【摘要】：目的:探討腸球菌心內膜炎抗原efaA與腸球菌生物膜形成之間的相關性,為深入研究腸球菌生物膜的致病性及耐藥性奠定基礎。 方法: 1.利用普通光學顯微鏡觀察efaA~+、efaA~-糞腸球菌在玻片上的生物膜形成過程,初步判定腸球菌生物膜形成各階段的時間點; 2.微量滴定板法比較efaA~+、efaA~-糞腸球菌的生物膜形成能力:細菌接種至96孔板中,培養(yǎng)不同時間后棄去培養(yǎng)液,結晶紫染色,乙醇-丙酮混合液溶解,酶標儀測定比較不同階段efaA~+、efaA~-菌的OD570值; 3. MTT法比較efaA~+、efaA~-糞腸球菌生物膜的活菌含量:細菌接種至96孔板中,培養(yǎng)不同時間后棄去培養(yǎng)液,MTT孵育后DMSO溶解,酶標儀測定比較不同階段efaA~+、efaA~-菌的OD492值; 4.共聚焦激光掃描顯微鏡觀察比較efaA~+、efaA~-糞腸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觀察,比較efaA~+、efaA~-菌所形成生物膜的厚度及生物膜內層、中層、外層的細菌密度和活菌比例,并做統計學分析。 5.采用SYBR GreenⅠ實時熒光定量PCR,檢測efaA~+、efaA~-糞腸球菌在細菌培養(yǎng)皿內培養(yǎng)不同時間后所形成生物膜的efaA mRNA的表達,與生物膜的厚度變化進行對比。 結果: 1.腸球菌生物膜黏附期為2h,基本形成期為6h,成熟期為24h,播散期為48h。 2.微量滴定板法比較生物膜形成能力,efaA~+糞腸球菌在各培養(yǎng)時間點的OD570均大于efaA~-菌,即efaA~+糞腸球菌的生物膜形成能力比efaA~-菌的強; 3. MTT法比較生物膜不同階段的活菌含量(OD492),efaA~+糞腸球菌在各培養(yǎng)時間點的OD492均大于efaA~-菌,即efaA~+糞腸球菌所形成的生物膜的活菌數比efaA~-菌的多; 4.共聚焦激光顯微鏡觀察,efaA~+糞腸球菌在不同階段所形成的生物膜的厚度、細菌密度及活菌比例均比efaA~-菌的大; 5. efaA~+糞腸球菌efaA mRNA的表達量隨培養(yǎng)時間的延長而增加,24h時達最高水平,隨后表達量降低,與生物膜平均厚度變化趨勢相平行;efaA~-糞腸球菌未檢測到efaA mRNA表達。
[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，

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