Significance: The global burden of antibiotic resistance has created a demand to better understand the basic mechanisms of existing antibiotics. Of significant interest is how antibiotics may perturb bacterial metabolism, and how bacterial metabolism may influence antibiotic activity. Here, we study the interaction of bacteriostatic and bactericidal antibiotics, the two major phenotypic drug classes. Interestingly, the two classes differentially perturb bacterial cellular respiration, with major consequences for their intrinsic activity both alone and in combination. Of note, bacteriostatic antibiotics decelerate cellular respiration, generating a metabolic state that is prohibitive to killing. Further, we show that the efficacy of bactericidal drugs can be improved by increasing basal respiration, and we identify a respiration-related drug target that potentiates the activity of bactericidal antibiotics.

Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies.

Physiologic and environmental factors can modulate antibiotic activity and thus pose a significant challenge to antibiotic treatment. The quinolone class of antibiotics, which targets bacterial topoisomerases, fails to kill bacteria that have grown to high density; however, the mechanistic basis for this persistence is unclear. Here, we show that exhaustion of the metabolic inputs that couple carbon catabolism to oxidative phosphorylation is a primary cause of growth phase-dependent persistence to quinolone antibiotics. Supplementation of stationary-phase cultures with glucose and a suitable terminal electron acceptor to stimulate respiratory metabolism is sufficient to sensitize cells to quinolone killing. Using this approach, we successfully sensitize high-density populations of Escherichia coli, Staphylococcus aureus, and Mycobacterium smegmatis to quinolone antibiotics. Our findings link growth-dependent quinolone persistence to discrete impairments in respiratory metabolism and identify a strategy to kill non-dividing bacteria.

James Collins and colleagues explore the role of the bacterial epigenome in antibiotic stress survival. They find that Escherichia coli survival under antibiotic pressure is strongly compromised in the absence of adenine methylation at GATC sites, suggesting that targeting adenine methylation might be a viable approach to enhance antibiotic activity. Antibiotic resistance is an increasingly serious public health threat1. Understanding pathways allowing bacteria to survive antibiotic stress may unveil new therapeutic targets2,3,4,5,6,7,8. We explore the role of the bacterial epigenome in antibiotic stress survival using classical genetic tools and single-molecule real-time sequencing to characterize genomic methylation kinetics. We find that Escherichia coli survival under antibiotic pressure is severely compromised without adenine methylation at GATC sites. Although the adenine methylome remains stable during drug stress, without GATC methylation, methyl-dependent mismatch repair (MMR) is deleterious and, fueled by the drug-induced error-prone polymerase Pol IV, overwhelms cells with toxic DNA breaks. In multiple E. coli strains, including pathogenic and drug-resistant clinical isolates, DNA adenine methyltransferase deficiency potentiates antibiotics from the β-lactam and quinolone classes. This work indicates that the GATC methylome provides structural support for bacterial survival during antibiotic stress and suggests targeting bacterial DNA methylation as a viable approach to enhancing antibiotic activity.

Nowadays, the emergence and spread of antibiotic resistance have become an utmost medical and economical problem. It has also become evident that subinhibitory concentrations of antibiotics, which pollute all kind of terrestrial and aquatic environments, have a non-negligible effect on the evolution of antibiotic resistance in bacterial populations. Subinhibitory concentrations of antibiotics have a strong effect on mutation rates, horizontal gene transfer and biofilm formation, which may all contribute to the emergence and spread of antibiotic resistance. Therefore, the molecular mechanisms and the evolutionary pressures shaping the bacterial responses to subinhibitory concentrations of antibiotics merit to be extensively studied. Such knowledge is valuable for the development of strategies to increase the efficacy of antibiotic treatments and to extend the lifetime of antibiotics used in therapy by slowing down the emergence of antibiotic resistance.