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 H2O2 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 H2O2 with KHS101 were insufficient to reproduce the enhanced efficacy observed with CAP. In contrast, H2O2 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 H2O2 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.

Purpose of Review The management of chronic wounds, a debilitating condition, presents a considerable challenge to healthcare professionals and a significant burden on services. When these wounds are exposed to the external environment, they are susceptible to microbial infection, which further complicates their management and worsens clinical outcomes. Recent Findings Bacteria typically exist in wounds as part of a biofilm, which is often polymicrobial in nature, alongside bacteria and fungi that are described as being more virulent and tolerant towards antimicrobials and antiseptics. Despite advancing knowledge in polymicrobial biofilm wound infections with respect to bacteria, the role of fungi is largely ignored, and their influence in chronicity and clinical management is not fully appreciated or understood. Summary The purpose of this review is to explore the significance of fungi within chronic wound environments and, in doing so, understand the importance of interkingdom interactions in wound management.

The global clinical and socioeconomic impact of chronic wounds is substantial. The main difficulty that clinicians face during the treatment of chronic wounds is the risk of infection at the wound site. Infected wounds arise from an accumulation of microbial aggregates in the wound bed, leading to the formation of polymicrobial biofilms that can be largely resistant to antibiotic therapy. Therefore, it is essential for studies to identify novel therapeutics to alleviate biofilm infections. One innovative technique is the use of cold atmospheric plasma (CAP) which has been shown to possess promising antimicrobial and immunomodulatory properties. Here, different clinically relevant biofilm models will be treated with cold atmospheric plasma to assess its efficacy and killing effects. Biofilm viability was assessed using live dead qPCR, and morphological changes associated with CAP evaluated using scanning electron microscopy (SEM). Results indicated that CAP was effective against Candida albicans and Pseudomonas aeruginosa, both as mono-species biofilms and when grown in a triadic model system. CAP also significantly reduced viability in the nosocomial pathogen, Candida auris. Staphylococcus aureus Newman exhibited a level of tolerance to CAP therapy, both when grown alone or in the triadic model when grown alongside C. albicans and P. aeruginosa. However, this degree of tolerance exhibited by S. aureus was strain dependent. At a microscopic level, biofilm treatment led to subtle changes in morphology in the susceptible biofilms, with evidence of cellular deflation and shrinkage. Taken together, these results indicate a promising application of direct CAP therapy in combatting wound and skin-related biofilm infections, although biofilm composition may affect the treatment efficacy.