請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57144
標題: | 非典型dnaJ基因型金黃色葡萄球菌之特性探討 Characterization of Unusual Staphylococcus aureus Lineage Revealed by Atypical dnaJ PCR-RFLP Pattern |
作者: | Hao Lee 李浩 |
指導教授: | 鄧麗珍 |
關鍵字: | 金黃色葡萄球菌,色素, S. aureus,pigment, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 金黃色葡萄球菌為臨床上常見的革蘭氏陽性菌,其與葡萄球菌屬其他菌種在凝固酶等生化特性上有較大的差異。金黃色葡萄球菌以其金黃色菌落得名,然而近年澳洲研究發現了一群不產生金黃色葡萄球菌色素的品系,其菌落呈現白色。
現行葡萄球菌屬菌種鑑定多以生化反應與菌落型態做為判斷依據,然而生化反應往往會有例外,在實驗室難以作為鑑定依據,進行16S rRNA定序卻又太耗費人力物力,因此有文獻報導可使用dnaJ 基因 PCR-RFLP作為葡萄球菌屬檢體菌種鑑定、再確認的方法。 本實驗在進行臨床菌種再確認時發現了會產生不同樣式(pattern) dnaJ 基因 PCR-RFLP結果的金黃色葡萄球菌分離株,以此為契機開始研究dnaJ基因PCR-RFLP在金黃色葡萄球菌產生的兩種非典型樣式,其在臺大醫院金黃色葡萄球菌檢體中分別所佔的比例,以及菌株特性。 篩選出具非典型樣式的金黃色葡萄球菌後,進行了dnaJ基因的分析與序列比對,發現第一型非典型樣式的金黃色葡萄球菌的基因序列與前述不產生金黃色色素的金黃色葡萄球菌幾乎相同,而與一般金黃色葡萄球菌相差較多。而經由進一步的分子分型確認,發現第一型非典型樣式金黃色葡萄球菌為CC75,而第二型非典型樣式金黃色葡萄球菌則屬CC45。在脈衝式電泳及MALDI-TOF的實驗中也可明確區分典型樣式、第一型非典型樣式與第二型非典型樣式金黃色葡萄球菌。 第一型非典型樣式金黃色葡萄球菌在dnaJ基因的序列上與不產生金黃色葡萄球菌色素的金黃色葡萄球菌品系幾乎相同,而經由色素基因、色素萃取,也發現的確無色素表現。金黃色葡萄球菌色素為一毒力因子,在抵抗氧化壓力以及嗜中性球毒殺上被證實有功效,然而由本實驗發現缺乏金黃色葡萄球菌色素的第一型與正常產生色素的第二型非典型樣式金黃色葡萄球菌在臺大醫院均不少見,因此進行了毒力因子與生物膜生成等各方面實驗,分析是否具有較有利感染的因素使此二型非典型dnaJ 基因 PCR-RFLP樣式金黃色葡萄球菌能在金黃色葡萄球菌檢體中占有一席之地。 在毒力因子上有部分第二型非典型樣式金黃色葡萄球菌具有毒性休克症候群毒素基因(60.9%)較具有顯著不同,而生物膜生成實驗發現第一型非典型樣式金黃色葡萄球菌具有較佳的合成力,可能與其良好感染力相關。而在生化反應的部分則是藉由自動化鑑定儀器發現了第一型非典型樣式金黃色葡萄球菌尿素酶活性具有明顯較高的陽性率,並經生化管確認,然而進一步的生長速率分析初步排除尿素酶活性對於其生長之影響。 Staphylococcus aureus is a common Gram positive bacteria, and is different from other Staphylococcus species by biochemical activities such as coagulase. Staphylococcus aureus is reputed by its golden colony type, nevertheless, a lineage of non-pigmented S. aureus displaying white colonies had been found recently. Currently, the major identification method for Staphylococcus species is by biochemical tests. But, biochemical activities usually have variation. Identification by 16S rRNA sequencing is more reliable, but it would cost more money and manpower. So, dnaJ gene PCR-RFLP would be a good choise for identification of Staphylococcus species. In the beginning of this study, we found a S. aureus isolate with an atypical dnaJ gene PCR-RFLP pattern, further , we tried to study on 2 atypical S. aureus dnaJ gene PCR-RFLP patterns in NTU hospital, trying to find out its proportion and bacterial characteristics. After the atypical pattern S. aureus had been selected out, we did the sequence blast and found out that atypical pattern 1 S. aureus is very similar to non-pigmented S. aureus mentioned above. Then we did some molecular typing, and found that atypical pattern 1 S. aureus isolates belongs to clonal complex 75, and atypical pattern 2 S. aureus isolates belongs to clonal complex 45. All of these three pattern could be separated by Pulsed-field gel electrophoresis and MALDI-TOF. Atypical pattern 1 S. aureus shows similarity in dnaJ gene sequence with the lineage of non-pigmented S. aureus. By pigment gene detection and the extraction of pigment, we found that atypical pattern 1 S. aureus truly was unable to produce pigment. The golden pigment is recognized as one of important virulence factor of S. aureus, which plays a great role in reducing the oxidative stress damage and resisting netrophil phagocytosis. Though lacking of this pigment, atypical pattern 1 S. aureus still had its stand in isolates in NTU hospital. Thus this study tried to examine virulence factors and biofilm activity on 2 atypical pattern S. aureus for studying its advantage during infection. The toxin shock syndrome toxin gene was found in many atypical pattern 2 S. aureus (60.9%), and so did it in biofilm activity in atypical pattern 1 S. aureus. These results might be an explanation for the well ability of infection. We then used Vitek 2 automated system to check the biochemical activities of 2 atypical pattern S. aureus, and found a dramatic high proportion on urease activity in atypical pattern 1 S. aureus. This result was confirmed by tube biochemical test, but after testing growth curve, we found it might not enhance the growth activity even in urea broth. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57144 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-103-1.pdf 目前未授權公開取用 | 4.75 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。