Development of a Three-Species Biofilm Model on Ex Vivo Porcine Skin Tissues

F. Tabatabaei, N. Byrnes-Shaver, T. Conti
United States

Keywords: wound infection, biofilm, 3D ex vivo


Non-healing chronic wounds afflict 1-3 million people per year in the United States. Despite numerous claims by commercially available dressings and treatments regarding antimicrobial and antibiofilm effects, clinical evidence supporting their efficacy remains limited. Notably, the lack of suitable preclinical models that accurately replicate the complex multispecies biofilms and chronic wound microenvironment observed in clinical practice further hinders the validation of novels, active compounds, and dressings for wound management. In this study we aimed to develop a three-species biofilm model and evaluate its longevity. Porcine skin explants sourced from commercial vendors were punched to a standardized diameter of 10mm. Wounds of 3mm were created before sterilization via supercritical CO2. To enhance explant maintenance, tissues were positioned within well-plate inserts within a gel-like matrix containing 1% agarose, facilitating epidermal contact with air. Medium was added to the bottom well. The explants underwent infection with various concentrations and combinations of bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus mutans) were prepared in wound liquid medium. The groups included a non-infected control group, those infected with S. Aureus, P. a, 1:1 ratios of S. a and P. a, 10:1 ratios of S. a and P. a, as well as those infected with S. mutans, followed by the addition of 1:1 or 10:1 ratios of S. a and P. a after 2 hours. Bacterial counts and histological staining were conducted at multiple time points to assess biofilm formation. The average total bacteria recovered from the explants after day 3 was 5.4-8.5 log CFU/ml (n = 3). Groups containing S. mutans were significantly more effective than the other combination in keeping the same survival rate for both bacteria. Histological staining of the model revealed microbial growth and biofilm formation on the surface of tissues. Our findings indicated that initiating biofilm formation with S. mutans proved conducive. Given P. aeruginosa's rapid growth, maintaining its inoculation at the lowest feasible concentration becomes crucial for sustaining S. aureus within the biofilm. In future investigations, we aim to include Candida albicans alongside S. aureus and P. aeruginosa to construct a more representative polymicrobial biofilm model of wounds.