华南地区质粒介导超广谱β-内酰胺酶的基因分型研究

Genotype characterization of plasmid-mediated extended-spectrum beta-lactamases in Southern China

目的 了解华南地区质粒介导超广谱 β 内酰胺酶 (ESBLs)的发生率及基因型特征。方法 收集 2 0 0 1年 4月～ 9月革兰阴性菌临床分离无重复株共 1184株 ,采用NCCLS表型筛选和确认试验进行ESBLs产酶株的识别 ,E test法检测各亚型ESBLs的MICs值 ,质粒接合及电转化实验、耐药质粒提取及酶切指纹分析、等电聚焦电泳、PCR通用引物扩增TEM、SHV、CTX M、VEB、PER、SFO基因及其克隆测序进行ESBLs基因分型和质粒定位。结果 革兰阴性菌ESBLs的检出率为 14 .6 % (173 1184 ) ;获得产ESBLs接合子 6 7株、电转化子 11株 ,其中产CTX M 14型ESBLs为 33.3% (2 6 78)、CTX M 3为 2 3.1% (18 78)、CTX M 9为 14 .1% (11 78)、CTX M 5为 6 .4 % (5 78)、CTX M 13为 2 .6 % (2 78)、SHV 5为 7.7% (6 78)、SHV 12为 5 .1% (4 78)及SHV 2a为 2 .6 % (2 78) ,未定型为 5 .1% (4 78) ;2 9.5 %(2 3 78)野生株伴产广谱酶TEM 1或SHV 1型 ;各型ESBLs基因约定位在 35～ 190kb大小的可接合性低拷贝数天然质粒上 ;CTX M型ESBLs以对头孢噻肟高水平耐药为特征。结论 华南地区质粒介导的ESBLs以CTX M型衍生酶为主 。

Objective To investigate the prevalance and genotype characteristics of epidemical gram negative bacteria producing plasmid mediated extended spectrum beta lactamases(ESBLs) in Southern China. Methods A total of 1184 clinical isolates of nonrepetitive gram negative bacteria were collected in 2001 from five different cities in Southern China. ESBLs producing isolates were identified by phenotypic confirmatory test based on NCCLS criteria. Plasmids conjugation and electroporation, plasmids extraction and their PstⅠ digest fingerprinting analysis, isoelectric focusing, universal primer PCR for TEM, SHV, CTX M, VEB, PER and SFO gene amplification and DNA sequencing were carried out for ESBLs gene typing, plasmid location, and E test for MICs determination of the different genotype. Results The prevalence of ESBLs producing strains in gram negative bacteria is 14.6%(173/1 184). 67 transcongants and 11 electro transformants obtained from ESBLs producing clinical isolates produce the following ESBLs genotype: 26(33.3%) CTX M 14, 18(23.1%) CTX M 3, 11(14.1%) CTX M 9, 5(6.4%) CTX M 5, 2(2.6%) CTX M 13, 6(7.7%) SHV 5, 4(5.1%) SHV 12, 2(2.6%) SHV 2a, and 4(5.1%) unidentified. Twenty three(29.5%) wild strains also carry broad spectrum beta lactamases TEM 1 or SHV 1. The above ESBLs genes are located on transferable plasmids with various size(from 35 to 190kb, respectively). CTX M type ESBLs are characterized by high level resistance to cefotaxime. Conclusion CTX M type is the most common genotype in plasmid mediated ESBLs produced by clinical isolates in Southern China.