Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods.
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.
Division of Environment Health, Osaka Prefectural Institute of Public Health, Osaka 537-0025, Japan. edagawa@iph.pref.osaka.jp.|Division of Microbiology, Department of Pathology and Microbiology, Faculty of Medicine, Saga University, Saga 849-8501, Japan. edagawa@iph.pref.osaka.jp.
Akiko
A
Edagawa
Division of Planning and Coordination, Osaka Prefectural Institute of Public Health, Osaka 537-0025, Japan. akkimura@iph.pref.osaka.jp.
Akio
A
Kimura
Division of Virology, Osaka Prefectural Institute of Public Health, Osaka 537-0025, Japan. kurata@iph.pref.osaka.jp.
Takako
T
Kawabuchi-Kurata
Division of Environment Health, Osaka Prefectural Institute of Public Health, Osaka 537-0025, Japan. adachi@iph.pref.osaka.jp.
Shinichi
S
Adachi
School of Life and Environmental Science, Azabu University, Kanagawa 252-5201, Japan. furuhata@azabu-u.ac.jp.
Katsunori
K
Furuhata
Division of Microbiology, Department of Pathology and Microbiology, Faculty of Medicine, Saga University, Saga 849-8501, Japan. miyamoth@cc.saga-u.ac.jp.
Hiroshi
H
Miyamoto
D048248:Acanthamoeba castellanii; D001494:Baths; D018920:Coculture Techniques; D045482:Hot Springs / Q000382:microbiology*; D007564:Japan; D007875:Legionella / Q000235:genetics; D016952:Legionella pneumophila / Q000302:isolation & purification*; D011633:Public Facilities; D060888:Real-Time Polymerase Chain Reaction
Acanthamoeba; Legionella; amoebic co-culture; bath water; intracellular growth