Permeability barriers of Gram-negative pathogens and approaches to bypass them A major source of resistance in Gram-negative species is the permeability barrier created by a synergistic action of slow uptake and active efflux of antibiotics across the outer membrane. The proposed research will address a critical need to understand penetration across the cell envelope of Gram-negative species and to develop antibiotics affective against these pathogens. In preliminary studies, we established novel mathematical and empirical approaches to analyze drug uptake across a two-membrane barrier with active efflux. Based on these results, we formulated a hypothesis that permeation and hence activities of antibiotics are affected by bacterial barriers and active efflux differently and that these differences are defined by physicochemical properties of antibiotics. To critically test this hypothesis, we will apply the new formalism and methods to analyze permeability barriers of Pseudomonas aeruginosa, Acinetobacter baumannii and Burkholderia spp. that differ significantly in the composition of their outer membranes and efflux pumps. These pathogens are notorious for their virtually unbreachable permeability barriers. Our goal is to establish structure-activity relationships between drug uptake, efflux and inhibitory activities for a select library of compounds in the context of barriers with different permeability properties. The proposed studies will define the limits of barriers and compounds and will create a framework for optimization of antibiotics targeting the “impermeable” pathogens.