临床分离屎肠球菌pilA和hylEfm基因流行病学研究

Prevalence of genes involved in pilA and hylEfm of clinical isolates of enterococcus faecium

摘要:

目的：检测屎肠球菌菌毛样结构蛋白基因pilA和类透明质酸酶基因hylEfm在临床感染株中的分布，探讨屎肠球菌感染率持续上升的原因。方法：采用多重PCR检测临床感染株、住院患者肠道定植株和健康志愿者肠道定植株的pilA和hylEfm基因的分布；然后通过接合实验确认pilA和hylEfm基因在屎肠球菌间的传递方式；最后采用分子杂交法确认转移后pilA和hylEfm的质粒或染色体定位。结果：137株屎肠球菌感染株中共筛查确认120株携带pilA基因，阳性率为88%；21株携带hylEfm基因，阳性率为15%。分离鉴定住院患者肠道定植株、健康志愿者肠道定植株各50株，pilA基因阳性菌株分别为35株（阳性率70%）、26株（阳性率52%），临床感染株与住院患者肠道定植株、健康志愿者肠道定植株pilA基因携带率差异具有统计学意义（P<0.000 1；P=0.008 0）。接合实验证实pilA和hylEfm基因均能通过质粒的接合在不同菌株间进行传递，并同时伴随耐药基因传递。Southern Slot杂交发现pilA与hylEfm基因可位于同一大质粒上，不同菌株间此质粒大小存在差异（145.5～291 kb）。结论：临床感染患者分离屎肠球菌大多携带pilA基因。pilA和hylEfm基因可通过同一个可移动大质粒在菌株之间进行传递，同时伴随耐药基因的转移。

Abstract:

Objective：To investigate the relationship between pilA and hylEfm genes and the pathogenic mechanism of enterococcus fae－cium. Methods：A multiplex PCR was applied to detect the distribution of hylEfm and pilA. The transfer mode between strains of ente－rococcus faecium was confirmed via filter matings. S1 nuclease and I-CeuI assay was performed to detect the position of pilA and hylEfm genes. Results：Totally 137 strains of enterococcus faecium were isolated，88% clinical isolates（120/137 isolates）carrying pilA genes and 15% clinical isolates（21/137 isolates）carrying hylEfm gene. Fifty strains of inpatients and 50 strains of health controls were isolated respectively；70% isolates（35/50 isolates） carrying pilA genes and 52% isolates（26/50 isolates） carriying pilA genes were respectively observed in inpatients and health controls. Highly significant differences in isolates carrying pilA genes among clinical group，inpatients group and health controls group were observed（P<0.000 1，P=0.008 0）. Filter mating confirmed that pilA and hylEfm genes can transfer between different strains through plasmid conjugation. Southern hybridization found both pilA and hylEfm genes located at a large plasmid with size running between 145.5 kb and 291 kb. Conclusion：Positive rate of Pilus-like structures protein genes pilA of enterococcus faecium isolated from our hospital is consistently high. pilA and hylEfm genes simultaneously encode on a large plasmid containing variable antibiotic resistance genes in different isolates of nosocomial enterococcus faecium strains.