Discovery of a novel polymyxin adjuvant against multidrug-resistant gram-negative bacteria through oxidative stress modulation

Antibiotic adjuvants offer a promising strategy for restoring antibiotic sensitivity, expanding antibacterial spectra, and reducing required dosages. Previously, compound <b>15</b> was identified as a potential adjuvant for Polymyxin B (PB) against multidrug-resistant (MDR) <i>Pseudomonas aeruginosa</i> DK2; however, its clinical utility was hindered by high cytotoxicity, uncertain <i>in vivo</i> efficacy, and an unclear synergetic mechanism. To address these challenges, we synthesized and evaluated a series of novel benzamide derivatives, with <b>A22</b> emerging as a particularly promising candidate. <b>A22</b> demonstrated potent synergistic activity to PB, minimal cytotoxicity, improved water solubility, and broad-spectrum synergism of polymyxins against various clinically isolated MDR Gram-negative strains. <i>In vivo</i> studies using <i>Caenorhabditis elegans</i> and mouse models further confirmed the efficacy of <b>A22</b>. Moreover, <b>A22</b> effectively suppressed the development of PB resistance in <i>Pseudomonas aeruginosa</i> DK2. Mechanistic investigations revealed that <b>A22</b> enhances polymyxins activity by inducing reactive oxygen species production, reducing ATP levels, increasing NOX activity, and inhibiting biofilm formation, leading to bacterial death. These findings position <b>A22</b> as a highly promising candidate for the development of polymyxin adjuvants, offering a robust approach to combating MDR Gram-negative bacterial infections.