台灣位處於亞熱帶地區，溫暖潮濕的氣候提供真、細菌良好的生長環境，且特定的環境氣象與空污事件如集中於特定月份的降雨、盛行於夏季的颱風、冬末春初的長程傳輸等，均可能影響其濃度分布；然而，過去研究受限於追蹤時間短且樣本採樣分析方法耗時，難以真正釐清上述事件對於大氣真細菌濃度的影響程度。因此，本研究透過長期性樣本收集，並以分子定量技術為本，探討空氣污染物及氣象因子之時間分布變化對於活性真、細菌濃度的影響，該結果將有助於瞭解大氣生物氣膠暴露的環境影響因子，做為後續在健康維護與預防介入之參考。
本研究利用2013年12月31日到2016年1月6日間以Burkard cyclone (流速16.7 l/min) 採集之空氣微粒樣本，採樣器設置於成功大學醫學院頂樓（高度15 m），每7天取得一樣本。本研究利用可同時萃取真、細菌DNA之ZR fungal/bacterial DNA MiniPrep套組，於萃取前需以propidium monoazide (PMA) 進行處理以利後續定量活性濃度，並建置適用於混合真、細菌樣本的最佳化PMA條件，最後以qPCR進行定量。分析所得之濃度將連結至環境資料，以廣義估計方程式估算其影響因子，並以Wilcoxon signed rank test分析在特定事件前後對活性真、細菌濃度的影響。
本研究建置之最佳化PMA條件為50 μM PMA、光照15分，經PMA-qPCR定量得活性真菌平均濃度為5.36 ± 0.92 (log spores/m3)，活性細菌則為4.17 ± 0.82 (log copies/m3)。當CH4、氣壓與降雨量每上升一單位，活性真菌濃度分別增加了0.673 log spores/m3/ppb、0.008 log spores/m3/hpa與0.091 log spores/m3/mm。而CH4每上升一單位，活性細菌濃度則增加0.840 log copies/m3/ppb。結果發現活性真、細菌的濃度於颱風期間濃度皆顯著高於颱風之前，然而雖然長程傳輸與強降雨發生期間活性濃度亦高於發生之前，但未達統計上的顯著差異。
結果顯示環境特殊事件的確與大氣中的活性真、細菌濃度變化有關。本研究亦進一步找出影響活性微生物濃度之可能因子，除可用於評估民眾之暴露風險與預防疾病之外，亦可做為建立預警系統之參考。

SUMMARY
Previous studies have focused on changing monthly and seasonal patterns of airborne microbes, but only limited evidence have addressed the impacts of special climatic events such as long-range transport, typhoon, and heavy rainfall. This study applied ZR fungal/bacterial DNA MiniPrep kit (ZR kit) to isolate fungal and bacterial DNA, simultaneously. The optimization of propidium monoazidein (PMA) treatment to distinguish viability of bacteria and fungi were determined before environmental samples analyses were conducted. Afterwards, sample extraction was completed by ZR kit and PMA-qPCR and viable fungal and bacterial concentrations were correlated with climatic factors and air pollution levels. This study identified that CH4, pressure, wind speed and rainfall positively correlated with viable fungal concentrations, and CH4, CO and rainfall positively correlated with viable bacterial concentrations. In addition, viable fungal and bacterial concentrations during long-range transport, typhoon and heavy rainfall were higher than levels found during non-event periods.

INTRODUCTION
Airborne fungal and bacterial concentrations are suggested to be correlated with the monthly and seasonal variations of various meteorological parameters and air pollutants. Taiwan is located in the subtropical region, with high temperature and humidity that favors microbial growth/survival. Therefore, the understanding of the temporal patterns in fungal and bacterial concentrations, and their associations with meteorology and air pollution ought to be apt for determining the exposure risk and clarifying the potential factors linked to their temporal distributions. Previous studies have focused on changing monthly and seasonal patterns of airborne microbes, but only limited evidence have addressed the impacts of special climatic events such as long-range transport, typhoon, and heavy rainfall. Moreover, the target microorganisms in such studies were mostly reported either as the “total” or “culturable” fungi and bacteria. However, there is only 1% microorganisms being culturable, and viable cells are considered to result in more serious threat in public health. The aims of this study are therefore to investigate the weekly, monthly and seasonal variations of microbial concentrations. In addition, environmental factors related to temporal variations of bioaerosols will also be characterized by analyzing the associations of microbial concentrations with air pollutants and meteorological factors.

MATERIALS AND METHODS
Two-year-long sampling (2013/12/31~2016/1/6) was performed on the 6th floor roof of National Cheng Kung University Medical College using Burkard cyclone (flow rate of 16.7 l / min). One sample was taken every seven days to analyze the ambient concentrations of bacteria and fungi. ZR kit was used to extract microbial DNA. However, for analyzing viable fungi and bacteria, samples should be treated with PMA before DNA extraction. The optimization of propidium monoazidein (PMA) treatment to distinguish viable and none viable viability of in bacteria and fungi were determined before environmental samples analyses were conducted by PMA-qPCR. Based on the results of qPCR assay, we acquired the weekly average concentrations of fungi and bacteria. Monthly and seasonal average concentrations were also calculated. In addition, this study also collected the daily information of meteorological factors (e.g. temperature, relative humidity, wind speed) and air pollutants (e.g. CO, SO2, O3, NOx, PM10, PM2.5) from Tainan Weather Station and Tainan Air Quality Monitoring Station, respectively. These daily data were further converted to weekly data. Finally, the associations of fungal and bacterial concentrations with meteorological factors and air pollutants were analyzed by generalized estimating equation. The effects of the events of typhoon, heavy rainfall and long-range transport were explored through use of Wilcoxon signed rank test to compare viable microbial concentration between events and controls.

RESULTS AND DISCUSSION
The average concentration of viable fungi was 5.36 ± 0.92 (log spores/m3) over the study period (fig.1), with the highest level in 2014/11/28 - 12/5 (6.56 log spores/m3) and 2015/12/30 - 1/6 (7.64 log spores/m3). Viable bacteria was 4.17 ± 0.82 (log copies/m3), and the highest level in 2014/12/19 - 12/26 (5.09 log copies/m3) and 2015/5/21 - 5/28 (6.04 log copies/m3) . During the study period, temperature and relative humidity were 24.34 ± 4.74 (℃) and 75.05 ± 5.36 (%), respectively.As showed in Table 1, CH4, pressure, wind speed and rainfall positively correlated with viable fungal concentration. CH4, CO and rainfall positively correlated with viable bacterial concentration. For the Table 2, viable fungal and bacterial concentration during long-range transport, typhoon and heavy rainfall were higher than those of non-event periods. Raindrops are thought to transport bioaerosols from higher altitude to the ground and the wind might then aersolize the microorganisms (Heo et al. 2014; Kang 2015). Besides, typhoon will carry heavy rainfall, leading to increased levels of microorganism (Raisi et al. 2010; Heo et al. 2014). The concentration of air pollutants during long-range transport are found higher than control periods and therefore the increased fungi and bacteria levels are found (Wu et al. 2004; Chao et al. 2012; Tam 2016).

CONCLUSION
This study presents the weekly temporal patterns of viable fungal and bacterial concentrations and identifies environmental factors which are cirtical to their levels and distributions. Viable fungal and bacterial concentrations during event periods are found to be higher than those of non-event periods. Residents are encouraged to stay indoors during event periods and refrain themselves from higher likelihood of exposure to viable microbes. Results could be considered as useful reference information for establishing the warning system in light of rapidly changing climate.

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