FIG 3.
ADO potentiates antibiotic lethality. (A) E. coli grown to stationary phase in LB broth was treated with very high concentrations of antibiotics for 0 to 24 h. Selected groups were treated with 80 μg/mL GEN, 3 μg/mL CIP, and/or 1 mM ADO. (B) E. coli downshifted from LB broth into PBS and treated at time zero with 1 mM ADO. Both control and treatment groups were established from one culture to ensure equal starting CFU for each group within an experiment. (C and D) Survival of E. coli after downshift from exponential-phase LB broth to PBS followed by an 18 h treatment with 5 μg/mL GEN or 500 ng/mL CIP. Selected samples were treated with 1 mM of the indicated nucleotides or metabolites. (E and F) Eighteen-hour survival experiment performed in an anaerobic chamber. Bacteria were passaged for at least 1 week to adapt to anaerobic conditions. Prior to treatment, bacteria were shifted from LB broth to PBS. Sodium nitrate (25 mM) was added at time of antibiotic treatment. Selected samples were treated with 5 μg/mL GEN, 500 ng/mL CIP, and/or 1 mM ADO. (G and H) Survival of aerobic E. coli after downshift from exponential-phase LB broth to PBS followed by a 3 h treatment with 5 μg/mL GEN or 500 ng/mL CIP. Selected samples were treated with 1 mM ADO and 50 μM CCCP at time of antibiotic treatment. Data are the mean of three biological replicates ± SEM. Dotted line indicates MDK99.99 threshold. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as assessed by Student's t test, Mann-Whitney U test, or one-way ANOVA with Fisher’s least significant difference (LSD) multiple comparison at each time point. ADE, adenine; ADO, adenosine; RIB, d-ribose; VEH, vehicle control.