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標題: class1integron在鮑氏不動桿菌臨床分離株中的盛行率與VIM-11之分子特性分析
Prevalence of class 1 integron and molecular characterization of VIM-11, a carbapenem-hydrolyzing metallo-β-lactamase gene cassettes in clinical Acinetobacter baumannii isolates.
作者: 陳琬雯
chen, wanwen
關鍵字: 鮑氏不動桿菌;Acinetobacter baumannii;class 1 integron;metallo-B-lactamase VIM11
出版社: 生命科學系
鮑氏不動桿菌(Acinetobacter baumannii)為近年來院內感染常見的致病菌。這類菌株在臨床上的重要性在於其常為多重抗藥性,治療這種細菌所導致感染症的首選抗生素主要是carbapenems類的藥物,然而近年來,在世界各國相繼出現了對carbapenems具抗性的鮑氏不動桿菌。integron是近年來被認為繼抗藥性質體和轉位子外,另一個與細菌獲得新抗藥基因及散佈抗藥基因有關的DNA元件,本研究自2003到2004年間,在台灣地區北、中、南、東部九家醫院病房與加護病房病患之檢體,共採集了178株菌株。利用CS-PCR(conserved-segments PCR)來偵測integron在鮑氏不動桿菌中的盛行率,在178株鮑氏不動桿菌中,有128株菌可增幅出class 1 integron的cassette array,約佔71.9% 。從這128株菌所增幅出來的cassette array呈現多樣性,共有6種不同大小,分別為:3000 bp、2500 bp、2000 bp、1500 bp、1200 bp、1000 bp。另一方面,在探討integron的過程中,發現來自台大醫院編號6915的菌株帶有藉由integron夾帶的blaVIM-11基因,可以轉譯出含有266個胺基酸的metallo-β-lactamase VIM 11,在分類上屬於Amber class B金屬乙內醯胺酶,與VIM-2有99%的相似性,只有一個核苷酸的不同,導致一個胺基酸的改變。我們也將其表現在E. coli BL21以觀察抗藥性的變化。藥敏試驗的結果證實VIM-11的確具有廣大的受質接受性,對於廣效性青黴素、頭孢子菌素類第一、二和三代都有很好的水解能力,而對於imipenem的抗性也有提升。此外,經由南方墨點法(Southern blotting)得知夾帶VIM-11基因卡匣的integron位於染色體上,也利用了Inverse PCR的方式完成了integrase基因的全長序列,但在其緊鄰的上游序列中並未發現轉位子的存在。這也在台灣第一次於鮑氏不動桿菌中發現金屬乙內醯胺酶的蹤跡。

Acinetobacter baumannii is an important nosocomial pathogen which is often resistant to multiple antimicrobial agents. In clinical therapy, carbapenems
(imipenem and meropenem) have retained better activity than other antimicrobial agents. Nevertheless, reports of carbapenem resistance among A. baumannii. In recent years, a third mechanism of resistance gene dissemination has been discovered. This novel DNA element , now called an integron.
Between 2003 and 2004, 178 unrelated Acinetobacter baumannii isolates randomly selected from the intensive care and word of nine different hospitals in Taiwan. Using conserved segment PCR (CS-PCR) to detect integron structures, to identity the integron prevalent in Taiwan.71.9% (128/178) of isolates were shown to be integron-positive, with inserted gene cassettes of various sizes. Six sizes of inserted regions of DNA(1,000, 1,200, 1,500, 2,000, 2,500, 3,000bp) were shown to be common among class I integrons.
The 1.0kb cassette array contains blaVIM-11, a metallo-β-lactamase gene from Acnetobacter baumannii 6915, a clinical isolate from National Taiwan university hospital in 2004. The deduced 266-amino acid protein was an ambler class B β-lactamase, It's only one nucleotide differences between blaVIM-2.
leading to one amino acid alterations. Expression of the integron-borne blaVIM-11 gene in Escherichia coli BL21 resulted in a significant decrease in susceptibility to a broad array of β-lactams (ampicillin, amoxicillin
,piperacillin, cefazolin, ceftazidime, cefotaxime , imipenem). Southern hybridization with the blaVIM-11-specific probes appeared to carry the MBL gene on chromosome. This study used molecular technology such as inverse PCR to finish the sequences of whole integrase gene and its upstream 1.0 kb, but don't find transposon exist. Our findings indicate that VIM-11 is the first carbapenem-hydrolyzing metalloenzyme characterized from a Acinetobacter baumannii isolate in Taiwan.
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