Abstract

Introduction: Chronic wounds cannot heal due to impaired tissue regeneration, caused by pathogenic biofilm-infections that provoke chronic inflammation, hypoxia and skin degradation. Biofilm-infections are persistent given their tolerance to broad-spectrum antibiotics and their multispecies composition, as shown by clinical isolates from chronic wounds. Representative in vitro multispecies biofilm models are crucial for screening new anti-biofilm strategies with therapeutic potential. Objective: Establish a tridimensional multispecies biofilm model over artificial skin scaffolds, with broad-spectrum antibiotic tolerance. Methodology: Bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis) were inoculated over artificial skin scaffolds, and incubated in M9 media during 24 h at 30°C. Samples were analyzed by fluorescent lectins binding analysis (FLBA) and CLSM, CFU counting, and MALDI-TOF mass spectrometry. For antibiotic tolerance, biofilm-containing scaffolds were incubated with ciprofloxacin 100 µg/ml or gentamicin 200 µg/ml during 24 h at 30°C, and XTT-reduction assay was performed. For statistical analysis, data were analyzed by two-way ANOVA and post-hoc test. Results: Cultures containing the three bacterial species formed a multispecies biofilm over the scaffold fibers after 24 h. Attached bacteria were not released from the biofilm, neither by PBS washes nor gauze drying steps. All three species were present, and total bacterial load ranged within 105-106 CFU/g. Compared to planktonic bacteria, biofilm-containing scaffolds tolerated both ciprofloxacin and gentamicin. Conclusions: Under specific conditions, three bacterial species interact with the scaffold fibers forming a tridimensional biofilm, with high bacterial load and broad-spectrum antibiotics tolerance. This biofilm model can be used for in vitro screening of novel treatments for chronic wounds. Acknowledgements: Beca Conicyt Doctorado, Fondecyt 1161007, CORFO Línea-2 14IDL2-30154, Fondecyt 1160917