Two canine urogenital isolates related to Trueperella bernardiae represent a distinct lineage

Trueperella ( T. ) bernardiae has been only rarely identified from animals, and its genomic diversity remains poorly characterised. This study analysed two canine-derived isolates initially suspected of representing T. bernardiae using phenotypic, molecular and whole-genome-based approaches. While the phenotypic profiles showed overall similarity to the type strain, several characteristics differed. The control strain was reliably identified phenotypically, whereas two isolates could not be identified beyond the genus level. Additionally, despite their high 16S rRNA gene similarity to the type strain, phylogenomic analyses consistently placed both isolates in a separate lineage. In the whole-genome sequencing (WGS)-based phylogeny, the two isolates formed a distinct cluster closest to T. bernardiae DSM 9152ᵀ and T. bernardiae UMB8254. Whole-genome comparisons supported this distinction, with average nucleotide identity values (ANIb and ANIm) of 93.98–94.69% and digital DNA–DNA hybridization (dDDH) values of 57.2–57.6% relative to T. bernardiae DSM 9152ᵀ. These findings indicate that the two isolates show genomic divergence from known Trueperella species, raising the possibility that they form a separate lineage. This study also provides the first whole-genome comparison of human- and animal-derived T. bernardiae isolates, expanding the limited genomic framework for this understudied genus. • First whole-genome-based comparison of human- and animal-derived Trueperella bernardiae isolates. • Whole-genome and multigene phylogenies placed the two investigated isolates in a distinct clade separate from human-derived Trueperella bernardiae strains. • Average Nucleotide Identity (ANI) and digital DNA–DNA hybridisation (dDDH) values confirmed clear genomic divergence from established Trueperella bernardiae lineages.