RESUMEN
Antibiotic-resistant Gram-negative bacteria remain a globally leading cause of bacterial infection-associated mortality, and it is imperative to identify novel therapeutic strategies. Recently, the advantage of using antibacterials selective against Gram-negative bacteria has been demonstrated with polymyxins that specifically target the lipopolysaccharides of Gram-negative bacteria. However, the severe cytotoxicity of polymyxins limits their clinical use. Here, we demonstrate that polymyxin B nonapeptide (PMBN), a polymyxin B derivative without the terminal amino acyl residue, can significantly enhance the effectiveness of commonly used antibiotics against only Gram-negative bacteria and their persister cells. We show that although PMBN itself does not exhibit antibacterial activity or cytotoxicity well above the 100-fold minimum inhibitory concentration of polymyxin B, PMBN can increase the potency of co-treated antibiotics. We also demonstrate that using PMBN in combination with other antibiotics significantly reduces the frequency of resistant mutant formation. Together, this work provides evidence of the utilities of PMBN as a novel potentiator for antibiotics against Gram-negative bacteria and insights for the eradication of bacterial persister cells during antibiotic treatment. IMPORTANCE: The significance of our study lies in addressing the problem of antibiotic-resistant Gram-negative bacteria, which continue to be a global cause of mortality associated with bacterial infections. Therefore, identifying innovative therapeutic approaches is an urgent need. Recent research has highlighted the potential of selective antibacterials like polymyxins, which specifically target the lipopolysaccharides of Gram-negative bacteria. However, the clinical use of polymyxins is limited by their severe cytotoxicity. This study unveils the effectiveness of polymyxin B nonapeptide (PMBN) in significantly enhancing the eradication of persister cells in Gram-negative bacteria. Although PMBN itself does not exhibit antibacterial activity or cytotoxicity, it remarkably reduces persister cells during the treatment of antibiotics. Moreover, combining PMBN with other antibiotics reduces the emergence of resistant mutants. Our research emphasizes the utility of PMBN as a novel potentiator to decrease persister cells during antibiotic treatments for Gram-negative bacteria.
Asunto(s)
Infecciones Bacterianas , Infecciones por Bacterias Gramnegativas , Polimixina B/análogos & derivados , Humanos , Polimixina B/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Polimixinas/química , Polimixinas/farmacología , Bacterias Gramnegativas , Lipopolisacáridos , Pruebas de Sensibilidad MicrobianaRESUMEN
The multidrug-resistant Gram-negative bacteria has evolved into a worldwide threat to human health; over recent decades, polymyxins have re-emerged in clinical practice due to their high activity against multidrug-resistant bacteria. Nevertheless, the nephrotoxicity and neurotoxicity of polymyxins seriously hinder their practical use in the clinic. Based on the quantitative structure-activity relationship (QSAR), analogue design is an efficient strategy for discovering biologically active compounds with fewer adverse effects. To accelerate the polymyxin analogues discovery process and find the polymyxin analogues with high antimicrobial activity against Gram-negative bacteria, here we developed PmxPred, a GCN and catBoost-based machine learning framework. The RDKit descriptors were used for the molecule and residues representation, and the ensemble learning model was utilized for the antimicrobial activity prediction. This framework was trained and evaluated on multiple Gram-negative bacteria datasets, including Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and a general Gram-negative bacteria dataset achieving an AUROC of 0.857, 0.880, 0.756, 0.895 and 0.865 on the independent test, respectively. PmxPred outperformed the transfer learning method that trained on 10 million molecules. We interpreted our model well-trained model by analysing the importance of global and residue features. Overall, PmxPred provides a powerful additional tool for predicting active polymyxin analogues, and holds the potential elucidate the mechanisms underlying the antimicrobial activity of polymyxins. The source code is publicly available on GitHub (https://github.com/yanwu20/PmxPred).
Asunto(s)
Infecciones por Bacterias Gramnegativas , Polimixinas , Humanos , Polimixinas/farmacología , Polimixinas/química , Antibacterianos/química , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Bacterias Gramnegativas , Farmacorresistencia Bacteriana Múltiple , Escherichia coli , Pruebas de Sensibilidad MicrobianaRESUMEN
Polymyxins, a last resort antibiotic, target the outer membrane of pathogens and are used to address the increasing prevalence of multidrug-resistant Gram-negative bacteria. The plasmid-encoded enzyme MCR-1 confers polymyxin resistance to bacteria by modifying the outer membrane. Transferable resistance to polymyxins is a major concern; therefore, MCR-1 is an important drug target. In this review, we discuss recent structural and mechanistic aspects of MCR-1 function, its variants and homologs, and how they are relevant to polymyxin resistance. Specifically, we discuss work on polymyxin-mediated disruption of the outer and inner membranes, computational studies on the catalytic mechanism of MCR-1, mutagenesis and structural analysis concerning residues important for substrate binding in MCR-1, and finally, advancements in inhibitors targeting MCR-1.
Asunto(s)
Proteínas de Escherichia coli , Polimixinas , Polimixinas/farmacología , Polimixinas/química , Colistina/química , Colistina/farmacología , Proteínas de Escherichia coli/metabolismo , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , BiologíaRESUMEN
Polymyxins are important last resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. However, pathogens have acquired resistance to polymyxins through a pathway that modifies lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N). Inhibition of this pathway is, therefore, a desirable strategy to combat polymyxin resistance. The first pathway-specific reaction is an NAD+-dependent oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) catalyzed by the dehydrogenase domain of ArnA (ArnA_DH). We present the crystal structure of Salmonella enterica serovar typhimurium ArnA in complex with UDP-GlcA showing that binding of the sugar nucleotide is sufficient to trigger a conformational change conserved in bacterial ArnA_DHs but absent in its human homologs, as confirmed by structure and sequence analysis. Ligand binding assays show that the conformational change is essential for NAD+ binding and catalysis. Enzyme activity and binding assays show that (i) UDP-GlcA analogs lacking the 6' carboxylic acid bind the enzyme but fail to trigger the conformational change, resulting in poor inhibition, and (ii) the uridine monophosphate moiety of the substrate provides most of the ligand binding energy. Mutation of asparagine 492 to alanine (N492A) disrupts the ability of ArnA_DH to undergo the conformational change while retaining substrate binding, suggesting that N492 is involved in sensing the 6' carboxylate in the substrate. These results identify the UDP-GlcA-induced conformational change in ArnA_DH as an essential mechanistic step in bacterial enzymes, providing a platform for selective inhibition.
Asunto(s)
NAD , Polimixinas , Humanos , Polimixinas/farmacología , Polimixinas/química , Ligandos , Uridina Difosfato Ácido Glucurónico/química , Uridina Difosfato Ácido Glucurónico/metabolismo , OxidorreductasasRESUMEN
Peptides can be converted to highly active compounds by introducing appropriate substituents on the suitable amino acid residue. Although modifiable residues in peptides can be systematically identified by peptide scanning methodologies, there is no practical method for optimization at the "scanned" position. With the purpose of using derivatives not only for scanning but also as a starting point for further chemical functionalization, we herein report the "scanning and direct derivatization" strategy through chemoselective acylation of embedded threonine residues by a serine/threonine ligation (STL) with the help of in situ screening chemistry. We have applied this strategy to the optimization of the polymyxin antibiotics, which were selected as a model system to highlight the power of the rapid derivatization of active scanning derivatives. Using this approach, we explored the structure-activity relationships of the polymyxins and successfully prepared derivatives with activity against polymyxin-resistant bacteria and those with Pseudomonas aeruginosa selective antibacterial activity. This strategy opens up efficient structural exploration and further optimization of peptide sequences.
Asunto(s)
Antibacterianos , Polimixinas , Polimixinas/farmacología , Polimixinas/química , Antibacterianos/farmacología , Antibacterianos/química , Bacterias , Relación Estructura-Actividad , Treonina , Pruebas de Sensibilidad MicrobianaRESUMEN
The exploration of therapies combining antimicrobial lung proteins and conventional antibiotics is important due to the growing problem of multidrug-resistant bacteria. The aim of this study was to investigate whether human SP-A and a recombinant trimeric fragment (rfhSP-A) have cooperative antimicrobial activity with antibiotics against pathogenic Gram-negative bacteria. We found that SP-A bound the cationic peptide polymyxin B (PMB) with an apparent dissociation constant (K D) of 0.32 ± 0.04 µM. SP-A showed synergistic microbicidal activity with polymyxin B and E, but not with other antibiotics, against three SP-A-resistant pathogenic bacteria: Klebsiella pneumoniae, non-typable Haemophilus influenzae (NTHi), and Pseudomonas aeruginosa. SP-A was not able to bind to K. pneumoniae, NTHi, or to mutant strains thereof expressing long-chain lipopolysaccharides (or lipooligosaccharides) and/or polysaccharide capsules. In the presence of PMB, SP-A induced the formation of SP-A/PMB aggregates that enhance PMB-induced bacterial membrane permeabilization. Furthermore, SP-A bound to a molecular derivative of PMB lacking the acyl chain (PMBN) with a K D of 0.26 ± 0.02 µM, forming SP-A/PMBN aggregates. PMBN has no bactericidal activity but can bind to the outer membrane of Gram-negative bacteria. Surprisingly, SP-A and PMBN showed synergistic bactericidal activity against Gram-negative bacteria. Unlike native supratrimeric SP-A, the trimeric rfhSP-A fragment had small but significant direct bactericidal activity against K. pneumoniae, NTHi, and P. aeruginosa. rfhSP-A did not bind to PMB under physiological conditions but acted additively with PMB and other antibiotics against these pathogenic bacteria. In summary, our results significantly improve our understanding of the antimicrobial actions of SP-A and its synergistic action with PMB. A peptide based on SP-A may aid the therapeutic use of PMB, a relatively cytotoxic antibiotic that is currently being reintroduced into clinics due to the global problem of antibiotic resistance.
Asunto(s)
Polimixina B , Polimixinas , Antibacterianos/química , Antibacterianos/farmacología , Antibióticos Antineoplásicos , Bacterias , Bacterias Gramnegativas/metabolismo , Humanos , Klebsiella pneumoniae , Polimixina B/metabolismo , Polimixina B/farmacología , Polimixinas/química , Polimixinas/metabolismo , Polimixinas/farmacología , Pseudomonas aeruginosa , Proteína A Asociada a Surfactante PulmonarRESUMEN
Research in the continuously increasing threat of polymyxin-resistant multidrug-resistant Pseudomonas aeruginosa, which causes severe infection in immunocompromised patients, has resulted in the development of several polymyxin-derived cyclic lipopeptides containing l-α-γ- diamino butyric acid-like FADDI-019 (F19). In this work, F19's insertion into a minimal model of the asymmetric outer membrane of the bacterium, which contained only penta-acylated lipid A (LipA) and lacked keto-d-octulosonic acid and O-antigens, in the top leaflet and phospholipids in the bottom leaflet, was studied. F19 exhibited all of the hallmarks of the self-uptake mechanism into the asymmetric bilayer. While a single monomer of the lipopeptide did not get partitioned into the inside of the bilayer, it competitively displaced Ca2+ from the membrane surface, observed as a decrease in Ca2+ coordination number with phosphate groups (1.89 vs 1.718), resulting in membrane destabilization. This resulted in an increment of the average defect size and the probability of interplay between lipid tails and hydrophobic residues of another F19. When more than one monomer was present in the system, the first monomer remained docked on the surface, while other monomers intercalated into the bilayer interior with their hydrophobic moieties "sleeved" by lipid acyl chains. The free energy barrier for partial insertion of the lipopeptide into a bilayer in the presence of surface-docked second F19 was recorded at â¼1.3 kcal/mol using two-dimensional (2D) well-tempered metadynamics, making it a low barrier process at 300 K. This study is an attempt to demonstrate the self-uptake mechanism of F19 during intercalation process into the bilayer interior, which may help in the design of better alternates for polymyxins to work against polymyxin resistance.
Asunto(s)
Lipopéptidos , Polimixinas , Humanos , Polimixinas/farmacología , Polimixinas/química , Lipopéptidos/farmacología , Polimixina B/farmacología , Lípido A/química , Antígenos O , Bacterias Gramnegativas , Antibacterianos/farmacología , Antibacterianos/química , FosfatosRESUMEN
Polymyxins are a class of antibiotics that were discovered in 1947 from programs searching for compounds effective in the treatment of Gram-negative infections. Produced by the Gram-positive bacterium Paenibacillus polymyxa and composed of a cyclic peptide chain with a peptide-fatty acyl tail, polymyxins exert bactericidal effects through membrane disruption. Currently, polymyxin B and colistin (polymyxin E) have been developed for clinical use, where they are reserved as "last-line" therapies for multidrug-resistant (MDR) infections. Unfortunately, the incidences of strains resistant to polymyxins have been increasing globally, and polymyxin heteroresistance has been gaining appreciation as an important clinical challenge. These phenomena, along with bacterial tolerance to this antibiotic class, constitute important contributors to polymyxin treatment failure. Here, we review polymyxins and their mechanism of action, summarize the current understanding of how polymyxin treatment fails, and discuss how the next generation of polymyxins holds promise to invigorate this antibiotic class.
Asunto(s)
Colistina , Polimixinas , Antibacterianos/química , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Colistina/farmacología , Colistina/uso terapéutico , Polimixina B/farmacología , Polimixina B/uso terapéutico , Polimixinas/química , Polimixinas/farmacología , Polimixinas/uso terapéutico , Insuficiencia del TratamientoRESUMEN
OBJECTIVES: Polymyxins are antibacterial polypeptides used as "last resort" therapy option for multidrug-resistant Gram-negative bacteria. The expansion of polymyxin-resistant infections has inspired development of novel polymyxin derivatives, and deacylation is one of the critical steps in generating those antibiotics. Deacylase from Actinoplanes utahensis hydrolyze the acyl moieties of echinocandins, and also efficiently deacylates daptomycin, ramoplanin and other important antibiotics. Here, deacylase was studied considering its potential usefulness in deacylating polymyxin B1. RESULTS: All the six recombinant strains containing the deacylase gene catalyzed hydrolysis of polymyxin B1, yielding cyclic heptapeptides. The efficiency of recombinant S. albus (SAL701) was higher than that of the others, and deacylation was the most efficient at 40 °C in 0.2 M Tris buffer (pH 8.0) with 0.2 M Mg2+. The optimal substrate concentration of SAL701 was increased from 2.0 to 6.0 g/L. SAL701 was highly thermostable, showing no loss of activity at 50 °C for 12 h, and the mycelia could be recycled at least three times without loss of catalytic activity. SAL701 could not deacylate ß-lactam substrate such as penicillin G and cephalosporin C. Deacylase catalyzes the amide bond 1 closest to the nucleus of polymyxin B1 rather than the other bond, suggesting that it has high catalytic site specificity. Homology modeling and the docking results implied that Thr190 in deacylase could facilitate hydrolysis with high regioselectivity. CONCLUSIONS: These results show that SAL701 is effective in increasing the cyclic heptapeptide moiety of polymyxin B1. These properties of the biocatalyst may enable its development in the industrial production of polymyxins antibiotics.
Asunto(s)
Streptomyces , Streptomyces/genética , Polimixinas/química , Antibacterianos/farmacología , Farmacorresistencia Bacteriana MúltipleRESUMEN
Polymyxin antibiotics are disfavored owing to their potential clinical toxicity, especially nephrotoxicity. However, the dry antibiotic development pipeline, together with the increasing global prevalence of infections caused by multidrug-resistant (MDR) gram-negative bacteria, have renewed clinical interest in these polypeptide antibiotics. This review highlights the current information regarding the mechanisms of resistance to polymyxins and their molecular epidemiology. Knowledge of the resistance mechanisms and epidemiology of these pathogens is critical for the development of novel antibacterial agents and rapid treatment choices.
Asunto(s)
Infecciones por Bacterias Gramnegativas , Polimixinas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana Múltiple , Enterobacteriaceae , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Polimixinas/química , Polimixinas/farmacología , Polimixinas/uso terapéuticoRESUMEN
Antimicrobial resistance (AMR) has been increasing unrelentingly worldwide, thus negatively impacting human health. The discovery and development of novel antibiotics is an urgent unmet need of the hour. However, it has become more challenging, requiring increasingly time-consuming efforts with increased commercial risks. Hence, alternative strategies are urgently needed to potentiate the existing antibiotics. In this context, short cationic peptides or peptide-based antimicrobials that mimic the activity of naturally occurring antimicrobial peptides (AMPs) could overcome the disadvantages of AMPs having evolved as potent antibacterial agents. Besides their potent antibacterial efficacy, short peptide conjugates have also gained attention as potent adjuvants to conventional antibiotics. Such peptide antibiotic combinations have become an increasingly cost-effective therapeutic option to tackle AMR. This Review summarizes the recent progress for peptide-based small molecules as promising antimicrobials and as adjuvants for conventional antibiotics to counter multidrug resistant (MDR) pathogens.
Asunto(s)
Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/química , Materiales Biomiméticos/química , Adyuvantes Inmunológicos , Secuencia de Aminoácidos , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Materiales Biomiméticos/farmacología , Permeabilidad de la Membrana Celular , Desarrollo de Medicamentos , Humanos , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Péptidos Cíclicos/química , Polimixinas/química , Relación Estructura-ActividadRESUMEN
Introduction. Colistin is one of the last-resort antibiotics for treating multidrug-resistant (MDR) or extensively drug-resistant (XDR) lactose non-fermenting Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii.Gap Statement. As the rate of colistin resistance is steadily rising, there is a need for rapid and accurate antimicrobial susceptibility testing methods for colistin. The Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test has recently been developed for rapid detection of colistin resistance in P. aeruginosa and A. baumannii.Aim. The present study aimed to evaluate the performance of the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test in comparison with the reference broth microdilution (BMD) method.Methodology. The Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test was performed using a total of 135 P. aeruginosa (17 colistin-resistant and 118 colistin-susceptible) and 66 A. baumannii isolates (32 colistin-resistant and 34 colistin-susceptible), in comparison with the reference BMD method.Results. The categorical agreement of the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test with the reference BMD method was 97.5â% with a major error rate of 0â% (0/152) and a very major error (VME) rate of 10.2â%. The VME rate was higher (23.5â%) when calculated separately for P. aeruginosa isolates. The overall sensitivity and specificity were 89.8 and 100â%, respectively.Conclusion. The Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test performed better for A. baumannii than for P. aeruginosa.
Asunto(s)
Antibacterianos/farmacología , Colistina/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/normas , Acinetobacter baumannii/efectos de los fármacos , Resistencia a Múltiples Medicamentos , Humanos , Indicadores y Reactivos/química , Oxazinas/química , Polimixinas/química , Pseudomonas aeruginosa/efectos de los fármacos , Xantenos/químicaRESUMEN
The outer membrane of Gram-negative bacteria is a formidable permeability barrier which allows only a small subset of chemical matter to penetrate. This outer membrane barrier can hinder the study of cellular processes and compound mechanism of action, as many compounds including antibiotics are precluded from entry despite having intracellular targets. Consequently, outer membrane permeabilizing compounds are invaluable tools in such studies. Many existing compounds known to perturb the outer membrane also impact inner membrane integrity, such as polymyxins and their derivatives, making these probes nonspecific. We performed a screen of â¼140â¯000 diverse synthetic compounds, for those that antagonized the growth inhibitory activity of vancomycin at 15 °C in Escherichia coli, to enrich for chemicals capable of perturbing the outer membrane. This led to the discovery that liproxstatin-1, an inhibitor of ferroptosis in human cells, and MAC-0568743, a novel cationic amphiphile, could potentiate the activity of large-scaffold antibiotics with low permeation into Gram-negative bacteria at 37 °C. Liproxstatin-1 and MAC-0568743 were found to physically disrupt the integrity of the outer membrane through interactions with lipopolysaccharide in the outer leaflet of the outer membrane. We showed that these compounds selectively disrupt the outer membrane while minimally impacting inner membrane integrity, particularly at the concentrations needed to potentiate Gram-positive-targeting antibiotics. Further exploration of these molecules and their structural analogues is a promising avenue for the development of outer membrane specific probes.
Asunto(s)
Antibacterianos/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Pared Celular/efectos de los fármacos , Vancomicina/química , Acinetobacter baumannii/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacología , Permeabilidad de la Membrana Celular , Pared Celular/metabolismo , Sinergismo Farmacológico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Ensayos Analíticos de Alto Rendimiento , Klebsiella pneumoniae/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Polimixinas/química , Polimixinas/metabolismo , Pseudomonas aeruginosa/metabolismo , Quinoxalinas/química , Quinoxalinas/metabolismo , Compuestos de Espiro/química , Compuestos de Espiro/metabolismo , Vancomicina/metabolismo , Vancomicina/farmacologíaRESUMEN
The periplasm of Gram-negative bacteria is a complex, highly crowded molecular environment. Little is known about how antibiotics move across the periplasm and the interactions they experience. Here, atomistic molecular dynamics simulations are used to study the antibiotic polymyxin B1 within models of the periplasm, which are crowded to different extents. We show that PMB1 is likely to be able to "hitchhike" within the periplasm by binding to lipoprotein carriers-a previously unreported passive transport route. The simulations reveal that PMB1 forms both transient and long-lived interactions with proteins, osmolytes, lipids of the outer membrane, and the cell wall, and is rarely uncomplexed when in the periplasm. Furthermore, it can interfere in the conformational dynamics of native proteins. These are important considerations for interpreting its mechanism of action and are likely to also hold for other antibiotics that rely on diffusion to cross the periplasm.
Asunto(s)
Antibacterianos/farmacología , Membrana Externa Bacteriana/efectos de los fármacos , Proteínas de Escherichia coli/química , Proteínas de Unión Periplasmáticas/química , Polimixinas/análogos & derivados , Antibacterianos/química , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Simulación de Dinámica Molecular , Periplasma/metabolismo , Proteínas de Unión Periplasmáticas/metabolismo , Polimixinas/química , Polimixinas/farmacologíaRESUMEN
One promising strategy to combat antibiotic-resistant bacteria is to develop compounds that block bacterial defenses against antibacterial conditions produced by the innate immune system. Salmonella enterica, which causes food-borne gastroenteritis and typhoid fever, requires histidine kinases (HKs) to resist innate immune defenses such as cationic antimicrobial peptides (CAMPs). Herein, we report that 2-aminobenzothiazoles block histidine kinase-dependent phenotypes in Salmonella enterica serotype Typhimurium. We found that 2-aminobenzothiazoles inhibited growth under low Mg2+ , a stressful condition that requires histidine kinase-mediated responses, and decreased expression of the virulence genes pagC and pagK. Furthermore, we discovered that 2-aminobenzothiazoles weaken Salmonella's resistance to polymyxin B and polymyxin E, which are last-line antibiotics and models for host defense CAMPs. These findings raise the possibilities that 2-aminobenzothiazoles can block HK-mediated bacterial defenses and can be used in combination with polymyxins to treat infections caused by Salmonella.
Asunto(s)
Antibacterianos/farmacología , Benzotiazoles/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Polimixinas/farmacología , Salmonella enterica/efectos de los fármacos , Antibacterianos/química , Benzotiazoles/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Polimixinas/química , Salmonella enterica/genética , Virulencia/efectos de los fármacosRESUMEN
A novel UHPLC-MS/MS method for the determination of polypeptide antibiotic residues in animal muscle, milk, and eggs was developed and validated. Bacitracin A, colistin A, colistin B, polymyxin B1, and polymyxin B2 were extracted from the samples with a mixture of acetonitrile/water/ammonia solution 25%, 80/10/10 (v/v/v), and put through further evaporation, reconstitution, and filtration steps. The chromatographic separation was performed on a C18 column in gradient elution mode. Mass spectral acquisitions were performed in selective multiple reaction monitoring mode by a triple quadrupole mass spectrometer. The method was validated according to the criteria of Commission Decision 2002/657/EC. The method quantifies polypeptides in a linear range from 10 to 1000 µg kg-1, where the lowest concentration on the calibration curve refers to the limit of quantification (LOQ). The recoveries ranged from 70 to 99%, the repeatability was below 13%, and within-laboratory reproducibility was lower than 15%. The decision limit (CCα) and detection capability (CCß) values were calculated, and ruggedness and stability studies were performed, to fulfill the criteria for confirmatory methods. Moreover, the developed method may also be used for screening purposes by its labor efficiency.
Asunto(s)
Antibacterianos/química , Leche/química , Músculos/química , Péptidos/química , Acetonitrilos/química , Animales , Antibacterianos/aislamiento & purificación , Bacitracina/química , Bacitracina/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Colistina/química , Colistina/aislamiento & purificación , Residuos de Medicamentos/química , Residuos de Medicamentos/aislamiento & purificación , Huevos/análisis , Péptidos/aislamiento & purificación , Polimixinas/análogos & derivados , Polimixinas/química , Polimixinas/aislamiento & purificación , Espectrometría de Masas en TándemRESUMEN
Polymyxins are used as the last-line therapy against multidrug-resistant bacteria. However, their further clinical development needs to solve problems related to the presence of heterogeneous analogs, but there is still no platform or methods that can regulate the biosynthesis of polymyxin analogs. In this study, we present an approach to swap domains in the polymyxin gene cluster to regulate the production of different analogs. Following adenylation domain swapping, the proportion of polymyxin B1 increased from 41.36 to 52.90%, while that of B1-1 decreased from 18.25 to 3.09%. The ratio of polymyxin B1 and B3 following starter condensation domain swapping changed from 41.36 and 16.99 to 55.03 and 6.39%, respectively. The two domain-swapping strains produced 62.96% of polymyxin B1, 6.70% of B3 and 3.32% of B1-1. This study also revealed the presence of overflow fluxes between acetoin, 2,3-butanediol and polymyxin. To our best knowledge, this is the first report of engineering the polymyxin synthetase gene cluster in situ to regulate the relative proportions of polymyxin analogs. This research paves a way for regulating lipopeptide analogs and will facilitate the development of novel lipopeptide derivatives.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple , Paenibacillus polymyxa/enzimología , Péptido Sintasas/química , Péptido Sintasas/genética , Polimixinas/análogos & derivados , Agar , Antibacterianos , Medios de Cultivo , Fermentación , Lipopéptidos , Ingeniería Metabólica , Paenibacillus polymyxa/genética , Polimixinas/biosíntesis , Polimixinas/química , Tensoactivos/químicaRESUMEN
Antibiotic resistance is a growing global challenge to public health. Polymyxin is considered to be the last-resort antibiotic against most gram-negative bacteria. Recently, discoveries of a plasmid-mediated, transferable mobilized polymyxin resistance gene (mcr-1) in many countries have heralded the increased threat of the imminent emergence of pan-drug-resistant super bacteria. MCR-1 is an inner membrane protein that enables bacteria to develop resistance to polymyxin by transferring phosphoethanolamine to lipid A. However, the mechanism associated with polymyxin resistance has yet to be elucidated, and few drugs exist to address this issue. Here, we review our current understanding regarding MCR-1 and small molecule inhibitors to provide a detailed enzymatic mechanism of MCR-1 and the associated implications for drug design.
Asunto(s)
Proteínas Bacterianas/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Proteínas de Escherichia coli/efectos de los fármacos , Polimixinas/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Colistina/química , Colistina/farmacología , Diseño de Fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Bacterianos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Humanos , Plásmidos/efectos de los fármacos , Polimixinas/químicaRESUMEN
To find novel polymycin analogues with high antimicrobial activities and low toxicity, 36 novel polymyxin analogues were synthesized, and in which TZ40-J and TZ40-K were evaluated for their antimicrobial activities using broth microdilution method and for their haemolytic toxicity with sterile sheep blood. Preliminary safety assessments of those two compounds were carried out via the MTT cell viability assay in vitro and acute toxicity assay in vivo. Experimental data demonstrated that TZ40-J and TZ40-K were less toxic and indicate higher activities against Pseudomonas aeruginosa than polymyxin B.
Asunto(s)
Polimixinas/síntesis química , Polimixinas/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Acinetobacter baumannii/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Células HEK293 , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Polimixinas/química , Polimixinas/toxicidad , Distribución AleatoriaRESUMEN
We designed a few polymyxin derivatives which exhibit broad-spectrum antimicrobial activity. Lead compound P1 could disrupt bacterial membranes rapidly without developing resistance, inhibit biofilms formed by E. coli, and exhibit excellent in vivo activity in an MRSA-infected thigh burden mouse model.