Using simple and complex wound in vitro biofilm models for testing of novel therapeutics

Abstract

Chronic wound infections, driven by resilient polymicrobial biofilms and escalating antimicrobial resistance (AMR), present a critical therapeutic challenge that requires alternative interventions. This thesis investigates innovative strategies to combat biofilm-associated infections, primarily focusing on cold atmospheric plasma (CAP) and repurposed compounds as alternatives to conventional therapies. Using in vitro models mimicking chronic wound microenvironments, including mono- and triadic-species biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, the study evaluates three key themes relating to antimicrobial testing.

CAP monotherapy (chapter 3) demonstrated time- and strain-dependent antimicrobial efficacy, with ≥3-log10 reductions in biofilm viability linked to hydrogen peroxide (H₂O₂) and reactive species generation. S. aureus exhibited strain-specific tolerance, while CAP disrupted polymicrobial community dynamics and induced oxidative damage visible via scanning electron microscopy (SEM). Dual therapies (chapter 4) combining CAP with repurposed agents (e.g., KHS 101 hydrochloride) or antiseptics H₂O₂, and povidone-iodine (PVP-I) overcame biofilm tolerance, achieving synergistic eradication (>3-log10 CFE reductions) in recalcitrant S. aureus-containing models with the novel dual intervention: KHS+CAP emerged as a lead strategy, destabilising biofilm matrices and enhancing oxidative stress. H₂O₂-antibiotic combinations (chapter 5) showed that H₂O₂ potentiated flucloxacillin and gentamicin against early-stage S. aureus biofilms, particularly in strain Newman. Synergy depended on treatment sequence, suggesting that such an intervention, if used clinically, would require careful consideration.

Key findings from this thesis have shown that such alternative therapies could be utilised for biofilm treatment in chronic wound management. In particular, the broad-spectrum activity of CAP and synergy with a repurposed agent (KHS) offers a potential replacement for antibiotic interventions during the care of such patients, which was demonstrated here using in vitro model systems. Alternatively, H₂O₂-mediated antibiotic re-sensitisation offers another pathway to mitigate AMR. Ultimately, multi-modal approaches rather than single-agent treatments, particularly those that “break” antimicrobial tolerance provide the most promising alternatives.