隨著科技的快速發展，抗生素被廣泛使用在治療人類細菌感染和飼養經濟動物上。其中家禽類因生產成本低，且沒有文化和宗教限制，是全世界消費最廣泛的肉類之一。因需求量逐年上升，養殖朝向密集飼養模式，加上氣候變遷劇烈，造成動物因「熱緊迫」和「冷緊迫」變得虛弱容易生病。因此，大多數國家會在飼料或水中加入不同種類的抗生素飼養家禽，用以預防或阻止疾病在食用動物間傳播，並促進食用動物生長。但有色雞種 (如烏骨雞) 對藥物的代謝率比白肉雞來得慢，再加上烏骨雞有「啄糞」的特性，較難掌控停藥期。而腸球菌 (Enterococcus) 是人和溫血動物胃腸道中的正常菌群，因此為家禽中最常檢出的抗生素抗藥性微生物之一，而臨床上分離率最高的腸球菌屬是糞腸球菌 (Enterococcus faecalis) 與屎腸球菌 (Enterococcus faecium)。另外腸球菌因生物膜形成能力強，可通過物理方式阻擋藥物滲透降低抗生素的敏感性；更可藉由水平基因移轉的方式，將遺傳抗藥性 DNA 片段轉移給其他的菌種，以上皆可提高腸球菌對抗生素耐受性，使抗生素抗藥性腸球菌 (Antibiotic-resistant Enterococci, ARE) 持續於環境中傳播，透過食物鏈轉移到人類生態系統。故本研究主要以臺灣市售烏骨雞作為研究目標，使用選擇性培養基分離樣品中的腸球菌，PCR 對菌種進行鑑定，並以鑑定後的分離株測試對抗生素敏感性和生物膜生成能力，最後針對實驗結果進行探討及分析。實驗結果表明，E. faecalis 和 E. faecium 是市售烏骨雞樣品中最常見的腸球菌菌種，並對於醫學或是動物養殖中使用的抗生素 (如 Tetracycline, Erythromycin 和 Kanamycin) 都具有高度抗藥性；另外在生物膜生成試驗中，83% 分離株皆可產生生物膜，其中更有 3 株 E. faecalis 具有強生物膜生成能力。根據上述得知，醫療及養殖上抗生素的使用，易使本身具有生物膜生成和基因移轉特性的腸球菌增加對抗生素的抗藥性，並透過食物鏈將抗藥性基因傳播到人類生態系統。因此，相關單位更應該制定相關防護措施，如限制或終止食用動物養殖中抗生素的使用，並監測食用肉製品中腸球菌的耐藥率，用以減輕消費性食品中 ARE 的傳播風險，維護從農場到餐桌整體供應鏈的食品安全。

With the rapid development of science and technology, antibiotics are widely used in the treatment of human bacterial infections and in raising economic animals. Among them, poultry is one of the most widely consumed meats in the world due to its low production cost and no cultural and religious restrictions. However, the demand has increased year by year, the farming has moved towards an intensive farming model, and the drastic climate change has caused animals to become weak and susceptible to diseases due to "heat stress" and "cold stress". Therefore, most countries feed poultry with different kinds of antibiotics in feed or water to prevent or stop the spread of disease among food animals and to promote the growth of food animals. But coloured broiler (such as silky fowls) have a slower metabolism than white broiler. In addition, silky chickens have the characteristic of "pecking manure", so it is difficult to control the withdrawal period. But colored chickens metabolized the drug more slowly than white broilers. Enterococcus is the normal flora in the gastrointestinal tract of humans and warm-blooded animals,it is the most commonly detected antibiotic-resistant microorganisms in poultry. Among, Enterococcus faecalis and Enterococcus faecalis are the Enterococcus with the highest clinical isolation rate. In addition, Enterococcus has a strong biofilm forming ability, which can physically block drug penetration to reduce antibiotic sensitivity; it can also transfer genetic drug-resistant DNA fragments to other strains by horizontal gene transfer. All of the above can increase the resistance of enterococci to antibiotics, so that antibiotic-resistant enterococci continue to spread in the environment and transfer to the human ecosystem through the food chain. Therefore, this study mainly took the silky fowls sold in Taiwan as the research target, using selective medium to isolate Enterococcus in the samples, identifying the bacterial species by PCR, and testing the antibiotic susceptibility and biofilm formation ability with the identified isolates, finally discuss and analyze the experimental results. Experimental results show that E. faecalis and E. faecium are the most common Enterococcus species in commercially available silky fowls samples and are highly resistant to antibiotics used in medicine or animal farming (such as Tetracycline, Erythromycin and Kanamycin). In addition, in the biofilm formation test, 83% of the isolates could produce biofilms, and 3 strains of E. faecalis had strong biofilm formation ability. According to the above, the use of antibiotics in medical treatment and animal feeding can easily make Enterococcus, which has the characteristics of biofilm formation and gene transfer, to increase the resistance to antibiotics, and spread the resistance genes to the human ecosystem through the food chain. Therefore, relevant units should formulate relevant protective measures, such as restricting or terminating the use of antibiotics in edible animal breeding, and monitoring the resistance rate of Enterococcus in edible meat products, to reduce the risk of transmission of ARE in consumer foods, and protect food safety across the entire supply chain from farm to fork.

謝誌 I
摘要 II
Abstract III
圖目錄 VII
表目錄 VIII
附錄目錄 IX
縮寫表 X
壹、 前言 1
貳、 文獻回顧 2
2.1抗生素 (Antibiotic) 2
2.1.1抗生素的歷史起源 2
2.1.2抗生素的種類&作用機制 3
2.1.3抗生素抗藥性 (Antimicrobial resistance, AMR) 5
2.1.4抗藥性機制 7
2.1.5抗藥性微生物傳播途徑 8
2.1.6抗藥性食源性微生物 10
2.2腸球菌 (Enterococcus) 13
2.2.1腸球菌歷史起源 13
2.2.2糞腸球菌 (Enterococcus faecalis) 14
2.2.3屎腸球菌 (Enterococcus faecium) 15
2.2.4食品中腸球菌之應用 15
2.2.5作為益生菌的相關風險 16
2.2.6腸球菌抗藥性(Antibiotic-resistant Enterococci, ARE) 18
2.2.7易具抗藥性之抗生素 18
2.2.8生物膜生成特性 22
2.2.9 ARE參與食源性疾病 23
2.3食用動物的養殖 25
2.3.1家禽使用的抗生素類別 25
2.3.2烏骨雞 (Silky fowl) 27
參、 實驗設計 29
肆、 材料與方法 30
4.1實驗材料 30
4.1.1實驗樣品來源 30
4.1.2實驗菌株 30
4.1.3引子對 30
4.1.3.1標準菌株 30
4.1.3.2抗藥性基因 31
4.1.4培養基 31
4.1.5反應緩衝液 32
4.1.6抗生素 33
4.1.7化學藥品 33
4.1.8試劑套組 33
4.1.9酵素 33
4.1.10實驗儀器與耗材 34
4.2實驗方法 35
4.2.1樣品處理 35
4.2.2菌株分離 35
4.2.2.1腸球菌 35
4.2.3菌株保存 35
4.2.4菌株活化 35
4.2.5生長曲線 36
4.2.6菌株鑑定 36
4.2.6.1 DNA萃取 36
4.2.6.2聚合酶鏈反應 37
4.2.6.3 DNA電泳 38
4.2.7 抗生素抗藥性試驗 38
4.2.8抗藥性基因檢測 38
4.2.9生物膜生成能力測試 40
4.2.10統計分析 40
伍、 結果與討論 41
5.1 烏骨雞樣品中腸球菌的盛行率 41
5.2 腸球菌分離株對抗生素的敏感性 42
5.3 腸球菌分離株的多重耐藥性 46
5.4 腸球菌分離株抗生素抗藥性基因 47
5.5 腸球菌分離株生物膜生成能力 48
5.6 腸球菌分離株抗藥性與生物膜生成間關係 49
陸、 結論 51
柒、 參考文獻 52
捌、 圖表 64
玖、 附錄 78

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