PROJECT SUMMARY Periprosthetic joint infection (PJI) after placement of an artificial joint is a devastating complication for both the patient and surgeon. Infection typically requires an arduous course of repeated surgeries with multiple courses of broad-spectrum antibiotics administered systemically or locally by antibiotic loaded bone cements. In many cases the infection cannot be resolved, even though the isolated pathogen is sensitive to the administered antibiotics, and amputation is not uncommon. Alarmingly a recent study reports that 26% of patients treated for PJI were dead within 5 years, twice the number of those being replaced for reasons other than infection and survivorship rates less than those with prostrate, melanoma and breast cancers. Although the infection incidence in total hip and total knee arthroplasties is relatively low (2.0% and 2.4% respectively), the number of procedures is forecast to grow exponentially and 15,000 hips and 50,000 knee PJIs are predicted in the US by 2020. Biofilm formation on the implant components by bacterial pathogens is associated with the intractability of chronic PJIs. Biofilms are attached communities of bacterial cells living within an extracellular polymeric substances (EPS) matrix composed of bacterial polymers and host components. Biofilm formation is a mechanism used for protection, and it confers high levels of tolerance to antibiotics and resistance to host immunity. However important unanswered clinical questions remain, how do biofilms become established from what is presumably a small numbers of cells entering the surgical site in the highly controlled environment of the modern OR and where do biofilms reside in the infected joint? Aggregation (or agglutination in blood) in staphylococci is a known virulence factor in sepsis but recently it has been shown that aggregates which form rapidly in human synovial fluid can attach to surfaces to form biofilms. Aggregation also affords rapid biofilm-like tolerance which may protect the bacteria before they attach to implants and establish mature biofilms. While in vitro models focus on interaction with single cells almost nothing is known about the kinetics of attachment and growth from aggregates. In Aim 1 we will determine the kinetics of aggregation in bovine synovial fluid to determine what size of aggregates are required to confer tolerance to antibiotics and neutrophil uptake. We will also assess the relative contribution of host factors, bacterial adhesion proteins and EPS components on aggregation kinetics and the use of surfactants as surgical irrigants to prevent and disperse aggregates. In Aim 2 we will quantify the attachment rate of aggregates and subsequent biofilm development on different orthopaedic materials under controlled shear stresses in flow-cell and chemostat models. In Aim 3 we will use a novel agar encasement culturing method to map and quantify biofilms to specific locations on entire implants from in situ outgrowth from the surface. An infected reconstructed joint has a complex distribution of materials and mechanical forces, and understanding how this leads to preferential niches for the attachment of cellular aggregates and subsequent biofilm formation is of profound interest for surgeons and for informed implant design.