Fig. 6: Cytoplasmic condensation and antibiotic killing vary with the supplementation of glutathione, an antioxidant, but not dithiothreitol, mercaptoethanol, or α-tocopherol.

a, b Survival curves of E. coli under kanamycin and ciprofloxacin treatment with and without exogenous supplementation of glutathione (10 mM), as determined by CFU plating and counting. Each point represents two biological replicates, error bars indicate one standard deviation, and data are presented as mean ± SEM. Positive survival values indicate increases in CFU/mL. Measurements at 1× and 10× MIC (a) and endpoint measurements (b) across a range of antibiotic concentrations are shown. Arrows highlight protection. All MICs used were with respect to their baseline values, with the exception of increased concentration values adjusted for growth inhibition effects (“normalized”), as summarized in Supplementary Table 1. c Phase-contrast microscopy images of control and antibiotic-treated E. coli (10× MIC) with and without exogenous supplementation of glutathione (10 mM), taken ~10 h after continual antibiotic treatment. The starting densities of cells are similar across all images, and results are representative of two biological replicates. Scale bar, 3 μm. d Same as (c), but for treatment with peroxynitrite, a reactive metabolic by-product. Cells were imaged 3 h after treatment with peroxynitrite. Results are representative of two biological replicates. e, f Same as (a, b), but for peroxynitrite treatment and exogenous supplementation of glutathione (10 mM), dithiothreitol (10 mM), and mercaptoethanol (10 mM). g Same as (a), but for kanamycin and ciprofloxacin treatment and exogenous supplementation of dithiothreitol (10 mM), mercaptoethanol (10 mM), and α-tocopherol (50 mM). h Fractions of all cells that are condensed or lysed 6 h after antibiotic treatment (10× MIC) or peroxynitrite treatment (1 mM) and antioxidant pretreatment (glutathione, dithiothreitol, mercaptoethanol: 10 mM; α-tocopherol: 50 mM). Data from two different fields of view from two biological replicates; individual data points from fields of view are shown. The number of cells in each field of view in each group are indicated in parentheses, and p-values for two-sample t-tests for differences in mean value compared to cases with no antioxidant are shown next to cell numbers.