Quantifying outer membrane permeability to β-lactam antibiotics using outer membrane vesicles that protect antibiotic-susceptible bacteria

The development of innovative methods for quantitatively assessing drug efficacy is essential for advancing effective treatments against antibiotic-resistant bacteria. A key determinant of drug activity against resistant Gram-negative bacteria is the permeability of the bacterial cell envelope, particularly that of the outer membrane, which is the first line of defense against antimicrobials. In this study, we report a method to quantitatively evaluate outer membrane permeability to β-lactams (nitrocefin, mecillinam, cloxacillin) and to a β-lactamase inhibitor (avibactam) by monitoring their entry into outer membrane vesicles (OMVs) produced by Escherichia coli and Pseudomonas aeruginosa. We also show that drug-susceptible bacteria are effectively shielded from β-lactams by OMVs emanating from resistant bacteria that contain a periplasmic β-lactamase captured upon their formation. Altogether, our work provides a comprehensive framework for understanding how antimicrobial molecules interact with bacterial OMVs and how this process contributes to the spread of antibiotic resistance. The outer membrane limits antibiotic entry into Gram-negative bacteria. Here, authors introduce an NMR-based outer membrane vesicle assay that quantifies β-lactam permeability and explore how vesicles protect susceptible bacteria.