The controversy about antibiotic lethality and reactive oxygen species

Highlights by José F. Silva Neto

Antibiotics are powerful compounds in our battle against bacterial diseases. Despite their miraculous efficacy over decades, nowadays we are faced with the global spreading of antibiotic resistance and the decrease of our antibiotic arsenal. For many years, we learned that antibiotics exert their effect by direct interaction with different primary bacterial targets, causing killing (bactericidal drugs) or growth inhibition (bacteriostatic drugs). In 2007, an influential paper from the Collins laboratory [1] placed reactive oxygen species (ROS) as central players in the mechanism of cell death induced by bactericidal antibiotics. Ever since, the hypothesis that clinically used antibiotics kill bacteria by stimulating the formation of oxidants has raised an intense scientific debate, with many groups generating experimental evidence that either support [1, 2, 3] or contradict [4, 5, 6] this hypothesis. At the heart of this controversy is the choice of assays used by those different groups, which resulted in ambiguous or contradictory data [7, 8].

The initial model of the Collins group proposed that bactericidal antibiotics of distinct classes, regardless of their primary targets, cause cell death by a common mechanism involving production of the reactive oxygen species hydroxyl radical. These conclusions were mainly supported by the use of a redox-sensitive dye (hydroxyphenyl fluorescein, HPF) intended to detect hydroxyl radical accumulation after antibiotic treatment. Accordingly, a hydroxyl radical quencher (thiourea) and an iron chelator (dipyridyl) suppressed the antibiotic-induced fluorescence [1]. Some years later, a series of works using classical genetic and biochemical strategies challenged this model of antibiotic lethality mediated by oxidants [4, 5, 6]. These papers found that: (i) many bactericidal antibiotics are equally effective against bacteria under aerobic and anaerobic conditions; (ii) the role of thiourea and dipyridyl in protecting cells from antibiotic killing is also observed anaerobically; (iii) antibiotic treatment does not create hydrogen peroxide stress or increase the levels of unincorporated iron; (iv) dye probes used to detect hydroxyl radicals could be nonspecifically oxidized. All these data led authors to refute the original ROS hypothesis of antibiotic toxicity [7]. More recently, a new paper from the Collins group addressed experimentally point-by-point most of the challenges to such ROS hypothesis [3], contributing to strengthen the relation between oxidants and antibiotic lethality [8]. Despite the opposite viewpoints, this controversy is functioning to accelerate our understanding of the complex mechanisms by which antibiotics kill bacteria, and inspiring redox-based strategies [2] to increase the efficacy of our available antibiotics.

1. M. A. Kohanski, D. J. Dwyer, B Hayete, C. A. Lawrence, J. J. Collins. A common mechanism of cellular death induced by bactericidal antibiotics Cell, 130 (5): 797-810, 2007 | doi: 10.1016/j.cell.2007.06.049
2. M. P. Brynildsen, J. A. Winkler, C. S. Spina, I. C. MacDonald, J. J. Collins. Potentiating antibacterial activity by predictably enhancing endogenous microbial ROS production Nature Biotechnology, 31 (2): 160-65, 2013 | doi: 10.1038/nbt.2458
3. D. J. Dwyer, P. A. Belenky, J. H. Yang, I. C. MacDonald, J. D. Martell, N. Takahashi, C. T. Chan, M. A. Lobritz, D. Braff, E. G. Schwarz, J. D. Ye, M. Pati, M. Vercruysse, P. S. Ralifo, K. R. Allison, A. S. Khalil, A. Y. Ting, G. C. Walker, J. J. Collins. Antibiotics induce redox-related physiological alterations as part of their lethality Proceedings of the National Academy of Sciences of the United States of America, 111 (20): E2100-9, 2014 | doi: 10.1073/pnas.1401876111
4. Y. Liu, J. A. Imlay. Cell death from antibiotics without the involvement of reactive oxygen species Science, 339 (6124): 1210-3, 2013 | doi: 10.1126/science.1232751
5. Keren, Y. Wu, J. Inocencio, L. R. Mulcahy, K. Lewis. Killing by bactericidal antibiotics does not depend on reactive oxygen species Science, 339 (6124): 1213-6, 2013 | doi: 10.1126/science.1232688
6. B. Ezraty, A. Vergnes, M. Banzhaf, Y. Duverger, A. Huguenot, A. R. Brochado, S. Y. Su, L. Espinosa, L. Loiseau, B. Py, A. Typas, F. Barras. Fe-S cluster biosynthesis controls uptake of aminoglycosides in a ROS-less death pathway Science, 340 (6140): 1583-7,340 (6140): 1583-7, 2013 | doi: 10.1126/science.1238328
7. J. A. Imlay. Diagnosing oxidative stress in bacteria: not as easy as you might think Current Opinion in MicrobiologyCurrent Opinion in Microbiology, 24: 124-131,24: 124-131, 2015 | doi: 10.1016/j.mib.2015.01.004
8. X. Zhao, K. Drlica. Reactive oxygen species and the bacterial response to lethal stress Current Opinion in Microbiology, 21: 1-6, 2014 | doi: 10.1016/j.mib.2014.06.008

José Freire da Silva Neto is a Professor of the Depto. de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, University of São Paulo, and a former post-doctoral fellow at the laboratory of Luis Eduardo Soares Netto, a principal investigator of our CEPID-Redoxoma.