Cefdinir reprograms Gram‐positive bacteria to synergize with lysozyme against superbugs

Abstract Multidrug‐resistant (MDR) bacteria pose a critical global health threat, urgently requiring solutions. Cefdinir, the highest‐selling third‐generation cephalosporin but one now facing clinical obsolescence due to escalating resistance, is conventionally classified as a nascent cell wall synthesis inhibitor. This study challenges this understanding by demonstrating that cefdinir renders existing cell walls negatively charged, thereby synergizing with ubiquitous lysozyme and simultaneously restoring both agents' efficacy against a broad spectrum of MDR Gram‐positive superbugs without driving resistance. Specifically, cefdinir is unexpectedly shown to post‐transcriptionally increase the levels of clustered enzymes in lipoteichoic acid (LTA) and wall teichoic acid (WTA) synthesis pathways, including TarA, TarB, TarD, TarF, TarH, TarK, TarL, TarS, and FmtA, thereby enhancing cell wall electronegativity to remodel existing cell wall into a lysozyme‐susceptible state, in which TarS is identified for the first time by mass spectrometry. The combination of cefdinir and lysozyme also significantly suppresses biofilm formation and minimizes de novo resistance mutations. Antibacterial efficacy of combination therapy is validated in both cell‐based infection models and rat skin infection models, demonstrating strong translational potential. Given the established safety profiles of both agents, this sensitization strategy can be applied to human clinical research to combat MDR Gram‐positive bacterial infections.