臺灣博碩士論文加值系統

Stenotrophomonas maltophilia 屬需氧性革蘭氏陰性桿菌，為重要的院內感染菌，其特徵在於對許多抗生素都具有抗藥性（如：β-actam類、aminoglycoside、quinolone……等），因此常造成臨床治療與院內感染管制上的困難。Polymyxin B是一種抗菌蛋白胜肽（antimicrobial peptides），利用其正電荷與細菌表面的負電荷產生交互作用，再藉由其兩性分子的特性穿透菌體外膜，提高細菌通透性，使細胞外物質大量進入細菌體內造成細菌死亡。根據之前文獻研究：polymyxin B對於臨床所分離之S. maltophilia大都為敏感性（>70% susceptible）。首先分析自2004-5年間由台大醫院所分離的70株多重抗藥性的臨床菌株對polymyxin B的MIC，發現其中85％為抵抗性（只有7%為敏感性）。故擬探討其機制。
我的研究主要針對S. maltophilia 對polymyxin B產生抗藥性的機制作探討，可分成四個部份論述:
第一部份、自發性突變研究：將臨床菌株S21利用自發性突變篩選出高抗藥性的突變株，研究其產生抗藥性的原因。
第二部分、S. maltophilia基因體研究：許多文獻提出polymyxin B抗性與雙組成調節系統（two-component system）PhoP/ PhoQ有關，但是沒有任何文獻證明S. maltophilia具有PhoP/ PhoQ基因，所以是利用目前已發表的PhoP/ PhoQ序列與S. maltophilia基因全長做比對，再藉由軟體分析並尋找其功能模組。並選殖PhoP/ PhoQ基因。
第三部分、PhoP/ PhoQ基因表現與polymyxin B感受性分析：先測試在不同鎂離子濃度下S. maltophilia對polymyxin B感受性之改變，再利用real-time PCR確認其PhoP表現量也會因鎂離子濃度而有所改變，最後在PhoP 大量表現菌株觀察到其對polymyxin B更具抗性來推測在S. maltophilia中會感受Mg2+ 的改變，來調控polymyxin B的抗性。
第四部份、Polymyxin B合併其他藥物研究:許多研究發現多重抗藥性的綠膿桿菌（Pseudomonas aeruginosa）藉由polymyxin B與其他抗生素（erythromycin, rifampine……等）合併使用可以提高其對抗生素的感受性。因此，最後是針對polymyxin B高抗藥性的臨床菌株做研究，探討polymyxin B與其他抗生素合併使用，是否可以提高S. maltophilia對抗生素的感受性。
本文是第一篇探討S. maltophilia 具有PhoP/ PhoQ系統且此PhoP/ PhoQ系統會感受鎂（或鈣）離子的變化來調控對polymyxin B的感受性。而對polymyxin B高抗藥性之菌株，可以利用polymyxin B與其他抗生素（如，rifampine）合併使用以提高治療效果。

Stenotrophomonas maltophilia is an aerobic, nonfermentative gram-negative bacterium. The organism has increasingly emerged as an important nosocomial pathogen, particularly for immunocompromised patients. It is characterized by its hegh-level resistance to a variety of structurally unrelated antimicrobials. Polymyxin B (PB) is a potent antibacterial lipopeptide composed of a positively charged cyclic peptide ring and a fatty acid containing tail. This capacity is due to its relatively high-affinity binding to lipid A of LPS（lipopolysaccharide）. The polymyxin B has activity a wide variety of Gram-negative bacilli. We detected MIC of polymyxin B in seventy clinical isolates of S.maltophilia from NTUH, the susceptibility rate was lower than those found in prior studies（＞70% susceptible）. To unravel the resistance mechanisms of which S.maltophilia against polymyxin B, we isolated spontaneous polymyxin-resistant mutants and analyzed their difference from the wild type .
The two-component regulatory system, PhoP-PhoQ, in many Gram-negative bacteria regulates resistance to cationic antimicrobial peptides, such as polymyxin B, in response to low Mg2+ conditions. Here we firstly have identified the PhoP/ PhoQ system, in S. maltophilia by bioinformatics and gene cloning. We in addition constructed the PhoP, PhoQ and PhoP/ PhoQ overexpression strains and investracted the effect of overexpression the polymyxin B resistance in S. maltophilia. Our result indicated that overexpression of PhoP affects polymyxin B resistance. For the first time, we identified the PhoP/ PhoQ system in S. maltophilia and demonstrated that the system is regulated by Mg2+ and involved in the polymyxin B resistance.
The relative impermeability of outer membrane of S. maltophilia produces intrinsic resistance to many antibiotics. Certain polycationic substances such as polymyxin B are known to make the outer membrane of Gram-negative bacterial permeable to solutes that normally are unable to penetrate outer membrane. We tested the susceptibility of mutants and 42 clinical isolates to some antibiotics (erythromycin, rifampine, imipenem, cefepime, gentamicin, ciprofloxacin and cefotaxime) by agar diffusion method on plates containing 56U/mL of polymyxin B. We found polymyxin B increase the susceptibility of the mutants and clinical isolates to rifampin. The clinical utility of this combination remains to be established.

中文摘要• • • • • • • • • • • • • •• • 1
英文摘要 • • • • • • • • • • • •• • •• 3
第一章 緒論
第一節 Stenotrophomonas maltophilia簡介• • •5
第二節 Polymyxin B及其抗藥機轉簡介• • • • •8
第三節 多重抗藥性菌株之治療模式-合併用藥•• 15
第四節 研究緣由與目的••• • • • • •••••••18
第五節 實驗設計• • • ••••••••••••••••• 20
第二章 研究材料與實驗方法
第一節 臨床菌株之分析與自發性突變• • • • 22
第二節 生物資訊學分析• • ••••••• • • • 31
第三節 PhoP/ PhoQ基因體試驗• • • • • • •40
第四節 抗生素合併使用• • • •••••• • • •46
第三章 實驗結果
第一節 臨床菌株之分析與自發性突變• • • • 47
第二節 生物資訊學分析• • • • • • • • ••50
第三節 PhoP/ PhoQ基因體試驗• • • • • • •53
第四節 抗生素合併使用• • • • • • • • • 56
第四章 討論•• • • • • • • • • • • • •57
第五章 表 • • • • • • • •••••• • • • •61
第六章 圖 • • • • • • • •••••••• • ••65
第七章 附錄 • • • • • • • • • • • • •83
第八章 參考文獻• • • • • • • • • • • •84

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