張靜文Chang, Ching-Wen臺灣大學:環境衛生研究所明凱文Ming, Kai-WenKai-WenMing2010-05-072018-06-302010-05-072018-06-302009U0001-1708200912472300http://ntur.lib.ntu.edu.tw//handle/246246/181429Acanthamoeba spp.及Hartmannella vermiformis為自營性阿米巴原蟲，常存在於自然及人工水體環境中。不僅本身具有致病性，且為其他致病菌繁殖增生的自然界宿主。本研究利用即時定量聚合酶鏈鎖反應 (Real-time quantitative polymerase chain reaction, Real-time qPCR) 建立此兩種原蟲數對應DNA量之檢量線，並在八間安養中心之冷卻水塔與熱水系統以及一間廢水處理廠中，進行此兩種原蟲環境調查，同時測量並記錄相關環4境因子，包括水質資料、系統維護與操作情況，以統計分析方法探究環境因子與此兩種原蟲的陽性檢出率及陽性樣本檢出濃度間之相關情形，藉以探討影響此兩種原蟲於不同環境中分布之顯著影響因子。 本研究發現，以real-time qPCR建立之檢量線可成功校正前處理對定量原蟲之影響，而PCR抑制現象則可透過適當稀釋DNA之方式獲得改善。據此，本研究環境測定的結果顯示，在冷卻水塔樣本中，Acanthamoeba spp.在水樣、水塔內空氣與水交界處之生物膜 (floating biofilm，FB) 及水塔內壁生物膜 (substrate biofilm，SB) 之陽性檢出率分別為70.4%、22.8% 及53.8%，而Acanthamoeba陽性樣本之平均濃度分別為965.3 cells/L、9 cells/cm2及1235.7 cells/g；H. vermiformis之陽性檢出率及陽性樣本平均濃度則分別為85.2%、66.7%、92.3%及1664 cells/L、49.3 cells/ cm2、14215 cells/g。而在熱水系統中，Acanthamoeba spp.之陽性檢出率及陽性樣本平均濃度在水樣，水龍頭生物膜 (faucet swab) 及蓮蓬頭生物膜 (showerhead swab)分別為60.3%、25%、40% 及 26.1 cells/L、3.51 cells/cm2、0.31 cells/cm2；H. vermiformis之陽性檢出率及陽性樣本平均濃度分別為69.1%、56.3%、60% 及 800.7 cells/L、39.8 cells/cm2、5.6 cells/cm2。結果顯示冷卻水塔及熱水系統中，H. vermiformis之陽性檢出率及陽性樣本平均濃度均高於Acanthamoeba spp.。相反地，Acanthamoeba spp.在在廢水處理廠進流水、二級生物沉澱池之出流水及加氯池之放流水的陽性檢出率 (88.9%、89.2%、83.8%) 及陽性樣本平均濃度 (2208.5 cells/L、5594.4 cells/L、3673.4 cells/L) 均高於H. vermiformis陽性檢出率 (77.8%、78.4%、78.4%) 及陽性樣本平均濃度 (1784.4 cells/L、576.8 cells/L、548.8 cells/L)。 以統計分析環境因子與Acanthamoebae spp.或H. vermiformis分布之相關性，綜合檢定結果發現，可培養之總細菌數在冷卻水塔水樣中對Acanthamoebae spp.之檢出濃度有顯著正面貢獻（P = 0.062），對於H. vermiformis在冷卻水塔水樣及熱水系統生物膜樣本也具有相同的趨勢（P = 0.003 and 0.001）。另外，總懸浮固體在熱水樣本當中，與Acanthamoeba spp. 之檢出濃度為正相關 (P = 0.09)，但與H. vermiformis呈現相反趨勢(P<0.0001)，顯示在相同的採樣環境中，兩種原蟲與環境因子之間的相關也有所差異。水溫經統計檢定後發現在冷卻水塔水樣及熱水系統生物膜樣本中對H. vermiformis 檢出濃度有顯著的負面影響 (P = 0.033 and 0.037)。退伍軍人菌之濃度則在熱水樣本及冷卻水塔FB樣本中發現分別和Acanthamoeba spp.及H. vermiformis之檢出濃度呈現負相關 (P = 0.032 and 0.095)。總體來看，相較於Acanthamoeba spp.而言，H. vermiformis對於環境因子的變化較敏感。Acanthamoeba spp.and Hartmannella vermiformis were free-living amoebae and distributed widely in the natural and man-made aquatic environments. These amoebae are not only infective to human but also the hosts for other pathogenic bacteria to multiply in the environments. This study applied the real-time qPCR (Real-time quantitative polymerase chain reaction, Real-time qPCR) and constructed the cell-based standard curves to quantify these two types of amoebae in the cooling towers and hot water systems of eight nursing homes, as well as a multiple wastewater treatment plant. The environmental factors including the water characteristics, maintenances of the facilities and operation conditions were also measured or recorded to determine the factors significantly affecting the presence and the concentrations of these two targeted amoebae by the statistical analysis. The results shown that the cell-based standard curves constructed by real-time qPCR could adjust the DNA loss which was caused during the sample pretreatments; and the PCR inhibition could be solved by the appropriate PCR dilution. According to this, our study indicated that the positive rates of Acanthamoeba spp. in the water, floating biofilm (FB) and substrate biofilm (SB) samples of cooling towers were 70.4%, 22.8% and 53.8%, respectively; and the cell concentrations of the Acanthamoeba-positive samples were 965.3 cells/L, 9 cells/cm2and 1235.7 cells/g, respectively. For H. vermiformis, the positive rates and concentrations of the positive samples were 85.2%, 66.7%, 92.3% and 1664 cells/L, 49.3 cells/ cm2, 14215 cells/g, respectively. In the hot water systems, the positive rates of Acanthamoeba spp. and the averaged concentrations of positive samples in the water, faucet swab and showerhead swab samples were 60.3%, 25%, 40% and 26.1 cells/L, 3.51 cells/cm2, 0.31 cells/cm2, respectively; For H. vermiformis, the positive rates and the concentrations of the positive samples were 69.1% and 800.7 cells/L in water samples, 56.3% and 39.8 cells/cm2 in the faucet swabs and 60% and 5.6 cells/cm2 in the showerhead swabs. The result revealed that the positive rates and cell concentrations of H. vermiformis wereboth higher than Acanthamoeba spp. in the cooling towers and the hot water systems. In contrast, the positive rates of Acanthamoeba spp. in the influent water, effluent water from secondary clarifier and chlorine contact tank (88.9%, 89.2% and 83.8%) and the averaged concentrations of the positive samples (2208.5 cells/L、5594.4 cells/L、3673.4 cells/L) were both higher than those of H. vermiformis (positive rates: 77.8%, 78.4% and 78.4%; mean concentrations: 1784.4 cells/L、576.8 cells/L、548.8 cells/L). The results of statistical analysis indicated that the levels of total culturable bacteria were positively contributed to the concentrations of Acanthamoebae spp. in the cooling tower water samples（P = 0.062）, and the trend also presented for H. vermiformis in the cooling tower water samples and the swab samples of the hot water systems（P = 0.003 and 0.001）. In addition, total suspended solids were positively related to the concentrations of Acanthamoeba spp. in the hot waters (P = 0.09), while the contrary trend was observed for H. vermiformis (P<0.0001). Water temperature was adversely associated with the concentrations of H. vermiformis in cooling tower waters and swabs of hot water systems (P = 0.033 and 0.037). Moreover, concentrations of total Legionella and viable Legionnella significantly affected the concentrations of Acanthamoeba spp. and H. vermiformis in the hot waters and FB samples, respectively (P = 0.032 and 0.095). In general, H. vermiformis appeared to be more sensitive to the variance of environmental factors than Acanthamoeba spp. in the aquatic habitats.Chapter 1 Introduction 1.1. Background 1.2. Literature Review 2.2.1. Distribution and health importance of Acanthamoeba spp. and Hartmannella vermiformis 2.2.2. Environmental factors affecting FLA 3.2.3. Detection and quantification methods for FLA 6.3. Rationales of this study 7hapter 2 Objectives of the Study 10hapter 3 Framework of the study 11hapter 4 Material and Methods 15.1. Microbial strains 15.1.1. Acanthamoeba castellanii 15.1.2. Hartmannella vermiformis 16.2. Culture medium and buffer solution 17.2.1. Preparation of ATCC medium 712 17.2.2. Preparation of ATCC medium 1034 18.2.3. Encystement medium 19.2.4. R2A agar 19.2.5. Preparation of Page’s Amoeba Saline (PAS) 20.2.6. Preparation of Phosphate buffered saline (PBS) 20.2.7. TE buffer solution 21.3. Construction of cell-based calibration curves and determination of detection limits and relative DNA recovery rates for A. castellanii and H. vermiformis 21.3.1. Preparation of trophozoites and cysts 21.3.2. Preparation of simulated samples 22.3.3. DNA extraction 25.3.4. Real-time qPCR 27.3.5. Cell-based calibration curves and detection limits 31.3.6. Relative recovery rates 32.4. Field sampling and amoebic analysis 33.4.1. Sampling strategy 33.4.2. Collection of environmental samples 34.4.3. Pretreatments of environmental samples 35.4.4. DNA extraction and DNA dilution 38.4.5. Real-time qPCR 39.4.6. Data analysis for environmental samples 40.5. Measurement of environmental factors 42.5.1. pH 42.5.2. Water temperature 43.5.3. Conductivity 43.5.4. Turbidity 43.5.5. Hardness 44.5.6. Free chlorine 44.5.7. Dissolved organic carbon 45.5.8. Total suspended solids 45.5.9. Total culturable bacteria 46.5.10. Legionella spp. 49.6. Statistical analysis 50hapter 5 Results 52.1. Cell-based standard curves, DNA recovery rates and detection limits for amoebic cells 52.1.1. Acanthamoeba spp. 52.1.2. H. vermiformis 61.2. Environmental investigation 70.2.1. Cooling towers 70.2.2. Hot water systems 101.2.3. Wastewater treatment plant 134.3. QA/QC of real-time qPCR 147.3.1. QA/QC of qPCR for Acanthamoeba spp. 147.3.2. QA/QC of qPCR for H. vermiformis 151hapter 6 Discussion 155.1. Comparison for DNA recovery rates, cell-based standard curves and detection limits of amoebae in different simulated sample types 155.1.1. Acanthamoeba spp. 155.1.2. H. vermiformis 156.1.3. Simulated SB samples 157.2. Environmental investigation of cooling towers 159.2.1. Dilution effect on amoebic detection and quantification in cooling towers 159.2.2. Association between environmental factors and amoebic cells in cooling towers 160.3. Environmental investigation of hot water systems 164.3.1. Dilution effect on amoebic detection and quantification of the hot water systems 164.3.2. Association between environmental factors and the distribution of amoebic cells in hot water systems 165.4. Environmental investigation of wastewater treatment plant 171.4.1. Dilution effect on amoebic detection and quantification in wastewater 171.4.2. Prevalence and quantification of amoebic cells in wastewater 171hapter 7 Conclusions and Suggestions 174eferences 177application/pdf1605612 bytesapplication/pdfen-USAcanthamoeba castellaniiHartmannella vermiformis即時定量聚合酶鏈鎖反應冷卻水塔熱水系統廢水處理廠環境因子陽性檢出率陽性檢出濃度real-time qPCRcooling towershot water systemswastewater treatment plantenvironmental factorspositive rateconcentrations of positive samplesthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/181429/1/ntu-98-R96844013-1.pdf