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Volume 23, Number 2—February 2017
Research Letter

Outbreak of Legionnaire’s Disease Caused by Legionella pneumophila Serogroups 1 and 13

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Toshiro Kuroki1Comments to Author , Junko Amemura-Maekawa1, Hitomi Ohya, Ichiro Furukawa, Miyuki Suzuki, Tomoka Masaoka, Kastuhiro Aikawa, Kazumi Hibi, Masatomo Morita, Ken-Ichi Lee, Makoto Ohnishi, and Fumiaki Kura
Author affiliations: Kanagawa Prefectural Institute of Public Health, Kanagawa, Japan (T. Kuroki, H. Ohya, I. Furukawa, M. Suzuki, T. Masaoka, K. Aikawa, K. Hibi); National Institute of Infectious Diseases, Tokyo, Japan (J. Amemura-Maekawa, M. Morita, K. Lee, M. Ohnishi, F. Kura)

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In Japan, hot springs and public baths are the major sources of legionellosis. In 2015, an outbreak of Legionnaires’ disease occurred among 7 patients who had visited a spa house. Laboratory investigation indicated that L. pneumophila serogroup 1 and 13 strains caused the outbreak and that these strains were genetically related.

Infection with Legionella bacteria is one of the major causes of community-acquired pneumonia (1). In Japan, the major sources of Legionella infection are hot springs and public baths (2). Among Legionella species, L. pneumophila serogroup 1 accounts for most human infections (3). Legionellosis outbreaks caused by a combination of L. pneumophila serogroup 1 or other serogroups have rarely been reported. We report an outbreak of Legionnaires’ disease caused by L. pneumophila strains of serogroup 1 and serogroup 13.

During June 1–17, 2015, the Health Centers in Kanagawa and Shizuoka Prefectures, Japan, were notified of 7 cases of Legionella pneumonia. All patients with pneumonia were admitted to 1 of 5 hospitals. All patients were male (mean age 66.3 years), 4 had diabetes mellitus, and 1 had hepatic cirrhosis and liver cancer. Diagnosis of pneumonia at the hospitals was based on clinical presentation and immunochromatographic detection of L. pneumophila serogroup 1 antigen in urine. All patients recovered and were discharged.

In epidemiologic interviews, all 7 patients stated that they had visited a spa house in Odawara, Kanagawa, Japan, before illness onset. The latent period was not accurately determined because 5 of the 7 patients frequently visited this spa house and some patients had visited it again after illness onset. The spa house had 7 circulating systems, including filtration and heating components, and 9 baths for men.

Sputum samples from 5 urinary antigen–positive patients and environmental samples from the spa house were collected for epidemiologic investigations and cultured for Legionella at the Kanagawa Prefectural Institute of Public Health. L. pneumophila serogroup 1 was detected in sputum from 4 patients (Table), and L. pneumophila serogroups 1 and 13 were detected in sputum from 1 patient (patient 2). Baths 1–5, but not baths 6–9, contained L. pneumophila. Epidemiologic investigation and laboratory results revealed that failure to adequately chlorinate the bath water and the circulating systems resulted in colonization of Legionella at the spa house.

Pulsed-field gel electrophoresis (PFGE) comparison (4) of clinical and environmental isolates revealed that the L. pneumophila serogroup1 strains produced 3 PFGE profiles: A and B, with a 1-band difference, and C (Table). Patient 7 was infected with 2 L. pneumophila serogroup 1 isolates possessing PFGE profiles A and B. The PFGE profile B of L. pneumophila serogroup 13 was identical to that of serogroup 1. We determined the sequence type (ST) of L. pneumophila strains (5,6) and identified 4 new STs: ST2113, ST2114, ST2115, and ST2121. ST2114 differed from ST2121 by only 1 nt in neuA and differed from ST2113 (serogroup 13) by only 2 alleles (mip and neuA), suggesting that these 3 STs are closely related and that 1 of 3 strains (ST2113, ST2114, or ST2121) may be derived from another by homologous recombination. All examined isolates lacked the lag-1 gene, a virulence-associated marker (7).

By using whole-genome sequencing, we applied core-genome multilocus sequence typing (cgMLST) with 50 genes (8) different from the 7 genes in sequence-based typing, thereby confirming the sequence-based typing data (Table). ST2114 and ST2115 isolates were each divided into 2 cgMLST profiles. cgMLST profile b differed from profile a by only 1 nt on lpg1503 and differed from profile d by 5 nt on lpg0812, near the lipopolysaccharide coding region, suggesting that the strain of profile d may be derived from another by homologous recombination. However, the remaining cgMLST profiles (c, e, and f) from strains not isolated from patients differed from profile b by 30, 42, and 43 alleles, respectively.

Multiple L. pneumophila strains with different genetic characteristics exist in the environment and pose infection risks (9). Among the strains studied, dual infections with L. pneumophila serogroup 1 and serogroup 13 strains (patient 2) and L. pneumophila serogroup 1 strains with different genomic subtypes (patient 7) were detected. Results from 3 genetic methods revealed that L. pneumophila serogroup 1 and 13 strains are closely related, although the serogroups differ. Results of this study were consistent with the hypothesis that multiple infections are more likely with less virulent strains and more likely in persons with medical conditions predisposing them to Legionnaires’ disease (10).

Our study of this outbreak suggests that the spa house was colonized by several L. pneumophila strains that were genetically related despite belonging to different serogroups and that 2 strains caused infection. Further analysis of the divergence of outbreak strains in genomes related to Legionella serogroup and sequence types is ongoing. This analysis clarifies the in-depth genetic relations among L. pneumophila strains, such as recombination sites and periods required for divergence. We recommend that the spa house provide high quality management and effective infection control practices according to an infection control manual (e.g., completion of documentation relating to infection control practices and training of employees) and that customers be aware of the sanitary status of spa houses.

Dr. Kuroki is a director of the Department of Microbiology at Kanagawa Prefectural Institute of Public Health, Kanagawa, Japan. His research interests include epidemiology of protozoan and bacterial infectious diseases.



We appreciate the help of the officers at Odawara Health and Welfare Center, who provided information on epidemiological studies.

This work was partly supported by Health and Labour Sciences Research (grant H25-kenki-009 to F. K.).



  1. Yu  VL, Stout  JE. Community-acquired legionnaires disease: implications for underdiagnosis and laboratory testing. Clin Infect Dis. 2008;46:13657. DOIPubMedGoogle Scholar
  2. National Institute of Infectious Diseases and Tuberculosis and Infectious Diseases Control Division, Ministry of Health, Labour and Welfare. Legionellosis, April 1999–July 2000. Infect Agents Surveill Rep. 2000;21:186–7 [cited 2016 May 9].
  3. Yu  VL, Plouffe  JF, Pastoris  MC, Stout  JE, Schousboe  M, Widmer  A, et al. Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey. J Infect Dis. 2002;186:1278. DOIPubMedGoogle Scholar
  4. Chang  B, Amemura-Maekawa  J, Watanabe  H. An improved protocol for the preparation and restriction enzyme digestion of pulsed-field gel electrophoresis agarose plugs for the analysis of Legionella isolates. Jpn J Infect Dis. 2009;62:54–6 [cited 2016 May 9].
  5. Gaia  V, Fry  NK, Afshar  B, Lück  PC, Meugnier  H, Etienne  J, et al. Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila. J Clin Microbiol. 2005;43:204752. DOIPubMedGoogle Scholar
  6. Ratzow  S, Gaia  V, Helbig  JH, Fry  NK, Lück  PC. Addition of neuA, the gene encoding N-acylneuraminate cytidylyl transferase, increases the discriminatory ability of the consensus sequence-based scheme for typing Legionella pneumophila serogroup 1 strains. J Clin Microbiol. 2007;45:19658. DOIPubMedGoogle Scholar
  7. Kozak  NA, Benson  RF, Brown  E, Alexander  NT, Taylor  TH Jr, Shelton  BG, et al. Distribution of lag-1 alleles and sequence-based types among Legionella pneumophila serogroup 1 clinical and environmental isolates in the United States. J Clin Microbiol. 2009;47:252535. DOIPubMedGoogle Scholar
  8. David  S, Mentasti  M, Tewolde  R, Aslett  M, Harris  SR, Afshar  B, et al. Evaluation of an optimal epidemiological typing scheme for Legionella pneumophila with whole-genome sequence data using validation guidelines. J Clin Microbiol. 2016;54:213548. DOIPubMedGoogle Scholar
  9. Qin  T, Yan  G, Ren  H, Zhou  H, Wang  H, Xu  Y, et al. High prevalence, genetic diversity and intracellular growth ability of Legionella in hot spring environments. PLoS One. 2013;8:e59018. DOIPubMedGoogle Scholar
  10. Balmer  O, Tanner  M. Prevalence and implications of multiple-strain infections. Lancet Infect Dis. 2011;11:86878. DOIPubMedGoogle Scholar




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DOI: 10.3201/eid2302.161012

1These authors contributed equally to this article.

Table of Contents – Volume 23, Number 2—February 2017

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Please use the form below to submit correspondence to the authors or contact them at the following address:

Toshiro Kuroki, Department of Microbiology, Kanagawa Prefectural Institute of Public Health, 1-3-1 Shimomachiya, Chigasaki, Kanagawa, 253-0087, Japan

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