多重抗藥性菌株造成無藥可醫的問題已經成為全世界迫切待解決的議題之一，其中一種解決策略是利用抗菌胜肽與傳統的抗生素共同處理以期能夠達到協同治療的效果。抗菌胜肽廣泛存在於植物與動物中，對於抵抗病原菌的入侵佔相當重要的角色，若能與抗生素藥物產生協同效果，可以有更廣效的抗菌效果、減少治療的劑量及副作用、縮短治療療程、減少產生抗藥性菌株的機率。在先前的研究中，S1系列的抗菌胜肽具有很好的抗菌活性、耐鹽性以及低溶血性，本實驗的目的是探討S1系列的抗菌胜肽與廣泛在臨床被使用的三大類抗生素Vancomycin 、Tetracycline、Ciprofloxacin，針對臨床分離的多重抗藥性菌株，包括革蘭氏陽性菌Enterococcus faecium以及革蘭氏陰性菌Acinetobacter baumannii、Escherichia coli，分析是否具有協同效果以及探討其協同機制。首先測試藥劑最小抑菌濃度試驗證實抗菌胜肽W5KA9W、S1Nal及S1Nal2對多重抗藥性菌株皆具有良好的殺抑菌功效；以棋盤格殺菌試驗(checkerboard method)試驗的結果顯示多數呈現協同效果，而且W5KA9W對於選用的抗生素具有更全面性的協同效果；以螢光標定的vancomycin證實抗菌胜肽處理的菌株確實能夠增加vancomycin 進入細菌標的位置作用。由於抗菌胜肽的抗菌機制是針對細菌細胞膜以其攜帶的正電性及疏水性達到擾動膜，甚至破膜的效果。我們以calcein-AM 試劑分析的結果顯示S1系列抗菌胜肽具有快速且優異的破膜能力，而且破膜的趨勢與協同能力具有相關性，推測其破膜能力可以破壞細菌膜細胞膜完整性並使抗生素易於進入作用的標的位置。

The rapid development and spread of bacterial resistance to conventional antibiotics has become a serious and urgent global issue. One of the strategies is to combine antimicrobial peptides (AMPs) with the conventional antibiotics, to achieve synergistic effect against multi-drug resistant bacteria. The advantages of synergistic treatment including killing a broader spectrum of microbes, decreasing the therapeutic dose, lowering side effects and treatment duration and reducing the risks of creating multi-drug resistant strains. In the previous studies, we designed a series of variants, W5KA9W, S1, S1-Nal and S1-Nal2, which revealed strong antibacterial activity, good salt resistant and low hemolytic activity.
In this study, we explore synergistic effects and mechanisms of S1 series AMPs and antibiotics (vancomycin, tetracycline, ciprofloxacin) against multi-drug resistant clinical bacteria, including Gram-positive bacteria: Enterococcus faecium, and Gram-negative bacteria: Acinetobacter baumannii and Escherichia coli. The Minimum inhibitory concentration assay results showed W5KA9W, S1Nal, and S1Nal2 have strong antibacterial activity against multi-drug resistant bacteria. And the checkerboard method demonstrated W5KA9W displayed more comprehensive synergism with antibiotics. Pre-treatment of resistant strains with AMPs led to increased uptake of bodipy labeled vancomycin.
AMPs characterized by their positive charges and amphipathic features, which enable them to cause the interference or even the disruption of the membrane. The result of calcein-AM assay demonstrated that S1 series AMPs have rapid and effective permeabilizing activity, and the activity is correlated with the synergism results. The result supports our hypothesis that the membrane-permeabilizing activity may destroy the integrity of bacteria membrane and lead to increased access of antibiotics to their target.

中文摘要 I
Abstract II
Contents IV
Chapter 1. Introduction 1
1.1 Antibiotic resistance 1
1.2 Antimicrobial peptides 2
1.3 Synergy effect 4
1.4 The aim of this study 5
Chapter 2. Materials and methods 7
2.1 Materials 7
2.2 Quantization of the peptides 8
2.3 Minimum inhibitory concentration assay 9
2.4 Checkerboard method 10
2.5 Calcein acetoxymethylester (calcein AM) 11
2.6 Fluorescence microscope 12
Chapter 3. Results 14
3.1 Bactericidal activity 14
3.2 FIC index 15
3.3 Peptides increased the uptake of labeled vancomycin 16
3.4 Combination effect in integrity of bacterial membrane 17
Chapter 4. Conclusion and Discussion 18
Figures and Tables 22
Figure 1. The structure of antibiotics 22
Figure 2. Fluorescence Microscopy: Uptake of labeled vancomycin in VRE strain 23
Figure 3 Fluorescence Microscopy: Uptake of labeled vancomycin in Acinetobacter baumannii strains 24
Figure 4. Fluorescence Microscopy: Uptake of labeled vancomycin in Escherichia coli strains 25
Figure 5. Permeabilization of the cytoplasmic membrane of VRE 26
Figure 6. Permeabilization of the cytoplasmic membrane of Acinetobacter baumannii strains 27
Figure 7. Permeabilization of the cytoplasmic membrane of Escherichia coli strains 28
Table 1. Antimicrobial peptide sequence 29
Table 2. Minimum inhibitory concentration of antimicrobial peptides and antibiotics. 30
Table 3. The fractional inhibitory concentration index range of peptides in combination with antibiotics against multidrug resistant bacteria. 31
References 32

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