粪肠球菌ace阳性和阴性菌株体外生物被膜形成能力比较

Biofilm formation ability of Enterococcus faecalis ace positive and negative strains in vitro

目的比较粪肠球菌ace基因阳性、阴性菌株的生物被膜形成能力,探讨粪肠球菌ace基因是否有利于细菌生物被膜的形成。方法微量滴定板法检测46株临床分离粪肠球菌的生物膜形成能力,PCR方法检测其ace基因,比较生物膜阳性组、阴性组粪肠球菌ace基因检出率;将粪肠球菌ATCC29212(ace^+)、野生株U8-ace^-、空质粒对照株EU8-ace^-、转化株ZU8-ace^+接种到96孔板,培养3h、6h、12h、24h、36h、48h,结晶紫染色,测定OD570,微量滴定板法比较ATCC29212(ace^+)、U8-ace^-、EU8-ace^-以及ZU8-ace^+的生物被膜形成能力;将ATCC29212(ace^+)、U8-ace^-、EU8-ace^-、ZU8-ace^+在含盖玻片的细胞培养皿内培养6h^72h,共聚焦激光扫描显微镜(CLSM)观察比较ace^+、ace^-粪肠球菌形成的生物膜平均厚度和密度。结果生物膜阳性组粪肠球菌ace基因检出率为76.67%,高于生物膜阴性组31.25%的检出率,差别有统计学意义(χ^2=9.04,P>0.05);微量滴定板法显示不同时间点ATCC29212(ace^+)以及转化株ZU8-ace^+的OD570值都大于U8-ace^-的OD570值,P<0.01,而EU8-ace^-与U8-ace^-比较,OD570值无统计学差别,P>0.05;CLSM观测的ace^+粪肠球菌在不同阶段所形成的生物膜的平均厚度、密度均高于ace^-粪肠球菌,P<0.01,空质粒对照株与野生株比较,生物膜的厚度、密度无统计学差别,P>0.05。结论粪肠球菌胶原黏附素ace基因或有利于肠球菌生物被膜的形成。

In this work, we explore whether the ace gene of E. faecalis is beneficial to the biofilm formation by comparing the biofilm formation ability of E. faecalis ace positive and negative strains. The ability of biofilm formation of 46 clinical isolates of E. faecalis was detected by microtitration plate method, and the ace gene of E. faecalis was detected by PCR. The detection rates of ace gene of E. faecalis in biofilm positive and negative groups were compared. E.faecalis strains ATCC29212- ace^+(clinical isolates),U8- ace^-,EU8- ace^-,ZU8- ace^+ were cultivated in 96-well culture plate for 3hours,6hours,12hours,24hours,36hours,48hours, OD 570 were detected after Gram crystal violet staining. The ability of biofilm formation of ace^+ or ace^- strains were detected and compared by microtiter plate assay;E. faecalis strains ATCC29212( ace^+),U8- ace^-,EU8- ace^- and ZU8- ace^+ strains were cultured on cover glasses in cell culture dish for 6-72 hours, the mean thickness and density of biofilms of ace^+ or ace^- E. faecalis strains at different incubation time were observed and compared by CLSM. It was shown that the detection rate of ace gene of Enterococcus in positive biofilm group was 76.67%, which was higher than that in negative biofilm group 31.25%(χ^2= 9.04, P >0.05);all the OD 570 value of wild strains ATCC29212( ace^+)and transformed plants ZU8- ace^+were greater than that of wild plants U8- ace^- respectively at different culture time points, P <0.01, while there was no significant difference between the empty plasmid control strain and the wild strain, P >0.05;the mean thickness,density of biofilms of ATCC29212( ace^+),ZU8- ace^+ observed by CLSM were all greater than that of wild strains U8- ace^- respectively ( P <0.01). There was no significant difference between control strains and wild strains ( P >0.05). It is concluded that E. faecalis ace might be in favor of the biofilm formation.