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研究生:李佩娟
研究生(外文):Pei-chuan Li
論文名稱:鼠傷寒沙門氏桿菌之baeSR訊息傳導系統之baeR基因表現ceftriaxone抗藥性
論文名稱(外文):Overexpression of the response regulator BaeR of the two-component system confer ceftriaxone resistance in Salmonella typhimurium
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:2003
畢業學年度:91
語文別:中文
論文頁數:74
中文關鍵詞:鼠傷寒沙門氏桿菌baeR訊息傳導系統頭孢子素抗藥性
外文關鍵詞:Salmonella typhimuriumbaeRtwo-component systemcephalosporinantibiotic resistance
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鼠傷寒沙門氏桿菌為重要的食物中毒菌株,且已知具有多種抗藥的特性,近年來也發現對頭孢子素有抗藥性增加的趨勢。目前臨床上以第三代頭孢子素最為廣泛使用,故本論文主要分析鼠傷寒沙門氏桿菌對第三代頭子素ceftriaxone(CRO)抗藥性的相關基因。本研究利用Tn5 mutagenesis的技術,將轉位子Tn5送入具有高抗CRO的S. typhimurium 菌株中,建立一個Tn5 mutant library。從這library篩選出46株對CRO-sensitive菌株,經由Southern blot分析出這些CRO-sensitive菌株,皆只有單一Tn5 插入,確定只有單一個基因遭受Tn5破壞後,進而分析這些菌株基因的破壞位置,再依各個基因產物的功能分成四大類,分別為:一、transporter and membrane proteins二、proteins related to LPS 三、enzyme 四、其它。我們從這些CRO-sensitive的菌株中,挑選baeR突變株做更深入的分析,主要是baeR 被破壞後抗藥程度下降5倍以上之多,且baeR為baeSR two-component system中的regulator,可調控其它的regulon。而在complementation 實驗中,我們overexpression BaeR可使baeR mutant菌株回復成原來對CRO的抗藥特性。我們更進一步地想知道baeR調控何種基因而導致對CRO的抗性,藉由2D電泳圖比較高抗菌株(WT200)與baeR mutant低抗菌株的outer-membrane proteins,發現到STM3031在baeR mutant中大量減少, OmpW則大量增加,而STM3031及OmpW的表現量是否為baeR直接或間接所調控則需要再更進一步地研究。
Salmonella enterica serovar Typhimurium is an important etiological agent causing human food-borne illness, and known with the property of multidrug resistantce. The third-generation cephalosporin is the popular drug which applied for treatment of infection. However, it has a tendency towards cephalosporin resistance, recently. In this study, we investigated genes are involved in Salmonella serova typhimurium to ceftriaxone(CRO) resistance. Tn5 tansposome was transformed into highly resistance strain of salmonella typhimuium by electroporation to construct Tn5 mutant library. Screening of this library, 46 CRO-sensitive mutant strains were obtained. Every mutant strain has confirmed to have a single Tn5 insertion by Southern hybridization assay, and then the inserted position in chromosomal gene was identified. Based on the function of protein, these genes were classified into four groups that include: transporter and membrane proteins; proteins related to LPS; enzyme and others. We selected baeR gene of CRO-sensitive mutants for further investigation.In addition, BaeR is a response regulator of baeSR two component signal transduction system and control regulon gene experession. In the complementation experiment, CRO-resistance was conferred by the introduction of expression plasmid carrying baeR gene. To understand which outer membrane protein can be regulated by BaeR, the outer membrane protein profile between highly resistant strain and baeR mutant strain was compared using two-dimensional gel electrophoresis. The result showed STM3031 were up-regulated and OmpW were down-regulated in baeR mutant strain. These proteins how to regulate by baeR either direct or indirect will be needed to further investigation in the future.
中 文 摘 要 ------------------------------------------------------------- 1
英 文 摘 要 ------------------------------------------------------------- 3
緒 論 ------------------------------------------------------------- 5
材料與方法 ------------------------------------------------------------- 14
結 果 ------------------------------------------------------------- 30
討 論 ------------------------------------------------------------- 36
圖 表 ------------------------------------------------------------- 44
參 考 文 獻 ------------------------------------------------------------- 69
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