Characteristics of Legionella pneumophila with heat treatment

• Main Authors: Chien-Hui Kao, 高千惠
• Other Authors: Ching-Wen Chang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/91965505800733436632

碩士 === 臺灣大學 === 環境衛生研究所 === 95 === 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.