BACKGROUND:Zoliflodacin, a first-in-class oral bacterial, DNA gyrase (GyrB) inhibitor, showed non-inferiority to ceftriaxone combined with azithromycin in a recent large international, phase 3, randomised controlled trial for treatment of uncomplicated urogenital gonorrhoea. The aim of this study was to describe the microbiological and whole-genome sequencing (WGS) analyses of paired baseline (pre-treatment) and test-of-cure (TOC) gonococcal isolates from the zoliflodacin phase 3, randomised controlled trial to further characterise and evaluate the protocol-specified microbiological failures with zoliflodacin (n=22) or ceftriaxone and azithromycin (n=1). METHODS:In this retrospective, genomic, observational study, results from antimicrobial susceptibility testing (agar dilution method) of isolates (n=960; 936 baseline isolates from 763 participants and 24 TOC isolates [23 with a paired baseline isolate in the same anatomical site] from 20 participants) collected during the zoliflodacin phase 3, randomised controlled trial done in 16 outpatient clinics in Belgium, the Netherlands, South Africa, Thailand, and the USA (Nov 6, 2019-March 16, 2023) are described. WGS analysis was performed on paired baseline and TOC isolates from participants with microbiological failures (zoliflodacin 44 isolates [19 participants]; ceftriaxone and azithromycin two isolates [one participant]), and the three baseline isolates with highest zoliflodacin minimum inhibitory concentration (MIC 0·5 mg/L). FINDINGS:All isolates were inhibited by the same zoliflodacin concentrations (MICs ≤0·008 to 0·5 mg/L) as wild-type strains cultured internationally in 2013-23. In participants with a microbiological failure after zoliflodacin treatment (n=22, 19 participants), zoliflodacin MIC values for baseline and TOC isolates were similar, and resistance selection was lacking. WGS showed that five (23%) of 22 infections (95% CI 10-43 [in four participants]) of zoliflodacin microbiological failures had different strains at TOC versus baseline. In 17 zoliflodacin microbiological failures (15 participants), isolates at baseline and TOC were indistinguishable. 13 of these 17 microbiological failures, corresponding to 59% (95% CI 39-77; 13 of 22) of all zoliflodacin microbiological failures, were in urogenital or rectal sites in 11 participants and the isolates had zoliflodacin MICs less than or equal to 0·008 to 0·25 mg/L. The single microbiological failure after ceftriaxone and azithromycin treatment had different strains at TOC versus at baseline. No sequenced isolates had mutations associated with elevated zoliflodacin MICs. INTERPRETATION:In the zoliflodacin phase 3, randomised controlled trial, 23% of the zoliflodacin microbiological failures and the single ceftriaxone and azithromycin microbiological failure had different gonococcal strains at TOC versus baseline, which suggests reinfections and not treatment failures. In addition, 59% of the zoliflodacin microbiological failures, all in anogenital sites, had no obvious microbiological explanation based on the low zoliflodacin MICs, previous pharmacodynamic studies, and no evidence of resistance selection after zoliflodacin therapy. A reinfection as the cause for these microbiological failures could not be excluded. We recommend that WGS is implemented in future randomised controlled trials for gonorrhoea treatment to further evaluate possible microbiological failures, exclude reinfections (to avoid underestimating the cure rates), and characterise antimicrobial resistance determinants. FUNDING:GARDP through grants from Germany BMFTR (03KA1831), UK DHSC as part of GAMRIF, Japan MHLW, the Netherlands' Ministry of Health, Welfare and Sport and Directorate-General for International Cooperation, the Federal Office of Public Health of Switzerland, the Canton of Geneva, Switzerland, and Örebro University Hospital, Sweden.

Antibacterial research and development (R&D) increasingly relies on public and philanthropic investments over private investments and on academia and small businesses over large pharmaceutical companies. To complement scientific reviews of the antibacterial pipeline, we examined global public and philanthropic funding for R&D of antibacterial therapeutics from 2017 to 2023 using data obtained from the Global AMR R&D Hub's Dynamic Dashboard. Projects were analysed considering funders and recipients, geographical location, R&D stage, mechanism of action, antibacterial class, clinical novelty, spectrum of activity, and alignment with the WHO bacterial priority pathogen list 2024. A total of US$2·51 billion was invested in antibacterial R&D by 130 funders, with a marked concentration among a small number of major sources. Funding peaked at $445 million in 2020 but declined by 18% to $363 million in 2023. Universities received the most awards, yet more than half of the total funding volume went to industry recipients. Investment broadly followed the WHO bacterial priority pathogens list, with Mycobacterium tuberculosis accounting for a fifth of the total. While the funding for clinical development remained stable, that for discovery and preclinical research declined. In this environment, public-private partnerships, such as Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator and the Global Antibiotic Research and Development Partnership, are crucial for attracting, channelling, and targeting funding; however, these partnerships alone will be insufficient. Enhanced strategic alignment in funding priorities and continued public and private investment will be essential for ensuring the discovery and development of effective new antibacterials meeting priority public health needs.

Using transcriptional GFP reporters, we previously found that most antibiotics tested induced acrAB via RamA, whilst other AraC/XylS family transcriptional activators, MarA, SoxS, or Rob, induced fewer signals. Surprisingly, we found that some antibiotics induced ramA with no subsequent acrAB induction. We postulated that expression of RamA, and subsequently AcrAB, must increase above basal levels to induce acrAB transcription. Furthermore, we hypothesized that a certain level of RamA is required to induce a measurable amount of acrAB, and likewise that a certain level of AcrAB is required to give a multidrug resistant (MDR) phenotype. The transcript levels of ramA and acrAB were measured in the presence of a range of concentrations of the ramA inducer, chlorpromazine. In parallel, the levels of RamA, AcrB, and antibiotic susceptibility were determined. Here, we show that a specific level of RamA, and subsequently AcrAB, must be reached before MDR is observed, and up to a maximum amount of RamA, there was enhanced production of AcrAB and MDR; higher RamA concentrations did not increase production of AcrAB or MDR. We postulate that this was due to saturation of the maximum number of RamA binding sites in acrB.

AntibioticDB (https://www.antibioticdb.com/), originally established in 2017 and since 2021 led by the Global Antibiotic Research & Development Partnership (GARDP), is a freely available database of antibacterial agents to facilitate research and development of new antibacterial therapeutics. Here, we describe a new release of AntibioticDB that has been significantly expanded and updated with the aid of user feedback and which offers additional functionality through a redesigned web portal. Improvements include reciprocal integration with the IUPHAR/BPS Guide to Pharmacology (https://www.guidetopharmacology.org), capturing of compound structure information in the form of standard chemical identifiers (canonical and isomeric SMILES, InChI, and InChI Key), chemical 2D structure images, and harmonizing terminology to optimize database searching. Ongoing curation efforts have increased the number of individual entries to >3,500, a process driven mostly by a significant expansion of historical natural product antibiotics that were previously under-represented in the database. The database is continuously updated by mining the published literature and capturing newly discovered antibacterial compounds as they are reported, making AntibioticDB the most complete global resource on antibacterial agents.

Background We showed that exposure of an AcrB D408A mutant to efflux inhibitors applied evolutionary pressure to select bacteria with the wild type acrB sequence. This suggested that reversion to wild type can differentiate between efflux inhibitors. Thus, we hypothesized that this experiment could identify inhibitors of the primary RND pump, AcrB or its homologues in other species. Objectives To construct three mutants, Escherichia coli AcrB D408A, Klebsiella pneumoniae AcrB D408A and Pseudomonas aeruginosa MexB D408A and expose the mutants to substrates and non-substrates of AcrB and/or MexB and determine the rate of reversion to wildtype acrB/mexB sequence. Methods Mutant Escherichia coli AcrB D408A, Klebsiella pneumoniae AcrB D408A and Pseudomonas aeruginosa MexB D408A were constructed with site-directed mutagenesis of the relevant nucleotide in the acrB/mexB gene. Mutants were exposed on agar to substrates and the mutation frequency and mutation rate determined. The MIC of antibiotics and the presence/absence of the D408A substitution was determined for mutants. Results Exposure to the AcrB substrates chlorpromazine and minocycline reverted the D408A genotypes to wild type in a species-dependent manner. Exposure to a non-AcrB substrate, spectinomycin, did not select wild type acrB. Chlorpromazine selected for wild type acrB K. pneumoniae as it had for S. Typhimurium, whereas minocycline selected for wild type E. coli acrB. None of the antibiotics selected wild type mexB, including the tested MexB substrates. Conclusions Evolutionary paths depend upon the genetic background of the species and availability of alternative routes/ genetic pathways that can confer resistance/ decreased susceptibility to antibiotics. ### Competing Interest Statement The authors have declared no competing interest.

Antibacterial resistance is a global challenge that requires a coordinated international response. The current clinical pipeline largely consists of derivatives of established antibiotic classes, whereas the discovery and preclinical pipeline is diverse and innovative including new direct-acting agents with no cross-resistance with existing antibiotics. These novel compounds target pathways such as lipoprotein synthesis, lipopolysaccharide biosynthesis and transport, outer membrane assembly, peptidoglycan biosynthesis, fatty acid biosynthesis and isoprenoid biosynthesis. If these agents can be developed into safe, effective and affordable drugs, they could address a broad range of infections worldwide, benefiting large patient populations without geographical limitations. However, strategies such as indirect-acting or pathogen-specific treatments are likely to benefit small patient groups, primarily in high-income countries that have advanced health-care systems and diagnostic infrastructure. Although encouraging, the discovery and preclinical pipeline remains insufficiently robust to offset the high attrition rates typical of early-stage drug innovation and to meet global health needs. Piddock and colleagues explore new antibacterial compounds in active late discovery phase and preclinical development focusing on innovative strategies, with a global health perspective.

Discovery and exploratory research can identify new antibiotics and biological targets. However, failure rates are high, and funding is insufficient to solve the scientific challenges and attract researchers to antibacterial R&D. Novel methods, including artificial intelligence, have been applied to early-stage research, but these have yet to deliver new antibiotics. The Global Antibiotic Research & Development Partnership (GARDP) is investing in discovery and exploratory research and an R&D education and outreach program. GARDP's efforts, including application of novel R&D methods and new global networks of R&D researchers to develop new antibiotics, is helping address antimicrobial resistance sustainably over the long-term.

BackgroundThe current pipeline for new antibiotics fails to fully address the significant threat posed by drug-resistant Gram-negative bacteria that have been identified by the World Health Organization (WHO) as a global health priority. New antibacterials acting through novel mechanisms of action are urgently needed. We aimed to identify new chemical entities (NCEs) with activity against Klebsiella pneumoniae and Acinetobacter baumannii that could be developed into a new treatment for drug-resistant infections.MethodsWe developed a high-throughput phenotypic screen and selection cascade for generation of hit compounds active against multidrug-resistant (MDR) strains of K. pneumoniae and A. baumannii. We screened compound libraries selected from the proprietary collections of three pharmaceutical companies that had exited antibacterial drug discovery but continued to accumulate new compounds to their collection. Compounds from two out of three libraries were selected using “eNTRy rules” criteria associated with increased likelihood of intracellular accumulation in Escherichia coli.FindingsWe identified 72 compounds with confirmed activity against K. pneumoniae and/or drug-resistant A. baumannii. Two new chemical series with activity against XDR A. baumannii were identified meeting our criteria of potency (EC50 ≤50 μM) and absence of cytotoxicity (HepG2 CC50 ≥100 μM and red blood cell lysis HC50 ≥100 μM). The activity of close analogues of the two chemical series was also determined against A. baumannii clinical isolates.InterpretationThis work provides proof of principle for the screening strategy developed to identify NCEs with antibacterial activity against multidrug-resistant critical priority pathogens such as K. pneumoniae and A. baumannii. The screening and hit selection cascade established here provide an excellent foundation for further screening of new compound libraries to identify high quality starting points for new antibacterial lead generation projects.FundingBMBF and GARDP.

The pipeline of new antibiotics is insufficient to keep pace with the growing global burden of drug-resistant infections. Substantial economic challenges discourage private investment in antibiotic research and development (R&D), with a decline in the number of companies and researchers working in the field. Compounding these issues, many countries (from low income to high income) face a growing crisis of antibiotic shortages and inequitable access to existing and emerging treatments. This has led to an increasing role for public and philanthropic funding in supporting antibiotic R&D via the creation of nonprofit public-private partnerships, including Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) and the Global Antibiotic Research and Development Partnership (GARDP), industry support for the AMR Action Fund, and pilot schemes to evaluate and reimburse antibiotics in innovative ways. Now is the time to raise the urgency, ambition and commitments of the world's leaders to fully support the antibiotic R&D ecosystem, incentivizing all sectors to conduct public health-driven antibiotic R&D and make effective antibiotics accessible to all who need them.

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria.

Efflux is a natural process found in all prokaryotic and eukaryotic cells that removes a diverse range of substrates from inside to outside. Many antibiotics are substrates of bacterial efflux pumps, and modifications to the structure or overexpression of efflux pumps are an important resistance mechanism utilized by many multidrug-resistant bacteria. Therefore, chemical inhibition of bacterial efflux to revitalize existing antibiotics has been considered a promising approach for antimicrobial chemotherapy over two decades, and various strategies have been employed. In this review, we provide an overview of bacterial multidrug resistance (MDR) efflux pumps, of which the resistance nodulation division (RND) efflux pumps are considered the most clinically relevant in Gram-negative bacteria, and describe over 50 efflux inhibitors that target such systems. Although numerous efflux inhibitors have been identified to date, none have progressed into clinical use because of formulation, toxicity, and pharmacokinetic issues or a narrow spectrum of inhibition. For these reasons, the development of efflux inhibitors has been considered a difficult and complex area of research, and few active preclinical studies on efflux inhibitors are in progress. However, recently developed tools, including but not limited to computational tools including molecular docking models, offer hope that further research on efflux inhibitors can be a platform for research and development of new bacterial efflux inhibitors.

ABSTRACT The aim of this study was to determine if acrAB induction in Salmonella Typhimurium relies solely on RamA or if other transcriptional activator pathways are also involved, and to better understand the kinetics of induction of both acrAB and ramA . We evaluated the expression of acrAB in S . Typhimurium in response to a variety of compounds that are known to induce the expression of one or more of the transcriptional activators, MarA, SoxS, RamA, and Rob. We utilized green fluorescent protein (GFP) transcriptional reporter fusions to investigate the changes in the expression of acrAB, ramA, marA, and soxS following exposure to sub-inhibitory concentrations of antimicrobial compounds. Of the compounds tested, 13 induce acrAB expression in S . Typhimurium via RamA, MarA, SoxS, and Rob-dependent pathways. None of the tested antibiotics induced acrAB expression, and compounds that induced acrAB expression also induced a general stress response. The results from this study show that the majority of compounds tested induced acrAB via the RamA-dependent pathway. However, none of the antibiotic substrates of the AcrB efflux pump directly increased the expression of AcrAB either directly or indirectly via the induction of one of the transcriptional activators. Using a dual GFP/RFP reporter, we investigated the kinetics of the induction of ramA and acrAB simultaneously and found that acrAB gene expression was transient compared to ramA gene expression. ramA gene expression increased with time and would remain high or decrease slowly over the course of the experiment indicating that RamA exerts a wider global effect and is not limited to efflux regulation alone.

Background: Efflux pump inhibitors (EPIs) offer an attractive therapeutic option when combined with existing classes. However, their optimal dosing strategies are unknown.Methods: MICs of ciprofloxacin (CIP)+/-chlorpromazine, phenylalanine-arginine beta naphthylamide (PA beta N) and a developmental molecule MBX-4191 were determined and the pharmacodynamics (PD) was studied in an in vitro model employing Escherichia coli MG1655 and its isogenic MarR mutant (I1147). Exposure ranging experiments were performed initially then fractionation. Changes in bacterial load and population profiles were assessed. Strains recovered after EPI simulations were studied by WGS.Results: The CIPMICs for E. coli MG1655 and I1147 were 0.08 and 0.03 mg/L. Chlorpromazine at a concentration of 60 mg/L, PA beta N concentrations of 30 mg/L and MBX-4191 concentrations of 0.5-1.0 mg/L reduced CIP MICs for I1147 and enhanced bacterial killing. Using CIP at an AUC of 1.2 mgh/L, chlorpromazine AUC was best related to reduction in bacterial load at 24 h, however, when the time drug concentration was greater than 25 mg/L (T > 25 mg/L) chlorpromazine was also strongly related to the effect. For Pa beta N with CIP AUC, 0.6 mgh/L Pa beta N AUC was best related to a reduction in bacterial load. MBX-4191T > 0.5-0.75 mgh/L was best related to reduction in bacterial load. Changes in population profiles were not seen in experiments of ciprofloxacin + EPIs. WGS of recovered strains from simulations with all three EPIs showed mutations in gyrA, gyrB or marR.Conclusions: AUC was the pharmacodynamic driver for chlorpromazine and PA beta N while T > threshold was the driver for MBX-4191 and important in the activity of chlorpromazine and PA beta N. Changes in population profiles did not occur with combinations of ciprofloxacin + EPIs, however, mutations in gyrA, gyrB and marR were detected.

Objectives Several recent studies highlight the high prevalence of resistance to multiple antibiotic classes used in current treatment regimens for neonatal sepsis and new treatment options are urgently needed. We aimed to identify potential new combination antibiotic treatment regimens by investigating the drug-resistance and genetic profiles of the most frequently isolated Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries (LMICs) in the NeoOBS study. Material and methods Gram-negative bacteria isolated from neonates with culture-confirmed sepsis from 13 clinical sites in nine countries, mainly LMICs, were analyzed. Culture-based identification was followed by whole-genome sequencing (WGS). Minimal inhibitory concentrations (MICs) for 8 antibiotics were determined for a representative subset of 108 isolates. Results Five bacterial species, Klebsiella pneumoniae (n=135), Acinetobacter baumannii (n=80), Escherichia coli (n=34), Serratia marcescens (n=33) and Enterobacter cloacae complex (ECC) (n=27) accounted for most Gram-negative bacterial isolates received (309/420, 74%). Extended-spectrum β-lactamases (ESBL) genes mostly belonging to CTX-M-15 were found in 107 (79%) K. pneumoniae isolates and 13 (38%) E. coli , as well as in 6 (18%) and 10 (37%) S. marcescens and ECC isolates, respectively. Carbapenem resistance genes were present in 41 (30%) K. pneumoniae, while 73 (91%) of A. baumannii isolates were predicted to be MDR based on carbapenem resistance genes. Apart from A. baumannii, in which two major pandemic lineages predominated, a wide genetic diversity occurred at the intraspecies level with different MDR clones occurring at the different sites. Phenotypic testing showed resistance to the WHO first- and second- line recommended treatment regimens: 74% of K. pneumoniae isolates were resistant to gentamicin and 85% to cefotaxime; E. coli isolates showed resistance to ampicillin, gentamicin and cefotaxime in 90%, 38% and 47%, respectively. For the novel antibiotic regimens involving different combinations of flomoxef, fosfomycin and amikacin, the overall predicted MIC-determined susceptibility for Enterobacterales isolates was 71% (n=77) to flomoxef-amikacin, 76% (n=82) to flomoxef-fosfomycin and 79% (n=85) to fosfomycin-amikacin combinations, compared to 31% and 22% isolates susceptible to ampicillin-gentamicin and cefotaxime, respectively. ESBL-producing Enterobacterales isolates were 100% susceptible both to flomoxef-fosfomycin and flomoxef-amikacin and 92% to fosfomycin-amikacin. Conclusion Enterobacterales carried multiple resistance genes to cephalosporins, carbapenems and aminoglycosides. ESBL-producing K. pneumoniae and E. coli isolates were highly susceptible to the three new antibiotic combination regimens planned to be evaluated in the currently recruiting GARDP-sponsored NeoSep1 trial. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was made possible with support from Bill & Melinda Gates Foundation; German Federal Ministry of Education and Research; German Federal Ministry of Health; Government of the Principality of Monaco; the Indian Council for Medical Research; Japanese Ministry of Health, Labour and Welfare; Netherlands Ministry of Health, Welfare and Sport; South African Medical Research Council; UK Department of Health and Social Care (UK National Institute of Health Research and the Global Antimicrobial Resistance Innovation Fund, GAMRIF); and Wellcome Trust. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Health Research Ethics Committee (HREC) of Stellenbosch University gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Background Resistance nodulation division (RND) family efflux pumps, including the major pump AcrAB-TolC, are important mediators of intrinsic and evolved antibiotic resistance. Expression of these pumps is carefully controlled by a network of regulators that respond to different environmental cues. EnvR is a TetR family transcriptional regulator encoded upstream of the RND efflux pump acrEF. Methods Binding of EnvR protein upstream of acrAB was determined by electrophoretic mobility shift assays and the phenotypic consequence of envR overexpression on antimicrobial susceptibility, biofilm motility and invasion of eukaryotic cells in vitro was measured. Additionally, the global transcriptome of clinical Salmonella isolates overexpressing envR was determined by RNA-Seq. Results EnvR bound to the promoter region upstream of the genes coding for the major efflux pump AcrAB in Salmonella, inhibiting transcription and preventing production of AcrAB protein. The phenotype conferred by overexpression of envR mimicked deletion of acrB as it conferred multidrug susceptibility, decreased motility and decreased invasion into intestinal cells in vitro. Importantly, we demonstrate the clinical relevance of this regulatory mechanism because RNA-Seq revealed that a drug-susceptible clinical isolate of Salmonella had low acrB expression even though expression of its major regulator RamA was very high; this was caused by very high EnvR expression. Conclusions In summary, we show that EnvR is a potent repressor of acrAB transcription in Salmonella, and can override binding by RamA so preventing MDR to clinically useful drugs. Finding novel tools to increase EnvR expression may form the basis of a new way to prevent or treat MDR infections.

The closely related transcription factors MarA, SoxS, Rob and RamA control overlapping stress responses in many enteric bacteria. Furthermore, constitutive expression of such regulators is linked to clinical antibiotic resistance. In this work we have mapped the binding of MarA, SoxS, Rob and RamA across the Salmonella Typhimurium genome. In parallel, we have monitored changes in transcription start site use resulting from expression of the regulators. Together, these data allow direct and indirect gene regulatory effects to be disentangled. Promoter architecture across the regulon can also be deduced. At a phylogenetic scale, around one third of regulatory targets are conserved in most organisms encoding MarA, SoxS, Rob or RamA. We focused our attention on the control of csgD, which encodes a transcriptional activator responsible for stimulating production of curli fibres during biofilm formation. We show that expression of csgD is particularly sensitive to SoxS that binds upstream to repress transcription. This differs to the situation in Escherichia coli, where MarA regulates csgD indirectly.

Advances in areas that include genomics, systems biology, protein structure determination and artificial intelligence provide new opportunities for target-based antibacterial drug discovery. The selection of a 'good' new target for direct-acting antibacterial compounds is the first decision, for which multiple criteria must be explored, integrated and re-evaluated as drug discovery programmes progress. Criteria include essentiality of the target for bacterial survival, its conservation across different strains of the same species, bacterial species and growth conditions (which determines the spectrum of activity of a potential antibiotic) and the level of homology with human genes (which influences the potential for selective inhibition). Additionally, a bacterial target should have the potential to bind to drug-like molecules, and its subcellular location will govern the need for inhibitors to penetrate one or two bacterial membranes, which is a key challenge in targeting Gram-negative bacteria. The risk of the emergence of target-based drug resistance for drugs with single targets also requires consideration. This Review describes promising but as-yet-unrealized targets for antibacterial drugs against Gram-negative bacteria and examples of cognate inhibitors, and highlights lessons learned from past drug discovery programmes.

The discovery of antibiotics started a new era in medicine. However, antimicrobial resistance (AMR) is now outpacing the development of new antimicrobials. New political and economic models are required to tackle the developing crisis. In this article I look at the challenges and how we can work to overcome them.

ObjectivesOptimal treatment of carbapenem-resistant Gram-negative bacteria (CR-GNB) infections is uncertain because of the lack of good-quality evidence and the limited effectiveness of available antibiotics. The aim of this survey was to investigate clinicians' prescribing strategies for treating CR-GNB infections worldwide.MethodsA 36-item questionnaire was developed addressing the following aspects of antibiotic prescribing: respondent's background, diagnostic and therapeutic availability, preferred antibiotic strategies and rationale for selecting combination therapy. Prescribers were recruited following the snowball sampling approach, and a post-stratification correction with inverse proportional weights was used to adjust the sample's representativeness.ResultsA total of 1012 respondents from 95 countries participated in the survey. Overall, 298 (30%) of the respondents had local guidelines for treating CR-GNB at their facility and 702 (71%) had access to Infectious Diseases consultation, with significant discrepancies according to country economic status: 85% (390/502) in high-income countries versus 59% (194/283) in upper-medium-income countries and 30% (118/196) in lower-middle-income countries/lower-income-countries). Targeted regimens varied widely, ranging from 40 regimens for CR-Acinetobacter spp. to more than 100 regimens for CR-Enterobacteriaceae. Although the majority of respondents acknowledged the lack of evidence behind this choice, dual combination was the preferred treatment scheme and carbapenem-polymyxin was the most prescribed regimen, irrespective of pathogen and infection source. Respondents noticeably disagreed around the meaning of ‘combination therapy’ with 20% (150/783) indicating the simple addition of multiple compounds, 42% (321/783) requiring the presence of in vitro activity and 38% (290/783) requiring in vitro synergism.ConclusionsManagement of CR-GNB infections is far from being standardized. Strategic public health focused randomized controlled trials are urgently required to inform evidence-based treatment guidelines.