臺灣博碩士論文加值系統

仔豬在離乳初期因消化系統尚未發展健全、離乳造成的生理緊迫及衛生條件不良而易感染病原菌，常見的病原菌為腸毒素型大腸桿菌 (enterotoxigenic Escherichia coli, ETEC)，其會引發仔豬下痢導致脫水，嚴重時甚至會死亡。然而在過去數十年間，由於抗生素的濫用進而加速多重抗藥性 (multidrug-resistant, MDR) 病原菌產生，因此找尋抗生素的替代藥物是重要的議題。許多研究皆指出抗菌胜肽 (antimicrobial peptides, AMPs) 能夠在短時間內殺死細菌且具有高選擇性毒殺能力，因此 AMPs 具有成為抗生素替代物的潛力。先前研究顯示本實驗室設計之AMP GW-Q4 及其截短與優化衍生物對革蘭氏陰性菌具有良好的抗菌效果，而在本篇研究中我們測試這些AMPs對MDR ETEC之抗菌活性，由最小抑菌濃度 (minimum inhibition concentrations, MIC) 測定結果顯示其皆具有良好的抗菌效果，MIC介於2~32 μg/mL之間，而其中又以AMP Q4-15a-1對MDR ETEC具有次佳抗菌活性 (MIC = 4 μg/mL) 及最低溶血活性 (hemolytic concentration, MHC= 256 μg/mL)，使其對MDR ETEC具有最佳的選擇性 (MHC/MIC = 64)，因此我們選用Q4-15a-1進行後續試驗。在與抗生素及天然AMPs組合試驗中，Q4-15a-1與Cephalothin、Pleurocidin、Pleurocidin-a、Magainin及Magainin-a組合使用皆具有加成 (additive) 效果，可有效降低抗生素與AMPs使用劑量。而在時間殺菌曲線 (time-kill curve) 實驗中，隨著 Q4-15a-1濃度提高，其抑制MDR ETEC的效力也隨之提升，因此Q4-15a-1對MDR ETEC的殺菌效果具有劑量依賴性。而在生物膜清除濃度 (minimum biofilm eradication concentration, MBEC) 試驗中，Q4-15a-1僅以4x MIC (16 μg/mL) 即能對MDR ETEC展現出良好的抗生物膜效力。而從兩項以螢光試驗分析Q4-15a-1處理MDR ETEC後在細菌的座落位置與對細胞膜完整性的實驗結果中可得知，Q4-15a-1能在短時間內與MDR ETEC作用，先出現於表面接著進入細菌，並在過程中造成其細胞膜受損。此外，我們也建立MDR ETEC 感染 IPEC-1細胞的感染模式，透過此感染模型發現Q4-15a-1能以32 μg/mL完全抑制MDR ETEC 感染IPEC-1細胞。綜上所述，這些實驗結果顯示 Q4-15a-1 具潛力作為治療受MDR ETEC感染之離乳仔豬的新穎抗菌製劑。

Post-weaning diarrhea due to enterotoxigenic Escherichia coli (ETEC) is a common disease of piglets and causes economic loss for swine industry. However, over the past decades, decreasing effectiveness of conventional antibiotics has caused serious problems because of the rapid emergence of multidrug-resistant (MDR) pathogens. Various researches indicated that antimicrobial peptides (AMPs) have potential to serve as an alternative to antibiotics owing to rapid killing action and high selective toxicity. Moreover, our previous studies have shown that AMP GW-Q4 and its derivatives possess effective antibacterial activities against the Gram-negative bacteria. Hence, in this research, we evaluate the antibacterial efficacy of GW-Q4 and its derivatives against MDR ETEC and their minimal inhibition concentration (MIC) values were determined to be around 2~32 μg/mL. Among them, AMP Q4-15a-1 with the second lowest MIC (4 μg/mL) and the highest hemolytic concentration (MHC= 256 μg/mL), thus showing the greatest selectivity (MHC/MIC = 64) was selected for further investigations. The combinations of Q4-15a-1 with cephalothin, Pleurocidin, Pleurocidin-a, Magainin and Magainin-a showed additive effect against MDR ETEC and improved antibacterial activity of conventional antibiotics and AMPs. Moreover, Q4-15a-1 showed dose-dependent activity against MDR ETEC in time-kill curve assays. According to the cellular localization and membrane integrity analyses using fluorescence assay, Q4-15a-1 can rapidly interact with the bacterial surface, disrupt the membrane and enter cytosol in only a few minutes. Minimum biofilm eradication concentration (MBEC) of Q4-15a-1 is 4x MIC (16 μg/mL), thus Q4-15a-1 is effective against MDR ETEC biofilm. Besides, we established an MDR ETEC infection model with IPEC-1 cell. In this infection model, 32 μg/mL Q4-15a-1 can completely inhibit ETEC adhesion on IPEC-1. Overall, these results suggested that Q4-15a-1 may be a promising antibacterial candidate for treatment of weaned piglets infected by MDR ETEC.

摘要 I
Abstract II
致謝 III
目錄 (Table of Contents) IV
圖目錄 (List of Figures) VI
表目錄 (List of Tables) VII
名詞縮寫表 (Abbreviations) VIII
第一章 前言 1
第一節 腸毒素型大腸桿菌 (Enterotoxigenic Escherichia coli) 1
第二節 抗生素 (Antibiotics) 4
第三節 多重抗藥性 (Multidrug-resistance, MDR) 6
第四節 抗菌胜肽 (Antimicrobial peptides, AMPs) 7
第二章 研究目的 11
第三章 實驗材料與儀器 12
第一節 生物材料與藥品試劑 12
第二節 儀器設備 14
第四章 實驗流程與方法 15
第一節 實驗之菌株與保存與培養 16
第二節 菌落數與濁度之關係 - 平板計數法 (Plate count) 16
第三節 最小抑菌濃度 (Minimal inhibitory concentration, MIC) 16
第四節 組合試驗 (Combination test) 17
第五節 時間殺菌曲線 (Time-kill assay) 18
第六節 最小生物膜清除濃度 (Minimum biofilm eradication concentration) 18
第七節 細胞培養與凍存 (Cell culture and freezing) 19
第八節 細胞活性分析 (Cell viability assay) 20
第九節 貼附感染試驗 (Infection assay of bacterial adhesion) 21
第十節 螢光試驗 (Fluorescence assay) 22
第五章 結果 23
第一節 腸毒素型大腸桿菌 23
一、 菌株來源、抗性資料與生長情形 23
二、 平板計數法 - 菌落數與濁度之間的關係 23
第二節 抗菌胜肽之抗菌活性 23
一、 AMPs對菌株之抑菌效力 23
二、 AMP Q4-15a-1對MDR腸毒素型大腸桿菌之時間殺菌曲線 24
三、 AMP Q4-15a-1與抗生素組合試驗 24
四、 AMP Q4-15a-1與天然AMPs組合試驗 25
五、 AMP Q4-15a-1 對MDR腸毒素型大腸桿菌之抗生物膜效力 25
第四節 抗菌胜肽對MDR腸毒素型大腸桿菌細胞膜影響 25
一、 AMP Q4-15a-1 於MDR腸毒素型大腸桿菌中定位 25
二、 AMP Q4-15a-1 對MDR腸毒素型大腸桿菌細胞膜之影響 26
第五節 抗菌胜肽應用於貼附型感染模式之效力 26
一、 IPEC-1與IPEC-J2細胞活性分析 26
二、 AMP Q4-15a-1 抑制MDR腸毒素型大腸桿菌感染試驗 26
第六章 討論 27
第一節 AMPs對MDR 腸毒素型大腸桿菌之抗菌能力 27
第二節 AMP Q4-15a-1之快速殺菌分析 27
第三節 AMP Q4-15a-1與抗生素及天然AMPs之組合抗菌效力 27
第四節 AMP Q4-15a-1抗生物膜效力 28
第五節 AMP Q4-15a-1抑制MDR ETEC感染效力 28
第七章 結論與未來展望 29
第八章 圖表 30
第九章 參考文獻 42

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