臺灣博碩士論文加值系統

嗜水性產氣單胞桿菌（Aeromonas hydrophila）是一種革蘭氏陰性的伺機性病原菌，廣泛分佈在各種水生環境中。此病原菌主要與淡水魚和兩棲動物中的疾病相關，對人類可能造成急性胃腸炎，蜂窩組織炎，腹膜炎和敗血症，患者多為免疫力低下的人。膽鹽是膽固醇的代謝產物，並且是膽汁的主要成分之一，膽鹽的主要作用是協助脂質乳化，增強其消化吸收。除此，膽鹽是有效的抗菌藥物，是腸內先天防禦的重要組成，可以保護免受微生物的侵害。因此，研究嗜水性產氣單胞桿菌和膽鹽之間的相互作用是理解其在宿主腸中定殖(colonization)能力的重要因素。生物膜是細菌生成的胞外聚合物(extracellular polymeric substances, EPS)，具有保護細菌免於被抗菌劑殺死之作用。本研究之目的在探討嗜水性產氣單胞桿菌受到膽鹽處理時，抗藥機制之調控是否受到改變。我們利用結晶紫染色細胞外基質證實膽鹽明顯增加了細菌生物膜的形成，以定量反轉錄聚合酶連鎖反應(RT-qPCR)分析生物膜相關基因的轉錄活性，顯示膽鹽使嗜水性產氣單胞桿菌生物膜相關基因表現量增加。膽鹽同樣促使嗜水性產氣單胞桿菌的運動性降低，而細菌運動性的減少可能與抑制了運動相關因子flgE與motY的轉錄有關。我們也發現膽鹽加強了嗜水性產氣單胞桿菌藥物RND幫浦基因的表現以及藥物排出的能力。綜合以上實驗結果，我們證實了膽鹽可以提高嗜水性產氣單胞桿菌的生物膜合成以及藥物幫浦基因的表現，這可能加強此一病菌適應消
化道中的壓力環境以及建立菌叢的能力。

Aeromonas hydrophila is a Gram-negative opportunistic pathogen that is widely distributed in a variety of aquatic environments. This pathogen is usually associated with diseases in freshwater fish and amphibians; however it may also cause acute gastroenteritis, cellulitis, peritonitis and sepsis in immunocompromised people. Bile salts are metabolites of cholesterol and are one of the primary components of bile. The main function of bile acids is to aid in the digestion and absorption of lipids and lipid-soluble nutrients in the intestine. In addition, bile salts are potent antimicrobial agents and are an important component of innate defenses in the intestine, giving protection against invasive organisms. Commensal or pathogenic microorganisms must resist the deleterious actions of bile in order to survive in the human digestive tract. Thus investigating the interaction between A. hydrophila and bile salts is an important factor in understanding its ability to colonize in the host intestine. Biofilms are aggregated microbial cells embedded in a matrix of extracellular polymeric substances (EPS) that protect bacteria from killing by
antimicrobials, including bile salts. The purpose of this study was to investigate whether the drug resistance mechanisms of A. hydrophila were altered upon bile salts exposure. Crystal violet staining of bacterial EPS showed that bile salts significantly increased the formation of biofilms. We used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to analyze the transcriptional activity of biofilm-related genes, and the results
showed that bile salts increased the expression of these genes. Bile salts also contribute to reduced the motility of A. hydrophila that may relate with the reduced transcription of flgE and motY. In addition, we also found that the transcription of RND efflux pump genes and the drug extrusion were increased by bile salts. Based on the above experimental results, we
confirmed that bile salts can improve the biofilm formation and efflux activity of A. hydrophila, which may enhance the ability of this pathogen to adapt to
the stressful condition and colonized in the digestive tract.

誌謝 -------------------------------------------------------------------------- i
中文摘要 -------------------------------------------------------------------------- ii
英文摘要 -------------------------------------------------------------------------- iv
目錄 -------------------------------------------------------------------------- vi
表目錄 -------------------------------------------------------------------------- viii
圖目錄 -------------------------------------------------------------------------- ix
符號說明 -------------------------------------------------------------------------- x
第一章 文獻探討-------------------------------------------------------------- 1
第一節 嗜水性產氣單胞桿菌介紹----------------------------------------- 1
一 嗜水性產氣單胞桿菌(Aeromonas hydrophila)----------------- 1
二 感染性疾病----------------------------------------------------------- 1
三 預防及治療----------------------------------------------------------- 1
四 致病毒力因子-------------------------------------------------------- 2
第二節 細菌與膽鹽之間的交互作用-------------------------------------- 3
一 膽鹽的生成與功能-------------------------------------------------- 3
二 細菌與膽鹽之關係-------------------------------------------------- 3
1 細菌的傷害----------------------------------------------------------- 3
1.1 蛋白質變性----------------------------------------------------------- 4
1.2 DNA損傷------------------------------------------------------------- 4
1.3 細胞膜損傷----------------------------------------------------------- 4
1.4 氧化壓力-------------------------------------------------------------- 5
2 細菌對抗膽鹽的機制----------------------------------------------- 5
2.1 代謝-------------------------------------------------------------------- 5
2.2 生物膜----------------------------------------------------------------- 6
2.3 幫浦系統-------------------------------------------------------------- 6
2.4 孔洞蛋白-------------------------------------------------------------- 7
3 細菌毒力因子之影響----------------------------------------------- 8
三 膽鹽對細菌基因調控機制之影響(在致病性)----------------- 9
第三節 研究動機與目的----------------------------------------------------- 11
第二章 材料與方法----------------------------------------------------------- 12
第一節 實驗菌株來源及保存方式----------------------------------------- 12
一 實驗菌株-------------------------------------------------------------- 12
二 細菌培養-------------------------------------------------------------- 12
三 細菌保存-------------------------------------------------------------- 12
第二節 核酸萃取方法及核酸引子----------------------------------------- 12
一 小量純化質體DNA ------------------------------------------------ 12
二 核酸引子-------------------------------------------------------------- 13
三 聚合酶連鎖反應(Polymerase chain reaction)------------------- 13
四 膠體電泳分析-------------------------------------------------------- 14
五 細菌之RNA萃取(傳統法及套組) ------------------------------ 14
(1) 傳統法----------------------------------------------------------------- 14
(2) 套組-------------------------------------------------------------------- 15
六 RNA反轉錄聚合酶連鎖反應（reverse transcription-PCR） 16
七 即時聚合酶連鎖反應（Real-time PCR）----------------------- 16
第三節 菌株特性分析-------------------------------------------------------- 17
一 細菌生長抑制試驗-------------------------------------------------- 17
二 細菌存活率試驗 ---------------------------------------------------- 17
三 細菌對膽鹽的感受性試驗 ---------------------------------------- 17
四 生物膜定量試驗 ---------------------------------------------------- 18
五 運動性分析 ---------------------------------------------------------- 18
六 蛋白酶水解活性 ---------------------------------------------------- 18
七 溶血試驗 ------------------------------------------------------------- 18
八 外排幫浦活性(efflux activity)試驗------------------------------- 19
第四節 生物資訊分析-------------------------------------------------------- 19
第三章 研究結果-------------------------------------------------------------- 21
第一節 菌株特性分析-------------------------------------------------------- 21
一 藥物感受性試驗----------------------------------------------------- 21
二 生長能力試驗-------------------------------------------------------- 21
第二節 生物膜、運動性及RND轉運蛋白同源基因之分析--------- 21
一 生物資訊分析-------------------------------------------------------- 22
二 基因反轉錄聚合酶連鎖反應（reverse transcription-PCR）之分析----------------------------------------------------------------- 22
第三節 生物膜合成之能力-------------------------------------------------- 22
第四節 運動性試驗----------------------------------------------------------- 24
第五節 RND外排幫浦------------------------------------------------------- 24
一 RND外排幫浦基因之表現---------------------------------------- 25
二 外排幫浦活性(efflux activity)試驗------------------------------- 25
第六節 蛋白酶試驗----------------------------------------------------------- 25
第七節 溶血試驗-------------------------------------------------------------- 26
第四章 討論-------------------------------------------------------------------- 27
參考文獻 -------------------------------------------------------------------------- 31

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