分離一株綠膿桿菌之噬菌體及鑑定可能的溶菌素

Abstract

綠膿桿菌（Pseudomonas aeruginosa）是一種革蘭氏陰性的需氧桿菌，常見於潮濕的環境，它也是最常見造成醫療相關感染的原因之一。近年來多重抗藥性綠膿桿菌已對公共衛生形成嚴重威脅，而噬菌體療法被視為最有潛力作為抗生素替代性療法的候選人。噬菌體是一種可以感染細菌的病毒，可專一性感染特定宿主菌株，依其生命週期可以分為潛溶型噬菌體以及裂解型噬菌體，而其中，裂解型噬菌體又被視為噬菌體療法中最理想的噬菌體種類。噬菌體療法在早期曾廣泛應用，但抗生素普及後逐漸被忽視。然而近年來由於抗藥性菌株的氾濫問題使噬菌體療法再度受到關注。 本研究首先從環境水源中分離並純化獲得數株噬菌體，經過測試後找到兩株擁有廣泛宿主範圍（Host range）的噬菌體，分別命名為P23以及P49。毒殺試驗（Phage killing assay）的結果顯示P23對臨床分離出的綠膿桿菌具有良好的殺菌效果，且對於部分臨床分離的綠膿桿菌具有清除生物膜的能力。另P23亦有進行進行動物實驗，然而由於感染複數（Multiplicity of infection，MOI）偏低，治療效果並不如預期。除此之外，噬菌體P23屬於RNA噬菌體，而目前在抽取噬菌體RNA仍較困難，因此未成功取得P23之基因組。P49則是在經過定序後，選擇針對其溶菌素（Endolysin）以及兩種尾纖維蛋白（Tail fiber proteins）進行基因重組蛋白的表現（Recombinant protein expression）。命名重組溶菌素為M16，尾纖維蛋白TFP1+2及TFP3+4。其中，兩種尾纖維蛋白皆為水溶性極低的內涵體（Inclusion bodies），而M16則是對26株綠膿桿菌菌株皆無直接溶菌的活性。

Pseudomonas aeruginosa is a Gram-negative bacterium commonly found in moist environments, it is also considered as one of the most important causes of healthcare-associated infections. In recent years, multidrug-resistant P. aeruginosa has become a serious issue in public health. Phages are viruses that can infect bacteria, with the specificity to infect their hosts, they do no harm to normal microbiota in human bodies, which makes them a promising alternative treatment strategy to antibiotics. According to their different life cycles, they can be classified as either lysogenic or lytic phages, with lytic phages being the most ideal candidates for phage therapy. Phage therapy was widely used in the past, but its popularity declined with the advent of antibiotics. However, due to the emergence of multidrug-resistant bacteria strains, phage therapy has regained its attention. This study first isolated and purified several phages from sewages from NTUH, two phages with the broad host range were selected and named P23 and P49. The results of the phage killing assay demonstrated that phage P23 could effectively kill clinical isolates of P. aeruginosa., it also proved its abilities of cleaning biofilm from a few clinical isolated P. aeruginosa. Additionally, in vivo animal experiment for phage P23 was also conducted. However, due to the low MOI, the result was not as effective as anticipated. However, due to the difficulties of extracting phage genomic RNA, the genomic sequence of phage P23 could not be obtained. As for P49, after whole-genome sequencing, efforts were focused on expressing recombinant proteins of its putative endolysin, and two tail fiber proteins. The recombinant endolysin was named M16, and the recombinant tail fiber proteins were named TFP1+2 and TFP3+4. Among the three of them, both TFP1+2 and TFP3+4 showed low solubilities and turned out to be inclusion bodies, and M16 showed no lytic activity against the 26 clinical isolated P. aeruginosa strains.