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Background and purpose — The preferred treatment of an acute prosthetic joint infection (PJI) is debridement, antibiotics, irrigation and retention of the prosthesis (DAIR). The antibiotic treatment consists of an empirical and targeted phase. In the empirical phase, intravenous antibiotics are started after surgery before micro-organisms are determined in microbiological cultures. Which empirical antibiotic is used differs between hospitals, partly reflecting geographic differences in susceptibility spectrums. We investigated whether flucloxacillin should remain the antibiotic of choice in our hospital for empiric treatment of acute PJI with DAIR. Patients and methods — We retrospectively analyzed 91 patients treated for PJI with DAIR between 2012 and 2016. The susceptibility of micro-organisms was determined in multiple cultures of periprosthetic tissue and synovial fluid for 3 antibiotics: amoxicillin/clavulanic acid, cefazolin, and flucloxacillin. Results — Positive microbiological cultures from 68 patients were analyzed. Staphylococcus aureus was the predominant pathogen, cultured in half of the patients. In one-third of patients more than 1 micro-organism was found. On a patient level, the data showed that 65% were responsive to flucloxacillin, 76% to amoxicillin/clavulanic acid, and 79% to cefazolin. Interpretation — Flucloxacillin appeared to be a suboptimal choice in our patient population treated with DAIR. We therefore changed our practice to cefazolin as the preferred antibiotic in the empirical treatment of acute PJI with DAIR.