應用分子生物技術偵測環境微生物

Abstract

生活中充滿的各種致病微生物。生物氣膠可能造成急性的疾病、感染及過敏反應；而水媒微生物則可能藉由飲用水系統影響大眾的健康，因此，對不同環境的微生物進行監測，成為近年來重要的議題。進行環境監測必須建立一套適當的採樣分析技術，目前常見的分析方法包括培養及非培養的方法。培養方法 (Culture-based Method)一直是主要的微生物分析方法，然而，此方法存在著許多限制如：部分菌種對培養條件相當挑剔、培養相當耗時以及某些菌本身具有活性但無法被培養，有鑑於此，對於微生物的分析有必要建立一個正確且快速的非培養方法 (Non-culture-based method)，以提供更完整的資訊。非培養的方法之中，螢光顯微鏡(epifluorescence microscopy, EFM)、流氏細胞儀(flow cytometric, FCM)、聚合酶連鎖反應(Real-Time quantitative polymerase chain reaction, real-time qPCR)以及螢光原位雜交(Fluorescence In Situ Hybridization, FISH)等方法可以評估微生物的總濃度以及活性，因此最有潛力可發展為替代培養方法的技術。至目前為止，使用非培養方法監測環境微生物的研究仍然很有限，因此，本研究將利用螢光原位雜交(FISH)與即時定量聚合酶連鎖反應 (real-time qPCR)，建立微生物分析之最佳化步驟，以應用於空氣及水體之微生物監測。 本研究首先建立螢光原位雜交(FISH)之最佳化條件，以大腸桿菌以及酵母菌為對象，配製不同死活的百分比，在實驗室評估FISH與傳統培養方法間的關係，結果顯示，各探針的雜交效率均高達90 ％，且兩種方法所得之微生物活性均呈高度相關，因此本研究指出FISH能對微生物活性測量提供相當快速與正確的資訊。此外本研究也成功的將FISH運用在養豬場、養雞場及大氣的空氣樣本分析，並與螢光染色(fluorochrome) 及培養方法比較，結果顯示，養豬場、養雞場及大氣樣本中的生物氣膠總濃度大約分別為9 x 10^6、5 x 10^8、8 x 10^5 cells/m3，但培養法所得的的濃度卻只有1 x 10^5、5 x 10^6、1 x 10^3 cells/m^3，而各空氣樣本之活性也顯著地高於可培養性。由此可知，傳統培養方法嚴重低估生物氣膠的濃度與活性。此外，養雞場中有最高的生物氣膠總濃度，在活性方面，養豬場與養雞場有較低的活性，但較高的可培養性，主要因為較長的清理間隔，以致有較多不具活性的微生物累積，而較高的可培養性則由於牲畜場所中動物的排泄物、食物可供微生物的生長。然而，大氣環境中的生物氣膠卻有較高的活性及較低的可培養性，其主要原因為大氣低營養鹽的惡劣環境，導致微生物進入具活性但不能培養的狀態(viable but non-culturable, VBNC)。整體上，FISH被證明能夠成功的定量空氣中之細菌與真菌之總濃度與活性。 此外，水中微生物偵測方面，real-time qPCR與FISH針對大腸桿菌之分析方法也成功的被建立，結果顯示，這兩種方法與傳統培養方法相較後，呈高度相關。此兩種方法亦成功應用於廢水處理廠與淨水廠中大腸桿菌之定量，結果顯示，廢水處理廠中進流水與放流水大腸桿菌的總濃度大約分別為5x 10^7 cells/100ml 與 2 x 10^7 cells/100ml，淨水廠則大約為2x 10^4 cells/100ml 與 8 x 10^2 cells/100ml。使用非培養方法所測得的活性高於培養方法的結果。整體而言，本研究所建立之非培養方法，FISH與real-time qPCR均為生物氣膠與水中微生物的研究提供了相當有利的分析工具，未來也能針對各環境中之特定致病菌，進行更進一步的研究與探討。

In the past decade, traditional culture-based methods are the major method for detecting microorganisms; however, culture-based methods for microorganism quantification are slow, tedious, and rather imprecise. In order to get more rapid, sensitive, and specific results for field microorganisms, non-culture-based methods for microorganism quantification should be therefore considered as an alternative. From non-culture-based methods, epifluorescence microscopy with fluorochrome (EFM/FL), flow cytometric with fluorochrome (FCM/FL), fluorescent in situ hybridization (FISH) and real-time polymerase chain reaction (real-time qPCR) for microorganisms quantification are the most promising methods to evaluate the viability, an important indicator for assessing potential health effects. To date, only limited data is available on the total cell concentration and viability of microorganisms in different environments. In this study, the optimal analysis methods of FISH and real-time qPCR for microorganism evaluation were established. The optimal conditions of FISH, and real-time qPCR were used to monitor the total cell concentration and viability of microorganisms in air and water environments. The results were then compared to those obtained using a commonly used culture-based method. For the FISH condition optimization, four different controlled-viability samples of E.coli and yeast were made to assess the relation between FISH and culture-based method. The result showed that hybridization efficiency of FISH with varies probes was higher than 90 %. In addition, FISH and culture-based method were highly associated for viability. In summary, FISH could provide rapid and accurate information about microorganism concentrations and viability. Moreover, the established optimal FISH with probes were validated for characterizing bioaerosol profiles from air samples in swine buildings, chicken houses, and ambient air. The results were then compared to those obtained using fluorochrome with dyes and a commonly used culture-based method. The total microbial cell concentrations measured by using non-culture-based methods averaged about 9 x 10^6 cells/m^3 for swine buildings, 5 x 10^8 cells/m^3 for chicken houses, and 8 x 10^5 cells/m^3 for ambient environment. The viabilities determined by using non-culture-based methods were much higher than the culturabilities. The total microbial cell concentration and viability in the atmosphere were highly underestimated by the culture-based method in the past. Moreover, the results revealed that there was lower viability, but higher culturability in the livestock environment. The lower viability might be associated with non-viable bioaerosols accumulated on the floor resulted from longer cleaning intervals. Furthermore, the higher culturability can be explained by the sufficient nutrients form animal dander, fecal matter, and feed materials for bioaerosol growth. However, the higher viability but lower culturability was observed in the atmosphere environment. The finding might be related to that bioaerosols entered a viable but non-culturable (VBNC) state because of insufficient nutrients in a harsh environment. In conclusion, the FISH successfully assessed the total concentration and vibility for bacterial and fungal microorganisms in environmental field samples. Regarding the microorganisms in water environments, FISH and real-time qPCR were successfully established and applied to E. coli quantification in drinking water and wastewater. Results of FISH, real-time qPCR and culture-based method indicated that viability was highly associated for culturability. By the non-culture-based methods, real-time qPCR with DNA gene probe, the total E. coli concentrations in raw sewage and final effluent averaged 5 x 10^7 cells/100 ml and 2 x 10^7 cells/100 ml, respectively. The viability determined by using non-culture-based methods was higher than those using culture-based method. In conclusion, FISH and real-time qPCR should be powerful tools to provide more insight in the area of bioaerosol and environmental microbiology.