Cell death from antibiotics without the involvement of reactive oxygen species

Abstract

Recent observations have suggested that classic antibiotics kill bacteria by stimulating the formation of reactive oxygen species (ROS). If true, this notion might guide new strategies to improve antibiotic efficacy. In this study, the model was directly tested. Contrary to the hypothesis, antibiotic treatment did not accelerate the formation of hydrogen peroxide in Escherichia coli and did not elevate intracellular free iron, an essential reactant for the production of lethal damage. Lethality persisted in the absence of oxygen, and DNA repair mutants were not hypersensitive, undermining the idea that toxicity arose from oxidative DNA lesions. We conclude that these antibiotic exposures did not produce ROS and that lethality more likely resulted from the direct inhibition of cell-wall assembly, protein synthesis, and DNA replication.

Figure 1. Antibiotic efficacy does not require oxygen or H₂O₂

(A–C) Wild type cells were treated with ampicillin (A), norfloxacin (B) or kanamycin (C) in the presence (solid squares) or absence (open squares) of oxygen. In panel C anoxic killing was also tested in the presence of 40 mM KNO₃ (gray squares). (D–F) Wild-type cells (solid squares, MG1655) and congenic Hpx⁻ cells (open diamonds, AL427) were treated with antibiotics. Results are representative of at least three biological replicates.

Figure 2. Antibiotics do not create H₂O₂ stress

(A) O₂ consumption rates were not substantially elevated by antibiotic treatment. Oxygen consumption was measured with a Clark-type electrode before and during exposure to 5 μg/ml kanamycin, 5 μg/ml ampicillin, or 250 ng/ml norfloxacin. Respiration rates were normalized to optical density. (B–C) The OxyR regulon was not induced by ampicillin or norfloxacin. (B) mRNA was collected from naïve and antibiotic-exposed cells, and the expression of OxyR-responsive genes was quantified by RT-PCR. Signals were normalized to that of the housekeeping gene gapA, and fold inductions were calculated. Where indicated, cultures were additionally treated with exogenous H₂O₂ at room temperature for 10 min prior to mRNA collection. (C, D) Expression of a katG′::lacZ fusion was measured in exponential-phase wild-type cells (AL441) before and after antibiotic incubations (C). As a positive control, expression was measured in wild-type and Hpx⁻ cells (AL494) before (−O₂) and after 1 hour aeration (+O₂) (D). (E–H) Classic antibiotics did not promote H₂O₂ formation. Aerobic Hpx⁻ cells (AL427) were treated with antibiotic, and at designated time points samples were collected and the ongoing rate of H₂O₂ production was measured. The redox-cycling compound paraquat (H) was included as a positive control; note that this dose was insufficient to cause cell death (viable counts increased for > 3 hours). Data in this figure are reported as the means and SEM from at least 3 independent experiments.

Figure 3. Antibiotic lethality does not correlate with levels of unincorporated iron

(A–C) The superoxide-sensitive enzyme Edd was not damaged by cell exposure to kanamycin or norfloxacin. Cells growing in aerobic LB with 0.2% gluconate were incubated with lethal doses of kanamycin (20 μg/ml, A) or norfloxacin (250 ng/ml, B), and Edd activity was measured before (open bar) and after (closed bar) in vitro cluster repair with ferrous iron/DTT. Panel C represents control data from an SOD-deficient strain (KI232). The asterisk denotes < 4% normal activity. (D–F) Norfloxacin and kanamycin did not increase intracellular iron levels. The levels of intracellular unincorporated iron were determined by whole-cell EPR analysis before and during antibiotic exposure. Gray bars represent iron levels in untreated cells at similar optical densities. (F) Levels of unincorporated iron were measured in untreated congenic wild type cells (MG1655) and Δfur mutants (Jem913). (G–I) DNA-repair mutants are not hypersensitive to antibiotic doses that kill wild-type cells. Wild-type cells (solid squares) and recA mutants (open circles) were treated with the indicated doses of kanamycin (G) or ampicillin (H) for 2 hours, or norfloxacin (I) for one hour. Fig. S3A demonstrates the hypersensitivity of recA mutants to authentic H₂O₂. Data in this figure are reported as the means and SEM from at least 3 independent experiments.