Sonoporation-enhanced the antibacterial efficacy of aminoglycosides against Gram-negative persisters.

Bacterial persisters pose a significant challenge to antibiotic therapy, often causing treatment failure and chronic infections. Improving antibiotic efficacy against persisters is thus clinically crucial. In this study, we developed a low-frequency and low-intensity ultrasound system (40 kHz, 0.2 W/cm) to induce sonoporation in bacterial persisters. Combining ultrasound with aminoglycosides (AGs) reduced the persister ratio of Gram-negative bacteria (E. coli, P. aeruginosa, and A. baumannii) by approximately 5- to 15-fold, and notably, this approach achieves eradication below the detection threshold (< 2 CFU/mL) in MG1655 persisters. Scanning electron microscopy (SEM), N-phenyl-1-naphthylamine (NPN) staining, propidium iodide (PI) staining, ortho-nitrophenyl-β-galactoside (ONPG) assays, and inhibition zone test revealed that ultrasound primarily enhances AG uptake by increasing outer membrane permeability without significantly damaging the inner membrane, thereby potentiating antibiotic influx in Gram-negative bacteria. Based on experiments of E. coli BW25113 and its mechanosensitive (MS) channel knockout mutants, we propose that ultrasound combined with hypotonic shock enhances the permeability of MscK and YbiO channels. These findings highlight the potential of ultrasound-aminoglycoside combination therapy as a promising strategy against persistent bacterial infections, especially those caused by Gram-negative pathogens. Furthermore, this study offers new insights into bacterial persister characteristics and their susceptibility to physical and chemical interventions.