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Int. J. Environ. Res. Public Health 2015, 12(10), 13118-13130; doi:10.3390/ijerph121013118

Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods

1
Division of Environment Health, Osaka Prefectural Institute of Public Health, Osaka 537-0025, Japan
2
Division of Microbiology, Department of Pathology and Microbiology, Faculty of Medicine, Saga University, Saga 849-8501, Japan
3
Division of Planning and Coordination, Osaka Prefectural Institute of Public Health, Osaka 537-0025, Japan
4
Division of Virology, Osaka Prefectural Institute of Public Health, Osaka 537-0025, Japan
5
School of Life and Environmental Science, Azabu University, Kanagawa 252-5201, Japan
*
Author to whom correspondence should be addressed.
Academic Editors: Nicholas Frederick Gray and Panagiotis Karanis
Received: 1 September 2015 / Revised: 13 October 2015 / Accepted: 14 October 2015 / Published: 19 October 2015
(This article belongs to the Special Issue Water Microbial Pollution and Disinfection)
View Full-Text   |   Download PDF [697 KB, uploaded 19 October 2015]

Abstract

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. View Full-Text
Keywords: Legionella; amoebic co-culture; Acanthamoeba; bath water; intracellular growth Legionella; amoebic co-culture; Acanthamoeba; bath water; intracellular growth
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Edagawa, A.; Kimura, A.; Kawabuchi-Kurata, T.; Adachi, S.; Furuhata, K.; Miyamoto, H. Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods. Int. J. Environ. Res. Public Health 2015, 12, 13118-13130.

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