There is growing evidence that bacteria form biofilm aggregates in synovial fluid (SF) and surface-attached biofilms on prostheses. If we want to study these biofilms in the context of prosthetic joint infections (PJI) we need better in vitro models. We also need better tools to detect and isolate relevant pathogens from PJI samples as well as novel approaches to better predict clinical success of an antimicrobial treatment. Our preliminary data show that it is possible to develop a synthetic synovial fluid (SSF) model that will support formation of biofilm aggregates by PJI pathogens and we have also shown that biofilm-based susceptibility testing is possible. In the present proposal we aim to build upon these promising preliminary data to obtain a better understanding of PJI biofilms and to translate these findings to innovative and clinically applicable approaches for diagnosis of PJI and susceptibility testing of the pathogens involved. To this end we propose the following specific objectives: -Development and validation of an in vitro SSF biofilm model that will increase our fundamental understanding of PJI biofilms and can be applied in clinical practice; -Development of SSF-based isolation and susceptibility testing protocols and comparing their performance to the current state-of-the-art in clinical practice; and -Identification of the most cost-efficient workflow for sampling and diagnosing PJI in the context of a clinical microbiology lab.