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研究生:謝沄蓁
研究生(外文):XIE, YUN-ZHEN
論文名稱:依布硒增強克痢黴素抗藥性克雷伯氏肺炎桿菌 對克痢黴素的敏感性並呈現協同效果
論文名稱(外文):Ebselen Enhances the Sensitization of Colistin Against Colistin-Resistant Klebsiella pneumoniae and Demonstrates Their Synergistic Effect
指導教授:曾嵩斌
指導教授(外文):TSENG, SUNG-PIN
口試委員:洪薇鈞楊宗穎
口試委員(外文):HUNG, WEI-CHUNYANG, TSUNG-YING
口試日期:2024-07-17
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學檢驗生物技術學系碩士班
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:2024
畢業學年度:112
語文別:中文
論文頁數:109
中文關鍵詞:克痢黴素
外文關鍵詞:colistin
相關次數:
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  • 點閱點閱:4
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中文摘要 1
Abstract 4
Graphical abstract 7
第一章 緒論 11
(一) 克雷伯氏肺炎桿菌 (Klebsiella pneumoniae)及其抗藥性 11
(二) 克痢黴素 (Colistin)之重要性及其抗藥性 14
(三) 具潛力的抗菌佐劑──依布硒 (Ebselen) 18
(四) 研究目的 19
第二章 材料方法 21
(一) 本篇研究使用菌株 21
(二) 菌株保存 21
(三) 抗菌活性偵測 22
1. 肉湯稀釋法 (Broth microdilution method) 22
2. 棋盤分析法 (Checkerboard assays) 24
(四) 抗菌機轉試驗 25
1. 時間-殺菌曲線 (Time-kill curves)測定 25
2. 溶菌酶通透性試驗 27
3. 外源性添加試驗 28
4. 掃描式電子顯微鏡 (Scanning Electron Microscope, SEM) 29
5. 即時定量聚合酶連鎖反應 (Real-time PCR, qPCR) 31
6. 活性氧生成試驗 36
7. 硫氧化還原蛋白還原酶活性試驗 (TrxR activity assays) 37
8. 還原型穀胱甘肽檢測試驗 (Reduced glutathione assay) 39
(五) 抗藥性偵測 41
1. 自發性抗藥頻率測定 (Spontaneous mutation frequency) 41
2. 連續傳代誘導抗藥性突變體 (Serial passage resistance induction) 42
(六) 線蟲體內試驗 43
1. 線蟲蟲種維持 43
2. 實驗用蟲製備 44
3. 藥物保護性試驗 46
第三章 實驗結果 47
(一) 克痢黴素與依布硒合藥之抗菌效果 47
(二) 克痢黴素與依布硒合藥之時間殺菌曲線 48
(三) 克痢黴素與依布硒合併使用下之作用標靶 49
1. 溶菌酶通透性試驗 49
2. 外源性添加試驗 50
3. 掃描式電子顯微鏡 51
(四) 依布硒重新致敏化克痢黴素之機轉 52
(五) 克痢黴素與依布硒合併使用下活性氧生成之機轉 53
1. 活性氧生成試驗 53
2. 硫氧化還原蛋白還原酶活性試驗 54
3. 還原型穀胱甘肽檢測試驗 55
(六) 克痢黴素與依布硒合併使用下抗藥性突變之偵測 56
1. 合藥作用之自發性抗藥突變率 56
2. 合藥存在下誘導抗藥性突變菌株出現 57
(七) 克痢黴素與依布硒合藥之體內抗菌效果 58
1. 合藥對於秀麗隱桿線蟲之保護性試驗 58
第四章 討論 59
第五章 實驗結果附表 80
【表一】Real-time PCR所使用之核酸引子 80
【表二】克痢黴素與依布硒對於41株克痢黴素抗藥性克雷伯氏肺炎桿菌的合藥抗菌效果 81
【表三】外源性添加脂多醣對於克痢黴素與依布硒合藥之克痢黴素MIC變化 83
【表四】外源性添加鈣離子對於克痢黴素與依布硒與合藥之克痢黴素MIC變化 83
第六章 實驗結果附圖 84
【圖一】克痢黴素與依布硒對於克痢黴素抗藥性克雷伯氏肺炎桿菌的時間殺菌曲線 (Time-kill curve)。 84
【圖二】克痢黴素與依布硒對於克痢黴素抗藥性克雷伯氏肺炎桿菌之溶菌酶通透性試驗。 85
【圖三】掃描式電子顯微鏡菌觀察菌體外觀變化之對照組 86
【圖四】掃描式電子顯微鏡菌觀察菌體外觀變化之克痢黴素單藥組 87
【圖五】掃描式電子顯微鏡菌觀察菌體外觀變化之依布硒單藥組 88
【圖六】掃描式電子顯微鏡菌觀察菌體外觀變化之克痢黴素與依布硒合藥組 89
【圖七】依布硒對於克痢黴素相關抗藥性基因的表現量變化 90
【圖八】克痢黴素與依布硒合藥共同作用之菌株體內活性氧生成量 91
【圖九】克痢黴素與依布硒合藥共同作用之菌株體內硫氧化還原蛋白還原酶活性 92
【圖十】克痢黴素與依布硒合藥共同作用之菌株體內還原型穀胱甘肽含量 92
【圖十一】克痢黴素與依布硒對於克痢黴素敏感性克雷伯氏肺炎桿菌之突變頻率偵測 93
【圖十二】克痢黴素與依布硒對於克痢黴素敏感性克雷伯氏肺炎桿菌之連續傳代下誘導抗藥性突變體 94
【圖十三】克痢黴素與依布硒保護秀麗隱桿線蟲免於克痢黴素抗藥性克雷伯氏肺炎桿菌感染之線蟲存活曲線 95
第七章 參考文獻 96
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