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研究生:李俊緯
研究生(外文):Chun-Wei Li
論文名稱:B群鏈球菌生物膜表現之分析
論文名稱(外文):Study on biofilm expression in group B streptococcus
指導教授:吳俊忠
指導教授(外文):Jiunn-Jong Wu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:醫學檢驗生物技術學系碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:72
中文關鍵詞:B群鏈球菌生物膜
外文關鍵詞:group B streptococcusbiofilm
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B群鏈球菌是最常見造成新生兒細菌性肺炎、敗血症及腦膜炎,也是引起懷孕婦女與免疫低落成年人重要傳染疾病的病原菌。目前B群鏈球菌主要以莢膜多醣體分成九種不同的血清型。本實驗室過去的研究發現,由成大醫院所收集的B群鏈球菌經過分型後,以第III型的比例最高,也與疾病的嚴重度有關,顯示第III型在感染上扮演著重要角色。目前已知許多重要病原菌其細菌生物膜的形成與疾病的嚴重度有關,由於B群鏈球菌目前在生物膜的研究所知有限,本論文主要分析臨床B群鏈球菌血清型及其環境因子與生物膜形成的關係。以45株B群鏈球菌不同血清型分析其生物膜的表現量,發現在血清型Ib的表現最高,其次依序為IV、V、II、VI、III及Ia。此外,也發現高抗藥性B群鏈球菌菌株其生物膜表現似乎並沒有較高的表現量。以不同來源的血清型第III型發現來自於婦女與懷孕婦女產道或生殖道的檢體,其生物膜的表現要高於來自非生殖道的檢體(p < 0.05)。以三株具有較高生物膜表現量的菌株來探討環境因子對其生物膜表現的影響,結果發現菌株在37°C的培養時,其生物膜的表現較30°C高;在10%二氧化碳的培養下,其生物膜的表現較5%二氧化碳高;生物膜表現在pH 7為最高,其次依序為pH 6、pH 5及pH 4,此表現與細菌生長有關;當額外加入碳源(葡萄糖、果糖、蔗糖)皆能增加生物膜的表現,但若以葡萄糖和果糖為碳源時,其生物膜表現比蔗糖來的高。在不同生物膜形成的時間點收取細菌萃取其蛋白質,以SDS-PAGE觀察,目前並無法找到具有專一性蛋白質的表現。本研究顯示B群鏈球菌在血清型Ib型具有較高的生物膜表現,而取自於生殖道的B群鏈球菌血清型III型比非生殖道的菌株有較高的生物膜表現。此外,溫度、二氧化碳濃度、pH及碳源都會對B群鏈球菌生物膜表現造成影響。
Group B streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates, and a cause of morbidity among pregnant women and immunocompromised adults. GBS have been classified into nine different serotypes (Ia, Ib and II through VIII) based on variations of the capsular polysaccharide. Previous data from our lab showed that GBS type III was the most common serotype isolated from NCKU hospital and was associated with severe infections. Biofilm formation is present in many important pathogens and it has been shown to be associated with severe human diseases. Currently, biofilm formation of GBS is poorly understood. The aims of my work were to analyze biofilm expression in different GBS serotypes and to understand how the environmental factors affected the biofilm formation in GBS. Forty-five GBS strains containing different serotypes were used to measure their biofilm formation. The results showed that serotype Ib had the highest biofilm expression followed by IV, V, II, VI, III and Ia. In addition, no significantly higher biofilm formation was found in the highly antibiotic resistant GBS clinical isolates. Serotype III GBS isolates from genitourinary tract and pregnant women had higher biofilm expression than those from non-genitourinary tract (p < 0.05). Three highly biofilm-expressing strains were used to demonstrate how environmental factors affected the biofilm expression. The results showed that the biofilm expression was higher at 37°C, 10% CO2 and pH 7. Additional carbon sources such as glucose, fructose and sucrose increased biofilm expression, but biofilm expression was higher in the presence of glucose and fructose than in the presence of sucrose. Total proteins of GBS were extracted at different time points of biofilm formation and analyzed by SDS-PAGE, and no significant protein expression was found during these periods. In summary, we demonstrated that serotype Ib of GBS is higher in biofilm expression and serotype III of GBS isolated from genitourinary tract showed higher biofilm expression. In addition, temperature, CO2 concentration, pH and carbon source could affect the biofilm expression in GBS clinical strains.
中文摘要…………………………………………………………………....…i
英文摘要……………………………………………………………...…...... iii
誌謝………………………………………………………………….……..…v
目錄………………………………………………………...……..……...…..vi
表目錄……………………………………………………………................viii
圖目錄……………………………………………………………..........……ix
符號及縮寫……………………………………………………….........….…xi
緒論……………………………………………………...………………....…1
材料與方法………………………………………………….…………....…..9
一、菌種來源…………..………………………………………….……...9
二、儀器與藥品…………………………………………………….…….9
三、細菌的培養與保存…………………………………………….…….9
四、B群鏈球菌生物膜表現的分析………..……………..………...…...9
五、溫度對B群鏈球菌生物膜表現測試…………………………..…..10
六、二氧化碳對B群鏈球菌生物膜表現測試….…………………..….10
七、不同碳源對B群鏈球菌生物膜表現測試………………..………..10
八、酸鹼值對B群鏈球菌生物膜表現測試………………………...….11
九、離子濃度對B型鏈球菌生物膜表現測試………………………….11
十、B群鏈球菌總蛋白的萃取…………………………………...……..11
十一、蛋白質定量(Bio-Rad Protein Assay)………….....................…...12
十二、蛋白質定量(Lowry assay)…………………..…………..…....12
十三、SDS-PAGE蛋白質膠體電泳………..………………….….….....13
十四、蛋白質膠體之銀染……………………………………...………..13
十五、生長曲線(Growth curve)的測定…………………..………….14
十六、統計分析方法…………………………………………………….14
結果:
一、B群鏈球菌生物膜的表現與莢膜血清型型別之分佈…………….15
二、B群鏈球菌生物膜的表現與莢膜血清型第三型菌種
來源之分佈…………………………………………………….…….15
三、B群鏈球菌生物膜的表現與環境因子的關係…………….......…..16
(1) 溫度試驗………………………………………………...……....16
(2) 二氧化碳濃度試驗…………………………………………...…16
(3) 碳源(carbon source)試驗…………………………………….17
(4) 酸鹼值酸鹼值(pH value)試驗………………………………..18
(5) 離子(ions)…………………………………………………….19
四、B群鏈球菌在生物膜表現時間總蛋白差異分析...………………..19
討論……………………………………………………...……………….….21
參考文獻……………………………………………………...………….….25
表……………………………………………………...……………...…..….32
圖……………………………………………………...………………….….34
附錄……………………………………………………...……………….….53
自述……………………………………………………...……………….….58
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