In vitro analysis of antiseptic solution effects on Staphylococcus aureus biofilms on orthopedic implant materials

Implant-related biofilm infections remain a major challenge in orthopedic surgery. Prior in vitro comparisons have focused on arthroplasty surfaces using static models; fracture fixation implants remain unstudied. We compared six antiseptic solutions against mature S. aureus biofilms on stainless steel, the most common fracture fixation metal, using a continuous-flow CDC biofilm reactor, more closely approximating hydrodynamic surgical conditions than static systems. Mature biofilms were created on stainless steel coupons using a continuous-flow reactor for 72 h, then washed for 3 min with one of six irrigation solutions: normal saline, 10 % PI (povidone-iodine), 0.35 % PI, a 10 % PI $+$ 3 % hydrogen peroxide mixture, hypochlorous acid, or 0.05 % chlorhexidine gluconate. Colony-forming units (CFUs) were quantified following standardized sonication and plating, and mixed-effects negative binomial regression was used to estimate treatment effects. All antiseptic solutions produced statistically significant reductions in biofilm-associated CFUs relative to untreated controls. Ten percent PI demonstrated the greatest reduction (3.46- ${\log }_{10}$ ), with 0.35 % PI producing a similar 3.42- ${\log }_{10}$ reduction. The PI-hydrogen peroxide mixture achieved a 3.24- ${\log }_{10}$ reduction without evidence of synergistic activity. Hypochlorous acid and chlorhexidine produced 2.95- ${\log }_{10}$ and 2.46- ${\log }_{10}$ reductions, respectively, while saline demonstrated minimal effect. These findings indicate that PI solutions, even at dilute concentrations, are highly effective at disrupting established biofilms on stainless steel. As an in vitro single-organism model, these findings establish a comparative efficacy baseline on a fracture-fixation-relevant surface but require validation in multispecies, multi-material, and mechanically realistic irrigation systems before clinical translation.