克雷伯氏肺炎桿菌對於磷黴素抗藥性之研究

Abstract

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.