Comparative Analysis of Single-Species and Polybacterial Wound Biofilms Using a Quantitative, In Vivo, Rabbit Ear Model

Abstract

IntroductionThe recent literature suggests that chronic wound biofilms often consist of multiple bacterial species. However, without appropriate in vivo, polybacterial biofilm models, our understanding of these complex infections remains limited. We evaluate and compare the effect of single- and mixed-species biofilm infections on host wound healing dynamics using a quantitative, in vivo, rabbit ear model.MethodsSix-mm dermal punch wounds in New Zealand rabbit ears were inoculated with Staphylococcus aureus strain UAMS-1, Pseudomonas aeruginosa strain PAO1, or both, totaling 10∧6 colony-forming units/wound. Bacterial proliferation and maintenance in vivo were done using procedures from our previously published model. Wounds were harvested for histological measurement of wound healing, viable bacterial counts using selective media, or inflammatory cytokine (IL-1β, TNF-α) expression via quantitative reverse-transcription PCR. Biofilm structure was studied using scanning electron microscopy (SEM). For comparison, biofilm deficient mutant UAMS-929 replaced strain UAMS-1 in some mixed-species infections.ResultsBacterial counts verified the presence of both strains UAMS-1 and PAO1 in polybacterial wounds. Over time, strain PAO1 became predominant (p<0.001). SEM showed colocalization of both species within an extracellular matrix at multiple time-points. Compared to each monospecies infection, polybacterial biofilms impaired all wound healing parameters (p<0.01), and increased expression of IL-1β and TNF-α (p<0.05). In contrast, mixed-species infections using biofilm-deficient mutant UAMS-929 instead of wild-type strain UAMS-1 showed less wound impairment (p<0.01) with decreased host cytokine expression (p<0.01), despite a bacterial burden and distribution comparable to that of mixed-wild-type wounds.ConclusionsThis study reveals that mixed-species biofilms have a greater impact on wound healing dynamics than their monospecies counterparts. The increased virulence of polybacterial biofilm appears dependent on the combined pathogenicity of each species, verified using a mutant strain. These data suggest that individual bacterial species can interact synergistically within a single biofilm structure.

Biofilm Models of Polymicrobial Infection

“…To maintain a 'biofilm-only' phenotype, antibiotic ointment was administered and antimicrobial absorbent dressings were applied to mature wounds to eliminate planktonic bacteria. The dual speciesinfected wounds demonstrated impaired healing and increased cytokine expression in comparison to monospecies infected wounds; however the investigators did note that P. aeruginosa tended to dominate the infection [117].…”

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.…”