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研究生：	張翔甯 Chang, Hsiang-Ning
論文名稱：	探討酪酸菌所產細菌素對困難梭狀桿菌的抑制效果 Inhibition of Clostridium difficile by Clostridium butyricum MIYAIRI produced bacteriocin
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：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	95
中文關鍵詞：	困難梭狀芽孢桿菌、酪酸菌、細菌素
外文關鍵詞：	Clostridium difficile, C. butyricum MIYAIRI 588, bacteriocin
相關次數：	點閱：42 下載：1
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困難梭狀芽孢桿菌是已知造成院內感染的致病菌，主要由抗生素使用而造成腸內共生菌不平衡，使得困難梭狀芽孢桿菌伺機於腸道內繁殖。糞便轉殖療法是目前有效治療困難梭狀芽孢桿菌感染性疾病的方法，已知細菌可透過產生代謝物或蛋白質等，以對抗其他菌種。在本篇研究中，我們試著了解有哪些有效物質被腸道菌株以及益生菌釋放,以對抗困難梭狀芽孢桿菌。在本篇研究中，實驗結果顯示酪酸菌所釋放的物質對於抑制困難梭狀芽孢桿菌有更好的效果。已知酪酸菌是會產生孢子的格蘭氏陽性菌，並且可治療或延緩腸道致病菌所引起的病徵，但對於酪酸菌如何有效對抗困難梭狀芽孢桿菌的相關研究很少，因此我們試著找出可能的成因。在先前文獻中，已發現酪酸菌可以表現細菌素，因此我們想了解在酪酸菌所釋放物質中，細菌素是否扮演有效抑制困難梭狀芽孢桿菌的角色，在體外實驗中我們證實酪酸菌確實可以表現並產生細菌素，此外，我們製造重組細菌素蛋白以進行後續實驗。首先我們了解重組細菌素蛋白(crude CBM-B)是一個對酸鹼值不敏感，但對蛋白酶敏感的蛋白，而在溫度的感受性上是相對的不敏感。進一步我們發現CBM-B可以有效抑制困難梭狀芽孢桿菌,並且對梭狀芽孢桿菌屬有相對專一性。雖然CBM-B可抑制細菌，但我們未發現CBM-B可有效作用於孢子，另外，我們發現CBM-B可抑制困難梭狀芽孢桿菌所產毒素的基因表現，且可能透過抑制毒素正向調控因子而調節。在體外動物實驗中，透過肛門給予CBM-B,我們進而證明CBM-B可對抗腸道中的困難梭狀芽孢桿菌。總結而言，我們推論酪酸菌可透過釋放CBM-B而有效抑制困難梭狀芽孢桿菌，並作為未來新型抑制劑以治療困難梭狀芽孢桿菌感染症狀。

Clostridium difficile (C. difficile) is a gram-positive, spore-forming nosocomial pathogen and Clostridium difficile infection (CDI) can lead to antibiotic-associated pseudo-membranous colitis. The success of fecal material transplantation in the treatment of C. difficile infections indicated that beneficial bacteria are capable of inhibiting C. difficile. Moreover, it has been known that bacteria secreted metabolites and proteins can inhibit other bacteria. In this study, we screened the supernatant of multiple gut commensals for their inhibitory ability to inhibit C. difficile. Supernatants of C. butyricum MIYAIRI 588 strain (CBM588), a commercially available probiotic was shown to have the highest inhibition compared to others. CBM 588 is an anaerobic spore forming gram-positive bacteria which has been used for the treatment of enteropathogens but its role in C. difficile infection has not been evaluated in detail. Characterization of the cultural supernatant from CBM 588 suggested that a proteinaceous substance is involved in inhibiting C. difficile growth in vitro. Based on bioinformatics analysis and literature search, we identified a bacteriocin-like gene in CBM 588. PCR and qRT-PCR analysis of CBM 588 demonstrated the same gene existed and the transcription of this gene is highest during late-log growth phase. Western blot analysis demonstrated that the bacteriocin is present in the supernatant of CBM 588. Importantly, recombinant CBM 588 bacteriocin (crude CBM-B) was demonstrated to be sufficient for inhibition of C. difficile. In addition, the crude CBM-B is pH stable, protease sensitive, and relatively temperature stable. Further analysis revealed a narrow spectrum of activity for bacteriocin (CBM-B). CBM-B was unable to alter spore germination efficiency and outgrowth. Interestingly, when applied at sub-lethal concentration, CBM-B was able to suppress tcdA and tcdB expression in part by decreasing the expression of a toxin regulator. Lastly, we demonstrated that intra-rectal administration of CBM-B was able to decrease the survival of C. difficile in colons of mice. Overall, we concluded that CBM-B from CBM 588 is a potential inhibitor against C. difficile which could be used as therapeutic treatment for CDI.

中文摘要	I
ABSTRACT	II
致謝	III
CONTENTS	IV
ABBREVIATION	VIII
CHAPTER I	1
INTRODUCTION	1
1.1 Clostridium difficile infection (CDI) and epidemiology	1
1.2 C. difficile pathogenesis	3
1.3 Therapeutic treatment of CDI	3
1.4 Possible mechanism behind probiotic inhibiting C. difficile	6
1.5 Bacteriocin	7
1.6 Characteristic of Clostridium butyricum MIYAIRI 588	8
1.7 Rationale	10
CHAPTER II	11
MATERIAL AND METHODS	11
2.1 Materials	11
2.1.1 Bacterial strains	11
2.1.2 Cell line	12
2.1.3 Animal	12
2.2 Methods	13
2.2.1 Bacterial culture	13
2.2.2 Co-culture assay	14
2.2.3 Cloning and purification of recombinant bacteriocin (crude CBM-B)	15
2.2.4 Fast performance liquid chromatography (FPLC)	16
2.2.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)	17
2.2.6 Western blot	17
2.2.7 Spot-on-lawn assay	18
2.2.8 Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) assay	19
2.2.9 Inhibitory kinetic assay and time kill assay	20
2.2.10 C. difficile spore preparation	21
2.2.11 C. difficile spore germination assay	21
2.2.12 Spore outgrowth assay	22
2.2.13 CBM-B and vancomycin co-treatment assay	22
2.2.14 Ribonucleic acid (RNA) extraction of bacteria	23
2.2.15 Quantitative real time transcription-polymerase chain reaction (qRT-PCR)	25
2.2.16 Cell culture	26
2.2.17 Cell viability assay	27
2.2.18 Animal model of CDI	28
2.2.19 CBM 588 treatment of mice in vivo	29
2.2.20 CBM-B treatment of mice ex vivo	29
2.2.21 Preparation of C. difficile suspension for rectal inoculation	30
2.2.22 Rectal administration of CBM-B in vivo	31
2.2.23 Polymerase chain reaction (PCR) product identification	31
2.2.24 Animal model for collection of anti-crude CBM-B	32
2.2.25 The enzyme-linked immunosorbent assay (ELISA)	32
2.2.23 Statistical analysis	33
CHAPTER III	34
RESULTS	34
3.1 Supernatant of C. butyricum MIYAIRI 588 and E. rectale could inhibit C. difficile growth in vitro	34
3.2 Temperature and pH decreased the inhibitory effect of CBM 588 and E. rectale supernatant	35
3.3 C. butyricum MIYAIRI 588 express ORF 3, a putative bacteriocin-like gene	36
3.4 Cloning, overexpression, and purification of recombinant CBM-B	36
3.5 Characterization of pH, protease, and heat effects on crude CBM-B inhibitory ability	37
3.6 CBM-B has narrow anti-bacterial spectrum	38
3.7 CBM-B elicits inhibitory effectiveness on C. difficile	39
3.8 CBM-B could inhibit C. difficile produced toxin A and toxin B	40
3.9 CBM-B has no ability to inhibit C. difficile spore germination and outgrowth	41
3.10 Combination of vancomycin and CBM-B provide superior effect against C. difficile	42
3.11 CBM-B is not cytotoxicity to Caco-2 cells	42
3.12 The effects of CBM 588 treatment on CDI	43
3.13 Therapeutic availability of CBM-B on CDI ex vivo and in vivo	43
CHAPTER IV	45
DISCUSSION	45
REFERENCES	53
TABLES	62
Table 1. Gut normal flora used in this study	62
Table 2. The strains of C. difficile used in this study	63
Table 3. CBM-B spectrum of gram-positive and gram-negative bacteria	64
Table 4. Oligonucleotide primers used in this study	65
Figures	66
Figure 1. The inhibitory effects of gut flora and CBM 588 supernatant on C. difficile growth	66
Figure 2. Temperature and pH decreased the inhibitory ability of CBM 588 and E. rectale supernatant	67
Figure 3. Further characterization of CBM588 supernatant	68
Figure 4. The transcriptional profile of bacteriocin (CBM-B) in CBM 588	69
Figure 5. Purification and protein sequence of recombinant bacteriocin (crude CBM-588)	70
Figure 6. Protein expression of CBM-B in the supernatant of CBM 588	71
Figure 7. Characterization of pH, protease, and heat effects on crude CBM-B inhibitory ability	72
Figure 8. Separation of crude CBM-B	73
Figure 9. Determination of the kinetic of CBM-B inhibitory effects on C. difficile	74
Figure 10. Evaluation of inhibitory effect of CBM-B on C. difficile	75
Figure 11. The killing effects of CBM-B on C. difficile	76
Figure 12. Effect of sub-lethal CBM-B on C. difficile toxin and toxin regulator gene expression	77
Figure 13. The effect of CBM-B treatment on C. difficile spore germination	78
Figure 14. The impact of CBM-B treatment on C. difficile spore outgrowth	79
Figure 15. The combination effect of vancomycin combined with CBM-B	80
Figure 16. Cytotoxicity of CBM-B toward Caco-2 cells	81
Figure 17. Animal model of C. difficile infection (CDI)	82
Figure 18. The effects of CBM 588 administration in vivo	83
Figure 19 The efficacy of CBM 588 treatment in vivo	84
Figure 20. The administration of CBM-B provides effectiveness on CDI ex vivo	85
Figure 21. Animal model of CDI and CBM-B administration in vivo	86
Figure 22. The protective role of CBM-B on CDI in vivo.	87
Supplementary material	88
A. Chemicals and reagents	88
B. Bacteria culture medium	89
C. SDS-PAGE and western blot used buffer and recipes	90
D. Protein purification buffer lists	92
E. The recipes of other buffers	93
SUPPLEMENTARY FIGURES	94
Figure A. The CBM 588 was examined for PCR amplification	94
Figure B. The ORF of pCBM	94
Figure C. Animal model for collection of anti-crude CBM-B	95
Figure D. The evaluation of antibody titers of submandibular blood collection	95
Figure E. The determination of crude CBM-B activity by Spot-on-lawn assay	95

                                    

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