臺灣博碩士論文加值系統

近年來，奈米材料在臨床治療及臨床診斷方面的應用也越來越廣泛。在我的研究中主要分為兩部分：一是探求用於治療艱難梭狀桿菌感染的奈米顆粒，另一是開發用於快速診斷念珠菌血症的磁性奈米顆粒。
艱難梭狀桿菌是一種會產生孢子的厭氧菌，其孢子形態被認為是環境傳播的主要來源。然而，臨床上目前針對此疾病的治療仍依賴於抗生素對菌體的傷害，而抗生素會破壞正常菌群，繼而導致感染復發風險變高。在本次研究中，我們篩選了13種不同的奈米粒子，用來抑制艱難梭狀桿菌孢子和細菌存活。其中，我們發現了一種奈米粒子不僅能抑制孢子的存活，還能抑制菌體的存活。從穿透式電子顯微鏡檢查顯示奈米顆粒能夠快速破壞艱難梭狀桿菌孢子和菌體。此外，在小鼠糞便離體試驗中，此新型奈米對艱難梭菌的殺菌能力與奈米材料濃度呈正相關性。在小鼠活體實驗中，新型奈米能夠有效減緩小鼠被艱難梭菌感染的病症，顯示新型奈米具有發展為治療艱難梭狀桿菌感染之全新型製劑的潛力。
念珠菌血症是常見造成血液感染性疾病的主要原因之一，可導致高死亡率。因此，念珠菌血症需要在短時間被正確性的檢測。在本次研究中，我們通過磁性奈米粒子濃縮病原體並以聚合酶連鎖反應檢測來縮短血液培養時間。我們發現鐵奈米可在短時間內與低濃度的念珠菌屬特異性地結合，直接檢測念珠菌血症的臨床樣本，其診斷結果與傳統診斷法的結果相符。此部分，我們開發了一種新型奈米用於念珠菌血症的快速檢測方法。

During recent years, there has been more interest in using nanoparticles for clinical diagnosis and therapy. In this study, my research is divided into two parts: one is searching for a new therapeutic nanoparticle to against Clostridium difficile infection (CDI) and the other is developing a rapid candidemia diagnostic magnetic nanoparticle.
C. difficile is a spore-forming anaerobe. The spores are considered a major source of transmission. However, current treatment against CDI relies on antibiotic usage, which also damaged the normal flora and raised a high CDI recurrence risk. Therefore, alternative therapies that are more effective against C. difficile bacteria or spores are needed. Here, we screened 13 different nanoparticles on inhibition of C. difficile spore- and bacterial-survival. We identified a new nanoparticle which inhibited the survival of not only spores but also vegetative cells. Transmission electron microscope examination revealed that the nanoparticle quickly disrupted C. difficile spores and vegetative cells. The survival of C. difficile in a fecal bench ex vivo test showed this nanoparticle exhibited bactericidal ability against C. difficile in a dose dependent manner. In a CDI murine model, this nanoparticle showed therapeutic efficacy through attenuating CDI. Taken together, these results suggest this nanoparticle possess great potential to be a new alternative therapeutic strategy to against CDI.
Candidemia is a one of common bloodstream infections worldwide causing high mortality rate. Here, we shorten the incubation time of blood cultures via condensing the pathogen by magnetic nanoparticles and identifying the pathogen by PCR. We found an iron nanoparticle can quickly and specifically bind to Candida spp. This condensation procedure revealed the same results as that in traditional diagnostic process in clinical candidemia specimen. Taken together, this study demonstrated a rapid candidemia identification process via magnetic nanoparticles.

CONTENTS
中文摘要 I
ABSTRACT II
致謝 III
CONTENTS IV
Part one: Inhibition of Clostridium difficile spore and bacterial survival by novel nanoparticles.
Chapter 1. INTRODUCTION 1
1.1 Clostridium difficile infection and epidemiology 1
1.2 Pathogenesis of Clostridium difficile infection 1
1.3 Clostridium difficile spores 2
1.4 Nanomaterials 3
1.5 Antibacterial activity of nanomaterials 3
Chapter 2. MATERIALS AND METHODS 5
2.1 Bacterial culture 5
2.2 Purification of spores 5
2.3 Spore staining viability assay 5
2.4 Spore viability assay 6
2.5 Spore germination assay 6
2.6 DPA release assay 6
2.7 Vegetative bacterial cells viability test 7
2.8 MIC of nanoparticles in against C. difficile 7
2.9 Trans-electron microscopic analysis of spores and vegetative cells 7
2.10 Spore integrity staining 7
2.11 Animal 7
2.12 C. difficile infection animal model 8
2.13 Inhibition of C. difficile in a fecal bench ex vivo test 8
2.14 The therapeutic efficacy of the nanoparticles in CDI murine
model 8
2.15 The effect of the nanoparticles on animal guts microbiota 9
2.16 Statistical analysis 9
Chapter 3. RESULTS 10
3.1 The sporicidal ability of nanoparticles against C. difficile spores 10
3.2 The effects of silver nanoparticles on C. difficile spore germination 10
3.3 The effects of silver nanoparticles on C. difficile spore DPA
releasing 10
3.4 The silver nanoparticles decrease the viability of C. difficile vegetative
cells 11
3.5 The new silver nanoparticles damage spore and vegetative
ultrastructures 11
3.6 The new silver nanoparticles disrupted integrities of spore and
vegetative cell 12
3.7 The new silver nanoparticles inhibit C. difficile growth mixed with
colonic microbiome 12
3.8 The new silver nanoparticles have the therapeutic effects on C. difficile
infection 13
Chapter 4. DISCUSSION 14
REFERENCES 16
APPENDIX 30
Appendix 1. List for nanoparticles used in this study 30
Appendix 2. List for primers used in this study 30
Appendix 3. Recipes of broth and agar 31

Part two: Magnetic nanoparticles for Candida concentration in Candidemia.
Chapter 1. INTRODUCTION 32
1.1 Candidemia 32
1.2 Epidemiology of candidemia 32
1.3 Limitation of blood culture for diagnosis of bloodstream infections 33
1.4 The application of magnetic nanoparticles in previous study 34
1.5 Iron nanoparticle (IONP) 34
Chapter 2. MATERIALS AND METHODS 35
2.1 Fungal culture 35
2.2 Bacterial culture 35
2.3 DNA extraction 35
2.4 Amplification of the ITS region and 16srRNA 36
2.5 Identification of yeast by ITS sequencing 36
2.6 Trans-electron microscopic analysis of fungi interacting with iron
nanoparticles 36
2.7 Spike assay 37
2.8 Fungal counting of clinical candidemia specimens 37
2.9 Data analysis 37
Chapter 3. RESULTS 38
3.1 Proposed working model for shorten identification process in blood
culture specimen 38
3.2 The iron nanoparticles (IONPs) magnetic condensed Candida spp.
which can be identified by PCR 38
3.3 TEM images of IONPs interacting with Candida tropicalis 39
3.4 IONPs specificity magnetic condensed Candida spp. 39
3.5 The optimum interacted condition of IONPs and Candida spp. 39
3.6 Condensation of Candida spp. in the blood culture condition with
IONPs 40
3.7 Detection accuracy of IONPs magnetic condensed and identification
by PCR in clinical candidemia specimens 40
Chapter 4. DISCUSSION 42
REFERENCES 44
APPENDIX 54
Appendix 1. List for primers used in this study 54
Appendix 2. Recipes of broth and agar 54

INDEX OF TABLES
Part one: Inhibition of Clostridium difficile spore and bacterial survival by novel nanoparticles.
Table 1. C. difficile growth inhibition by silver nanoparticles 29

Part two: Magnetic nanoparticles for Candida concentration in Candidemia.
Table 1. The colony forming units of Candida spp. from candidemia specimens 53

 
INDEX OF FIGURES
Part one: Inhibition of Clostridium difficile spore and bacterial survival by novel nanoparticles.
Figure 1. The sporicidal activity of 13 different nanoparticles on C. difficile spore 19
Figure 2. The silver nanoparticles inhibit C. difficile spore germination 20
Figure 3. The silver nanoparticles inhibit C. difficile spore DPA releasing 21
Figure 4. The silver nanoparticles inhibit C. difficile growth 22
Figure 5. The new silver nanoparticles damage spore and vegetative
ultrastructures 23
Figure 6. The new silver nanoparticles damaged spore and vegetative cell integrity 24
Figure 7. The new silver nanoparticles inhibit C. difficile growth mixed with colonic microbiome 25
Figure 8. The new silver nanoparticles have therapeutic effects on C. difficile infection 26
Figure 9. The new silver nanoparticles altered the microbiome in a dose-dependent manner 27
Figure 10. The new silver nanoparticles inhibit human colon cancer cell line HT29 growth 28
Part two: Magnetic nanoparticles for Candida concentration in Candidemia.
Figure 1. Proposed working model for shorten identification process in blood culture specimen 46
Figure 2. The iron nanoparticles (IONPs) magnetic condensed Candida spp. which can be identified by PCR 47
Figure 3. TEM images of IONPs interacting with Candida tropicalis 48
Figure 4. IONPs specificity magnetic condensed Candida spp. 49
Figure 5. The optimum interacted condition of IONPs and Candida spp. 50
Figure 6. Condensation of Candida spp. in the blood culture condition with IONPs 51
Figure 7. Detection accuracy of IONPs magnetic condensed and identification by PCR in clinical candidemia specimens 52

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