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Legionella Prevalence and Risk of Legionellosis in Japanese Households

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Legionella Prevalence and Risk of Legionellosis in Japanese Households Epidemiol. Infect. (2017), 145, 1398–1408. © Cambridge University Press 2017 doi:10.1017/S0950268817000036 Legionella prevalence and risk of legionellosis in Japanese households T. KUROKI1*, Y. WATANABE1†,H.TERANISHI2,S.IZUMIYAMA3, 4 4 J. AMEMURA-MAEKAWA AND F. KURA 1 Department of Microbiology, Kanagawa Prefectural Institute of Public Health, Chigasaki, Kanagawa, Japan 2 Department of Regional Hygiene Inspection, Kanagawa Prefectural Institute of Public Health, Chigasaki, Kanagawa, Japan 3 Department of Parasitology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan 4 Department of Bacteriology I, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan Received 6 September 2016; Final revision 28 December 2016; Accepted 28 December 2016; first published online 7 February 2017 SUMMARY This study determined the occurrence of legionellae in private houses for which there were no available data on aquatic environments other than the water supply system. From June 2013 to November 2014, we collected 138 water and 90 swab samples from aquatic environments in 19 houses. Legionella DNA was detected via a loop-mediated isothermal amplification assay in 66 (47·8%) water and 17 (18·9%) swab samples. High Legionella DNA detection rates were observed in water samples from washing machines and aquariums. Legionella spp. was isolated from 9 (6·5%) water and 3 (3·3%) swab samples. Legionella pneumophila SG 1 was detected from the outlet water of a bathtub spout and a bath sponge. Use of amoebic co-culture effectively increased legionellae and Legionella DNA detection rates from all sample types. A logistic regression analysis revealed that the heterotrophic plate count was significantly related to Legionella contamination. Our findings indicate that there is a risk of legionellosis from exposure to Legionella spp. in a variety of aquatic environments in residential houses. Control measures for legionellae in houses should include frequent cleaning and disinfecting to reduce heterotrophic bacteria in water and, where possible, preventing aerosolization from aquatic environments. Key words: Legionella, household, logistic regression analysis, washing machine, aquarium. INTRODUCTION Legionnaires’ disease and Pontiac fever [2]. Sporadic Legionella Legionellae, Gram-negative bacteria found in aquatic infections, rather than outbreak cases, Legionella environments are causative agents of legionellosis [1]. account for most infections [3]. A substan- This disease is acquired by inhalation or aspiration of tial proportion of sporadic cases may be residentially aerosols containing legionellae from an environmental acquired [4], and these infections have been documen- source. Legionellosis has two distinct forms: ted worldwide. Several cases of legionellosis linked to home water supplies were previously reported [5, 6]. Previously, approximately 700–900 cases of * Author for correspondence: T. Kuroki, Department of Legionella infection were reported in Japan each Microbiology, Kanagawa Prefectural Institute of Health, 1-3-1 year [7], but in 2014 the number of reported cases Shimomachiya, Chigasaki, Kanagawa 253-0087, Japan. (Email: [email protected]) increased to 1248 (http://www.nih.go.jp/niid/ja/survei/ † The author has retired. 2085-idwr/ydata/5672-report-ja2014-20.html). Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.219, on 28 Sep 2021 at 11:55:14, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817000036 Legionella prevalence in households 1399 Sporadically occurring cases of community-acquired years old, respectively. No members of any of these legionellosis accounted for most of these infections homes contracted Legionnaires’ disease or Pontiac [7]. While most cases of Legionella infection are asso- fever before and during the investigation period. ciated with spas in public bath facilities and accom- Overall, 138 water samples (Table 1) and 90 swab modations [8], some sporadic cases associated with samples (Table 2) were analyzed in this study. Water baths involved residential infections [9]. samples (up to 1000 ml) were collected in sterile vessels Control measures for preventing Legionella infec- containing 0·5 g sodium thiosulphate/1000 ml sample tion from water supply systems have been reported water to neutralize residual chlorine. The samples previously. Chlorination of potable water, increasing were collected as follows: water samples were taken water heater temperatures, and limiting exposure to from the tap outlet of cold water supplies immediately aerosols in private residences were recommended [4]. after the valve was opened. Residual water in shower Regular flushing of showers decreased Legionella to hoses and showerheads or garden hoses in the house below detectable levels [10]. Because Legionella yard was collected. To collect a water sample from a requires an appropriate temperature and pH to washing machine, about 10 liters of tap water from a grow, extreme water temperatures and pH can be tap directly connected to the washing machine was used to prevent Legionella proliferation [11]. added into a drum, the washing machine was run for Despite the occurrence of Legionella in a wide range 10 min, and then 1000 ml of water was collected in a of aquatic environments, a limited number of studies sterile container. Tap water was used for this proced- have analyzed Legionella contamination in the water ure instead of sterile water to analyze the contamin- supply systems of houses [12], and, to our knowledge, ation of washing machines under their normal use no information on Legionella contamination in other conditions. Bath sponges that were used for cleaning aquatic environments within houses is available. The bathtubs were squeezed by hand with sterile gloves to aim of this study was to investigate residential collect water samples. The swab samples were taken Legionella contamination. Additionally, the relation- using a sterile Dacron swab (JCB Industry Limited, ship between Legionella contamination and the charac- Tokyo, Japan) at each sampling site and were each teristics of the water samples collected in households, immersed in 1·0 ml of sterilized phosphate-buffered including the number of heterotrophic bacteria, saline (PBS) that had been diluted 50-fold. residual-free chlorine concentration, pH, and tempera- ture, was investigated to determine whether these fac- tors could be utilized to prevent the colonization of Physical and chemical analyses Legionella in household aquatic environments. The temperature and pH of water samples were measured on site. The residual-free chlorine concentrations were measured at our laboratory by the DPD (N, METHODS N-diethyl-p-phenylenediamine) method using AQUAB AQ-201 (Shibata Scientific Technology, Saitama, Japan) Sample collection in accordance with the manufacturer’s instructions. Samples were collected from 19 private homes during the period between July 2013 and November 2014. Heterotrophic plate count The private homes were selected from those of collea- gues of our Institute and associates. House members The number of heterotrophic bacteria was determined were asked to collaborate and provide consent for by heterotrophic plate count (HPC). Water samples their house samples to be collected and tested for were inoculated with 10-fold serial dilutions. Serially Legionella. Fourteen of the 19 homes, which were diluted samples (100 µl) were inoculated onto R2A located in Kanagawa Prefecture, provided a variety agar plates (BD, Tokyo, Japan), and the colonies of samples, while the remaining five homes provided were counted after the plates had been incubated at only aquarium water samples. These 14 houses con- 25 °C for 7 days. sisted of five condominium units and nine single- family houses. All houses studied received chlorinated tap water that met the Japanese water quality stand- Legionella detection ard. The ages of the condominium units and houses Water samples (500 ml) were filtered through a 0·2-μm ranged from 1 to 30 years old and from 4 to 55 porosity polycarbonate membrane filter (Advantec Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.219, on 28 Sep 2021 at 11:55:14, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817000036 https://www.cambridge.org/core/terms Downloaded from https://www.cambridge.org/core Table 1. Household Legionella contamination in water samples 1400 T. Kuroki and others Residual chlorine Temperature (°C) pH e conc. (mg/l) HPC (CFU/ml) Amoeba co-culture Combined . https://doi.org/10.1017/S0950268817000036 LAMP- Culture- LAMP- Culture- LAMP- Culture- No. of No. of positive positive (CFU/ positive positive positive positive Samples houses samples Mean Range Mean Range Mean Range Mean Range (%) (%) 100 ml) (%) (%) (%) (%) . IPaddress: Samples from water systems Kitchen tap 10 14 26·4 11·0–42·0 7·3 7·1–7·8 0·27 0–0·80 353 0–1·1 × 105 2 (14·3) 0 3 (21·4) 1* (7·1) 4 (28·6) 1 (7·1) 4 170.106.40.219 Bathroom tap 11 12 25·9 18·5–36·0 7·4 7·1–7·8 0·26 0–0·80 205 1–1·0 × 10 1 (8·3) 0 1 (8·3) 0 1 (8·3) 0 Bathtub spout 12 17 33·7 28·0–39·0 7·4 7·1–7·8 0·17 0–0·80 2432 0–1·7 × 106 8 (47·1) 1† (5·9) 15 7 (41·2) 0 9 (52·9) 1 (5·9) Shower 7 8 25·9 23·0–31·5 7·4 7·1–7·8 0·15 0–0·49 7759 0–4·7 × 106 1 (12·5) 0 1 (12·5) 0 2 (25·0) 0 ‡ Hand-wash 7 8 25·7 22·0–30·0 7·3 7·2–7·4 0·27 0·02–0·80 302 4–4·6 × 105 1 (12·5) 1 (12·5) 370 1 (12·5)
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