Preparing a Dual-Species <i>In Vitro</i> Biofilm Model for Testing Antibiofilm Efficacy

All wounds are contaminated, and there is a risk of developing an infection. Furthermore, most wounds contain biofilm and are contaminated by two bacteria, termed dual-species, or more bacteria, termed polybacterial biofilms. New antibacterial and antibiofilm wound care products are constantly being developed to combat this problem. There is a need to develop more biorelevant and reproducible models to test the efficacy of these wound care products. We used an electrospun (ES) gelatin-glucose matrix (Gel-Gluc) as an artificial skin substrate for dual-species biofilm formation using wound pathogens <i>Staphylococcus aureus</i>, <i>Escherichia coli</i>, and <i>Pseudomonas aeruginosa</i>, combining them in pairs. When analyzing the biofilms, selective agars were used to differentiate various bacteria from one another while counting. The developed method supported the growth of dual-species biofilm that contained both bacteria up to 10⁸ CFU/Gel-Gluc after 24 h. Over 48 h, there was a decrease in the number of <i>S. aureus</i> in the biofilms. Confocal microscopy imaging allowed monitoring of the location of bacteria in the Gel-Gluc and proved that different species were located closely together. ES polycaprolactone (PCL) fibrous wound dressings containing chloramphenicol (CAM) or ciprofloxacin (CIP), or their pristine analogs, were used to test the model. Both ES fibrous wound dressings were effective in preventing dual-species biofilm formation. PCL-CIP fibrous dressing was also effective in treating biofilms. The efficacy of treatment of <i>E. coli</i> varied in different dual-species combinations of <i>E. coli</i>. The developed dual-species biofilm model on artificial skin (Gel-Gluc) supported the successful growth of different bacterial combinations and proved to be suitable for testing the efficacy of ES fibrous wound dressings in preventing and treating biofilms.