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研究生(外文):Wen-Yin Liou
論文名稱(外文):Correlation between expression of hfq and rpmE genes and ceftriaxone-resistance in Klebsiella pneumoniae
指導教授(外文):Wensi S. Hu
外文關鍵詞:Klebsiella pneumoniaeantibiotic resistanceceftriaxone
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克雷伯氏肺炎桿菌(Klebsiella pneumoniae;K. p)為一種常引起院內感染的重要病原菌,其中有許多屬於ESBL strain,且具多重抗藥性,這在台灣已是嚴重的問題。近年來,由K. p引起的化膿性肝膿瘍的病例無論國內外皆有逐漸增加的趨勢,在台灣地區更高達82.1 %。
本研究為探討除了ESBL所引起的抗藥性外,還有哪些基因與抗藥性相關,故利用Tn5 transposon mutagensis送入對CRO (ceftriaxone)高抗藥的K. p去建立一個Tn5突變株圖庫,從library篩選出13株對CRO- less resistance的菌株,經由southern blot確定有12株只有單一個基因遭受Tn5破壞,進而分析破壞基因名稱。
在2E11、9D4兩株突變菌株中,其破壞基因分別為hfq、及rpmE,再經由pGEM-T載體將完整的基因送入2E11及9D4兩突變株中,可回復對CRO的抗藥性。Hfq 為RNA binding protein,可調控RpoS的轉錄,RpoS為sigma factor,可經由外界環境壓力所誘導的因子,進而去調控下游基因的表現,RpmE為50S ribosomal protein L31,與蛋白質合成相關,也許可以成為抗細菌的新標的。為求何基因受到影響,藉由2D電泳來分析外膜蛋白,發現hfq及rpmE兩基因的突變,皆引起兩個外膜蛋白的減少,包括:conserved hypothetical protein 及 outer membrane protein K 17,這其中的機制還需更進一步的研究。
Klebsiella pneumoniae is a common nosocomial pathogen, characterized by ESBL-producing with multi-drug resistance. In addition, this microorganism is a significant cause of liver abscess in Taiwan. In this study, we are searching for genes, but β-lactamase -encoded gene, or proteins which involving in ceftriaxone (CRO) resistance. A clinical isolate of K. pneumoniae, with resistance to CRO but susceptible to kanamycin (KAN), was chosen to generate insertion mutants using Tn5 transposome mutagenesis. Among 1500 insertion mutants we obtained, the MICs of thirteen mutants have 3-fold reduction of CRO resistance. Twelve out of thirteen mutants had single Tn5 insertion by southern blot assay and their inserted sites in the chromosome were identified by DNA sequencing method.
Insertion mutants 2E11 and 9D4 were selected in this study. Mutant 2E11 has transposon inserted in hfq gene, encoded for a RNA binding protein which can regulate sigma factor RpoS translation. Mutant 9D4 has transposon inserted in rpmE gene, encoded for 50S ribosomal protein L31. The wild type genes hfq and rpmE were inserted into pGEM-T expression vector to construct pGEM-spt-hfq and pGEM-spt-rpmE and transformed in 2E11 and 9D4 mutants, respectively, for complementation experiment. The result showed both of genes were able to restore CRO resistance in their own mutants. To investigate expression of outer membrane protein regulated by hfq and rpmE, the outer membrane protein profile between parental strains and insertion mutant 2E11 or 9D4 was compared using two-dimensional gel electrophoresis(2-DE). Two protein spots with molecular mass about 18kDa were up-regulated in parental strain, but down-regulated in both insertion mutants. These two proteins are further identified by MALDI-TOF as conserved hypothetical protein (18567Da) and outer membrane protein K17 (18452Da), respectively. These two protein involving in CRO resistance or not and how to regulate need further investigation in the future.
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