万古霉素耐药肠球菌的分子特征及同源性分析

Molecular characterization of vancomycin-resistant Enterococci isolates

目的研究北京朝阳医院临床分离的万古霉素耐药肠球菌（VRE）的基因型及同源性。方法采用WHONET软件调查北京朝阳医院2003年6月至2007年3月VRE的发生率，收集这段时间分离的、来自不重复患者的38株VRE，采用含6mg／L万古霉素筛选平皿确定VRE。多重PCR及序列分析确定糖肽类耐药基因的基因型，脉冲场凝胶电泳（PFGE）确定耐药株的亲缘关系。结果VRE较多出现在重症监护病房（34．2％，13／38）。2003至2007年，VRE的分离率如下：2003年2．6％（5／190），2004年1．8％（6／340），2005年0．7％（2／271），2006年6．8％（20／294），2007年1至3月3．3％（5／153）。38株VRE中，PCR和序列分析证明15株为vanB基因型，均为粪肠球菌，万古霉素最低抑菌浓度（MIC）在16～48mg／L，替考拉宁MIC在0．4～0．5mg／L；23株为vanA基因型，均为屎肠球菌，万古霉素MIC≥256mg／L（除E31为32mg／L），替考拉宁MIC在2～32mg／L。23株vanA基因型屎肠球菌中有12株表现为vanA基因型、vanB表型。PFGE共分为10型，15株粪肠球菌中有14株均来自同一克隆（B型），1株分型未明；23株屎肠球菌中21株分为9型，主要是A、D、E、F型，2株分型未明。结论北京朝阳医院流行的VRE主要有vanA、vanB两种基因型。vanA基因型屎肠球为多克隆传播，vanB基因型粪肠球为单克隆传播。部分vanA基因型屎肠球存在基因型与表型不一致现象。

Objective To investigate the prevalence and genotype of vancomycin-resistant enterococci （VRE） faecium and faecalis isolates. Methods Thirty-eight non duplicate vancomycin -resistant enterococci isolates were collected from 2003 to 2007 in Beijing Chaoyang hospital. The prevalence of VRE was analyzed using WHONET software. These strains were processed by brain heart infusion agar screening in the presence of vancomycin （6 mg/L）, and were analyzed for genotypic characteristics using multiplex PCR. The homology of the isolates was determined by pulsed-field gel electrophoresis （ PFGE ）. Results Most VRE strains were isolated from the ICU patients （34.2%, 13/38 ）. The vancomycin resistance rate （vancomycin MIC≥16 mg/L） was 2.6% in 2003, 1.8% in 2004, 0. 7% in 2005, 6. 8% in 2006, and 3.3% in Jan - Mar 2007. Fifteen vancomycin-resistant Enterococci faecalis isolates were identified as vanB genotype by PCR and sequencing. （ the vancomycin MIC ranged from 16 to 48 mg/L and the teicoplanin MIC ranged from 0. 38 to 0. 5 mg/L）. The vanA gene was confirmed by PCR and sequencing in twenty-three vancomycin-resistant Enterococusfaecium （vancomycin MIC ≤256 kg/L, teicoplanin MIC = 2-32 mg/L）. Twelve vancomycin-resistant Enterococus faecium isolates showed incongruence between phenotype and genotype for glycopeptides resistance （vanA genotype and vanB phenotype）. The PFGE analysis revealed 10 different PFGE types. Fourteen vancomycin-resistant Enterococcifeacalis isolates belonged to the same clone. 21 vancomycin-resistant Enterococci feacium isolates belonged to 9 PFGE types. The four most prevalent clones was A, D, E and F. Conclusion The VRE strains isolated from Beijing Chaoyang Hospital belong to vanA and vanB genotypes. Vancomycin -resistant Enterococci feacalis is monoclonal, and vancomycin- resistant Enterococci feacium is polyclonal. Some of the vancomycin-resistant Enterococus faecium isolates show incongruence between phenotype and genotype.