Quantitative comparison and analysis of species-specific wound biofilm virulence using an in vivo, rabbit-ear model

Although bacterial biofilm is recognized as an important contributor to chronic wound pathogenesis, differences in biofilm virulence between species have never been studied in vivo.

Dermal punch wounds in New Zealand white rabbit ears were inoculated with Klebsiella pneumoniae, Staphylococcus aureus, or Pseudomonas aeruginosa, or left uninfected as controls. In vivo biofilm was established and maintained using procedures from our previously published wound biofilm model. Virulence was assessed by measurement of histologic wound healing and host inflammatory mediators. Scanning electron microscopy (SEM) and bacterial counts verified biofilm viability. Extracellular polymeric substance (EPS)-deficient P aeruginosa was used for comparison.

SEM confirmed the presence of wound biofilm for each species. P aeruginosa biofilm-infected wounds showed significantly more healing impairment than uninfected, K pneumoniae, and S aureus (p < 0.05), while also triggering the largest host inflammatory response (p < 0.05). Extracellular polymeric substance-deficient P aeruginosa demonstrated a reduced impact on the same quantitative endpoints relative to its wild-type strain (p < 0.05).

Our novel analysis demonstrates that individual bacterial species possess distinct levels of biofilm virulence. Biofilm EPS may represent an integral part of their distinct pathogenicity. Rigorous examination of species-dependent differences in biofilm virulence is critical to developing specific therapeutics, while lending insight to the interactions within clinically relevant, polybacterial biofilms.