万古霉素非敏感肠球菌的筛选和基因型分析

Screening and genotype analysis of vancomycin-non-susceptible enterococci

目的对实验室保存的325株肠球菌进行万古霉素非敏感肠球菌筛选,并对筛选出的菌株进行耐药表型、耐药基因型、菌株同源性分析。方法采用琼脂平皿二倍稀释法筛选万古霉素非敏感肠球菌,并检测菌株对替考拉宁的敏感性;采用PCR方法检测万古霉素非敏感肠球菌的耐药基因型;通过肠道细菌基因间重复一致序列(ERIC)PCR技术、脉冲场凝胶电泳(PFGE)技术、多位点序列分型(MLST)技术进行菌株的同源性分析。结果从325株临床分离肠球菌中共筛选出17株万古霉素非敏感肠球菌,包括1株粪肠球菌、11株屎肠球菌和5株鹑鸡肠球菌。其中1株粪肠球菌和11株屎肠球菌对万古霉素显示高水平耐药,对替考拉宁耐药或中介耐药,PCR检测结果显示van A型;5株鹑鸡肠球菌对万古霉素中介耐药,对替考拉宁敏感,PCR检测结果显示van C1型。ERIC同源性分析显示同基因型的大多数菌株显示相似的分型;PFGE同源性分析显示,17株临床万古霉素非敏感肠球菌分型不同,不属于单克隆流行播散;MLST同源性分析显示,1株临床万古霉素非敏感粪肠球菌属于ST4,11株临床万古霉素非敏感屎肠球菌共分为6个ST型,其中4株属于ST78,是屎肠球菌出现频率最高的ST型。结论我国万古霉素耐药菌在粪肠球菌中分离率较低,但屎肠球菌中万古霉素耐药情况较严重,并且耐药菌株携带易于传播和转移的van A基因。同源性分析结果显示万古霉素耐药存在同源性传播的可能性。

Objective To screen vancomycin-non-susceptible enterococci from 325 strains of clinical enterococci isolated from 2006 to 2012, and to further conduct the resistance phenotype, resistance genotype and homology analysis of the vancomycin-non-susceptible enterococci.Methods Screening of the vancomycin-non-susceptible enterococci and detection of the susceptibility to teicoplanin were conducted by blood agar dilution method. Resistance genotypes were determined by PCR method. Homology analysis was carried out by ERIC-PCR, PFGE and MLST technology. Results Totally 17 strains of vancomycin-non-susceptible enterococci were screened out from 325 strains of clinical enterococci, including 1 strain of E. faecalis, 11 strains of E. faecium, and 5 strains of E. gallinarum. 1 strain of E. faecalis and 11 strains of E. faecium showed high level resistance to vancomycin, and resistance or intermediate resistance to teicoplanin, PCR results suggesting van A genotype. 5 strains of E. gallinarum demonstrated intermediate resistance to vancomycin, and susceptibility to teicoplanin, PCR results suggesting van C1 genotype. ERIC homology analysis results showed that most of isolates with the same genotype demonstrated similar ERIC profiles. Pulsed-field gel electrophoresis results of 17 strains of vancomycin-non-susceptible enterococci showed differences in PFGE type. 1 strain of E. faecalis displayed ST4 in multilocus sequence typing analysis, and 11 strains of E. faecium isolates were typed into six different sequence types（STs）, including ST17, ST78, ST203, ST323, ST571, ST262. Four of the E. faecium isolates belonged to ST78, which was the most prevalent ST type in E. faecium of this study.Conclusions In our country, vancomycin resistance rate is low in E. faecalis, but vancomycin resistance is relatively a serious problem in E. faecium. And the resistant isolates usually carry the van A gene favoring resistance transfer between different isolates. Homology analysis of vancomycin resistant isolates suggests the possibility of homologic transfer of vancomycin resistance.