Comparison of ceftobiprole 5 μg disk diffusion, MIC test strip, and broth microdilution for susceptibility testing of Staphylococcus aureus clinical isolates

Ceftobiprole is a new-generation cephalosporin with activity against Staphylococcus aureus, but a comprehensive evaluation of MIC Test Strip (MTS) and disk diffusion (DD) methods using EUCAST and FDA breakpoint is lacking. Using broth microdilution (BMD) as the reference standard, we assessed the performance of MTS and DD for determining ceftobiprole susceptibility among 422 clinical S. aureus isolates. Ceftobiprole retained potent activity, with MIC₅₀/MIC₉₀ of 0.5/1 mg/L and an overall susceptibility rate of 98.8%; methicillin-resistant Staphylococcus aureus (MRSA) showed 97.6% susceptibility. MTS demonstrated high agreement with BMD, achieving 98.8% categorical agreement (CA), 1.2% major error (ME), and zero very major error (VME) under European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria, meeting the acceptability limits of ISO 20776-2:2021 and CLSI M52. DD performance was breakpoint-dependent: under EUCAST criteria, the overall CA was 89.6%, with 10.6% ME and 0 VME. The 40.3% of MRSA that fell into the EUCAST-defined Area of Technical Uncertainty (ATU) zone were all shown to be susceptible by the reference BMD method. Under Food and Drug Administration (FDA) breakpoints, the CA rose to 94.1% and ME fell to 1.7%. Consequently, MTS offers a reliable routine option; DD is suitable for routine testing under FDA breakpoints, while for EUCAST users, MRSA isolates in the ATU zone should be confirmed by BMD or MTS. VME was not determined under FDA breakpoints due to the absence of resistant isolates. Importance: Ceftobiprole offers a β-lactam option for Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA); however, its susceptibility testing remains insufficiently examined across both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Food and Drug Administration (FDA) interpretive criteria. We systematically compared MIC Test Strip (MTS) and disk diffusion (DD) with broth microdilution (BMD), demonstrating breakpoint-specific performance for methicillin-susceptible Staphylococcus aureus (MSSA) and MRSA and providing practical recommendations that allow laboratories to report accurate results regardless of the guidelines they follow.