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Investigations on Contamination of Environmental Water Samples by Legionella Using Real-Time Quantitative PCR Combined with Amoebic Co-Culturing

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Investigations on Contamination of Environmental Water Samples by Legionella Using Real-Time Quantitative PCR Combined with Amoebic Co-Culturing Biocontrol Science, 2019, Vol. 24, No. 4, 213—220 Original Investigations on Contamination of Environmental Water Samples by Legionella using Real-Time Quantitative PCR Combined with Amoebic Co-Culturing AKIKO EDAGAWA1＊, AKIO KIMURA1, AND HIROSHI MIYAMOTO2 1Department of Environment Health, Osaka Prefectural Institute of Public Health, Osaka, 1-3-69 Nakamichi, Higashinari-ku, Osaka 537-0025, Japan 2Department of Pathology and Microbiology Faculty of Medicine, Saga University. Japan, 5-1-1 Nabeshima, Saga, 849-8501, Japan. Received 23 May, 2019/Accepted 8 August 2019 We analyzed the contamination of environmental water samples with Legionella spp. using a conventional culture method, real-time quantitative PCR（ qPCR）, and real-time qPCR combined with an amoebic co-culture method. Samples（ n = 110） were collected from 19 cooling towers, 31 amenity water facilities, and 60 river water sources of tap water in Japan. Legionella was detected in only three samples（ 3/110, 2.7%） using the culture method. The rate of Legionella detection using amoebic co-culture followed by qPCR was 74.5%, while that using qPCR without amoebic co-culture was 75.5%. A higher than 10-fold bacterial count was observed in 19 samples（ 19/110, 17.3%） using real-time qPCR subsequent to amoebic co-culture, compared with identical samples analyzed without co-culture. Of these 19 samples, 13 were identified as Legionella spp., including L. pneumophila and L. anisa, and the non-culturable species were identified as L. lytica and L. rowbothamii. This study showed that the detection of Legionella spp., even in those samples where they were not detected by the culture method, was possible using real-time qPCR and an amoebic co-culture method. In addition, this analytical test combination is a useful tool to detect viable and virulent Legionella spp.. Key words : Legionella species / Amoebic co-culture / Acanthamoeba castellanii / Environmental water. INTRODUCTION in environmental waters, especially during a disease outbreak, is important for public health. Legionella species are Gram-negative bacteria that are The culture method is routinely used to test Legionella widely present in both natural and artificial water bodies spp. in environmental samples（ Totaro et al., 2017; （Fields et al., 2002）. Infection with Legionella spp. Furuhata et al., 2013）. However, this method has certain leads to clinical manifestations, including Legionnaires’ limitations associated with the culture characteristics disease and Pontiac fever. Legionella spp. proliferates in of Legionella spp. These bacteria exhibit a slow growth aquatic environments within free-living protozoal hosts rate, and growth can be inhibited in the presence of （Greub et al., 2004）. Infection is passed on to humans other microorganisms, thereby transforming the culture via inhalation, aspiration, or micro-aspiration of legionel- into a viable but non-culturable（ VBNC） form under la-carrying aerosols. Legionnaires’ disease is prevented certain environmental conditions. Recently, quanti- mainly by monitoring and controlling the contamina- tative real-time polymerase chain reaction（ real-time tion of environmental waters with Legionella spp. Also, qPCR） specific for Legionella 16S rRNA or macrophage accurately identifying the source of Legionella infection infectivity potentiator genes have been widely applied to overcome the limitations of standard culture meth- ＊Corresponding author. Tel & Fax: +81-6-6972-1321, E-mail : ods.（ Touron-Bodilis et al., 2011; Bonetta et al., 2010; edagawa（a）iph.osaka.jp Edagawa et al., 2008）. Real-time qPCR can detect 214 A. EDAGAWA ET AL. VBNC Legionella types; however, it cannot distinguish Ltd.）. After culture for 3 days at 37°C, the isolates that between viable and dead cells. Further, although real- grew on BCYE-alpha but not on blood agar were exam- time qPCR is significantly more sensitive in detecting ined by Gram staining. A final Gram-negative staining lower levels of contamination than the culture method, it was considered suggestive of the presence of Legionella cannot detect low levels of Legionella contamination in a spp. Such colonies were observed under UV light and natural environment. 1–5 colonies were next randomly selected for a latex Recently, the amoebic co-culture technique has been agglutination test（ Kanto Chemical Co., Tokyo, Japan） used for to detect bacterial species from clinical and and an immune serum agglutination test（ Denka Seiken environment samples（ Inoue et al., 2019; Edagawa Co. Ltd., Tokyo, Japan） to identify the serogroups of et al., 2015; La Scola et al., 2001）. Acanthamoeba, a L. pneumophila, L. bozemanii, L. dumoffii, L. gormanii, genus of amoeba, serves as a host for Legionella pneu- and L. micdadei.. For Legionella strains not identified by mophila. Amoebic co-culture using Acanthamoeba is these tests, Legionella spp. were identified by 16S rRNA used to detect Legionella spp., including VBNC and PCR and nucleotide sequencing as described below. Legionella-like amoebal pathogen（ LLAP） types, after Acanthamoeba castellanii strain ATCC 30234 was their co-proliferation within the amoebae. grown in 75-cm2 culture flasks at 30°C for 4 days on 50 In this study, we used the culture method, real-time mL of peptone/yeast extract/glucose（ PYGC） medium qPCR, and real-time qPCR combined with amoebic （10 g proteose peptone, 10 g yeast extract, 10.1 g co-culture to analyze the contamination of various envi- glucose, 5 g NaCl, 0.95 g L-cysteine hydrochloride, 1.74 ronmental water samples with Legionella spp. Samples g Na2HPO4, and 1.36 g KH2PO4 in 1 L of distilled water; were collected from cooling towers, water-amenity facil- the pH was adjusted to 6.8）. Cells were harvested by ities, and tap water sources in Kansai area of Japan. centrifugation and re-suspended in PYGC medium at a density of approximately 1 × 105 cells/mL. The amoebal MATERIALS AND METHODS suspension was distributed into each well of 12-well micro-plates at 30°C until the cells formed monolayers. In total, 110 water samples were collected from 19 Just before processing the water samples, the PYGC cooling towers, 31 water-amenity facilities, and 60 river medium was removed from each well, and the cells were water sources of tap water from the Osaka Prefecture washed twice with 1 mL of Neff’s amoeba saline（ 120 of the Kansai area in Japan. All samples（ 400 mL mg NaCl, 3 mg MgCl2, 3 mg CaCl2, 142 mg Na2HPO4, each） were collected in sterile bottles containing 0.01% and 136 mg KH2PO4 in 1 L of distilled water）. sodium thiosulfate. Samples were immediately trans- A volume of 1.5 mL of the processed environmental ported to a laboratory for further processing. water sample was inoculated into the amoebal micro- Each sample was concentrated by filtration through plate wells（ amoebic co-culture）. After incubating for a 0.22-μm-pore size polycarbonate filter（ Advantec 7 days at 30°C, 1 mL of the suspension was stored at Tokyo Co. Ltd., Tokyo, Japan）. The membrane was then −20°C for DNA extraction. DNA was extracted using a immersed in 4 mL of sterile deionized water, vortexed QIAamp DNA Mini Kit（ Qiagen K.K., Tokyo, Japan） and for 1 min, and shaken vigorously 50 times. From Legionella spp. were detected using real-time qPCR. this suspension, 1 mL was stored at −20°C for DNA The supernatant was stored at −20°C until use. extraction and 1.5 mL was used for amoebic co-culture. Real-time qPCR was performed according to the A 0.2-mL aliquot of the suspension was used for acid manufacturer’s instructions as using a Cycleave PCR treatment culture method, while the remaining suspen- Legionella（ 16S rRNA） Detection Kit（ Takara Bio Inc., sion was heated in a water bath at 50°C for 30 min and Shiga, Japan）. PCRs with duplicate standards, positive used for the heat treatment culture method. and negative controls, and samples were performed A volume of 1 mL of the above sample suspension using an ABI PRISM 7900HT Real-time qPCR System was mixed with 1 ml of 0.2M acid-phosphate buffer （Applied Biosystems） according to the manufacturer’s （pH2.2）, and 0.2 mL of this acid-treated sample was instructions. The PCR mixtures（ 20 µL） were prepared inoculated after 5 min onto Wadowsky-Yee-Okuda agar according to the manufacturer’s protocol and reactions plates（ WYO-alpha plates; Eiken Chemical Co. Ltd., were performed under the following amplification condi- Tokyo, Japan）. From the heat-treated sample, 0.1 mL tions: 95°C for 10 s, then 45 cycles of 95°C for 5 s, was inoculated onto an WYO-α plate. After incuba- 55°C for 10 s, and 72°C for 25 s. DNA amplification was tion for 5–7 days at 37°C, 1–50 colonies resembling detected by monitoring the fluorescence at two wave- Legionella spp. were selected and cultured on blood lengths, viz., FAM and ROX. The amplified 16S rRNA of agar plates（ Eiken Chemical Co. Ltd.） and on buffered Legionella and the internal control gene were detected charcoal–yeast extract agar plates supplemented with by FAM and ROX, respectively. The samples whose DNA alpha-ketoglutarate（ BCYE-alpha; Eiken Chemical Co. was not amplified due to the presence of inhibitors were LEGIONELLA DETECTION BY AMOEBIC CO-CULTURE 215 TABLE 1. Positive samples of Legionella species in environmental water detecting by real-time qPCR methods with or without amoebic co-culture techniques and plate culture method Real-time qPCR Plate culture method Total with co-culture without co-culture Cooling tower 17 18 0 19 Amenity water facilities 18 22 3 31 River as source of tap water 47 43 0 60 Total 82 83 3 110 determined from internal standard ROX analysis data. More than 10-fold higher copy numbers of Legionella RESULTS AND DISCUSSION 16S rRNA gene were observed by real-time qPCR with the co-culture technique in 19 samples（ 19/110, The detection of Legionella spp.
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