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Here we report that 1 min co-treatment with glycerol markedly enhances aminoglycoside efficacy against stationary-phase\n                      <jats:italic toggle=\"yes\">Staphylococcus aureus<\/jats:italic>\n                      cells. This rapid combined treatment also effectively eliminates\n                      <jats:italic toggle=\"yes\">S. aureus<\/jats:italic>\n                      biofilms and persister cells, methicillin-resistant\n                      <jats:italic toggle=\"yes\">S. aureus<\/jats:italic>\n                      clinical isolates\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      , and\n                      <jats:italic toggle=\"yes\">S. aureus<\/jats:italic>\n                      in a mouse skin infection model. The potentiation is achieved through rapid enhancement of proton motive force-dependent aminoglycoside uptake. Atmospheric and room temperature plasma (ARTP)-based genome-wide mutagenesis screens further reveal the genetic basis of this potentiation, highlighting a central role for glycerol kinase (GlpK). Mechanistically, GlpK initiates glycerol catabolism and boosts cellular energy metabolism, thereby increasing proton motive force-driven aminoglycoside uptake. Unlike widely reported long-term adjuvant treatments, our approach offers an immediate potentiation strategy and may open avenues to develop glycerol as an aminoglycoside adjuvant, given its \u201cgenerally recognized as safe\u201d characteristic. Our study also illustrates the potential of ARTP-based genome-wide mutagenesis in antibiotic resistance and stress biology research.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aac.00938-25","type":"journal-article","created":{"date-parts":[[2025,10,23]],"date-time":"2025-10-23T13:11:23Z","timestamp":1761225083000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":3,"title":["Genome-wide screen reveals glycerol-induced aminoglycoside potentiation against\n                    <i>Staphylococcus aureus<\/i>\n                    via boosting GlpK-initiated energy metabolism"],"prefix":"10.1128","volume":"69","author":[{"given":"Yaqin","family":"Xing","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/020azk594","id-type":"ROR","asserted-by":"publisher"}],"name":"Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, College of Life 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