Non-Traditional Antibacterial Screening Approaches for the Identification of Novel Inhibitors of the Glyoxylate Shunt in Gram-Negative Pathogens

Antibacterial compounds that affect bacterial viability have traditionally been identified, confirmed, and characterized in standard laboratory media. The historical success of identifying new antibiotics via this route has justifiably established a traditional means of screening for new antimicrobials. The emergence of multi-drug-resistant (MDR) bacterial pathogens has expedited the need for new antibiotics, though many in the industry have questioned the source(s) of these new compounds. As many pharmaceutical companies'' chemical libraries have been exhaustively screened via the traditional route, we have concluded that all compounds with any antibacterial potential have been identified. While new compound libraries and platforms are being pursued, it also seems prudent to screen the libraries we currently have in hand using alternative screening approaches. One strategy involves screening under conditions that better reflect the environment pathogens experience during an infection, and identifying in vivo essential targets and pathways that are dispensable for growth in standard laboratory media in vitro. Here we describe a novel screening strategy for identifying compounds that inhibit the glyoxylate shunt in Pseudomonas aeruginosa, a pathway that is required for bacterial survival in the pulmonary environment. We demonstrate that these compounds, which were not previously identified using traditional screening approaches, have broad-spectrum antibacterial activity when they are tested under in vivo-relevant conditions. We also show that these compounds have potent activity on both enzymes that comprise the glyoxylate shunt, a feature that was supported by computational homology modeling. By dual-targeting both enzymes in this pathway, we would expect to see a reduced propensity for resistance development to these compounds. Taken together, these data suggest that understanding the in vivo environment that bacterial pathogens must tolerate, and adjusting the antibacterial screening paradigm to reflect those conditions, could identify novel antibiotics for the treatment of serious MDR pathogens.     

Identification and analysis of novel small molecule inhibitors of RNase E: Implications for antibacterial targeting and regulation of RNase E

Increasing resistance of bacteria to antibiotics is a serious global challenge and there is a need to unlock the potential of novel antibacterial targets. One such target is the essential prokaryotic endoribonuclease RNase E. Using a combination of in silico high-throughput screening and in vitro validation we have identified three novel small molecule inhibitors of RNase E that are active against RNase E from Escherichia coli, Francisella tularensis and Acinetobacter baumannii. Two of the inhibitors are non-natural small molecules that could be suitable as lead compounds for the development of broad-spectrum antibiotics targeting RNase E. The third small molecule inhibitor is glucosamine-6-phosphate, a precursor of bacterial cell envelope peptidoglycans and lipopolysaccharides, hinting at a novel metabolite-mediated mechanism of regulation of RNase E.     

Downregulation of yidC in Escherichia coli by Antisense RNA Expression Results in Sensitization to Antibacterial Essential Oils Eugenol and Carvacrol

Background

The rising drug resistance in pathogenic bacteria and inefficiency of current antibiotics to meet clinical requirements has augmented the need to establish new and innovative approaches for antibacterial drug discovery involving identification of novel antibacterial targets and inhibitors. Being obligatory for bacterial growth, essential gene products are considered vital as drug targets. The bacterial protein YidC is highly conserved among pathogens and is essential for membrane protein insertion due to which it holds immense potential as a promising target for antibacterial therapy.

Methods/Principal Findings

The aim of this study was to explore the feasibility and efficacy of expressed antisense-mediated gene silencing for specific downregulation of yidC in Escherichia coli. We induced RNA silencing of yidC which resulted in impaired growth of the host cells. This was followed by a search for antibacterial compounds sensitizing the YidC depleted cells as they may act as inhibitors of the essential protein or its products. The present findings affirm that reduction of YidC synthesis results in bacterial growth retardation, which warrants the use of this enzyme as a viable target in search of novel antibacterial agents. Moreover, yidC antisense expression in E. coli resulted in sensitization to antibacterial essential oils eugenol and carvacrol. Fractional Inhibitory Concentration Indices (FICIs) point towards high level of synergy between yidC silencing and eugenol/carvacrol treatment. Finally, as there are no known YidC inhibitors, the RNA silencing approach applied in this study put forward rapid means to screen novel potential YidC inhibitors.

Conclusions/Significance

The present results suggest that YidC is a promising candidate target for screening antibacterial agents. High level of synergy reported here between yidC silencing and eugenol/carvacrol treatment is indicative of a potential antibacterial therapy. This is the first report indicating that the essential gene yidC is a therapeutic target of the antibacterial essential oils eugenol and carvacrol in E. coli.     

Antibacterial activity of marine macroalgae against fish pathogenic Vibrio species

In mariculture, diseases of microbial origin can cause significant economic losses worldwide; the evolution of microorganism resistance to antibiotics has resulted in a growing need for new antibacterial compounds that are effective in veterinary medicine and characterized by limited undesirable side effects. Increased attention has recently been turned to seaweeds as a promising source for metabolites with antimicrobial activity. Vibriosis is a common disease, caused by bacteria of the genus Vibrio, that can result in high mortality in aquaculture. The aim of this study was to identify seaweeds with antibacterial activity against some pathogenic Vibrio species, in order to identify a possible alternative to the commonly used antibiotics in aquaculture. Chloroform/methanol lipidic extracts of six seaweed species (Chaetomorpha linum, Cladophora rupestris, Gracilaria dura, Gracilaria gracilis, Gracilariopsis longissima, Ulva prolifera) were tested for their antibacterial activities against six fish pathogenic Vibrio species using the disc diffusion method. Different susceptibilities to lipidic algal extracts were observed. All six of the seaweed extracts tested demonstrated inhibition of Vibrio ordalii. The best was that from Gracilariopsis longissima, showing activity against Vibrio ordalii, Vibrio salmonicida, Vibrio alginolyticus and Vibrio vulnificus. The results confirmed the potential use of seaweed extracts as a source of antibacterial compounds or as a health-promoting feed for aquaculture.     

Identification of cellular targets of a series of boron heterocycles using TIPA II—A sensitive target identification platform

One of the hurdles in the discovery of antibiotics is the difficulty of linking antibacterial compounds to their cellular targets. Our laboratory has employed a genome-wide approach of over-expressing essential genes in order to identify cellular targets of antibacterial inhibitors. Our objective in this project was to develop and validate a more sensitive disk diffusion based platform of target identification (Target Identification Platform for Antibacterials version 2; TIPA II) using a collection of cell clones in an Escherichia coli mutant (AS19) host with increased outer membrane permeability. Five known antibiotics/inhibitors and 28 boron heterocycles were tested by TIPA II assay, in conjunction with the original assay TIPA. The TIPA II was more sensitive than TIPA because eight boron heterocycles previously found to be inactive to AG1 cells in TIPA assays exhibited activity to AS19 cells. For 15 boron heterocycles, resistant colonies were observed within the zones of inhibition only on the inducing plates in TIPA II assays. DNA sequencing confirmed that resistant clones harbor plasmids with fabI gene as insert, indicating that these boron heterocycles all target enoyl ACP reductase. Additionally, cell-based assays and dose response curved obtained indicated that for two boron heterocycle inhibitors, the fabI cell clone in AG1 (wild-type) host cells exhibited at least 11 fold more resistance under induced conditions than under non-induced conditions. Moreover, TIPA II also identified cellular targets of known antibacterial inhibitors triclosan, phosphomycin, trimethoprim, diazaborine and thiolactomycin, further validating the utility of the new system.     

Novel targets of pentacyclic triterpenoids in Staphylococcus aureus: A systematic review

BackgroundStaphylococcus aureus is an important pathogen both in community-acquired and healthcare-associated infections, and has successfully evolved numerous strategies for resisting the action to practically all antibiotics. Resistance to methicillin is now widely described in the community setting (CMRSA), thus the development of new drugs or alternative therapies is urgently necessary. Plants and their secondary metabolites have been a major alternative source in providing structurally diverse bioactive compounds as potential therapeutic agents for the treatment of bacterial infections. One of the classes of natural secondary metabolites from plants with the most bioactive compounds are the triterpenoids, which comprises structurally diverse organic compounds. In nature, triterpenoids are often found as tetra- or penta-cyclic structures.AimThis review highlights the anti-staphylococcal activities of pentacyclic triterpenoids, particularly α-amyrin (AM), betulinic acid (BA) and betulinaldehyde (BE). These compounds are based on a 30-carbon skeleton comprising five six-membered rings (ursanes and lanostanes) or four six-membered rings and one five-membered ring (lupanes and hopanes).MethodsElectronic databases such as ScienceDirect, PubMed and Scopus were used to search scientific contributions until March 2018, using relevant keywords. Literature focusing on the antimicrobial and antibiofilms of effects of pentacyclic triterpenoids on S. aureus were identified and summarized.ResultsPentacyclic triterpenoids can be divided into three representative classes, namely ursane, lupane and oleananes. This class of compounds have been shown to exhibit analgesic, immunomodulatory, anti-inflammatory, anticancer, antioxidant, antifungal and antibacterial activities. In studies of the antimicrobial activities and targets of AM, BA and BE in sensitive and multidrug-resistant S. aureus, these compounds acted synergistically and have different targets from the conventional antibiotics.ConclusionThe inhibitory mechanisms of S. aureus in novel targets and pathways should stimulate further researches to develop AM, BA and BE as therapeutic agents for infections caused by S. aureus. Continued efforts to identify and exploit synergistic combinations by the three compounds and peptidoglycan inhibitors, are also necessary as alternative treatment options for S. aureus infections.     

Alterations in the antibacterial potential of Synechococcus spp. PCC7942 under the influence of UV-B radiations on skin pathogens

Marine organisms are seen as a source of novel drugs and the discovery of new pharmaceutical is increasingly in demand. Cyanobacteria are regarded as a potential target for this as antibacterial, antiviral, antifungal, algicide and cytotoxic activities have been reported in these organisms. They have been identified as a new and rich source of bioactive compounds belonging to diversified groups. Radiation in the UV-B range interferes with various metabolic reactions by generating free radicals and active oxygen species. These deleterious compounds are inactivated by antioxidants. Among them are the carotenoids and phycocyanin which protect against photodynamic action in different ways. Stress plays an important role in the production of bioactive metabolites from organisms. Synechococcus spp. PCC7942 was studied for antibacterial activity against various pathogenic bacteria resistant to a number of available antibiotics after being exposed to UV-B radiation. The antibacterial activity of Synechococcus spp. PCC7942 was studied on five potent skin pathogens. The highest antibacterial activity was seen the methanol extracts of 24 h UV-B exposed cultures of Synechococcus spp. PCC7942. It can be concluded that there was moderate antibacterial activity. Results showed stress, solvent and dose-dependent activity. This antibacterial activity might be due to the enhanced synthesis of carotenoids and phycocyanin under UV-B stress. The purpose of the present study was to relate the inhibitory effects of the cyanobacterial compounds specifically on skin pathogens with exposure to UV-B radiation as UV protecting compounds are already reported in these organisms.     

Relacin,a Novel Antibacterial Agent Targeting the Stringent Response

Finding bacterial cellular targets for developing novel antibiotics has become a major challenge in fighting resistant pathogenic bacteria. We present a novel compound, Relacin, designed to inhibit (p)ppGpp production by the ubiquitous bacterial enzyme RelA that triggers the Stringent Response. Relacin inhibits RelA in vitro and reduces (p)ppGpp production in vivo. Moreover, Relacin affects entry into stationary phase in Gram positive bacteria, leading to a dramatic reduction in cell viability. When Relacin is added to sporulating Bacillus subtilis cells, it strongly perturbs spore formation regardless of the time of addition. Spore formation is also impeded in the pathogenic bacterium Bacillus anthracis that causes the acute anthrax disease. Finally, the formation of multicellular biofilms is markedly disrupted by Relacin. Thus, we establish that Relacin, a novel ppGpp analogue, interferes with bacterial long term survival strategies, placing it as an attractive new antibacterial agent.     

Marine bacteria: potential sources for compounds to overcome antibiotic resistance

Methicillin-resistant Staphylococcus aureus (MRSA) is the most problematic Gram-positive bacterium in the context of public health due to its resistance against almost all available antibiotics except vancomycin and teicoplanin. Moreover, glycopeptide-resistant S. aureus have been emerging with the increasing use of glycopeptides. Recently, resistant strains against linezolid and daptomycin, which are alternative drugs to treat MRSA infection, have also been reported. Thus, the development of new drugs or alternative therapies is clearly a matter of urgency. In response to the antibiotic resistance, many researchers have studied for alternative antibiotics and therapies. In this review, anti-MRSA substances isolated from marine bacteria, with their potential antibacterial effect against MRSA as potential anti-MRSA agents, are discussed and several strategies for overcoming the antibiotic resistance are also introduced. Our objective was to highlight marine bacteria that have potential to lead in developing novel antibiotics or clinically useful alternative therapeutic treatments.     

Discovery of antimicrobial compounds targeting bacterial type FAD synthetases

The increase of bacterial strains resistant to most of the available antibiotics shows a need to explore novel antibacterial targets to discover antimicrobial drugs. Bifunctional bacterial FAD synthetases (FADSs) synthesise the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These cofactors act in vital processes as part of flavoproteins, making FADS an essential enzyme. Bacterial FADSs are potential antibacterial targets because of differences to mammalian enzymes, particularly at the FAD producing site. We have optimised an activity-based high throughput screening assay targeting Corynebacterium ammoniagenes FADS (CaFADS) that identifies inhibitors of its different activities. We selected the three best high-performing inhibitors of the FMN:adenylyltransferase activity (FMNAT) and studied their inhibition mechanisms and binding properties. The specificity of the CaFADS hits was evaluated by studying also their effect on the Streptococcus pneumoniae FADS activities, envisaging differences that can be used to discover species-specific antibacterial drugs. The antimicrobial effect of these compounds was also evaluated on C. ammoniagenes, S. pneumoniae, and Mycobacterium tuberculosis cultures, finding hits with favourable antimicrobial properties.     

3-Phenyl substituted 6,7-dimethoxyisoquinoline derivatives as FtsZ-targeting antibacterial agents

The emergence of multidrug-resistant bacteria has created an urgent need for antibiotics with a novel mechanism of action. The bacterial cell division protein FtsZ is an attractive target for the development of novel antibiotics. The benzo[c]phenanthridinium sanguinarine and the dibenzo[a,g]quinolizin-7-ium berberine are two structurally similar plant alkaloids that alter FtsZ function. The presence of a hydrophobic functionality at either the 1-position of 5-methylbenzo[c]phenanthridinium derivatives or the 2-position of dibenzo[a,g]quinolizin-7-ium derivatives is associated with significantly enhanced antibacterial activity. 3-Phenylisoquinoline represents a subunit within the ring-systems of both of these alkaloids. Several 3-phenylisoquinolines and 3-phenylisoquinolinium derivatives have been synthesized and evaluated for antibacterial activity against Staphylococcus aureus and Enterococcus faecalis, including multidrug-resistant strains of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). A number of derivatives were found to have activity against both MRSA and VRE. The binding of select compounds to S. aureus FtsZ (SaFtsZ) was demonstrated and characterized using fluorescence spectroscopy. In addition, the compounds were shown to act as stabilizers of SaFtsZ polymers and concomitant inhibitors of SaFtsZ GTPase activity. Toxicological assessment of select compounds revealed minimal cross-reaction mammalian β-tubulin as well as little or no human cytotoxicity.     

Antibacterial activity of 3-methylbenzo[d]thiazol-methylquinolinium derivatives and study of their action mechanism

The increasing incidence of multidrug resistant bacterial infection renders an urgent need for the development of new antibiotics. To develop small molecules disturbing FtsZ activity has been recognized as promising approach to search for antibacterial of high potency systematically. Herein, a series of novel quinolinium derivatives were synthesized and their antibacterial activities were investigated. The compounds show strong antibacterial activities against different bacteria strains including MRSA, VRE and NDM-1 Escherichia coli. Among these derivatives, a compound bearing a 4-fluorophenyl group (A2) exhibited a superior antibacterial activity and its MICs to the drug-resistant strains are found lower than those of methicillin and vancomycin. The biological results suggest that these quinolinium derivatives can disrupt the GTPase activity and dynamic assembly of FtsZ, and thus inhibit bacterial cell division and then cause bacterial cell death. These compounds deserve further evaluation for the development of new antibacterial agents targeting FtsZ.     

The decline of antibiotic era--new approaches for antibacterial drug discovery

Infectious diseases still remain the main cause of human premature deaths; especially in developing countries. The emergence and spread of pathogenic bacteria resistant to many antibiotics (multidrug-resistant strains) have created the need for the development of novel therapeutic agents. Only two new classes of antibiotics of novel mechanisms of action (linezolid and daptomycin) have been introduced into the market during the last three decades. The recent progress in molecular biology and bacterial genome analysis has had an enormous impact on antibacterial drug research. This review presents new achievements in searching a new bacterial essential genes, a potential targets for antibacterial drugs. Application of metagenomics strategy is also shown. Some recent technologies aimed at development of anti-pathogenic drugs such as inhibitors of quorum sensing process or histidine kinases are also discussed. Extensive research efforts have provided many details concerning structure of bacterial proteins playing an important role in pathogenesis such as adherence proteins or toxins, what allowed searching for antitoxin drugs or drugs interfering with bacterial adhesion. As an example, the review focuses on anthrax therapies under development. Additionally, the article presents the progress in phage therapy; using bacteriophages or their products such as lysins in antibacterial therapy.     

Chalcone derivatives and their antibacterial activities: Current development

The increase in antibiotic resistance due to various factors has encouraged the look for novel compounds which are active against multidrug-resistant pathogens. In this framework, chalcone-based compounds showed a diversity of pharmacological properties, and its derivatives possess a high degree of structural diversity, and it is helpful for the discovery of new therapeutic agents. The growing resistance to antibiotics worldwide has endangered their efficacy. This has led to a surging interest in the discovery of new antibacterial agents. Thus, there is an urgent need for new antibacterial drug candidates with increased strength, new targets, low cost, superior pharmacokinetic properties, and minimum side effects. The present review concluded and focuses on the recent developments in the area of medicinal chemistry to explore the diverse chemical structures of potent antibacterial agents and also describes its structure-activity relationships studies. The various synthetic structures leading to this class of neutral protective compound is common and additional structural optimization is promising for potential drug discovery and development.     

Development of ebsulfur analogues as potent antibacterials against methicillin-resistant Staphylococcus aureus

Antibiotic resistance is a worldwide problem that needs to be addressed. Staphylococcus aureus is one of the dangerous “ESKAPE” pathogens that rapidly evolve and evade many current FDA-approved antibiotics. Thus, there is an urgent need for new anti-MRSA compounds. Ebselen (also known as 2-phenyl-1,2-benzisoselenazol-3(2H)-one) has shown promising activity in clinical trials for cerebral ischemia, bipolar disorder, and noise-induced hearing loss. Recently, there has been a renewed interest in exploring the antibacterial properties of ebselen. In this study, we synthesized an ebselen-inspired library of 33 compounds where the selenium atom has been replaced by sulfur (ebsulfur derivatives) and evaluated them against a panel of drug-sensitive and drug-resistant S. aureus and non-S. aureus strains. Within our library, we identified three outstanding analogues with potent activity against all S. aureus strains tested (MIC values mostly ⩽2 μg/mL), and numerous additional ones with overall very good to good antibacterial activity (1–7.8 μg/mL). We also characterized the time-kill analysis, anti-biofilm ability, hemolytic activity, mammalian cytotoxicity, membrane-disruption ability, and reactive oxygen species (ROS) production of some of these analogues.     

Optimization of physicochemical properties and safety profile of novel bacterial topoisomerase type II inhibitors (NBTIs) with activity against Pseudomonas aeruginosa

Type II bacterial topoisomerases are well validated targets for antimicrobial chemotherapy. Novel bacterial type II topoisomerase inhibitors (NBTIs) of these targets are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. We now disclose the optimization of a class of NBTIs towards Gram-negative pathogens, especially against drug-resistant Pseudomonas aeruginosa. Physicochemical properties (pK_a and log D) were optimized for activity against P. aeruginosa and for reduced inhibition of the hERG channel. The optimized analogs 9g and 9i displayed potent antibacterial activity against P. aeruginosa, and a significantly improved hERG profile over previously reported analogs. Compound 9g showed an improved QT profile in in vivo models and lower clearance in rat over earlier compounds. The compounds show promise for the development of new antimicrobial agents against drug-resistant Pseudomonas aeruginosa.     

Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps.     

Insights into the Function of YciM,a Heat Shock Membrane Protein Required To Maintain Envelope Integrity in Escherichia coli

The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function.     

The Naphthoquinone Diospyrin Is an Inhibitor of DNA Gyrase with a Novel Mechanism of Action

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.     

Hidden antibiotics: Where to uncover?

The struggle of humans versus pathogens is a never ending battle. Since the discovery of antibiotics humans have tipped the scales in their favour, but today bacteria are nullifying this advantage by developing resistance mechanisms against these molecules. The plethora of different antibiotics active against pathogens is shrinking while the discovery of new molecules is arduous. Especially the development of drugs active against Gram^? pathogens continues slowly. New strategies to discover novel, potent antibiotics are hence needed. Adopting the optimistic view of technological singularity, innovative and disruptive approaches are required and hence proposed to lift the current conundrum. In this review, questions are answered on where and how to look for new natural product hit molecules with antibacterial activity, on how the field of synthetic biology can aid the contemporary pharmaceutical challenge and whether we are ready to make the transition towards other approaches, such as narrow-spectrum antibiotics and phage therapy.