126. Comparative Genomics of Fusobacterium Necrophorum Reveals Subspecies-specific Antimicrobial Resistance Gene Patterns and Virulence Factor Distribution Across Bovine and Human Clinical Isolates.

Abstract Fusobacterium necrophorum is an important opportunistic pathogen implicated in necrotizing infections in livestock and humans, including liver abscesses, calf diphtheria, mastitis, and foot rot in cattle, as well as Lemierre’s syndrome and tonsillopharyngitis in humans. However, increasing evidence suggests that F. necrophorum may also exist as a commensal member of the bovine microbiota, particularly within reproductive tract and gastrointestinal environments. Understanding the genomic features that enable F. necrophorum to colonize diverse hosts and anatomical niches as either a commensal or pathogen is critical for improving control strategies and for understanding its ecological versatility. We performed comparative genomic analysis of F. necrophorum isolates recovered from multiple hosts and anatomical niches. A total of 137 genomes, including 80 isolates sequenced in this study and 57 publicly available genomes, were analyzed. These genomes represented both recognized subspecies, F. necrophorum subsp. funduliforme (FNF) and F. necrophorum subsp. necrophorum (FNN) and were recovered from bovine and human hosts across seven anatomical sites including bull semen, liver abscesses, lungs, rumen fluid, calf diphtheria lesions, healthy liver, and mediastinal lymph nodes. Whole-genome sequencing and comparative genomic approaches were used to characterize pangenome structure, phylogenetic relationships, functional gene repertoires, virulence factors, and antimicrobial resistance genes. The F. necrophorum pangenome comprised 5,820 predicted genes, indicating an open pangenome with extensive accessory genomic diversity. Phylogenetic analysis resolved two distinct subspecies clades with high genomic similarity within subspecies. Functional profiling revealed substantial metabolic divergence between subspecies, with FNN enriched for carbohydrate transport systems and advanced glycation end-product degradation pathways, including raffinose/stachyose transport genes (msmE, msmF, msmG) and fructoselysine metabolism genes (frlB, frlD). In contrast, FNF genomes were enriched for genes associated with threonate metabolism (pdxA2, dtnK) and hemolysin-related systems (fhaC). Virulence gene analysis identified 84 variants across multiple functional categories, including leukotoxins, hemagglutinins, autotransporter outer membrane proteins, and lipopolysaccharide biosynthesis enzymes, with subspecies- and host-associated distribution patterns. Antimicrobial resistance genes were detected in 22.6% of genomes, primarily tetracycline resistance genes (tet(O), tet(M), tet(40)) and the macrolide resistance gene erm(B), with higher prevalence observed in bovine isolates. Together, these findings demonstrate extensive genomic and functional diversity within F. necrophorum, highlighting genomic characteristics associated with subspecies divergence, host adaptation, and niche specialization that may contribute to its ability to persist as both a commensal and opportunistic pathogen. These findings provide a genomic foundation for advancing our understanding of the ecological plasticity of F. necrophorum, including efforts to distinguish pathogenic from commensal strains and to explore its potential contribution as a beneficial member of the reproductive tract microbiome associated with favorable pregnancy outcomes. They also support improved surveillance, prevention, and management strategies for fusobacterial infections in livestock within a broader One Health framework encompassing antimicrobial resistance and zoonotic risk.