Toward an improved in vitro model of prosthetic joint infection for Staphylococcus aureus biofilm characterization.

Biofilm formation on orthopedic implants is often implicated in chronic prosthetic and joint infections (PJI) that are complex to manage. To date, no current bacterial in vitro model can fully simulate the PJI environment leading to a lack of knowledge to develop diagnosis tool and adapted treatment. Our project aims to set up an innovative in vitro model to characterize Staphylococcus aureus clinical strains biofilms in a PJI context, focusing on several parameters: culture media, incubation time, atmospheric conditions and support for biofilm growth. Biofilm formation was evaluated in various culture media, by counting both planktonic and adherent bacteria (CFU) and quantifying biofilm biomass using crystal violet staining. A mature biofilm was obtained after 72 h of incubation with a similar proportion of planktonic and adherent bacteria whereas a variable dispersion was observed at 96 h. Comparing two different oxygen concentrations (Hypoxia 2.5 % like in bone site vs Anoxia) revealed that a slight variation had a strong impact on biofilm formation, underlining the fact that the physiological conditions are highly necessary to set a mimetic model. A medium has therefore been developed, the modified Bone-Like Environment (BLE+) allowing a consistent biofilm growth. When studying bacterial adhesion, planktonic bacteria can gather and form aggregates that are distinct from mature biofilms. To avoid this phenomenon, a suspended pegs was used. By holding the pegs in the medium, we specifically drove active bacterial adhesion related to biofilm formation, eliminating interference from sedimented aggregates. Moreover, to limit the interaction between planktonic bacteria and biofilm over the 72 h, a medium renewal was applied at 8 h of incubation with a low impact on biofilm biomass. This method allowed the observation of differences between the USA300 (MRSA) and SH1000 (MSSA) strains: the MSSA showed more adherent bacteria and bigger aggregates than the MRSA strain. In conclusion, the parameters for an in vitro biofilm model simulating PJI context have been validated. These parameters include 2.5 % dioxygen, BLE + supplementation, and 72-h incubation on suspended titanium pegs with a renewal media after a primo bacteria adhesion of 8 h.