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研究生:張瑋哲
研究生(外文):Wei-Che Chang
論文名稱:不動桿菌屬乙內醯胺酶OXA-58其基因啟動子與imipenem抗藥性之研究
論文名稱(外文):Characterization of blaOXA-58 promoter associated with imipenem resistance in Acinetobacter spp.
指導教授:馮長風馮長風引用關係陳德禮陳德禮引用關係
指導教授(外文):Chang-Phone FungTe-Li Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:臨床醫學研究所
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:2009
畢業學年度:97
語文別:中文
論文頁數:74
中文關鍵詞:不動桿菌屬乙內醯胺酶啟動子
外文關鍵詞:blaOXA-58Acinetobacter spp.imipenem
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Acinetobacter spp.不動桿菌屬近年來造成的院內感染日益增加,且對於多種抗生素產生抗藥性,包括廣效性��-lactams,aminoglycosides,和fluoroquinolones等,其中carbapenem類的藥物是治療Acinetobacter spp.感染最重要的藥物,然而具有抗藥性的菌株比例正急遽增加。從過去的論文研究發現blaOXA58週邊常存在有插入序列(insertion sequence)影響其抗藥性表現,因此我的研究目的想探討這些Acinetobacter spp.其blaOXA58周邊基因結構及這些基因結構對於抗藥性的貢獻。
本實驗從台灣北、中、南三家醫學中心收集得到208株Acinetobacter spp.,利用PCR篩選出其中48株帶有blaOXA58的菌株;脈衝式電泳及限制酶分析這些Acinetobacter spp.之親源性和質體DNA圖譜後,篩選出17株不同基因型態的菌株。利用PCR偵測blaOXA58週邊是否存在特殊的插入序列,結果發現這些基因的上游有3種不同插入序列組合;其中一株Acinetobacter baumannii具有IS1008-△ISAba3-blaOXA58,2株Acinetobacter 13TU具有△ISAba3-IS1006- △ISAba3-blaOXA58,剩下的14株則為ISAba3-blaOXA58;而在這些blaOXA58的下游則皆存在一個ISAba3。其中IS1006是首次發現存在blaOXA58上游,因此針對這段特殊的序列做後續的研究。
I-Ceu I搭配Southern blot的實驗發現這段IS1006-△ISAba3-blaOXA58位於這株細菌的質體上。將這段IS1006-△ISAba3-blaOXA58轉殖到穿梭載體並利用電穿孔送入不具有blaOXA58的標準菌株ATCC17903 (A.13TU),結果顯示經過轉殖的ATCC17903(A.13TU) imipenem之MIC從0.19μg/ml提升至≧32μg/ml,並增加對於meropenem、piperacillin-tazobactam、piperacillin、cefuroxime之抗藥性。而這個現象在DH5α (E.coli)一樣可以被觀察到,但其抗藥性提升幅度相對低於ATCC17903 (A.13TU);特別是針對carbapenem類的抗生素,雖然抗藥性有些微的提升,但仍具有感受性。5’RACE發現IS1006-△ISAba3-blaOXA58序列的轉錄起點位於blaOXA58上游ISAba3內;配合軟體分析其啟動子的位置,結果顯示在這株A.13TU內ISAba3及IS1006分別提供了-10及-35的複合啟動子(hybrid promoter)給下游的blaOXA58。Promoter assay確認這些啟動子的位置及對於抗藥性的貢獻,發現當刪除了IS1006提供的-35啟動子,將使這株經轉殖的ATCC17903對imipenem產生異質性(heteroresistance);如果同時刪除-10(ISAba3所提供)和-35(IS1006所提供)這兩個啟動子則使imipenem之MIC從≧32μg/ml下降至0.38μg/ml倍。這些實驗結果證明這個ISAba3及IS1006一起組成的複合啟動子確實能夠驅動下游的blaOXA58表現,進而使A.13TU對imipenem產生抗藥性。
In recent years, several outbreaks of nosocomial infections caused by Acinetobacter spp. strains were resistant to a wide range of antibiotics, including broad-spectrum ��-lactams, aminoglycosides, and fluoroquinolones. Carbapenems are the most important drugs for treating Acinetobacter infections. However, this resistance rate is increasing. blaOXA58 gene have been shown to contribute significantly to carbapenem resistance in Acinetobacter spp.,and it has been shown that the blaOXA58 gene is often associated with insertion sequences involved in its expression.
The aim of this study is to investigate the genetic structures surrounding the blaOXA58 genes in Acinetobacter spp. isolates in Taiwan and their contributions to antibiotic resistance. A total of 208 Acinetobacter isolates, collected from 3 medical centers and a local hospital in northern, middle and southern of Taiwan were screened, and 48 of them were blaOXA58 positive. Pulsed-field gel electrophoresis (PFGE) and plasmid pattern analysis were used to observe their correlation. A total of 17 different DNA pulsotypes of Acinetobacter spp. were gained. Different IS primers were used to PCR with blaOXA58 primers, and 3 different kinds of IS elements (ISAba3, IS1006, IS1008) were found in upstream of blaOXA58. There was a ISAba3 downstream of blaOXA58 in all strains.
It was the first time to find two copies of blaOXA-58 DNA fragment
△-ISAba3-IS1006-△ISAba3-blaOXA58-ISAba3-blaOXA58-ISAba3 in A.13TU. Therefore, a further detail study had been performed. Southern blot showed that was a plasmid borne DNA fragment. The cloning experiment showed that IS1006-△ISAba3-blaOXA58 can increase imipenem(IMP) resistance from 0.19μg/ml to ≧32μg/ml folds, and also increase meropenem(MEM), cefuroxime(CXM), piperacillin-tazobactam(TZP), and piperacillin(PIP) resistance in A.13TU. The phenomenon was also found in DH5α(E.coli), but the increment of carbapenem resistance was much lower than in A.13TU. 5’RACE found that the transcription start site was in ISAba3. Promoter assay and RT-PCR were used to observe the promoter contribution and mRNA expression. The result, showed that IS1006 and ISAba3 provide -35 and -10 hybrid promoters, respectively to drive downstream blaOXA58 expression. When deleted the P2 promoter which provided by IS1006, the mRNA level decreased, and IMP MIC became heteroresistance; if deleted -35 and -10 promoters, it seems that no mRNA expression, and IMP minimum inhibitory concentration(MIC) decreased from ≧32μg/ml to 0.38μg/ml.
In conclusion, this study demonstrated blaOXA58 is a transposon-borne gene. ISAba3-blaOXA58-ISAba3 can be found in many Acinetobacter species. A plasmid borne△ISAba3-IS1006-△ISAba3-blaOXA58-ISAba3-blaOXA58- ISAba3 was found in a A. 13TU. The insertion of IS1006 provide a new hybrid promoter to increase the transcription of the blaOXA58, and also increase IMP MEM,CXM,TZP, PIP resistance in A.13TU.
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目錄
中文摘要……………………………....................................................................................i
英文摘要…………………………………………………………………………………..iii
目錄………………………………………………………………………………………...v
第壹章、緒論……………………………………………………….……………………..1
一、Acinetobacter不動桿菌屬之簡介………………………………….…………………1
二、細菌之抗藥機制…………………………………………………….………………....2
三、碳青黴烯酶carbapenemase……………………………………………..……………..3
四、抗生素imipenem………………………………………………………………………5
五、跳躍子transposon…………………………………………………………………..….6
六、研究目的………………………………………………………………………….……7
第貳章、材料與方法…………………………………….………………………………..8
一、實驗菌株………………………………………………………………………….…..8
二、引子序列………………………………………………………………………….…..8
三、抗生素最低抑制濃度MIC…………………………………………………………....8
1 E-test………………………………………………………..……………………….8
2 Agar dilution method……………………………………………………………..…9
3 Vitek 2 Compact…………………………………………..………………………...9
四、脈衝式膠體電泳PFGE……………………………………………………………….10
1 菌體培養………………………………………………………………………….10
2 菌體包埋及菌體溶解…………………………………………………………….10
3 膠體清洗………………………………………………………………………….11
4 限制酵素切割…………………………………………………………………….12
5 脈衝式電場電泳………………………………………………………………….12
6 GelCompar V4.x親源性分析……………………………………………………..13
五、質體DNA純化……………………………………………………………………….13
六、聚合酶連鎖反應(polymerase chain reaction;PCR)……...………………………......14
七、序列資料分析………………………………………………………………………...15
八、南方雜交法Southern blot………………………………...…………………………..15
1 薄膜轉印………………………………………………………………………….16
2 探針製作………………………………………………………………………….17
3 雜交與呈色……………………………………………………………………….17
九、DNA轉殖(DNA cloning)…………………………………………………………….18
十、質體電穿孔轉移試驗electroporation……………………………….……………….19
十一、RNA之萃取純化(RNA extraction)……………………………………………19
十二、5’端基因快速放大技術 ( Rapid Amplification of cDNA End;5’RACE )……..20
十三、反轉錄-聚合酵素連鎖反應(Reverse-transcription PCR;RT-PCR)…………21
第參章、結果……………………………………………………………………….…….23一、臨床blaOXA58 PCR陽性的實驗菌株及其imipenem MIC測試………………….23
二、脈衝式膠體電泳(PFGE) …………………………………………………………….23
三、實驗菌株基因親原性分析………………………………………………………......23
四、利用膠體電泳SDS-PAGE分析實驗菌株的plasmid ………………………………24
五、利用膠體電泳SDS-PAGE分析經過限制酶切割實驗菌株的plasmid …………....24
六、以PCR檢測blaOXA-58上下游基因結構……………………………………………24
七、blaOXA58 週邊基因結構定序………………………………………………………24
八、南方雜交法(Southern Blot)分析plasmid上的blaOXA-58………………………...…25
九、南方雜交法(Southern Blot)搭配I-Ceu I分析細菌內blaOXA-58位置……………...25
十、Clone IS1006-△ISAba3- blaOXA58並利用電穿孔送入ATCC17908表現…………26
十一、Clone IS1006-△ISAba3- blaOXA58並利用電穿孔送入E.coli (DH5α)表現....…26
十二、E-test、Agar dilution method、Vitek 2 Compact偵測細菌抗藥性變化………..26
十三、5’RACE (Rapid Amplification of cDNA End)分析……………………………….27
十四、Promoter assay…………………………………………………………………….27
十五、RT-PCR觀察blaOXA58 mRNA表現……………………………………………...28
第肆章、討論……………………………………………………...…...…………………29
圖表……………………………………………………………………………………….34
參考文獻………………………………………..……………...………………………....61
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