1-(1-naphthylmethyl)piperazine and phenylalanine-arginine-beta-naphthylamide

Multidrug efflux protein complexes such as AcrAB-TolC from Escherichia coli are paramount in multidrug resistance in Gram-negative bacteria and are also implicated in other processes such as virulence and biofilm formation. Hence efflux pump inhibition, as a means to reverse antimicrobial resistance in clinically relevant pathogens, has gained increased momentum over the past two decades. Significant advances in the structural and functional analysis of AcrB have informed the selection of efflux pump inhibitors (EPIs). However, an accurate method to determine the kinetics of efflux pump inhibition was lacking. In this study we standardised and optimised surface plasmon resonance (SPR) to probe the binding kinetics of substrates and inhibitors to AcrB. The SPR method was also combined with a fluorescence drug binding method by which affinity of two fluorescent AcrB substrates were determined using the same conditions and controls as for SPR. Comparison of the results from the fluorescent assay to those of the SPR assay showed excellent correlation and provided validation for the methods and conditions used for SPR. The kinetic parameters of substrate (doxorubicin, novobiocin and minocycline) binding to AcrB were subsequently determined. Lastly, the kinetics of inhibition of AcrB were probed for two established inhibitors (phenylalanine arginyl β-naphthylamide and 1-1-naphthylmethyl-piperazine) and three novel EPIs: 4-isobutoxy-2-naphthamide (A2), 4-isopentyloxy-2-naphthamide (A3) and 4-benzyloxy-2-naphthamide (A9) have also been probed. The kinetic data obtained could be correlated with inhibitor efficacy and mechanism of action. This study is the first step in the quantitative analysis of the kinetics of inhibition of the clinically important RND-class of multidrug efflux pumps and will allow the design of improved and more potent inhibitors of drug efflux pumps. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Topics: Anti-Bacterial Agents; Antibiotics, Antineoplastic; Dipeptides; Doxorubicin; Drug Resistance, Multiple, Bacterial; Escherichia coli; Escherichia coli Proteins; Kinetics; Minocycline; Molecular Structure; Multidrug Resistance-Associated Proteins; Naphthalenes; Novobiocin; Piperazines; Protein Binding; Surface Plasmon Resonance

The multidrug efflux system in bacteria can reduce antibiotic concentration inside the cell, leading to failure in the treatment of bacterial diseases. This study evaluated the influence of two efflux pump inhibitors (EPIs), phenylalanine arginyl ß-naphthylamide (PAßN) and 1-(1-naphthylmethyl)-piperazine (NMP), on the gene expression of three multidrug efflux systems, AcrAB, AcrEF and EmrAB in Escherichia coli bovine mastitis isolates resistant to ampicillin and sulfamethoxazole/trimethoprim simultaneously. Each isolate had at least three multidrug efflux system genes. The acrA and acrB had the lowest expression levels in all treatments, while the emrA or emrB showed the highest expression levels in the presence of ampicillin, sulfamethoxazole/trimethoprim, PAβN and NMP. EPIs also contributed to the decrease in arcF expression when used in combination with ampicillin treatment. Since PAßN showed stronger effects than NMP, it may serve as an alternative to assist in the antimicrobial therapy of mastitis.

Topics: Ampicillin; Animals; Anti-Infective Agents; Bacterial Proteins; Cattle; Colony Count, Microbial; Dipeptides; Drug Resistance, Multiple, Bacterial; Escherichia coli; Female; Gene Expression Regulation, Bacterial; Mastitis, Bovine; Membrane Transport Proteins; Microbial Sensitivity Tests; Piperazines; Polymerase Chain Reaction; Trimethoprim, Sulfamethoxazole Drug Combination

Campylobacter are the most commonly reported bacterial causes of human gastroenteritis, and they are becoming increasingly resistant to antibiotics, including macrolides and fluoroquinolones, those most frequently used for the treatment of campylobacteriosis. Active efflux mechanisms are involved in resistance of Campylobacter to a broad spectrum of antimicrobials, and are also essential for Campylobacter colonization in the animal intestine, through mediation of bile resistance. Acquisition of antibiotic resistance through resistance-conferring mutations can impose a fitness cost of Campylobacter. The aim of the present study was to determine whether macrolide and fluoroquinolone resistance in Campylobacter affects their tolerance to bile salts and sodium deoxycholate through the most frequent resistance-conferring mutations. Antimicrobial efflux was studied on the basis of restored sensitivity in the presence of the efflux-pump inhibitors (EPIs) phenylalanine-arginine beta-naphthylamide (PAβN) and 1-(1-naphthylmethyl)-piperazine. In the 15 Campylobacter jejuni and 23 Campylobacter coli strains examined here, both of these EPIs partially reversed the resistance to bile salts and sodium deoxycholate. Erythromycin-sensitive C. coli strains were more resistant to bile salts and sodium deoxycholate than erythromycin-resistant strains. PAβN had greater effects on bile salt and sodium deoxycholate resistance in these erythromycin-resistant strains compared to erythromycin-sensitive strains. However, no differences were seen between the ciprofloxacin-sensitive and ciprofloxacin-resistant strains.

Topics: Animals; Anti-Bacterial Agents; Bile Acids and Salts; Campylobacter coli; Campylobacter Infections; Campylobacter jejuni; Ciprofloxacin; Deoxycholic Acid; Dipeptides; Drug Resistance, Bacterial; Erythromycin; Fluoroquinolones; Humans; Macrolides; Microbial Sensitivity Tests; Mutation; Piperazines; Poultry; Swine

Topics: Acinetobacter baumannii; Acinetobacter Infections; Anti-Bacterial Agents; Bacterial Proteins; Dipeptides; Drug Resistance, Multiple, Bacterial; Humans; Membrane Transport Proteins; Microbial Sensitivity Tests; Piperazines

The aim was to assess the potency of the efflux pump inhibitors (EPIs) phenylalanine-arginine ß-naphthylamide (PAßN) and 1-(1-naphthylmethyl)-piperazine (NMP) and the putative natural EPI phenolic (-)-epigallocatechin gallate (EGCG) for the reversal of erythromycin, ciprofloxacin, and tetracycline resistance in Campylobacter jejuni and Campylobacter coli isolates. We investigated target mutations and resistant genes involved in erythromycin and tetracycline resistance and determined the roles of the bacterial drug efflux systems (cmeB, cmeF, and cmeR) in antimicrobial resistance. Our data show that most of the high-level erythromycin resistance and all of the tetracycline resistance can be explained through mutations in 23S rRNA and the presence of the tetO gene, respectively. The EPIs show the ability to partly reverse drug resistance in these Campylobacter isolates. Based on a fourfold or greater reduction in the erythromycin minimal inhibitory concentration (MIC), PAßN and NMP had clear effects in almost of all of the isolates tested. PAßN had a highly selective action on the ciprofloxacin and tetracycline MICs. Inactivation of cmeB increased susceptibility to all of the antimicrobials tested, whereas inactivation of cmeF and cmeR had no effects. A notable decrease in resistance to erythromycin and ciprofloxacin in the presence of subinhibitory concentrations of EGCG demonstrates the resistance-modifying activities of this natural EPI, and indicates its potential use in the control of Campylobacter spp. in the food chain.

Topics: Animals; Anti-Bacterial Agents; Campylobacter coli; Campylobacter jejuni; Catechin; Ciprofloxacin; Dipeptides; Drug Resistance, Multiple, Bacterial; Erythromycin; Humans; Membrane Transport Proteins; Microbial Sensitivity Tests; Piperazines; Tetracycline Resistance

The aim of the study was to test the hypothesis that the efflux pump inhibitors (EPIs) 1-(1-naphthylmethyl)-piperazine (NMP) and phenyl-arginine-beta-naphthylamide (PAbetaN) can inhibit the Vibrio cholerae resistance-nodulation-division (RND) family efflux systems, and thereby render V. cholerae susceptible to antimicrobial agents and inhibit the production of the virulence factors cholera toxin (CT) and the toxin coregulated pilus (TCP).. The susceptibility of V. cholerae to antimicrobial compounds was determined in the presence or absence of NMP and PAbetaN. Transcriptional reporters were used to assess the effects of NMP and PAbetaN on the expression of the genes encoding the virulence factor regulators TcpP and ToxT, whereas CT and TCP production were determined by ELISA using GM1 ganglioside-coated microtitre plates and TcpA Western immunoblotting, respectively.. NMP and PAbetaN potentiated antimicrobial compounds that were substrates for the V. cholerae RND efflux systems. PAbetaN exhibited complete inhibition of the RND efflux systems for Triton X-100 and deoxycholate, but partial inhibition of the efflux systems for cholate and erythromycin. NMP exhibited partial inhibition for all compounds tested except for SDS. The presence of NMP reduced the MIC of SDS to a level that was lower than that observed in an RND efflux-deficient strain, whereas the SDS MIC was unaffected by the presence of PAbetaN. Neither EPI potentiated polymyxin B, penicillin, ampicillin or chloramphenicol. Both NMP and PAbetaN inhibited the production of CT and the TCP and appeared to have additional virulence gene repressing activity independent of RND efflux inhibition.. RND efflux inhibitors represent potential novel therapeutics for the treatment of cholera.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Cholates; Deoxycholic Acid; Dipeptides; Enzyme Inhibitors; Erythromycin; Microbial Sensitivity Tests; Octoxynol; Piperazines; Sodium Dodecyl Sulfate; Transcription Factors; Vibrio cholerae; Virulence Factors

Topics: Anti-Bacterial Agents; Cystic Fibrosis; Dipeptides; Humans; Microbial Sensitivity Tests; Piperazines; Pseudomonas aeruginosa; Pseudomonas Infections

The CmeABC efflux pump plays an important role in the antimicrobial resistance of Campylobacter jejuni and Campylobacter coli. The aim of this investigation was to study the effect of putative efflux pump inhibitors, phenyl-arginine-beta-naphthylamide (PAbetaN) and 1-(1-naphthylmethyl)-piperazine (NMP), as well as the effect of putative efflux pump inducers, sodium salicylate and sodium deoxycholate, on the MIC levels of erythromycin, ciprofloxacin, kanamycin, tetracycline and rifampicin for C. jejuni and C. coli. Our results indicated that susceptibility to erythromycin and rifampicin increased, respectively, 8- to 32- and 8- to 64-fold in the presence of PAbetaN and to a lesser extent in the presence of NMP. Salicylate produced a 2- to 4-fold increase in ciprofloxacin MIC values, whereas little effect was observed in the presence of deoxycholate.

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Campylobacter coli; Campylobacter jejuni; Deoxycholic Acid; Dipeptides; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Humans; Membrane Transport Proteins; Microbial Sensitivity Tests; Piperazines; Sodium Salicylate

1-(1-Naphthylmethyl)-piperazine (NMP) has been shown to reverse multidrug resistance (MDR) in Escherichia coli overexpressing RND-type efflux pumps but there are no data on its activity in non-fermenters like Acinetobacter.. Antimicrobial susceptibility in the absence and presence of NMP and, for comparison, phenyl-arginine-beta-naphthylamide (PAbetaN), another putative efflux pump inhibitor (EPI), was tested in laboratory and mutant strains with differing intracellular dye accumulation and expression of adeB, and in clinical isolates of Acinetobacter baumannii.. Based on a 4-fold or greater MIC reduction, the effects of both EPIs at low concentrations (25 mg/L) were limited. PAbetaN had a highly selective action on the reduction in the MIC of rifampicin and clarithromycin. At a higher concentration of the putative EPIs (100 mg/L), NMP was more active than PAbetaN. This effect was not limited to strains with adeB overexpression, but affected the susceptibility to linezolid, chloramphenicol and tetracycline most, and was enhanced in clinical isolates with reduced fluoroquinolone susceptibility.. NMP can partially reverse MDR in A. baumannii and differs substantially in its activity from that of PAbetaN.

Topics: Acinetobacter baumannii; Anti-Bacterial Agents; Bacterial Proteins; Dipeptides; Drug Resistance, Multiple, Bacterial; Membrane Transport Proteins; Microbial Sensitivity Tests; Piperazines