In vitro analysis of antiseptic solution effects on <i>Staphylococcus aureus</i> biofilms on orthopedic implant materials

Abstract. 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.