Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5529
標題: 飲用水中非結核性分枝桿菌分析方法之研究
Isolation and Identification of Non-tuberculous Mycobacteria in Drinking Water by traditional and Molecular Methods
作者: 郭詠琪
Kuo, Yung-Chi
關鍵字: Nontuberculous mycobacteira;非結核性分枝桿菌;Drinking water;Polymerase chain reaction;Denaturing gradient gel electrophoresis;Acid-fast stain;飲用水;聚合酶;連鎖反應;變性梯度凝膠電泳;酸性染色
出版社: 環境工程學系
摘要: 
自來水雖然經過一連串的淨化和處理程序,但其中尚可能存在許多不同的致病性微生物,這些微生物通常是存在於原水中,在進入淨水廠的淨化程序後,卻因為其特殊的性質,而沒有完全被去除。然而另一方面,也可能是因為台灣地區的供水水管常有破裂或滲漏的情形,造成微生物進入管線中,或使一些微生物可利用的基質進入管線,提供微生物在管線中生長的必須營養源等,都可能造成微生物的污染,例如在美國威斯康辛州引起極大影響的隱胞子蟲和梨型鞭毛蟲等等。在我國在目前的水質標準中,所監測的項目僅有大腸桿菌及總菌數兩種,而在其他國家已有學者重視到普遍存在於環境中的非結核性分枝桿菌(Non-Tuberculous Mycobacterium ; NTM),這些非結核性分枝桿菌所引起的疾病和結核菌非常相似,有部分的報告指出,可能會對於癌症、免疫不全的患者、無脾臟或切除脾臟者造成傷害,這類型的病菌存在於飲用水中,將對於病患有相當的威脅性。但是在台灣地區相關的研究以及報導卻相當缺乏,並且有許多研究仍然沿用傳統的檢測方式,較為耗時且研究的範圍都是限定在醫學或結核菌上,而且對於環境中的菌種分析幾乎沒有統計資料。
有鑑於此,本研究的目的為採用分子生物的技術,來建立有效且準確的NTM檢測方法,並與傳統分析方法所得結果作比較。在建立有效的分析方法後,將針對由不同場所所取得之自來水樣進行分析,來瞭解台灣地區的飲用水中分枝桿菌的存在情形,並且與國外目前現有的文獻相比較。
本研究中發現,傳統培養基中所含的營養鹽對於分枝桿菌並無促進生長的效果,需要長時間培養才有結果,而使用商用液體培養基Middelbrook 7H9,可使培養時間縮短至兩週。於傳統分析方法上,酸性染色之檢出率僅達72%,若針對環境樣本進行分析時,僅使用酸性染色作為非結核性分枝桿菌存在與否的判斷便非常不適合。於分子生物方法分析中,使用聚合酶連鎖反應及變性梯度凝膠電泳兩種,結果顯示,聚合酶連鎖反應方法可於24小時內快速偵測出水中非結核性分枝桿菌的存在,但對於所存在NTM之種類則無法區別,為進一步了解菌相分佈,研究中選擇兩組引子進行變性梯度凝膠試驗分析,分別為:引子My 264和引子My 285,引子My 259與引子My 285,結果顯示兩組引子皆可放大分枝桿菌族群,但引子My 259和引子My 285所放大之區域因變異度不足,使用變性梯度凝膠電泳分析時,以目前的實驗設計尚無法有效區別菌種間的不同。經定序結果得知本實驗所分離之非結核性分枝桿菌為M. porcinum、M. mucogenicum、M. duvalii及M. wolinskyi,皆為快速生長族群,其中M. porcinum及M. duvalii對於人體健康具有危害性。此結果與目前國內外文獻中所提及於飲用水中分離出的菌種並不相同,可能原因為台灣中部地區因其地質、氣候及環境等因素較適合此類NTM生長。

Environmental mycobacteria which are also called atypical mycobacteria or nontbuerculosis mycobacteria (NTM), are common on saprophytes in all ecosystems, including water, soil, food, and dust. Some species are pathogenic for humans and animals, including pulmonary and cutaneous, lymphadenitis, especially in the growing immunodeficient population. NTM infections are transmitted by ingestion, inhalation, and inoculation from environmental sources. There is increasing evidence which suggestes that drinking water is the vehicle by which mycobacteria infect or colonize the human body. In a number of causes, mycobacterial species (M. kansasii, M. fortuitum, M. avium, and M. porcinum) have been recovered from drinking water or patients. Mycobacteria have been isolate from public systems, hot and cold water taps, and ice machines. NTM can colonize, survive, persist, grow, and multiply in drinking water.
In present drinking water management statutes, the numbers of E. coli cannot confirm the relationship of public health. Mycobacteria have high resistance of chlorine, can across the water treatment lines in to the water distribution systems. In this study, we collect drinking water in eight health center and a house in Taichung. According to the results, it show that the growth of NTM were necessary to cultivate for 4-8 weeks in the traditional medium. The cultivated time could become to 2 weeks in the commercial medium - Middelbrook 7H9. The accuracy of acid-fast stain was only 72%. It can be explained that the microorganism are very complex in the environment. The phenomenon indicated that this method was not suitable for analyzing the NTM in the environmental samples. In the results of molecular biological methods, it indicated the existence of NTM could be detected by using polymerase chain reaction (PCR) technique in 24 hours. Primer My 264 and My 285 can be successful identification of mycobacterial. In primer My 259 and My 285 to amplify the fragment is to similarity to unsuccessful identification the mycobactial.
In this study, mycobacteria isolated from the drinking water which included M. porcinum, M. wolinskyi, M. dvualii, and M. mucogenicum, respectively. All of these four species of mycobacteria belong to the fast-growth group. M porcinum and M. dvualii were saprophytic mycobacteria. No significant correlations were found among the presence of mycobacteria, the origin of water, pH, DO, TOC, and age of sample.
URI: http://hdl.handle.net/11455/5529
Appears in Collections:環境工程學系所

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