以噬菌體治療感染克雷白氏肺炎桿菌之小鼠

Abstract

克雷白氏肺炎桿菌造成的社區性化膿性肝膿瘍是全球性的新興感染症。目前已知其致病力與莢膜型具有密切關連，莢膜血清分型研究顯示K1與K2二種分型菌株在台灣為最常見的致病菌株。根據報導，台灣10-20% 的院內感染菌株具有extended spectrum β-lactamase (ESBL)抗藥性基因，對第三代頭孢子素產生抗性，近來發現的NDM-1抗藥型基因則可分解廣效型抗生素carbapenem，在治療上造成極大的困擾。噬菌體療法自1930年代已在東歐及前蘇聯用於人類感染疾病的治療至今，但仍有許多限制性及爭議性，此方法便逐漸沒落。近年來細菌抗藥性問題日益嚴重，噬菌體療法重新成為具有潛力的治療方法。由於噬菌體可隨著克雷白氏肺炎桿菌的存在而增生，並具有專一性，因此本研究希望了解噬菌體療法是否具有清除克雷白氏肺炎桿菌深部聚集以達到治療感染之潛力。目前本實驗室已分離可涵蓋所有莢膜型的噬菌體，其中NTUH-K2044-K1-1能夠特異性感染K1莢膜型並具有全基因定序，因此先以此噬菌體評估噬菌體療法。首先純化出低內毒素汙染噬菌體並證實對小鼠不具毒性。藥物動力學結果發現，噬菌體進入小鼠體內6小時在各組織達到最高濃度，在脾臟停留時間最長，也發現噬菌體可通過血腦屏障。免疫反應研究則發現噬菌體進入體內24小時後，血中TNF-α及IL-6的濃度無顯著改變，但小鼠經過重複給予噬菌體卻發現體內清除速率卻顯著提高。以腹腔注射感染克雷白氏肺炎桿菌的小鼠在感染16小時或24小時後，接受單劑噬菌體 (108 PFU)療法可提高75 %存活率。進一步以動物核磁共振造影 (animal MRI)系統確認小鼠發病後，再驗證噬菌體治療的效果。目前完成8隻治療組，4隻對照組觀察顯示，在發病後連續兩週一天給予二次噬菌體 (108 PFU)，可延長小鼠帄均壽命。這些結果顯示噬菌體療法可能可用為抗藥性細菌或深部感染之替代或輔助治療。

Community-acquired pyogenic liver abscess (PLA) caused by Klebsiella pneumoniae has become an emerging infectious disease. Capsular serotypes K1 and K2 are predominant virulent strains of isolates in Taiwan. Phage therapy has several limitations before, but now it is one of potential therapeutic approaches due to antibiotic resistance. The phage has infection specificity and can be propagated in the presence of its host. Therefore, we evaluated the therapeutic potential of phage in PLA mice model. NTUH-K2044-K1-1 with full genome sequenced was chosen from different phage isolates covering all capsular types to do the pioneer study, which specifically infects K1 strains. Toxicity testing in animals revealed no rectal temperature rise. Pharmacokinetic analysis showed phage concentration reached its peak among different tissues at 6 h and retention of phage in spleen was still observed at 72 h. Moreover, phage potentially crossed blood-brain barrier. The level of TNF- The level of TNF-α and IL-6 in serum beared no difference after phage administration, however, the clearance rates among tissues significantly increased after repeated administrations. Intraperitoneal administration of a single dose of 108 PFU phage at 16 h or 24 h after intraperitoneal K. pneumonia infection was sufficient to improve 75% survival. The efficacy of phage therapy was evaluated in mice with image-documented abscess observed by animal MRI. The results of 8 phage-treated mice and 4 untreated mice illustrated that administration of 108 PFU phage twice a day for 2 weeks could prolong the life span of infected mice. These data suggested that phage therapy have the potential to become alternative or adjuvant agents in treating antibiotic-resistant or deep tissue infections.