Comparative Genomics of Bovine and Human Fusobacterium necrophorum Strains Reveal Subspecies- and Host-associated Differences in Virulence and Antimicrobial Resistance

Fusobacterium necrophorum (FN) is an important opportunistic pathogen implicated in necrotizing infections, including liver abscesses, calf diphtheria, metritis, and foot rot in cattle, and tonsillopharyngitis in humans. However, FN also exists as a commensal member of the bovine reproductive microbiota with potential negative, as in metritis, and even positive associations with pregnancy outcomes. The genomic features that enable FN to colonize diverse hosts and anatomical niches as either a commensal or a pathogen is poorly understood. We addressed these knowledge gaps by performing comparative genomic analysis of 137 FN strains (80 newly sequenced, 57 publicly available) from clinical and non-clinical sources across human and bovine hosts. We investigated the pangenome structure, virulence gene repertoire, antimicrobial resistance genes (ARG) prevalence, as well as host-and subspecies-associated genomic signatures of two FN subspecies: subsp. necrophorum (FNN) and subsp. funduliforme (FNF). Comparative genomics revealed an open pangenome with high accessory diversity, and phylogenetic analysis separated the strains into two distinct subspecies clades. Functional profiling revealed substantial metabolic divergence between subspecies, with FNN showing higher prevalence of carbohydrate transport systems and advanced glycation-related pathways, while FNF showed enrichment in threonate metabolism and hemolysin-related systems. Virulence gene analysis identified 84 variants across multiple functional categories with subspecies- and host-specific distributions. Antimicrobial resistance genes, primarily tetracycline resistance genes [tet(O), tet(M), tet(40)] and the macrolide resistance gene erm(B), were detected in 22.6% of strains, with higher prevalence in bovine than human strains. Overall, our results suggest that pathogenic potential of FN appears to be determined by the interplay between an open pangenome, subspecies-specific metabolic and virulence repertoires, host-associated adaptation, and niche specialization. IMPORTANCEFusobacterium necrophorum, comprising two subspecies, necrophorum (FNN) and funduliforme (FNF), is a major pathogen in cattle and humans, yet it also occurs as a commensal inhabitant in healthy cattle, particularly in the rumen, hindgut, semen, and the female reproductive tract. However, emerging evidence indicates it also occurs as a non-clinical inhabitant, particularly in the bovine reproductive tract, where it may be associated with improved pregnancy outcomes. We conducted a comparative genomic analysis of 137 F. necrophorum strains, including FNN (n = 12) and FNF (n = 125), sourced from humans (n=53) and cattle (n=84) across seven anatomical niches spanning both healthy and diseased sources. We identified subspecies- and host-specific metabolic pathways, antimicrobial resistance profiles, and distinct virulence gene distributions that underpin the ecological versatility of Fusobacterium necrophorum. Overall, these findings provide a genomic framework for understanding its host adaptation and niche specialization across bovine and human hosts.