Legionella-like isolates, HB10 and PATHC038^T, were isolated from a cold water distribution system on a cruise ship in 2017. The strains have been characterized by employing discriminatory genome typing, matrix-assisted laser desorption/ionization time-of-flight MS technique, fatty acid profiling and biochemical tests, thus identifying a new species in the genus Legionella. Colonies displayed phenotypic characteristics typical of the family Legionellaceae, including a requirement for l-cysteine and testing catalase positive. Fatty acid methyl ester analysis showed that the predominant fatty acids detected in the studied strains are C16:0 iso, C16:1 ω7c, C16:0 and C15:0 anteiso. The obtained MIC values showed antimicrobial susceptibility to the antimicrobial drugs ciprofloxacin and erythromycin (0.8 and 2 µg ml⁻¹, respectively). The bacteria were also Gram-negative, rod-shaped, grew aerobically on buffered charcoal yeast extract agar and weakly grew on glycine-vancomycin-polymyxin-cycloheximide media at 36 °C with the ability to autofluoresce a blue-white colour when placed under a long-wavelength UV light (365 nm). The whole-genome sequencing performed displayed a G+C content of 38.2 mol%. The digital DNA–DNA hybridization analysis demonstrated a separation from the phylogenetically most related Legionella cherrii, with 54.7% DNA–DNA relatedness. The identity percentage measured by average nucleotide identity between the PATHC038^T strain and its respective closest species, L. cherrii, was 93.9%, also confirming the distinctiveness of the novel species. The 16S rRNA gene, mip and rpoB sequences showed a similarity of 98.7, 98.4 and 96.2%, respectively, with L. cherrii NCTC 11976^T. Additionally, average amino acid identity and percentage of conserved proteins analyses further supported their classification as a novel species. The results obtained in this study confirm the status of an independent species. The name proposed for this species is Legionella sheltonii sp. nov. with PATHC038^T (CCUG 76918T, ATCC TSD-370) as the type strain.

Accurate species-level identification of Legionella during routine environmental surveillance remains challenging, as standard proteomic screening methods like MALDI-TOF MS often lack the resolution to distinguish closely related taxa. This study introduces a novel, cost-effective, and stepwise taxonomic framework that bridges routine environmental monitoring with multi-layered, genome-resolved analysis. We expanded upon our previous screening of four novel candidate lineages by focusing on the three remaining, uncharacterized environmental isolates, while using the recently validly described Legionella sheltonii (strain PATHC038) as a reference framework control. By applying nucleotide- and protein-based overall genomic relatedness indices (ANI, dDDH, AAI, and POCP) alongside core-genome phylogenomics, our framework independently confirmed the genomic distinctiveness of the three isolates, which exhibited AAI values of 94.3–96.6% and POCP values of 86.5–91.0%, with POCP values lying close to the proposed species delineation threshold. By defining clear criteria for escalating isolates from culture to targeted sequencing, this workflow prevents diagnostic misidentification during epidemiological outbreak investigations and provides public health agencies with a scalable tool to monitor hidden Legionella diversity, ultimately enhancing proactive water safety and risk assessment.

Environmental surveillance of Legionella bacteria relies on standardized culture-based methods that enable reliable detection; however, accurate species-level identification remains challenging. Phenotypic, serological, and proteomic methods, including latex agglutination and MALDI-TOF mass spectrometry, enable rapid screening but often lack sufficient resolution to distinguish closely related species. This study proposes a structured framework for the identification and taxonomic characterization of environmental Legionella isolates. Environmental isolates were obtained using standard culture methods and initially identified by MALDI-TOF MS, followed by molecular characterization using PCR, 16S rRNA gene sequencing, and whole-genome sequencing. Phylogenomic analyses were conducted using overall genomic relatedness indices, including Average Amino Acid Identity (AAI) and Percentage of Conserved Proteins (POCP), and a phylogenomic tree was constructed based on 400 universal marker genes. The framework was applied to four environmental isolates (PATHC032, PATHC035, PATHC038, and PATHC039), including one previously described species, Legionella sheltonii. Results showed AAI values ranging from 94.3% to 96.6% and POCP values from 86.5% to 91.0%, supporting their genomic distinctiveness from any validly described Legionella species, which was confirmed by phylogenomic analysis. These findings demonstrate that the proposed framework enables consistent species-level identification and provides guidelines for recognizing candidate novel Legionella species through the integration of environmental surveillance and genome-based taxonomy.