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研究生:陳沛諭
研究生(外文):Pei-Yu Chen
論文名稱:評估compound X對colistin和carbapenem抗藥性克雷伯氏肺炎桿菌的殺菌效果
論文名稱(外文):Bactericidal activity of compound X against colistin & carbapenem resistant Klebsiella pneumoniae isolates
指導教授:曾嵩斌
指導教授(外文):Sung-Pin Tseng
口試委員:洪薇鈞鄧麗珍
口試委員(外文):Wei-Chun HungLee-Jene Teng
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:2017
畢業學年度:105
語文別:中文
論文頁數:73
中文關鍵詞:克雷伯氏肺炎桿菌compound X克痢黴素碳青黴烯
外文關鍵詞:Klebsiella pneumoniaecompound Xcolistincarbapenem
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克雷伯氏肺炎桿菌 (Klebsiella pneumoniae)是一種伺機性感染的病原菌,對於免疫力低下者可以造成許多感染包括菌血症、肺炎、肝膿瘍、尿路感染等等。 Carbapenem屬於β-lactam類抗生素,用來治療腸內菌感染,隨著藥物使用量上升目前carbapenem抗藥性的克雷伯氏肺炎桿菌在全球皆有很高的流行率。目前colistin是治療這些抗藥菌株的最後一線用藥,然而近幾年也開始發現對colistin產生抗藥性的克雷伯氏肺炎桿菌。在可用藥物越來越少的情況下,開發新型且有用的抗菌劑治療多重抗藥性細菌感染的問題已是目前迫切所需。本研究所採用的compound X在先前的研究已指出具有抗腫瘤、抗菌、抗發炎等效果。為了評估compound X對此難以治療的抗藥性克雷伯氏肺炎桿菌殺菌效果,採用全台灣醫院所收集到對colistin和carbapenem呈現抗藥性的克雷伯氏肺炎桿菌臨床菌株。以克雷伯氏肺炎菌標準菌株 BAA1705做為對照組,測試compound X對細菌的最低抑菌濃度 (minimum inhibitory concentration)、Time-kill菌落計數推測此藥物的抗菌機轉、microarray推測藥物可能殺菌機制,最後以秀麗隱桿線蟲 (in vivo assay)評估compound X對於線蟲感染此菌的保護效果。實驗結果發現感受性濃度最高為32 μg/ml,顯示compound X對於此菌的抗菌效果穩定而卓越。Time-kill菌落計數測試中可得知compound X的抗菌機制屬於直接殺死細菌 (bactericidal agent)而非抑菌 (bacteriostatic agent)。Microarray實驗結果推測殺菌可能機制為compound X與細菌作用後產生活性氧化物質 (Reactive oxygen species)進而殺死細菌。線蟲實驗則發現32 μg/ml的compound X濃度對於感染克雷伯氏肺炎桿菌的線蟲具有保護效果,甚至使用至64 μg/ml線蟲存活率也沒有下降,進一步表示在此濃度下compound X對線蟲不具毒性反應。於本研究結果顯示compound X為一極具潛力對抗多重抗藥性克雷伯氏肺炎桿菌的藥物,值得未來再深入研究。
Klebsiella pneumoniae is an important pathogen that causes various infections including bacteremia, pneumonia, liver abscesses, and urinary tract infections. The prevalence of carbapenem-resistant K. pneumoniae (CRKP) has been increasing globally, making antimicrobial treatment difficult and causing higher disease-related mortality rates. Colistin is one of the few remaining last-resort antibiotics that can be used to treat CRKP infection. However, recent reports indicate increasing colistin resistance among clinical isolates of K. pneumoniae. Antimicrobial resistance is a global public health concern contributing to increased morbidity and mortality particularly. Thus, development of new antibacterial agents against K. pneumoniae, especially strains resistant to multiple antibiotics, has become an urgent public health need. The non-toxic immunomodulatory compound X is a low molecular weight (312 Da) synthetic organo-tellurium compound. The antibacterial effect of the compound X on the multiple-resistant K. pneumoniae is shown in this study. A total of 185 clinical strains of K. pneumoniae were studied in this research. Antimicrobial susceptibility testing as determined by broth dilution method. The MIC range of carbapenem-resistant and colistin-resistant strains were 1 to 32 μg/ml. Compound X at concentration of 2× and 4× MIC reduce the bacterial population by >99.9%. Based on these findings, compound X can be classified as bactericidal. Then we employed a transcriptomic, microarray based strategy to investigate how the antimicrobial characteristics and gene expression of the strain. Our results suggest that expression of three TCA cycle genes were increased, including sdhCDAB, mqo and lpdA, and regulated NADH and FADH2 which caused ROS increase to lead bacterial death. To investigate whether compound X can suppress infection by K. pneumoniae in C. elegans. The survival of worms receiving compound X at 2× MIC was even significantly increased. The results indicate that compound X was not acutely toxic to C. elegans and possesses antibacterial activity that is capable of suppressing K. pneumoniae infection in host animals. In light of its high potency in suppressing K. pneumoniae in both in vitro and in vivo models, compound X represents a potential lead agent for continued preclinical development as a therapeutic intervention against K. pneumoniae.
第一章 第一章 緒論 ................................ ................................ ............. 7
一、 克雷伯氏肺炎桿菌( 克雷伯氏肺炎桿菌( 克雷伯氏肺炎桿菌( Klebsiella pneumoniae)簡介 7
二、 抗生素藥性發展 抗生素藥性發展 抗生素藥性發展 ................................ .................. 7
三、 碳青黴烯( 碳青黴烯( carbapenem) ................................ ....... 9
四、 克痢黴素( 克痢黴素( colistin) ................................ ............ 11
五、 Compound X ................................ .......................... 14
第二章 第二章 材料與方法 材料與方法 ................................ ................................ . 16
一、 實驗菌株 實驗菌株 ................................ ................................ 16
二、 菌株保存 菌株保存 ................................ ................................ 16
三、 最小抑菌濃度檢測之肉湯微量稀釋法 最小抑菌濃度檢測之肉湯微量稀釋法 最小抑菌濃度檢測之肉湯微量稀釋法 最小抑菌濃度檢測之肉湯微量稀釋法 最小抑菌濃度檢測之肉湯微量稀釋法 ................. 17
四、 Time-kill curves之瓊脂稀釋法 之瓊脂稀釋法 之瓊脂稀釋法 ............................. 18
五、 萃取核醣酸( 萃取核醣酸( 萃取核醣酸( RNA)-kit法 ............................. 19
1. 基因表現分析( 基因表現分析( 基因表現分析( Microarray) .............................. 21
六、 掃描式電子顯微鏡 掃描式電子顯微鏡 掃描式電子顯微鏡 ................................ ................ 21
七、 穿透式電子顯微鏡 穿透式電子顯微鏡 穿透式電子顯微鏡 ................................ ................ 22
八、 秀麗隱桿線蟲之體內試驗 秀麗隱桿線蟲之體內試驗 秀麗隱桿線蟲之體內試驗 秀麗隱桿線蟲之體內試驗 ................................ .... 23
1. 相關試劑配置 相關試劑配置 相關試劑配置 ................................ ........................ 23
2. 線蟲培養與維持 線蟲培養與維持 線蟲培養與維持 ................................ .................... 25
6
3. 同步線蟲生長階段 同步線蟲生長階段 同步線蟲生長階段 ................................ ................ 26
4. 藥物毒性測試之培養皿分析法( 藥物毒性測試之培養皿分析法( 藥物毒性測試之培養皿分析法( 藥物毒性測試之培養皿分析法( 藥物毒性測試之培養皿分析法( Plate toxicity assay) ................................ ................................ .......... 27
第三章 第三章 實驗結果 實驗結果 ................................ ................................ .... 29
一、 收集 carbapenem與 colistin抗藥的克雷伯氏肺炎 抗藥的克雷伯氏肺炎 抗藥的克雷伯氏肺炎 抗藥的克雷伯氏肺炎 菌之臨床株,檢測抗生素活性 菌之臨床株,檢測抗生素活性 菌之臨床株,檢測抗生素活性 菌之臨床株,檢測抗生素活性 菌之臨床株,檢測抗生素活性 ................................ ...... 29
二、 評估 compound X對 carbapenem與 colistin抗藥的 抗藥的 克雷伯氏肺炎菌之臨床株的殺效果 克雷伯氏肺炎菌之臨床株的殺效果 克雷伯氏肺炎菌之臨床株的殺效果 克雷伯氏肺炎菌之臨床株的殺效果 克雷伯氏肺炎菌之臨床株的殺效果 .......................... 30
三、 Compound X抗菌機制探討 抗菌機制探討 抗菌機制探討 ................................ .. 31
四、 RNA微陣列( microarray)分析推測 )分析推測 )分析推測 compound X的殺菌路徑 的殺菌路徑 ................................ ................................ .......... 32
五、 掃描式電子顯微鏡( 掃描式電子顯微鏡( 掃描式電子顯微鏡( SEM)菌相觀察 )菌相觀察 ................ 34
六、 穿透式電子顯微鏡( 穿透式電子顯微鏡( 穿透式電子顯微鏡( TEM)菌相觀察 )菌相觀察 )菌相觀察 ................ 34
七、 秀麗隱桿 秀麗隱桿 線蟲( C. elegans)培養基測試 )培養基測試 ............ 35
八、 Compound X對於 秀麗隱桿 秀麗隱桿 線蟲 (in vivo)感染克雷 感染克雷 伯氏肺炎桿菌的保護效果 伯氏肺炎桿菌的保護效果 伯氏肺炎桿菌的保護效果 伯氏肺炎桿菌的保護效果 ................................ .................. 36
第四章 第四章 討論 ................................ ................................ ............ 38
第五章 第五章 附圖 ................................ ................................ .............. 46
第六章 第六章 參考文獻 參考文獻 ................................ ................................ ...... 67
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