雨刷水相關清潔用品對嗜肺性退伍軍人菌抑菌效能評估

Antibacterial activities of Windscreen wiper fluid (WWF) against Legionella pneumophila

洪于茹(Yu-Ju Horng)

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(2017年)

https://doi.org/10.6342/NTU201704234

摘要

過往研究退伍軍人病 (Legionellosis)零星個案報告中，發現職業駕駛人所占比例甚高，並於汽車內雨刷水箱檢測出退伍軍人菌Legionella pneumophila (L. pneumophila)。雨刷精具有維護雨刷水箱及保護車窗的作用，國內研究發現駕駛人員不僅習慣添加雨刷精 (Commercial screenwash,CS)，亦會添加家用清潔劑 (Household detergent, HD)。國外研究指出添加雨刷精可降低退伍軍人菌的檢出，且雨刷精與家用清潔劑兩類型雨刷水清潔用品添加至雨刷水箱內之退伍軍人菌檢出情形不一致。本研究為探討雨刷水清潔用品對L. pneumophila影響，以12種市售雨刷精及10種家用清潔劑探討不同清潔用品種類、不同清潔用品濃度 (0.1及0.01 g/mL)、接觸時間 (2時及30日)及不同水溫 (25及37℃)，同時亦探討pH及硬度對L. pneumophila影響。
本研究以添加雨刷水清潔用品之實驗組及未添加雨刷水清潔用品之控制組分別進行三階段之抑菌實驗：(1)25℃短期抑菌實驗；(2)37℃短期抑菌實驗；(3)37℃長期追蹤實驗，並以推盤法及EMA-qPCR分析L. pneumophila培養力及活性濃度，同時於短時間120分鐘時及長期追蹤30日時測量pH及硬度。
統計結果顯示，於市售雨刷精品牌中，以TM效果最佳，對L. pneumophila培養力及活性皆有顯著較佳抑制作用 (P-value<0.0001)。另外對於抑制活性顯著較佳者為GD(P-value<0.0001)。就家用清潔劑而言，以MB對培養力抑制顯著較佳(P-value<0.0001)，WD、DW及MA則對活性抑制較佳，具顯著影響 (P-value<0.0001)。本研究共探討22種雨刷水清潔用品，相較於家用清潔劑，添加市售雨刷精抑菌效果較佳。另外顯示雨刷水清潔用品之添加濃度為0.1 g/mL時，相對於0.01 g/mL較能有效抑制L. pneumophila培養力。對於探討接觸時間之影響方面，本研究於短期及長期時間觀察結果包含：(1)雨刷水清潔用品能立即性抑制L. pneumophila培養力及活性；(2)隨著接觸時間增加，雨刷水清潔用品抑制L. pneumophila培養力及使活性下降之趨勢緩慢；(3)到接觸結束時間點仍雨刷水清潔用品中有L. pneumophila培養力及活性濃度。上述結果顯示了不同雨刷水清潔用品的抑菌效果之優劣勢。於探討水溫部分，結果顯示於37℃下退伍軍人菌可能較能抵抗雨刷水清潔用品抑制影響或延遲抑菌效果。
探討pH值之影響方面，結果顯示pH高於8者對L. pneumophila培養力較具影響，對其活性影響力較低；對家用清潔劑而言，結果顯示愈鹼性的環境，對L. pneumophila培養力愈不利，對其活性則無明顯影響。另外，針對雨刷水清潔用品硬度影響之結果則顯示硬度介於3-12 mg/L者對L. pneumophila培養力較具影響，對其活性影響力較低；就家用清潔劑而言，硬度介於3-12 mg/L可使L. pneumophila培養力上升，硬度介於30-195 mg/L則使該菌培養力下降，然而硬度高，L. pneumophila活性濃度亦高。
另外針對具活性但不具培養性 (Viable but nonculture, VBNC)之L. pneumophila進行探討，對22種雨刷水清潔用品而言，於接觸3日能有效抑制L. pneumophila培養力及活性者佔18.2% (4/22)，含低濃度VBNC之L. pneumophila者佔13.6% (3/22)，含高濃度VBNC之L. pneumophila者佔50% (11/22)，於接觸8日培養力完全抑制且含VBNC之L. pneumophila者佔18.2% (4/22)。
本研究證實不同成分雨刷水清潔用品對L. pneumophila培養力及活性有影響具差異，且不同清潔用品濃度、接觸時間及不同水溫皆有不同抑菌效果之影響，可供未來作為駕駛人員添加雨刷水清潔用品建議方法參考依據，以降低社區零星式退伍軍人菌病之發生。

關鍵字

嗜肺性退伍軍人菌 ； 雨刷水清潔用品 ； 抑菌實驗

並列摘要

Literature showed that drivers possess greater risk of acquiring legionellosis, and windscreen wiper systems (WWS) of vehicles are considered as the potential contamination source. Windscreen wiper fluid (WWF) is used to clean the windscreen, research showed that some people use commercial screenwash (CS) and some use household detergent (HD). Previous study had suggest that adding WWF may affect the Legionella growth, and adding household detergent may had lower detection rate of Legionella pneumophila of WWS. The aim of this study was to determine the antimicrobial activity of WWF against L. pneumophila, and to assess the influence on the culturable and viable L. pneumophila of several factors, including the type and the concentration of detergent, contact time, water temperature, pH value and hardness.
In this study, three experiments were carried out on the experimental group with the addition of WWF and the control group without WWF. First was short-term antibacterial test at 25 ℃; second was short-term test at 37 ℃; third was long-term test at 37 ℃. Culture method and EMA-qPCR analysis were used for analyzing the concentration of culturable and viable L. pneumophila. The pH value and hardness of WWF were measured at 120 minutes for short term and 30 days for long term test.
The results showed that TM had significant effect on the culturable and viable L. pneumophila (P-value <0.0001). In addition, GD has better effect inhibition of viable L. pneumophila (P-value <0.0001). WD, DW and MA had significant effect inhibition of viable L. pneumophila (P-value <0.0001). Moreover, the antibacterial effect of commercial screenwash was better than household detergent. Results of the influence of concentration of detergentshowed that 0.1 g/mL inhibited more L. pneumophila than 0.01 g/mL. On the other hand, there were three key point of influence of contact time, including short-term and long-term: (1) immediate inhibition to the culturable and viable L. pneumophila; (2) inhibition of the culturable and viable L. pneumophila decreased slowly as time increased; (3) at the end of the time point, the culturable and viable L. pneumophila still can be detected (Culturable >1 or 5 CFU/mL；Viable> 620 cells/mL). Above all, results showed the advantages and disadvantages of different WWF. Influence of the water temperature showed that L. pneumophila may be more resistant the antibacterial effect at 37 °C.
For commercial screenwash, results showed that pH value above 8 may have impact on the culturable L. pneumophila, but less for the viable L. pneumophila. As for household detergents, when the environment was more alkaline, the culture of L. pneumophila was more likely to get impaired, but there was no significant effect on the viable L. pneumophila. For commercial screenwash, results showed that hardness between 3-12 mg/L may have more impact on the culturable L. pneumophila, but less on viable L. pneumophila. As for household detergents, the hardness of 3-12 mg/L may increase the culture capacity, and hardness between 30-195 mg/L may decline theculturalbility; besides, the higher the hardness, the higher the concentration of viable L. pneumophila.
In addition, there were 18.2% (4/22) of WWF could inhibit the culture and viable L. pneumophila, 13.6% (3/22) had lower viable but nonculture (VBNC), 50% (11/22) had high level concentration of VBNC L. pneumophila, and 18.2 % (4/22) had completely inhibition and had VBNC L. pneumophila at 8 days .
Above all, this study confirmed that different ingredient of WWF have different effects on the culture and viable L. pneumophila. Different concentration of detergent, contact time and the water temperature may have different the antibacterial effect, which can provide further recommendation of adding WWF in order to reduce the occurrence of community sporadic disease.

並列關鍵字

Legionella pneumophila ； windscreen wiper fluids ； antibacterial activity

參考文獻

Al‐Bana, B. H., Haddad, M. T., & Garduño, R. A. (2014). Stationary phase and mature infectious forms of Legionella pneumophila produce distinct viable but non‐culturable cells. Environmental microbiology, 16(2), 382-395.

Alexandropoulou, I. G., Konstantinidis, T. G., Parasidis, T. A., Nikolaidis, C., Panopoulou, M., & Constantinidis, T. C. (2013). First report of Legionella pneumophila in car cabin air filters. Are these a potential exposure pathway for professional drivers? Scand J Infect Dis, 45(12), 948-952. doi:10.3109/00365548.2013.840917

Allegra, S., Grattard, F., Girardot, F., Riffard, S., Pozzetto, B., & Berthelot, P. (2011). Longitudinal evaluation of the efficacy of heat treatment procedures against Legionella spp. in hospital water systems by using a flow cytometric assay. Applied and Environmental Microbiology, 77(4), 1268-1275.

Alleron, L., Khemiri, A., Koubar, M., Lacombe, C., Coquet, L., Cosette, P., . . . Frere, J. (2013). VBNC Legionella pneumophila cells are still able to produce virulence proteins. Water Research, 47(17), 6606-6617.

Alleron, L., Merlet, N., Lacombe, C., & Frere, J. (2008). Long-term survival of Legionella pneumophila in the viable but nonculturable state after monochloramine treatment. Curr Microbiol, 57(5), 497-502. doi:10.1007/s00284-008-9275-9