Resolving taxonomic uncertainties in the genus Haemophilus: a genomics-based approach for the reclassification of species within genera of the family Pasteurellaceae and proposal of four novel genera and one novel species

Introduction: genus. Methods: We used whole-genome sequencing, core proteome phylogeny and genome-wide similarity analyses to assess and define genus and species boundaries. Results: , revealing several misclassified species. Comparative POCP and AAI analyses, using genomic sequence data, showed that traditional genus-level thresholds (≥50% for POCP and 60-80% for AAI) of calculated protein content are insufficient to resolve species of genera with extensive horizontal gene transfer, whereas more stringent cutoffs aligned better with phylogenomic groupings. ANI and dDDH analyses effectively delineated species-level boundaries but offered limited detail for higher taxonomic ranks. Analyses of virulence factors found conserved sets of core genes known to be crucial for colonization, immune evasion and iron uptake, along with genus- and species-specific factors, indicating ecological adaptations. Functional annotation and metabolic pathway analysis highlighted universal processes and phylogenetic lineage-specific features. Discussion: . These results establish the basis for accurate species identification, clinical diagnostics, evolutionary research and functional studies within this medically and veterinarily important family.