Nutrient-limited conditions reveal the activity of a minor groove binder, MGB-BP-3, against Escherichia coli

Abstract

BackgroundAntimicrobial resistance demands new antibiotics, but conventional screening in nutrient-rich media often fails to predict in vivo efficacy, especially against Gram-negative bacteria. Minor groove binders (MGB), such as MGB-BP-3, are a promising new antibacterial with potent activity against Gram-positive bacteria but thought to have limited potency against Gram-negative bacteria. ObjectivesThis study evaluated the antibacterial activity of MGB-BP-3 under nutrient-limited, physiologically relevant conditions to better mimic in vivo environments. MethodsMinimum inhibitory concentrations (MICs) were determined against a panel of ESKAPEE pathogens in both rich (Cation Adjusted Mueller-Hinton Broth, CA-MHB) and nutrient-limited media (Roswell Park Memorial Institute, RPMI-1640 + 10% Luria-Bertani, LB). Synergy with efflux pump inhibitors and membrane permeabilizers was evaluated via checkerboard assays. Permeability was assessed using a fluorescent DNA-binding cell permeant (Hoechst 33342) accumulation assay. MICs were determined in the presence of different concentrations of ions, using both wild-type and efflux-compromised strains. In vivo efficacy was assessed using the Galleria mellonella infection model. ResultsThe activity of MGB-BP-3 against Gram-negative bacteria was improved by shifting the medium from rich to nutrient-limited conditions. Specifically, it showed potent activity against E. coli in nutrient-limited media (MIC 3.1 {micro}M) but was inactive in rich media (MIC 700 {micro}M). Synergy with efflux inhibitors occurred mainly in rich media, indicating barriers to intracellular accumulation were not present in nutrient-limited conditions. Increased permeability correlated with enhanced susceptibility and efflux-deficient strains were more susceptible, confirming the role of efflux pumps in resistance. MGB-BP-3 provided dose-dependent protection in G. mellonella larvae infected with E. coli or S. aureus. For example, at concentrations of 12.5 {micro}M and 100 {micro}M, MGB-BP-3 was able to protect the larvae by more than 70% over 5 days of treatment, respectively. ConclusionsNutrient-limited media better reveal MGB-BP-3s activity against Gram-negative bacteria and align with in vivo efficacy, highlighting the need for host-mimicking conditions in antibiotic screening.