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標題: | 分離金黃色葡萄球菌與黏附侵襲性大腸桿菌之噬菌體 Isolation of phages infecting Staphylococcus aureus and adherent-invasive E. coli |
作者: | 廖婉婷 Wan-Ting Liao |
指導教授: | 王錦堂 Jin-Town Wang |
關鍵字: | 金黃色葡萄球菌,噬菌體,tarM基因,tarS基因,克隆氏症,黏附侵襲性大腸桿菌, Staphylococcus aureus,bacteriophage,tarM gene,tarS gene,Crohn’s disease,Adherent-invasive E. coli ( AIEC), |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 金黃色葡萄球菌 (Staphylococcus aureus) 為革蘭氏陽性球菌,是院內及社區感染常見的重要病原菌之一。在抗生素長期篩選壓力下,出現了多重抗藥性菌株,使治療更為棘手,因此噬菌體治療成為治療多重抗藥性菌株的替代方法。噬菌體對其宿主具有專一性,然而對於金黃色葡萄球菌及其噬菌體之間特異性結合的詳細機制仍不清楚。本研究的目標是想找到以不同附著位置感染宿主的金黃色葡萄球菌噬菌體,期盼未來能應用於噬菌體治療上。從先前文獻得知金黃色葡萄球菌噬菌體44AHJD及P68屬於短尾噬菌體科 (Podoviridae) ,對金黃色葡萄球菌有很好的感染力,且宿主範圍廣,因此本實驗室選用此兩株噬菌體,以台灣的40株臨床金黃色葡萄球菌菌株測試其感染力及宿主範圍,結果發現這2株噬菌體只能感染其中的6株,感染力只有15%,因此接著嘗試從環境中分離出金黃色葡萄球菌的噬菌體。本實驗室從各種環境中共搜集了74個樣本,結果分離出1株具有感染力的噬菌體,命名為ФSA1468-1,其對宿主MRSA1468可產生溶菌斑,然而其溶菌斑十分細小,在純化、增殖噬菌體過程中較困難。另一方面,先前文獻指出,短尾噬菌體科的噬菌體感染金黃色葡萄球菌和tarM及tarS基因調控的壁磷酸 (wall teichoic acids) 醣基化有關。本實驗室以聚合酶連鎖反應確認臨床菌株是否具有tarM、tarS基因,預期有正常的tarM或是無tarS皆不會被短尾噬菌體科的噬菌體感染。實驗結果與文獻大致上吻合,但仍有幾株與預期結果不符,因此推測除了tarM和tarS基因外,還有其他影響金黃色葡萄球菌噬菌體感染性的因素。
克隆氏症 (Crohn's disease, CD) 是一種慢性的腸道發炎疾病,為發炎性大腸疾病 (Inflammatory bowel disease, IBD) 的一種,疾病會反覆復發。雖然克隆氏症在西方國家的盛行率顯著高於亞洲國家,但其症狀的嚴重程度與西方相似,且近年來台灣的患者有逐漸增加的趨勢。許多研究指出,黏附侵襲性大腸桿菌(Adherent-invasive E. coli, AIEC) 與克隆氏症有關。目前克隆氏症的治療方法都是針對這些疾病中的過度免疫反應,但也有針對腸內菌的標靶療法被提出,噬菌體療法為其中之一。在此研究中,本實驗室想以AIEC噬菌體移除在老鼠發炎腸道中的AIEC。首先,我們從污水中分離出三株AIEC的噬菌體,命名為ФLF82-1、ФLF82-2及ФLF82-3,這些噬菌體可感染AIEC LF82,其為AIEC的人類參考菌株。接著測試這三株噬菌體對LF82的感染效率。結果顯示,噬菌體LF82-1、LF82-2和LF82-3幾乎可殺死全部的LF82,除此之外,具LF82-1噬菌體抗性的菌株LF82-1R也可被噬菌體LF82-2和LF82-3殺死。我們更進一步地透過次世代定序分析這三株噬菌體的全基因組序列。噬菌體LF82-1屬於長尾噬菌體科 (Siphoviridae); 噬菌體LF82-2和LF82-3是相同噬菌體,屬於肌尾噬菌體科 (Myoviridae)。另外,我們取得一株從小鼠腸胃道分離出的AIEC — LI60C3,並從污水中分離出可感染LI60C3的噬菌體,命名為ФLI60C3-1。測試其殺菌能力發現,ФLI60C3-1可殺掉約69 % 的LF82及100 % LI60C3。本實驗室共分離出四株AIEC的噬菌體,未來可應用到噬菌體療法,將人體腸道內的AIEC去移生化 (decolonization)。 Staphylococcus aureus is a Gram-positive, round-shaped bacterium, which is one of the most important pathogens that cause nosocomial and community infections. Today, multiple drug resistant strains are more common due to long-term use of antibiotics, making treatment more difficult. Therefore, phage therapy has become an alternative method for treating multiple drug resistant strains. Bacteriophages are specific to their hosts, however the mechanism for specific binding between S. aureus and its phage remains unclear. In this study, we aim to find S. aureus phages that infect the host at different adhesion sites and use these phages to eliminate S. aureus. It is known from references that S. aureus phage 44AHJD and P68 have good infectivity and a wide range of hosts. Therefore, we used 40 clinical isolate of S. aureus in Taiwan to test the infectivity and host range of the two phages. Result showed that, the two phages could only infect 6 of 40 clinical isolate, the infectivity was only 15%. So we tried to isolate other phages of S. aureus from the environment. We collected 74 samples from various environments, isolated an infectious phage and named as ФSA1468-1, which produced plaque against host MRSA1468. However, its plaque was very tiny and was difficult to purify and proliferate. On the other hand, references indicate that phages of Podoviridae infecting S. aureus is associated with glycosylation of wall teichoic acids, which is regulated by tarM and tarS. We examined whether the clinical isolate have tarM and tarS genes by PCR, expected that the clinical isolate with normal tarM or without tarS would not be infected by phages of Podoviridae. Most of our results were corresponded to the references, but still several results were not. Therefore, we speculated that there are other factors affecting the infectivity of S. aureus phage. Crohn’s disease (CD) is a chronic relapsing and remitting inflammatory disease of the gastrointestinal tract. It is a type of inflammatory bowel disease (IBD). Although the prevalence of Crohn's disease in Western countries is significantly higher than in Asian countries, the severity of symptoms is similar to that of the West. Moreover, the number of patients in Taiwan has gradually increased in recent years. Many studies indicate that adherent-invasive Escherichia coli (AIEC) are pathogenic bacteria associated with human Crohn's disease. There are targeted therapies directed against intestinal bacteria were proposed, and phage therapy is one of them. In this study, we aim to use phage therapy to eliminate AIEC from the inflamed intestinal mucosa in mice. First, we isolated three phages from sewage and named as ФLF82-1, ФLF82-2 and ФLF82-3. These phages could infect E. coli LF82, the reference strain of AIEC. Next, we examined the infection efficiency of these isolated phages. Our results showed that all of LF82 (almost 100%) was killed by phage LF82-1, LF82-2 and LF82-3, respectively. The LF82-1 phage-resistant strain LF82-1R was total killed by either phage LF82-2 or LF82-3. Moreover, the whole genome sequences of these three phages were analyzed by the Next-Generation Sequencing. Phage LF82-1 belongs to the Family Siphoviridae; phage LF82-2 and LF82-3 seems to be the same phage which belongs to Myoviridae. In addition, we obtained a strain of AIEC-LI60C3, which was isolated from the gastrointestinal tract of mice. Then a bacteriophage which can infect LI60C3 was isolated, and named as ФLI60C3-1. This phage can kill about 69% of LF82 and 100% of LI60C3. We have isolated four AIEC phages, which has the potential to be applied to phage therapy to decolonize AIEC in the human intestinal tract. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78997 |
DOI: | 10.6342/NTU201803624 |
全文授權: | 未授權 |
電子全文公開日期: | 2023-10-11 |
顯示於系所單位: | 微生物學科所 |
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