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,...

Bacteriostatic and bactericidal antibiotic treatments result in two fundamentally different phenotypic outcomes-the inhibition of bacterial growth or, alternatively, cell death. Most antibiotics inhibit processes that are major consumers of cellular energy output, suggesting that antibiotic treatment may have important downstream consequences on bacterial metabolism. We hypothesized that the specific metabolic effects of bacteriostatic and bactericidal antibiotics contribute to their overall efficacy. We leveraged the opposing phenotypes of bacteriostatic and bactericidal drugs in combination to investigate their activity. Growth inhibition from bacteriostatic antibiotics was associated with suppressed cellular respiration whereas cell death from most bactericidal antibiotics was associated with accelerated respiration. In combination, suppression of cellular respiration by the bacteriostatic antibiotic was the dominant effect, blocking bactericidal killing. Global metabolic profiling of bacteriostatic antibiotic treatment revealed that accumulation of metabolites involved in specific drug target activity was linked to the buildup of energy metabolites that feed the electron transport chain. Inhibition of cellular respiration by knockout of the cytochrome oxidases was sufficient to attenuate bactericidal lethality whereas acceleration of basal respiration by genetically uncoupling ATP synthesis from electron transport resulted in potentiation of the killing effect of bactericidal antibiotics. This work identifies a link between antibiotic-induced cellular respiration and bactericidal lethality and demonstrates that bactericidal activity can be arrested by attenuated respiration and potentiated by accelerated respiration. Our data collectively show that antibiotics perturb the metabolic state of bacteria and that the metabolic state of bacteria impacts antibiotic efficacy.

Summary Understanding how antibiotics impact bacterial metabolism may provide insight into their mechanisms of action and could lead to enhanced therapeutic methodologies. Here, we profiled the metabolome of Escherichia coli after treatment with three different classes of bactericidal antibiotics (beta-lactams, aminoglycosides, quinolones). These treatments induced a similar set of metabolic changes after 30 minutes that then diverged into more distinct profiles at later timepoints. The most striking changes corresponded to elevated concentrations of central carbon metabolites, active breakdown of the nucleotide pool, reduced lipid levels, and evidence of an elevated redox state. We examined potential end-target consequences of these metabolic perturbations and found that antibiotic-treated cells exhibited cytotoxic changes indicative of oxidative stress, including higher levels of protein carbonylation, malondialdehyde adducts, nucleotide oxidation, and double-strand DNA breaks. This work shows that bactericidal antibiotics induce a complex set of metabolic changes that are correlated with the buildup of toxic metabolic by-products.

Summary Microbial drug persistence is a widespread phenomenon in which a sub-population of microorganisms is able to survive antimicrobial treatment without acquiring resistance-conferring genetic changes. Microbial persisters can cause recurrent or intractable infections, and like resistant mutants, they carry an increasing clinical burden. In contrast to heritable drug resistance, however, the biology of persistence is only beginning to be unraveled. Persisters have traditionally been thought of as metabolically dormant, non-dividing cells. However, as discussed in this review, increasing evidence suggests that persistence is in fact an actively maintained state, triggered and enabled by a network of intracellular stress-responses that can accelerate processes of adaptive evolution. Beyond shedding light on the basis of persistence, these findings raise the possibility that persisters behave as an evolutionary reservoir from which resistant organisms can emerge. As persistence and its consequences come into clearer focus, clinically relevant eradication strategies are urgently needed.

SUMMARY Metabolically dormant bacteria present a critical challenge to effective antimicrobial therapy because these bacteria are genetically susceptible to antibiotic treatment but phenotypically tolerant. Such tolerance has been attributed to impaired drug uptake, which can be reversed by metabolic stimulation. Here, we evaluate the effects of central carbon metabolite stimulations on aminoglycoside sensitivity in the pathogen Pseudomonas aeruginosa. We identify fumarate as a tobramycin potentiator that activates cellular respiration and generates a proton motive force by stimulating the tricarboxylic acid (TCA) cycle. In contrast, we find that glyoxylate induces phenotypic tolerance by inhibiting cellular respiration with acetyl-coenzyme A diversion through the glyoxylate shunt, despite drug import. Collectively, this work demonstrates that TCA cycle activity is important for both aminoglycoside uptake and downstream lethality and identifies a potential strategy for potentiating aminoglycoside treatment of P. aeruginosa infections.

Antibiotics target energy-consuming processes. As such, perturbations to bacterial metabolic homeostasis are significant consequences of treatment. Here, we describe three postulates that collectively define antibiotic efficacy in the context of bacterial metabolism: (1) antibiotics alter the metabolic state of bacteria, which contributes to the resulting death or stasis; (2) the metabolic state of bacteria influences their susceptibility to antibiotics; and (3) antibiotic efficacy can be enhanced by altering the metabolic state of bacteria. Altogether, we aim to emphasize the close relationship between bacterial metabolism and antibiotic efficacy as well as propose areas of exploration to develop novel antibiotics that optimally exploit bacterial metabolic networks.

Continual human immunodeficiency virus type 1 (HIV-1) evolution and expansion within the human population have led to unequal distribution of HIV-1 group M subtypes. In particular, recent outgrowth of subtype C in southern Africa, India, and China has fueled speculation that subtype C isolates may be more fit in vivo. In this study, nine subtype B and six subtype C HIV-1 isolates were added to peripheral blood mononuclear cell cultures for a complete pairwise competition experiment. All subtype C HIV-1 isolates were less fit than subtype B isolates (P < 0.0001), but intrasubtype variations in HIV-1 fitness were not significant. Increased fitness of subtype B over subtype C was also observed in primary CD4؉ T cells and macrophages from different human donors but not in skin-derived human Langerhans cells. Detailed analysis of the retroviral life cycle during several B and C virus competitions indicated that the efficiency of host cell entry may have a significant impact on relative fitness. Furthermore, phyletic analyses of fitness differences suggested that, for a recombined subtype B/C HIV-1 isolate, higher fitness mapped to the subtype B env gene rather than the subtype C gag and pol genes. These results suggest that subtype B and C HIV-1 may be transmitted with equal efficiency (Langerhans cell data) but that subtype C isolates may be less fit following initial infection (T-cell and macrophage data) and may lead to slower disease progression.

Human lymphocyte antigen (HLA)-restricted CD8+ cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escape mutations emerging in seven CTL epitopes in the gp120 Env and p24 Gag coding regions of an individual followed longitudinally from the time of acute HIV-1 infection, as well as some of these same epitopes recognized in other HIV-1-infected individuals. Nine dominant mutations appeared in five gp120 epitopes within the first year of infection, whereas all four mutations found in two p24 epitopes emerged after nearly two years of infection. These mutations were introduced individually into the autologous gene found in acute infection and then placed into a full-length, infectious viral genome. When competed against virus expressing the parental protein, fitness loss was observed with only one of the nine gp120 mutations, whereas four had no effect and three conferred a slight increase in fitness. In contrast, mutations conferring CTL escape in the p24 epitopes significantly decreased viral fitness. One particular escape mutation within a p24 epitope was associated with reduced peptide recognition and high viral fitness costs but was replaced by a fitness-neutral mutation. This mutation appeared to alter epitope processing concomitant with a reduced CTL response. In conclusion, CTL escape mutations in HIV-1 Gag p24 were associated with significant fitness costs, whereas most escape mutations in the Env gene were fitness neutral, suggesting a balance between immunologic escape and replicative fitness costs.