近年來由於抗生素之濫用，多重抗藥性菌株對於臨床上釀成極大危害。可以利用抗菌胜肽與傳統的抗生素共同使用，達到協同治療的療效。
　　實驗室先前研究發現，萬古黴素原對革蘭氏陰性菌菌株沒有抑菌能力，在與設計的S1系列抗菌胜肽合併使用後，萬古黴素產生抑菌的能力。本篇研究是根據三色矛頭蝮之蛇毒蛋白片段PEM2保留其蛋白二級螺旋結構及雙極性的特色所設計出WLK系列的蛋白片段。運用WLK、WLK01及WLK11三條不同電荷分布之蛋白片段，探討將這些抗菌胜肽分別與三大類抗生素Vancomycin、Tetracycline、Ciprofloxacin，對於臨床抗藥性菌株，包括革蘭氏陽性菌Enterococcus faecium以及革蘭氏陰性菌Escherichia coli、Acinetobacter baumannii，抑菌能力(MIC assay)與蛋白片段中電荷分佈差異對協同機制之影響。
　　首先我們先做了單株菌對於抗生素以及抗菌胜肽的抑菌試驗(MIC assay)，顯示挑選的菌對於抗生素皆有抗性;接著進行棋盤格試驗(Checkerboard assay)，發現在合併使用後，多數有著協同的效果，而較特別的是Vancomycin本對於陰性菌沒有抑菌效果，在加入低濃度的抗菌胜肽合併使用後，有著很好的抑菌效果;接著，利用了螢光標定的Vancomycin在螢光顯微鏡下證實加入我們的抗菌胜肽後，可以幫助Vancomycin在細菌的作用位置作用;最後我們利用了Calcein AM試劑測試，結果顯示使用的抗菌胜肽對於選用的抗藥性菌株皆有著優異的擾膜破膜效果，而抗菌胜肽的正電荷分佈推測其影響不是最主要影響協同機制的效果。

Recently, due to the abuse of antibiotics, the multi-drug resistant strains are caused the great harmfulness to clinical. To solve these problems, the previous study show that can use the antimicrobial peptide combination with the traditional antibiotics to achieve synergy effect.
　　Vancomycin, which had no therapeutic effect on the Gram-negative bacteria. Our laboratory previous studies have found that Vancomycin combined with S1 series antimicrobial peptides could be against Gram-negative bacteria. In this study, we used the WLK series of peptides which were designed from the PEM2 which was derived from the C-terminal of enzyme myotoxin II in the venom of Bothrops asper [1]. WLK series peptides retain the secondary helix structure and bipolar characteristics. We choose three different protein fragments of charge distribution WLK、WLK01、WLK11 and three widely used antibiotics, Vancomycin, Tetracycline and Ciprofloxacin to against the multi-drug resistant clinical bacteria, including Gram positive bacteria：Enterococcus faecium, and Gram negative bacteria：Escherichia coli and Acinetobacter baumannii to explore the ability and the effect between the difference in charge distribution in protein fragment and explore the synergy mechanism.
　　The Minimum inhibitory concentration assay results showed that the selected strains are resistant to antibiotics. And the checkerboard assay demonstrated that antimicrobial peptides combined with antibiotics showed good synergy. Co-treatment of resistant strains with antimicrobial peptides led to increased uptake of BODIPY labeled vancomycin. The result of calcein-AM assay demonstrated that WLK series antimicrobial peptides have rapid and effective permeabilizing activity, and the activity is correlated with fluorscence experiment. 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. According to the sequence of antimicrobial peptide, positive charge distribution speculates that its effect is not the most important effect on the synergistic mechanism.

中文摘要 ........................................................I
Abstract .......................................................II
Acknowledgement ................................................IV
Contents .......................................................V
Chapter 1 Introduction .........................................1
1-1 Challenge of antimicrobial peptide and drug resistance .....1
1-2 Antibiotic resistance ......................................2
1-3 Synergy effect .............................................5
1-4 Aim of this study ..........................................6
Chapter 2 Materials and Methods ................................7
2-1 Materials ..................................................7
2-2 Quantization of the peptide ................................8
2-3 Bioassay for antimicrobial activity ........................8
2-4 Checkerboard assay for antimicrobial activities ............9
2-5 Calcein acetoxymethyl ester (Calcein AM) --- dye leakage ...10
2-6 Combination effect evaluated by using fluorescence microscope ................................................................11
Chapter 3 Result ...............................................13
3-1 Antimicrobial activities in MIC assays .....................13
3-2 Combination therapy analysis ...............................13
3-3 Peptides increase Vancomycin effect in multidrug resistant strains ........................................................15
3-4 Integrity of bacterial membrane ............................16
Chapter 4 Discussion ...........................................17
Figure and Tables ..............................................21
Table 1. Primary structure of WLK and its analogues. ...........21
Table 2. Minimum inhibitory concentration results of the antibiotics and peptides against multi-drug resistant strains. .22
Table 3. The fractional inhibitory concentration index range of peptides in combination with antibiotics against multidrug resistant bacteria. ............................................23
Figure 1. Fluorescence Microscopy：Uptake of labeled vancomycin in Enterococcus faecium - VRE132 (BCRC 15B0132) strain. ...........24
Figure 2. Fluorescence Microscopy: Uptake of labeled vancomycin in Acinetobacter baumannii - Ab97 (BCRC 14B0097) strain. ..........25
Figure 3. Fluorescence Microscopy：Uptake of labeled vancomycin in Escherichia coli – E.coli 207 (BCRC 13B0207) strain. ...........26
Figure 4. Permeabilization of the cytoplasmic membrane of Enterococcus faecium strain. ...................................27
Figure 5. Permeabilization of the cytoplasmic membrane of Acinetobacter baumannii strains. ...............................28
Figure 6. Permeabilization of the cytoplasmic membrane of Escherichia coli strains. ......................................29
Reference ......................................................30

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