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Treatment of Pseudomonas aeruginosa Biofilm-Infected Wounds with Clinical Wound Care Strategies: A Quantitative Study Using an In Vivo Rabbit Ear Model
Akhil K. Seth, MD1, Anandev N. Gurjala, MD, MS1, Matthew R. Geringer, BS1, Seok K. Hong, PhD1, Robert D. Galiano, MD1, Kai P. Leung, PhD2, Thomas A. Mustoe, MD1.
1Northwestern University, Feinberg School of Medicine, Chicago, IL, USA, 2US Army Dental and Trauma Research, Institute of Surgical Research, Microbiology Branch, San Antonio, TX, USA.

Bacterial biofilms, most prominently Pseudomonas aeruginosa (PAO1), are increasingly being recognized as a major detriment to chronic wound healing. The efficacy of traditional wound care methods against biofilm has never been studied. We evaluate the effects of these clinical strategies on biofilm-infected wounds in a quantitative, reproducible in vivo model.
Six mm dermal punch wounds in New Zealand rabbit ears were inoculated 3 days post-wounding with 10^6 colony-forming units (cfu) of PAO1, or left uninfected as controls. Planktonic bacteria were killed by topical antibiotic, creating an in vivo biofilm infection on day 6. PAO1 wounds acted as treatment controls or underwent 1 of 5 treatments (n=18-24/group): every other day (QOD) sharp debridement (1), QOD lavage (2), QOD topical silvadene (3), QOD lavage and silvadene (4), one-time debridement with daily lavage and silvadene (5). Wounds were harvested on day 12 with measurement of histological wound healing parameters (epithelial gap (EG), granulation tissue gap (GG), total granulation tissue area (TGA)) or viable bacterial counts. Biofilm structure was studied using scanning electron microscopy (SEM).
Uninfected control wounds healed better than PAO1 biofilm-infected wounds (EG: 0.29 mm v 4.29 mm, GG: 0.38 mm v 5.05 mm, TGA: 332.5 mm^2 v 49.3 mm^2; p<0.01). Groups 1, 2 and 3 did not demonstrate a difference in EG, GG, TGA or viable bacterial counts compared to untreated PAO1 wounds. SEM before and after treatment showed only temporary disruption of biofilm structure, reestablishing within 24 hours. Groups 4 and 5 had improved healing (EG: 3.46 mm and 2.89 mm, GG: 3.95 mm and 3.35 mm, TGA: 109.0 mm^2 and 155.9 mm^2, respectively; p<0.05) and decreased viable bacterial counts (1.81 x 10^5 cfu/ml and 5.84 x 10^3 cfu/ml, respectively) when compared to untreated PAO1 wounds (2.29x10^7 cfu/ml) (p<0.05).

Pseudomonal biofilm significantly impairs cutaneous wound healing, demonstrated quantitatively using a reproducible in vivo biofilm model. Despite popular clinical practice, traditional wound care methods cannot adequately restore biofilm-infected wounds to a healing phenotype when used alone or infrequently. The pervasiveness and durability of biofilm requires strict and aggressive multimodal therapy directed at reducing bacterial burden, thus helping to eliminate the chronicity of non-healing wounds. Our novel and rigorous study validates critical principles applicable to all clinical wound care.

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