CAJ | 학술논문

目的：分析携带blaIMP‐4或blaKPC‐2的产新德里金属β‐内酰胺酶‐1（NDM‐1）的肠杆菌科菌的基因型及其质粒转移传播机制。方法收集2012年4月至2014年10月间浙江省温州市和杭州市5家医院细菌培养非重复菌株中碳青霉烯类耐药（如亚胺培南、美罗培南、厄他培南等耐药）肠杆菌科菌33株，应用全自动微生物分析仪对该类菌株进行鉴定和药物敏感试验。并用改良 Hogde试验、EDTA协同初筛金属酶试验、超广谱β‐内酰胺酶（ESBL ）检测（采用美国临床和实验室标准协会推荐的双纸片方法）、高产头孢菌素（AmpC ）酶检测（采用头孢西丁三维试验），对试验菌基因包括 blaNDM‐1在内及ISAba125‐NDM连锁基因进行 PCR检测，并将扩增产物纯化、克隆后测序，对部分菌株及2株携带blaIMP‐4或blaKPC‐2的产 blaNDM‐1基因的肺炎克雷伯菌进行质粒转移、接合与消除试验。结果33株碳青霉烯类耐药的肠杆菌科菌分别为肺炎克雷伯菌28株，产酸克雷伯菌1株，大肠埃希菌2株，植生乌拉尔菌1株，阴沟肠杆菌1株；分别来自浙江大学医学院附属邵逸夫医院13株、温州市中医院13株、温州市人民医院1株、温州医科大学附属第一医院3株、温州市中西医结合医院3株。有31株确定为产碳青霉烯酶，包括 blaKPC‐224株、blaNDM‐13株、blaNDM‐51株、blaIMP‐43株，其中 blaNDM‐1阳性菌株中同时携带blaIMP‐4或 blaKPC‐2的各1株，将携带部分阳性基因的菌株与大肠埃希菌 EC600或肺炎克雷伯菌ATCC13833进行质粒转移结合试验，获得结合子进行相应基因检测，均获得相应 blaNDM‐1、ISAba125‐N DM等基因。结论碳青霉烯类耐药肠杆菌科菌中最常见的是 blaKPC‐2型；blaNDM‐1、blaNDM‐5基因与ISAba125‐NDM连锁基因可能有相关；2株 blaNDM‐1型阳性菌株中有1株同时携带 blaKPC‐2型基因、另1株同时携带金属酶blaIMP‐4型基因。由于这些菌株为多重耐药且基因大部分在质粒上，容易在菌株间发生水平传播，应引起临床高度关注。目的：分析攜帶blaIMP‐4或blaKPC‐2的產新德裏金屬β‐內酰胺酶‐1（NDM‐1）的腸桿菌科菌的基因型及其質粒轉移傳播機製。方法收集2012年4月至2014年10月間浙江省溫州市和杭州市5傢醫院細菌培養非重複菌株中碳青黴烯類耐藥（如亞胺培南、美囉培南、阨他培南等耐藥）腸桿菌科菌33株，應用全自動微生物分析儀對該類菌株進行鑒定和藥物敏感試驗。併用改良 Hogde試驗、EDTA協同初篩金屬酶試驗、超廣譜β‐內酰胺酶（ESBL ）檢測（採用美國臨床和實驗室標準協會推薦的雙紙片方法）、高產頭孢菌素（AmpC ）酶檢測（採用頭孢西丁三維試驗），對試驗菌基因包括 blaNDM‐1在內及ISAba125‐NDM連鎖基因進行 PCR檢測，併將擴增產物純化、剋隆後測序，對部分菌株及2株攜帶blaIMP‐4或blaKPC‐2的產 blaNDM‐1基因的肺炎剋雷伯菌進行質粒轉移、接閤與消除試驗。結果33株碳青黴烯類耐藥的腸桿菌科菌分彆為肺炎剋雷伯菌28株，產痠剋雷伯菌1株，大腸埃希菌2株，植生烏拉爾菌1株，陰溝腸桿菌1株；分彆來自浙江大學醫學院附屬邵逸伕醫院13株、溫州市中醫院13株、溫州市人民醫院1株、溫州醫科大學附屬第一醫院3株、溫州市中西醫結閤醫院3株。有31株確定為產碳青黴烯酶，包括 blaKPC‐224株、blaNDM‐13株、blaNDM‐51株、blaIMP‐43株，其中 blaNDM‐1暘性菌株中同時攜帶blaIMP‐4或 blaKPC‐2的各1株，將攜帶部分暘性基因的菌株與大腸埃希菌 EC600或肺炎剋雷伯菌ATCC13833進行質粒轉移結閤試驗，穫得結閤子進行相應基因檢測，均穫得相應 blaNDM‐1、ISAba125‐N DM等基因。結論碳青黴烯類耐藥腸桿菌科菌中最常見的是 blaKPC‐2型；blaNDM‐1、blaNDM‐5基因與ISAba125‐NDM連鎖基因可能有相關；2株 blaNDM‐1型暘性菌株中有1株同時攜帶 blaKPC‐2型基因、另1株同時攜帶金屬酶blaIMP‐4型基因。由于這些菌株為多重耐藥且基因大部分在質粒上，容易在菌株間髮生水平傳播，應引起臨床高度關註。
목적：분석휴대blaIMP‐4혹blaKPC‐2적산신덕리금속β‐내선알매‐1（NDM‐1）적장간균과균적기인형급기질립전이전파궤제。방법수집2012년4월지2014년10월간절강성온주시화항주시5가의원세균배양비중복균주중탄청매희류내약（여아알배남、미라배남、액타배남등내약）장간균과균33주，응용전자동미생물분석의대해류균주진행감정화약물민감시험。병용개량 Hogde시험、EDTA협동초사금속매시험、초엄보β‐내선알매（ESBL ）검측（채용미국림상화실험실표준협회추천적쌍지편방법）、고산두포균소（AmpC ）매검측（채용두포서정삼유시험），대시험균기인포괄 blaNDM‐1재내급ISAba125‐NDM련쇄기인진행 PCR검측，병장확증산물순화、극륭후측서，대부분균주급2주휴대blaIMP‐4혹blaKPC‐2적산 blaNDM‐1기인적폐염극뢰백균진행질립전이、접합여소제시험。결과33주탄청매희류내약적장간균과균분별위폐염극뢰백균28주，산산극뢰백균1주，대장애희균2주，식생오랍이균1주，음구장간균1주；분별래자절강대학의학원부속소일부의원13주、온주시중의원13주、온주시인민의원1주、온주의과대학부속제일의원3주、온주시중서의결합의원3주。유31주학정위산탄청매희매，포괄 blaKPC‐224주、blaNDM‐13주、blaNDM‐51주、blaIMP‐43주，기중 blaNDM‐1양성균주중동시휴대blaIMP‐4혹 blaKPC‐2적각1주，장휴대부분양성기인적균주여대장애희균 EC600혹폐염극뢰백균ATCC13833진행질립전이결합시험，획득결합자진행상응기인검측，균획득상응 blaNDM‐1、ISAba125‐N DM등기인。결론탄청매희류내약장간균과균중최상견적시 blaKPC‐2형；blaNDM‐1、blaNDM‐5기인여ISAba125‐NDM련쇄기인가능유상관；2주 blaNDM‐1형양성균주중유1주동시휴대 blaKPC‐2형기인、령1주동시휴대금속매blaIMP‐4형기인。유우저사균주위다중내약차기인대부분재질립상，용역재균주간발생수평전파，응인기림상고도관주。
Objective To analyze the genotype and plasmid transfer of Enterobacteriaceae carring blaNDM‐1 with blaIMP‐4 or blaKPC‐2 .Methods From April 2012 to October 2014 ,a total of 33 non‐repeatitive carbapenem‐resistant Enterobacteriaceae ( including Imipenem‐resistant , meropenem‐resistant or Ertapenem‐resistant) were isolated from 5 hospitals in Wenzhou and Hangzhou . Identification and antimicrobial susceptibility test were performed using Vitek 2 Compact automatic microbiology analyzer . Phenotypes of carbapenemase were screened using modified Hodge test and ethylenediamine tetraacetic acid‐disk synergy test .Extended spectrum βlactamase test was determined by the double disk combination test which was recommended by Clinical and Laboratory Standards Institute .AmpC activity was tested by a three‐dimensional Cefoxitin method .Drug resistant genes including blaNDM‐1 and linkage of ISAba125‐NDM were detected by polymerase chain reaction (PCR) .The purified PCR products were cloned and sequenced .Plasmid conjugation experiment and elimination method were carried out to test partial bacterial strain and K . pneumoniae carrying blaNDM‐1 with blaIMP‐4 or blaKPC‐2 .Results Of the 33 non‐repeatitive carbapenem‐resistant Enterobacteriaceae ,28 were strains of K .pneumoniae ,1 strain of K . oxytoca,2strainsof Escherichiacoli,1strainof K.planticolaand1strainof E.cloacae.Thirteenstrains were isolated from Hospital of Sir Run Run Shaw of Zhejiang University ,thirteen from Wenzhou Hospital of Traditional Chinese Medicine ,one from Wenzhou People′s Hospital ,three from the First Affiliated Hospital of Wenzhou Medical University and three from Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine .Thirty‐one strains were confirmed as carbapenemase‐producing with 24 of blaKPC‐2 ,3 of blaNDM‐1 ,1 of blaNDM‐5 and 3 of blaIMP‐4 .Among them ,one strain carried blaNDM‐1 with blaIMP‐4 and one strain carried blaNDM‐1with blaKPC‐2 ,respectively .The plasmid transfer and conjugation experiment was performed between strains carrying blaNDM‐1 and Escherichia coli EC600 or K . pneumoniae ATCC13833 and genes of blaNDM‐1 and ISAba125‐NDM were obtained .Conclusions blaKPC‐2 gene is the popular carbapenemase genotype .blaNDM‐1 or blaNDM‐5 may be correlated with linkage gene of ISAba125‐N DM .Coexistence of blaNDM‐1 carrying blaIMP‐4 or blaKPC‐2 is detected in the same strain , respectively . Enough importance should be attached to the strains ,because most of them are multiple drug resistance with related genes located in the plasmid which is easily spread between strains .