武汉市儿童医院临床分离大肠埃希菌与肺炎克雷伯菌中产质粒介导AmpC酶和ESBLs的研究

The plasmid-mediated AmpC beta-lactamase and extra-extended-spectrum β lactamase in the Escherichia coli and Klebsiella pneumoniae strains isolated from Wuhan Children's Hospital

目的研究武汉市儿童医院临床分离高产质粒AmpC酶和超超广谱β内酰胺酶(SSBL)肺炎克雷伯菌与大肠埃希菌的分子流行病学特征。方法收集我院2005年8月—2006年8月住院患儿临床分离的头孢西丁中介或耐药的大肠埃希菌和肺炎克雷伯菌161株。采用三维试验检测AmpC酶,表型确认试验检测ESBLs,质粒转化试验定位耐药基因;采用PCR技术扩增质粒AmpC与CTX-M基因;使用限制性片段长度多态性(RFLP)技术确定CTX-M基因簇的特征;利用基因测序确定AmpC酶及CTX-M的基因亚型。结果DHA-1型和ACT-1型为主要的质粒AmpC酶基因型,发现1种新的ACT型质粒AmpC酶,其与ACT-1的同源性仅为84%。CTX-M-14型和CTX-M-3型为主要的ESBLs基因型。最常见的大肠埃希菌SSBL基因组合为CTX-M-14+DHA-1+ACT-1型,肺炎克雷伯菌的为CTX-M-3+DHA-1+ACT-1。结论我院临床分离的肺炎克雷伯菌和大肠埃希菌中,产SSBL菌株所占比例显著高于单产质粒AmpC酶菌株;CTX-M-14/CTX-M-3+DHA-1+ACT-1是大肠埃希菌和肺炎克雷伯菌中最常见的SSBL基因型。

Objective To study the production of plasmid-mediated AmpC β-lactamases and extra-extended-spectrum β-lactamase （SSBLs） in Escherichia coli and Klebsiella pneumoniae strains isolated in Wuhan. Methods A total of 161 strains of cefoxitin-resistant or -intermediate E. coli and K. pneumoniae were collected at Wuhan children＇s hospital from August 2005 to August 2006. Three-dimensional extract test was used for primary analysis of plasmid-mediated AmpC-lactamases. Phenotypic confirmatory test was applied for detecting ESBLs. Transformation experiment was done to position drug resistant gene. Universal primer PCR for AmpC and CTX-M gene amplification and DNA sequencing were carried out to identify the genotype of β- lactamases, bla-CTX M genes were detected and subtyped by restriction fragment length polymorphism （RFLP） analysis. Results DHA-1 and ACT-1 genotypes were identified as the primary plasmid-mediated-AmpC β-lactamase genes in the 161 isolates. CTX-M-14 and CTX-M-3 genotypes were proved to be the predominant ESBLs. A new ACT genotype was identified. CTX-M-14＋ DHA-1 ＋ ACT-1 genotype was the most common SSBL in E. coll. However, in K. pneumoniae, CTX-M-3 ＋ DHA-1 ＋ ACT-1 genotype was the most common SSBL. Conclusions The proportion of SSBL-producing E. coli or K. pneumoniae was much higher than that producing single plasmid-mediated AmpC enzyme in Wuhan Children＇s Hospital. CTX-M-14 or CTX-M-3 ＋ DHA-1 ＋ ACT-1 is the primary SSBL genotype.