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研究生：	鄭雅蓮 Cheng, Ya-Lien
論文名稱：	探討淡水與棘阿米巴是否能成為產毒性困難梭狀芽孢桿菌的潛在宿主 Fresh water source and Acanthamoeba castellanii as a potential reservoir for toxigenic Clostridium difficile
指導教授：	黃一修 Huang, I-Hsiu
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	58
中文關鍵詞：	困難梭狀芽孢桿菌、棘阿米巴、吞噬作用
外文關鍵詞：	Clostridium difficile, Acanthamoeba castellanii, phagocytosis
相關次數：	點閱：46 下載：0
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棘阿米巴（A. castellanii）是一種發現在土壤和水等環境中的變形蟲，可以通過吞噬作用將細菌當作食物，但是其中一種叫做變形蟲抗性的細菌（ARB）可以抵抗吞噬作用。困難梭狀芽孢桿菌(C. difficile）是引起抗生素相關性腹瀉的主要原因，是革蘭氏陽性，會形成孢子的厭氧細菌。在困難梭狀芽孢桿菌感染（CDI）的過程中，困難梭狀芽孢桿菌經過孢子形成並將孢子釋放到結腸環境中。在我們研究的前半部，我們從動物養殖場和醫院附近所收集的河水樣本中分離和鑑定出產毒性的困難梭狀芽孢桿菌，進而確定困難梭狀芽孢桿菌能夠在水源當中存活。重要的是，這些河流所分離出的困難梭狀芽孢桿菌的核醣體與從台灣動物和人類分離物中發現的核醣體是相同的。而在我們研究的後半部，我們假設棘阿米巴和困難梭狀芽孢桿菌可以共同存活在一個環境中，那麼困難梭狀芽孢桿菌可能會將棘阿米巴當作一個傳播媒介。在這項研究中，我們試著表現困難梭狀芽孢桿菌孢子和棘阿米巴之間的交互作用。我們確定困難梭狀芽孢桿菌孢子可以通過棘阿米巴的吞噬作用而內化。為了進一步研究棘阿米巴體內的環境對內化後細菌存活力的影響，而進行了存活試驗。存活試驗結果顯示困難梭狀芽孢桿菌孢子可以被吞噬並且存活在棘阿米巴內，有趣的是在吞噬12小時後發現孢子從棘阿米巴中釋放出來。然而與困難梭狀芽孢桿菌的共同培養卻不會影響棘阿米巴滋養體的存活力。簡而言之，我們的初步結果表明，淡水和棘阿米巴是困難梭狀芽孢桿菌傳播的潛在來源，這也可能對公眾健康產生重大的影響。

Acanthamoeba castellanii (A. castellanii) is a genus of amoeba found in environments such as soil and water, and is capable of feeding on bacteria by phagocytosis, but several bacteria, called Amoebae-resisting bacteria (ARB) can resist killing. Clostridium difficile (C. difficile), the leading cause of antibiotic associated diarrhea, is a gram-positive, spore-forming anaerobic bacterium. During the course of C. difficile infections (CDI), C. difficile undergo sporulation and releases spores into the colonic environment. In the first half of the project, we were able to isolate and identify toxigenic C. difficile from river water samples collected near animal farms and hospitals, thus suggesting that C. difficile is also capable of surviving in water source. Importantly, many of these river-borne C. difficile isolates was found to belong to the same ribotype as one found in animal and human isolates here in Taiwan. For the second half of the project, we hypothesized that since A. castellanii and C. difficile can both co-exist in the same environment, C. difficile might use A. castellanii as a transmission vehicle. In this study, we attempted to characterize the interactions between C. difficile spores and A. castellanii. We determined that C. difficile spores were internalized by phagocytosis. To further investigate the impact of intracellular environment on the viability of bacteria, a survival assay was performed. The survival assay showed that C. difficile spores was readily phagocytosed and survived inside A. castellanii. Interestingly, 12 hours after phagocytosis, spores were found to be released out of A. castellanii. On the other hand, co-incubation with C. difficile did not affect the viability of A. castellanii. In short, our preliminary results indicate that fresh water and A. castellanii is a potential source for C. difficile contamination which could have a great impact on public health.

CONTENTS
中文摘要 ................ I
ABSTRACT ............... II
致謝 ............... III
ABBREVIATIONS ............. IX
Chapter 1 INTRODUCTION ........... 1
1.1 Clostridium difficile infection (CDI) and epidemiology ...... 1
1.2 The Pathogenicity Loci (PaLoc) of C. difficile ........ 2
1.3 Diagnosis and treatment of CDI ........... 2
1.4 The life cycle of Acanthamoeba castellanii (A. castellanii) ...... 3
1.5 Amoebae-resisting bacteria (ARB) .......... 4
1.6 The stages of A. castellanii ............ 4
1.7 Rationale .............. 5
Chapter 2 MATERIALS AND METHODS .......... 6
2.1 Isolation of environment samples........... 6
2.2 Bacteria strains, and growth conditions ......... 6
2. 3 Genomic deoxyribonucleic acid (DNA) extraction of bacteria ..... 7
2.4 Polymerase chain reaction (PCR) primer and program ........ 7
2.5 Agarose gel electrophoresis ........... 8
2.6 Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) ..... 8
2.7 Antimicrobial susceptibility assay.......... 9
2.8 Detection of Toxin A and B by ELISA .......... 9
2.9 Ribonucleic acid (RNA) extraction of bacteria ........ 9
2.10 Quantitative real time transcription-polymerase chain reaction (qRT-PCR) ... 11
2.11 Biofilm formation assay ........... 11
2.12 Motility assay ............. 12
2.13 C. difficile spore preparation ............ 12
2.14 Amoebal culture ............. 13
2.15 Amoebal infections ............ 13
2.16 Immunofluorescence assay (IFA) .......... 13
2.17 Transmission Electron Microscopy (TEM) ........ 14
2.18 C. difficile survival assay ........... 14
2.19 Amoeba survival assay ............. 15
2.20 Spore release assay ............. 15
2.21 Time course of amoeba encystation assay ......... 16
2.22 Adhesion assay to lenses ............ 16
2.23 Stress resistance assay ............ 17
2.24 Statistical analyses ............ 17
Chapter 3 RESULTS............ 18
3.1 Isolation and characterization of C. difficile from water sources .... 18
3.1.1 Isolation of toxigenic C. difficile from environmental water sources in Taiwan .. 18
3.1.2 The toxin gene content and ribotype of toxigenic C. difficile isolates .... 18
3.1.3 Minimum spanning tree analysis of C. difficile RT 126 isolates by MLVA ... 19
3.1.4 Gyrase gene mutation affect susceptibility to Fluoroquinolones in C. difficile isolates .. 19
3.1.5 C. difficile isolates can produce toxin in the environmental ..... 20
3.1.6 Characterization of virulence phenotypes ......... 21
3.2 The interaction of C. difficile with A. castellanii ...... 21
3.2.1 Engulfment by A. castellanii affect the viability of C. difficile vegetative cells but not
spores ................ 21
3.2.2 C. difficile spores are internalized by A. castellanii through phagocytosis ... 22
3.2.3 Survival of engulfed spores and spore-containing A. castellanii trophozoites .. 22
3.2.4 Spore engulfment does not affect virulence factors of A. castellanii .... 23
3.2.5 Engulfed spores are gain a survival advantage by A. castellanii .... 23
Chapter 4 DISCUSSION ............. 25
REFERENCES .............. 29
TABLES .............. 34
Figures ............... 39
Supplementary material ............. 56
Table list
Table 1. Strains used in this study ........... 34
Table 2. Sequences of oligonucleotides primers used in this study ..... 35
Table 3. The toxin gene content and ribotype of toxigenic C. difficile isolates .. 37
Table 4. Antimicrobial susceptibility and gyrase mutations of toxigenic C. difficile isolates
................. 38
Figure list
Figure 1. Isolation of toxigenic C. difficile from environmental water sources in Taiwan. 39
Figure 2. A schematic diagram of pathogenicity locus (PaLoc) in toxigenic C. difficile strains.
................. 40
Figure 3. Minimum spanning tree analysis of reference RT 126 and C. difficile RT 126
isolates by MLVA. ............. 42
Figure 4. Toxin production by environmental C. difficile isolates. ..... 43
Figure 5. Transcriptional analysis of tcdA and tcdB in the environmental C. difficile isolates.
................. 44
Figure 6. C. difficile isolates have high biofilm formation ability as strain R20291... 45
Figure 7. The motility ability of C. difficile isolates. ........ 46
Figure 8. C. difficile spores but not vegetative cells are able to survive co-incubation with A.
castellanii. ............... 47
Figure 9. C. difficile spores are internalized by A. castellanii. ...... 48
Figure 10. C. difficile spores are internalized by A. castellanii ..... 49
Figure 11. Engulfment by A. castellanii affect the viability of C. difficile spore at 12 hours.
................. 50
Figure 12. Co-culture with C. difficile spore did not affect the viability of A. castellanii
trophozoites. .............. 51
Figure 13. C. difficile spores were released from A. castellanii at 12 hours. .... 52
Figure 14. Phagocytosis of C. difficile spores does not affect A. castellanii encystation. .. 53
Figure 15. Phagocytosis of C. difficile spores does not affect the contact lens adhesion ability
of A. castellanii. ............. 54
Figure 16. Engulfed spores are resistance to hydrogen peroxide but sensitive to sodium
hypochlorite and antibiotic. ........... 55
                                    

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