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研究生:廖玉婷
研究生(外文):Yu-Ting Liao
論文名稱:抗藥性蛋白Oxacillinase58在鮑氏不動桿菌及大腸桿菌之特性分析
論文名稱(外文):Characterization of drug resistance protein Oxacillinase 58 in Acinetobacter baumannii and Escherichia coli
指導教授:卓文隆
指導教授(外文):Wen-Long Cho
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:92
中文關鍵詞:鮑氏不動桿菌大腸桿菌Oxacillinase 58
外文關鍵詞:Acinetobacter baumanniiEscherichia coliOxacillinase 58
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鮑氏不動桿菌(Acinetobacter baumannii)是一種伺機性感染致病原,常見於院內感染並且大多具有多重抗藥性。近年來碳青黴醯類(carbapenem)抗生素,包含imipenem及meropenem,成為用來治療具多重抗藥性之鮑氏不動桿菌的主要首選,然而卻也導致鮑氏不動桿菌產生了抗藥性,最主要的機制就是產生D類的乙內醯胺酶。
2004年於台北榮民總醫院分離出一株臨床抗藥性鮑氏不動桿菌株TVICU53,在其帶有的質體上發現blaOXA-58基因,將此基因與穿梭載體接合後轉型至勝任細胞株:鮑氏不動桿菌(Ab290)及大腸桿菌(DH5α),發現這兩個轉型株的抗藥表現不盡相同,在本研究中,主要針對OXA-58蛋白在鮑氏不動桿菌及大腸桿菌的功能差異進行分析。首先發現以不同劑量的ticarcillin及imipenem處理Ab290-OXA-58或DH5α-OXA-58,相同的OXA-58質體,在Ab290-OXA-58中OXA-58蛋白的表現量高於在DH5α-OXA-58;改以pET表現系統將大腸桿菌內的OXA-58蛋白表現量提高仍然無法提高imipenem的最小抑制濃度,並且Ab290的蛋白萃取物也無法幫助DH5α-OXA-58抗imipenem。然而當DH5α-OXA-58培養到生長固定期,其死亡的菌體會釋放出OXA-58蛋白而讓imipenem感受性菌株存活於含imipenem的上清液中。
Ab290-OXA-58表現的OXA-58蛋白被發現會主動釋出菌體外,並且會被imipenem所刺激而大量釋出;其與imipenem作用後最佳的釋出環境分別為:培養溫度30℃、在含有0.2 M NaCl或pH 7的培養基,並且在培養8小時可偵測到最大量的胞外OXA-58蛋白。有趣的是,Ab290-OXA-58釋出的OXA-58蛋白能夠幫助感受性Ab290及DH5α生長於含有imipenem的上清液,並且將Ab290 -OXA-58以腹腔注射感染BALB/c 小鼠於十天後可在其血清中偵測到抗OXA-58的抗體。
以程式分析發現OXA-58蛋白的N端帶有可能的訊號胜肽,將該訊號胜肽刪除後的signal peptide deleted OXA-58(ΔSP OXA-58)蛋白仍然可以在Ab290-ΔSP OXA-58中大量表現卻無法對抗imipenem。將Ab290-ΔSP OXA-58的胞內ΔSP OXA-58蛋白以及DH5α-OXA-58的胞內OXA-58蛋白萃取出,發現它們都具有抗藥的功能,並幫助感受性菌株存活於含有imipenem的培養基,而在兩菌株的胞外週質皆無法偵測到ΔSP OXA-58蛋白或OXA-58蛋白,由此可知OXA-58蛋白要到達胞外週質才能夠讓細菌本身抗imipenem。
自Ab290-OXA-58釋出的胞外OXA-58蛋白能夠幫助碳青黴醯類敏感的菌株存活,此現象不論在in vivo及in vitro已都被證實。而且這是第一次發現OXA-58蛋白能夠從鮑氏不動桿菌中釋出,而這也說明了何以抗碳青黴醯類的鮑氏不動桿菌菌株感染病人體內偶而也會伴有感受性菌株共同感染。
A. baumannii is an important opportunistic pathogen responsible for a variety of nosocomial infections and usually has multiple drug resistance. Carbapenems are β-lactam antibiotics including meropenem and imipenem which have been the drugs of choice to treat severe infections caused by A. baumannii. An efficient mechanism for A. baumannii to resist carbapenem is by producing Class D β-lactamase ( Carbapenem hydrolyzing oxacillinases, CHDLs ).
An OXA-58 gene was isolated from a clinical isolate TVICU53 and characterized extensively by Chen et al., 2008. An blaOXA-58 gene was cloned into a shuttle plasmid and transformed into A. baumannii (Ab290) and E. coli (DH5α). The antibiotic performance of OXA-58 in Ab290-OXA-58 and DH5α-OXA-58 were quite different. In this study, the molecular and functional difference of OXA-58 in A. baumannii and E. coli was investigated. The expression of OXA-58 in Ab290-OXA-58 or DH5α-OXA-58 was constant and independent to different dosages of imipenem and ticarcillin treatments, but that of Ab290-OXA-58 was higher than that of DH5α-OXA-58.To increase the expression OXA-58 by changing to pET expression system in E .coli could not increase the MIC of imipenem. And the protein extracts from imipenem susceptible Ab290 could not make DH5α harbored pOXA-58 grow in the broth with imipenem. In addition, OXA-58 of DH5α-OXA-58 could be released when DH5α-OXA-58 were at stationary phase.
OXA-58 protein was found to be liberated from Ab290-OXA-58 and the liberation was inducible by imipenem. The optimal liberation of OXA-58 in Ab290-OXA-58 is at 30℃, in LB broth containing 0.2 M salt, at pH7 and peaks at 8 hours. Interestingly, the liberated OXA-58 in broth could help susceptible Ab290 and DH5α to grow in the broth with imipenem. The anti OXA-58 antibody in serum could be detected 10 days post injection of Ab290- OXA-58 into BALB/c mice.
A putative signal peptide-deleted OXA-58 (ΔSP OXA-58) could still be highly expressed in Ab290-ΔSP OXA-58, but this strain could not resist to imipenem. The protein extracts from Ab290-ΔSP OXA-58 or DH5α-OXA-58 could help Ab290 and DH5α grow in the broth with imipenem 1μg/ml. However, ΔSP OXA-58 and OXA-58 could not be detected in periplasm of these two strains. It indicates that the translocation of OXA-58 to the periplasm is essential for host cells to be resistant to imipemem. The ΔSP OXA-58 produced by Ab290/ΔSP-OXA-58 might not translocate to the right place resulting in loss of resistance, but extracted OXA-58 andΔSP-OXA-58 both were functionally resistant to imipenem.
The liberated OXA-58 helps imipemem or meropenem susceptible bacteria growth was examined in vitro and in vivo. This is the first finding that OXA-58 could be liberated from A. baumanni and it also explains that why both Carbapenem resistant A. baumannii amd Carbapenem susceptible bacteria other than A. baimannii could be isolated from abscess of the same patient treated by Carbapenem for some time.
致謝………………………………………………………………… i
縮寫表……………………………………………………………… ii
中文摘要…………………………………………………………… iii
英文摘要…………………………………………………………… v
目錄………………………………………………………………… vii
壹、 緒論 …………………………………………………… 1
貳、 材料與方法 …………………………………………… 12
參、 結果 …………………………………………………… 24
肆、 討論 …………………………………………………… 37
伍、 參考文獻 ……………………………………………… 44
陸、 圖表 …………………………………………………… 49
柒、 附錄 …………………………………………………… 84
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