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研究生:曾麗芸
研究生(外文):TSENG LI YUN
論文名稱:抗藥性鮑氏不動桿菌對ciprofloxacin及imipenem之抗藥性機制分析
論文名稱(外文):Mechanism of Ciprofloxacin- and Imipenem- Resistance in Multidrug-Resistant Acinetobacter baumannii
指導教授:劉淑瑛 ; 邱政洵
指導教授(外文):Shu-Ying Liu ; Cheng-Hsun Chiu
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:61
中文關鍵詞: 多重抗藥性鮑氏不動桿菌 及時定量聚合酶鏈鎖反應 藥物輸出幫浦 抗藥基因 ciprofloxacin
外文關鍵詞:multidrug-resistant Acinetobacter baumannii (MDRAB)real-time quantitative PCRefflux pumpdrug resistance geneciproflo
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多重抗藥性的鮑氏不動桿菌(multidrug-resistant Acinetobacter baumannii, MDRAB)所造成的感染日益增加,已成為重要的院內感染致病菌。目前在林口長庚醫院篩選出35株臨床分離菌株,經由real-time quantitative PCR(及時定量聚合酶鏈鎖反應)分析藥物輸出幫浦Ade drug transporter的表現,發現與抗生素ciprofloxacin及ampicillin-sulbactam之抗藥性有高度的相關性。進一步以ciprofloxacin抗藥菌株AB-1227分析抗生素的影響,及啟動菌株中排出藥物的基因,結果則顯示Ade drug transpoter為持續性的表現,但此現象的原因未明。而基因突變的分析中,則發現gyrA及parC的突變與A. baumannii對ciprofloxacin造成之抗藥性有關,如果菌株同時有藥物輸出的能力,將會進而增加其抗藥性。另外在對imipenem抗藥基因的分析方面,35株臨床菌株中有8株帶有blaIMP-1,且基因片匣(gene cassette)中所攜帶的抗藥基因其排列組合分別為:5'CS-blaIMP-1-aadA4-3'CS,5'CS- aacA4-aadA1-3'CS,及5'CS-aacC1- aadA1-3'CS。此外,所有菌株皆不帶有抗藥基因blaVIM-1 或blaVIM-2。這些基因片匣中帶有多種不同之抗藥基因,其中一類還帶有blaIMP-1;先前的研究曾顯示,blaIMP-1之抗藥基因會以基因片匣的方式存在於細菌的質體中,而細菌間質體之轉移則可造成抗藥基因之傳播。綜合結果顯示,造成A. baumannii之多重抗藥性的原因乃是透過多種抗藥機制,很可能是細菌適應外界環境的改變而演化的結果。
The infections caused by multidrug-resistant Acinetobacter baumannii (MDRAB) have been increasing in recent years, ever since MDRAB became major sources for nosocomial infections in many countries. In this study, 35 strains of A. baumannii isolated from Chang-Gung Memorial Hospital (CGMH)were tested. We analyzed the expression of ade in these clinical isolates’ efflux pump systems by real-time quantitative PCR, and found that the expression of this drug transporter were most significantly associated with drug-resistance to ciprofloxacin and ampicillin-sulbactam. Among these isolates, ciprofloxacin-resistant AB-1227 constitutively expressed ade in the presence or absence of ciprofloxacin, but the mechanism remained to be elucidated. Furthermore, mutation analyses of GyrA and ParC showed that the change of few residues, together with ciprofloxacin-output by Ade transporter, highly increased the capacity of resistance to ciprofloxacin. In the studies of imipenem-resistance genes, we found blaIMP-1 in 8 of 35 isolates. Besides blaIMP-1, the combination of drug-resistance genes located in the gene cassette including 5'CS-blaIMP-1-aadA4-3'CS, 5'CS-aacA4-aadA1-3'CS, and 5'CS- aacC1- aadA1-3'CS. No blaVIM-1 or blaVIM-2 was found in these resistant isolates. It had been shown that blaIMP-1 can be inserted into the plasmids, so the drug-resistance among pathogens could spread through plasmids transfer. In conclusion, the multidrug-resistance of A. baumannii was not due to drug resistance genes only, but also some other drug-resistance mechanisms (ex. efflux pump). All together, these mechanisms make pathogens more drug-resistant.
第一章 緒論
1.1 Acinetobacter baumannii簡介……………………………..1
1.1.1 鮑氏不動桿菌之生物特性及其臨床表徵…………….1
1.1.2 抗生素之分類及其作用機制………………………….3
1.1.3 細菌的抗藥機制……………………………………….4
1.1.4 多重抗藥性菌株藥物輸出幫浦之結構及分類……….6
1.1.5 針對鮑氏不動桿菌之抗藥性研究…………………….7
1.2 及時定量聚合酶鏈鎖反應…………………………….…....9
1.3 抑制減除雜合法…………………………………………...10
1.4 實驗目的…………..……………………………………….13

第二章 材料與方法………………………………………………......14
2.1 菌株來源及分群…………………………………………...14
2.2 病原菌Genomic DNA的萃取…………………………….14
2.3 聚合酶鏈鎖反應(PCR)……………………………………14
2.4 純化PCR產物……………………………………………....16
2.5 凝膠萃取PCR產物…………………………………………17
2.6 定序分析…………………………………………………….17
2.7 Total RNA萃取……………………………………………...17
2.8 製備cDNA (Reverse Transcription) ………………………..18
2.9 即時定量聚合酶鏈鎖反應………………………………….19
2.10 抑制減除雜合法(SSH)…………………………………20
2.10.1 抽取基因組DNA…………………………………..20
2.10.2 限制酶切割及產物回收……………………………21
2.10.3 連接tester DNA與adaptor…………………………22
2.10.4 第一次雜合反應……………………………………22
2.10.5 第二次雜合反應……………………………………23
2.10.6 PCR增幅反應………………………………………23
2.10.7 PCR產物的選殖(PCR cloning)…………………25
2.10.8 轉形(transformation)…………………………….25
2.11抽取質體DNA……………………………………………...26
2.12 限制酶切割……………………………………………….27
第三章 結果與討論…………….………………………….…..........28
3.1 adeB抗藥性基因的表現與抗生素的關係………………..28
3.2 抗生素的誘導與adeB表現量之關係…………………….29
3.3 分析gyrA及parC基因突變與CIP抗藥性之相關性……31
3.4 Integron I攜帶之抗藥基因及其序列分析………………..33
3.5 SSH減除後序列比對之結果……………………………...35
第四章 結論….………………………………………………..........37
參考文獻……….……………………………………………………...38
附錄….………………………………………………...........................44
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