A Modern Framework for Identifying Novel Environmental Legionella Species
Journal article, 2026
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.
percentage of conserved proteins (POCP)
novel Legionella species
phylogenomics
whole genome sequencing
average amino acid identity (AAI)
Author
Karla Vasari
PathCon Laboratories
University of Rijeka
Maja Kovačević
University of Zagreb
PathCon Laboratories
Niko Kasalo
Ruder Boskovic Institute
Snjezana Kazazic
Ruder Boskovic Institute
Ivan Mijakovic
Technical University of Denmark (DTU)
Chalmers, Life Sciences, Systems and Synthetic Biology
Goran Klobucar
University of Zagreb
D. Franjevic
University of Zagreb
Josip Skejo
University of Zagreb
Tomislav Domazet-Lošo
Ruder Boskovic Institute
Catholic University of Croatia
Brian G. Shelton
PathCon Laboratories
Marina Santic
Ruder Boskovic Institute
University of Rijeka
Roberta Sauerborn Klobucar
PathCon Laboratories
Life
0024-3019 (ISSN) 2075-1729 (eISSN)
Vol. 16 7 1187Subject Categories (SSIF 2025)
Microbiology in the Medical Area
DOI
10.3390/life16071187
PubMed
42514254