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

Abstract

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 highresolution 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.

Genomic Resolution of Outbreak-Associated Legionella pneumophila Serogroup 1 Isolates from New York State

“…Our finding extends the known geographical distribution of ST1204 isolates. Similar to other reports utilizing WGS approaches for L. pneumophila subtyping, an ST could not be assigned to some isolates due to the presence of multiple copies of the mompS allele (25). In these cases, either sequence assemblies failed to cover the entire allele or multiple alleles were identified within the genome sequence.…”

A Primer on Infectious Disease Bacterial Genomics

“…As bacterial typing is enhanced by the finer resolution of genome-wide data, revealing novel alleles and gene functions, it provides opportunities for new protocol development (209,210). Furthermore, the ability to mine WGS data and extract multiple typing schemes makes global comparisons achievable, which is essential for global surveillance and epidemiological investigations.…”

Commentary: Next-Generation Epidemiology: Using Real-Time Core Genome Multilocus Sequence Typing To Support Infection Control Policy

“…The cgMLST methods address this problem by comparing only genes common to a set of bacterial isolates (2). These methods are now mature and have been applied successfully to the analysis of a number of bacterial outbreaks (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).Given the developments in WGS-based strain typing described above, there is much excitement about applying these methods to real-time hospital epidemiology, but the actual cost-benefit equation, the feasibility of achieving results on an actionable time scale, and the technical requirements for implementation in a "real world" clinical microbiology laboratory are difficult to study. In work published in this issue of the Journal of Clinical Microbiology, …”

“…The cgMLST methods address this problem by comparing only genes common to a set of bacterial isolates (2). These methods are now mature and have been applied successfully to the analysis of a number of bacterial outbreaks (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”