The increasing isolation of penicillin-susceptible Staphylococcus aureus (PSSA) has renewed interest in benzylpenicillin (penicillin G, PCG) for targeted therapy. However, blaZ-encoded inducible penicillinase may not be detected by routine susceptibility testing. This study evaluated the diagnostic performance of phenotypic penicillinase detection methods applicable to routine clinical laboratories, using blaZ polymerase chain reaction (PCR) as the reference standard. A total of 379 non-duplicate S. aureus isolates with a PCG minimum inhibitory concentration (MIC) ≤0.12 μg/mL were collected at a single institution between January 2020 and July 2024. The blaZ gene was detected by PCR. Phenotypic methods included low-range MIC determination by Etest, disk diffusion using PCG 10 U and PCG 1 U disks, zone-edge testing, and nitrocefin-based assays. Sensitivity and specificity were calculated relative to blaZ PCR results. Among the 379 isolates, 20 (5.3%) were blaZ-positive despite being classified as PCG-susceptible by both CLSI and EUCAST criteria. Neither low-range MIC values nor inhibition zone diameters reliably differentiated blaZ-positive from blaZ-negative isolates. The zone-edge test demonstrated the highest diagnostic performance, with a sensitivity of 95.0% and specificity of 93.3% when performed using a PCG 1 U disk according to EUCAST methodology. Nitrocefin-based assays showed poor sensitivity (<50%). MIC- and zone diameter–based methods alone are insufficient to detect penicillinase-producing S. aureus among PCG-susceptible isolates. The zone-edge test using a PCG 1 U disk is a practical and reliable phenotypic confirmatory method suitable for routine clinical microbiology laboratories to support safe PCG therapy. • A subset of blaZ-positive Staphylococcus aureus isolates was identified among strains categorized as penicillin-susceptible by routine MIC- and zone diameter–based methods. • Low-range MIC values and inhibition zone diameters alone were insufficient to reliably distinguish penicillinase-producing from non-producing isolates. • The zone-edge test using a penicillin G 1 U disk demonstrated the highest diagnostic performance among evaluated phenotypic methods. • Nitrocefin-based assays showed poor sensitivity even when performed after penicillinase induction and were unsuitable as standalone confirmatory tests. • A simple, cost-free stepwise algorithm incorporating the PCG 1 U zone-edge test can improve the safe clinical use of benzylpenicillin in routine laboratories.
Yonetani et al. (Sun,) studied this question.