Beyond <i>mecA</i> : a two-tiered mechanism and regulatory rewiring drive high-level ceftaroline resistance in clinical MRSA

High-level resistance to ceftaroline, a fifth-generation β-lactam critical for treating methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), is an emerging threat to global health. While resistance is traditionally attributed to <i>mecA</i>-mediated expression of PBP2a, our study reveals a previously unrecognized mechanism. We show that high-level resistance to ceftaroline can arise independently of ceftaroline exposure through a collateral pathway triggered by carbapenems typically used to treat Gram-negative infections. Our findings reveal a two-tiered adaptive process. First, meropenem selects non-synonymous mutations in <i>rpoB</i>, a core transcriptional regulator, which primes resistance by reprogramming gene expression. These changes consistently co-occur with a key substitution in <i>pbp1</i> (H499R), an essential protein for cell division, and specific <i>mecA</i> variants (Y446H, E447K) following ceftaroline exposure. Second, resistance is stabilized through regulatory and signaling adaptations, with elevated basal levels of the oxidative stress regulator Spx and its adaptor protein TrfA supporting the altered cellular state. Proteomic and biophysical studies revealed direct binding of TrfA to GdpP, the phosphodiesterase for cyclic-di-AMP, linking this regulatory circuit to elevated c-di-AMP levels and resistance maintenance. Our findings challenge the assumption that ceftaroline resistance is driven solely by PBP2a alterations and reveal how collateral resistance pathways can be activated by broad-spectrum antibiotic use. This study highlights the evolutionary capacity of MRSA to circumvent antibiotic pressure and underscores the need for improved antimicrobial stewardship.