- 學位論文
分離一株金黃色葡萄球菌之噬菌體及其內溶素之功能鑑定
Isolation of a Staphylococcus aureus Phage and Functional Characterization of Its Endolysin
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摘要
金黃色葡萄球菌（Staphylococcus aureus）是一種革蘭氏陽性細菌，在人體的鼻咽、皮膚和腸道等部位常見。然而，它也是引發多種院內感染的主要病原體，包括呼吸性肺炎、術後傷口感染等。此外，金黃色葡萄球菌也具有多重抗藥性，導致治療上的困難，特別是耐甲氧西林金黃色葡萄球菌（MRSA, Methicillin-resistant Staphylococcus aureus）等耐藥菌株的出現。噬菌體是一種僅感染細菌並在其內複製的病毒，可用於治療細菌感染，稱為噬菌體療法。噬菌體療法具有多項優勢，如高度特異性、對人體無害等，且在多個領域都已有應用，如食品、農業和臨床診斷等。而在金黃色葡萄球菌的噬菌體，也有多項研究證實其在臨床治療上的應用潛力。 鑑於臺灣金黃色葡萄球菌院內感染問題嚴峻，且耐藥性菌株比例居高不下，迫切需要開發新的治療方法。因此，本研究旨在分離並鑑定金黃色葡萄球菌噬菌體，以應用於治療金黃色葡萄球菌感染。實驗在廢水樣品中成功分離出金黃色葡萄球菌噬菌體ϕS12，並對其宿主範圍及噬菌體毒殺能力進行測試。結果表明，ϕS12的宿主範圍廣泛，且對多數宿主表現出較強的溶菌圈。然而，在對臨床菌株進行的噬菌體毒殺試驗中，ϕS12需要達到較高的感染倍數（MOI）才能有效殺滅細菌。接著，我們進行ϕS12噬菌體的全基因定序，以深入了解其基因組結構，並在基因序列中尋找可能具有裂解細菌功能的蛋白。成功從候選基因中表現出具有活性的內溶素蛋白Endo17，並進行該蛋白的表現與純化。最後測試Endo17蛋白的功能，發現Endo17蛋白具有廣泛的宿主範圍以及有效的細菌毒殺功能，並且能夠防止金黃色葡萄球菌生物膜的形成，顯示出其在治療金黃色葡萄球菌感染方面的潛力。 綜合以上結果，本研究成功從環境中分離出一株金黃色葡萄球菌噬菌體，並初步證實該噬菌體及其內溶素蛋白在治療金黃色葡萄球菌感染方面的潛力。然而，針對生物膜的治療仍需要進一步研究和評估。希望這項研究能夠應用於臺灣院內感染的防治，尤其在耐藥性問題日益嚴重的情況下，使噬菌體療法成為一種有效的治療方式。
並列摘要
Staphylococcus aureus is a Gram-positive bacterium commonly found in the human nasopharynx, skin, and intestines. However, it is also a major pathogen responsible for various nosocomial infections, including respiratory pneumonia and postoperative wound infections. Furthermore, S. aureus exhibits multiple drug resistance, complicating treatment, particularly with the emergence of methicillin-resistant Staphylococcus aureus (MRSA). Bacteriophages, viruses that infect and replicate within bacteria, can be used to treat bacterial infections through phage therapy. Phage therapy offers several advantages, such as high specificity and being harmless to humans, and has applications in various fields like food, agriculture, and clinical diagnostics. Multiple studies have shown the potential of S. aureus phages in clinical treatment. Given the severe issue of nosocomial S. aureus infections in Taiwan and the high proportion of drug-resistant strains, there is an urgent need to develop new treatment methods. This study aims to isolate and characterize S. aureus bacteriophages for treating S. aureus infections. Experimental results demonstrated the successful isolation of the S. aureus phage ϕS12 from wastewater samples and tested its host range and bactericidal activity. Results indicated that ϕS12 has a broad host range and exhibits strong lysis against most of the hosts available in our laboratory. However, in bactericidal assays on clinical strains, ϕS12 required a high multiplicity of infection (MOI) to effectively kill the bacteria. Subsequently, whole-genome sequencing of ϕS12 was conducted to understand its genomic structure and identify potential tail proteins and endolysins with lytic functions. The active endolysin protein Endo17 was successfully expressed and purified from candidate genes. Functional tests of Endo17 revealed a broad host range, effective bactericidal activity, and the ability to prevent S. aureus biofilm formation, demonstrating its potential in treating S. aureus infections. In summary, this study successfully isolated an S. aureus phage from the environment and preliminarily demonstrated the potential of the phage and its endolysin protein Endo17 in treating S. aureus infections. However, further research and evaluation of its therapeutic efficacy and application scope are needed. It is hoped that this research can be applied to the prevention and control of nosocomial infections in Taiwan, particularly under the increasing problem of drug resistance, making phage therapy an effective treatment method.
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