分離與鑑定二株金黃色葡萄球菌之噬菌體

Abstract

金黃色葡萄球菌是一種革蘭氏陽性球菌，也是人類皮膚及鼻咽的常在菌叢之一，在免疫低下時會造成伺機性感染，甚至是引起嚴重的菌血症、感染性心內膜炎、骨髓炎和壞死性肺炎等。後續出現的抗甲氧西林金黃色葡萄球菌（Methicillin-resistant Staphylococcus aureus, MRSA）及抗萬古黴素菌株（Vancomycin-resistant Staphylococcus aureus，VRSA），更加深了治療的困難度。因為噬菌體對其宿主有專一性，應用在人體治療也較安全，所以人們除了尋找新的抗生素外，對開發噬菌體治療也越來越關注。從文獻中得知大多數金黃色葡萄球菌噬菌體都屬於尾狀噬菌體目（Caudovirales），其中已知的短尾噬菌體科（Podoviridae）、長尾噬菌體科（Siphoviridae）和肌尾噬菌體科（Myoviridae）受體皆為磷壁酸（wall teichoic acid, WTA），但對於噬菌體與金黃色葡萄球菌之間特異性辨認的詳細機制仍未明瞭。本研究的目標是想找到金黃色葡萄球菌噬菌體，以期能應用在開發相關疫苗或噬菌體治療。本實驗使用了27株金黃色葡萄球菌，分別為19株MRSA（Methicillin-resistant Staphylococcus aureus）及8株MSSA（Methicillin- susceptible Staphylococcus aureus），我們以cap基因進行莢膜分型，其中11株為莢膜第5型，16株為莢膜第8型，結果顯示莢膜型與抗藥與否並無關聯性。接著為了尋找金黃色葡萄球菌噬菌體，從環境中採集了20個樣本，嘗試從中分離噬菌體，結果分離出2株宿主涵蓋範圍較多的噬菌體，命名為ɸS3及ɸS5，在27株菌中可感染比例為63%和59%。接著以限制片段長度多型性（Restriction Fragment Length Polymorphism, RFLP）方法，初步分辨ɸS3、ɸS5和參考株ɸ44AHJD應為不同種噬菌體，再進行全基因定序（Whole-Genome Sequencing, WGS）。根據基因序列結果推測ɸS3和ɸS5為長尾噬菌體科的溫帶噬菌體（temperate phage）。另一方面，金黃色葡萄球菌BD55是ɸS3的宿主，能被多株噬菌體感染，而且有相對較高的轉型效率，所以我們挑選該菌株進行WGS，建立本土分離株的基因定序資料。由於文獻表示多數金黃色葡萄球菌噬菌體會辨認磷壁酸，所以我們建構磷壁酸的初始合成基因tagO的缺失菌株，測試我們分離出的ɸS1至ɸS10對該菌株的感染力，結果證實此10株噬菌體都是辨認磷壁酸結構。往後將可以用tagO的剔除株篩選辨認其他構造之噬菌體。

Staphylococcus aureus is a Gram-positive coccus, which is the common flora of human skin and nasopharynx. As an opportunistic human pathogen, it can invade into human body when the immune system gets weakened. S. aureus infections even cause bacteremia, infective endocarditis (IE), osteomyelitis and necrotizing pneumonia. The rapid increase in Methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant Staphylococcus aureus (VRSA) has become the challenge in dealing with these infections. Due to the specificity of bacteriophages and their corresponding host, they do not target other normal microbiome in the body and are safe to use in human therapy. Therefore, in addition to finding new antibiotics, people are paying more attention to phages as an alternative treatment. Many studies indicate that S. aureus phages belong to the order Caudovirales (tailed phages). The three major families: Podoviridae, Siphoviridae, and Myoviridae all require wall teichoic acid (WTA) for infection. However, the mechanism of specific recognition between phages and S. aureus is still unclear. In this study, we aim to find S. aureus phages and apply to the development of relative vaccine or phage therapy. We used 27 clinical isolate strains in Taiwan, which include 19 MRSA (Methicillin-resistant Staphylococcus aureus) and 8 MSSA (Methicillin-susceptible Staphylococcus aureus). The capsule typing showed that among which 11 strains were type 5 and 16 were type 8. The result shows no relevance between capsule type and drug resistance. We collected 20 samples from environment, isolated 2 infectious phages with wider host range named ɸS3 and ɸS5, which infectivity are 63% and 59% among 27 strains. Restriction Fragment Length Polymorphism (RFLP) and Whole-Genome Sequencing (WGS) were utilized to examine ɸS3, ɸS5 and reference strain ɸ44AHJD. The results showed both ɸS3 and ɸS5 belong to temperate phages of Siphoviridae. On the other hand, we also selected S. aureus strain BD55, due to its sensitivity to multiple phages and high transformation efficiency for WGS to establish the genome data of local isolates. It is known from reference that the tagO gene is the first gene in the WTA biosynthetic pathway. Therefore, we constructed a tagO deletion mutant strain to test the infectivity. The results confirmed that all 10 phages recognized the structure of wall teichoic acid. In the future, phages targeting other surface structures could be isolated using the deletion mutant.