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研究生:林恆加
研究生(外文):Heng-Jia Lin
論文名稱:台灣具超廣效性乙醯胺酶之克雷白氏菌之質體特性
論文名稱(外文):Characterization of ESBL carrying plasmids in Klebsiella pneumoniae in Taiwan
指導教授:張仲羽
指導教授(外文):Chung-Yu Chang
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:70
中文關鍵詞:克雷白氏肺炎桿菌超廣效性乙醯胺酶質體
外文關鍵詞:Klebsiella pneumoniaeExtended-spectrum β-lactamasesPlasmid
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本研究分析臨床克雷白氏肺炎桿菌帶有超廣效性乙醯胺酶 (ESBLs, extended spectrum beta-lactamases)的質體其質體類型、質體大小、質體片段多型性等質體特性,並探討ESBL types與質體特性的相關性,以及其散佈情形。
實驗樣本為TSAR III計劃 (Taiwan''s Surveillance of Antimicrobial Resistance)中51株來自臨床ESBLs克雷白氏肺炎桿菌之E. coli接合轉移菌株,研究方法包括: 從菌株萃取出來的DNA進行多對引子聚合酶連鎖反應 (multiplex PCR) 來測定各菌株質體的互不相容群 (incompatibility groups) 類型。分別以Kado & Liu方法抽取質體及S1 nuclease-PFGE來測得質體大小並比較結果。並以BamHI和PstI做質體的限制片段長度多形性分析 (RFLP)以比較質體之相關性。
本研究菌株的ESBLs以blaCTX-M-3最多,佔54.9%。研究菌株的質體互不相容群類型以A/C type最多,有31株 (佔60.8%),另有6株菌株質體不能以multiplex PCR分型。攜帶blaCTX-M-3、 blaCTX-M-14、 blaCTX-M-15的菌株質體,都以A/C type質體所佔比例最多,各有46.4% (13/28)、83.3% (10/12)及72.7% (8/11),但各blaCTX-M基因也存在於其他互不相容群質體上。以S1 nuclease-PFGE所測得之ESBLs菌株質體大小介於126 kb至 241 kb,大部份為160~170 kb (19/51; 37.3%)。 質體RFLP結果顯示以BamHI可將38株菌質體中的21株分辨為7個clusters,而PstI可將51株質體中的27株分辨為10個clusters,同一個cluster內的質體相似度達80%,且都屬於相同的互不相容群 (PstI RFLP的C6除外),未歸於cluster內的質體RFLP相似度多低於75%。
本研究結果顯示,ESBLs基因已散佈於不同的質體上,而質體的演化使得同一互不相容群的質體其RFLP型式可能相異,研究發現有超過50%的菌株質體可歸屬於cluster內 (55.3% by BamHI and 52.9% by PstI, respectively),顯示質體藉水平轉移已在菌株間傳遞散佈開來。以PCR based replicon typing與質體RFLP兩種方法探討質體流行病學,有助於了解質體攜帶ESBL基因的散佈情形。

Objective: This research analyzed the replicon type, size, and restriction fragment length polymorphism of ESBLs (extended spectrum beta-lactamases) carrying plasmid from Klebsiella pneumoniae clinical isolates.
Methods: A total of 51 E. coli transconjugant strains obtained from clinical ESBLs Klebsiella pneumoniae collected in the TSAR III (Taiwan''s Surveillance of Antimicrobial Resistance) were included in the study. Multiplex PCR was performed to determine the incompatibility groups of plasmids. Using Kado & Liu''s method and S1 nuclease-PFGE, plasmid size was determined and compared. Plasmid similarity was analyzed by plasmid RFLP using BamHI and PstI restriction enzyme.
Results: The most prevalent plasmid type in our experiment is A/C type (31/51; 60.8%) and six strains could not be typed. The most strain (28/51; 54.9%) carried CTX-M-3. Plasmid size ranged from 126 kb to 241 kb by S1 nuclease-PFGE and most plasmid size (37.3%) was 160~170 kb. Plasmid RFLP identified 21 from 38 plasmids belonging to 7 clusters by BamHI and 27 from 51 plasmids belonging to 10 clusters by PstI. Except the cluster C6, plasmids in the same clusters belonged to the same incompatibility groups and had 80% similarity. Most plasmids not divided into clusters had <75% similarity.
Conclusion: This study indicated that ESBL genes have been spread among different plasmids. Plasmid evolution may lead to plasmids with distinct RFLP patterns even though they belonged to the same incompatibility group. RFLP indicated that more than 50% of plasmids were divided into clusters (55.3% by BamHI and 52.9% by PstI, respectively), suggesting the horizontal transfer of plasmids contributed to the plasmid dissemination. Molecular typing with PCR based replicon typing and plasmid RFLP is helpful to understand the role of plasmids in the dispersion of ESBL genes and its epidemiology implication.

中文摘要 1
英文摘要 3
第一章 緒論 5
1.1 前言 5
1.2 克雷白氏肺炎桿菌 ( Klebsiella pneumoniae ) 6
1.2.1 克雷白氏肺炎桿菌之特性 6
1.2.2 克雷白氏肺炎桿菌引起之疾病 6
1.2.3 克雷白氏肺炎桿菌之抗藥機制 7
1.2.4 克雷白氏肺炎桿菌之流行病學 8
1.3 超廣效性乙內醯胺酶 (Extended-spectrum β-lactamases;ESBLs ) 9
1.3.1 ESBLs之起緣 9
1.3.2 ESBLs之分類 10
1.3.3 ESBLs之種類 11
1.3.4 ESBLs之流行病學 11
1.4 微生物抗藥基因水平散播機制 13
1.5 抗藥性質體種類及不相容群(incompatibility groups, Inc groups) 14
1.6 研究目的 16
第二章 材料與方法 19
2.1 實驗菌株 19
2.2帶有ESBLs質體之互不相容群分型 19
2.2.1 Total DNA萃取 19
2.2.2 Multiplex PCR 21
2.2.3 瓊脂醣明膠電泳 ( Agarose gel electrophoresis ) 22
2.3 ESBLs質體大小(plasmid size)分析 23
2.3.1 Kado & Liu質體抽取 23
2.3.2 Kado & Liu質體電泳 24
2.3.3 Pluse field gel electrophoresis (PFGE) and S1 nuclease treatment 25
2.4質體片段多型性分析 28
2.4.1 QIAGEN Plasmid Midi kits 抽質體 28
2.4.2 限制酶digest質體DNA 29
第三章 結果 31
3.1 帶有ESBLs的質體 31
3.1.1 ESBLs菌株的質體類型 31
3.1.2質體攜帶的 ESBLs 31
3.1.3 ESBLs分佈於各種質體類型的情形 32
3.1.4統計分析 32
3.2質體大小 33
3.2.1以Kado & Liu方法測得之質體大小 33
3.2.2以S1-nuclease PFGE測得之質體大小 33
3.2.3 質體類型在不同質體大小的分佈 34
3.2.4 ESBLs在不同質體大小的分佈 34
3.3 ESBLs菌株質體RFLP片段相似度比較 35
第四章 討論 37
第五章 圖 42
第六章 表 49
第七章 參考文獻 62

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