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系統識別號 U0026-2908201609010400
論文名稱(中文) 以次氯酸與SC5005化合物抑制困難梭狀芽孢桿菌孢子的發芽
論文名稱(英文) Inhibiting of Clostridium difficile spore germination by hypochlorous acid and SC5005
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 104
學期 2
出版年 105
研究生(中文) 謝旻容
研究生(英文) Min-Jung Hsieh
學號 t36034076
學位類別 碩士
語文別 英文
論文頁數 41頁
口試委員 指導教授-蔡佩珍
口試委員-鄧景浩
口試委員-陳振暐
口試委員-張松彬
中文關鍵字 C. difficile  孢子發芽  次氯酸 
英文關鍵字 C. difficile  spore germination  Hypochlorous acid 
學科別分類
中文摘要 困難梭狀芽孢桿菌(Clostridium difficile) 是一種會產生孢子的專性厭氧革蘭氏陽性菌,困難梭狀芽孢桿菌感染是由於孢子型態的困難梭狀芽孢桿菌引起造成的,最常造成院內感染腹瀉。在預防疾病傳染而言,接觸困難梭狀芽孢桿菌的孢子以及孢子的在腸道內的發芽,對於困難梭狀芽孢桿菌致病途徑有著獨特的意義。困難梭狀芽孢桿菌孢子對於醫院最常用的消毒劑酒精具有抗性,然而次氯酸(hypochlorous acid;HOCl)成份的消毒劑已經被報導對於多種細菌具有殺菌效果,其中也包含困難梭狀芽孢桿菌,然而並未深入探討。除此之外,以抗癌分子索拉非尼衍生的小化合物SC5005已經被報導可以有限抑制methicillin-resistant S. aureus (MRSA),初期也測試出可以抑制困難梭狀芽孢桿菌。在本研究中,我們進一步探討與次氯酸消毒劑或小分子SC5005抑制困難梭狀芽孢桿菌孢子發芽的作用。在次氯酸水部分,我們發現次氯酸具有劑量效應的影響困難梭狀芽孢桿菌孢子發芽與滋養細胞(vegetative cell)的生長,並藉由破壞困難梭狀芽孢桿菌孢子的微結構抑制孢子發芽。由於次氯酸的活性在有機物質存在下失去活性,我們應用次氯酸由於在不同材質表面抑制困難梭狀芽孢桿菌孢子生長,支持了次氯酸可以作為抗困難梭狀芽孢桿菌孢子消毒劑在健康照護上。在SC5005部分,我們發現SC5005能夠藉由競爭型抑制(competitive inhibition)抑制以牛磺膽為媒介困難梭狀芽孢桿菌孢子的發芽,並也發現SC5005在小鼠模式的動物實驗上可以阻止CDI疾病的病程。這些結果提供SC5005具有作為預防困難梭狀芽孢桿菌感染的潛力。
英文摘要 Clostridium difficile is a Gram-positive obligate anaerobic spore-forming bacterium. C. difficile infection (CDI) which initiated by infection with the spore is the most frequent nosocomial infectious diarrhea. Spores exposure and spore germination in the gut are the most important steps of CDI. The C. difficile spores are resistant to alcohol. Another disinfectant, hypochlorous acid (HOCl), has been shown bactericidal activity with several species, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and C. difficile. In addition, a sorabenib-based small compound, SC5005, has been shown to suppress methicillin-resistant S. aureus (MRSA) and C.difficile. Here, we further characterized the effects of HOCl disinfectant and the small compound SC5005 on C.difficile spores. At first, we found that HOCl has the potential to inhibit C. difficile spore germination and the bacterial growth in a dose-dependent manner. We showed that HOCl inhibits C. difficile spore germination by damaging ultrastructure of spore. Nevertheless, this inhibition of spore germination was blocked by organics. We demonstrated that HOCl can suppress C. difficile spores on difference surfaces, suggesting it has the potential to be an ideal anti-C. difficile sporicidal agent for health care. Second, we found SC5005 also has the ability to inhibit taurocholate-mediated germination but in a competitive inhibition mechanism. We demonstrated that SC5005 prevents CDI disease progression on CDI-mice animal model. These results suggest that SC5005 has the potential to be an alternative strategy to prevent C. difficile infection.
論文目次 中文摘要 II
ABSTRACT III
致謝 IV
CONTENTS V
TABLE INDEX VII
FIGURE INDEX VIII
INTRODUCTION 1
1.1 Clostridium difficile 1
1.2 Pathogenesis of C. difficile infection 1
1.3 Nosocomial infection of C. difficile 2
1.4 Ultrastructure of spore 2
1.5 Mechanism of C. difficile spore germination 3
1.6 Hypochlorous acid 3
1.7 Sorabenib-based small compound SC5005 3
METHODS 5
2.1 Bacterial strains and spore preparation. 5
2.2 Sporcidal activity 5
2.3 C. difficile spore germination 5
2.4 Spore germination was monitored by phase-contrast microscopy 6
2.5 Spore integrity 6
2.6 Ultrastructure of spore 6
2.7 DPA release assay 6
2.8 Artificial germination 7
2.9 The sporicidal activity on different material surfaces 7
2.10 Hemolysis assay 7
2.11 C. difficile infection animal model 8
2.12 Statistical analysis 8
RESULTS 9
3.1 Hypochlorous acid inactivates C. difficile spores. 9
3.2 The effects of hypochlorous acid on spore germination. 9
3.3 Inhibition kinetics of hypochlorous acid on C. difficile spore germination. 10
3.4 Hypochlorous acid causes spore released dipicolinic acid fast during germination. 10
3.5 Hypochlorous acid damages spore integrity. 10
3.6 Spore cortex, coat and exosporium are damaged by hypochlorous acid. 11
3.7 Hypochlorous acid inhibits C. difficile spore germination is taurocholate-independent. 11
3.8 The sporicidal activity of hypochlorous acid on different material surfaces. 12
3.9 SC5005 inhibits C. difficile spores growth. 12
3.10 SC5005 inhibits spore germination. 12
3.11 SC5005 is not able to disrupt red blood cells. 13
3.12 Inhibition kinetics of SC5005 on C. difficile spore germination. 13
3.13 SC5005 does not damage the spore integrity 13
3.14 SC5005 inhibits TA-dependent spore germination. 14
3.15 The effect of compound SC5005 on C. difficile infection in vivo 14
DISCUSSION 15
REFERENCE 20
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