应用DHPLC技术分析浙江沿海地区ESBLs基因类型的研究

Investigate genetype of distribution of extend spectrum β lactamases by denaturing high performance liquid chromatography in littoral of Zhejiang

目的应用变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)技术检测超广谱β-内酰胺酶(extend spectrumβ-lactamases,ESBLs)基因型,了解浙江沿海地区ESBLs基因型分布情况。方法收集171株多重耐药肠杆菌及标准菌株采用多重PCR进行头孢克肟-M型(CTX-M)扩增,扩增产物经DHPLC分析,确定临床菌株基因分型。结果171株多重耐药肠杆菌经表型试验有142株临床菌株ESBLs表型阳性,经多重PCR扩增109株携带CTX-M基因,其中52株为CTX-M-1产物,57株为CTX-M-9产物,检出率达76.8%(109/142)。其中大肠埃希菌携带CTX-M基因比例最高(92.3%),其次是肺炎克雷伯菌(72.3%)和阴沟肠杆菌(60.0%)。109株临床菌株PCR产物经DHPLC分析检测出4种CTX-M基因型,33株携带CTX-M-3、19株携带CTX-M-15、5株携带CTX-M-9、52株携带CTX-M-14。结论 CTX-M型ESBLs菌株检测出4种基因型,CTX-M-3、CTX-M-15、CTX-M-9和CTX-M-14,其中CTX-M-14型是CTX-M型ESBLs主要型别。浙江沿海地区携带CTX-M型ESBLs的细菌以大肠埃希菌、肺炎克雷伯菌、阴沟肠杆菌为多见。

Objective To investigate the genotype distribution of CTX-M-type extend spectrum β-1actamases （ES- BLs） by denaturing high-performance liquid chromatography （DHPLC） in littoral of Zhejiang. Methods A total of 171 muhidrug resistant Enterobacteriaceae isolates were collected. The CTX-M genes of standard strains and clinical ES- BLs-producing isolates were amplified by multiplex PCR, and then the products were analysed by DHPLC to determine the genotype. Results A total of 142 ESBLs-producing isolates were confirmed by phenotypie confirmatory tests from 171 multidrug resistant Enterobacteriaceae isolates, 109 isolates （76.8%） carried CTX-M genes among 142 ESBLs-produc- ing isolates, The 109 clinical isolates included 52 belonging to group CTX-M-1, 57 belonging to group CTX-M-9. The proportion of the E.eoIi carting CTX-M gene was the highest, followed by Klebs;ella pneumoniae and Enterobacter cloacae, they were 92.3%,72.3%,60.0%. The 109 clinical isolates were found belonging to 4 different CTX-M genotypes by DHPLC, including CTX-M-3 （33/109）, CTX-M-15 （19/109）,CTX-M-9 （5/109） and CTX-M-14 （52/109）. Conclusion Four dif- ferent subtypes of CTX-M-type ESBLs are detected, which are CTX-M-3, CTX-M-15, CTX-M-9 and CTX-M-14. A- mong them, CTX-M-14 is the main subtype. Many species of Euterobacteriaceae isolates were found containing CTX-M ESBLs in littoral of Zhejiang. E.eoli, Klebsiella pneumoniae and Enterobacter cloaca were the most common CTX-M ES- BLs-produeing species.