水中分離之克雷伯氏肺炎桿菌噬菌體特性分析

Abstract

過去二十年來，在台灣陸續出現新型侵襲性克雷伯氏肺炎桿菌病例。此菌會造成原發性肝膿瘍、敗血性休克、菌血症、轉移性眼內炎、腦膜炎等嚴重病徵。克雷伯氏肺炎桿菌菌體最外層的莢膜與其黏附、毒性有極大關聯，特別是毒性較強的K1和K2莢膜血清型菌株。施打疫苗是疾病預防重要策略之一。市售肺炎鏈球菌疫苗即為複數種莢膜多醣寡分子（capsular polysaccharide oligomers）混合成多效價疫苗。故尋找能夠切割巨大莢膜成小分子，又不影響其免疫原特性之方法是我們努力的目標。已有研究指出噬菌體能對莢膜多醣產生切割降解效果（CPS depolymerization）。因此，在本研究中，我們收取未經處理的原水與克雷伯氏肺炎桿菌K1莢膜血清型菌株NTUH-K2044共同培養，分離出能夠感染此菌株之18個噬菌體候選。經噬菌體純化、DNA萃取與限制性核酸內切酶（EcoRI、HindIII與PstI）剪切反應，得知這些噬菌體均為雙股DNA噬菌體，再由其相同限制性片段長度多態性（restriction fragment length polymorphism, RFLP）判別為相同噬菌體，並依據片段大小預估其基因組大小約為40Kbp。取噬菌體DNA片段序列與NCBI BLAST資料庫裡的DNA與胺基酸序列做比對，推斷為一全新未知噬菌體，且屬於ΦKMV噬菌體群一員。此外，使用穿透式電子顯微鏡（TEM）可觀察到此噬菌體型態。透過塗點試驗（Spot test）進行此噬菌體宿主範圍分析，發現其對感染克雷伯氏肺炎桿菌K1莢膜血清型菌株具有專一性。目前已完成於噬菌體DNA大片段定序（42.5 Kbp），與進行莢膜多醣降解實驗，結果顯示莢膜多醣可能降解成以數個單醣鏈結之分子存在。在未來，此噬菌體將有機會應用於菌種或菌株之鑑定，以及複合疫苗發展。

Klebsiella pneumoniae caused community-acquired pyogenic liver abscess is an emerging infectious disease and is often complicated with metastatic endophthalmitis and septic meningitis. Capsular serotypes, especially K1 and K2, were reported to play an important role in pathogenesis of K. pneumoniae causing pyogenic liver abscess. In this study, bacteriophages were isolated from the co-culture of serotype K1 K. pneumoniae NTUH-K2044 strain and sewage. We randomly selected eighteen bacteriophages for further study. By experiments of restriction enzyme digestion, we have known that the genome of bacteriophages were double-stranded DNA and the length of restriction fragments among these eighteen bacteriophages were identical. The genome size of bacteriophage was predicted to be about 40 kbp, and a rough morphology of bacteriophage was observed by transmission electron microscope. Our preliminary result revealed that the sequences of this bacteriophage was novel by comparison with NCBI BLAST databases, and we had a DNA frament covering about 95% area of bacteriophage genome. With spot test for host range, we found the new bacteriophage specifically infected K. pneumoniae serotype K1. In addition, it’s been known that capsular polysaccharide degraded to oligomers after phage treatment could be detected by Western blotting and mass spectrometry. This bacteriophage might be a good candidate for typing, and helps the development of conjugate vaccine.