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.

Vernal keratoconjunctivitis (VKC) is a chronic allergic ocular surface disease with seasonal recurrences and severe forms showing vision threatening complications. The purpose of the study is to understand the prevalence and diversity of biofilm-forming bacteria and antimicrobial resistance in VKC compared to healthy individuals (HC). For this, conjunctival swab samples were collected from VKC (n=26) and HC (n=23), of which culture positive samples were 77% and 78.26% respectively. The 16S rRNA gene sequencing revealed a significant increase in bacterial diversity in VKC compared to HC (p<0.05), identifying 16 and 9 bacterial species, respectively. Staphylococcus epidermidis emerged as the predominant bacterium in both groups, with relative abundances of 52.8% in HC and 30.2% in VKC (p<0.001). Biofilm formation was observed in 64.15% of bacterial species in VKC and 31% in HC (p<0.001). Scanning electron microscopy analysis confirmed temporal biofilm formation by Staphylococcus spp. in both groups. Minimum inhibitory concentration testing showed that biofilm forming Staphylococcus spp. from VKC exhibited multidrug resistance (>2 antibiotics) more frequently than those from HC. Additionally, Staphylococcus spp. in VKC demonstrated higher resistance to fluoroquinolones compared to HC. These findings indicate a significantly greater prevalence of biofilm-forming and antimicrobial resistant Staphylococcus bacteria in VKC Patients compared with HC.