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However, how metabolic heterogeneity underpins collective antibiotic tolerance within biofilms remains poorly understood, particularly at single-cell resolution. Here, we apply single-cell RNA sequencing to dissect the response of Staphylococcus aureus ATCC 12600 biofilms to vancomycin. We show that vancomycin induces heterogeneous upregulation of the fructose metabolism genes fruA and fruK, which spatially segregates the biofilm into metabolically active and metabolically reduced subpopulations. Active cells exhibit increased fructose consumption, which fuels peptidoglycan biosynthesis and promotes cell-wall thickening, as a mechanism that directly enhances vancomycin tolerance. In contrast, certain metabolically reduced subpopulations potentiate oxidative stress repair programs, representing an alternative survival strategy. In a diabetic mouse model characterized by impaired fructose clearance, elevated systemic fructose levels exacerbate pulmonary S. aureus infection, indicating that fructose utilization may reflect clinically relevant features of host susceptibility. Together, our findings uncover a fundamental link between metabolic heterogeneity and antibiotic tolerance in S. aureus biofilms, opening new avenues for the rational design of anti-biofilm therapeutics.<\/jats:p>","DOI":"10.1093\/ismejo\/wrag265","type":"journal-article","created":{"date-parts":[[2026,10,10]],"date-time":"2026-10-10T11:50:35Z","timestamp":1791633035000},"source":"Crossref","is-referenced-by-count":0,"title":["Fructose metabolic heterogeneity drives vancomycin tolerance in clonal\n                    <i>Staphylococcus aureus<\/i>\n                    biofilms"],"prefix":"10.1093","volume":"20","author":[{"given":"Zhichen","family":"Jiang","sequence":"first","affiliation":[{"name":"MOE Key Laboratory of Evolution and Marine Biodiversity and Institute of Evolution and Marine Biodiversity, Ocean University of China , Qingdao 266000 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,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi","family":"Yu","sequence":"additional","affiliation":[{"name":"College of Oceanography and Ecological Science, Shanghai Ocean University , Shanghai 201306 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiejie","family":"Hao","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Marine Drugs and School of Medicine and Pharmacy, Ocean University of China , Qingdao 266000 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weipeng","family":"Zhang","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Evolution and Marine Biodiversity and Institute of Evolution and Marine Biodiversity, Ocean University of China , Qingdao 266000 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