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標題: Imipenem抗性包氏不動桿菌臨床分離株之分子特性
Molecular Characterization of Clinical Imipenem-resistant Acinetobacter baumannii isolates
作者: 吳敏華
Wu, Min-Hua
關鍵字: Imipenem-resistant;Imipenem抗性;Acinetobacter baumannii;包氏不動桿菌
出版社: 生命科學院碩士在職專班
包氏不動桿菌(Acinetobacter baumannii),為革蘭氏陰性之球桿菌,為一常見的環境微生物,亦是人類皮膚與黏膜上的正常菌叢之一,近年來卻因其抗藥性所帶來之問題趨嚴重,而成為院內感染的重要病原菌,且已陸續出現對最後一線抗生素imipenem具抗藥性的菌株(imipenem-resistant Acinetobacter baumannii ;IMRAB) 。近年來在台灣也頻頻出現IMRAB,且其對他種抗生素抗藥性的普遍性也日益嚴重,因而喚起國內外對包氏不動桿菌抗藥性之問題的注意與關切。
本研究以台中澄清醫院中所收集的IMRAB臨床菌株之流行病學資料為基礎,分析其表現型與基因型的相關性,並探討其抗藥性基因是否經水平傳播的方式散佈於各菌株間之可能性。使用的臨床菌株是民國九十一年六月一日到民國九十二年五月三十一日止,在台中澄清醫院分離出的12株imipenem-resistant包氏不動桿菌為材料,分別再以Disk diffusion experiment與VITEK 2 Advance Expert System等抗生素藥物敏感性測試來測定這12株菌株的抗生素抗藥性表現型。另經脈衝電場電泳(PFGE)電泳後,用圖譜分析軟體-BioNumerics進行比對,描繪菌株親緣關係。PFGE結果顯示可將這12株IMRAB菌株,分成2個群組(cluster A、B)。
此外,以聚合連鎖反應(PCR)方法進行Carbapenem的抗藥基因(blaOXA23、 blaOXA24 、 blaIMP-1 、blaVIM-1、blaVIM-2)與水平傳播平台Integron內含的抗藥基因來分析,結果顯示,這12株菌中並未發現有與Carbapenem的抗藥基因有關之特異性產物,但這12株IMRAB菌株均帶有一個約2.5 Kb的第一類integron,其中的cassette排列方式也完全相同,這結果顯示出抗藥基因水平傳播的可能性。在藉由轉位子移動的Integron之探討實驗中,以Tn21反向重複序列所設計的引子進行single primer PCR之結果,N29、N33、N34、N38、N40在約8 kb處得到了一個大小相似的產物,但經南方墨點法顯示本transposon-like的區域中,並無上述2.5kb的第一類integron存於其中。本研究再利用mating的方式,檢討抗藥性基因水平傳播之移動性,結果發現ampicillin抗性基因亦可能藉由質體來進行水平傳播。

Acinetobacter baumannii, a coccobacilli, is a common environmental microbe as well as one of the normal floras on the skin and mucous membranes of human bodies. Due to the serious problems resulted from drug resistance of such germ for the past few years, it becomes a critical pathogenic bacteria causing infections in the hospitals. Imipenem-resistant Acinetobacter baumannii (IMRAB), a strain that resists imipenem, the ultimate antibiotic, has appeared. Recently, IMRAB also discovered in Taiwan very frequently and caused a deteriorating problem of drug resistance against other antibiotics. Therefore, awareness of such problems and finding out proper solutions have become critical issues both in our country and overseas.
Based on the epidemic information on clinical IMRAB strains collected by Cheng Ching Hospital, Taichung, the correlation between their phenotype and genotype is analyzed in this research. In addition, the possibility of whether the drug-resistant gene diffuses among strains in a horizontal way is explored. The twelve imipenem-resistant Acinetobacter baumannii strains used in this study were clinical strains isolated from in Cheng Ching Hospital, Taichung from June 1, 2002 to May 31, 2003. They were further conducted an antibiotic susceptibility test by the Disk diffusion experiment and VITEK 2 Advance Expert System to explore the phenotype of these twelve imipenem-resistant strains. Furthermore, the “BioNumerics” analysis software was applied after PFGE to compare and locate the genetic/phylogenesis relationship among the strains. The PFGE result indicated these twelve IMRAB strains could be divided into two clusters (Cluster A & Cluster B).
Besides, drug-resistant genes of Carbapenem (blaOXA25, bla OXA24, bla IMP-1, bla VIM-1 & bla VIM-2) and those of Integron, a horizontal diffusion platform, were analyzed with the PCR method. No specificity PCR products were found to the Carbapenem-resistant related genes of the twelve isolated strains. However, there was a type 1 Integron of about 2.5 Kb existed in all these twelve IMRAB strains and the arrangement of cassette was the same. The result revealed the possibility of horizontal transfer of the drug-resistant genes. A single primer PCR method by using Tn21 inverted repeat sequence was performed to found mobile element such as Tn21 that can carry our type 1 integron. A product of a similar size was obtained at about 8 kb for N29, N33, N34, N38 and N40. However, no type 1 integron of 2.5 kb existed in the transposon-like region by using Southern blotting and type 1 integron probe. On the other hand , the approach by mating experiments was used to probe whether the mobility of drug-resistant genes in a horizontal diffusion way or not. he result indicated drug-resistant genes of ampicillin could be diffused horizontally via conjugation.
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