1Chronic diabetic foot ulcers are frequently colonised and infected by 2 polymicrobial biofilms that ultimately prevents healing. In this study, we aimed 3 to create a novel in vitro inter-kingdom wound biofilm model on complex 4 hydrogel-based cellulose substrates to test commonly used topical wound 5 treatments. Inter-kingdom triadic biofilms composed of Candida albicans, 6Pseudomonas aeruginosa, and Staphylococcus aureus were shown to be 7 quantitatively greater in this model compared to a simple substrate when 8 assessed by conventional culture, metabolic dye and live dead qPCR. These 9 biofilms were both structurally complex and compositionally dynamic in 10 response to topical therapy, so when treated with either chlorhexidine or 11 povidone iodine principal component analysis revealed that the 3-D cellulose 12 model was minimally impacted compared to the simple substrate model. This 13 study highlights the importance of biofilm substrate and inclusion of relevant 14 polymicrobial and inter-kingdom components, as these impact penetration and 15 efficacy of topical antiseptics.

Diabetic foot ulcer treatment currently focuses on targeting bacterial biofilms, while dismissing fungi. To investigate this, we used an biofilm model containing bacteria and fungi, reflective of the wound environment, to test the impact of antimicrobials. Here we showed that while monotreatment approaches influenced biofilm composition, this had no discernible effect on overall quantity. Only by combining bacterium- and fungus-specific antibiotics were we able to decrease the biofilm bioburden, irrespective of composition.

Calcium sulfate (CS) has been used clinically as a bone or void filling biomaterial, and due to its resorptive properties have provided the prospect for its use as a release mechanism for local antibiotics to control biofilms. Here, we aimed to test CS beads loaded with three antifungal drugs against planktonic and sessile fungal species to assess whether these antifungal beads could be harnessed to provide consistent release of antifungals at biofilm inhibitive doses. A panel of different fungal species (n=15) were selected for planktonic broth microdilution testing with fluconazole (FLZ), amphotericin B (AMB) and caspofungin (CSP). After establishing planktonic inhibition, antifungal CS beads were introduced to fungal biofilms (n=5) to assess biofilm formation and cell viability through a combination of standard quantitative and qualitative biofilm assays. Inoculation of a hydrogel substrate, packed with antifungal CS beads, was also used to assess diffusion through a semi-dry material, to mimic active infection in-vivo. In general, antifungals released from CS loaded beads were all effective at inhibiting the pathogenic fungi over 7-days within standard MIC ranges for these fungi. We observed a significant reduction of pre-grown fungal biofilms across key fungal pathogens following treatment, with visually observable changes in cell morphology and biofilm coverage provided by scanning electron microscopy. Assessment of biofilm inhibition also revealed reductions in total and viable cells across all organisms tested. These data show that antifungal loaded CS beads produce a sustained antimicrobial effect, which inhibits and kills clinically relevant fungal species in-vitro as planktonic and biofilm cells.

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