Genetic relatedness between clinical and environmental Legionella isolates reveals limited within-system diversity and differences between hot- and cold-water systems

Legionnaires’ disease (LD) is a severe pneumonia increasingly linked to building water systems. Although Legionella colonizes both hot- and cold-water networks, the precise source of infection often remains unclear, and the interpretation of minor genomic differences between related isolates is not well established. We aimed to assess patterns of genetic variation between sample points within water systems and evaluate whether they are consistent among the most likely sources of infection using whole-genome sequencing (WGS). We retrospectively analyzed LD cases in Finland from 2017 to 2024 with available WGS data from both patient and epidemiologically linked environmental isolates. Genetic relatedness was assessed using cgMLST (≤ 4 allele differences defining close relatedness). Sequence types, clustering patterns and allele differences were compared across sampling points and water system types. WGS of 22 clinical and 65 epidemiologically associated environmental isolates identified 14 sequence types (STs) and cgMLST grouped isolates accordingly. Isolates were genetically diverse between cases and showed no clustering beyond known outbreak-related strains, except in a single case. 73% were caused by Legionella pneumophila serogroup 1 and 23% by other L. pneumophila serogroups. Clinical and environmental isolates were closely related in 28/30 cases (93%), and at least one environmental isolate was cgMLST-identical to the patient isolate in 25 cases (83%) supporting genetic link between clinical and environmental sources. Minor allelic differences (typically 1–2 alleles) were observed in 60% of cases, with identical and closely related variants often co-occurring within the same water system. In addition, hot-water isolates were significantly more likely to match patient isolates than cold-water isolates (85% vs 44%, p = 0.001), although matching strains were also frequently detected in cold-water systems. WGS demonstrated high concordance between clinical and epidemiologically associated environmental isolates and supports source attribution in most LD investigations. Minor allelic differences between related isolates were common and reflected limited within-system microdiversity rather than conflicting evidence of source attribution. Integrating genomic and epidemiological data is essential for accurate interpretation of LD investigations. Hot-water isolates were more frequently identical to patient strains than cold-water isolates, although both water systems may harbor infection-associated populations.