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研究生:黃哲經
研究生(外文):Che-Ching Huang
論文名稱:不動桿菌屬genomicspecies13TU其β-lactamase在抗藥機制中所扮演的角色
論文名稱(外文):The role of β-lactamase in Acinetobacter genomic species 13TU in imipenem resistance
指導教授:胡文熙
指導教授(外文):Wensi S. Hu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
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:65
中文關鍵詞:imipenem不動桿菌屬genomic species 13TU水解酵素OXA-58IMP-1CENTA
外文關鍵詞:imipenemAcinetobacter genomic species 13TUβ-lactamaseOXA-58IMP-1CENTA
相關次數:
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不動桿菌(Acinetobacter genomic species 13TU)抗imipenem 臨床菌株︰ 22、25、
1245、h34 以及不抗imipenem 菌株︰5、6、10、13、14、15、17、18、19、21,
以multiplex PCR 去探測class D β-lactamase 基因,在經過定序確認得知
imipenem 抗藥菌株皆有blaOXA-58 基因,而在不抗imipenem 菌株之中只有A5 有
OXA-58 基因。此外發現在blaOXA-58 基因上游序列發現有ISAba3,且在偵測
OXA-58 之mRNA 中除了在imipenem 抗藥菌株有表現之外,在imipenem 不抗
藥菌株A5 幾乎相同表現量。接著用MBLs strip 去偵測class B β-lactamases 基
因,只有在四株抗imipenem 臨床菌株中發現存在blaIMP-1 基因,且RT-PCR 實驗
證實RNA level 幾乎相同,我們分別建構了帶有blaOXA-58 以及blaIMP-1 的表現載
體,在大腸桿菌系統大量表現蛋白,以瞭解OXA-58 與IMP-1 是否俱有抗藥效
能,經E-test strip 測MIC 之值,發現帶有blaOXA-58 基因與blaIMP-1 基因在大腸桿
菌,分別上升了三倍和八倍。另外我們以CENTA 為受質做酵素活性分析實驗,
發現IMP-1 的活性為OXA-58 活性的兩倍以上。以上兩項實驗結果說明IMP-1
的抗藥效能較OXA-58 有效。此外分離Acinetobacter 抗imipenem 臨床菌株與不
抗imipenem 臨床菌株的periplasmic fraction,以SDS-PAGE 去比較分析,發現在
分子量約27kDa 的蛋白質且抗imipenem 臨床菌株顯著增加,經質譜儀分析發現
為IMP-1 的蛋白酵素卻無OXA-58 的蛋白酵素。這些結果說明了IMP-1 的生成
及送至periplasm 而導致Acinetobacter13TU 臨床抗藥菌株俱抗imipenem 的能力。
Fourteen clinical isolate of Acinetobacter genomic species 13TU were used in
this study. These isolates are imipenem-resistant strains(A22、A25、A1245、Ah34)
and imipenem-susceptible strains (A5、A6、A10、A13、A14、A15、A17、A18、
A19、A21)strains were used in this study.
All imipenem-resistant strains and one imipenem-susceptible strain A5 contain
class D β-lactamase gene, blaOXA-58 using multiplex PCR. An insertion sequence,
ISAba3, located in the upstream of blaOXA-58 in the all above strains. Also the mRNA
of blaOXA-58 gene of these strains are similar.
Subsequently, we detected the class B β-lactamase may also exist in the
imipenem-resistant strains but not imipenem-susceptible strains using MBLs E-test
strip. Furthermore, PCR showed blaIMP-1 gene present in the four imipenem-resistant
strains, and RT-PCR results demonstrated that the RNA expression levels in these
strains were similar. To elucidate the contribution of OXA-58 and IMP-1 in imipenem
resistance, we constructed pBAD /Myc-His-OXA-58 and the pBAD /Myc-His-IMP-1
clones and then introduced into E.coli Top10 system. The MIC value of pBAD
/Myc-His-IMP-1 strain was 8-folds increased compared to pBAD /Myc-His-OXA-58
was 3-folds increased. Besides, the enzyme activity assay of the OXA-58 and IMP-1
using the CENTA as the substrate, the results showed that the activity of IMP-1 has
about 2-folds higher than those of OXA-58. Then, we purified the periplasmic
fraction of clinical isolate and was analyzed using the SDS-PAGE. The results showed
that a protein band about 27kDa was revealed in the imipenem-resistant strains but
not in the imipenem-susceptible. The result of mass spectrometry demonstrated that
only IMP-1 was identified in these 27KDa protein bands. Taken together, these results
6
suggest that IMP-1 rather than to OXA-58 hydrolyze imipenem and resulted in
imipenem resistence of A22, A25, A1245 and Ah34.
中文摘要............................................................4
英文摘要............................................................5
緒論................................................................7
ㄧ、不動桿菌屬簡介............................................................................................7
二、細菌之抗藥機制............................................................................................8
(1)細菌對抗生素的通透性(membrane permeability)改變:............8
(2)細菌運用efflux pump system 將抗生素排出體(active efflux)
........................................................................................................................9
(3)改變抗生素與細菌結合的部位(altered target)....................11
(4) 產生酵素將抗生素分解..................................................................11
三、抗生素Carbapenem.....................................................................................15
材料與方法.........................................................17
一、細菌培養基.....................................................17
二、引子序列(primers)............................................17
三、藥品和酵素.....................................................17
四、菌種貯存.......................................................18
五、檢測β-lactamase 活性所需試劑..................................18
六、最低抑菌藥物濃度Minimum Inhibitory Concentration(MIC)測定...18
七、DNA 選殖(DNA cloning) ........................................19
八、RNA 之萃取純化.................................................22
九、反轉錄-聚合酵素連鎖反應 (Reverse-transcription PCR)..........23
十、蛋白質定量(Protein Determination)............................23
十一、蛋白質膠體電泳製備(SDS-PAGE)...............................24
十二、蛋白質質譜分析法 (In gel digestion)..........................25
十三:抽取periplasm 蛋白..........................................25
十四:利用超音波破菌法(Sonicator method)收集β-lactamase.........26
十五:β-lactamase 活性分析........................................26
實驗結果...........................................................27
ㄧ︰class D oxacillinase 與其上下游序列定序.........................................27
二︰Class B β-lactamase..............................................................................28
三︰RT-PCR 分析IMP-1 及OXA-58 在抗藥與不抗藥株的表現量...................28
四︰imipenem 抗藥性在大腸桿菌中分別大量表現OXA-58 與IMP-1 蛋白質
..............................................................................................................................29
五︰OXA-58 與IMP-1 酵素活性分析.................................................................31
六︰The periplasmic fraction of 13TU......................................................32
討論...............................................................34
參考文獻...........................................................60
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