Investigation of a Legionella pneumophila Outbreak at a Bath Facility in Japan Using Whole-Genome Sequencing of Isolates from Clinical and Environmental Samples

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Abstract

Exposure to aerosols containing Legionella from artificially made water systems has been established as a primary cause of Legionnaires’ disease. In this study, we investigated an outbreak of L. pneumophila serogroup 1 sequence type 138 which occurred at a bath facility in 2022. The whole-genome sequencing of isolates revealed that the colonization of L. pneumophila at the bath facility had occurred before 2013, and the patients had been exposed to multiple genetic lineages of the strain. Our study demonstrates the importance of performing a careful comparative genetic analysis of clinical and environmental isolates from LD outbreaks in order to effectively investigate and prevent future LD outbreaks.

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Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins

Nicholas Croucher, Andrew J Page, Thomas R. Connor … (2014)

The emergence of new sequencing technologies has facilitated the use of bacterial whole genome alignments for evolutionary studies and outbreak analyses. These datasets, of increasing size, often include examples of multiple different mechanisms of horizontal sequence transfer resulting in substantial alterations to prokaryotic chromosomes. The impact of these processes demands rapid and flexible approaches able to account for recombination when reconstructing isolates’ recent diversification. Gubbins is an iterative algorithm that uses spatial scanning statistics to identify loci containing elevated densities of base substitutions suggestive of horizontal sequence transfer while concurrently constructing a maximum likelihood phylogeny based on the putative point mutations outside these regions of high sequence diversity. Simulations demonstrate the algorithm generates highly accurate reconstructions under realistically parameterized models of bacterial evolution, and achieves convergence in only a few hours on alignments of hundreds of bacterial genome sequences. Gubbins is appropriate for reconstructing the recent evolutionary history of a variety of haploid genotype alignments, as it makes no assumptions about the underlying mechanism of recombination. The software is freely available for download at github.com/sanger-pathogens/Gubbins, implemented in Python and C and supported on Linux and Mac OS X.

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M Pastoris, Andreas F. Widmer, T File … (2002)

This international collaborative survey identified culture-confirmed legionellosis in 508 patients with sporadic community-acquired legionellosis. Legionella pneumophila constituted 91.5% of the isolates. Serogroup 1 was the predominant serogroup (84.2%), and serogroups 2-13 (7.4%) accounted for the remaining serogroups. The Legionella species most commonly isolated were L. longbeachae (3.9%) and L. bozemanii (2.4%), followed by L. micdadei, L. dumoffii, L. feeleii, L. wadsworthii, and L. anisa (2.2% combined). L. longbeachae constituted 30.4% of the community-acquired Legionella isolates in Australia and New Zealand.

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Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila.

Valeria Gaia, Norman Fry, Baharak Afshar … (2005)

A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of > 0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.