An integrated genomic framework for Aeromonas genomic species delineation using average nucleotide identity, core-genome phylogeny and digital DNA–DNA hybridisation

Aeromonas taxonomy has long been complicated by overlapping phenotypic, biochemical and protein profiles. Here, we establish a robust genome-based framework for Aeromonas genomic species delineation. We analysed average nt identity (ANI) across 4,366 available Aeromonas genomes and demonstrated that at a 96% ANI threshold, skANI and fastANI generated too many clusters (65 and 57, respectively) and these clusters were not supported by core-genome phylogeny. We identified 95.4% skANI (equivalent to 95.6% fastANI) as an operational threshold for delineating Aeromonas genomic species. Using the 95.4% skANI threshold, we identified 44 ANI clusters among the 4,366 genomes, of which 43 clusters were genomic species supported by the core-genome phylogeny. Thirty-four of the 43 genomic species corresponded to existing taxonomic species, whilst the remaining 9 are currently not recognised as taxonomic species. All recognised taxonomic species represented in the dataset retained their existing species designation except Aeromonas mytilicola , which was not separated from Aeromonas rivipollensis in both ANI clusters and the core-genome phylogeny. The digital DNA–DNA hybridisation values between the genomic species were below 70%, further supporting genomic species delineation. We further developed AeromonasGStyper, a genomic species typing tool that assigns query genomes based on ANI similarity to medoid genomes. In conclusion, this study establishes a genomic species framework for genome-based classification of Aeromonas and provides a practical approach for future genomic surveillance.