A large outbreak investigation of Legionnaires’ disease associated with a public bath facility in Hiroshima, Japan, using PFGE, SBT, MLVA, and whole-genome sequencing

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In 2017, a large outbreak of Legionnaires' disease, involving 58 patients, occurred in a public bath facility in Hiroshima Prefecture, Japan. We analyzed 94 Legionella pneumophila strains isolated from patients and the public bath facility using molecular typing methods, including Pulsed-Field Gel Electrophoresis, sequence-based typing, multi-locus variable number tandem repeat analysis, and Whole-Genome Sequencing (WGS). Genotypes obtained using these molecular epidemiological typing methods were highly correlated with each other. L. pneumophila strains of various genotypes were isolated from the public bath facility, of which only ST2398 and ST2399 were isolated from patients. ST2398 and ST2399, isolated from patients, bath water, and swabs, derived from one common circulating system at the bath facility out of seven were found to be novel genotypes and a highly clonal genetic lineage by single nucleotide variant (SNV) analysis based on WGS. The result of haplotype network analysis based on SNVs showed that ST2398 and ST2399 differed only approximately 30-42 SNVs, and some environmental strains that differed by only 0-3 SNVs from patient strains were isolated. These results demonstrated that this outbreak was caused by L. pneumophila assigned to the ST2398 and ST2399 clades. We found that at least three patients were co-infected with different clusters of L. pneumophila serogroup 1. Our results show that several strains must be isolated from a single sample to consider the accumulation of mutations in water and co-infection when investigating outbreaks.

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