Biofilm sensitization to break Staphylococcus aureus tolerance to cold atmospheric plasma therapy

Introduction. Biofilm-associated infections present a major therapeutic challenge due to their intrinsic tolerance to conventional antimicrobials. Cold atmospheric plasma (CAP) has shown promise as a non-antibiotic antimicrobial modality; however, some bacteria including Staphylococcus aureus can exhibit tolerance to plasma exposure. Gap Statement. Strategies that sensitize CAP-tolerant biofilms to plasma treatment may improve CAP efficacy, but suitable adjunctive compounds and mechanisms remain poorly defined. Aim. This study aimed to determine whether repurposed bioactive compounds could enhance CAP activity against S. aureus biofilms. Methodology. Selected compounds from the Tocriscreen™ bioactive compound library were initially screened, followed by treatment of S. aureus biofilms with KHS101 ±CAP therapy. Biofilm viability was quantified using live/dead qPCR. To probe mechanisms of sensitization, biofilms were exposed to H 2 O 2 at concentrations equivalent to those generated by CAP, either alone, or in combination with KHS101 or conventional antibiotics. Various microscopy techniques were used to visualize the cellular impacts of KHS101, while metabolic activity and cell viability of dual therapies were determined using AlamarBlue ® assay and plate count assays, respectively. Results. Short-term KHS101 treatment alone displayed modest antibiofilm activity at concentrations that inhibited planktonic growth. However, pre-treatment with KHS101 followed by CAP therapy resulted in significant reductions in viable populations in S. aureus -containing biofilms. Microscopy revealed structural perturbations consistent with cellular stress following KHS101 exposure, but also showed intact cellular ultrastructure. Mechanistic probing demonstrated that equivalent concentrations of H 2 O 2 with KHS101 were insufficient to reproduce the enhanced efficacy observed with CAP. In contrast, H 2 O 2 enhanced flucloxacillin activity in a strain-dependent manner, sensitizing S. aureus biofilms to otherwise sub-lethal concentrations of antibiotic. Conclusion. These findings demonstrate that tolerance of S. aureus biofilms to CAP can be overcome through dual-therapy strategies. Treatment with the repurposed compound KHS101 was associated with enhancement of CAP efficacy via an unknown mechanism. However, the inability of H 2 O 2 to reproduce this effect highlights the importance of additional plasma-derived reactive species in mediating this dual-action killing. Together, these findings position biofilm sensitization as a central concept emerging from this study, whereby a non-lethal adjunct can lower the threshold for CAP-mediated killing without acting primarily as a direct antimicrobial.