引起血流感染的耐万古霉素肠球菌基因型、毒力及MLST分型研究

Molecular Characterization of Drug Resistance, Virulence and MLST in Vancomycin-resistant Enterococci Isolated from Blood Stream Infection

调查引起血流感染的耐万古霉素肠球菌(VRE)的检出情况,并对其进行耐药基因型、毒力和多位点序列分型(MLST)研究。方法:收集2012年7月~2015年7月从笔者医院血培养阳性标本中分离的68株屎肠球菌和28株粪肠球菌,采用含6μg/ml万古霉素的脑心浸液平皿筛选VRE。采用多重PCR法检测vanA、vanB及粪、屎肠球菌种特异基因,另一种多重PCR法检测常见5种毒力基因(esp、hyl、gelE、asa1和cylA)。采用MLST技术分析菌株序列型(ST)从而进行同源性分析。结果: 96株肠球菌中共检出22株VRE (22/96, 22.9%),包括20株屎肠球菌(20/68, 29.4%)和2株粪肠球菌(2/28, 7.1%)。所有VRE耐药表型与基因型一致,属vanA型。20株屎肠球菌VRE仅esp (16/20, 80.0%)和hyl (10/20, 50.0%)阳性;2株粪肠球菌VRE仅gelE和 asa1阳性。20株屎肠球菌VRE分属7个ST型,包括8株ST78 (8/20, 40.0%)、6株ST17 (6/20, 30.0%)以及2株ST18和ST389、ST414、ST571、ST564各1株。结论:笔者医院VRE耐药问题严重,耐药基因和毒力基因携带率高。20株屎肠球菌VRE经eBURST软件分析均属于与医院感染相关的CC17克隆复合群。

To investigate the prevalence of vancomycin-resistant Enterococci (VRE) isolated from blood stream infection, and to determine the genotype, virulence genes and clonality by Multilocus sequence typing (MLST) scheme. Methods 68 Entercoccus faecium and 28 Entercoccus faecalis isolates were collected from blood culture of inpatients between July 2012 and July 2015. VRE presence was confirmed by growth in brain heart infusion agar that contained 6μg/ml vancomycin. The vanA,vanB and species-specific genes for Entercoccus faecalis and Entercoccus faecium were analyzed using a multipex PCR method. The occurrence of the esp, hyl, gelE, asa1 and cylA were evaluated by another multiplex PCR. MLST scheme was used to assess the clonality. Results Of 96 isolates, 22 (22.9%) VRE were screened, including 20 (20/68, 29.4%) Entercoccus faecium (VREfm) and two (2/28, 7.1%) Entercoccus faecalis (VREfs). All VRE isolates exhibited vanA phenotype and harbored vanA genes. The VREfm isolates only carried esp (16/20, 80.0%) and hyl (10/20, 50.0%), while gelE and asa1 were positive in two VREfs isolates. MLST analysis demonstrated seven STs (sequence type) in 20 VREfm strains, including ST78 (8/20, 40.0%), ST17 (6/20, 30.0%), two for ST18, and one each for ST389, ST414, ST571 and ST564. Conclusion High prevalence of VRE was a serious problem in our hospital, and most isolates harbored vanA and virulence genes. All STs in VREfm isolates were designated as CC17 by eBURST algorithm,which was considered to be associated with nosocomial infections.