Validation of an innovative tissue-cage/beads rabbit model for ex vivo determination of minimum levels of Staphylococcus aureus biofilm eradication.

To offset limitations of current methods of antibiofilm activity determination, including in vitro technical variations and the limited number of conditions assessable in animals, we describe a rabbit tissue-cage model allowing ex vivo screening of antimicrobials against in vivo-formed S. aureus biofilm. Cages containing six beads of polyethylene, titanium or steel served as support for S. aureus SH1000 biofilm formation. In vitro validation steps included: (i) quantification of bead-adherent bacteria after 24 h incubation in bacterial suspension, measured at 4.7 (95%CI, 4.5-4.9) log10 CFU/bead, with no difference between biomaterials, allowing the selection of polyethylene for animal experiments due to its lighter weight; and (ii) minimum concentration eradicating 90% of biofilm-embedded bacteria (MBEC90) determination by bead incubation in increasing concentrations of vancomycin, daptomycin and rifampicin, which were not significantly different compared to the reference method (MBECAssay®). In vivo validation included: (i) subcutaneous implantation of six cages per rabbit; (ii) infection with a range of different inocula (500/1000/2000 CFU/cage) before harvesting on day 7, 14 or 28 for quantification of bead-adherent bacteria. The 500 CFU/cage inoculum with a 14-day harvesting time provided the best balance between reproducibility and infection persistence and was therefore selected for ex vivo MBEC90 determination against in vivo-formed biofilms. Vancomycin, daptomycin, and rifampin MBEC90 values were 1.0, 1.5, and < 0.016 mg/L, respectively, and were significantly lower than those obtained against in vitro-formed biofilms. Compared with existing in vitro and in vivo studies, this innovative model of ex vivo determination of MBEC90 on in vivo-formed biofilm is likely to be more predictive of the in vivo anti-staphylococcal antibiofilm activity and requires a limited number of animals.