Design and application of a core genome multilocus sequence typing scheme for investigation of Legionnaires' disease incidents

Sequence-based typing (SBT) for Legionella pneumophila (Lp) has dramatically improved Legionnaires' disease (LD) cluster investigation. Microbial whole genome sequencing (WGS) is a promising modality for investigation but sequence analysis methods are neither standardised, nor agreed. We sought to develop a WGS-based typing scheme for Lp using de novo assembly and a genome-wide gene-by-gene approach (core genome multilocus sequence typing, cgMLST). We analysed 17 publicly available Lp genomes covering the whole species variation to define a core genome (1,521 gene targets) which was validated using 21 additional published genomes. The genomes of 12 Lp strains implicated in three independent cases of paediatric humidifier-associated LD were subject to cgMLST together with three 'outgroup' strains. cgMLST was able to resolve clustered strains and clearly identify related and unrelated strains. Thus, a cgMLST scheme was readily achievable and provided high-resolution analysis of Lp strains. cgMLST appears to have satisfactory discriminatory power for LD cluster analysis and is advantageous over mapping followed by single nucleotide polymorphism (SNP) calling as it is portable and easier to standardise. cgMLST thus has the potential for becoming a gold standard tool for LD investigation. Humidifiers pose an ongoing risk as vehicles for LD and should be considered in cluster investigation and control efforts. .