RESUMEN
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.
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Antibacterianos , Descubrimiento de Drogas , Antibacterianos/farmacología , Humanos , Inteligencia Artificial , Farmacorresistencia BacterianaRESUMEN
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.
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Acinetobacter baumannii , Antibacterianos , Bacterias Gramnegativas , Infecciones por Bacterias Gramnegativas , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , Sepsis Neonatal , Humanos , Antibacterianos/uso terapéutico , Antibacterianos/farmacología , Sepsis Neonatal/microbiología , Sepsis Neonatal/tratamiento farmacológico , Recién Nacido , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/aislamiento & purificación , Acinetobacter baumannii/genética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/aislamiento & purificación , Klebsiella pneumoniae/genética , Amicacina/farmacología , Amicacina/uso terapéutico , Fosfomicina/farmacología , Fosfomicina/uso terapéutico , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Países en Desarrollo , Farmacorresistencia Bacteriana Múltiple/genética , Quimioterapia Combinada , Serratia marcescens/efectos de los fármacos , Serratia marcescens/genética , Serratia marcescens/aislamiento & purificación , Enterobacter cloacae/efectos de los fármacos , Enterobacter cloacae/genética , Enterobacter cloacae/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
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.
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Antibacterianos , Farmacorresistencia Bacteriana Múltiple , Bacterias Gramnegativas , Proteínas de Transporte de Membrana , Antibacterianos/farmacología , Antibacterianos/química , Bacterias Gramnegativas/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , HumanosRESUMEN
BACKGROUND: The 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. METHODS: We 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. FINDINGS: We 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. INTERPRETATION: This 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. FUNDING: BMBF and GARDP.
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Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas , Humanos , Bibliotecas de Moléculas Pequeñas/farmacología , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Escherichia coli , Farmacorresistencia Bacteriana MúltipleRESUMEN
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.
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Antibacterianos , Transactivadores , Antibacterianos/farmacología , Antibacterianos/metabolismo , Transactivadores/genética , Salmonella typhimurium , Serogrupo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
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.
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Antibacterianos , Infecciones por Bacterias Gramnegativas , Humanos , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antibacterianos/química , Inteligencia Artificial , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Bacterias , Bacterias GramnegativasRESUMEN
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.
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Proteínas de Unión al ADN , Proteínas de Escherichia coli , Proteínas de Unión al ADN/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Proteínas de Escherichia coli/genética , Regulón , Filogenia , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Biopelículas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
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.
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Proteínas Bacterianas , Salmonella typhimurium , Antibacterianos/farmacología , Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Farmacorresistencia Microbiana , Salmonella typhimurium/genética , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Transcripción GenéticaAsunto(s)
Antibacterianos , Farmacorresistencia Bacteriana , Clima , Cambio Climático , Calentamiento Global , HumanosRESUMEN
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.
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Antibacterianos , Antiinfecciosos , Antibacterianos/uso terapéutico , Desarrollo de MedicamentosRESUMEN
BACKGROUND: Neonatal sepsis is a serious bacterial infection of neonates, globally killing up to 680 000 babies annually. It is frequently complicated by antimicrobial resistance, particularly in low- and middle-income country (LMIC) settings with widespread resistance to the WHO's recommended empirical regimen of ampicillin and gentamicin. OBJECTIVES: We assessed the utility of flomoxef and fosfomycin as a potential alternative empirical regimen for neonatal sepsis in these settings. METHODS: We studied the combination in a 16-arm dose-ranged hollow-fibre infection model (HFIM) experiment and chequerboard assays. We further assessed the combination using clinically relevant regimens in the HFIM with six Enterobacterales strains with a range of flomoxef/fosfomycin MICs. RESULTS: Pharmacokinetic/pharmacodynamic modelling of the HFIM experimental output, along with data from chequerboard assays, indicated synergy of this regimen in terms of bacterial killing and prevention of emergence of fosfomycin resistance. Flomoxef monotherapy was sufficient to kill 3/3 strains with flomoxef MICs ≤0.5â mg/L to sterility. Three of three strains with flomoxef MICs ≥8â mg/L were not killed by fosfomycin or flomoxef monotherapy; 2/3 of these were killed with the combination of the two agents. CONCLUSIONS: These data suggest that flomoxef/fosfomycin could be an efficacious and synergistic regimen for the empirical treatment of neonatal sepsis in LMIC settings with prevalent antimicrobial resistance. Our HFIM results warrant further assessment of the flomoxef/fosfomycin combination in clinical trials.
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Fosfomicina , Sepsis Neonatal , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Cefalosporinas , Farmacorresistencia Bacteriana , Fosfomicina/farmacología , Fosfomicina/uso terapéutico , Humanos , Recién Nacido , Pruebas de Sensibilidad Microbiana , Sepsis Neonatal/tratamiento farmacológicoRESUMEN
OBJECTIVES: To determine whether expression of efflux pumps and antibiotic susceptibility are altered in Escherichia coli in response to efflux inhibition. METHODS: The promoter regions of nine efflux pump genes (acrAB, acrD, acrEF, emrAB, macAB, cusCFBA, mdtK, mdtABC, mdfA) were fused to gfp in pMW82 and fluorescence from each reporter construct was used as a measure of the transcriptional response to conditions in which AcrB was inhibited, absent or made non-functional. Expression was also determined by RT-qPCR. Drug susceptibility of efflux pump mutants with missense mutations known or predicted to cause loss of function of the encoded efflux pump was investigated. RESULTS: Data from the GFP reporter constructs revealed that no increased expression of the tested efflux pump genes was observed when AcrB was absent, made non-functional, or inhibited by an efflux pump inhibitor/competitive substrate, such as PAßN or chlorpromazine. This was confirmed by RT-qPCR for PAßN and chlorpromazine; however, a small but significant increase in macB gene expression was seen when acrB is deleted. Efflux inhibitors only synergized with antibiotics in the presence of a functional AcrB. When AcrB was absent or non-functional, there was no impact on MICs when other efflux pumps were also made non-functional. CONCLUSIONS: Absence, loss-of-function, or inhibition of E. coli AcrB did not significantly increase expression of other efflux pump genes, which suggests there is no compensatory mechanism to overcome efflux inhibition and supports the discovery of inhibitors of AcrB as antibiotic adjuvants.
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Proteínas de Escherichia coli , Escherichia coli , Adyuvantes Farmacéuticos , Antibacterianos/metabolismo , Antibacterianos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismoRESUMEN
OBJECTIVES: Optimal 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. METHODS: A 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. RESULTS: A 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. CONCLUSIONS: Management 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.
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Antibacterianos , Carbapenémicos , Farmacorresistencia Bacteriana , Infecciones por Bacterias Gramnegativas , Antibacterianos/uso terapéutico , Carbapenémicos/uso terapéutico , Estudios Transversales , Países Desarrollados , Países en Desarrollo , Bacterias Gramnegativas , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , HumanosRESUMEN
Antibiotics underpin modern medicine and are critical for pandemic preparedness. Push funding has revitalized the preclinical antimicrobial resistance (AMR) pipeline and government funding via CARB-X and BARDA, as well as private sector-led investment via the AMR Action Fund, will help several new antibiotics obtain regulatory approval. Nevertheless, revenues generated by new antibiotics are not considered sufficiently profitable by commercial developers to address unmet need. The question remains: Who could viably fund development and secure global equitable access for new antibiotics? Public health need should be the primary driver for antibiotic development. Improved prioritization and government oversight by funders who allocate public resources are a needed first step. In this framework, nonprofit research and development organizations, with support from public funders, and unconstrained by commercial profitability requirements are well positioned to work with public and private actors to viably provide new antibiotics to all in need.
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Antibacterianos , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Desarrollo de Medicamentos , HumanosRESUMEN
BACKGROUND: Effective treatment of sepsis due to carbapenem-resistant Gram-negative bacteria (CR-GNB) remains a challenge for clinicians worldwide. In recent years, the combination of antibiotics has become the preferred treatment strategy for CR-GNB infection. However, robust evidence to support this approach is lacking. This systematic review aimed at critically evaluating all available antibiotic options for CR-GNB sepsis with particular focus on combination. METHODS: We systematically searched published literature from January 1945 until December 2018 for observational comparative and non-comparative studies and randomized trials examining any antibiotic option for CR-GNB. Studies were included if reporting microbiologically-confirmed infection caused by Acinetobacter baumannii, Enterobacteriaceae/Klebsiella spp., or Pseudomonas aeruginosa, reporting at least one of the study outcomes, and definitive antibiotic treatment. Carbapenem-resistance was defined as phenotypically-detected in vitro resistance to at least one of the following carbapenems: doripenem, ertapenem, imipenem, meropenem. Each antibiotic regimen was classified as "defined" when at least the molecular class(es) composing the regimen was detailed. Primary outcomes were 30-day and attributable mortality. Bayesian network meta-analysis (NMA) approach was selected for quantitative synthesis to explore feasibility of pooling data on antibiotic regimens. RESULTS: A total of 6306 records were retrieved and 134 studies including 11,546 patients were included: 54 studies were on Acinetobacter, 52 on Enterobacteriaceae/Klebsiella, 21 on mixed Gram-negative, and 7 on Pseudomonas. Nine (7%) were RCTs; 19 prospective cohorts (14%), 89 (66%) retrospective, and 17 (13%) case series. Forty-one studies (31%) were multicentric. Qualitative synthesis showed an heterogeneous and scattered reporting of key-clinical and microbiological variables across studies. Ninety-two distinct antibiotic regimens were identified with 47 of them (51%, 5863 patients) not reporting any details on numbers, type, dosage and in vitro activity of the included antibiotic molecules. The NMAs could not be performed for any of the selected outcome given the presence of too many disconnected components. CONCLUSION: The existing evidence is insufficient to allowing for the formulation of any evidence-based therapeutic recommendation for CR-GNB sepsis. Future studies must provide a standardized definition of antibiotic regimen to drive recommendations for using combination of antibiotics that can be reliably applied to clinical practice.
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Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana , Quimioterapia Combinada , Bacterias Gramnegativas , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Acinetobacter baumannii , Carbapenémicos , Estudios Clínicos como Asunto , Enterobacteriaceae , Humanos , Pseudomonas aeruginosaRESUMEN
The superiority of combination therapy for carbapenem-resistant Gram-negative bacilli (CR-GNB) infections remains controversial. In vitro models may predict the efficacy of antibiotic regimens against CR-GNB. A systematic review and meta-analysis was performed including pharmacokinetic/pharmacodynamic (PK/PD) and time-kill (TK) studies examining the in vitro efficacy of antibiotic combinations against CR-GNB [PROSPERO registration no. CRD42019128104]. The primary outcome was in vitro synergy based on the effect size (ES): high, ES ≥ 0.75, moderate, 0.35 < ES < 0.75; low, ES ≤ 0.35; and absent, ES = 0). A network meta-analysis assessed the bactericidal effect and re-growth rate (secondary outcomes). An adapted version of the ToxRTool was used for risk-of-bias assessment. Over 180 combination regimens from 136 studies were included. The most frequently analysed classes were polymyxins and carbapenems. Limited data were available for ceftazidime/avibactam, ceftolozane/tazobactam and imipenem/relebactam. High or moderate synergism was shown for polymyxin/rifampicin against Acinetobacter baumannii [ES = 0.91, 95% confidence interval (CI) 0.44-1.00], polymyxin/fosfomycin against Klebsiella pneumoniae (ES = 1.00, 95% CI 0.66-1.00) and imipenem/amikacin against Pseudomonas aeruginosa (ES = 1.00, 95% CI 0.21-1.00). Compared with monotherapy, increased bactericidal activity and lower re-growth rates were reported for colistin/fosfomycin and polymyxin/rifampicin in K. pneumoniae and for imipenem/amikacin or imipenem/tobramycin against P. aeruginosa. High quality was documented for 65% and 53% of PK/PD and TK studies, respectively. Well-designed in vitro studies should be encouraged to guide the selection of combination therapies in clinical trials and to improve the armamentarium against carbapenem-resistant bacteria.
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Antibacterianos/farmacología , Sinergismo Farmacológico , Bacterias Gramnegativas/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Amicacina/farmacología , Compuestos de Azabiciclo/farmacología , Carbapenémicos/farmacología , Ceftazidima/farmacología , Cefalosporinas/farmacología , Colistina/farmacología , Combinación de Medicamentos , Farmacorresistencia Bacteriana , Quimioterapia Combinada , Fosfomicina/farmacología , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Imipenem/farmacología , Técnicas In Vitro , Pruebas de Sensibilidad Microbiana , Polimixinas/farmacología , Rifampin/farmacología , Tazobactam/farmacología , Tobramicina/farmacologíaRESUMEN
In the fight against antibiotic resistance, drugs that target resistance mechanisms in bacteria can be used to restore the therapeutic effectiveness of antibiotics. The multidrug resistance efflux complex AcrAB-TolC is the most clinically relevant efflux pump in Enterobacterales and is a target for drug discovery. Inhibition of the pump protein AcrB allows the intracellular accumulation of a wide variety of antibiotics, effectively restoring their therapeutic potency. To facilitate the development of AcrB efflux inhibitors, it is desirable to discover the native substrates of the pump, as these could be chemically modified to become inhibitors. We analyzed the native substrate profile of AcrB in Escherichia coli MG1655 and Salmonella enterica serovar Typhimurium SL1344 using an untargeted metabolomics approach. We analyzed the endo- and exometabolome of the wild-type strain and their respective AcrB loss-of-function mutants (AcrB D408A) to determine the metabolites that are native substrates of AcrB. Although there is 95% homology between the AcrB proteins of S. Typhimurium and E. coli, we observed mostly different metabolic responses in the exometabolomes of the S. Typhimurium and E. coli AcrB D408A mutants relative to those in the wild type, potentially indicating a differential metabolic adaptation to the same mutation in these two species. Additionally, we uncovered metabolite classes that could be involved in virulence of S. Typhimurium and a potential natural substrate of AcrB common to both species.IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a global threat to human health. The AcrB efflux pump confers inherent and evolved drug resistance to Enterobacterales, including Escherichia coli and Salmonella enterica serovar Typhimurium. We provide insights into the physiological role of AcrB: (i) we observe that loss of AcrB function in two highly related species, E. coli and S. Typhimurium, has different biological effects despite AcrB conferring drug resistance to the same groups of antibiotics in both species, and (ii) we identify potential natural substrates of AcrB, some of which are in metabolite classes implicated in the virulence of S. Typhimurium. Molecules that inhibit multidrug efflux potentiate the activity of old, licensed, and new antibiotics. The additional significance of our research is in providing data about the identity of potential natural substrates of AcrB in both species. Data on these will facilitate the discovery of, and/or could be chemically modified to become, new efflux inhibitors.
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Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Salmonella typhimurium/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Metabolómica , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genéticaRESUMEN
Efflux pumps are a mechanism of intrinsic and evolved resistance in bacteria. If an efflux pump can expel an antibiotic so that its concentration within the cell is below a killing threshold the bacteria are resistant to the antibiotic. Efflux pumps may be specific or they may pump various different substances. This is why many efflux pumps confer multi drug resistance (MDR). In particular over expression of the AcrAB-TolC efflux pump system confers MDR in both Salmonella and Escherichia coli. We consider the complex gene regulation network that controls expression of genes central to controlling the efflux associated genes acrAB and acrEF in Salmonella. We present the first mathematical model of this gene regulatory network in the form of a system of ordinary differential equations. Using a time dependent asymptotic analysis, we examine in detail the behaviour of the efflux system on various different timescales. Asymptotic approximations of the steady states provide an analytical comparison of targets for efflux inhibition.
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Proteínas de Escherichia coli , Redes Reguladoras de Genes , Modelos Biológicos , Antibacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/metabolismo , Proteínas de Transporte de Membrana/genética , Salmonella/genética , Salmonella/metabolismo , TiempoRESUMEN
Resistance-nodulation-division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics.
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Ciprofloxacina/farmacología , Dipéptidos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Quinasas/química , Antibacterianos/química , Antibacterianos/farmacología , Sitios de Unión/genética , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/genética , Ciprofloxacina/química , Dicroismo Circular , Deuterio/química , Dipéptidos/química , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ligandos , Espectrometría de Masas/métodos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Simulación de Dinámica Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismoRESUMEN
Active efflux of antibiotics preventing their accumulation to toxic intracellular concentrations contributes to clinically relevant multidrug resistance. Inhibition of active efflux potentiates antibiotic activity, indicating that efflux inhibitors could be used in combination with antibiotics to reverse drug resistance. Expression of ramA by Salmonella enterica serovar Typhimurium increases in response to efflux inhibition, irrespective of the mode of inhibition. We hypothesized that measuring ramA promoter activity could act as a reporter of efflux inhibition. A rapid, inexpensive, and high-throughput green fluorescent protein (GFP) screen to identify efflux inhibitors was developed, validated, and implemented. Two chemical compound libraries were screened for compounds that increased GFP production. Fifty of the compounds in the 1,200-compound Prestwick chemical library were identified as potential efflux inhibitors, including the previously characterized efflux inhibitors mefloquine and thioridazine. There were 107 hits from a library of 47,168 proprietary compounds from L. Hoffmann La Roche; 45 were confirmed hits, and a dose response was determined. Dye efflux and accumulation assays showed that 40 Roche and three Prestwick chemical library compounds were efflux inhibitors. Most compounds had specific efflux-inhibitor-antibiotic combinations and/or species-specific synergy in antibiotic disc diffusion and checkerboard assays performed with Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella Typhimurium. These data indicate that both narrow-spectrum and broad-spectrum combinations of efflux inhibitors with antibiotics can be found. Eleven novel efflux inhibitor compounds potentiated antibiotic activities against at least one species of Gram-negative bacteria, and data revealing an E. coli mutant with loss of AcrB function suggested that these are AcrB inhibitors.IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a serious threat to human and animal health. Molecules that inhibit multidrug efflux offer an alternative approach to resolving the challenges caused by antibiotic resistance, by potentiating the activity of old, licensed, and new antibiotics. We have developed, validated, and implemented a high-throughput screen and used it to identify efflux inhibitors from two compound libraries selected for their high chemical and pharmacological diversity. We found that the new high-throughput screen is a valuable tool to identify efflux inhibitors, as evidenced by the 43 new efflux inhibitors described in this study.