臺灣博碩士論文加值系統

大腸桿菌 (Escherichia coli)為人體腸道正常菌叢之一，即使大多數大腸桿菌是無害的，但當其獲得毒力因子時仍可造成嚴重的感染包括食物中毒及菌血症等等，由於過度的使用抗生素於大腸桿菌感染症的治療，目前抗藥性問題在大腸桿菌中快速增加，值得注意的是大腸桿菌對最後一線用藥”碳青黴烯”的抗藥性也日趨嚴重，可怕的是此種抗藥性基因能夠藉由質體在細菌間廣泛的傳播，造成治療困難與感染後的高死亡率。故找尋新的藥物治療碳青黴烯抗藥性大腸桿菌是目前迫切需要的項目之一，然而新藥的開發需要投資大量時間及金錢，使得抗生素新藥進度緩慢，因此老藥新用的方式成為一個重要的替代方式。在本研究中選用了具有免疫調節功能之無毒性合成有機化合物Compound X做為候選藥物測試對於多重抗藥性產碳青黴烯酶大腸桿菌之抗菌活性並進一步探討其抗菌機轉。
本研究蒐集2012至2015間全台各地區6家醫院所分離之多重抗藥性產碳青黴烯酶大腸桿菌 (CPEC)臨床菌株，抗菌活性以肉湯微量稀釋法測定Compound X之最小抑菌濃度，整體MIC範圍為0.5 至 32 μg/ml，此數值遠低於其CC50數值 (150 μg/ml)及最高血漿濃度，並以脈衝場凝膠電泳及多位點序列分析臨床菌株親緣性呈現相當高之異質性，顯示在本研究中CPEC菌株並無單一clone擴散之現象。
Compound X之抗菌機制首先使用Time-kill assays測定，在1、2及4倍藥物MIC下觀察到99.9%殺菌能力，故可判斷其抗菌機制為一種殺菌劑。接著採用post-DCF fluorescence及化學製劑偵測此化合物的抗菌機轉，並以DNA片段化試驗及掃描式電子顯微鏡觀察藥物對細菌之影響。發現投予1、2及4倍MIC藥物後ROS生成皆有大幅上升，同時有著DNA降解及細胞壁損傷。
體內試驗則選用秀麗隱桿線蟲及小鼠敗血症模式來測試Compound X對於CPEC菌株治療效果，與安慰劑組相比，經過Compound X治療後大幅提升了線蟲及小鼠隻存活率，同時亦降低小鼠器官載菌量。
總結上述實驗結果表明Compound X是一個極具潛力之新興抗微生物製劑，提供對抗多重抗藥性CPEC菌株之不同治療選項，值得更進一步之研究。

Escherichia coli is an important organism in the human lower intestine that usually harmless, but some pathogen E. coli can cause serious infections including food poisoning and bacteremia. Owing to the overuse of antibiotics, the number of antibiotic-resistant E. coli has rapidly increased, especially for carbapenemase-producing E. coli (CPEC). Furthermore, plasmid and transposable elements carrying multiple antibiotic resistance genes could be transferred horizontally, leading treatment options to become limited and increased mortality. In this situation, developments of new antibiotic are urgently needed whereas this process requires a long lead period and a large investment of resources. Due to the high cost of antibiotic development, drug repurposing was considered as an alternative approach. For this project, we re-evaluated the organic compound X, which was reported as an immunomodulation agent, as a novel antimicrobial agent.
Among a total of 14 multidrug resistant CPEC clinical isolates was collected from 6 hospitals in Taiwan during 2012 to 2015, the broth microdilution method was used to determine the minimum inhibitory concentration of compound X. Genotype analysis was conducted by PFGE (Pulsed Field Gel Electrophoresis) and MLST (Multilocus sequence typing). The bactericidal activity of compound X against the CRE-415 isolate was tested using time-kill assays. The bactericidal mechanism of compound X was detected by the post-DCF fluorescence and the other chemical agents. Scanning electron microscope was used to observe compound X-treated bacterial cells morphology. Caenorhabditis elegans and mice septicemia model was employed as the in vivo model to investigate the treatment effect of compound X against CPEC.
The MIC range of the CPEC isolates were from 0.5 to 32 μg/ml, well below its 50% cytotoxicity (nearly 150 μg/ml) and maximum human plasma concentration. Following genotype analysis showed no outbreak phenomenon. 99.9% of the bacterial reduction was observed at 1x, 2x and 4x MIC of compound X. Three possible bactericidal mechanisms were found. ROS levels were significantly increased in the group of 1x, 2x and 4x MIC in comparison with that of the control group. DNA degradation and cell damage were found in compound X-treated bacterial cells. Compared to the placebo, compound X demonstrated the robust treatment effects with increased median nematode survival time and bacteria-infected mice survival. Furthermore, compound X significantly rescued the lethal-infected mice via the significant eradication of bacterial organ loads.
In conclusion, our works indicated that compound X is a potentially novel antimicrobial agent worthy of further research.

第一章 緒論 10
1. 大腸桿菌 (Escherichia coli) 簡介 10
2. 抗藥性的發展 11
3. 碳青黴烯 (Carbapenem)簡介及其抗藥性問題 14
4. Compound X簡介 19
第二章 材料與方法 21
第一節 研究用菌株 21
第二節 臨床菌株親緣性分析 22
第三節 毒力基因偵測 26
第四節 Compound X抗菌活性偵測 30
第五節 Compound X抗菌機制試驗 31
第六節 Compound X抗藥性偵測 40
第七節 Compound X 體內投藥試驗 41
第三章 實驗結果 48
1. 台灣14株產碳青黴烯酶大腸桿菌 (carbapenemase-producing Escherichia coli；CPEC)抗生素感受性試驗分析結果 48
2. 台灣14株產碳青黴烯酶大腸桿菌 (carbapenemase-producing Escherichia coli；CPEC)毒力因子偵測 49
3. 台灣14株產碳青黴烯酶大腸桿菌 (carbapenemase-producing Escherichia coli；CPEC)親源性分析 50
4. 評估Compound X對於台灣14株產碳青黴烯酶大腸桿菌(carbapenemase-producing Escherichia coli；CPEC)之抗菌效果 50
5. Compound X對於台灣產碳青黴烯酶大腸桿菌(carbapenemase-producing Escherichia coli；CPEC)之抗菌機制探討 51
6. Compound X抗菌機轉探討 (mechanism of action) 52
7. Compound X投藥後活性氧化物質 (Reactive oxygen species, ROS) 生成試驗 53
8. DNA片段化試驗 53
9. 掃瞄式電子顯微鏡 (Scanning Electron Microscopy；SEM)菌相觀察 54
10. Compound X抗藥性頻率 55
11. Compound X對於秀麗隱桿線蟲感染產碳青黴烯酶大腸桿菌的治療效果 (in vivo) 55
13. Compound X對於感染產碳青黴烯酶大腸桿菌引起小鼠敗血症治療效果 (in vivo) 56
14. Compound X對於產碳青黴烯酶大腸桿菌感染小鼠器官載菌量測試 (organ load) 57
第四章 討論 58
第五章 實驗結果附表 71
【表一】毒力因子偵測所使用核酸引子 71
【表二】台灣14株產碳青黴烯酶大腸桿菌所攜帶碳青黴烯酶基因及乙內醯胺酶基因 72
【表三】Compound X對台灣14株產碳青黴烯酶大腸桿菌之最小抑菌濃度及毒力因子偵測結果 72
【表四】Compound X抗菌機轉探討。 73
【表五】小鼠器官載菌量。 73
第六章 實驗結果附圖 74
【圖一】台灣14株產碳青黴烯酶大腸桿菌抗生素感受性試驗分析結果 74
【圖二】台灣14株產碳青黴烯酶大腸桿菌親源性分析 75
【圖三(a)】Compound X抗菌機制偵測之抑菌劑對照組 76
【圖三(b)】Compound X抗菌機制偵測之殺菌劑對照組 76
【圖三(c)】Compound X抗菌機制偵測結果。 77
【圖四】 Compound X抗菌機轉偵測 78
【圖五】 DNA片段化試驗 79
【圖六(a)】掃描式電子顯微鏡菌相觀察之對照組 80
【圖六(b)】掃描式電子顯微鏡菌相觀察之對照組 80
【圖六(c)】掃描式電子顯微鏡菌相觀察之投予Compound X組 81
【圖六(d)】掃描式電子顯微鏡菌相觀察之投予Compound X組 81
【圖七】 Compound X抗藥性頻率偵測 82
【圖八】 秀麗隱桿線蟲體內試驗 83
【圖九】 小鼠敗血症模式體內試驗 84
【圖十(a)】 小鼠器官載菌量之肝臟組 84
【圖十(b)】 小鼠器官載菌量之腎臟組 85
【圖十(c)】 小鼠器官載菌量之脾臟組 85
第七章 參考文獻 86

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