屎肠球菌高水平庆大霉素耐药基因研究

Analysis of high-level gentamicin resistant genes in Enterococcus faecium

目的研究我国临床分离高水平庆大霉素耐药屎肠球菌中庆大霉素耐药基因分类及其水平传递情况。方法采用PCR法检测庆大霉素耐药基因aac(6′)-Ie-aph(2″)-Ia、aph(2″)-Ib、aph(2″)-Ic、aph(2″)-Id和aph(3″)-Ⅲa,采用质粒酶切图谱分析耐药菌同源性,采用液体传递和固体传递法研究庆大霉素耐药基因水平传递情况。结果105株临床分离屎肠球菌中,76.2%(80/105)为高水平庆大霉素耐药,其中aac(6′)-Ie-aph(2″)-Ia基因阳性率为95.0%(76/80),aph(3″)-Ⅲa基因阳性率为78.8%(63/80),aph(2″)-Ib、aph(2″)-Ic、aph(2″)-Id基因皆阴性。这些高水平庆大霉素耐药屎肠球菌间大多无明显同源性。21.1%(16/76)菌株携带的aac(6′)-Ie-aph(2″)-Ia基因在屎肠球菌间固体传递,13.2%(10/76)菌株携带的aac(6′)-Ie-aph(2″)-Ia基因在屎肠球菌间液体环境中水平传递。结论本次研究中屎肠球菌中高水平庆大霉素耐药主要由aac(6′)-Ie-aph(2″)-Ia和aph(3″)-Ⅲa基因介导,这些菌株间没有明显同源性,且该基因可在屎肠球菌间水平传递。

Objective To analyze the prevalence and horizontal transfer of gentamicin resistant genes in Enterococcus faecium. Methods PCR technique was applied to identify genes including aac（6＇）-Ie-aph （2＂）-Ia, aph（2＂）- Ib, aph（2＂）- Ic, aph（2＂）- Id and aph（3＂）-Ⅲ a, Plasmid DNA was analyzed with restriction enzymes and agarose gel electrophoresis. The transferability of the high-level gentamicin resistance trait of the strains was examined by broth mating and solid mating. Results Of the 105 clinical isolates tested, 80 （76.2 % ） were high-level-resistant to gentamicin（HLGR）. In the HLGR E. faecium, 95.0 % （76/ 80） were positive for aac（6＇）-Ie-aph （2＂）-Ia and 78.8% （63/80） positive for aph（3＂）Ⅲ a gene. aph（2＂）- Ib, aph（2＂）- Ic and aph （2＂）- Id were not detected. Majority of the plasmid patterns were identified in single isolates. The gentamicin resistances were transferred in 16 （21.1% ） of 76 isolates at frequency of 10^-4 and 10^-5 per donor cell between E. faecium strains during broth mating and horizontal transfer was found in 10 （13.2%） of 76 isolates between E. faecium during solid mating. Conclusions The spread of aac（6＇）-Ie-aph （2＂）-Ia and aph （3＂）Ⅲa genes were responsible for high-level resistance to gentamicin. These genes can transfer horizontally between E. faecium. There is no obvious homology in HLGR E. faecium isolates.