Establishment of a multi-species biofilm model to evaluate chlorhexidine efficacy

Background: Chronic infections, for example, diabetic foot ulcers, have a large impact in terms of patient morbidity and mortality. These wounds are characterized by complex polymicrobial communities of bacteria, which may include a number of difficult-to-eradicate multidrug-resistant pathogens. Aim: To establish a multi-species biofilm model to test the efficacy of chlorhexidine and chlorhexidine-containing formulas in eradication of polymicrobial biofilms. Methods: A Centers for Disease Control and Prevention bioreactor was used to establish a multi-species biofilm incorporating Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis with equal numbers of each pathogen. This model was used to test the effectiveness of chlorhexidine at controlling the pre-formed biofilm. Findings: Chlorhexidine digluconate (CHD) was added to the bioreactor at a range of concentrations. K. pneumoniae and P. aeruginosa survived within multi-species biofilms, up to and including 4% CHD, whereas S. aureus was reduced to below the level of detection at 1%. Wiping the biofilm-containing coupons from the bioreactor with chlorhexidine-containing medical wipes resulted in >3 to <4log₁₀ reduction after 24h, for all species. When the coupons were embedded in a simulated wound bed, formed in an agar plate, CHD-containing medical dressings completely eliminated S. aureus (>8log₁₀ reduction), but had minimal effect (<3log₁₀) against the other species tested. Conclusion: The study demonstrates that the effectiveness of chlorhexidine may be limited in settings where it is required to act on multi-species biofilms. This may compromise the ability of chlorhexidine to control the infection and spread of these pathogens.