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研究生:洪宗林
研究生(外文):Tzung-Lin Hong
論文名稱:豬隻大腸桿菌抗藥性調查及其相關基因
論文名稱(外文):Antimicrobial Susceptibility of Swine Escherichia coli and Related Genes
指導教授:張紹光張紹光引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:71
中文關鍵詞:大腸桿菌抗藥性乙內醯胺酶
外文關鍵詞:E. coliantimicrobial resistanceβ-lactamasefloRcmlAintegron
相關次數:
  • 被引用被引用:2
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  長久以來,使用抗菌劑已是獸醫臨床上用來控制疾病、預防傳染病或促進家畜、禽生長的主要手段之一,但經研究證實,不當或長期低劑量使用,均易因抗菌劑篩選壓力而造成抗藥性,各種抗藥性基因更可經由質體或異種細菌間的轉移或交換而迅速散播,明顯降低治療效能與增加公共衛生上的困擾。
  本研究自2006年6月至2007年5月由臺灣北、中、南部三地區,逢機選取30個一貫式養豬場,以棉棒拭子採取哺乳、保育、肥育與母豬直腸糞便樣本共300個,在純化後,除採API 20E進行生化鑑定外,另以聚合酶鏈鎖反應與大腸桿菌標準菌株(ATCC 25922)比對,結果共分離出256株大腸桿菌(Escherichia coli),其中自北、中、南部三地區分離數分別為75、86及95株。經藥物敏感性試驗的結果發現:所分離得之大腸桿菌對amoxicillin、 ampicillin、oxytetracycline、florfenicol、chloramphenicol、nalidixic acid等6種抗菌劑已產生高程度的抗藥性(>70%),僅對colistin具有感受性。根據敏感試驗結果探討不同地區之抗藥性差異與豬隻飼養階段-抗藥性基因之分布特性,結果發現中、南部地區豬隻分離株對於上述的amoxicillin等6種抗菌劑之抗藥性顯著高於北部地區。
  進一步分析抗藥性圖譜、blaTEM-1、blaSHV-1及blaCTX-M-1乙內醯胺酶基因、floR、cmlA及class I integron所挾帶之dfr12-orfF-aadA2、dfr17-aadA5、aadA1及aadA2基因匣之盛行率與其抗藥性之間的關係。經聚合酶鏈鎖反應檢測抗藥性基因,其陽性率高低依序為floR(90.3%)、cmlA(91.1%)、blaTEM-1(70.7%)、blaSHV-1(67.2%)、class I integron(51.6%)及blaCTX-M-1(38.7%)。此亦解釋為何部份藥品的感受性偏低。本研究結果除可瞭解健康豬隻之大腸桿菌抗藥性現況外,亦可作為獸醫師臨床用藥之參考;另藉由逐年調查累積監測資料,應可供作為動物用藥品管理、風險評估等政策參考。
  Antimicrobials had been used for therapy and prevention of infectious disease in human and domestic animals as well as to promote growth in animals. Studies indicated that increasing of antimicrobial resistance in bacteria was caused by selection pressure from drug-abuse and sub-therapeutic dosage used in a long-term manner. Resistance genes could transfer among bacteria by plasmids, increasing the probability of transfering resistance genes form human to animals and vice versa, which could result in therapy failure in clinical practice.
  300 rectal feces swabs were collected from farrow-to-feedlot swine herds by stratified random sampling in the northern, middle and southern Taiwan from June 2006 to May 2007. A total of 256 E. coli isolates were identified by API 20E kit, and reconfirmed with polymerase chain reaction (PCR) comparing to ATCC 25922 E. coli. There were 75, 86 and 95 isolates collected from northern, middle and southern Taiwan respectively. All E. coli isolates were analyzed for their antibiogram and prevalence of blaTEM-1, blaSHV-1, blaCTX-M-1, floR, cmlA, and gene cassettes of class I integron including dfr12-orfF-aadA2, dfr17-aadA5, aadA1 and aadA2 by PCR. The susceptibility test results showed that more than 70% of the isolates resistant to ampicillin, amoxicillin, oxytetracycline, florfenicol and chloramphenicol, while sensitive to colistin. The prevalence results were that floR 90.3% and cmlA 91.1%; blaTEM-1 70.7%, blaSHV-1 67.2%, class I integron 51.6% and blaCTX-M-1 38.7%, respectively.
  This study demonstrated that the genes of floR, cmlA, blaTEM-1, blaSHV-1 and class I integron were widely distributed. In addition, both middle and southern swine herds had significantly higher resistances to amoxicillin, ampicillin, oxytetracycline, florfenicol, chloramphenicol and nalidixic acid than those swine herds in northern Taiwan. The antimicrobial susceptibility results could be useful for veterinary clinical practice in the selection of antimicrobials. It would be necessary for a long period surveillance and monitoring of the antibiogram and resistance genes of indicator bacteria such as E. coli. The determinant factors for the development of resistance genes also need to be clarified.
口試委員會審書........................................ i
誌謝.................................................. ii
中文摘要.............................................. iii
英文摘要.............................................. iv
第一章 緒言.........................................  1
第二章 文獻探討.....................................  3
第一節 大腸桿菌.....................................  3
1.1 大腸桿菌形態學、生化特性.....................  3
1.2 大腸桿菌之抗藥性機制.........................  3
第二節 抗藥性機制及基因傳遞方式.....................  7
2.1 胺基醣苷類抗菌劑.............................  7
2.2 phenicol類抗菌劑.............................  8
2.3 奎諾酮類抗菌劑...............................  9
2.4 頭孢子素類抗生素.............................  9
2.5 多黏菌素..................................... 10
第三節 乙內醯胺酶................................... 11
3.1 乙內醯胺酶的特性............................. 11
3.2 超廣效性乙內醯胺酶........................... 13
第四節 超廣效性乙內醯胺酶之檢測..................... 16
4.1 MIC檢測.......................................... 16
4.2 雙紙錠協同檢測................................... 17
4.3 E-test........................................... 17
4.4 等電點檢測....................................... 17
4.5 聚合酶鏈鎖反應................................... 18
4.6 核酸定序分析..................................... 18
第五節 ESBLs、class 1 integron常見之抗藥性基因...... 19
第六節 臺灣大腸桿菌抗藥性之流行病學................. 20
第三章 材料與方法................................... 21
第一節 實驗材料..................................... 21
1.1 培養基....................................... 21
1.2 染色體DNA之萃取.............................. 21
1.3 聚合酶鏈鎖反應............................... 22
1.4 器具......................................... 22
1.5 儀器設備..................................... 22
第二節 實驗方法...................................... 24
2.1 大腸桿菌之蒐集............................... 24
2.2 細菌增值與純化............................... 24
2.3 鑑定與保存................................... 24
2.4 藥物感受性試驗............................... 25
2.5 染色體DNA之萃取.............................. 26
2.6 抗藥基因之偵測............................... 27
2.7 瓊脂膠體電泳分析、染色、照膠................. 28
第三節 統計分析...................................... 29
第四章 結果......................................... 30
第一節 大腸桿菌之培養與分離結果..................... 30
第二節 藥物感受性試驗結果........................... 31
第三節 抗藥基因檢測結果............................. 33
  3.1 blaTEM、blaSHV及blaCTX-M-1基因............... 33
  3.2 floR及cmlA基因............................... 33
  3.3 Class I integron............................. 34
第五章 討論......................................... 35
第一節 大腸桿菌之抗藥性比較......................... 35
第二節 抗藥基因對抗藥性之影響....................... 38
2.1 乙內醯胺酶對抗藥性之影響......................... 38
2.2 floR及cmlA對抗藥性之影響......................... 41
2.3 Class I integron對抗藥性之影響................... 42
第六章 結論.......................................... 43
參考文獻.............................................. 45
圖表目錄.............................................. 60
表一 大腸桿菌對各抗菌劑臨界點之參考濃度(MIC breakpoints)
................................................ 60
表二 聚合酶鏈鎖反應引子序列、反應條件與產物大小...... 61
表三 不同地區與檢體來源之大腸桿菌分離數量統計表...... 62
表四 不同檢體來源之大腸桿菌分離株對13種抗菌劑之感受性
試驗結果........................................ 63
表五 不同地區之大腸桿菌分離株對13種抗菌劑之感受性試驗
   結果............................................ 64
表六 大腸桿菌之抗藥性基因鑑定統計及分佈表............ 65
圖一 不同檢體來源之大腸桿菌分離株對13種抗菌劑之抗藥性
比例............................................ 66
圖二 不同地區之大腸桿菌分離株對13種抗菌劑之抗藥性比例 67
圖三 哺乳豬之大腸桿菌抗藥性樹狀圖.................... 68
圖四 保育豬之大腸桿菌抗藥性樹狀圖.................... 69
圖五 肥育豬之大腸桿菌抗藥性樹狀圖.................... 70
圖六 母豬之大腸桿菌抗藥性樹狀圖...................... 71
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