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.
Highlights
Legionella are gram-negative bacteria and the causative agent of legionellosis, a group of related illnesses that include severe pneumonia and non-pneumonic Pontiac fever [1]
When using the conventional culture method combined with the amoebic co-culture technique, L. pneumophila was detected in only two of the 68 samples (#42 and #51 in Table 1). #51 had a bacterial load of 2.5 × 105 colony-forming units (CFU)/L, and #42 had too many Legionella colonies to count
We could not evaluate the presence of Legionella in 14 samples because the plates were overgrown with other bacterial species and Legionella colonies could not be identified
Summary
Legionella are gram-negative bacteria and the causative agent of legionellosis, a group of related illnesses that include severe pneumonia and non-pneumonic Pontiac fever [1]. Infection by Legionella occurs through the inhalation or aspiration of aerosols generated from contaminated environmental water found in structures such as cooling towers, evaporative condensers of large air-conditioning systems, whirlpool spas, showers, and hot water tanks [1]. Among the Legionella, Legionella pneumophila (L. pneumophila) is the major disease causative agent, other species such as L. anisa, L. bozemanii, L. dumoffii, L. gormanii, and L. longbeachae are human pathogens [1]. More than cases of legionellosis in Japan, caused by contaminated artificial whirlpool spas or natural hot springs, were reported in Infectious Agents Surveillance Report 2014 [2]. Legionella infection caused by contaminated bath water is an important public health concern in Japan [4,5,6], and sensitive detection and identification of Legionella from bath water samples is crucial for legionellosis control
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