Pathogen genome enrichment sequencing to support a city-wide Legionnaires' disease outbreak investigation.

Pathogen genomics can provide high-confidence source attribution for epidemiological investigations of Legionnaires' disease (LD) outbreaks. However, the utility of culture-based genomics can be undermined by long lead times for Legionella pneumophila culture or a lack of isolates when dealing with non-culturable bacteria or low-biomass specimens. Here, during a large 2024 LD outbreak in Melbourne, Australia, we compared direct specimen, hybridization-based enrichment sequencing with traditional culture-based whole-genome sequencing of 18 clinical and 5 environmental L. pneumophila specimens. Informed by the L. pneumophila global population structure, the method used 28,061 RNA bait hybridization probes to enrich >170,000 known genome-wide informative SNP sites directly from clinical and environmental specimens. Genome enrichment sequencing detected L. pneumophila with high sensitivity, achieving genome-wide coverage at 100 c.f.u. ml-1. Using sequence enrichment data with phylogenomic and machine learning approaches, we identified that a single cooling tower was likely responsible for the outbreak. Genome enrichment sequencing has the potential to enable faster and more comprehensive source tracking, thereby enhancing public health responses to outbreaks of environmental pathogens, permitting earlier, more confident interventions.