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采用菌落杂交法检测364株不同脉冲场电泳(PFGE)型的屎肠球菌中透明质酸酶(hyl)基因的分布。用自杀质粒pTX4577通过体内同源重组,筛选获得hyl基因的突变株,体外研究hyl基因缺失对突变株生长能力的影响。采用小鼠腹膜炎模型体内研究hyl基因突变株的毒力下降情况。结果显示,在临床分离菌株中hyl基因阳性率为32.76%,明显高于非临床分离菌株(5.30%)。经同源重组,利用卡那霉素抗性筛选,PCR、PFGE和Southern印迹进行鉴定获得hyl基因突变株,命名为*hyl。突变株在体外的生长能力低于野生株。小鼠腹膜炎模型中,在相同细菌感染浓度(3.7×109cfu)下,野生株TX2466组小鼠的存活率为0,而*hyl组小鼠的存活率为50%,差异有显著性。hyl基因主要出现于临床感染的屎肠球菌中,在屎肠球菌致病中起着重要的作用,可能是屎肠球菌的毒力因子之一。
The distribution of hyl gene in 364 strains of Enterococcus faecium with different pulsed field electrophoresis (PFGE) was detected by colony hybridization. The mutant of hyl gene was screened by homologous recombination in suicide plasmid pTX4577, and the effect of hyl gene deletion on the growth ability of mutant was investigated in vitro. The murine peritonitis model was used to study the decrease of virulence of hyl gene mutant in vivo. The results showed that the positive rate of hyl gene in clinical isolates was 32.76%, significantly higher than that of non-clinical isolates (5.30%). The homologous recombination, kanamycin resistance screening, PCR, PFGE and Southern blot identification identified hyl gene mutant, named * hyl. Mutant strains in vitro growth capacity than wild strains. In mouse peritonitis model, the survival rate of wild-type strain TX2466 mice was 0 at the same bacterial infection concentration (3.7 × 109 cfu), while the survival rate of mice in * hyl group was 50%, the difference was significant. The hyl gene is mainly found in clinically infected Enterococcus faecium and plays an important role in the pathogenesis of Enterococcus faecium. It may be one of the virulence factors of Enterococcus faecium.