Piperine reverses colistin resistance in multidrug resistant Gram-negative pathogens by membrane disruption and ROS damage

The global spread of colistin (COL) resistance, particularly mediated by mcr-1, threatens the efficacy of this last-line antibiotic against multidrug-resistant (MDR) Gram-negative pathogens. This development underscores the urgency of developing innovative therapeutic strategies to counteract antimicrobial resistance mechanisms. This study demonstrates that piperine (PIP), a natural alkaloid, functions as a potent adjuvant that restores COL efficacy through a multi-target mechanism. The combination of PIP and COL exhibits significant synergism against clinically relevant pathogens, including mcr-1-positive strains (FICI: 0.07–0.281), and was not susceptible to the development of drug resistance. Additionally, this combination effectively inhibits and eradicates biofilms. Mechanism studies and omics analysis confirmed that PIP combined with COL could cause bacterial membrane disruption, proton motive force (PMF) disruption, efflux pumps inhibition, resulting in accumulation of bacterial reactive oxygen species (ROS) and finally cell death. Furthermore, PIP suppresses MCR-1 through dual suppression of gene expression and protein function. The therapeutic potential was confirmed in murine infection models, where the combination significantly improved survival rates, reduced bacterial loads, and attenuated inflammatory responses. This study provides the first comprehensive elucidation of the multi-target synergy between COL and PIP, offering a promising therapeutic strategy that simultaneously overcomes existing resistance and impedes resistance development.