Abstract

Introduction: Chronic wounds are those that cannot heal due to impairment of the tissue regeneration process. This medical problem affects 6,7million people and costs over 50billion dollars for the US health system. In our current knowledge, these wounds fail to heal because of the persistent infection caused by a multispecies pathogenic biofilm that drives chronic inflammation, evades host’s immune response, induces hypoxia, causes loss of the skin barrier function and tissue degradation by enzymes. In several studies, strains of Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis bacteria have been found on clinical isolates from different wounds such as diabetic foot and pressure ulcers. In order to evaluate novel antimicrobial strategies, investigation calls for an in vitro robust model of biofilm formation that resembles the diversity and antibiotic tolerance that biofilms exhibit in chronic wounds. Objective: Characterize the complex combination of Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis forming biofilm over a scaffold, using different staining techniques. Materials and methods: Bacteria were seeded at equal proportions over an artificial skin scaffold, and allowed to form biofilm in minimal culture media, during 24 hours or 7 days. The use of fluorescent conjugated lectins, inmunofluorescence, and lipophilic cell tracers was evaluated as a staining technique for pathogen visualization. Results: Combination of the three pathogens form a complex biofilm over the scaffold fibers, with higher biofilm formation capacity and different structural features compared to the respective monospecies biofilms. Conjugated lectins stained all bacteria present in the multispecies biofilm, while cell tracers allowed to distinguish each pathogen and track them until the 7 days of biofilm formation process. Conclusions: Combination of these three pathogens can form a complex biofilm over an artificial skin scaffold, which is more robust than the respective monospecies biofilms, and it is possible to visualize each pathogen using cell tracers.