Antibiotic therapy against an established biofilm often fails without removal of the infected implant, despite the use of drugs which are highly active in standard in vitro susceptibility tests. In contrast to antibiotic therapy of many bacterial infections, standard in vitro susceptibility tests are not predictive of the therapeutic outcome. To study this complex phenomenon we developed a simple cellulose dialysis membrane model system to evaluate the penetration of antimicrobial agent namely vancomycin on mixed culture biofilm formed by Staphylococcus epidermidis RP62A and Listeria monocytogenes EDG-e. Biofilm formation was optimized at 30 C on cellulose membrane with tryptic soy broth (TSB) supplemented with 1% glucose and 0.6% yeast extract as the culture medium and cultivation duration of 48 h so as to mimic an infected biomaterial or food processing environment. Biofilm development on the membranes was confirmed by scanning election microscopy and penetration of antibacterial agent was demonstrated by a simple diffusion cell bioassay of antibiotic-affected cells of the biofilm. The results showed that planktonic mixed culture bacteria was rapidly killed by treatment with 0.1 ml of vancomycin and in contrast, failed to effectively kill the bacteria in mixed biofilms. Acquisition of resistance to antibiotics would represent a major therapeutic problem. Thus it is very important to test for antibiotic resistance in food-borne and clinical isolates and to reconsider critically the use of antibiotics
