Development of a tri-species wound model for studying fungal-bacterial interactions and antimicrobial therapies

Chronic wounds are increasing in numbers and biofilm-producing bacteria are highly prevalent in these wounds and often create resilient polymicrobial infections. Moreover, estimates suggest that up to 23 % of wounds contain fungi, particularly Candida albicans. Currently, inter-kingdom chronic wound models are scarce; thus, this study presents one of the few in vitro models that incorporate both bacterial and fungal species in a wound-relevant environment, addressing a critical gap in current biofilm research. The newly developed model contained the commonly isolated wound bacteria Pseudomonas aeruginosa and Staphylococcus aureus, and the fungus Candida albicans. Inter-species interactions were investigated through selective plate counting and pH and oxygen measurements, as well as confocal microscopy. Investigations were carried out before and after exposure to commonly used clinical antimicrobial treatments, including silver-infused bandages. When grown in a tri-species consortium, P. aeruginosa and S. aureus exhibited a higher tolerance towards silver-infused bandages than when they were grown individually. This suggests that C. albicans plays a protective role for the bacteria. In addition, the treatment also caused a shift in species ratios, moving from a P. aeruginosa-dominated consortium to a S. aureus-dominated consortium. Moreover, confocal microscopy revealed a change in biofilm architecture when comparing single-species models to tri-species models. Finally, we observed that silver-infused bandages increased the pH in the tri-species model as well as partially restoring the oxygenation within the wound model. In conclusion, our novel model exemplifies how inter-kingdom interactions in fungal-bacterial infections can complicate both the microenvironment and treatment efficacy.