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研究生:王傳儒
研究生(外文):Chuan-Ru Wang
論文名稱:高鹽度影響綠膿桿菌生物膜形成與pel操縱子基因表現之研究
論文名稱(外文):Studies on High Salinity Affecting Biofilm Formation and Expression of pel Operon Genes in Pseudomonas aeruginosa
指導教授:黃小萍黃小萍引用關係
指導教授(外文):Huang Shiao-ping
口試委員:王紹鴻尤慧玲
口試委員(外文):Shao-Hung WangHuey-Ling You
口試日期:2014-06-23
學位類別:碩士
校院名稱:輔英科技大學
系所名稱:醫學檢驗生物技術系碩士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:89
中文關鍵詞:綠膿桿菌生物膜薄膜pel
外文關鍵詞:Pseudomonas aeruginosaBiofilmPelliclepel
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綠膿桿菌為革蘭氏陰性的伺機性病原菌,在潮濕的環境可以發現。多重抗生素抗性與生物膜的形成使綠膿桿菌感染在治療上倍受重視。綠膿桿菌形成的生物膜有三個主要的細胞外多醣體參與,包括Pel、Psl與Alginate。其中Pel在氣體-液體介面形成薄膜上扮演重要的角色,並且提供結構支架幫助早期生物膜的發展。由於綠膿桿菌於高鹽環境下會抑制生物膜的生合成,並且對其生物膜基質細胞外多醣體的影響仍是不清楚。因此本研究利用各種鹽度的培養液去探討鹽度滲透壓對細菌生長特徵、生物膜形成、薄膜的形成、薄膜中細菌群的差異與其相關細胞外多醣體Pel基因表現的影響。結果發現:一、綠膿桿菌無論在不添加鹽或添加鹽環境的培養下,細菌生長良好,但細菌生長的對數期會隨著鹽度的提高,而有延遲的現象。二、在生物膜的形成因broth中鹽度提高而有顯著的下降,分別下降17% (300 mM NaCl)、40% (500 mM NaCl)、54% (700 mM NaCl)。三、氣體-液體介面的薄膜形成厚度因培養液中鹽度提高而有顯著的下降,48小時分別下降18% (300 mM NaCl)、50% (500 mM NaCl)、56% (700 mM NaCl)。四、進一步對綠膿桿菌胞外多醣體相關基因pel的表現進行分析,結果顯示在添加700 mM NaCl的高鹽環境下pelA、pelB、pelD、pelF與pelG的表現顯著被抑制。在高鹽環境下可導致綠膿桿菌生理特性的改變,包括pel操縱子的表現與生物膜的形成。
Pseudomonas aeruginosa are Gram-negative opportunistic pathogens and can be found in the environments including soil, freshwater, and seawater. Multiple antibiotic resistance and biofilm formation are major problems in the treatment of Pseudomonas aeruginosa infection. Pseudomonas aeruginosa produces extracellular polysaccharides for biofilm development including alginate, Pel, and Psl. Pel play an important role in pellicle formation at the air-liquid interface and providing a structural scaffold for biofilm formation at early stages. The genes associated with Pel formation are pel operon including pelA-G. Since the high salt environment would inhibit the biofilm formation of Pseudomonas aeruginosa and the effect on extracellular polysaccharide in biofilm matrix is unclear, this study investigated the effects of salinity on bacterial growth, biofilm formation, pellicle formation, and mRNA expression of relevant genes in Pseudomonas aeruginosa. The obtained results were analyzed. First, the Pseudomonas aeruginosa grew well in medium either with or without supplemented NaCl. However, the growth curve showed a log-phase-delay in medium with supplemented NaCl. Second, the results of the biofilm formation indicated that the decreases of biofilm were proportional to the concentrations of supplemented NaCl including 17% in 300 mM NaCl, 40% in 500 mM NaCl, and 54% in 700 mM NaCl, respectively. Third, the results of the pellicle formation showed that the thickness in pellicle were decreased by the supplemented with NaCl including 18% in 300mM NaCl, 50% in 500mM NaCl, and 56% in 700mM NaCl, respectively. Fourth, the mRNA expression of pel operon was measured and the results showed that the low mRNA expression of pel operon in medium with supplemented NaCl. In conclusion, this study revealed that the supplemented NaCl in medium may cause the physiological changes in Pseudomonas aeruginosa including gene expression of pel operon, and biofilm formation.
目錄
致謝 i
中文摘要 iii
Abstract v
目錄 vii
表目錄 x
圖目錄 xi
名詞縮寫說明 xii
第一章 緒論 1
第一節 綠膿桿菌 1
一、 綠膿桿菌生長特性 1
二、 綠膿桿菌PAO1基因體 2
三、 環境分佈 2
四、 臨床感染 3
第二節 生物膜 (Biofilm) 4
一、 生物膜概述 4
二、 生物膜形成的發展 5
三、 綠膿桿菌生物膜的細菌抗藥性 7
四、 群體感應系統(Quorum Sensing, QS) 9
五、 細胞外聚合物質(Extracellular Polymeric Substances, EPS) 11
第四節 高滲透壓對細菌形成生物膜與胞外多醣體的影響 18
第五節 研究動機與目的 19
第六節 研究架構圖 20
第二章 材料與方法 21
第一節 實驗材料與設備 21
一、 菌株 21
二、 培養基與藥品試劑 21
第二節 實驗方法 24
一、 細菌培養與保存 24
二、 生長曲線分析 24
三、 綠膿桿菌生物膜形成測定 25
四、 薄膜多醣體生成與螢光染色 25
五、 引子設計及合成 26
六、 RNA的萃取與反轉錄的作用 27
七、 即時定量聚合酶連鎖反應分析 28
八、 統計分析 29
第三章 研究結果 30
第一節 分析綠膿桿菌在高鹽環境下的生長情形 30
第二節分析綠膿桿菌在高鹽環境下生物膜的形成 32
第三節 分析綠膿桿菌在高鹽環境氣體-液體介面薄膜的形成 33
第四節 分析胞外多醣體相關基因pel在不同鹽度環境的mRNA表現 35
一、 pelA基因於高鹽環境下的變化 35
二、 pelB基因於高鹽環境下的變化 35
三、 pelD基因於高鹽環境下的變化 37
四、 pelF基因於高鹽環境下的變化 38
五、 pelG基因於高鹽環境下的變化 39
第四章 討論 41
一、 高鹽對綠膿桿菌生長的影響 42
二、 高鹽對於生物膜形成的影響 42
三、 高鹽對於細菌薄膜形成的影響 43
四、 高鹽對於基因表現的影響 44
參考文獻 47
附錄 65
附錄一、綠膿桿菌野生株PAO1的基因體 65
附錄二、生物膜進展的過程 66
附錄三、Pel蛋白質在細胞膜上的位置與其蛋白質功能 67
附錄四、原始的生長曲線 68
附錄五、AlgC於高鹽環境下的表現 69
表目錄
表一、綠膿桿菌中調控EPS基因生合成相關基因序列資訊 55
表二、綠膿桿菌Pel多醣體合成相關基因的引子序列 56
表三、綠膿桿菌在700mM NaCl鹽度環境下胞外多醣體pel基因表現的變化 67
圖目錄
圖一、測定綠膿桿菌在各種鹽度環境下的生長曲線。 57
圖二、測定綠膿桿菌在各種鹽度環境下生物膜的形成。 58
圖三、觀察綠膿桿菌在各種鹽度環境下氣體-液體介面間薄膜的形成。 59
圖四、綠膿桿菌在各種鹽度環境下,薄膜形成的厚度。 60
圖五、為了解氣體-液體介面間薄膜中細菌聚集的現象。 61
圖六、綠膿桿菌在LB broth(170 mM NaCl)與700 mM NaCl LB broth環境下胞
外多醣體pelA基因表現的變化。 62


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