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研究生：	李雅茹 Li, Ya-Ru
論文名稱：	從台南的污水與海鮮嘗試分離困難梭狀芽孢桿菌並分型 Isolation and characterization of Clostridioides difficile from wastewater and seafoods in Tainan
指導教授：	陳振暐 Chen, Jenn-Wei
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：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	62
中文關鍵詞：	困難梭狀芽孢桿菌、污水處理廠、廢水、海鮮、核醣體分型
外文關鍵詞：	C. difficile, wastewater treatment plant, wastewater, seafood, ribotyping
相關次數：	點閱：145 下載：0
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困難梭狀芽孢桿菌是一種革蘭氏陽性、會產孢子的厭氧桿菌，是世界上廣為人知，引起抗生素相關性腹瀉的主要原因。困難梭狀芽孢桿菌的主要毒力因子是毒素A和毒素B，以及二元毒素 CDT。此外，困難梭狀芽孢桿菌的孢子對環境壓力具有很強的抵抗力，並會通過糞口、食物來源和潛在的人畜共通途徑傳播給人類。過去的研究表明，困難梭狀芽孢桿菌可以從各種環境樣本中分離出來，這些樣本可能是人類感染的潛在宿主。污水處理廠和醫院排放的廢水被認為是多種腸道病原體所造成水污染的重要來源。因此，廢水中的環境病原體可能會污染在河口附近養殖的海鮮。在這項研究中，我們從台南海岸附近的多個污水處理廠、醫院和海鮮的多個水樣中分離出了困難梭狀芽孢桿菌。本研究共鑑定出 97 株困難梭狀芽孢桿菌分離株。我們進行了基因型分析，包括毒素基因的多重聚合酶鏈鎖反應和核醣體分型。我們還進行了表現型分析，包括抗生素敏感性測定、運動性測定和生物膜形成能力的測定。有趣的是，所有分離株中有 62% (60/97) 屬於已知對氟喹諾酮類抗生素具有抗性的高毒力核醣型 027 或 078 相關核醣型。57% (55/97) 來自醫院的分離株屬於高毒力 RT127 核醣型。表型分析表明，與其他表現毒素和未表現毒素分離株相比，屬於 RT078 相關核醣型的分離株表現出顯著較低的游泳運動。然而，就生物膜形成能力而言，所有分離株之間沒有觀察到顯著差異。簡而言之，本研究在台灣首次從各種水源和海產品中分離到表現毒素的困難梭狀芽孢桿菌，表明即使經過廢水處理，困難梭狀芽孢桿菌仍然可以持續存在。

Clostridiodes difficile (C. difficile) is a gram-positive, spore-forming anaerobic bacillus and is the major cause of antibiotic-associated diarrhea (AAD) worldwide. The major virulence factors of C. difficile are TcdA (toxin A) and TcdB (toxin B), and the binary toxin CD. In addition, C. difficile spores are highly resistant to environmental stress and is responsible for transmission to human via fecal-oral, food source, and potentially zoonotic routes. Past studies have indicated that C. difficile can be isolated from various environmental samples which might serve as potential reservoirs for human infection. Wastewater released by wastewater treatment plant (WWTP) and hospitals are considered as an important source of water contamination by multiple intestinal pathogens. Therefore, it is possible that hardy pathogens within wastewater might contaminate seafood that are raised near estuaries. In this study, we isolated C. difficile from multiple water samples from multiple WWTPs, hospital, and seafood near the Tainan coast. A total of 97 C. difficile isolates were identified in this study. We performed genotypic analysis, which included multiplex PCR for toxin genes, and ribotyping. We also performed phenotypic analysis which included antibiotic susceptibility assays, motility assay, and biofilm formation assay. Interestingly, 62% (60/97) of all total isolates belonged to either the hypervirulent ribotype 027 or 078 lineage which are known to be resistant to fluoroquinolone antibiotics. 57% (55/97) of isolates from the hospital belonged to the hypervirulent RT127 ribotype. Phenotypic analysis suggested that isolates belonging to RT078 lineage exhibited significant lower swimming motility when compared to other toxigenic and non-toxigenic isolates. No significant differences however were observed among all isolates in terms of biofilm formation ability. In summary, for the first time ever in Taiwan, toxigenic C. difficile were isolated from various water source as well as from seafood, indicating that C. difficile can persist even after wastewater treatment. We hope the result of this research could let the audiences know that the sources of C. difficile spores, and pay attention to remove them from these sources.

中文摘要 I
ABSTRACT II
致謝 IV
CONTECTS VI
ABBREVIATIONS IX
Chapter 1 1
INTRODUCTION 1
1.1 C. difficile epidemiology 1
1.2 The mechanism of C. difficile infection (CDI) 1
1.3 Hypervirulent ribotype 027 and 078 2
1.4 Background on the sources of spores 2
1.5 Virulence factors of C. difficile 2
1.5.1 Factors involved in resistance to host innate defenses 2
1.5.2 Flagella 3
1.5.3 Biofilm formation 3
1.5.4 Toxins 4
1.6 Rationale 5
Chapter 2 6
MATERIALS AND METHODS 6
2.1 Collection of wastewater samples from sewage treatment plants 6
2.2 Water Sampling method 6
2.3 Isolation of isolates from seafood samples 7
2.4 Bacteria strains and growth conditions 7
2.5 Genomic deoxyribonucleic acid (DNA) extraction of bacteria 7
2.6 Polymerase chain reaction (PCR) primer and program 8
2.7 Agarose gel electrophoresis 9
2.8 PCR product purification 9
2.9 Antimicrobial susceptibility assay 9
2.10 Biofilm formation assay 10
2.11 Swimming Motility Assay 10
2.12 Cell culture 11
2.13 Cytotoxicity assay	11
2.14 Statistical analysis 12
Chapter 3 13
RESULTS 13
3.1 Toxin gene and ribotype characterization of C. difficile isolates from wastewater and seafood	13
3.2 Sequencing of gyrA and gyrB genes for fluoroquinolones-non-susceptible isolates 13
3.3 Virulent effect of biofilm formation by C. difficile isolates 14
3.4 Motility measurement of C. difficile isolates with different ribotypes 15
3.5 Phase variation of C. difficile isolates with different ribotypes 15
3.6 The typing of flagella operon in RT126s by PCR 16
3.7 Cytotoxicity of C. difficile isolates with different genotypes 16
Chapter 4 18
DISCUSSION 18
REFERENCES 23
TABLES 33
Table 1. Control strains used in this study 33
Table 2. Sequences of oligonucleotide primers used in this study 34
Table 3. Characterization of C. difficile isolates by multiplex PCR 36
Table 4. Antimicrobial susceptibility of C. difficile isolates by E-test strips 37
Table 5. Phase variation screening of C. difficile isolates 39
Table 6. Cytotoxicity of environmental isolates tested by Vero cell rounding 40
FIGURES 41
Figure 1. Isolation of C. difficile from environmental sources in Tainan 41
Figure 2. Schematic diagram of sewage disposal steps in wastewater plants 42
Figure 3. Schematic diagrams of pathogenicity locus (PaLoc), binary toxin encoding locus (CdtLoc) and tpi marker in C. difficile 44
Figure 4. Isolation and ribotyping of C. difficile from various sources 45
Figure 5. Amino acid alignment of GyrA and GyrB of C.difficile strains used in this study.	46
Figure 6. The comparison for biofilm formation of C. difficile strains.	48
Figure 7. The swimming motility of C. difficile strains 50
Figure 8. Phase variation of C. difficile isolates by orientation-specific PCR 52
Figure 9. Schematic diagrams of the flagellar operons, and the upstream and downstream genes of them 54
Figure 10. The screening of genes expression about RT126s by gel electrophoresis 55
Figure 11. Cytotoxicity determination by Alamar blue staining 56
SUPPLEMENTARY MATERIAL 57
A. Bacteria culture medium 57
B. Buffer 58
SUPPLEMENT DATA 59
Supplementary table 1. The CPE figures of each isolate 59
                                    

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2026-08-13公開

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