gltA基因对粪肠球菌生物膜形成能力影响的初步研究

A preliminary study of the effects of gltA gene on the biofilm formation of Enterococcus faecalis

目的:探讨粪肠球菌(E.faecalis)谷氨酸合酶基因A(gltA)对生物膜形成的影响。方法:采用激光共聚焦扫描电镜(CLSM)观察临床分离的4株E.faecalis的生物膜形成能力、生物膜体积及活菌数;用RT-qPCR检测4株临床菌株的gltA基因表达水平的变化;构建gltA基因高表达E.faecalis菌株,以标准菌V583为对照,分别观察两者生长曲线的变化及24 h内的生物膜形成情况,同时检测其生物膜的体积及活菌数。结果:4株E.faecalis临床菌株均能形成结构致密的生物膜,其生物膜体积及其活菌数均显著高于标准株V583(P<0.05);4株临床菌株的gltA基因表达量均随着培养时间的增加而明显降低,在培养6、12 h时,4株临床菌株的gltA基因表达显著高于标准株V583(P<0.05);培养24 h时,4株临床菌株的gltA基因表达量与标准株V583相比无统计学差异(P>0.05)。gltA基因高表达株的生长速率与标准株相比无统计学差异(P>0.05);gltA基因高表达株生物膜形成较快,结构更加致密且厚度较厚,而标准株V583的生物膜形成较慢,较薄,且gltA基因高表达株在12、24 h时生物膜体积及其活菌数均显著高于对照组(P<0.05)。结论:gltA基因能在E.faecalis生物膜形成的早期阶段发挥关键的调控作用,是E.faecalis生物膜形成的重要基因。

AIM： To investigate the effects of glutamate synthase gene A（ gltA） on the biofilm formation of Enterococcus faecalis（ E. faecalis）. METHODS： CLSM was used to observe the biofilm formation ability of 4 strains of E. faecalis from clinical isolates. The number of viable cells and biofilm volume were compared with that of the standard strain V583. gltA gene expression in 4 clinical isolates were detected by RT-qPCR. The gltA-highly expressed strain was constructed and its growth curve was detected. The formation of biofilm the viable cell number in the biofilm and biofilm volume were observed by LSCM,after 24 h culture. RESULTS： All the 4 isolates could form compact biofilms,and the viable cells and biofilm volume of the isolate groups were significantly higher than those of the control group（ P〈0.05）. gltA gene expression in the 4 clinical isolates and control group were decreased with culture time.At 6 h and 12 h,the expression level of gltA gene in 4 isolates groups was significantly higher than that in the control group（ P〈0.05）,but no significant difference was found at 24 h（ P〉0.05）. There was no difference of growth between the gltA-highly expressed strain and the control group（ P〉0.05）. The cells of gltA-highly expressed strain aggregated into distinct clusters and relatively dense structures,the control cells grew slowly with loose structures. The viable cell number and biofilm volume of gltA-highly expressed strain at 12 h and 24 h were significantly bigger than those of the control（ P〈0.05）. CONCLUSION： The high expression of gltA gene in E. faecalis can enhance the biofilm formation at early stage,gltA is an important gene in the regulation of biofilm formation of E. faecalis.