In vitro interaction of chronic wound bacteria in biofilms.
To use in vitro biofilm models of wound bacterial isolates and compare the biofilms produced for different combinations of wound bacterial species. In vitro biofilms, generated by Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus oralis and Micrococcus luteus in microtitre plates and a constant depth film fermentor (CDFF), were studied. The tested isolates all originated from chronic venous leg ulcers. Biofilms of individual and dual combinations of these species were generated in microtitre plate wells at 37°C for 24-96 hours and also in the CDFF for 7 days. The extent of biofilm formation from these systems was then measured using crystal violet staining and/or total viable counts. All the chronic wound bacteria formed biofilms (both individually and in mixed culture) in these models. In mixed species microtitre plate biofilms, both P. aeruginosa and S. aureus appeared to antagonise biofilm formation by S. oralis and M. luteus, with P. aeruginosa completely inhibiting the growth of these organisms. Similar effects were evident in the CDFF model, when all four bacterial species were added simultaneously, with M. luteus being 'out-competed' by the other organisms present and occurring at numbers at the limits of detection; however, there was an apparent increase in the numbers of S. oralis compared with its single culture equivalent. The study highlighted differences in biofilm formation ability for the tested species in both closed and open model systems. Using dual species biofilms, distinct species antagonism was observed with apparent antagonism of pathogenic species over 'commensal' ones. Such a finding provides insight into possible bacterial interactions during development of 'non-healing' wound biofilms.