評估compound X對colistin和carbapenem抗藥性克雷伯氏肺炎桿菌的殺菌效果

Bactericidal activity of compound X against colistin & carbapenem resistant Klebsiella pneumoniae isolates

陳沛諭(Pei-Yu Chen)

指導教授 ： 曾嵩斌

高雄醫學大學/健康科學院/醫學檢驗生物技術學系碩士班/碩士(2017年)

摘要

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。

關鍵字

克雷伯氏肺炎桿菌 ； compound X ； 克痢黴素 ； 碳青黴烯

並列摘要

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.

並列關鍵字

Klebsiella pneumoniae ； compound X ； colistin ； carbapenem

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