致羔羊脑炎粪肠球菌生物被膜形成能力及相关基因检测研究  

Detection on Biofilm and Biofilm-associated Genes of Enterococcus faecalis Inducing Lamb Encephalitis

作　　者：孔静雅[1] 郭强强[1] 孔科委 李劼[1] 周霞[1] 齐亚银[1] KONG Jing-ya GUO Qiang-qiang KONG Ke-wei LI Jie ZHOU Xia QI Ya-yin(College of Animal Science and Technology , Shihezi University, Shihezi 832000, Chin)

机构地区：[1]石河子大学动物科技学院,石河子832000

出　　处：《中国畜牧兽医》2017年第3期679-685,共7页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31160507);国家自然科学基金(31260610)

摘　　要：为观察致羔羊脑炎粪肠球菌XJ05株生物被膜(BF)的形成能力并检测致羔羊脑炎粪肠球菌BF形成相关基因的携带情况,本研究以XJ05为研究对象,运用96孔板建立不同时间BF模型,染色后酶标仪测定D595nm值,并用倒置显微镜观察BF形态。同时检测11株菌中与BF形成相关基因(gelE、esp、ace、asa1、asa373、efaA、ef0591、ef3314、ahrc、eep)的携带情况,并对相关基因片段做同源性分析。结果显示,在不同时间段XJ05株BF的生成量与空白对照组相比差异显著(P<0.05),24h时D595nm值最高且与其他各个时间段相比均差异显著(P<0.05)。显微镜下观察6h时BF初具规模,可见散落的网状结构,12~24h矩阵网格结构明显,24h时形成高度有序的网格结构,24h后网状结构逐渐脱落,BF开始降解。11株菌中10种相关基因的携带情况不同,有6株携带8种基因,1株携带7种基因,1株携带6种基因,1株携带2种基因,有2株未检测到10种基因的任何一种。检出的基因片段同源性均在93.3%~100.0%之间。结果表明,XJ05株能形成完整的BF,11株分离的致羔羊脑炎粪肠球菌中有9株携带部分与BF形成相关的基因。The purpose of this study was to observe and analyze the ability of biofilm formation of the Enterococcus faecalis（E.faecalis）XJ05and to detect if the E.faecalis which could induce lamb encephalitis carried the biofilm-associated genes.The E.faecalis XJ05 was used as the sample.The biofilm model was established by the 96-well microtiter plate and read by microplate reader in the D595 nmat the same time observed by the inverted microscope.The primers were designed to examine if the 11 strains of E.faecalis carried the biofilm related genes and then the amplified products were sequenced and analyzed for homology.The results showed that the amount of BF produce in XJ05 strain was significantly different from blank control group at different time（P〈0.05）,and the highest D595 nmvalue was at 24 hand significant difference compared with other time（P〈0.05）.The biofilm of 6hwas scattered when observed under the microscope and the time between 12 to 24hbiofilm to be more denser,after 24 hthe biofilm became to degradate and loose gradually.It turned out that the 11 strains of bacteria carried different biofilm-associated genes,8biofilm-associated genes were found in 6strains of bacteria,7biofilm-associated genes were find in 1strain of bacterium,6biofilm-associated genes were found in 1strainof bacterium,2biofilm-associated genes were found in 1strain of bacterium,2strains of bacteria could not text out anyone of the detected genes.The homology of all detected genes were all between 93.3%to 100.0%.It concluded that the E.faecalis XJ05 could form the completed biofilm and there were 9kinds carried the biofilm-associated genes in 11 strains of E.faecalis.

关 键 词：粪肠球菌 生物被膜 生物被膜相关基因 同源性分析 

分 类 号：S855.1[农业科学—临床兽医学]