Antibiotic prophylaxis is standard for patients undergoing surgical procedures, yet despite the wide use of antibiotics, breakthrough infections still occur. In the setting of total joint arthroplasty, such infections can be devastating. Recent findings have shown that synovial fluid causes marked staphylococcal aggregation, which can confer antibiotic insensitivity. We therefore asked in this study whether clinical samples of synovial fluid that contain preoperative prophylactic antibiotics can successfully eradicate a bacterial challenge by pertinent bacterial species. This study demonstrates that preoperative prophylaxis with cefazolin results in high antibiotic levels. Furthermore, we show that even with antibiotic concentrations that far exceed the expected bactericidal levels, Staphylococcus aureus bacteria added to the synovial fluid samples are not eradicated and are able to colonize model implant surfaces, i.e., titanium pins. Based on these studies, we suggest that current prophylactic antibiotic choices, despite high penetration into the synovial fluid, may need to be reexamined.I nfection remains a major concern with orthopedic procedures, although infection rates are generally Ͻ1% (1). Because of the high prophylactic antibiotic levels within synovial fluid, the use of minimally invasive techniques, and short surgical times, infection rates remain low. However, with increasing numbers of total knee arthroplasties, infections are projected to exceed 60,000 to 70,000 cases in the United States by 2020 (2, 3). When hardware, such as a prosthesis, is present, infection is recalcitrant to antibiotic treatment. Once established, infection can cause prolonged disability, multiple operations, and increased health care costs (3, 4). According to the WHO, patients with a surgical site infection have twice the mortality rate, are twice as likely to spend time in an intensive care unit, and are five times more likely to be readmitted to the hospital than uninfected patients (5).To avoid the establishment of infection, antibiotic prophylaxis has become the standard of care (6, 7). For orthopedic procedures, cefazolin (CFZ), which is bactericidal for staphylococci, streptococci, and Escherichia coli (8, 9), is commonly used in the absence of a penicillin allergy (10). Alternative prophylactic antibiotics include fusidic acid, cloxacillin (11), cefixime (12), vancomycin, and gentamicin (13). Interestingly, synovial fluid has been suggested to possess antibacterial properties (14-16) that are attributed to hyaluronic acid within the synovial fluid (17), the induction of bactericidal/permeability-increasing protein (15), or unknown compounds, including antimicrobial peptides, within the synovial fluid (14). We recently reported that this ostensible decrease in bacterial numbers, which is perceived to be an "antibacterial" effect, is actually due to the clumping of bacteria within synovial fluid, which masks the true microbial load levels (18).The recognition of this behavior also helps to elucidate the difficulties ...