Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods.
Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods.
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DOI：
10.3390/ijerph121013118
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发表时间：
2015-10-19
影响因子：
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通讯作者：
Miyamoto H
中科院分区：
文献类型：
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作者：
Edagawa A;Kimura A;Kawabuchi-Kurata T;Adachi S;Furuhata K;Miyamoto H
通过培养、与阿米巴共培养、实时定量PCR （qPCR）和与阿米巴共培养的实时定量PCR，对日本68个洗浴设施的洗浴水样品中的军团菌污染情况进行了调查。常规培养法检出嗜肺军团菌11份（11/68,16.2%）。与我们的预期相反，与阿米巴共培养技术的培养方法并没有提高军团菌的检出率（4/68,5.9%）。相比之下，将阿米巴共培养技术与qPCR相结合，比单独使用实时qPCR（46/68, 67.6%）成功地提高了检出率（57/68,83.8%）。经与阿米巴共培养后的实时qPCR检测，30份样品的细菌数量比未共培养的样品增加了10倍以上（30/68,44.1%）。32份样品经变形虫繁殖后细菌数量未见增加（32/68,47.1%）。其余6份标本（6/68,8.8%）均未检出军团菌。这些结果表明，在实时qPCR之前应用阿米巴共培养技术可能有助于对洗澡水样品中的军团菌进行灵敏检测。此外，阿米巴虫共培养和实时qPCR的结合可能有助于检测活的和致命的军团菌，因为它们在自由生活的阿米巴虫体内入侵和繁殖的能力被认为与它们对人类的致病性有关。这是评价阿米巴共培养技术在洗澡水样品中检测军团菌效果的第一份报告。
We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.