Antimicrobial paeonol modulates efflux pumps and membrane permeability in Aeromonas hydrophila: Proteomic insights and application in grass carp meat preservation

The growing drug resistance of the fish pathogen Aeromonas hydrophila places a significant strain on aquaculture production and food safety. Paeonol, a plant-derived compound, exhibits substantial antibacterial potential. Our study shows that paeonol inhibits 12 bacterial strains, with an IC 50 of 256.8 μg/mL against A. hydrophila . Its antibacterial mechanisms include reducing bacterial motility, inhibiting biofilm formation, enhancing cell membrane permeability, and decreasing efflux pump activity. Proteomics revealed that efflux pump-related proteins were upregulated, while ABC transporter proteins were downregulated after paeonol treatment. Combining paeonol with efflux pump inhibitor (CCCP) enhances its antibacterial effects. Furthermore, the deletion of oppA , which encodes the periplasmic oligopeptide-binding protein in the ABC transport system, led to reduced survival of A. hydrophila under paeonol treatment, suggesting that paeonol interferes with the expression or function of OppA. A cell thermal shift assay (CETSA) further confirmed that paeonol binds to OppA, leading to the destabilization of the target protein. Notably, paeonol has low toxicity, minimal resistance induction, and high protective efficacy both in vitro and in vivo . It also reduces bacterial counts in fish meat, improving freshness. Overall, paeonol is a promising natural antimicrobial agent and food preservative for aquaculture production and food preservation. • Paeonol shows broad-spectrum antibacterial activity against various bacteria. • Paeonol disrupts biofilm formation and inhibits efflux pumps in A. hydrophila. • Paeonol binds to and destabilizes OppA, a key protein in bacterial resistance. • Paeonol combined with CCCP significantly enhances antibacterial efficacy. • Paeonol effectively reduces bacterial load in fish meat, preserving its freshness.