肠球菌心内膜炎抗原efaA在粪肠球菌生物膜形成中的作用
发布时间：2019-04-07 16:55
【摘要】：目的:探讨肠球菌心内膜炎抗原efaA与肠球菌生物膜形成之间的相关性,为深入研究肠球菌生物膜的致病性及耐药性奠定基础。 方法: 1.利用普通光学显微镜观察efaA~+、efaA~-粪肠球菌在玻片上的生物膜形成过程,初步判定肠球菌生物膜形成各阶段的时间点; 2.微量滴定板法比较efaA~+、efaA~-粪肠球菌的生物膜形成能力:细菌接种至96孔板中,培养不同时间后弃去培养液,结晶紫染色,乙醇-丙酮混合液溶解,酶标仪测定比较不同阶段efaA~+、efaA~-菌的OD570值; 3. MTT法比较efaA~+、efaA~-粪肠球菌生物膜的活菌含量:细菌接种至96孔板中,培养不同时间后弃去培养液,MTT孵育后DMSO溶解,酶标仪测定比较不同阶段efaA~+、efaA~-菌的OD492值; 4.共聚焦激光扫描显微镜观察比较efaA~+、efaA~-粪肠球菌所形成的生物膜:各菌株在玻片上形成生物膜,AO/EB染色后CLSM观察,比较efaA~+、efaA~-菌所形成生物膜的厚度及生物膜内层、中层、外层的细菌密度和活菌比例,并做统计学分析。 5.采用SYBR GreenⅠ实时荧光定量PCR,检测efaA~+、efaA~-粪肠球菌在细菌培养皿内培养不同时间后所形成生物膜的efaA mRNA的表达,与生物膜的厚度变化进行对比。 结果: 1.肠球菌生物膜黏附期为2h,基本形成期为6h,成熟期为24h,播散期为48h。 2.微量滴定板法比较生物膜形成能力,efaA~+粪肠球菌在各培养时间点的OD570均大于efaA~-菌,即efaA~+粪肠球菌的生物膜形成能力比efaA~-菌的强; 3. MTT法比较生物膜不同阶段的活菌含量(OD492),efaA~+粪肠球菌在各培养时间点的OD492均大于efaA~-菌,即efaA~+粪肠球菌所形成的生物膜的活菌数比efaA~-菌的多; 4.共聚焦激光显微镜观察,efaA~+粪肠球菌在不同阶段所形成的生物膜的厚度、细菌密度及活菌比例均比efaA~-菌的大; 5. efaA~+粪肠球菌efaA mRNA的表达量随培养时间的延长而增加,24h时达最高水平,随后表达量降低,与生物膜平均厚度变化趋势相平行;efaA~-粪肠球菌未检测到efaA mRNA表达。
[Abstract]:Aim: to investigate the relationship between enterococcus endocarditis antigen efaA and enterococcus biofilm formation, and to lay a foundation for further study on the pathogenicity and drug resistance of enterococcus biofilm. Methods: 1. The biofilm formation process of Enterococcus faecalis efaA~ and efaA~- on slide was observed by ordinary optical microscope, and the time point of biofilm formation of Enterococcus faecalis was determined. The biofilm-forming ability of efaA~ and efaA~- Enterococcus faecalis were compared by microtitration plate method: bacteria were inoculated into 96-well plate, after culture for different time, the culture medium was discarded, crystal violet staining, ethanol-acetone mixed solution dissolved. The OD 570 values of efaA~ and efaA~- strains in different stages were measured and compared by enzyme labeling instrument. 3. MTT method was used to compare the living bacteria content of enterococcus faecalis biofilm between efaA~ and efaA~-: the bacteria were inoculated into 96-well plate, the culture medium was discarded after culturing for different time, the DMSO was dissolved after MTT incubation, and the OD 492 values of efaA~ and efaA~- in different stages were measured by enzyme standard instrument; 4. The biofilm formed by efaA~ and efaA~- Enterococcus faecalis were observed by confocal laser scanning microscope. The biofilm was formed on slide by each strain, and observed by CLSM after AO/EB staining. The thickness of biofilm formed by efaA~ and efaA~- bacteria and the inner layer of biofilm were compared. The density of bacteria and the proportion of living bacteria in the middle layer and outer layer were analyzed statistically. 5. SYBR Green 鈪，
本文编号：2454247
[Abstract]:Aim: to investigate the relationship between enterococcus endocarditis antigen efaA and enterococcus biofilm formation, and to lay a foundation for further study on the pathogenicity and drug resistance of enterococcus biofilm. Methods: 1. The biofilm formation process of Enterococcus faecalis efaA~ and efaA~- on slide was observed by ordinary optical microscope, and the time point of biofilm formation of Enterococcus faecalis was determined. The biofilm-forming ability of efaA~ and efaA~- Enterococcus faecalis were compared by microtitration plate method: bacteria were inoculated into 96-well plate, after culture for different time, the culture medium was discarded, crystal violet staining, ethanol-acetone mixed solution dissolved. The OD 570 values of efaA~ and efaA~- strains in different stages were measured and compared by enzyme labeling instrument. 3. MTT method was used to compare the living bacteria content of enterococcus faecalis biofilm between efaA~ and efaA~-: the bacteria were inoculated into 96-well plate, the culture medium was discarded after culturing for different time, the DMSO was dissolved after MTT incubation, and the OD 492 values of efaA~ and efaA~- in different stages were measured by enzyme standard instrument; 4. The biofilm formed by efaA~ and efaA~- Enterococcus faecalis were observed by confocal laser scanning microscope. The biofilm was formed on slide by each strain, and observed by CLSM after AO/EB staining. The thickness of biofilm formed by efaA~ and efaA~- bacteria and the inner layer of biofilm were compared. The density of bacteria and the proportion of living bacteria in the middle layer and outer layer were analyzed statistically. 5. SYBR Green 鈪，
本文编号：2454247
本文链接：https://www.wllwen.com/xiyixuelunwen/2454247.html
最近更新
教材专著
