CAJ | 학술논문

目的：探讨我院肠杆菌科细菌对碳青霉烯类抗生素的耐药机制及其流行特点。方法收集杭州市富阳区第一人民医院2013年1月至2014年8月分离的对碳青霉烯类抗生素（厄他培南）敏感性下降的肠杆菌科细菌，采用K-B纸片法及E-test法进行药敏试验，改良Hodge试验、EDTA抑制试验及超广谱β-内酰胺酶（ ESBLs）表型筛选试验进行耐药表型筛选；采用PCR法及测序技术检测耐药基因、KPC基因周围序列和肺炎克雷伯菌7个管家基因，多位点序列分型进行7个管家基因的序列分析；运用PFGE对鉴定为同一菌种的细菌进行同源性分析；采用S1-PFGE联合Southern杂交对已明确的碳青霉烯耐药基因进行基因定位。结果共收集到19株厄他培南敏感性下降的肠杆菌科细菌，所有菌株呈多重耐药现象，且同一菌株常携带有多种耐药基因，其中 blaKPC-2、blaIMP-4、blaSHV-1、blaCTX-M-65、blaCTX-M-15、blaTEM-1、rmtB为最常见共同携带的耐药基因。 PFGE结果提示14株肺炎克雷伯菌呈多克隆分布，MLST分型以ST11型为主。11株肺炎克雷伯菌和1株大肠埃希菌产KPC-2酶， blaKPC-2基因分别定位于4种大小不同的质粒上（大小分别约为95 kb、140 kb、200 kb及240 kb），所有菌株blaKPC-2周围基因结构从上游至下游均为ISKpn8、blaKPC-2和ISKpn6-like元件；1株产酸克雷伯菌和1株肺炎克雷伯菌产 IMP-4型碳青霉烯酶， blaIMP-4基因定位于大小约为300 kb 的质粒上。结论KPC-2型及IMP-4型碳青霉烯酶是造成我院肠杆菌科细菌对碳青霉烯类抗生素耐药的主要原因；我院碳青霉烯类耐药肺炎克雷伯菌（ CRKP）菌株呈多克隆散在播散的流行特点，未发现优势克隆。
목적：탐토아원장간균과세균대탄청매희류항생소적내약궤제급기류행특점。방법수집항주시부양구제일인민의원2013년1월지2014년8월분리적대탄청매희류항생소（액타배남）민감성하강적장간균과세균，채용K-B지편법급E-test법진행약민시험，개량Hodge시험、EDTA억제시험급초엄보β-내선알매（ ESBLs）표형사선시험진행내약표형사선；채용PCR법급측서기술검측내약기인、KPC기인주위서렬화폐염극뢰백균7개관가기인，다위점서렬분형진행7개관가기인적서렬분석；운용PFGE대감정위동일균충적세균진행동원성분석；채용S1-PFGE연합Southern잡교대이명학적탄청매희내약기인진행기인정위。결과공수집도19주액타배남민감성하강적장간균과세균，소유균주정다중내약현상，차동일균주상휴대유다충내약기인，기중 blaKPC-2、blaIMP-4、blaSHV-1、blaCTX-M-65、blaCTX-M-15、blaTEM-1、rmtB위최상견공동휴대적내약기인。 PFGE결과제시14주폐염극뢰백균정다극륭분포，MLST분형이ST11형위주。11주폐염극뢰백균화1주대장애희균산KPC-2매， blaKPC-2기인분별정위우4충대소불동적질립상（대소분별약위95 kb、140 kb、200 kb급240 kb），소유균주blaKPC-2주위기인결구종상유지하유균위ISKpn8、blaKPC-2화ISKpn6-like원건；1주산산극뢰백균화1주폐염극뢰백균산 IMP-4형탄청매희매， blaIMP-4기인정위우대소약위300 kb 적질립상。결론KPC-2형급IMP-4형탄청매희매시조성아원장간균과세균대탄청매희류항생소내약적주요원인；아원탄청매희류내약폐염극뢰백균（ CRKP）균주정다극륭산재파산적류행특점，미발현우세극륭。
Objective To investigate the mechanism of carbapenem-resistant in Enterobacteriaceae strains isolated from Fuyang First People′s Hospital and to analyze their epidemiological features. Methods The Enterobacteriaceae strains with reduced ertapenem susceptibility were isolated from the Fuy-ang First People′s Hospital during January 2013 to August 2014.K-B disk diffusion and E-test were per-formed to detect the antimicrobial susceptibilities of those strains.The modified Hodge test, ethylenediami-netetraacetic acid ( EDTA) disk synergy test and extended-spectrumβ-lactamases ( ESBLs) confirmation test were used to screen out the carbapenem-resistant phenotypes.PCR analysis and gene sequencing were used to analyze drug resistance genes, genetic structures surrounding the blaKPC-2 gene and seven house-keeping genes of Klebsiella pneumonia ( K.pneumoniae) strains.The sequences of the seven house-keeping genes were analyzed with multilocus sequence typing ( MLST) .Pulsed field gel electrophoresis ( PFGE) was per-formed for homology analysis within the same species.S1-PFGE in combination with Southern blot analysis was used to determine the location of carbapenem resistance genes.Results A total of 19 Enterobacteriace-ae isolates with reduced susceptibility to ertapenem were screened out.Each of them was resistant to multiple antibiotics and harbored several resistance genes.Seven genes including the blaKPC-2 , blaIMP-4 , blaSHV-1 , blaCTX-M-65 , blaCTX-M-15 , blaTEM-1 and rmtB genes were the prevalent drug resistance genes.Fourteen out of the nineteen strains were identified as K.pneumoniae strains, mainly belonged to the ST11 type according to the results of MLST.Among the nineteen strains, eleven K.pneumoniae isolates and one Escherichia coli isolate carried the blaKPC-2 gene, located on plasmids varying in size (95 kb, 140 kb, 200 kb and 240 kb) .The ge-netic structures of all isolates were ISKpn8, blaKPC-2 and ISKpn6-like from upstream to downstream.The blaIMP-4 gene was detected in one Klebsiella oxytoca isolate and one K.pneumoniae isolate, located on a plas-mid about 300 kb in size.Conclusion Carbapenemases KPC-2 and IMP-4 were closely related to the car-bapenem resistance in carbapenem-resistant Enterobacteriaceae strains isolated form the Fuyang First People′s Hospital.No predominant clone was found in those carbapenem-resistant K.pneumoniae isolates.