Stenotrophomonas maltophilia 誘發L1及L2乙內醯胺酶基因表現之探討; Induction studies of L1 and L2 β-lactamases in            Stenotrophomonas maltophilia

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 310903500/874

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題名:	Stenotrophomonas maltophilia 誘發L1及L2乙內醯胺酶基因表現之探討;Induction studies of L1 and L2 β-lactamases in Stenotrophomonas maltophilia
作者:	蕭盈如;Ying-Ju Hsiao
貢獻者:	中國醫藥大學：醫學檢驗生物技術學系碩士班
關鍵詞:	乙內醯胺酶;β-lactamases
日期:	2007-07-06
上傳時間:	2009-08-12 17:09:54 (UTC+8)
摘要:	Stenotrophomonas maltophilia 為多重抗藥性的細菌，其位於染色體上的 L1, L2 二基因可經由誘發(induction) 產生高活性 β-lactamase，可水解 β-lactam 類的抗生素。本實驗菌株來源為中國醫院大學附設醫院細菌檢驗室所提供臨床菌株，S. maltophilia N。利用基因替換方式(Gene replacement mutagenesis )突變N菌的 L1， L2 二基因取得NL1-及NL2-之突變株，分別在L1，L2 二基因起始氨基酸 (Met)下游置入一個和L1， L2 二基因轉錄方向一致的報導基因, xlyE。因此可以觀察報導基因, xlyE的表現來評估L1，L2 二基因啟動子分別被誘發(induction)的能力。為了評估L1及L2基因是否會因為在培養基中加入5%人類血清或因改變生長環境中的碳源而誘發(induction)或影響二基因表現，我們在培養NL1-及NL2-突變株過程中加入5 %人類血清及在培養NL1-及NL2-突變株過程中分別加入1%的碳源，不會對L1及L2基因產生誘發情形。為了評估clavulanate是否會誘發S. maltophilia 的Class B L1 ??-lactamase， Class A L2 ??-lactamase二基因表現而導致clavulanate 和其他β-lactam抗生素一起治療時產生拮抗(antagonism)或協同(synergism)作用，因此分別測試2、8、10、16、50 ?慊/ml clavulanate 對 L1， L2 基因誘發的能力，分別得知clavulanate 對L1，L2 二基因表現的誘發能力為concentration dependent。因此我們利用抗生素感受性的結果並分別測試在不同濃度的clavulanate及搭配不同β-lactams抗生素而產生共同或拮抗誘發L1， L2 二基因啟動子表現。得知NL1-在濃度為Cab/CA(8/4) μg/ml ，對L1 基因有induction synergism的情形。NL2-在濃度Azt /CA(32/16) μg/ml ，對L2 基因有induction synergism的情形發生。因clavulanate/β-lactams抗生素組裝方式所引起的L1， L2 二基因induction表現，可提供在選擇治劑上會影響療效的因素：1.選擇適當β-lactams抗生素與clavulanate的組裝2.選擇治劑組裝的適當比例。 Stenotrophomonas maltophilia has assumed an increasingly important role as a nosocomial pathogen in compromised patients.。S. maltophilia isolates are resistant to many clinically useful antibiotics，with β-lactam resistance mediated through two β-lactamases，L1 and L2，whose expression is induced when cells are exposed to β-lactam antibiotics。Little is known about the induction of L1 and L2 β-lactamases。A clinical S. maltophilia isolate, S. maltophilia N, was obtained in China Medical University Hospital. The PCR amplicons containing L1 and L2 ??-lactamase genes, respectively, were sequenced and analyzed. The L1- and L2- mutants of S. maltophilia N were constructed by gene replacement strategy, respectively. A reporter gene, xylE, is inserted into the L1 and L2 gene, down-strand the promoter of L1 and L2 gene and with the same orientation as that of L1 and L2 gene, respectively. The induction of L1 and L2 gene promoters can be monitored by analyzing the catechol 2,3-dioxygenase (C23O) activities in different induced conditions. The studies in the thesis were designed to elucidate the factors that affect the induction of L1 and L2 gene promoters. The induction potency of cefuroxime against NL1- and NL2- is independent on the type of carbon source in growth medium and the addition of 5% human serum. Clavulanic acid acts as an inducer toward the L1 and L2 gene promoters, and its induction potency against L1 and L2 genes is concentration dependent. The susceptibility test of S. maltophilia N against different combined regimens of ??-lactams/clavulanate was evaluated.The factors affected the effect of combination regimens were elucidated using the isogeneic mutants, NL1- and NL2-, as the assayed systems. To be a successful combined regimen, three important elements must be considered: (1) The hydrolytic ability of L1 toward the selected ??-lactam. (2) The minimal concentration of clavulanate needed to inhibit the activity of induced L2 enzyme. (3) The occurrence of induction synergy by the combination of ??-lactam and clavulanate.
顯示於類別:	[醫學檢驗生物技術學系暨碩士班 ] 博碩士論文

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