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Objectives: We conducted an in vitro investigation of the activity of rifamycins against planktonic and biofilm states of Staphylococcus aureus and Staphylococcus epidermidis isolates from patients with prosthetic joint infections (PJIs), characterised their rpoB gene mutations, and analysed the clinical outcomes of rifampicin-resistant isolates., Methods: A total of 110 staphylococcal isolates were collected from patients with PJI. Antimicrobials tested using the broth microdilution method included rifampicin, rifabutin, rifapentine and rifaximin. We evaluated rpoB gene mutations to identify rifampicin resistance mechanisms. Clinical outcomes were assessed in rifampicin-resistant isolates., Results: The 110 staphylococcal isolates included 85 S. aureus (55% methicillin-resistant) and 25 S. epidermidis (100% methicillin-resistant). Seven S. aureus isolates and two S. epidermidis isolates were resistant to rifampicin [minimum inhibitory concentration (MIC) ≥2 μg/mL] and these isolates exhibited rpoB gene mutations. Among the 78 rifampicin-susceptible S. aureus isolates and 23 S. epidermidis isolates, 76 S. aureus isolates (97.4%) and all S. epidermidis isolates (100%) were highly susceptible (MIC ≤ 0.06 μg/mL) to other rifamycins. The minimum biofilm bactericidal concentrations for ≥50% of isolates (MBBC 50 ) to rifampicin, rifabutin, rifapentine and rifaximin were 4, 1, 2 and 4 μg/mL for S. aureus and 1, 0.125, 0.25 and 0.5 μg/mL for S. epidermidis, respectively, among rifampicin-susceptible isolates. Among nine patients bearing rifampicin-resistant isolates, only three (33%) had successful outcomes., Conclusion: Rifamycins other than rifampicin show promising antistaphylococcal activity, including antibiofilm activity. Rifamycin-resistant staphylococci exhibit rpoB gene mutations., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)