Phylogenetic analysis of Legionella pneumophila from clinical and environmental isolates in a public health surveillance context

Background Legionella pneumophila (Lp) is a ubiquitous pathogen of significant public health relevance and a leading cause of severe community-acquired pneumonia. Its ability to colonize artificial water systems and survive intracellularly within human alveolar macrophagescomplicates clinical management and epidemiological control. Methods A retrospective analysis was performed on clinical (n=78) and environmental (n=14) Legionella pneumophila isolates collected in Milanby Niguarda Hospital and ATS. Whole Genome Sequencing (WGS) was carried out on 84 frozen strains, and core-genome multilocussequence typing (cgMLST) was used to assess phylogenetic relationships and identify potential infection clusters; following casenotifications, environmental sampling targeted facilities visited by culture-positive patients (Figure1). A subset of 31 isolates alsounderwent antimicrobial susceptibility testing using the Sensititre system (Thermo Fisher), with genomic data integrated to explorecorrelations between genotype and antimicrobial response. Results The demographic analysis of cases showed a predominance of male patients (58.7%) with a median age of 65 years, consistent with thepopulation most susceptible to Lp. The predominant serogroup (68/92) was Lp1, mainly of clinical origin, while serogroups 2–15 weremore represented among environmental isolates. cgMLST analysis revealed 56 distinct allelic profiles and 10 molecular clusters.Notably, a genetic match between environmental and clinical strains was found only in 2 cases (Figure2A, C) while no direct linkemerged in the other 3 cases (Figure2B, D, E) although the environmental isolates from D and E were genetically related to each other.Antimicrobial susceptibility testing on 31 isolates confirmed overall sensitivity to fluoroquinolones and macrolides, with MICs consistentwith the reference strain, whereas elevated MICs observed in a few isolates suggest the potential presence of emerging resistancemechanisms (Figure3). The integration of genomic, clinical, and environmental data highlighted a heterogeneous population of Lp atthe genetic level, yet with similar phenotypic profiles. Conclusions These findings emphasize the importance of integrating advanced molecular methods such as WGS and cgMLST into epidemiologicsurveillance, supporting rapid diagnosis, outbreak tracing, and patient management, and underscore the need for a multidisciplinaryapproach in clinical microbiology for effective disease control.