臺灣博碩士論文加值系統

由於近年來抗生素的大量廣泛使用，抗藥細菌已經出現並且已經成為一個全球的健康議題。因此，目前迫切需要開發具有不同作用機制的新型抗菌藥物。最近，在生物體中發現的一些抗菌胜肽（antimicrobial peptides, AMPs）被發現會透過過破壞膜完整性或者影響細胞內重要的合成途徑，例如DNA或蛋白質的合成，進而殺死細菌。在這個研究中，我們將鑑定能有效對抗並抑制抗藥細菌的AMP，包含抗甲氧西林金黃葡萄球菌（Methicillin resistant Staphylococcus aureus, MRSA）和多重抗藥鮑曼氏不動桿菌（Multi-drug resistant Acinetobacter baumannii, MDRAB）。在已測試的AMP中，SMAP-29對許多包括MRSA和MDRAB的細菌具有廣效性的抑菌能力。其他AMP如Nigrocin和OdP1a對金黃葡萄球菌生物膜形成具有抑制能力。另外我們發現，幾乎所有的AMP都對MDRAB具有較高的抑菌能力，而MDRAB是目前在院內感染中的相當棘手的問題。這些抗菌肽不僅可以抑制細菌生長，而且可以減少生物膜形成。我們也建立了一個小鼠肺炎模型來進一步評估這些AMP在體內的效用，期望具有體內功效的AMP能夠在將來具有臨床應用的潛力。

Antibiotics-resistant bacteria have emerged due to the widespread usage of antibiotics. Thus, development of new antimicrobial agents with different action mechanisms is acute and urgent. Recently, some antimicrobial peptides (AMPs) found in organisms have shown to have bactericidal abilities by disrupting the membrane integrity or by inhibiting important pathways inside the cell such as DNA replication and protein synthesis. In this project, we will identify the AMPs exhibiting efficient antimicrobial activity against antibiotic-resistant bacteria, including methicillin- resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MDRAB). Among the tested AMPs, SMAP-29 and TP4 have a broad-spectrum bactericidal effect toward many kinds of bacteria, including MRSA and MDRAB. Other AMPs such as Nigrocin and OdP1a, have inhibitory effect on S. aureus biofilm formation. Interestingly, all the AMPs exhibit antimicrobial activities against MDRAB, which is a refractory issue in intensive care unit or nosocomial infection. These AMPs can not only inhibit the bacteria growth but also decrease the biofilm formation. We also established a mouse pneumonia model to evaluate the effect of these tested AMPs. The AMPs, which have the in vivo efficacy, will be anticipated to have the potential for the clinical application in the future.

口試委員會審定書 i
致謝 ii
摘要 iii
Abstract iv
Table of Contents v
List of tables viii
List of figures ix
Appendixes x
Chapter 1 Introduction 1
1.1 Staphylococcus aureus 1
1.2 Methicillin resistant Staphylococcus aureus (MRSA) 1
1.3 Acinetobacter baumannii 2
1.4 Multidrug resistant Acinetobacter baumannii (MDRAB) 2
1.5 Biofilm 4
1.6 Antimicrobial peptides (AMPs) 4
1.7 Mechanism of Antimicrobial peptides 5
Chapter 2 Purpose and aim 7
Chapter 3 Material and Method 8
3.1 Mice 8
3.2 Bacteria strains and growth conditions 8
3.3 Antimicrobial peptides 9
3.4 Preparation of broth, agar and solutions 10
3.5 Minimal inhibition concentration (MIC) and Minimal bactericidal concentration (MBC) testing 11
3.6 Biofilm bacteria killing test 12
3.7 Biofilm inhibition test 12
3.8 Biofilm quantification 13
3.9 Mouse intratracheal inoculation and sample collections 13
3.10 Bacteria count 14
3.11 Paraffin embedded sample preparation 14
3.12 Immunohistochemistry (IHC) 15
3.13 Hemolysis assay 15
3.14 MTT assay 16
Chapter 4 Result 17
4.1 The antimicrobial activities of tested AMPs 17
4.2 Effect of AMPs toward MRSA 17
4.3 The ability of AMPs to eradicate MRSA formed biofilm 18
4.4 The ability of AMPs to prevent MRSA biofilm formation 18
4.5 Effect of AMPs toward clinical MDRAB strain 19
4.6 The ability of AMPs to eradicate A. baumannii formed biofilm 19
4.7 Establishing mouse pneumonia model 19
4.8 Effect of AMPs in mouse pneumonia model 20
4.9 The toxicity and hemolytic activity of tested AMPs 21
Chapter 5 Discussion 22
Reference 27
Table 39
Figure 46
Appendix 55

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