CAJ | 학술논문

目的调查我院产ESBL肺炎克雷伯菌临床分离株16S rRNA甲基化酶基因的分布以及与耐药谱的关系,并初步探讨其在分子流行病学分析中的作用.方法收集我院临床2010年3月至9月分离出69株非重复产ESBL肺炎克雷伯菌,采用PCR法检测16S rRNA 甲基化酶基因,并对阳性菌株进行ESBL基因及整合子基因分析,通过DNA直接测序确定.质粒接合试验和质粒消除试验确定16S rRNA甲基化酶基因的传播途径,利用ERIC-PCR技术进行基因分型.结果 69株产ESBL肺炎克雷伯菌中有rmtB阳性菌株20株(28.9％),其中2株同时携带有rmtB和armA.在20株产16SrRNA甲基化酶菌株中,均携带有CTX-M基因,测序显示14株CTX-M-14基因,6株CTX-M-15基因；14株携带有TEM-1基因；8株携带有SHV基因,测序显示5株SHV-12基因,3株SHV-11基因；3株携带有OXA-10基因；3株携带有VBE-1基因.另有12株携带有int1阳性,含有5种不同的耐药基因盒,分别携带drfA25、drfA1、drfA12、aadA1、aadA2、sat和blaVEB-1基因.ERIC-PCR法显示20株16SrRNA甲基化酶基因阳性的肺炎克雷伯菌主要分为5型,A型为优势流行克隆株.质粒接合和消除试验发现A型克隆株KP5和KP16 rmtB均位于一质粒上并通过接合传播.结论本院产ESBL肺炎克雷伯菌临床分离株中存在16S rRNA甲基化酶基因rmtB的普遍流行,导致对多种氨基糖苷类抗生素高水平耐药.rmtB可通过水平基因传播和克隆传播的两种方式进行播散,并且存在同时产ESBLs、16S rRNA甲基化酶和Ⅰ类整合子的肺炎克雷伯菌的传播.
목적 조사아원산ESBL폐염극뢰백균림상분리주16S rRNA갑기화매기인적분포이급여내약보적관계,병초보탐토기재분자류행병학분석중적작용.방법 수집아원림상2010년3월지9월분리출69주비중복산ESBL폐염극뢰백균,채용PCR법검측16S rRNA 갑기화매기인,병대양성균주진행ESBL기인급정합자기인분석,통과DNA직접측서학정.질립접합시험화질립소제시험학정16S rRNA갑기화매기인적전파도경,이용ERIC-PCR기술진행기인분형.결과 69주산ESBL폐염극뢰백균중유rmtB양성균주20주(28.9％),기중2주동시휴대유rmtB화armA.재20주산16SrRNA갑기화매균주중,균휴대유CTX-M기인,측서현시14주CTX-M-14기인,6주CTX-M-15기인；14주휴대유TEM-1기인；8주휴대유SHV기인,측서현시5주SHV-12기인,3주SHV-11기인；3주휴대유OXA-10기인；3주휴대유VBE-1기인.령유12주휴대유int1양성,함유5충불동적내약기인합,분별휴대drfA25、drfA1、drfA12、aadA1、aadA2、sat화blaVEB-1기인.ERIC-PCR법현시20주16SrRNA갑기화매기인양성적폐염극뢰백균주요분위5형,A형위우세류행극륭주.질립접합화소제시험발현A형극륭주KP5화KP16 rmtB균위우일질립상병통과접합전파.결론 본원산ESBL폐염극뢰백균림상분리주중존재16S rRNA갑기화매기인rmtB적보편류행,도치대다충안기당감류항생소고수평내약.rmtB가통과수평기인전파화극륭전파적량충방식진행파산,병차존재동시산ESBLs、16S rRNA갑기화매화Ⅰ류정합자적폐염극뢰백균적전파.
Objective To investigate the prevalence and distribution of 16S rRNA methylase gene and research the relationship with drug resistant spectrum.And preliminary explore its role in molecular epidemiology analysis.Methods Collected 69 clinical isolates of non repetitive ESBL-producing Klebsiella pneumoniae in our hospital from Mar to Sep 2010.Detection 16S rRNA methylation enzyme gene by PCR,and analyze ESBL genetype and integron gene of the positive strains.All PCR products were sequenced for determination.Plasmid conjugation test and plasmid elimination method to determine dissemination of 16S rRNA methylase gene.Then we used ERIC-PCR genotyping technology for the establishment of DNA fingerprinting.Results In sixty-nine strains,twenty isolates were rmtB positive (28.9％),two isolates were armA positive,and two strains coproduce rmtB and armA.All positive isolates carried the CTX-M gene,detemined by sequencing,14 strains of CTX-M-14 gene,6 strains of CTX-M-15 gene,14 strains carried TEM1 gene,8 strains carried SHY gene,sequencing showed that 5 strains of SHV-12 gene,3 strains of SHV-11 gene,3 strains carried OXA-10 gene,3 strains carried VBE-1 gene.In addition,the intl gene was found in 12 isolates of 20 rmtB positive strains.All the intl gene positive strains were divided into five kinds gene cassettes,which contained drfA25,drfA1,drfA12,aadA1,aadA2,sat and blaVEB-1 genes.Respectivily,16S rRNA methylase gene positive strains were divided into five genetypes using ERIC-PCR technology.A genetype was the advantage popular clones.Conjugative plasmid and elimination test found that rmtB gene was located in a plasmid in KP5 and KP16 isolates with A genetype,and can disseminate by conjugation.Conclusion A high prevalence of 16S rRNA methylase gene-rmtB was found among clinical ESBL-producing K.pneumoniae isolates in our hospital,which could lead to resistant to almost all aminoglycoside at a high level.Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB gene.In addition,K.pneumoniae co-producing ESBLs,16S rRNA methylation enzymes and class Ⅰ integron existed and were spreading.