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The looming antibiotic-resistance crisis has penetrated the consciousness of clinicians, researchers, policymakers, politicians and the public at large. The evolution and widespread distribution of antibiotic-resistance elements in bacterial pathogens has made diseases that were once easily treatable deadly again. Unfortunately, accompanying the rise in global resistance is a failure in antibacterial drug discovery. Lessons from the history of antibiotic discovery and fresh understanding of antibiotic action and the cell biology of microorganisms have the potential to deliver twenty-first century medicines that are able to control infection in the resistance era.

Antimicrobial resistance continues to be a public threat on a global scale. The ongoing need to develop new antimicrobial drugs that are effective against multi-drug-resistant pathogens has spurred the research community to invest in various drug discovery strategies, one of which is drug repurposing—the process of finding new uses for existing drugs. While still nascent in the antimicrobial field, the approach is gaining traction in both the public and private sector. While the approach has particular promise in fast-tracking compounds into clinical studies, it nevertheless has substantial obstacles to success. This Review covers the art of repurposing existing drugs for antimicrobial purposes. We discuss enabling screening platforms for antimicrobial discovery and present encouraging findings of novel antimicrobial therapeutic strategies. Also covered are general advantages of repurposing over de novo drug development and challenges of the strategy, including scientific, intellectual property and regulatory issues. This Review describes the potential opportunities for finding new uses as antimicrobials for existing drugs, the approaches used for screening and the scientific, intellectual property and regulatory challenges to be overcome.

The spread of antibiotic resistance is an urgent threat to global health that necessitates new therapeutics. Treatments for Gram-negative pathogens are particularly challenging to identify due to the robust outer membrane permeability barrier in these organisms. Recent discovery efforts have attempted to overcome this hurdle by disrupting the outer membrane using chemical perturbants and have yielded several new peptides and small molecules that allow the entry of otherwise inactive antimicrobials. However, a comprehensive investigation into the strengths and limitations of outer membrane perturbants as antibiotic partners is currently lacking. Herein, we interrogate the interaction between outer membrane perturbation and several common impediments to effective antibiotic use. Interestingly, we discover that outer membrane disruption is able to overcome intrinsic, spontaneous, and acquired antibiotic resistance in Gram-negative bacteria, meriting increased attention toward this approach. Disruption of the outer membrane (OM) barrier allows for the entry of otherwise inactive antimicrobials into Gram-negative pathogens. Numerous efforts to implement this approach have identified a large number of OM perturbants that sensitize Gram-negative bacteria to many clinically available Gram-positive active antibiotics. However, there is a dearth of investigation into the strengths and limitations of this therapeutic strategy, with an overwhelming focus on characterization of individual potentiator molecules. Herein, we look to explore the utility of exploiting OM perturbation to sensitize Gram-negative pathogens to otherwise inactive antimicrobials. We identify the ability of OM disruption to change the rules of Gram-negative entry, overcome preexisting and spontaneous resistance, and impact biofilm formation. Disruption of the OM expands the threshold of hydrophobicity compatible with Gram-negative activity to include hydrophobic molecules. We demonstrate that while resistance to Gram-positive active antibiotics is surprisingly common in Gram-negative pathogens, OM perturbation overcomes many antibiotic inactivation determinants. Further, we find that OM perturbation reduces the rate of spontaneous resistance to rifampicin and impairs biofilm formation. Together, these data suggest that OM disruption overcomes many of the traditional hurdles encountered during antibiotic treatment and is a high priority approach for further development. IMPORTANCE The spread of antibiotic resistance is an urgent threat to global health that necessitates new therapeutics. Treatments for Gram-negative pathogens are particularly challenging to identify due to the robust outer membrane permeability barrier in these organisms. Recent discovery efforts have attempted to overcome this hurdle by disrupting the outer membrane using chemical perturbants and have yielded several new peptides and small molecules that allow the entry of otherwise inactive antimicrobials. However, a comprehensive investigation into the strengths and limitations of outer membrane perturbants as antibiotic partners is currently lacking. Herein, we interrogate the interaction between outer membrane perturbation and several common impediments to effective antibiotic use. Interestingly, we discover that outer membrane disruption is able to overcome intrinsic, spontaneous, and acquired antibiotic resistance in Gram-negative bacteria, meriting increased attention toward this approach.

Plasmid-borne colistin resistance mediated by mcr-1 may contribute to the dissemination of pan-resistant Gram-negative bacteria. Here, we show that mcr-1 confers resistance to colistin-induced lysis and bacterial cell death, but provides minimal protection from the ability of colistin to disrupt the Gram-negative outer membrane. Indeed, for colistin-resistant strains of Enterobacteriaceae expressing plasmid-borne mcr-1 , clinically relevant concentrations of colistin potentiate the action of antibiotics that, by themselves, are not active against Gram-negative bacteria. The result is that several antibiotics, in combination with colistin, display growth-inhibition at levels below their corresponding clinical breakpoints. Furthermore, colistin and clarithromycin combination therapy displays efficacy against mcr-1 -positive Klebsiella pneumoniae in murine thigh and bacteremia infection models at clinically relevant doses. Altogether, these data suggest that the use of colistin in combination with antibiotics that are typically active against Gram-positive bacteria poses a viable therapeutic alternative for highly drug-resistant Gram-negative pathogens expressing mcr-1 . The plasmid-borne mcr-1 gene confers resistance to the antibiotic colistin. Here, MacNair et al. show that mcr-1 positive bacteria are however susceptible to colistin-mediated disruption of the outer membrane, and can be killed in vitro and in vivo by combining colistin with other antibiotics.

The systematic exploration of off-patent drugs in combination with the antibiotic minocycline uncovers unexpected synergies in antibiotic-nonantibiotic pairs. These interactions are exemplified by the nonantibiotic loperamide, which finds a new function in facilitating tetracycline uptake. Combinations of antibiotics are commonly used in medicine to broaden antimicrobial spectrum and generate synergistic effects. Alternatively, combination of nonantibiotic drugs with antibiotics offers an opportunity to sample a previously untapped expanse of bioactive chemical space. We screened a collection of drugs to identify compounds that augment the activity of the antibiotic minocycline. Unexpected synergistic drug combinations exhibited in vitro and in vivo activity against bacterial pathogens, including multidrug–resistant isolates.

Salmonella Typhimurium ( S . Tm) establishes systemic infection in susceptible hosts by evading the innate immune response and replicating within host phagocytes. Here, we sought to identify inhibitors of intracellular S . Tm replication by conducting parallel chemical screens against S . Tm growing in macrophage-mimicking media and within macrophages. We identify several compounds that inhibit Salmonella growth in the intracellular environment and in acidic, ion-limited media. We report on the antimicrobial activity of the psychoactive drug metergoline, which is specific against intracellular S . Tm. Screening an S . Tm deletion library in the presence of metergoline reveals hypersensitization of outer membrane mutants to metergoline activity. Metergoline disrupts the proton motive force at the bacterial cytoplasmic membrane and extends animal survival during a systemic S . Tm infection. This work highlights the predictive nature of intracellular screens for in vivo efficacy, and identifies metergoline as a novel antimicrobial active against Salmonella . Salmonella Typhimurium replicates within host phagocytes. Here, the authors show that a known psychoactive drug (metergoline) inhibits growth of the pathogen in macrophages, disrupts the proton motive force at the bacterial cytoplasmic membrane, and extends animal survival during systemic infection.

Exposure of Escherichia coli to sub-inhibitory antibiotics stimulates biofilm formation through poorly characterized mechanisms. Using a high-throughput Congo Red binding assay to report on biofilm matrix production, we screened ~4000 E . coli K12 deletion mutants for deficiencies in this biofilm stimulation response. We screened using three different antibiotics to identify core components of the biofilm stimulation response. Mutants lacking acnA , nuoE , or lpdA failed to respond to sub-MIC cefixime and novobiocin, implicating central metabolism and aerobic respiration in biofilm stimulation. These genes are members of the ArcA/B regulon–controlled by a respiration-sensitive two-component system. Mutants of arcA and arcB had a ‘pre-activated’ phenotype, where biofilm formation was already high relative to wild type in vehicle control conditions, and failed to increase further with the addition of sub-MIC cefixime. Using a tetrazolium dye and an in vivo NADH sensor, we showed spatial co-localization of increased metabolic activity with sub-lethal concentrations of the bactericidal antibiotics cefixime and novobiocin. Supporting a role for respiratory stress, the biofilm stimulation response to cefixime and novobiocin was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of a gene encoding part of the machinery for respiring nitrate abolished its ameliorating effects, and nitrate respiration increased during growth with sub-MIC cefixime. Finally, in probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors, unlike other antibiotic classes, was minimally affected by nitrate supplementation, suggesting that targeting the ribosome stimulates biofilm formation in distinct ways. By characterizing the biofilm stimulation response to sub-MIC antibiotics at a systems level, we identified multiple avenues for design of therapeutics that impair bacterial stress management.

The increasing use of polymyxins 1 in addition to the dissemination of plasmid-borne colistin resistance threatens to cause a serious breach in our last line of defence against multidrug-resistant Gram-negative pathogens, and heralds the emergence of truly pan-resistant infections. Colistin resistance often arises through covalent modification of lipid A with cationic residues such as phosphoethanolamine—as is mediated by Mcr-1 (ref. 2 )—which reduce the affinity of polymyxins for lipopolysaccharide 3 . Thus, new strategies are needed to address the rapidly diminishing number of treatment options for Gram-negative infections 4 . The difficulty in eradicating Gram-negative bacteria is largely due to their highly impermeable outer membrane, which serves as a barrier to many otherwise effective antibiotics 5 . Here, we describe an unconventional screening platform designed to enrich for non-lethal, outer-membrane-active compounds with potential as adjuvants for conventional antibiotics. This approach identified the antiprotozoal drug pentamidine 6 as an effective perturbant of the Gram-negative outer membrane through its interaction with lipopolysaccharide. Pentamidine displayed synergy with antibiotics typically restricted to Gram-positive bacteria, yielding effective drug combinations with activity against a wide range of Gram-negative pathogens in vitro , and against systemic Acinetobacter baumannii infections in mice. Notably, the adjuvant activity of pentamidine persisted in polymyxin-resistant bacteria in vitro and in vivo . Overall, pentamidine and its structural analogues represent unexploited molecules for the treatment of Gram-negative infections, particularly those having acquired polymyxin resistance determinants. A screen for outer-membrane permeating compounds finds that the clinically approved antiprotozoal drug pentamidine sensitizes drug-resistant Gram-negative pathogens to new antibiotic classes.

While there has been much study of bacterial gene dispensability, there is a lack of comprehensive genome-scale examinations of the impact of gene deletion on growth in different carbon sources. In this context, a lot can be learned from such experiments in the model microbe Escherichia coli where much is already understood and there are existing tools for the investigation of carbon metabolism and physiology (1). Gene deletion studies have practical potential in the field of antibiotic drug discovery where there is emerging interest in bacterial central metabolism as a target for new antibiotics (2). Furthermore, some carbon utilization pathways have been shown to be critical for initiating and maintaining infection for certain pathogens and sites of infection (3–5). Here, with the use of high-throughput solid medium phenotyping methods, we have generated kinetic growth measurements for 3,796 genes under 30 different carbon source conditions. This data set provides a foundation for research that will improve our understanding of genes with unknown function, aid in predicting potential antibiotic targets, validate and advance metabolic models, and help to develop our understanding of E. coli metabolism. Central metabolism is a topic that has been studied for decades, and yet, this process is still not fully understood in Escherichia coli , perhaps the most amenable and well-studied model organism in biology. To further our understanding, we used a high-throughput method to measure the growth kinetics of each of 3,796 E. coli single-gene deletion mutants in 30 different carbon sources. In total, there were 342 genes (9.01%) encompassing a breadth of biological functions that showed a growth phenotype on at least 1 carbon source, demonstrating that carbon metabolism is closely linked to a large number of processes in the cell. We identified 74 genes that showed low growth in 90% of conditions, defining a set of genes which are essential in nutrient-limited media, regardless of the carbon source. The data are compiled into a Web application, Carbon Phenotype Explorer (CarPE), to facilitate easy visualization of growth curves for each mutant strain in each carbon source. Our experimental data matched closely with the predictions from the EcoCyc metabolic model which uses flux balance analysis to predict growth phenotypes. From our comparisons to the model, we found that, unexpectedly, phosphoenolpyruvate carboxylase ( ppc ) was required for robust growth in most carbon sources other than most trichloroacetic acid (TCA) cycle intermediates. We also identified 51 poorly annotated genes that showed a low growth phenotype in at least 1 carbon source, which allowed us to form hypotheses about the functions of these genes. From this list, we further characterized the ydhC gene and demonstrated its role in adenosine efflux. IMPORTANCE While there has been much study of bacterial gene dispensability, there is a lack of comprehensive genome-scale examinations of the impact of gene deletion on growth in different carbon sources. In this context, a lot can be learned from such experiments in the model microbe Escherichia coli where much is already understood and there are existing tools for the investigation of carbon metabolism and physiology (1). Gene deletion studies have practical potential in the field of antibiotic drug discovery where there is emerging interest in bacterial central metabolism as a target for new antibiotics (2). Furthermore, some carbon utilization pathways have been shown to be critical for initiating and maintaining infection for certain pathogens and sites of infection (3–5). Here, with the use of high-throughput solid medium phenotyping methods, we have generated kinetic growth measurements for 3,796 genes under 30 different carbon source conditions. This data set provides a foundation for research that will improve our understanding of genes with unknown function, aid in predicting potential antibiotic targets, validate and advance metabolic models, and help to develop our understanding of E. coli metabolism.

Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks andmechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase inStaphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis inS. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery.