Lineage-associated genomic features and phenotypic heterogeneity in clinical borderline oxacillin-resistant Staphylococcus aureus

ABSTRACT Borderline oxacillin-resistant Staphylococcus aureus (BORSA) represents a mecA / mecC -negative phenotype characterized by oxacillin minimum inhibitory concentrations (MICs) of 1–8 mg/L. The molecular basis of this resistance, particularly regarding lineage-associated differences in induction profiles, remains incompletely understood. In this study, 11 clinical BORSA isolates from pneumonia and bloodstream infections were identified among 374 clinical S. aureus isolates. Resistance-associated features were investigated using phenotypic profiling, whole-genome sequencing (WGS), RT-qPCR of blaZ expression, and time-kill assays. All BORSA isolates were cefoxitin-susceptible, β-lactamase-positive, and distributed across six clonal backgrounds, with ST25 and ST7 each represented by three isolates. WGS identified a shared GdpP N105D substitution in all isolates, together with lineage-associated variations in pbp1 and pbp4 . Following oxacillin exposure, blaZ was significantly upregulated in all three ST25 isolates (all P < 0.001), accompanied by accelerated nitrocefin hydrolysis, whereas the remaining isolates showed weaker or more heterogeneous responses. While oxacillin monotherapy (1× MIC) failed to prevent bacterial regrowth, oxacillin/sulbactam combinations produced sustained synergistic killing. This effect was most consistently observed among the ST25 isolates, whereas the ST7 isolates showed greater phenotypic variation. These findings suggest that the BORSA phenotype in this cohort may involve a multifactorial resistance profile, including candidate gdpP variation, inducible β-lactamase activity, and alterations in penicillin-binding proteins (PBPs). The cefoxitin susceptibility of these isolates presents a diagnostic challenge that may lead to therapeutic misclassification and subsequent inappropriate therapy. β-lactam/β-lactamase inhibitor combinations demonstrated promising in vitro activity and warrants further investigation for BORSA isolates exhibiting strong inducible β-lactamase activity. IMPORTANCE Borderline oxacillin-resistant Staphylococcus aureus (BORSA) is difficult to detect because it lacks mecA / mecC and remains susceptible to cefoxitin, increasing the risk of misclassification and inappropriate β-lactam therapy. This study suggests that BORSA resistance in this cohort is multifactorial, involving candidate gdpP variation, lineage-associated variations in penicillin-binding proteins, and inducible β-lactamase activity. Importantly, the ST25 isolates in this cohort exhibited rapid blaZ induction and stronger responses to oxacillin/sulbactam than to oxacillin alone. These findings improve understanding of BORSA biology, highlight a significant diagnostic blind spot in routine susceptibility testing, and support further evaluation of β-lactam/β-lactamase inhibitor combinations as a promising treatment strategy for BORSA isolates exhibiting strong inducible β-lactamase activity.