SmeVWX多重藥物輸出幫浦之過度表現導致Stenotrophomonas maltophilia多重抗藥性之表型

中國醫藥大學機構典藏 China Medical University Repository, Taiwan > 醫學院 > 醫學檢驗生物技術學系暨碩士班 > 博碩士論文 > Item 310903500/41423

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题名:	SmeVWX多重藥物輸出幫浦之過度表現導致Stenotrophomonas maltophilia多重抗藥性之表型 Overexpression of Resistance Nodulation Division Efflux Pump SmeVWX Confers the Multidrug Resistance Phenotype of Stenotrophomonas maltophilia
作者:	黃姜敬
贡献者:	醫學檢驗生物技術學系碩士班
关键词:	抗藥性;多重藥物輸出幫浦 Efflux Pump;Multidrug Resistance
日期:	2011-07-28
上传时间:	2011-10-17 16:57:03 (UTC+8)
出版者:	中國醫藥大學
摘要:	Stenotrophomonas maltophilia 是一株具有多重抗藥性(MDR)之特性的重要伺機性感染致病菌株。在革蘭氏陰性桿菌中，多重藥物輸出幫浦系統的過度表現是一個造成多重抗藥性表型的重要因素。在本研究論文中，探討利用一個 chloramphenicol抗生素所篩選到之 S. maltophilia 突變菌株KJ09C之表型。這株突變菌株KJ09C 除了對chloramphenicol 抗生素有抗性外，更能夠對 quinolone類和tetracyclines類抗生素有抗性。令人驚訝的是突變菌株KJ09C 相較於原生菌株KJ對aminoglycoside類抗生素之感受性卻上升。由即時定量PCR（qRT-PCR）的結果說明突變菌株KJ09C之 smeU1-V-W-U2-X operon 有過度表現的現象。突變菌株KJ09C之SmeVWX 多重藥物輸出幫浦失去活性後會使突變菌株KJ09C的感受性恢復至原生菌株一樣的層級，顯示突變菌株KJ09C之SmeVWX 多重藥物輸出幫浦的過度表現有助於多重抗藥性之表型。在smeU1-V-W-U2-X 基因上游有一屬於 LysR 家族之轉錄調控基因smeRv，並藉由轉錄融合分析的結果顯示這調控基因所轉譯之調控蛋白SmeRv在smeU1-V-W-X-U2 基因的過度表現中扮演正調控的角色。在原生菌株KJ的背景下，調控蛋白SmeRv扮演一個自我負調控的角色；在突變菌株KJ09C的背景下，則是扮演一個自我正調控的角色。為了更進一步了解smeU1-V-W-U2-X 基因中每一個基因在突變菌株KJ09C中所扮演的角色，分別構築KJ09C△SmeU1、KJ09C△SmeVW、KJ09C△SmeU2、KJ09C△SmeX 等突變菌株。結果顯示突變菌株KJ09C之 smeU1 基因與多重抗藥性表型的關係不大，而且突變菌株KJ09C的smeV-W 基因失活會破壞SmeVWX 多重藥物輸出幫浦將chloramphenicol 、quinolone 類和tetracycline 類藥物排出的功能。為了進一步釐清smeU2和smeX基因對抗藥性所扮演的角色，分別將smeU2和 smeX基因在KJ09C△5菌株 ( 菌株KJ09C之smeU1-V-W-U2-X 基因刪除突變菌株) 中過度大量表現來評估其意義。結果發現SmeU2的角色似乎在SmeVWX 多重藥物輸出幫浦過度表現的情況下才有意義；只要突變菌株KJ09C的 smeX 基因單獨過度表現就能夠讓突變菌株KJ09C對aminoglycoside 類的感受性下降。 Stenotrophomonas maltophilia is an important opportunistic pathogen characterized by the phenotype of multidrug resistance (MDR). Overexpression of the resistance nodulation division (RND) efflux systems is a critical cause of the MDR phenotype in gram-negative bacteria. A chloramphenicol-selective S. maltophilia MDR mutant, KJ09C, was characterized in this study. In addition to chloramphenicol, KJ09C was cross-resistant to quinolones and tetracyclines. Surprisingly, mutant KJ09C increased aminoglycoside susceptibility compared to wild-type KJ. The results of qRT-PCR demonstrated that SmeVWX pump was overexpressed in mutant KJ09C. Inactivation of smeU1-V-W-U2-X operon of mutant KJ09C restored the antimicrobial susceptibility of KJ09C to the level as that of wild-type KJ, indicating that overexpression of SmeVWX pump contributes to the MDR phenotype of KJ09C. A LysR-type transcriptional regulator gene, smeRv, divergently located upstream of smeU1-V-W-U2-X operon. The results of transcriptional fusion assay showed that the SmeRv plays a positive role in the overexpression of smeU1-V-W-X-U2 operon, and that SmeRv has a characteristic of negative autoregulation in wild-type background and positive autoregulation in KJ09C background. To elucidate the role of each component of smeU1-V-W-U2-X operon in the resistance, a series of mutants were constructed, including KJ09C△SmeU1, KJ09C△SmeVW, KJ09C△SmeX, and KJ09C△SmeU2. The results showed the smeU1 of KJ09C had no effect on antibiogram resistance, and inactivation smeVW of KJ09C abolished SmeVWX pump activity for extrusion of chloramphenicol, quinolone, and tetracycline. To further clarify the role of smeU2 and smeX on the antibiotics resistance, smeU2 and smeX overexpression mutants were constructed in KJ09C△5, including KJ09C△5L2::SmeU2 and KJ09C△5L2::SmeX. The role of SmeU2 seems significant only when the SmeVWX pump is overexpressed. The smeX overexpression of KJ09C is responsible for the decreased aminoglycoside resistance of KJ09C.
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