Legionella petroniana sp. nov., a novel species isolated in Bologna, Italy: taxonomic, genomic and ecological insights in the era of environmental change

This study presents the characterization of a novel Legionella species isolated in Italy over three different years from one company and two hospitals. Starting from standard techniques such as culture of water samples, agglutination test, MALDI-TOF MS and gene sequencing analysis used to identify the isolates, genomic and metabarcoding approaches were subsequently employees to further characterize the species. Legionella contamination ranged from 400 to 700 CFU/L. The tiny colonies displayed atypical morphology compared to typical Legionella features, although they grew on BCYE medium supplemented with L-cysteine. Microscopic and phenotypic analyses revealed Gram-stain negative, Ziehl-Neelsen-negative, rod-shaped, motile cells capable of growing at 32-37 °C, including on selective media such as GVPC and MWY. The isolates tested positive for oxidase and gelatinase activity. Fatty acid profiling identified the dominant components as Summed Features 3 (C16:1 ω7c/C16:1 ω6c, 28.9%), C16:0 iso (18.4%), and C15:0 anteiso (15.4%). Ubiquinone Q13 was the major quinone. Sequence analysis of the mip and rpoB genes showed 98.2% and 95.1% similarity, respectively, to L. feeleii (WO-44C ATCC 35072^T). Whole genome sequencing (WGS) revealed a GC content of 41.5%, a dDDH value of ≤54.9%, and an ANI of 94.06% with L. feeleii (WO-44C ATCC 35072^T), supporting the classification of a novel species within the genus Legionella. Furthermore, taxonomic resolution of water samples revealed the presence of 168 bacterial genera, including several respiratory, opportunistic, and zoonotic pathogens, as well as seven Legionella species. The name Legionella petroniana sp. nov. is proposed, with strain 31fI33^T (=DSM 114357^T=CCUG 76442^T) designated as type strain.