Abstract
The antimicrobial mechanisms of high-frequency ultrasound (HFUS) under low-power conditions were investigated using Escherichia coli as a model foodborne pathogen, with oxidative and physical contributions evaluated through radical scavenger assays, intracellular ROS quantification, flow cytometry, and scanning electron microscopy. Bacterial inactivation was strongly frequency-dependent. 760 kHz induced greater intracellular ROS accumulation and membrane permeabilisation than 500 kHz, and radical scavenger experiments confirmed a major ROS contribution to inactivation. The combination of HFUS (760 kHz, 30 W) with epigallocatechin gallate (EGCG; 0.2 mg/mL) produced synergistic antibacterial efficacy, accompanied by enhanced membrane depolarisation, permeabilisation, and ultrastructural damage. Pre-exposure to ultrasound increased bacterial sensitivity to subsequent lethal thermal (56°C) and oxidative (10 mM H2O2) challenges. Mutant analysis of eight E. coli K-12 strains revealed that antioxidant defence systems, particularly glutathione reductase (gor), were critical for survival, while deletion of the general stress regulator rpoS or oxidoreductase grxA increased tolerance, indicating complex interacting stress-response mechanisms. Overall, HFUS inactivation is driven primarily by a combination of chemical and physical mechanisms, encompassing ROS-mediated oxidative damage and cavitation-induced membrane disruption, enhancing bacterial susceptibility to additional antimicrobial stresses, and therefore holds greater potential as a complementary technology than as a stand-alone treatment.