肉桂醛對嗜肺性退伍軍人菌抑菌效能評估

Abstract

嗜肺性退伍軍人菌(Legionella pneumophila)是造成退伍軍人病和龐提亞克熱的致病菌，從肉桂葉中萃取的精油主成分肉桂醛(cinnamaldehyde)已被證實對於懸浮狀態的退伍軍人菌具有良好的殺菌效果。在環境中，退伍軍人菌普遍存在於熱水系統或冷卻水塔的生物膜中。已知生物膜中的細菌會增加對殺菌劑的抵抗性，且從生物膜中剝落下來的細菌會持續不斷的汙染水體，增加人類感染風險。但截至目前為止，並無研究利用肉桂醛針對退伍軍人菌形成的生物膜進行殺菌效能的評估,也無研究探討pH值和溫度的改變對於肉桂醛抑制生物膜效能的影響。因此，此研究的目的是評估肉桂醛對以靜置或流動的方式培養而形成之嗜肺性退伍軍人菌生物膜的殺菌效能，另外也探討pH值(7-9)和溫度(42°C和28.5°C)對於肉桂醛抑制嗜肺性退伍軍人菌生物膜效能的影響。除利用培養法觀察肉桂醛對細菌可培養性的影響，本研究也利用Ethidium monoazide (EMA)結合real-time quantitative PCR (EMA-qPCR)之技術探討其對細胞膜的破壞。結果顯示,肉桂醛能有效的抑制嗜肺性退伍軍人菌形成的生物膜，且抑菌效能隨濃度和時間的增加而增加(P<0.05)，展現劑量-效應關係。當嗜肺性退伍軍人菌與1000 μg/ml以上的肉桂醛接觸60分鐘時，可完全抑制此菌的可培養性。暴露於125 μg/ml下之肉桂醛10分鐘，即可觀察到肉桂醛對細胞膜的破壞，對於靜置培養生物膜的殺菌效能為10.25%，流動培養生物膜的殺菌效能為7.37%。此外當兩種生物膜暴露於濃度小於500 μg/ml肉桂醛60分鐘時，其感受性具顯著差異(P<0.05)，利用雷射掃描式共軛焦顯微鏡 (confocal laser scanning microscopy)觀察後發現結構上的差異是影響對肉桂醛感受性的主要原因。另外也觀察到肉桂醛對於生物膜的殺菌效能隨著pH值和溫度的增加而顯著上升。 以上結果顯示，肉桂醛除了對懸浮態的退伍軍人菌具有良好的殺菌效果之外，也可顯著破壞固著態的退伍軍人菌(退伍軍人菌生物膜)之可培養性與細胞膜。未來可考量將肉桂醛應用在水溫較高、水質偏鹼的環境中，以控制懸浮態及固著態的嗜肺性退伍軍人菌。

Cinnamaldehyde is a major component in mature leaves of Cinnamomum osmophloeum. It has been proved to inactive planktonic Legionella pneumophila, an organism causing Legionnaires’ disease and Pontiac fever. L. pneumophila usually survives in water environment as habitants, living within biofilms that commonly found in distribution systems and cooling towers. Bacteria in biofilms are generally considered to be more resistant against disinfectants, and detachment of biofilm cells becomes a continual source providing microbial contamination to the bulk water. There was no research focused on the disinfection efficiency of L. pneumophila biofilms by cinnamaldehyde, and fewer studies focused on the effect of pH value and temperature on cinnamaldehyde’s effect on sessile cells. Therefore, the purpose of the study was to investigate the antibacterial effect of cinnamaldehyde against sessile L. pneumophila which were in static and continuous-flow culture, and to evaluate the pH and temperature effects on the disinfection efficacy of cinnamaldehyde on sessile L. pneumophila. In addition to observing the effect of cinnamaldehyde on cellular culturability by culture assay, ethidium monoazide coupled with real-time quantitative PCR (EMA-qPCR) was used to observe the effect of cinnamaldehdye on cell membrane integrity. The results shows that cinnamaldehyde was effective in disinfection of sessile L. pneumophila, and the antibacterial effect increased with the concentration and contact time (P<0.05), indicating a dose-response biocidal effect against sessile L. pneumophila. Contacted with cinnamaldehyde at concentration higher than 1000 μg/ml for 60 min, sessile L. pneumophila totally lost their culturability on BCYEα agar. Expose to 125 μg/ml of cinnamaldehyde for 10 min, cell membrane damage measured by EMA-qPCR assay was observed. The inactivation rate was 10.25% for static cultured bilfilms and 7.37% for continuous-flow culured biofilms. However, the susceptibility to cinnamaldehyde at concentration lower than 500 μg/ml was significantly different between two kinds of sessile cells (P<0.05). The different structure of two kinds of sessile cells was further proved by confocal laser scanning microscopy (CLSM), supporting that different structural feature between two kinds of sessile cells may contribute to different susceptibility to cinnamaldehyde. Moreover, the disinfection efficacy was enhanced against sessile cells at higher pH levels and at higher temperatures. In conclusion, in this study, it was found that cinnamaldehyde is an effective disinfectant not only against planktonic L. pneumophila but also sessile L. pneumophila by destroying their cell membrane or cellular culturability. In the future, cinnamaldehyde possesses the potential to be used to control Legionella in aquatic environment with high pH values and high temperature, e.g., air-conditioning cooling towers in the summer.