臺灣博碩士論文加值系統

豬霍亂沙門氏菌 (Salmonella enterica serovar Choleraesuis, S. Choleraesuis) 是近年來在台灣被受關注的一種重要傳染病原菌，在人類感染病例中造成敗血症導致死亡。常見原因是誤食被沙門氏菌汙染的食物而造成人類及動物以腸胃症狀為主的疾病；雖然通常可以自行痊癒，但對免疫力較低者，如老人、小孩、先天性免疫缺乏的病人、全齡豬隻，或是離乳至4月齡豬，則可能造成嚴重的症狀。由於近年來抗生素的濫用造成抗藥性菌株大量出現，多重抗藥性 (multidrug-resistant, MDR) S. Choleraesuis已成為公共衛生的隱憂；如何尋覓或開發新穎抗生素替代製劑已成為當務之急，具有抗菌、抗癌等多元宿主防禦活性的抗菌胜肽 (antimicrobial peptides, AMPs) 被認為極具潛力。本實驗室前人亦自行設計合成了一系列強效且具高度選擇性的AMPs。本研究主要目的在於探討AMPs與現行抗生素合併使用時對於MDR- S. Choleraesuis的抗菌效力，使用由行政院農委會家畜衛生試驗提供的菌株進行測試，經最小抑菌濃度 (minimal inhibitory concentration, MIC) 分析得知，來自非洲比目魚的AMP- pleurocidin (Ple) 的C端醯胺基(amide)修飾產物 Ple-a與本實驗室自行設計合成的Q4-amide (Q4-a)具有最佳的抗菌活性。進一步將Ple-a = 8 μg/ml和Q4-a = 16 μg/ml與革蘭氏陰性菌抗生素藥物做組合試驗，計算其分量抑菌係數 (fractional inhibitory concentration index, FICI)，結果顯示Ple-a = 8μg/ml與Ampicillin以2:1組合時FICI = 0.34具有協同作用 (synergy)；而Ple-a = 8μg/ml與Kanamycin以1:2組合時FICI = 0.71具有加成作用(additivity)；而Ple-a = 8 μg/ml與Nalidixic acid以2:1組合時FICI = 0.69具有加成作用；而Ple-a = 8 μg/ml與Ciprofloxacin以1:2組合時FICI = 0.83具有加成作用；而Q4-a = 16 μg/ml與Ciprofloxacin以1:1, 1:2, 2:1組合時FICI = 0.63, 0.75, 1具有加成作用；而Ple-a = 8 μg/ml與Enrofloxacin以2:1組合時FICI = 0.67具有加成作用；而Q4-a = 16 μg/ml與Enrofloxacin以1:2, 2:1組合時FICI = 0.75, 1具有加成作用。本研究初步證實，AMPs與抗生素的組合製劑具潛力可對抗MDR- S. Choleraesuis具有殺菌效果；而臨床上的使用方式及其安全性，則有待進一步審慎評估。

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) has been Concerned to be an important pathogen in Taiwan in recent years, it causes sepsis in human infections and may lead to death. The most common cause of S. Choleraesuis infection is having contaminated food and cause to humans and animals with gastrointestinal symptoms. Antibiotics were applied to prevent bacterial infections, however, the extensive use has led to the growing emergence of multidrug-resistant (MDR) strains. MDR-S. Choleraesuis has become a public health problem. Previous studies have proven that antimicrobial peptides (AMPs) are of greatest potential to serve as a new class of antibiotics. In our previous studies, we have designed and synthesized a series of potent AMPs with high selectivity. The aim of this study is to investigate the antibacterial effect of the combination use of AMPs and antibiotics against MDR-S. Choleraesuis. Minimal inhibitory concentration (MIC) analsysis revealed that the C-terminal amidated derivative of pleurocidin (Ple)- Ple-a and our self-designed Q4-a showed the best antibacterial activity. Fractional inhibitory concentration index (FICI) was also calculated from combination test using Ple-a = 8 μg/ml and Q4-a = 16 μg/ml with clinically used antibiotics. Synergy effect was monitored when Ple-a = 8μg/ml and Ampicillin were applied in a 2:1 molar ratio FICI = 0.34. Furthermore, Additivity effect was monitored when Ple-a = 8μg/ml and Kanamycin were applied in a 1:2 molar ratio FICI = 0.71. Furthermore, Additivity effect was monitored when Ple-a = 8μg/ml and Nalidixic acid were applied in a 2:1 molar ratio FICI = 0.69. Furthermore, Additivity effect was monitored when Ple-a = 8μg/ml and Ciprofloxacin were applied in a 1:2 molar ratio FICI = 0.83. Furthermore,
Additivity effect was monitored when Q4-a = 16 μg/ml and Ciprofloxacin were applied in 1:1, 1:2, 2:1 molar ratio FICI = 0.63, 0.75, 1. Furthermore, Additivity effect was monitored when Ple-a = 8μg/ml and Enrofloxacin were applied in a 2:1 molar ratio FICI = 0.67.
Furthermore, Additivity effect was monitored when Q4-a = 16 μg/ml and Enrofloxacin were applied in 1:2, 2:1 molar ratio FICI = 0.75, 1. Based on the above findings, we preliminarily proved that combinatory agents consist of AMPs and antibiotics are of greatest potential to fight against MDR- S. Choleraesuis. However, the clinical application methods and the safety issues await further evaluation.

目錄(Table of contents)
摘要 I
Abstract II
目錄(Table of contents) III
圖目錄(List of figures) V
表目錄(List of tables) VI
名詞縮寫表(Abbreviations) VII
第一章、前言 1
第一節、豬霍亂沙門氏菌(Salmonella enterica serovar Choleraesuis, S. Choleraesuis)介紹 1
第二節、抗生素 2
第三節、多重抗藥性機制之產生 4
第四節、抗菌胜肽(Antimicrobial peptides, AMPs) 5
第二章、實驗目的 10
第三章、實驗器材與儀器 11
第一節、生物材料、藥品及試劑 11
第二節、儀器設備 11
第四章、實驗流程與方法 12
第一節、設計抗菌胜肽 13
第二節、菌種保存培養 13
第四節、使用VITEK ®2全自動微生物分析儀再次確認菌種 13
第五節、菌落數與濁度之計算(平板計數法) 14
第六節、最小抑菌濃度(Minimum inhibitory concentration, MIC) 14
第七節、組合試驗 14
第五章、結果 16
第一節、豬霍亂沙門氏菌再次鑑定與藥敏試驗 16
第二節、平板計數法_菌落數與濁度之相對關係 16
第三節、AMPs的MIC 16
第四節、AMPs與抗生素的組合試驗之FICI評估 16
第六章、討論 18
第一節、AMPs對MDR S. Choleraesuis之抗菌能力 18
第二節、AMPs對MDR S. Choleraesuis之抗菌機制 18
第三節、AMPs對Ampicillin之作用機制 18
第四節、AMPs與抗生素的組合藥物對MDR S. Choleraesuis的抗菌活性 18
第五節、VITEK ®2全自動微生物分析儀與傳統鑑定法之差異 19
第七章、結論與未來展望 20
第八章、參考文獻 35

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