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Hydrogen peroxide drainage is an effective antimicrobial treatment that has been adopted to combat <jats:italic>S. aureus<\/jats:italic> infections. Previous investigations have indicated that the antisense RNA (asRNA) strategy negatively modulates <jats:italic>S. aureus<\/jats:italic> YycFG TCS, and it significantly disrupts biofilm formation. However, the effects of the antisense <jats:italic>yycG<\/jats:italic> RNA (AS<jats:italic>yycG<\/jats:italic>) strategy on the susceptibility of biofilm-producing <jats:italic>S. aureus<\/jats:italic> to hydrogen peroxide and the mechanisms underlying this effect have not been elucidated to date.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>Overexpression of AS<jats:italic>yycG<\/jats:italic> inhibited the transcription of biofilm formation-related genes, including <jats:italic>sarA<\/jats:italic> and <jats:italic>icaA<\/jats:italic>. Additionally, the CFU counts and the live bacterial ratios of AS<jats:italic>yycG<\/jats:italic> biofilm-producing <jats:italic>S. aureus<\/jats:italic> treated with H<jats:sub>2<\/jats:sub>O<jats:sub>2<\/jats:sub> were notably reduced across the groups. Notably, the predicted promoter regions of the <jats:italic>sarA<\/jats:italic> and <jats:italic>icaA<\/jats:italic> genes were directly regulated by YycF.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>AS<jats:italic>yycG<\/jats:italic> was observed to sensitize biofilm-producing <jats:italic>S. aureus<\/jats:italic> to H<jats:sub>2<\/jats:sub>O<jats:sub>2<\/jats:sub> intervention synergistically via the <jats:italic>sarA<\/jats:italic> and thus may represent a supplementary strategy for managing osteomyelitis. 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