耐甲氧西林金黄色葡萄球菌（MRSA）是全球重大的公共卫生威胁。联合疗法，特别是抗生素与非抗生素药物联用，已成为一种应对日益严重的抗生素耐药性危机的有前景的策略。磷霉素（FOS）在临床实践中越来越多地用于治疗耐药细菌感染，但作为单一疗法疗效有限。本研究发现，美国食品药品监督管理局（FDA）批准的抗癌药物5-氟尿嘧啶（5-FU）能有效增强FOS对MRSA（包括生物膜内MRSA细胞）的抗菌活性。机制上，5-FU靶向胞苷三磷酸（CTP）合酶，该酶负责在三磷酸腺苷（ATP）存在下，将尿苷三磷酸（UTP）转化为CTP，是嘧啶合成途径中的限速步骤。此外，我们证明5-FU和FOS的协同效应源于对嘧啶代谢的扰动，这种扰动诱导了膜损伤、质子动力（PMF）耗散、ATP合成增强和活性氧积累，最终导致细菌死亡。在大蜡螟（G. mellonella）和小鼠感染模型中，5-FU和FOS联用显著提高了生存率并降低了细菌负荷。总之，我们的工作证明了5-FU联合FOS在治疗MRSA感染的治疗潜力，并强调了扰动嘧啶代谢在恢复细菌对抗生素敏感性中的关键作用。

Methicillin-resistant Staphylococcus aureus (MRSA) represents a significant global public health threat. Combination therapy, particularly the use of antibiotics in conjunction with non-antibiotic agents, has emerged as a promising strategy to address the growing crisis of antibiotic resistance. Fosfomycin (FOS), increasingly utilized in clinical practice for treating drug-resistant bacterial infections, exhibits limited efficacy as a monotherapy. Here, we find that 5-fluorouracil (5-FU), a Food and Drug Administration (FDA)-approved anticancer drug, effectively enhances the antibacterial activity of FOS against MRSA, including biofilm-embedded MRSA cells. Mechanistically, 5-FU targets cytidine triphosphate (CTP) synthase, a rate-limiting enzyme responsible for the adenosine triphosphate (ATP)-dependent conversion of uridine triphosphate (UTP) to CTP. Moreover, we demonstrate that the synergistic effect of 5-FU and FOS arises from the perturbation of pyrimidine metabolism, which induces membrane damage, dissipation of the proton motive force (PMF), enhanced ATP synthesis, and accumulation of reactive oxygen species, culminating in bacterial death. In both Galleria mellonella (G. mellonella) and murine infection models, the combination of 5-FU and FOS markedly improves survival and reduces bacterial burdens. Collectively, our work demonstrates the therapeutic potential of 5-FU combined with FOS for tackling MRSA infections and highlights the pivotal role of perturbing pyrimidine metabolism in restoring antibiotic susceptibility.