Antibiotics induce redox-related physiological alterations as part of their lethality

Abstract

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H 2 O 2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H 2 O 2 . We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.reactive oxygen species | DNA repair | mutagenesis T he increasing incidence of antibiotic-resistant infections coupled with a declining antibiotic pipeline has created a global public health threat (1-6). Therefore there is a pressing need to expand our conceptual understanding of how antibiotics act and to use insights gained from such efforts to enhance our antibiotic arsenal. It has been proposed that different classes of bactericidal antibiotics, regardless of their drug-target interactions, generate varying levels of deleterious reactive oxygen species (ROS) that contribute to cell killing (7,8). This unanticipated notion, built upon important prior work (9-11), has been extended and supported by multiple laboratories investigating wide-ranging drug classes (e.g., β-lactams, aminoglycosides, and fluoroquinolones) and bacterial species (e.g., Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baumannii, Burkholderia cepecia, Streptococcus pneumonia, Enterococcus faecalis) using independent lines of evidence (12-39). Importantly,...

Generation of reactive oxygen species by lethal attacks from competing microbes

“…A subsequent paper by Dwyer et al (27) provided further evidence that ROS production was a typical consequence of the cellular disruption caused by antibiotics, and thus could enhance the lethality of antibiotics beyond their primary modes of action. The results presented here resonate with all four of these studies in that the lethal insults we studied (T6SS effectors, P1vir phage, and polymyxin B) induced ROS production but cell killing could occur by nonoxidative (cell wall hydrolysis, phage replication, and envelope disruption, respectively) as well as oxidative mechanisms depending upon the insult.…”

“…Previous reports suggested that the expression of soxS could be up-regulated by antibiotics (16,27), volatile organic compounds (39), and hydrogen peroxide (40). Interestingly, all of these compounds can be produced by various bacterial species.…”

“…These observations include the fact that antibiotics kill under anaerobic conditions, oxidation of the hydroxyphenyl fluorescein fluorescence dye used to measure ROS levels is nonspecific, and the extracellular level of H 2 O 2 is not elevated by antibiotic treatment (24,26). To address these concerns, Dwyer et al (27) used a panel of ROS-detection fluorescence dyes, a defined growth medium under stringent anaerobic conditions, and an in vivo H 2 O 2 enzymatic assay to study the effects of antibiotics on cells. The results further support that antibiotics induce ROS generation, which contributes to the efficacy of antibiotics in addition to their primary lethal actions (18,27,28).…”

“…To address these concerns, Dwyer et al (27) used a panel of ROS-detection fluorescence dyes, a defined growth medium under stringent anaerobic conditions, and an in vivo H 2 O 2 enzymatic assay to study the effects of antibiotics on cells. The results further support that antibiotics induce ROS generation, which contributes to the efficacy of antibiotics in addition to their primary lethal actions (18,27,28).…”

Antibiotic treatment enhances the genome-wide mutation rate of target cells

“…Under sustained exposure to norfloxacin, mutation rates revealed by reporter constructs can be elevated 2-9 times (18,23). Norfloxacin is also thought to elevate intracellular reactive oxygen species (ROS) levels (24,25), which may further increase the rate of genomic mutation and the subsequent rate of acquisition of multidrug resistance (23,26).…”