Pangenome Architecture and Accessory Gene-Driven Population Structure of Staphylococcus aureus Revealed by a...

Staphylococcus aureus is a globally distributed bacterium that spans interconnected human, animal, and environmental niches and is a major driver of antimicrobial resistance. Environmental and wildlife-associated isolates from hospital-surrounding settings remain underrepresented in comparative genomic studies. To address this gap, we integrated a newly sequenced environmental isolate recovered from pigeon fecal samples collected around a hospital into a standardized pangenome framework composed of 99 reproducibly selected RefSeq genomes plus the environmental isolate S_S3. Using uniform genome annotation and orthologous gene family clustering, we identified an open pangenome of 8366 gene families (Heaps' law γ = 0.275), consistent with the high genomic plasticity previously reported for S. aureus. The core genome stabilized at approximately 1757 genes, including 1651 genes conserved across all genomes. Gene frequency spectra showed a dominant cloud genome and a structured shell fraction contributing to interstrain differentiation. Jaccard-based gene content similarity resolved clusters shaped mainly by accessory gene composition. The environmental isolate retained the complete core genome, carried only 15 isolate-specific gene families (0.18% of the pangenome), and clustered within an established lineage. Its unique content included a lincosamide resistance-associated locus and efeB, a gene potentially related to heme or iron metabolism and oxidative stress response. These findings highlight a conserved genomic backbone over a dynamic accessory reservoir and support One Health genomic surveillance that includes wildlife-associated niches, while indicating that the environmental isolate fits within the broader gene content diversity observed in the analyzed dataset.