加熱處理之嗜肺性退伍軍人菌特性研究

Characteristics of Legionella pneumophila with heat treatment

高千惠(Chien-Hui Kao)

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(2007年)

https://doi.org/10.6342/NTU.2007.03097

摘要

為了解自來水中退伍軍人菌於37℃或受熱60℃時之反應，本研究利用培養法及螢光染色法評估退伍軍人菌之可培養性、細菌活性及總細菌濃度之變化，並以此二種方法探討受熱細菌與兩種原蟲宿主- A. castellanii 和H. vermiformis共同培養3日內細菌之變化。除此短期觀察外，另以即時定量聚合酶連鎖反應法取代螢光染色法評估與原蟲共存27日之細菌總濃度變化，藉此了解退伍軍人菌的增生能力，細菌之可培養性亦同步評估。此外，在27日觀察期中，也探討以加熱（60℃,30分鐘）處理的兩種原蟲是否可成為退伍軍人菌營養源，及其對此菌可培養性與增生能力之影響。
結果顯示，退伍軍人菌（108 cells/ml）於60℃受熱5分鐘與30分鐘後，可培養濃度分別下降了4 log CFU/ml與6 log CFU/ml，而細胞膜完整性則分別下降56 %與83 %，但未造成總細菌濃度之改變，故可知受熱60℃主要影響細菌的可培養性。而37℃之退伍軍人菌與原蟲共同培養的3日內，總細菌濃度最大增生量可達2.5 log cells/ml，且以經A. castellanii之增生能力較高。當細菌在60℃受熱達30分鐘時，即使經兩種原蟲培養，72小時內仍無法觀察到具可培養性之細菌，總細菌濃度亦無增生現象；但與H. vermiformis共同培養時卻可觀察到受熱60℃的退伍軍人菌出現活性增生的情況。
27日長期觀察的結果則顯示，37℃ 5分鐘之退伍軍人菌若僅與兩種原蟲營養源共存時，營養源比例高低與營養源種類對細菌之增生能力無顯著影響，且可培養細菌濃度亦隨觀察時間而下降；但若退伍軍人菌與高濃度營養源(細菌：營養源=1:100)共存1日後再與活性A. castellanii及H. vermiformis宿主共同培養時，退伍軍人菌總濃度則出現上升現象。當將受熱60℃ 5分鐘退伍軍人菌直接與A. castellanii共同培養時，亦發現其總細菌濃度在27日內最高可上升至6 log cells/ml，且較經H. vermiformis共同培養的退伍軍人菌有較高的增生能力。但若僅有營養源而無原蟲宿主時，反而出現總細菌濃度下降之現象，且營養源濃度越高下降情形更為顯著；即使於營養源共存1或3日後亦加入活性原蟲宿主，退伍軍人菌總濃度仍趨於下降，且下降幅度亦隨營養源濃度增加而提高；至於可培養性方面，受熱於60℃之細菌僅在立即與原蟲共同培養時可偵測到具可培養性之細菌。
整體而言，受熱溫度顯著影響退伍軍人菌之可培養性與細胞膜完整性，且以可培養性所受之影響較大；受熱細菌與原蟲宿主共同培養三日之結果顯示，增加共同培養與受熱時間可分別顯著提高與降低細菌之增生性、可培養性與細胞膜完整性(p < 0.05與 < 0.001)；而在27日共同培養的觀察中發現，營養源與受熱溫度皆為顯著抑制退伍軍人菌總濃度(p < 0.001)與可培養濃度 (p < 0.001) 之主要因子。而不論觀察期間之長短，原蟲種類均顯著影響退伍軍人菌的總濃度與可培養濃度，且A. castellanii均優於H. vermiformis。

關鍵字

退伍軍人菌 ； A. castellanii ； H. vermiformis ； 加熱法 ； 可培養性 ； 細胞膜完整性 ； 細菌增生性

並列摘要

In this study, conventional standard culture method and fluorescent LIVE/DEAD BacLight staining assay were performed for evaluating the culturability, viability and total cell concentration of Legionella pneumophila after heat exposure (37℃ and 60℃) in tap water. Heated L. pneumophila in 3-day coculture with A. castellanii and H. vermiformis, respectively, were also evaluated. Real-time PCR assay was used to determine total concentration of L. pneumophila in 27-day coculture with amoebae; culturability was evaluated simultaneously. Moreover, we investigated the culturability and proliferation ability of L. pneumophila while serving the heat treated (60℃, 30 min) A. castellanii and H. vermiformis as the nutrient during this time period.
First, we found that after exposure to 60℃ for 5 and 30 min ,culturable L. pneumophila concentration reduced 4 and 6 log CFU/mL, and the loss of cell membrane integrity was 56 % and 83 %, respectively. However, the total concentration was not affected. These results indicate heat exposure decreases the culturability of L. pneumophila significantly.
Second, heated L. pneumophila (37℃) could proliferate to the maximum of total concentration at 2.5 log cells/mL in coculture with amoebae in 3 days. Higher proliferation ability was observed in coculture with A. castellanii. After 30 min exposure at 60℃ for 30min culturable L. pneumophila was not detected and no cellular proliferation was found in coculture with amoebae. However, increase in cellular membrane integrity of L. pneumophila was observed in H. vermiformis coculture.
Third, in the 27 observation days without coculture with viable amoebae, both nutrient contents and types of amoebae used as nutrients presented no significant effect on proliferation ability of 37℃ L. pneumophila. Also, the culturability of L. pneumophila decreased over time period. However, total concentration of L. pneumophila increased in high levels of nutrients when adding viable A. castellanii. As for 60℃ heated L. pneumophila, total concentration of L. pneumophila showed 6 log cells/ml increase when cocultured with A. castellanii, and its reproducibility is higher than that cocultured with H. vermiformis. When L. pneumophila grew with nutrient alone without viable amoebae as hosts, total concentration of L. pneumophila was decreasing, and higher nutrient contents caused more decrease. Even amoebic hosts added after L. pneumophila co-existed with nutrients for 1 day or 3 days, total concentration of L. pneumophila still decreased. And higher nutrient concentration would cause more decrease. On the other hand, the culturability of 60℃ heated L. pneumophila can be detected only when cocultured with amoebae immediately right after heating treatment.
In conclusion, the culturability and membrane integrity of L. pneumophila were affected by heating temperature. Results showed that higher temperature would significantly decrease the producibility, culturability, and membrane integrity of heated L. pneumophila cocultured with protozoa for 3 days (p<0.05). However, the longer coculture time with amoebae would significantly increase the producibility, culturability, and membrane integrity of L. pneumophila (p<0.001). And we revealed that nutrients (heated amoebae) and heating temperature are the most important factors which significantly decrease the total and culturable concentration of L. pneumophila in 27-day coculture period (p<0.001). No matter how long the period was observed, amoebic genera significantly affect the total and culturable concentration of L. pneumophila.

並列關鍵字

L. pneumophila ； A. castellanii ； H. vermiformis ； heat disinfection ； culturability ； membrane integrity ； reproducibility

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