Genomic and phenotypic characterization of a novel recombinant Legionella pneumophila lineage associated with long-term persistence in a building water system and recurrent legionellosis outbreaks.

Understanding Legionella pneumophila microevolution within building water systems is crucial for effective long-term water safety plans. We investigated an exceptional 20-year contamination case within a closed, state-managed residential facility where routine engineering controls, including hyperchlorination and thermal shocks, systematically failed. Integrating genomic surveillance, transcriptomics, 3D biofilm characterisation and cell infection models, we show this persistence was driven by the in situ emergence of a novel recombinant strain (ST3043), which achieved over 80% dominance. Phylodynamic modelling revealed that this recombination event, involving the acquisition of a 225-kb fragment that rewired a cyclic-di-GMP hub, occurred at the time of the building's commissioning. This proves that the infrastructure acted as an evolutionary sieve. Importantly, while the ancestral parental lineages remained confined to minor environmental niches, ST3043 underwent sharp phenotypic divergence. Biofilm architectural characterisation revealed that, whereas parental strains produced thin, compact layers (∼24 µm), ST3043 developed a complex 3D matrix featuring a non-adherent, loose fraction (∼90 µm), which occupied 95% of the total biofilm space. Viability assays showed that cells within this dispersal compartment had high metabolic activity and acted as an efficient seeding source in the liquid phase. Furthermore, detached ST3043 cells displayed optimised fitness, with robust intracellular replication inside human macrophage and epithelial models, compared to impaired parental cells. This combination of enhanced environmental persistence, efficient dispersal and intracellular fitness may have contributed to the predominance of ST3043 among clinical isolates linked to the facility, with its high environmental abundance likely increasing the opportunity for human exposure. Overall, localised, infrastructure-driven recombination may contribute to the emergence and persistence of successful clones by combining environmental dominance with traits that may facilitate infection, thereby exposing specific vulnerabilities.