Antibiofilm activity of chitosan/epsilon-poly-L-lysine hydrogels in a porcine ex vivo skin wound polymicrobial biofilm model.

Abstract

As antibiotic resistance continues to increase globally, there is an urgency for novel, non-antibiotic approaches to control chronic drug-resistant infections, particularly those associated with polymicrobial biofilm formation in chronic wounds. Also needed are clinically relevant polymicrobial biofilm models that can be utilized to assess the efficacy of innovative therapeutics against mature biofilms. We successfully developed a highly reproducible porcine ex vivo skin wound polymicrobial biofilm model using clinical isolates of multidrug-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Candida albicans. This ex vivo biofilm model was then used to assess the antimicrobial and antibiofilm properties of an easily fabricated chitosan hydrogel incorporating the natural antimicrobial peptide epsilon-poly-L-lysine. Antimicrobial activity was evaluated against planktonic cultures in vitro and against mature biofilms ex vivo. The antibiofilm efficiency of the hydrogels was especially pronounced against Pseudomonas aeruginosa, whose counts were reduced by 99.98% after 2 hours in vitro and by 99.94% after treatment for 24 hours when applied to 24 hour ex vivo polymicrobial wound biofilms. The activity of the hydrogels was lower against Staphylococcus aureus and ineffective against Candida albicans. Gram, Hucker-Twort staining of paraffin sections revealed balanced polymicrobial communities in mature 48 hour untreated biofilms. Treatment of 48 or 72 hour biofilms for 2 or 3 days with hydrogels that were applied within 5 hours after inoculation resulted in an impressive 96% and 97% reduction in biofilm thickness compared to untreated biofilms, respectively (P < .001). Likewise, topical gel treatment for 24 hours reduced biofilm thickness by 84% and 70%, respectively, when applied to mature biofilms at 24 and 48 hours after inoculation (P < .001). Thus, this ex vivo wound biofilm model provides a useful means to assess the efficacy of novel treatments to prevent and eradicate polymicrobial biofilms consisting of multidrug-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Candida albicans.