Fine-scale structural diversification among closely related Legionella pneumophila isolates from a bath-associated...

Legionella pneumophila populations within environmental reservoirs can exhibit genomic diversity, complicating source attribution during outbreak investigations. Core genome single-nucleotide variant (cgSNV)-based approaches are widely used for identifying closely related lineages during molecular epidemiological investigations; however, they primarily focus on nucleotide-level variation and may overlook structural variation among closely related isolates. In this study, we investigated genomic diversity among clinical and environmental L. pneumophila isolates obtained during an epidemiological investigation of a legionellosis case. By analyzing multiple colonies isolated from individual samples, we integrated cgSNV analysis with comparative genome structural analysis to characterize insertion sequence (IS)-mediated structural variation within a highly related population. A total of 138 serogroup 1 isolates belonging to sequence type 138 were analyzed. cgSNV analysis revealed that clinical isolates formed a clonal lineage, whereas environmental isolates from the same bath system exhibited substantial diversity despite differing by no more than 20 cgSNVs. Complete genome analysis of 48 representative isolates demonstrated nearly identical genome synteny with limited large-scale rearrangements. In contrast, IS insertion patterns showed considerable variability, particularly for IS256, which exhibited extensive diversity in insertion sites across isolates. Differences in IS insertion patterns were observed even among isolates sharing identical cgSNV genotypes, indicating that IS-mediated structural variation can occur independently of core genome SNV variation. More than half of the coding sequence differences relative to the reference genome were associated with IS insertion sites, predominantly IS256-associated events. These findings suggest that closely related L. pneumophila populations within environmental reservoirs undergo fine-scale diversification associated with dynamic IS activity.IMPORTANCEIn this study, we analyzed a large collection of Legionella pneumophila isolates obtained from a patient and a bath-associated environmental source and identified extensive insertion sequence-associated structural variation even among isolates sharing highly similar core genome single-nucleotide variant genotypes. Despite this structural diversification, the closely related isolate population retained highly conserved genome organization and broadly similar insertion sequence distribution patterns, supporting the interpretation that it remained highly clonal rather than undergoing large-scale genomic divergence. These findings highlight the importance of analyzing multiple colonies from individual samples to better characterize within-source population diversity. They further indicate that structural genome variation, particularly mobile insertion sequences such as IS256, provides complementary resolution beyond conventional nucleotide-based approaches for resolving fine-scale relationships among closely related isolates. Our study provides new insight into the microevolution of L. pneumophila populations within environmental reservoirs relevant to public health investigations.