Functional Diversity of Gram-Negative Permeability Barriers Reflected in Antibacterial Activities and Intracellular Accumulation of Antibiotics

Gram-negative bacteria are notoriously more resistant to antibiotics than Gram-positive bacteria, primarily due to the presence of the outer membrane and a plethora of active efflux pumps. However, the potency of antibiotics also varies dramatically between different Gram-negative pathogens, suggesting major mechanistic differences in how antibiotics penetrate permeability barriers. Gram-negative bacteria are notoriously more resistant to antibiotics than Gram-positive bacteria, primarily due to the presence of the outer membrane and a plethora of active efflux pumps. However, the potency of antibiotics also varies dramatically between different Gram-negative pathogens, suggesting major mechanistic differences in how antibiotics penetrate permeability barriers. Two approaches are used broadly to analyze how permeability barriers affect intracellular accumulation of antibiotics. One compares the antibacterial activities of compounds, while the other measures the total intracellular concentrations of compounds in nongrowing cells, with both approaches using strains harboring wild-type or genetically modified efflux systems and permeability barriers. Whether the two assays provide similar mechanistic insights remains unclear. In this study, we analyzed the intracellular accumulation and antibacterial activities of antibiotics representative of major clinical classes in three Gram-negative pathogens of high clinical importance, Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii. We found that both assays are informative about properties of permeability barriers, but there is no quantitative agreement between the assays. Our results show that the three pathogens differ dramatically in their permeability barriers, with the outer membrane playing the dominant role in E. coli and P. aeruginosa but efflux dominating in A. baumannii. However, even compounds of the same chemotype may use different permeation pathways depending on small chemical modifications. Accordingly, a classification analysis revealed limited conservation of molecular properties that define compound penetration into the three bacteria.

Automated summary

Gram-negative bacteria are more resistant to antibiotics due to their outer membrane and efflux pumps. Different Gram-negative pathogens have different permeability barriers and mechanisms for antibiotics penetration. Two methods, comparing antibacterial activities and measuring intracellular concentrations, are used to analyze the effect of permeability barriers on antibiotics. However, there is no quantitative agreement between these assays. The three pathogens analyzed in this study, Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii, have different permeability barriers, with the outer membrane playing a dominant role in E. coli and P. aeruginosa, while efflux dominates in A. baumannii. Additionally, compounds of the same chemotype may use different permeation pathways depending on small chemical modifications.