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標題: | 克雷伯氏肺炎桿菌對於磷黴素抗藥性之研究 Fosfomycin Resistance Mechanism(s) in Klebsiella pneumoniae |
作者: | I-Cheng Hung 洪一琤 |
指導教授: | 王錦堂(Jin-Town Wang) |
關鍵字: | 克雷伯氏肺炎桿菌,磷黴素,GlpT,UhpT,FosA,MurA,GST, Klebsiella pneumoniae,fosfomycin,GlpT,UhpT,MurA,FosA,GST, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 細菌抗藥性在全世界日趨嚴重,早期開發的磷黴素(fosfomycin)也重新被檢視。磷黴素毒性低、殺菌力強,對產生廣效性乙內醯胺酶(extended spectrum beta-lactamase, ESBL)分解酶的腸內菌具有高度感受性。我們測試50株由社區型原發性肝膿瘍(community-acquired pyogenic liver abscess)分離出的克雷伯氏肺炎桿菌以及52株carbapenem-resistant Klebsiella pneumoniae (CRKP),發現共72株(70.6%)對磷黴素具感受性(MIC≦64μg/ml),其中社區型肝膿瘍菌株100%(50/50)為感受型,而CRKP僅42.3%(22/52)為感受型。檢查18株抗磷黴素的CRKP帶有何種已知的抗藥機制,以功能分析證實4株帶有缺失的磷黴素運輸蛋白GlpT,藉由比對分析序列,發現此4株抗藥菌株的glpT確實有序列上的變異,並證實GlpT點突變(Glu299Asp)與磷黴素的抗藥性及sn-glycerol 3-phosphate (G3P)的運輸相關。挑選一株對磷黴素有高度抗藥性(MIC ≥2048 μg/ml)的菌株9921,建構突變株庫(transposon mutant library) 和基因表現庫(expression library)以尋找可能的未知抗藥機制。利用磷黴素篩選9921突變株庫後,得到一個突變株對磷黴素的最小抑菌濃度有下降,定序分析後得到跳躍子破壞的基因為pstA。篩選9921基因表現庫後,定序分析得到四段基因片段可能與磷黴素的抗藥性相關,並在大腸桿菌DH10B表現篩選到的基因片段和在磷黴素感受型臨床菌株NTUH-K2044中置換抗藥性候選基因,發現過量表現穀胱甘肽轉移酶(glutathione transferase)會造成磷黴素最小抑菌濃度上升。 Bacterial infections caused by antibiotic-resistant isolates have become a major health problem in recent years. Retrieval of old antibiotics, like fosfomycin, may be a short-term solution to the difficulties. Fosfomycin was recently revived as an antibiotic that could be effective against extended-spectrum-β-lactamase (ESBL) producers. We examined 50 Klebsiella pneumonia isolates causing community-acquired pyogenic liver abscess and 52 carbapenem-resistant K. pneumoniae (CRKP) isolates. There are 72 isolates(72/102, 70.6%) susceptible to fosfomycin (MIC≦64μg/ml) including 50 isolates causing community-acquired pyogenic liver abscess (100%) and 22 CRKP isolates (22/52, 42.3%). Among 18 fosfomycin-resistant strains, 4 strains are found to have defective sn-glycerol 3-phosphate transporter (GlpT) and harbor mutations in glpT gene. A single amino acid substitution (Glu299Asp) was demonstrated to contribute to fosfomycin resistance. In order to find novel fosfomycin-resistant mechanism(s), a transposon mutant library and expression library of a highly fosfomycin-resistant strain 9921 were constructed. Screening by fosfomycin revealed 1 mutant with decreased MIC and 5 expression clones which may contain fosfomycin-resistant genes. The gene pstA encoding phosphate transporter subunit was identified to be disrupted by transposon, and its functions in fosfomycin resistance need further studies. Overexpression of fosfomycin-resistant gene candidates in E. coli DH10B and replacement of fosfomycin-resistant gene candidates in NTUH-K2044, we found that overexpression of glutathione transferase causes the increase of fosfomycin MIC. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19369 |
DOI: | 10.6342/NTU201600750 |
全文授權: | 未授權 |
顯示於系所單位: | 微生物學科所 |
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