生物氣膠採樣器效能評估-暴露艙研究

A Chamber Study on Performance Evaluation of Bioaerosol Samplers

周芳禛(Fang-Chen Chou)

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/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00623

摘要

吸入空氣中具可培養性或活性的嗜肺性退伍軍人菌可能會導致退伍軍人症的發生,因此定量空氣中具可培養性或活性的嗜肺性退伍軍人菌濃度是重要的環境衛生議題。然而當細菌被氣膠化或是被生物氣膠採樣器採下來後，其可培養性可能因過程中壓力的影響而降低，但卻不影響細菌的活性。過去生物氣膠採樣器評估之研究，分析方法多以定量可培養性及總退伍軍人菌為主，欠缺評估空氣中活性退伍軍人菌的研究。而且目前並無研究探討以培養基收集的採樣器間、液體收集的採樣器間及濾紙收集的採樣器間採集空氣中嗜肺性退伍軍人菌之效能。
本研究以培養法、即時定量聚合酶鏈鎖反應(Real-time quantitative polymerase chain reaction)及ethidium monoazide結合qPCR (EMA-qPCR)評估以培養基收集的採樣器 (MAS-100及Andersen一階採樣器)、液體收集的採樣器(BioSampler, AGI-30及MAS-100)及以濾紙收集的採樣器(IOM採樣器結合明膠濾紙(gelatin filter)及濾紙匣結合聚碳酸酯樹脂濾紙(polycarbonate filter))採集空氣中可培養性、活性及總嗜肺性退伍軍人菌(Legionella pneumophila)或大腸桿菌(Escherichia coli)之效能。根據採樣效能以培養法及EMA-qPCR偵測的結果得知，部分空氣中嗜肺性退伍軍人菌及大腸桿菌採樣後喪失了可培養性，但仍保持其細胞膜完整性。採樣器評估比較結果發現: Andersen一階採樣器採集可培養性細菌之效能優於MAS-100。採樣時間小於4分鐘時，液體收集的BioSampler和AGI-30優於以培養基收集的採樣器。BioSampler和AGI-30以去離子無菌水採樣60分鐘並每採樣15分鐘補充一次收集液時，採集可培養性或活性嗜肺性退伍軍人菌的效能優於BioSampler及AGI-30僅以Tween 80混合收集液或以去離子無菌水採樣但採樣過程中不補充液體之效能，而且也優於濾紙收集的採樣器。然而濾紙收集的採樣器採樣30或60分鐘時，採集總嗜肺性退伍軍人菌的效能優於液體收集的採樣器。此外，IOM採樣器結合明膠濾紙(gelatin filter)採樣270分鐘時，其採集可培養性及活性嗜肺性退伍軍人菌的效能優於濾紙匣結合聚碳酸酯樹脂濾紙的採樣效能，但其採集總嗜肺性退伍軍人菌之效能劣於濾紙匣結合聚碳酸酯樹脂濾紙之效能。對採集可培養性及活性細菌而言，BioSampler、AGI-30及MAS-100採集嗜肺性退伍軍人菌的效能一致優於大腸桿菌。
總結來說，EMA-qPCR可應用於環境定量空氣中具活性嗜肺性退伍軍人菌之濃度，採樣器部分，因BioSampler及AGI-30輔以補充液體的步驟可以有效的保持採樣後嗜肺性退伍軍人菌之可培養性及活性，建議以此方法進一步於環境中進行長達60分鐘的採樣。而空氣中總細菌濃度之定量，則建議使用濾紙收集的採樣器。

關鍵字

空氣中的 ； 嗜肺性退伍軍人菌 ； 大腸桿菌 ； 可培養性 ； 活性 ； 效能 ； 生物氣膠採樣器 ； 補充收集液 ； 即時定量聚合酶鏈鎖反應 ； ethidium monoazide ； EMA-qPCR

並列摘要

It is expected that inhalation of culturable or viable L. pneumophila may result in Legionnaires’ disease. Thus, quantification of culturable or viable airborne L. pneumophila is an important environmental health issue. However, as the bacteria are aerosolized or sampled by bioaerosol samplers, the sampling stresses might decrease cellular culturability but not affect the viability. Previous studies evaluated the performance of samplers for airborne Legionella have presented the results by quantification of culturable or total cells, whereas the level of viable cells remains unknown. Moreover, no study has been conducted to determine the difference in performance among different agar-based samplers, liquid-based samplers or filter-based samplers for airborne L. pneumophila.
In this study, the performance of agar-based samplers (MAS-100 and Andersen one stage), liquid-based samplers (BioSampler, AGI-30 and MAS-100) and filter-based samplers (IOM with gelatin filter and cassette with polycarbonate filter) for collecting culturable, viable or total L.egionella pneumophila or E.scherichia coli determined by culture assay, real-time quantitative polymerase chain reaction (qPCR) and ethidium monoazide combined with qPCR (EMA-qPCR) were evaluated. According to the performance of samplers for L. pneumophila and E. coli determined by culture and EMA-qPCR, it was observed that a portion of L. pneumophila and E. coli was nonculturable but membrane-intact after being collected from the air. Evaluating and comparing the performance of samplers, the results showed that Andersen one stage performed better than MAS-100 for collecting both culturable L. pneumophila and E. coli. The performances of liquid-based BioSampler and AGI-30 for culturable L. pneumophila were greater than that of agar-based samplers in less than 4-min sampling time. In addition, for BioSampler and AGI-30, it was more efficient to use sterile and filtered de-ionized water (DW) to collect both culturable and viable L. pneumophila for 60 min and replenish the water every 15 min than to use Tween 80 mixture or DW without replenishment for collection. And the performance of BioSampler and AGI-30 with DW and replenishment for culturable and viable L. pneumophila were better than that of filter-based samplers. However, the performance for total L. pneumophila was better in filter-based samplers than in any liquid-based samplers for 30- and 60-min sampling time. Furthermore, IOM sampler with gelatin filter showed greater performance for collecting culturable and viable L. pneumophila than cassette with polycarbonate filter for 270-min sampling time. As for collecting total cells, the result was opposite. For sampling viable and total L. pneumophila and E. coli, the performance of BioSampler, AGI-30 and MAS-100 were consistently better for L. pneumophila than for E. coli.
In conclusion, EMA-qPCR could be applied to quantify the concentration of viable airborne L. pneumophila. The refilling process on BioSampler and AGI-30 may be effective to remain the culturability and viability of L. pneumophila, and it was further recommended monitoring and sampling for 60-min in field. As for quantification of total cells, filter-based samplers were suggested.

並列關鍵字

airborne ； Legionella pneumophila ； Escherichia coli ； culturable ； viable ； performance ； bioaerosol samplers ； replenishment ； real-time qPCR ； ethidium monoazide ； EMA-qPCR

參考文獻

Agranovski, V., Ristovski, Z., Hargreaves, M., Blackall, P. J. and Morawska, L. (2003). Real-time measurement of bacterial aerosols with the UVAPS: performance evaluation. Journal of Aerosol Science 34:301-317.

Angenent, L. T., Kelley, S. T., St Amand, A., Pace, N. R. and Hernandez, M. T. (2005). Molecular identification of potential pathogens in water and air of a hospital therapy pool. Proc Natl Acad Sci U S A 102:4860-4865.

Blatny, J. M., Reif, B. A., Skogan, G., Andreassen, O., Hoiby, E. A., Ask, E., Waagen, V., Aanonsen, D., Aaberge, I. S. and Caugant, D. A. (2008). Tracking airborne Legionella and Legionella pneumophila at a biological treatment plant. Environ Sci Technol 42:7360-7367.

Blatt, S. P., Parkinson, M. D., Pace, E., Hoffman, P., Dolan, D., Lauderdale, P., Zajac, R. A. and Melcher, G. P. (1993). Nosocomial Legionnaires' disease: aspiration as a primary mode of disease acquisition. American Journal of Medicine 95:16-22.

Chang, B., Sugiyama, K., Taguri, T., Amemura-Maekawa, J., Kura, F. and Watanabe, H. (2009). Specific detection of viable Legionella cells by combined use of photoactivated ethidium monoazide and PCR/real-time PCR. Appl Environ Microbiol 75:147-153.