介导为主,膜孔蛋白OprD2缺失以及主动外排泵MexAB-OprM过度表达为辅。

Objective To investigate the gene distribution of imipenem-resistant Pseudomonas aeruginosa(IRPA)β-lactamase,deletion of porin protein OprD2 and expression of efflux pump MexAB-OprM in this hospital.Methods IRPA in this hospital from April 2015 to July 2016 screened out by the automatic bacteriological analyzer were collected and conducted the drug resistance phenotype confirmation by adopting the K-B method.Metalloenzyme gene IMP,VIM,SIM,GIM,SPM,extended-spectrumβ-lactamase gene TEM,VEB,SHV,PER,OXA-2 and OXA-10 in IRPA were detected by adopting PCR;the products of amplification positive conducted the gene sequencing.Pseudomonas membrane porin OprD2 deletion and expression level of mexA were detected by adopting real time fluorescence quantitative PCR.Results Metalloenzyme genes of IRPA were not detected in this hospital,and 20(83.3%)strains were positive for extended-spectrumβ-lactamase TEM.Other types were not detected.The positive products gene sequencing by comparison was confirmed as the type TEM-1,there were 6 strains(25%)of membrane pore proteins OprD2 deletion and 6 strains(25%)of efflux pump MexAB-OprM overexpression positive.Conclusion The drug resistance mechanism of IRPA toβ-lactams in this hospital is mainly mediated by extended-spectrumβ-lactamase gene TEM-1 type,membrane pore protein OprD2 deletion and is supplemented by active efflux pump MexAB-OprM.