耐碳青霉烯铜绿假单胞菌耐药表型及基因型分析

Genotypes and phenotypes analysis of carbapenem-resistant Pseudomonas aeruginosa

目的了解深圳福田人民医院铜绿假单胞菌对碳青霉烯类抗生素的耐药机制,为临床合理使用抗生素提供参考依据。方法用E-test试剂条检测铜绿假单胞菌对哌拉西林、头孢他啶、亚胺培南、美罗培南、庆大霉素、妥布霉素、环丙沙星7种抗生素的最小抑菌浓度,用三维实验对产AmpC、KPC酶菌株的耐药表型进行确认,金属酶的表型确证实验则采用EDTA双纸片扩散法。用PCR方法检测AmpC、VIM-1、VIM-2、IMP-1、SPM、KPC及OprD7个基因型,并分析表型和基因型之间的关系,质粒接合实验用来证实耐药基因的传播性。结果 29例铜绿假单胞菌均为多重耐药菌株,且对亚胺培南、美罗培南及庆大霉素耐药率最高,3种药物最小抑菌浓度分别为32、32、256μg/mL。26株携带有AmpC基因的铜绿假单胞菌仅有5株为持续高表达菌株,18株金属酶阳性菌中有15株与VIM-2基因型检测一致,其余3株VIM-2基因型阴性,并有1株为VIM-2基因型阳性但金属酶阴性。29株中检测出8株携带OprD基因,外膜蛋白缺失率为72%(21/29),其余基因型均阴性。结论该院铜绿假单胞菌耐碳青霉烯类抗生素的耐药机制主要为产金属酶及外膜蛋白缺失,碳青霉烯类抗生素的不合理使用将加剧该类药物的耐药性。

Objective To explore the present levels and major drug-resistant mechanisms to Carbapenems of Pseudomonas aeruginosa in People’s Hospital of Futian Shenzhen,and to provide references for reasonable usage of antibiotics.Methods Minimal inhibitory concentration（MIC） of Pseudomonas aeruginosa to 7antibiotic agents,including piperacillin,ceftazidime,imipenem,meropenem,gentamicin,tobramycin and ciprofloxacin were determined by E-test strips.MBLs,KPC and AmpC were detected by double disks synergy tests,3-D tests and 3-D tests respectively.7genes,including AmpC,VIM-1,VIM-2,IMP-1,SPM,KPCand OprD,were detected by polymerase chain reaction.The correlation between phenotype and genotype were analyzed.Transmission of resistant genes were proved by plasmid conjugation test.Results All 29Carbapenem-resistant Pseudomonas aeruginosa were multidrugresistant strains,with high resistant rate to imipenem,meropenem and gentamicin,with MIC of 32,32and 256 μg /mL respectively.5of 26Pseudomonas aeruginosa,carrying AmpCresistant gene,were with persistent hyperexpression of AmpC beta-lactamase.18 strains were positive with MBLs,15strains of which were coincident with the VIM-2genotypes,and the other 3 MBLs positive strains were negative with VIM-2gene,but 1strain was VIM-2gene positive and MBLs negative.8strains of 29Pseudomonas aeruginosa were positive with OprDgene,and the miss rate of outer membrane protein was 72%（21 /29）.Other genes could not been detected in this research.Conclusion Major drug-resistant mechanisms to Carbapenems were producing MBLs and loss of outer membrane protein.Unreasonable usage of Carbapenems might aggravate their drug-resistance.