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系統識別號 U0026-2408201815105900
論文名稱(中文) 困難梭狀桿菌RT017與其他毒性菌株之表現型比較
論文名稱(英文) Phenotypic comparison of Clostridium dificile RT017 strains with other toxigenic strains
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 106
學期 2
出版年 107
研究生(中文) 郭明渝
研究生(英文) Ming-Yu Guo
學號 T36051028
學位類別 碩士
語文別 英文
論文頁數 58頁
口試委員 指導教授-蔡佩珍
口試委員-柯文謙
口試委員-洪元斌
中文關鍵字 困難梭狀桿菌  核醣型017  自誘導物-2  秀麗隱桿線蟲 
英文關鍵字 Clostridium. difficile  Ribotype 017  Autoinducer-2  Caenorhabditis. elegans 
學科別分類
中文摘要 困難梭狀桿菌為全世界新興的院內感染之重要病原,引起的臨床症狀特別是在長期使用廣效性抗生素的患者引發嚴重的腹瀉和偽膜性結腸炎。因此困難梭狀桿菌的預防及控制成為目前最重要的公共衛生議題。過去已知在歐美地區核醣型027為主要流行且具高毒性的菌株。然而,在其他世界地區卻觀察到有不同種的核醣型菌株的流行趨勢,在亞洲分佈的產毒株中核醣型017是主要的分型,比例約10%。許多研究發現核醣型017所引起困難梭狀桿菌感染的嚴重程度跟核醣型027很相似,然而,與核醣型027相比,核醣型017只具有tcdB一種毒素基因。在2015年至2016年的疾病管制署監測計畫中,我們實驗室從台灣五家醫院收集了1,112株困難梭狀桿菌,其中842株(75.8%)為產毒菌株,270株(24.2%)為非產毒菌株,其中核醣型017在產毒菌株中所佔的比例大約是9.97%。然而,目前尚不清楚RT017菌株為何會引起嚴重的疾病症狀,本研究,我們利用小鼠活體試驗,評估了RT017菌株的毒力,發現RT017菌株比CD630(RT012)菌株導致更嚴重的疾病症狀,與英國流行菌株R20291(RT027)相仿。通過體外的實驗發現,RT017之tcdB表達和其毒素葡糖基轉移酶活性均不如R20291高。與R20291相比,RT017菌株生物膜的形成與孢子形成率與R20291相似,而RT017的細菌聚集能力更強。此外,已知困難梭狀桿菌可產生自誘導物-2(AI-2),我們以哈氏弧菌MM32測量AI-2誘導生物發光的結果評估AI-2的表現量,發現RT017菌液中的AI-2表現量與R20291相似。將之誘導低毒力且低AI-2表現量之菌株(CD630),可誘導孢子形成能力增加。最後,以秀麗隱桿線蟲為模型生物,檢測菌液的毒性,發現RT017的菌液感染線蟲可降低其存活率,而加熱處理後的菌液則無法回復線蟲的存活率。綜合以上,我們發現RT017具高致病毒力、孢子形成能力及高表達量的AI-2,顯示可能與集中程度有關。
英文摘要 Clostridium. difficile causes nosocomial infection worldwide, especially in the patients with long-term usage of broad spectrum antibiotics. Clinical symptoms of C. difficile infections may present as diarrhea and pseudomembranous colitis (PMN). Therefore, controlling of C. difficile infection (CDI) is the most important public-health issues. In the United States and Europe, RT027 was known to be the epidemic and hypertoxigenic clone. However, the different epidemiologic trends in ribotype strain were observed in other continents. In Asia, RT017 is the predominant typeable ribotype in toxigenic strains, the prevalence rate is around 10% in Asia. Many studies demonstrated that the severity of CDI caused by RT 017 is similar to that caused by RT027. Compared to RT027, RT017 encodes only one toxin gene, tcdB. In a CDC surveillance program, 1,112 C. difficile isolates were collected from five hospitals from 2015 to 2016 in Taiwan. In this study, 842 (75.8%) strains were toxin-producing strains and 270 (24.2%) were non-toxigenic strains, where the proportion of RT017 in toxigenic strains was approximately 9.97%. However, it is unclear whether the RT017 strains cause severe diseases. Here, we evaluated the virulence of RT017 strain in vivo and found RT017 strain induced more severe disease symptoms than CD630 (RT012) did, but as well as a clinical UK epidemic C. difficile strain R20291 (RT027) did. By in vitro testing, both tcdB expression and toxin glucosyltransferase activity were not as high as R20291. Compared to R20291, biofilm formation in RT017 strains was similar to that in R20291, the aggregation ability in RT017 was stronger, spore formation rate of RT017 was similar to R20291. In addition, C. difficile produces small molecule, autoinducer-2 (AI-2), which can influence gene expression such as virulence, luminescence, and biofilm formation across different species. The levels of AI-2 that induces bioluminescence in Vibrio harveyi MM32 were measured. The AI-2 levels in RT017 spent medium is similar to that in R20291. Using spent medium of a less virulent strain (CD630), the spore formation was increased. Finally, used C. elegans as a model to examine the toxicity of spent medium, the survival rate was decreased when treating worms with spent medium of RT017 which cannot be reversed by heating the spent medium. Taken together, RT017 has higher virulence in vivo, ability in formation of spore, and higher AI-2 production, which might contribute to the disease progression.
論文目次 中文摘要 I
ABSTRACT Ⅱ
致謝 Ⅲ
TABLE OF CONTENTS Ⅳ
TABLE INDEX Ⅶ
FIGURE INDEX Ⅷ
ABBREVIATION Ⅸ

CHAPTER 1 INTRODUCTION 1
1.1 Clostridium. difficile infection1
1.2 Virulence factors of CDI 2
1.3 Clinical diagnosis and treatment for Clostridium difficile infection 5
1.4 The epidemiology of C. difficile infection worldwide 6
1.5 PCR ribotype 017 (RT017) strain 7
1.6 The epidemiology of CDI in East Asia 9
1.7 The rationale in this study 10

CHAPTER 2 MATERIALS AND METHODS 11
2.1 Bacteria Strains 11
2.1.1 Clinical C. difficile isolates 11
2.1.2 Reference strains of C. difficile 11
2.2 CDI animal model 11
2.2.1 NF-κB-RE-luc transgenic (FVB) mice 11
2.2.2 In vivo imaging 12
2.3 TcdB mRNA level 12
2.3.1 Purified RNA 12
2.3.2 Reverse transcriptase PCR 14
2.3.3 RT-qPCR analysis 14
2.4 Toxin B activity 15
2.4.1 Cell line 15
2.4.2 Sample preparation and examine 15
2.4.3 Glucosyltransferase activity by western blot 15
2.5 Model animal toxicity test in C. elegans 17
2.5.1 Sample preparation 17
2.5.2 C. elegans preparation 17
2.5.3 Toxicity of C. elegans 18
2.6 Biofilm formation analysis 19
2.7 Aggregation ability 19
2.8 Spore formation 20
2.9 Autoinducer-2 (AI-2) expression 20
2.9.1 Sample collecting 20
2.9.2 Quorum sensing 21
2.10 Autoinducer-2 biological function 21
2.10.1 Autoinducer 2 (AI-2) effect on biofilm formation of low toxigenic CD630 strain 21

CHAPTER 3 RESULT
3.1 The epidemiology of C. difficile RT017 in Taiwan 23
3.2 RT017 strain induces NF-κB inflammatory responses as high as hypertoxigenic R20291 strain does 24
3.3 Toxin B levels in RT017 is not as high as that in hypertoxigenic R20291 strain 24
3.4 Biofilm-forming ability of RT017 is similar to hypertoxigenic R20291 strain and aggregation ability of RT017 is higher than hypertoxigenic R20291 strain 25
3.5 Spore formation rate of RT017 was as high as the hypertoxigenic R20291 strain 25
3.6 The AI-2 level in spent medium of RT017 is as high as that of other well-known hypertoxigenic strains 26
3.7 The AI-2 higher producer has the potential in induce more biofilm formation and spore formation but not tcdB expression 26
3.8 Spent medium of RT017 has the ability to inhibit Caenorhabditis elegans survival 27
CHAPTER 4 DISCUSSION 28
REFERANCE 32
APPENDIX 54
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