Electrostatic Modulation of Antibiotic Susceptibility by the Bacterial Donnan Potential

Abstract Antibiotic resistance in Gram-negative bacteria is a major global health threat, in part because of the permeability barriers of their double-membrane envelope and active efflux systems. While porins, lipopolysaccharides, and efflux pumps have been extensively studied, the role of membrane energetics, particularly the Donnan potential (DP), in antibiotic susceptibility remains poorly understood. The DP arises from negatively charged osmoregulated periplasmic glucans (OPGs), which generate an electrostatic potential in the periplasm that is capable of influencing how molecules traverse the outer membrane. Here, we systematically examine how OPG-dependent electrostatic changes associated with the DP shape antibiotic susceptibility in Escherichia coli. By altering the environmental osmolarity, we generated conditions that changed OPG abundance and were expected to alter associated periplasmic electrostatics, revealing that low-salt conditions enhance the potency of cationic antibiotics but reduce the activity of anionic compounds, whereas high-salt conditions or OPG deficiency attenuate cationic drug activity. Strains producing neutral OPGs phenocopied OPG-deficient mutants, implying that the OPG charge is involved in this effect. Clinical E. coli isolates varied in sensitivity to osmolarity- and OPG-dependent electrostatic effects, and similar trends were observed across multiple Gram-negative pathogens. Finally, we show that dietary salt intake modulates antibiotic efficacy in vivo in a manner consistent with the salt-dependent susceptibility observed in vitro. Together, these findings systematically quantify antibiotic susceptibility phenotypes across a broad compound panel and under environmental and genetic perturbations expected to alter OPG-associated periplasmic electrostatics and suggest new principles for antibiotic design and therapeutic optimization against multidrug-resistant pathogens.