CLONALITY OF STAPHYLOCOCCUS AUREUS IN FRACTURE-RELATED AND PROSTHETIC JOINT INFECTIONS AND IMPLICATIONS FOR THERAPEUTIC PHAGE DEVELOPMENT

Introduction & Aim Biofilm-associated musculoskeletal infections, such as fracture-related infection (FRI) and prosthetic joint infection (PJI), are commonly caused by Staphylococcus aureus and are difficult to eradicate with conventional antibiotics. Precision approaches, including bacteriophage therapy, depend on whether infections are driven by a single bacterial clone or comprise multiple coexisting strains. We aimed to determine the clonality of S. aureus in FRIs and PJIs and to assess intra-infection variation in wall teichoic acid (WTA) glycosylation genes critical for bacteriophage attachment to S. aureus. Methods Clinical S. aureus isolates were obtained intraoperatively from patients with confirmed FRIs and PJIs. For each infection, a minimum of eight morphologically distinct or randomly selected colonies were submitted for whole-genome sequencing. Genomic variants were identified by alignment to a reference genome to assess intra-infection relatedness. Clonality was evaluated by comparing shared variants across colonies within each infection and assessing overlap between individual colony and merged variant profiles. The presence and sequence variation of WTA glycosyltransferase genes (tarS, tarM, tarP) were analyzed to assess potential intra-infection heterogeneity in WTA glycosylation capacity. Results Across all FRIs and PJIs, isolates from each patient demonstrated a high degree of genetic relatedness consistent with clonal infections. FRI isolates demonstrated >92% shared whole-genome variants among colonies and >98% overlap between individual colony and merged variant profiles. The majority of isolates contained tarS, fewer harbored tarM, and tarP was absent. When present, WTA glycosyltransferase genes showed minimal to no sequence variation within individual infections, indicating uniform glycosylation potential across bacterial populations despite differing clinical contexts and infection chronicity. Conclusions Both S. aureus FRIs and PJIs are predominantly clonal infections with limited intra-infection genomic and WTA glycosylation variability. These findings support the use of a single representative clinical isolate for microbiological and phage therapeutic testing. Understanding infection clonality provides a critical foundation for precision anti-biofilm strategies, including bacteriophage therapy, and informs the rational development of targeted adjunctive treatments for musculoskeletal infections.