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

<h4>Introduction</h4>The taxonomy of species within the genus <i>Haemophilus</i>, within the family <i>Pasteurellaceae</i>, shows extensive polyphyly, causing long-standing taxonomic ambiguities that complicate clinical, epidemiological and evolutionary research. Traditional classifications based on phenotypes and comparative 16S rRNA gene sequence analyses have resulted in discrepancies in defining genera and species, necessitating a thorough genome-based reevaluation. In this study, we have conducted an integrated phylogenomic analysis of the type strains of 90 species, covering all genera with validly published names within the family <i>Pasteurellaceae</i>, focusing on the historically-problematic <i>Haemophilus</i> genus.<h4>Methods</h4>We used whole-genome sequencing, core proteome phylogeny and genome-wide similarity analyses to assess and define genus and species boundaries.<h4>Results</h4>Results from phylogenomic analyses identified four well-supported species-clades within <i>Haemophilus</i>, 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.<h4>Discussion</h4>Overall, our comprehensive genomic approach has elucidated a reliable phylogenetic-based taxonomy of <i>Haemophilus</i>, detected misclassified species, recognized new genera and supports a biologically meaningful taxonomy for <i>Pasteurellaceae</i>. These results establish the basis for accurate species identification, clinical diagnostics, evolutionary research and functional studies within this medically and veterinarily important family.