Hyperforin potentiates polymyxin B against multidrug-resistant Gram-negative pathogens via membrane disruption, biofilm eradication, and oxidative stress

The escalating spread of multidrug-resistant (MDR) Gram-negative pathogens, particularly <i>Pseudomonas aeruginosa</i>, <i>Klebsiella pneumoniae</i>, and <i>Acinetobacter baumannii</i>, has severely undermined the efficacy of polymyxin B, one of the few remaining last-line antibiotics. Here, we identify hyperforin, a natural product derived from <i>Hypericum perforatum</i>, as a potent polymyxin B adjuvant capable of restoring its antibacterial activity against clinical Gram-negative isolates with high-level polymyxin resistance. Using a suite of <i>in vitro</i> assays, including fractional inhibitory concentration index, time-kill kinetics, biofilm eradication, membrane integrity assays, and ultrastructural imaging of the bacterial outer membrane, we show that hyperforin significantly enhances polymyxin B-mediated bacterial killing. Microscopy and <i>N</i>-phenyl-1-naphthylamine uptake are consistent with membrane perturbation, and increased intracellular reactive oxygen species is consistent with oxidative stress under combination treatment. Taken together, these orthogonal readouts support a working model of complementary membrane perturbation and oxidative stress. Remarkably, the observed synergistic effects occur at concentrations potentially achievable in the epithelial lining fluid of the lungs, providing a testable rationale for localized lung delivery (e.g., via a dry-powder inhaler). These findings unveil a promising therapeutic strategy that repurposes a bioactive phytochemical to potentiate polymyxins against otherwise untreatable polymyxin-resistant MDR Gram-negative infections.