退伍軍人菌環境監測技術研究

Development of Techniques on Environmental Monitoring for Legionellae

陳乃慈(Nai-Tzu Chen)

指導教授 ： 張靜文

國立臺灣大學/公共衛生學院/環境衛生研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.01137

摘要

即時定量聚合酶鏈鎖反應技術 (qPCR) 廣泛應用於定量退伍軍人菌。然而，其會受到環境qPCR抑制物的干擾，且無法區分具活性及不具活性的細菌。為了改善此qPCR的限制，本研究首先進行DNA分離處理程序最佳化，以添加腐植酸 (≤ 126.8 mg L-1)、鐵離子 (≤ 3 mg L-1) 或腐植酸/鐵離子 (≤ 3/100 mg L-1) 之樣本進行評估，並發現QiAamp DNA Mini Kit (Q) 為以qPCR定量退伍軍人菌之最佳DNA萃取方法。方法Q可有效將腐植酸由1.9-126.8 mg L-1移除至低於偵測下限 (< 1 mg L-1)，並可獲得最高之DNA產量。在稀釋處理 (10或100倍) 後，方法Q可去除qPCR抑制，且具有最高之退伍軍人菌檢測濃度。進一步的冷卻水塔水樣驗證試驗則發現，以qPCR檢測Legionella pneumophila及檢測/定量Legionella spp.，樣本應先以方法Q進行DNA萃取，所得DNA於10倍稀釋後以qPCR分析。而L. pneumophila陽性樣本則將DNA進行100倍稀釋處理，並重新進行qPCR分析以獲得準確的L. pneumophila濃度。
本研究並以含有L. pneumophila DNA的樣本添加或不添加腐植酸或鐵離子進行試驗，以評估internal inhibition control (IIC) 定量qPCR抑制程度的能力。結果顯示IIC方法所測得的qPCR抑制程度與qPCR測得的L. pneumophila DNA量呈現顯著負相關 (r = -0.917)。另外，此劑量效應關係亦顯現於含有可測得的退伍軍人菌且具有> 5%以上qPCR抑制程度的冷卻水塔及熱水系統樣本 (r = -0.635 and -0.826)。這樣的結果證明了IIC方法具有定量> 5%以上qPCR抑制程度的能力。此外，DNA稀釋處理顯著降低環境樣本的qPCR抑制程度，並增加qPCR測得的退伍軍人菌濃度，顯現了IIC方法監測處理方法減緩qPCR程度的能力。
為了定量具活性的退伍軍人菌，本研究進行了結合ethidium monoazide (EMA, 0.9-45.5 μg mL-1) 與qPCR的技術 (EMA-qPCR) 最佳化，並評估其於冷卻水塔及熱水系統水樣和生物膜的應用性。qPCR結合2.3 μg mL-1的EMA為檢測退伍軍人菌之最佳EMA-qPCR條件，其可準確定量具活性之L. pneumophila、L. anisa和legionellae-like amoebal pathogens 6，且不會受到樣本中所含加熱處理過之退伍軍人菌、未加熱處理之其他細菌以及冷卻水塔水樣基質的干擾 (P > 0.05)。另外，將EMA-qPCR應用於冷卻水塔及熱水系統水樣和生物模，其測得的具活性退伍軍人菌濃度於大部分的樣本都高於培養法測得具可培養性的退伍軍人菌濃度，而低於qPCR測得的總退伍軍人菌濃度。
本研究亦進行了安養院環境調查，發現當冷卻水塔水中溶解性有機碳 (DOC) 濃度高於20 mg L-1和導電度大於2,000 μs cm-1，以及距離水塔上次清洗時間大於50天會導致顯著較高之退伍軍人菌盛行率 (all p < 0.05)。而於未操作的水塔，及水塔水樣具有較高之硬度和總懸浮固體物 (TSS) 時可測得較高之退伍軍人菌濃度 (all p < 0.05)。對於熱水系統，退伍軍人菌污染的風險因子為系統中存在Hartmannella vermfiormis、屋齡大於24年、水龍頭未加裝過濾接頭、水中硬度大於50 mg L-1 as CaCO3、pH > 7.5、餘氯濃度 ≤ 0.1 mg L-1、以及生物膜中含有的異營性細菌濃度 (heterotrophic plate counts, HPC) 高於7 log CFU per cm2 (all p < 0.05)。而熱水系統中的退伍軍人菌濃度則會隨著屋齡及熱水pH、導電度、TSS的增加而上升 (all p < 0.05)。另外，熱水系統中含高濃度HPC亦為高退伍軍人菌濃度之風險因子 (p < 0.05)。然而，生物膜中的退伍軍人菌盛行率及檢出濃度則與水中TSS呈顯著負相關 (both p < 0.05)。為能有效降低退伍軍人菌感染的風險，上述這些導致嚴重退伍軍人菌污染的風險因子均需於研擬冷卻水塔及熱水系統中退伍軍人菌的控制策略時納入考量。
總結而言，本研究成功的發展/最佳化DNA分離處理程序和IIC方法以解決qPCR抑制問題，並證明了以EMA-qPCR定量具活性退伍軍人菌的可環境應用性。此外，退伍軍人菌污染冷卻水塔和熱水系統的風險因子亦於本研究中清楚闡明，提供了控制退伍軍人菌及防治退伍軍人症的有效資訊。

關鍵字

退伍軍人菌 ； 即時定量聚合酶鏈鎖反應 ； qPCR抑制 ； 抑制內標 ； ethidium monoazide ； 安養院

並列摘要

Real-time quantitative PCR (qPCR) is popularly used to quantify legionellae; however, it may be interfered by environmental qPCR inhibitors and cannot differentiate viable and non-viable cells. This study tried to overcome such problems by optimizing DNA isolation procedure first. Evaluation was performed using samples spiked with humic acid (HA, ≤126.8 mg L-1), ferric ion alone (Fe, ≤3 mg L-1) or Fe/HA (≤3/100 mg L-1). QiAamp DNA Mini Kit (Q) is the appropriate DNA isolation method for quantifying legionellae by qPCR because it removed HA from 1.9-126.8 mg L-1 to an undetectable level (< 1 mg L-1), obtained the highest DNA yield, and relieved qPCR inhibition and acquired the highest cell recovery under 10- or 100-fold dilution. Further validation with cooling tower (CT) water shows that for detecting Legionella pneumophila and detecting/quantifying Legionella spp. by qPCR, Q with post 10-fold dilution is suggested to perform first. Afterward, L. pneumophila-positive samples should be re-analyzed a 100-fold dilution to acquire accurate cell concentrations.
Secondly, the capability of an internal inhibition control (IIC) to quantify the level of qPCR inhibition was assessed. Evaluation of L. pneumophila DNA-spiked samples, with or without the addition of HA or Fe, shows a significant and negative correlation between qPCR inhibition levels and qPCR-determined quantities of L. pneumophila DNA (r = -0.917). Such a dose-response relationship was also found in samples taken from CT and hot water systems (HWS) with detectable legionellae and > 5% partial qPCR inhibition levels (r = -0.635 and -0.826). These findings demonstrate that the IIC assay can reliably quantify > 5% partial qPCR inhibition levels. Moreover, for environmental samples, dilution treatment can significantly reduce qPCR inhibition levels and increase legionellae concentrations, highlighting the ability of the IIC assay to monitor the effectiveness of an intervention on the relief of inhibition.
Thirdly, this study optimized the technique of combining ethidium monoazide (EMA, 0.9-45.5 μg mL-1) with qPCR (i.e. EMA-qPCR) and evaluated its environmental applicability on quantifying viable legionellae in water and biofilm of CT and HWS. qPCR with EMA at 2.3 μg mL-1 was determined as the optimal EMA-qPCR assay, which can accurately quantify viable L. pneumophila, L. anisa and legionellae-like amoebal pathogens 6 without interferences by heated legionellae, unheated non-legionellae cells, and CT water matrix (p > 0.05). For water and biofilm samples of CT and HWS, the viable legionellae counts determined by EMA-qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR.
A nursing home survey conducted found a higher prevalence of legionellae in CT with a time interval between sampling and last cleaning > 50 days, dissolved organic carbon (DOC) > 20 mg L-1, and conductivity > 2,000 μs cm-1 (all p < 0.05), while legionellae concentrations were positively associated with non-operating CT and an increase in water hardness and total suspended solids (TSS) (all p < 0.05). Moreover, legionellae were more found in HWS with hardness > 50 mg L-1 as CaCO3, pH > 7.5, free chlorine ≤ 0.1 mg L-1, presence of Hartmannella vermfiormis, heterotrophic plate counts (HPC) in swab > 7 log CFU per cm2, building age > 24 years, and faucets without filters (all p < 0.05), while their abundance was increased with building age, pH, conductivity, and TSS (all p < 0.05). A higher level of HPC in HWS also increased legionellae concentration (p < 0.05). Furthermore, presence of legionellae in hot water was enhanced for DOC > 1 mg L-1 (p < 0.05), whereas legionellae prevalence and counts in swab were negatively correlated with water TSS (both p < 0.05). These risk factors should be considered in the control of legionellae contamination in CT and HWS to minimize the risk of legionellae infection.
In conclusion, this study successfully optimizes DNA isolation procedure and develops the IIC assay for overcoming qPCR inhibition and demonstrates the applicability of EMA-qPCR for quantifying viable legionellae. Moreover, the risk factors for legionellae in CT and HWS are clearly identified, which would help to control legionellae and prevent legionellosis.

並列關鍵字

legionellae ； real-time quantitative PCR ； qPCR inhibition ； internal inhibition control ； ethidium monoazide ； nursing home

參考文獻

Adeleke, A., Pruckler, J., Benson, R., Rowbotham, T., Halablab, M. and Fields, B. (1996) Legionella-like amebal pathogens--phylogenetic status and possible role in respiratory disease. Emerg Infect Dis 2, 225-230.

Allegra, S., Berger, F., Berthelot, P., Grattard, F., Pozzetto, B. and Riffard, S. (2008) Use of flow cytometry to monitor Legionella viability. Appl Environ Microbiol 74, 7813-7816.

Alleron, L., Merlet, N., Lacombe, C. and Frere, J. (2008) Long-term survival of Legionella pneumophila in the viable but nonculturable state after monochloramine treatment. Curr Microbiol 57, 497-502.

Arbeli, Z. and Fuentes, C.L. (2007) Improved purification and PCR amplification of DNA from environmental samples. FEMS Microbiol Lett 272, 269-275.

Bachoon, D.S., Otero, E. and Hodson, R.E. (2001) Effects of humic substances on fluorometric DNA quantification and DNA hybridization. J Microbiol Methods 47, 73-82.