Abstract Total knee arthroplasty (TKA) is the largest major surgical procedure by volume for Medicare, and infection is the largest reason for TKA revision. Irrigation and debridement (I&D) with long-term antibiotics is the preferred method to manage periprosthetic joint infection (PJI; infected TKA). I&D fails in approximately 60% of cases. The high failure rate of I&D is a result of the formation of biofilm on the implant and the subsequent high tolerance of biofilm to antibiotics. There is a large gap in knowledge in how biofilm develops antibiotic tolerance, how it is regulated, and there are no strategies to disrupt this tolerance in PJI. The hypothesis of this proposal is that biofilm formation, regulated by toxin antitoxin systems, are a major factor responsible for the formation of antibiotic tolerant biofilm. In other diseases of chronic infection (ie tuberculosis and cystic fibrosis), biofilm formation and antibiotic tolerance have been well-recognized to decrease antibiotic efficacy. Understanding the initial events that lead to the development of antibiotic tolerant biofilm in PJI is the first step in developing treatment strategies. The aims include establishing the relationship between biofilm formation and antibiotic tolerance. Preliminary results in this proposal demonstrate that biofilm formation is a critical step in the development of antibiotic tolerant biofilm. In Aim 1, we have collected a library of PJI clinical isolates, and will identify genetic and phenotypic differences in binding and antibiotic tolerant biofilm formation in these isolates. In Aim 2, we will determine the role of toxin-antitoxin systems in PJI biofilm formation and association with antibiotic tolerance in vitro and in the animal model we have developed. This will add further evidence to the role of antibiotic tolerant biofilm in PJI to help direct improved the development of treatment strategies.