A cell-based infection assay identifies efflux pump modulators that reduce bacterial intracellular load
(A, B) Salmonella was incubated with Hoechst 33342 and the indicated compound. (A) Fluorescence was normalized to the maximum of heat-killed bacteria (100%). Mean and SEM from three biological replicates. (B) Maximum fluorescence over 60 minutes of exposure normalized to the maximum fluorescence of heat-killed bacteria (100%). The EC50s in the key were established using a nonlinear four-parameter fit. Mean and SEM from three biological replicates, each performed in duplicate. (C, D) Nitrocefin [100 μM] hydrolysis in a membrane-compromised E. coli strain treated with the indicated concentrations of drugs. Hydrolysis increases when efflux is blocked. (C) Absorbance 486 nm of RAM121 E. coli. Mean and SD are representative of three independent biological replicates. (D) Slope of the linear region of the A486 plot from at least three experiments. Data is normalized to A486/minute. ** p < 0.001, **** p < 0.0001 by one-way ANOVA with Dunnett’s post-test. (E) Structures of the three EPMs.