In Vitro Polymicrobial Wound Biofilm Model Systems for Assessing Antimicrobial Treatment Efficacy and Host-pathogen Interactions

Abstract

Background: Chronic wounds such as diabetic foot ulcers often become infected with polymicrobial biofilms which can interfere with sufficient healing and repair. Alleviating biofilm growth is often the primary aim of clinicians. To do this, antiseptic wound washes can be used. However, there remains a need for pre-clinical studies investigating the efficacy of such topical treatments against complex polymicrobial biofilms, alongside consideration of how the host responds. Materials: A complex polymicrobial biofilm model was created under different environmental conditions utilising microorganisms commonly isolated from infected wound milieu. Biofilm composition was assessed using genus and species-specific primers, and ultrastructure visualised with scanning electron microscopy. These polymicrobial biofilms were treated with three conventional anti-biofilm wound washes, chlorhexidine (CHX), povidone-iodine (PVP-I) and hydrogen peroxide (H2O2), and efficacy against the microorganisms assessed using live/dead qPCR. Next, treated and untreated biofilms were exposed to human skin epidermis in a unique co-culture model system. A detailed transcriptional and proteomic signature of the epidermis was gathered following biofilm stimulation using gene expression profiling and novel Olink technology. Results: Compositional analysis of the complex polymicrobial revealed colonization of aerobic and anaerobic bacteria with the fungus, Candida albicans. Biofilm treatment efficacy varied amongst the antiseptics, as H2O2 was found to be the most effective treatment modality. In the co-culture model, at the transcriptional level the polymicrobial biofilm stimulated an inflammatory response in the tissue, with elevated expression of genes such as IL6, IL8 and colony stimulating factors 1, 2 and 3. All biofilm treatments reduced expression of inflammatory markers back to levels comparable with untreated tissue controls. Olink technology revealed a unique proteomic response in the tissue following stimulation with untreated and CHX-treated biofilms. Conclusions: Results from this study highlights that antiseptic treatment choice to combat wound-related polymicrobial biofilms can influence the host response. To the authors knowledge, this study is the first to attempt to decipher the host skin response to complex polymicrobial biofilms in vitro