Nagasaki Prefectural Institute for Environmental Research and Public Health, 2-1306-11 Ikeda, Omura, Nagasaki 856-0026, Japan.
J Microbiol Methods. 2011 Jul;86(1):25-32. doi: 10.1016/j.mimet.2011.03.012. Epub 2011 Apr 2.
Legionella species are the causative agents of human legionellosis, and bathing facilities have been identified as the sources of infection in several outbreaks in Japan. Researchers in Japan have recently reported evidence of significant associations between bacterial counts and the occurrence of Legionella in bathing facilities and in a hot tub model. A convenient and quantitative bacterial enumeration method is therefore required as an indicator of Legionella contamination or disinfection to replace existing methods such as time-consuming Legionella culture and expensive Legionella-DNA amplification. In this study, we developed a rapid detection method (RDM) to monitor the risk of Legionella using an automated microbial analyzing device based on flow cytometry techniques to measure the total number of bacteria in water samples within two minutes, by detecting typical patterns of scattered light and fluorescence. We first compared the results of our RDM with plate counting results for five filtered hot spring water samples spiked with three species of bacteria, including Legionella. Inactivation of these samples by chlorine was also assessed by the RDM, a live/dead bacterial fluorescence assay and plate counting. Using the RDM, the lower limit of quantitative bacterial counts in the spiked samples was determined as 3.0×10(3)(3.48log)counts mL(-1). We then used a laboratory model of a hot tub and found that the RDM could monitor the growth curve of naturally occurring heterotrophic bacteria with 1 and 2 days' delayed growth of amoeba and Legionella, respectively, and could also determine the killing curve of these bacteria by chlorination. Finally, samples with ≥3.48 or <3.48log total bacterial counts mL(-1) were tested using the RDM from 149 different hot tubs, and were found to be significantly associated with the positive or negative detection of Legionella with 95% sensitivity and 84% specificity. These findings indicated that the RDM can be used for Legionella control at bathing facilities, especially those where the effectiveness of chlorine is reduced by the presence of Fe(2+), Mn(2+), NH(4)(+), skin debris, and/or biofilms in the water.
军团菌物种是人类军团病的病原体，沐浴设施已被确定为日本几起暴发感染的源头。日本的研究人员最近报告了细菌计数与沐浴设施和热水浴缸模型中军团菌发生之间存在显著关联的证据。因此，需要一种方便且定量的细菌计数方法作为军团菌污染或消毒的指标，以取代现有的方法，如费时的军团菌培养和昂贵的军团菌-DNA 扩增。在这项研究中，我们开发了一种快速检测方法 (RDM)，使用基于流式细胞术技术的自动微生物分析设备，通过检测散射光和荧光的典型模式，在两分钟内测量水样中的总细菌数，从而监测军团菌的风险。我们首先将我们的 RDM 结果与平板计数结果进行了比较，该结果针对五种过滤温泉水样本，这些样本中加入了三种细菌，包括军团菌。我们还通过 RDM、活菌/死菌荧光测定和平板计数评估了这些样本的氯灭活情况。使用 RDM，确定了添加样本中定量细菌计数的下限为 3.0×10(3)(3.48log)counts mL(-1)。然后，我们使用实验室模型的热水浴缸，发现 RDM 可以监测自然发生的异养菌的生长曲线，其中变形虫的生长分别延迟了 1 天和 2 天，还可以确定这些细菌的氯化杀菌曲线。最后，使用 RDM 对 149 个不同的热水浴缸的样本进行了测试，这些样本的总细菌计数≥3.48 或<3.48log，结果发现与军团菌的阳性或阴性检测具有显著相关性，具有 95%的敏感性和 84%的特异性。这些发现表明，RDM 可用于沐浴设施的军团菌控制，特别是在水中存在 Fe(2+)、Mn(2+)、NH(4)(+)、皮肤碎屑和/或生物膜会降低氯的有效性的情况下。
