Microscopic and physiologic evidence for biofilm-associated wound colonization in vivo.

A biofilm is a collection of microbial cells that are attached to a surface and embedded in a self-produced extrapolymeric substance. The understanding of the biofilm phenotype is important in the understanding of bacteria in vitro but it has been difficult to translate biofilm science to the clinical setting. More recently, preliminary criteria for defining biofilm associated diseases have been proposed and the purpose of this study was to create a biofilm-associated wound model based on these criteria. Using a porcine model, partial thickness wounds were inoculated with a wound isolate Staphylococcus aureus strain. Wounds were then treated with either one of two topical antimicrobial agents (mupriocin cream or triple antibiotic ointment) within 15 minutes to represent planktonic bacteria or 48 hours after initial inoculation to represent biofilm-associated wound infection. Using light microscopy, scanning electron microscopy and epifluorescence microscopy, we were able to observe biofilm-like structures in wounds after 48 hours of inoculation and occlusion. The in vivo antimicrobial assay was used to demonstrate that both mupirocin cream and the triple antibiotic ointment were effective in reducing planktonic S. aureus but had reduced efficacy against biofilm-embedded S. aureus. Our results demonstrated that S. aureus form firmly attached microcolonies and colonies of bacteria encased in an extracellular matrix on the surface of the wounds. These biofilm-like communities also demonstrated increased antimicrobial resistance when compared with their planktonic phenotype in vivo. The structural and physiological results support the hypothesis that bacterial biofilms play a role in wound colonization and infection.