Antibiotic mode-of-action classification is based upon drug-target interaction and whether the resultant inhibition of cellular function is lethal to bacteria. Here we show that the three major classes of bactericidal antibiotics, regardless of drug-target interaction, stimulate the production of highly deleterious hydroxyl radicals in Gram-negative and Gram-positive bacteria, which ultimately contribute to cell death. We also show, in contrast, that bacteriostatic drugs do not produce hydroxyl radicals. We demonstrate that the mechanism of hydroxyl radical formation induced by bactericidal antibiotics is the end product of an oxidative damage cellular death pathway involving the tricarboxylic acid cycle, a transient depletion of NADH, destabilization of iron-sulfur clusters, and stimulation of the Fenton reaction. Our results suggest that all three major classes of bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage, including proteins involved in triggering the DNA damage response, e.g., RecA.

PrefaceAntibiotic drug-target interactions, and their respective direct effects, are generally wellcharacterized. In contrast, the bacterial responses to antibiotic drug treatments that contribute to cell death are not as well understood and have proven to be quite complex, involving multiple genetic and biochemical pathways. Here, we review the multi-layered effects of drug-target interactions, including the essential cellular processes inhibited by bactericidal antibiotics and the associated cellular response mechanisms that contribute to killing by bactericidal antibiotics. We also discuss new insights into these mechanisms that have been revealed through the study of biological networks, and describe how these insights, together with related developments in synthetic biology, may be exploited to create novel antibacterial therapies. IntroductionOur understanding of how antibiotics induce bacterial cell death is centered on the essential cellular function inhibited by the primary drug-target interaction. Antibiotics can be classified based on the cellular component or system they affect, in addition to whether they induce cell death (bactericidal drugs) or merely inhibit cell growth (bacteriostatic drugs). Most current bactericidal antimicrobials, which are the focus of this review, inhibit DNA synthesis, RNA synthesis, cell wall synthesis, or protein synthesis 1 .Since the discovery of penicillin was reported in 1929 2 , other, more effective antimicrobials have been discovered and developed by elucidation of drug-target interactions, and by drug molecule modification. These efforts have significantly enhanced our clinical armamentarium. Antibiotic-mediated cell death, however, is a complex process that begins with the physical interaction between a drug molecule and its bacterial-specific target, and involves alterations to the affected bacterium at the biochemical, molecular and ultrastructural levels. The increasing prevalence of drug-resistant bacteria 3 , as well as the means of gaining resistance, has made it crucial that we better understand the multilayered mechanisms by which currently available antibiotics kill bacteria, as well as explore and find alternative antibacterial therapies.Antibiotic-induced cell death has been associated with the formation of double-stranded DNA breaks following treatment with DNA gyrase inhibitors 4 , with the arrest of DNA-dependent RNA synthesis following treatment with rifamycins 5 , with cell envelope damage and loss of structural integrity following treatment with cell-wall synthesis inhibitors 6 , and with cellular energetics, ribosome binding and protein mistranslation following treatment with protein * Corresponding author: James Collins, jcollins@bu.edu, phone: (617) 353-0390. NIH Public Access Author ManuscriptNat Rev Microbiol. Author manuscript; available in PMC 2010 December 1. Published in final edited form as:Nat Rev Microbiol. 2010 June ; 8(6): 423-435. doi:10.1038/nrmicro2333. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript sy...

Summary Antibiotic resistance arises through mechanisms such as selection of naturally occurring resistant mutants and horizontal gene transfer. Recently, oxidative stress has been implicated as one of the mechanisms whereby bactericidal antibiotics kill bacteria. Here we show that sub-lethal levels of bactericidal antibiotics induce mutagenesis, resulting in heterogeneous increases in the minimum inhibitory concentration for a range of antibiotics, irrespective of the drug target. This increase in mutagenesis correlates with an increase in ROS, and is prevented by the ROS scavenger thiourea and by anaerobic conditions, indicating that sub-lethal concentrations of antibiotics induce mutagenesis by stimulating the production of ROS. We demonstrate that these effects can lead to mutant strains that are sensitive to the applied antibiotic but resistant to other antibiotics. This work establishes a radical-based molecular mechanism whereby sub-lethal levels of antibiotics can lead to multidrug resistance, which has important implications for the widespread use and misuse of antibiotics.