Legionella pneumophila serogroup 1 (SG1) is the most frequent cause of legionellosis. Our previous genetic analysis indicated that SG1 environmental isolates represented 8 major clonal complexes, consisting of 3 B groups, 2 C groups, and 3 S groups, which included major environmental isolates derived from bath water, cooling towers, and soil and puddles, respectively. Here, we surveyed clinical isolates collected from patients with legionellosis in Japan between 2008 and 2016. Most strains belonging to the B group were isolated from patients for whom bath water was the suspected or confirmed source of infection. Among the isolates derived from patients whose suspected infection source was soil or dust, most belonged to the S1 group and none belonged to the B or C groups. Additionally, the U group was discovered as a new group, which mainly included clinical isolates with unknown infection sources.
KEYWORDS: Legionella species, Legionella pneumophila, legionellosis, sequence-based typing, genetic variability
ABSTRACT
The Legionella Reference Center in Japan collected 427 Legionella clinical isolates between 2008 and 2016, including 7 representative isolates from corresponding outbreaks. The collection included 419 Legionella pneumophila isolates, of which 372 belonged to serogroup 1 (SG1) (87%) and the others belonged to SG2 to SG15 except for SG7 and SG11, and 8 isolates of other Legionella species (Legionella bozemanae, Legionella dumoffii, Legionella feeleii, Legionella longbeachae, Legionella londiniensis, and Legionella rubrilucens). L. pneumophila isolates were genotyped by sequence-based typing (SBT) and represented 187 sequence types (STs), of which 126 occurred in a single isolate (index of discrimination of 0.984). These STs were analyzed using minimum spanning tree analysis, resulting in the formation of 18 groups. The pattern of overall ST distribution among L. pneumophila isolates was diverse. In particular, some STs were frequently isolated and were suggested to be related to the infection sources. The major STs were ST23 (35 isolates), ST120 (20 isolates), and ST138 (16 isolates). ST23 was the most prevalent and most causative ST for outbreaks in Japan and Europe. ST138 has been observed only in Japan, where it has caused small-scale outbreaks; 81% of those strains (13 isolates) were suspected or confirmed to infect humans through bath water sources. On the other hand, 11 ST23 strains (31%) and 5 ST120 strains (25%) were suspected or confirmed to infect humans through bath water. These findings suggest that some ST strains frequently cause legionellosis in Japan and are found under different environmental conditions.
IMPORTANCE Legionella pneumophila serogroup 1 (SG1) is the most frequent cause of legionellosis. Our previous genetic analysis indicated that SG1 environmental isolates represented 8 major clonal complexes, consisting of 3 B groups, 2 C groups, and 3 S groups, which included major environmental isolates derived from bath water, cooling towers, and soil and puddles, respectively. Here, we surveyed clinical isolates collected from patients with legionellosis in Japan between 2008 and 2016. Most strains belonging to the B group were isolated from patients for whom bath water was the suspected or confirmed source of infection. Among the isolates derived from patients whose suspected infection source was soil or dust, most belonged to the S1 group and none belonged to the B or C groups. Additionally, the U group was discovered as a new group, which mainly included clinical isolates with unknown infection sources.
INTRODUCTION
Legionellosis is an infectious disease whose severity varies from a mild febrile illness to a serious and sometimes fatal form of pneumonia (1); it is caused by Legionella spp., Gram-negative bacteria that multiply in free-living, ubiquitous, freshwater and soil amoebae (2). The bacteria also contaminate manmade water systems, such as cooling towers, hot and cold water systems, and whirlpool spas, from which humans contract the disease via inhalation of contaminated aerosols (3, 4).
In Japan, legionellosis is an infectious disease that has required mandatory notification to the National Epidemiological Surveillance of Infectious Diseases, under the Diseases Control Law, since April 1999. The notified number of legionellosis patients increased from 154 to 1,602 between 2000 and 2016 (Fig. 1) (https://www.niid.go.jp/niid/ja/survei/2085-idwr/ydata/7310-report-ja2016-20.html), with the diagnosis of urinary antigen being prevalent. Several outbreaks of Legionnaires' disease associated with public bath facilities have occurred (5–10), and bathing facilities were found to be the main source of infection in Japan (8), although most such facilities had circulation systems fitted with filters and used hot spring water. Even for sporadic cases, infection source surveys were conducted by some public health centers, and bathing facilities could be identified as the source of infection by using molecular epidemiology (11, 12). Identification of the sources of infection has led to the strengthening of sanitary management to prevent legionellosis (13, 14).
FIG 1.
Annual incidence of legionellosis cases recorded by the National Epidemiological Surveillance of Infectious Diseases and the numbers of isolates submitted to the Legionella Reference Center in Japan between 2008 and 2016.
The Legionella Reference Center, comprising the National Institute of Infectious Diseases and the regional institutes of public health in Japan, has performed voluntary collection and analysis of clinical isolates of Legionella. We were able to collect isolates from approximately 4.4% patients with legionellosis. We previously reported 86 unrelated clinical Legionella pneumophila strains isolated in Japan between 1980 and February 2008 (15). The objectives of the present study were to determine the sequence type (ST) and/or serogroup (SG) of clinical isolates from legionellosis patients in Japan between March 2008 and December 2016 (Fig. 1) and to compare them with those reported earlier.
RESULTS AND DISCUSSION
Characteristics of patients and confirmed/suspected infection sources.
The mean age of the 415 legionellosis patients (374 men and 41 women) was 65.2 years (men: mean, 65.6 years [range, 29 to 96 years]; women: mean, 67.6 years [range, 30 to 89 years]). Except for 9 patients with nosocomial infections, all cases were community infections, including travel-related cases. The suspected/confirmed sources of infection were water related for 165 (40%) of the 415 patients. Several of the sources were bathing facilities (146 cases [35%]), such as public baths, hot spring inns, sports gyms, hospitals, and other bathing facilities. Examples of other suspected water-related infection sources were pools, tank waters, and tap water. A total of 43 patients (10%) were suspected of contracting legionellosis through inhalation of soil or dust. The sources of infection for the remaining 207 patients (50%), including 6 nosocomial cases and 5 travel-related cases, were unknown (see Table S1 in the supplemental material).
Among the 415 legionellosis patients, the source of infection for 32 patients was confirmed by comparing the pulsed-field gel electrophoresis (PFGE) DNA patterns (16) of the clinical isolates with those of environmental isolates from the suspected source of infection at prefectural/municipal public health institutes. The confirmed cases included 26 from bathing facilities (25 from bath water and 1 from shower water), 2 from home baths, 2 from hospital cooling towers, 1 from a nursing home bath (occurrence in 2 patients), and 1 from a garden hose (17). Among the 7 isolates from outbreaks (Table 1), the source of infection was confirmed for 5.
TABLE 1.
Outbreaks of culture-positive L. pneumophila cases in Japan between 2008 and 2016
| Year | Laboratory no. | Infection source | Confirmation statusa | Prefecture | No. of patients | Serogroup(s) | ST | Groupb |
|---|---|---|---|---|---|---|---|---|
| 2009 | NIIB2569 | Hot spring bathing facilities (nursing home) | Suspected | Osaka | 2 | 1 (lag-1-positive) | ST23 | S1 |
| 2011 | NIIB2838 | Hot spring bathing facilities (sports gym) | Confirmed | Kanagawa | 9 | 1 (lag-1-positive) | ST256 | (B1) |
| 2012 | NIIB2997 | Hot spring bathing facilities (hotel) | Confirmed | Yamagata | 3 | 1 (lag-1-positive) | ST138 | B3 |
| 2013 | NIIB3201 | Bath (nursing home) | Confirmed | Miyazaki | 2 | 1 (lag-1-positive) | ST23 | S1 |
| 2015 | NIIB3389, NIIB3390 | Public bath | Confirmed | Iwate | 13 | 1 (lag-1-positive) | ST679, ST23 | S1 |
| 2015 | NIIB3425, NIIB3429 | Hot spring bathing facilities | Confirmed | Kanagawa | 7 | 1 (lag-1-positive), 13 | ST2114, ST2113 | B1 |
| 2016 | NIIB3706 | Hot spring bathing facilities | Suspected | Nagano | 2 | 1 (lag-1-positive) | ST89 | (S1) |
“Confirmed” indicates that some clinical isolates and some environmental isolates had PFGE patterns indistinguishable from each other.
Groups for which STs differed at 3 loci are indicated in parentheses.
Legionella species and SGs from patients.
Most isolates belonged to L. pneumophila (419 isolates [98%]), of which 372 were in SG1 (87%) and 47 (11%) were in a SG other than SG1. The remaining 8 isolates (2%) were other Legionella spp. (Table 2).
TABLE 2.
Distribution of 427 clinical Legionella sp. isolates in Japan between 2008 and 2016
| Species | No. (%) of isolates |
|---|---|
| Legionella pneumophila | 419 (98.1) |
| SG1 | 372 (87.1) |
| lag-1-positive/lag-1-negative | 333 (90)/37 (10)a |
| SG2 | 7 (1.6) |
| SG3 | 11 (2.6) |
| SG4 | 1 (0.2) |
| SG5 | 4 (0.9) |
| SG6 | 6 (1.4) |
| SG8 | 1 (0.2) |
| SG9 | 7 (1.6) |
| SG10 | 3 (0.7) |
| SG12 | 2 (0.5) |
| SG13 | 2 (0.5) |
| SG14 | 1 (0.2) |
| SG15 | 1 (0.2) |
| Untypeable | 1 (0.2) |
| Legionella longbeachae SG2 | 3 (0.7) |
| Legionella bozemanae SG2 | 1 (0.2) |
| Legionella dumoffii | 1 (0.2) |
| Legionella feeleii SG1 | 1 (0.2) |
| Legionella londiniensis SG1 | 1 (0.2) |
| Legionella rubrilucens | 1 (0.2) |
Two SG1 isolates were not assessed.
Distribution of STs and lag-1 gene.
Among the 419 L. pneumophila isolates, 187 STs (including 126 singletons) were detected (Table 3) (index of discrimination [IOD] of 0.984) (18). The IOD for the 419 clinical isolates (IOD of 0.984) was similar that that obtained by analyzing the 86 clinical isolates in our previous study (IOD of 0.979) (15) and was similar to the value obtained in a study conducted in Portugal (IOD of 0.972) (19). In the case of SG1 only, 372 isolates were divided into 159 STs, with the IOD being 0.980, which was similar to the finding for all SGs included and was greater than the IOD of 0.924 obtained in a study of sporadic cases conducted in the United States (20) and the IOD of 0.879 obtained in a study conducted in Belgium (21). The major STs were ST23 (35 isolates [8.4%]), ST120 (20 isolates [4.8%]), ST138 (16 isolates [3.8%]), ST89 (13 isolates [3.1%]), ST1 (11 isolates [2.6%]), and ST42 (10 isolates [2.4%]). ST23 was the most prevalent ST and the most causative ST for outbreaks. ST138 was detected not only in sporadic cases but also in small outbreaks. Of the 9 nosocomial isolates, ST1 accounted for 44% (4 isolates), and the proportion of ST1 was greater than that of the clinical isolates, although the number of nosocomial isolates was fewer. According to a previous survey conducted with 86 clinical isolates of L. pneumophila collected between 1980 and 2008 (15), ST1 was the most common (7 isolates [8.1%]), followed by other major STs, including ST306 (6 isolates [7.0%]), ST120 (5 isolates [5.8%]), ST138 (5 isolates [5.8%]), and ST23 (4 isolates [4.7%]). The incidence of ST1 was significantly lower between 1980 and 2016 (P = 0.0002), while ST306, ST120, ST138, and ST23 incidences did not change significantly. Two ST42 isolates and 1 ST89 isolate were also detected among 86 isolates in a previous study (15). Among the 6 major STs in Japan, ST1 and ST42 are the major STs in Europe and North America (20, 22, 23), and ST23 is the most prevalent ST in Europe and Japan but not in the United Kingdom and North America (20, 24). ST120 and ST89, which are the other 2 major STs in Japan, have rarely been detected in other countries, and ST138 has been detected only in Japan. On the other hand, among the major STs in Europe, no isolates of ST37 and ST47 (21, 24–26) were detected in Japan.
TABLE 3.
Distribution of STs among 419 clinical L. pneumophila isolates in Japan between 2008 and 2016
| No. of isolates | ST |
|---|---|
| 35 | ST23 |
| 20 | ST120 |
| 16 | ST138a |
| 13 | ST89 |
| 11 | ST1 |
| 10 | ST42 |
| 9 | ST93,b ST353,a ST384,a ST507a |
| 8 | ST550a |
| 7 | ST679a |
| 6 | ST132,a ST502,a ST609 |
| 5 | ST354,b ST1346a |
| 4 | ST142,a ST505,a ST644,a ST687,a ST876,a ST973 |
| 3 | ST131,a ST211, ST256, ST566,a ST642, ST701, ST1077, ST1186,a ST1480a |
| 2 | ST2, ST36, ST39,b ST48, ST59, ST68,b ST114, ST129, ST224, ST294, ST306,a ST530,a ST537,a,b ST552,a ST553, ST622,a ST624, ST739, ST850,a ST905,a ST1023, ST1032,b ST1136, ST1187,a ST1275,a ST1427,b ST1798, ST2114, ST2128a |
| 1 | 126 kinds of STs (including 82 kinds of new STs) |
STs unique to Japan as of 28 August 2017.
STs derived from groups other than SG1.
The SG1-specific gene lag-1 is known to be a pathogenic marker, and 75% of SG1 clinical isolates and 8% of SG1 environmental isolates collected from water were positive for lag-1 in the United States (27). In Japan, 90% of the SG1 clinical isolates were positive for lag-1 by PCR in the present study (Table 2). In our previous survey of environmental isolates, the proportion of isolates positive for MAb 3/1 (a phenotype of lag-1-positive isolates) was 2% among isolates from cooling towers, 15% among isolates from soil, and 26% among isolates from bath water (28). Because the main infection source of legionellosis in Japan is bath water, the proportion of lag-1-positive clinical isolates in Japan may be greater than that in the United States. SG1 isolates of the major STs, i.e., ST23, ST120, ST138, ST89, and ST42, were lag-1 positive, while all ST1 isolates were lag-1 negative.
MST analysis.
In a previous study (28), minimum spanning tree (MST) analysis of SG1 environmental isolates revealed 8 major clonal complexes, including 3 B groups, 2 C groups, and 3 S groups; these groups included major environmental isolates derived from bath water (B), cooling towers (C), and soil and puddles (S), respectively. All 8 groups also included clinical isolates. MST analysis using 419 L. pneumophila clinical isolates also showed 9 major groups, i.e., B1 (for example, ST129), B2 (for example, ST1346, which differed from ST128 in 1 locus), B3 (ST138), C1 (ST1), C2 (ST154), S1 (ST610), S2 (ST48), S3 (ST739), and U (Fig. 2).
FIG 2.
MST analysis of 419 L. pneumophila isolates collected in Japan between 2008 and 2016. The major ST numbers are shown beside the circles. The size of each circle indicates the number of isolates. Short thick branches connect single-locus variants, thin branches connect double- or triple-locus variants, dashed branches connect 4-locus variants, and thinner branches connect 5- or 6-locus variants. The numbers of locus variants are proportional to the lengths of the branches. Groups generated with single- and double-locus variants are indicated by shaded backgrounds. The group names corresponding to groups of environmental isolates are shown. Isolates other than SG1, shown by SG numbers in Roman numerals, are enclosed by thick lines.
Most infection sources of SG1 strains belonging to the U group were unknown (18/25 isolates [72%]); 20% (5/25 isolates) had bath water as the infection source, and the remainder had shower water or air conditioning as the source (Fig. 2; also see Table S1 in the supplemental material). Most strains (60/86 isolates [70%]) belonging to the B1, B2, and B3 groups were isolated from patients for whom the suspected or confirmed source of infection was bath water. For example, in ST138, which belonged to the B3 group, 84% of the strains (16/19 isolates) were derived from patients for whom bath water was the suspected or confirmed infection source. Sometimes routine examinations detected the ST138 strain in bath water (28). Among the 372 isolates in SG1, 32 were obtained from patients for whom the suspected infection source was soil or dust; 24 of those isolates belonged to the S1 group, and none belonged to the C1, C2, B1, B2, or B3 groups. The B1′ group, which is in close proximity to the S1 group, originally belonged to the B1 group (Fig. 2), although the proportion of clinical isolates from suspected or confirmed bath water infection sources belonging to the B1′ group was lower than that of clinical isolates belonging to the original B1 group (3/11 isolates [27%] versus 32/40 isolates [80%]). The isolates belonging to the C2 group, and a close small group belonging to L. pneumophila subsp. fraseri (Fig. 2), possessed pilE14; we confirmed that some isolates with pilE14 belonged to L. pneumophila subsp. fraseri by using DNA-DNA hybridization on a microplate (J. Amemura-Maekawa, unpublished results). The source of infection for clinical isolates belonging to the C2 group either was unknown or was estimated to be bath water. All 11 isolates belonging to the C1 group (ST1) were lag-1 negative, and the remaining 26 lag-1-negative isolates were scattered among 22 different STs belonging to groups other than the S1 and B3 groups.
The ST23, ST120, and ST384 strains belonging to the S1 group were isolated from puddles (29) and were rarely detected in bath water or cooling water in routine environmental inspections. ST1 strains have been isolated frequently from cooling tower water (28), although it is rare that cooling towers are presumed to be the source of legionellosis in Japan. We did not experience large outbreaks of legionellosis caused by cooling towers. Most environmental isolates belonging to the C2 group were isolated from cooling tower water (28), but the estimated source of infection in the C2 group was bath water or an unknown source. These findings suggest that the ST groups in the MST analysis with environmental isolates are useful for identifying the sources of infection.
Some genes that were used for sequence-based typing (SBT) coded for proteins, e.g., flagellum, pilus, outer membrane protein, infectivity macrophage potentiator protein, and zinc metalloproteinase, that interact with the external environment (and host amoebae adapted to the environment). Therefore, isolates adapted to the environment may have particular STs suitable for each environment. Several clinical isolates for which bath water was considered to be the source of infection belonged to groups B1, B2, or B3. However, some clinical isolates belonged to groups S1, S2, and S3, which contained isolates from soil and puddles, and to groups C1 and C2, which contained most strains derived from cooling towers; these were also identified as causative strains isolated from bath water. Such strains from the original habitat contaminate bath water temporarily and can survive and proliferate in the bathtub to infect patients. It may be necessary not only to manage the cleaning of bathing facilities but also to prevent environmental contamination.
MATERIALS AND METHODS
Legionella strains.
A total of 427 Legionella isolates from 415 legionellosis patients were collected from the Legionella Reference Center, which comprises the National Institute of Infectious Diseases and 6 representative prefectural/municipal public health institutes in each district in Japan. The clinical isolates included representative isolates from each of the Legionella outbreaks. Of the 427 isolates, 407 had no known epidemiologic linkage; some isolates derived from clusters that were not identified as being dispersed might have been included. The remaining 20 isolates were obtained from 8 patients from whom more than 2 different species, SGs, and/or STs were obtained (see Table S1 in the supplemental material).
Legionella species and SG identification.
Serogrouping of Legionella isolates was performed using a latex agglutination test kit (Oxoid, Hampshire, UK) and slide agglutination tests with monovalent antisera (Denka Seiken, Tokyo, Japan). Non-L. pneumophila isolates were identified by sequencing of 16S rRNA and mip (30, 31). The 16S rRNA and mip sequences obtained were queried against the DNA Data Bank of Japan and the Legionella mip sequence database (http://bioinformatics.phe.org.uk/cgi-bin/legionella/mip/mip_id.cgi), respectively, using the Basic Local Alignment Search Tool (BLAST).
SBT and lag-1 gene detection.
The ST of L. pneumophila was determined in accordance with the European Working Group for Legionella Infections (EWGLI) SBT protocol (http://www.hpa-bioinformatics.org.uk/legionella/legionella_sbt/php/sbt_homepage.php), as described previously (32, 33). A MST with categorical coefficients of similarity and the priority rule of the highest number of single-locus variants was constructed using BioNumerics software (version 6.5; Applied Maths, Sint-Martens-Latem, Belgium). PCR was performed to detect lag-1, using the primers lag-F and lag-R (27).
Statistical analysis.
The Cochran-Armitage trend test was used to assess whether there was a significant change in the frequency of ST isolation over years. Statistical analysis was conducted using the R software package. P values of <0.05 were considered statistically significant.
Accession number(s).
The sequence of mip76 obtained in this study was deposited in the GenBank database under accession number LC415301.
Supplementary Material
ACKNOWLEDGMENTS
We thank Norman K. Fry, Massimo Mentasti, Baharak Afshar, and Timothy G. Harrison (Respiratory and Vaccine Preventable Bacteria Reference Unit, Public Health England) for assigning the newly identified alleles and STs. We also thank the public health centers and hospitals in Japan. We thank Enago for the English language review.
This work was supported by Health and Labor Sciences research grants (grant H25-Kenki-Ippan-009 to F.K. and grants H28-Kenki-Ippan-006 and H28-Shinko-Ippan-006 to J.A.-M).
The following are the members of the Working Group for Legionella in Japan and their affiliations at that time: F. Kodama, Teine Keijinkai Hospital; K. Iwabuchi and S. Fujii, Research Institute for Environmental Sciences and Public Health of Iwate Prefecture; Y. Yamaguchi, Miyagi Prefectural Institute of Public Health and Environment; N. Numata, Sendai City Institute of Public Health; T. Konno, Akita Research Center for Public Health and Environment; A. Kaneko, J. Seto, and Y. Suzuki, Yamagata Prefectural Institute of Public Health; R. Kikuchi and N. Tomita, Fukushima Institute for Public Health; T. Hakuta, Ibaraki Prefectural Institute of Public Health; F. Nagashima and Y. Tokoi, Utsunomiya City Institute of Public Health and Environment Science; K. Goto and H. Kurosawa, Gunma Prefectural Institute of Public Health and Environmental Sciences; Y. Tamura and S. Kanazawa, Sagamihara City Institute of Public Health; S. Kobori and K. Kikuchi, Saitama City Institute of Health Science and Research; T. Tomita and M. Nakamura, Chiba Prefectural Institute of Public Health; T. Kitahashi, Chiba City Institute of Health and Environment; E. Ogura, Kashiwa City Health Center; R. Okuno, Tokyo Metropolitan Institute of Public Health; I. Nakamura, Tokyo Medical University Hospital; D. Kurai, Kyorin University Hospital; Y. Watanabe, Kanagawa Prefectural Institute of Public Health; Y. Kojima, Y. Miyashita, C. Matsuo, and E. Yuzawa, Kawasaki City Institute for Public Health; M. Shindo, Yokosuka Institute of Public Health; M. Hosoya and Y. Kimura, Niigata Prefectural Institute of Public Health and Environmental Sciences; K. Yamamoto, Niigata City Institute of Public Health and Environment; K. Kawakami and E. Kitagawa, Ishikawa Prefectural Institute of Public Health and Environmental Science; K. Yanagimoto, Yamanashi Institute for Public Health and Environment; Y. Igawa, H. Kasahara, H. Ueda, and T. Koyama, Nagano Environmental Conservation Research Institute; K. Sahara, Shizuoka Institute of Environment and Hygiene; A. Tomita and Y. Kanazawa, Shizuoka City Institute of Environmental Sciences and Public Health; M. Hikida, Hamamatsu City Health Environment Research Center; Y. Kadokura, M. Noda, and Y. Shiraki, Gifu Prefectural Research Institute for Health and Environmental Sciences; M. Suzuki, Aichi Prefectural Institute of Public Health; N. Agata, Nagoya City Public Health Research Institute; C. Matsuo and M. Shimizu, Kyoto City Institute of Health and Environmental Sciences; N. Fujita and Y. Kimura, University Hospital, Kyoto Prefectural University of Medicine; C. Katsukawa, Osaka Prefectural Institute of Public Health; J. Ogasawara, Osaka City Institute of Public Health and Environmental Sciences; T. Fushiwaki, Osaka Anti-Tuberculosis Association, Osaka Hospital; H. Tsuji, Hyogo Prefectural Institute of Public Health and Environmental Sciences; Y. Kanazawa, H. Ekawa, T. Nishiyama, and M. Hirooka, Wakayama City Institute of Public Health; Y. Hanabara, Tottori Prefectural Institute of Public Health and Environmental Science; J. Uchida, Kagawa Prefectural Research Institute for Environmental Sciences and Public Health; T. Karasudani, Ehime Prefectural Institute of Public Health and Environmental Science; K. Murakami, Fukuoka Institute of Health and Environmental Sciences; Y. Shimizu, Kitakyushu City Institute of Environmental Sciences; Y. Miyamoto, H. Yoshida, and N. Matsunaga, Fukuoka City Institute for Hygiene and the Environment; M. Kawano and T. Taguri, Nagasaki Prefectural Institute for Environmental Research and Public Health; Y. Harada, Shunkaikai Inoue Hospital; I. Fukushiyama and J. Toda, Kumamoto Prefectural Institute of Public Health and Environmental Science; T. Yasaka and W. Sugitani, Kumamoto City Environmental Research Center; M. Sasaki and K. Ogata, Oita Prefectural Institute of Health and Environment; K. Kawano, Miyazaki Prefectural Institute for Public Health and Environment.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.00721-18.
Contributor Information
Christopher A. Elkins, Centers for Disease Control and Prevention.
Working Group for Legionella in Japan:
F. Kodama, K. Iwabuchi, S. Fujii, Y. Yamaguchi, N. Numata, T. Konno, A. Kaneko, J. Seto, Y. Suzuki, R. Kikuchi, N. Tomita, T. Hakuta, F. Nagashima, Y. Tokoi, K. Goto, H. Kurosawa, Y. Tamura, S. Kanazawa, S. Kobori, K. Kikuchi, T. Tomita, M. Nakamura, T. Kitahashi, E. Ogura, R. Okuno, I. Nakamura, D. Kurai, Y. Watanabe, Y. Kojima, Y. Miyashita, C. Matsuo, E. Yuzawa, M. Shindo, M. Hosoya, Y. Kimura, K. Yamamoto, K. Kawakami, E. Kitagawa, K. Yanagimoto, Y. Igawa, H. Kasahara, H. Ueda, T. Koyama, K. Sahara, A. Tomita, Y. Kanazawa, M. Hikida, Y. Kadokura, M. Noda, Y. Shiraki, M. Suzuki, N. Agata, C. Matsuo, M. Shimizu, N. Fujita, Y. Kimura, C. Katsukawa, J. Ogasawara, T. Fushiwaki, H. Tsuji, Y. Kanazawa, H. Ekawa, T. Nishiyama, M. Hirooka, Y. Hanabara, J. Uchida, T. Karasudani, K. Murakami, Y. Shimizu, Y. Miyamoto, H. Yoshida, N. Matsunaga, M. Kawano, T. Taguri, Y. Harada, I. Fukushiyama, J. Toda, T. Yasaka, W. Sugitani, M. Sasaki, K. Ogata, and K. Kawano
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