A Methylazanediyl Bisacetamide Derivative Sensitizes Staphylococcus aureus Persisters to a Combination of Gentamicin And Daptomycin

Infections caused by Staphylococcus aureus, notably methicillin‐resistant S. aureus (MRSA), pose treatment challenges due to its ability to tolerate antibiotics and develop antibiotic resistance. The former, a mechanism independent of genetic changes, allows bacteria to withstand antibiotics by altering metabolic processes. Here, a potent methylazanediyl bisacetamide derivative, MB6, is described, which selectively targets MRSA membranes over mammalian membranes without observable resistance development. Although MB6 is effective against growing MRSA cells, its antimicrobial activity against MRSA persisters is limited. Nevertheless, MB6 significantly potentiates the bactericidal activity of gentamicin against MRSA persisters by facilitating gentamicin uptake. In addition, MB6 in combination with daptomycin exhibits enhanced anti‐persister activity through mutual reinforcement of their membrane‐disrupting activities. Crucially, the “triple” combination of MB6, gentamicin, and daptomycin exhibits a marked enhancement in the killing of MRSA persisters compared to individual components or any double combinations. These findings underscore the potential of MB6 to function as a potent and selective membrane‐active antimicrobial adjuvant to enhance the efficacy of existing antibiotics against persister cells. The molecular mechanisms of MB6 elucidated in this study provide valuable insights for designing anti‐persister adjuvants and for developing new antimicrobial combination strategies to overcome the current limitations of antibiotic treatments. This study presents MB6, a methylazanediyl bisacetamide derivative, as a potent antimicrobial agent selectively targeting MRSA membranes. MB6 demonstrates triple synergistic killing against MRSA persisters by promoting gentamicin uptake and enhancing the membrane‐disrupting activity of daptomycin. This positions MB6 as a promising adjuvant in combating antibiotic‐resistant and ‐tolerant infections, offering innovative strategies in antimicrobial treatments.