Development of a novel, highly quantitative in vivo model for the study of biofilm‐impaired cutaneous wound healing

Abstract

A growing body of evidence suggests that in addition to hypoxia, ischemia-reperfusion injury, and intrinsic host factors, bacterial biofilms represent a fourth major pillar in chronic wound pathogenesis. Given that most studies to date rely on in vitro or observational clinical data, our aim was to develop a novel, quantitative animal model enabling further investigation of the biofilm hypothesis in vivo. Dermal punch wounds were created in New Zealand rabbit ears, and used as uninfected controls, or inoculated with green fluorescent protein-labeled Staphylococcus aureus to form wounds with bacteria predominantly in the planktonic or biofilm phase. Epifluorescence and scanning electron microscopy revealed that S. aureus rapidly forms mature biofilm in wounds within 24 hours of inoculation, with persistence of biofilm viability over time seen through serial bacterial count measurement and laser scanning confocal imaging at different time points postwounding and inoculation. Inflammatory markers confirmed that the biofilm phenotype creates a characteristic, sustained, low-grade inflammatory response, and that over time biofilm impairs epithelial migration and granulation tissue in-growth, as shown histologically. We have established and validated a highly quantitative, reproducible in vivo biofilm model, while providing evidence that the biofilm phenotype specifically contributes to profound cutaneous wound healing impairment. Our model highlights the importance of bacterial biofilms in chronic wound pathogenesis, providing an in vivo platform for further inquiry into the basic biology of bacterial biofilm-host interaction and high-throughput testing of antibiofilm therapeutics.

Prevalence and antibiotic susceptibility of bacteria from acute and chronic wounds in Malaysian subjects

“…Virulence factors, including lipopolysaccharide (LPS), enterotoxin A, and ADP ribosylating enzymes excreted by P. aeruginosa have proven to be cytotoxic and have potent inhibitory effects on the proliferation of human granulocytes and macrophage progenitor cells during the wound healing process [36,37]. In vivo studies of mice and rabbits have further confirmed that S. aureus and P. aeruginosa evade host immunity and establish persistent infections via biofilm formation [38][39][40]. When compared to planktonic cells, bacteria in biofilm promote higher resistance towards antibiotics and other antimicrobial agents [6,41].…”

Chronic Wound Biofilm Model

“…Outcomes such as impairment of epithelialization, overabundance of granulation tissue, and a hyperinflammatory state are interesting. 8,26,[40][41][42][43] However, reported studies involve short-term infection disallowing prolonged interaction between polymicrobial pathogens and the host. In that respect, the rabbit ear model suffers from limitations comparable to those discussed for the rodent models.…”

Rabbit M1 and M2 macrophages can be induced by human recombinant GM‐CSF and M‐CSF

“…Moreover, owing to similarities of rabbit ear skin to that of human skin, New Zealand white rabbit ear models have been established for wound healing research. Rabbits are also used to determine inflammatory responses of wounds to bacterial biofilm infection 6, 7, 8 and diabetic neuroischemic wound healing 9.…”