Limonene‐Rich Citrus sinensis Essential Oil Modulates Antimicrobial Resistance and Virulence Traits of Foodborne Pseudomonas aeruginosa : Mechanistic Eviden

ABSTRACT

The increasing prevalence of Pseudomonas aeruginosa in food systems, coupled with its antimicrobial resistance and expression of multiple virulence traits, poses an important challenge for food safety and spoilage control. In this context, essential oils (EOs) rich in monoterpenes have emerged as promising natural alternatives for microbial control in food matrices. This study evaluated the antimicrobial, antivirulence, and resistance‐modulating effects of limonene‐rich Citrus sinensis essential oil (CS‐EO) against food‐associated P. aeruginosa , including multidrug‐resistant strains, and validated its efficacy in a fresh‐produce model. CS‐EO exhibited antimicrobial activity against all tested strains, with minimum inhibitory concentrations (MICs) ranging from 3.12 to 12.5 mg/mL. Time–kill assays demonstrated a concentration‐dependent bacteriostatic effect at inhibitory concentrations (1× and 2× MIC), while exposure to ½ MIC modulated antibiotic susceptibility profiles in a strain‐dependent manner. Membrane integrity assays indicated increased cellular permeability and structural damage following CS‐EO treatment. In addition, CS‐EO reduced important virulence‐associated traits, including motility, biofilm formation (up to 77.01%), and pyocyanin production (27.4%–58.8%). Thermal treatment partially reduced antimicrobial activity. In a refrigerated fresh‐cut carrot model treated with 1× MIC, CS‐EO reduced bacterial populations by up to 2.5 log CFU/mL while limiting spoilage‐associated visual changes. Limonene‐rich Citrus sinensis essential oil exhibited multitarget activity against P. aeruginosa , affecting bacterial viability, virulence‐associated traits, and antimicrobial susceptibility. Its efficacy in a fresh‐produce system highlights its potential as a natural complementary strategy for controlling spoilage‐associated and antimicrobial‐resistant bacteria in food matrices.