scholarly journals In vitro synergy between Sodium Deoxycholate and Furazolidone against Enterobacteria via Inhibition of Multidrug Efflux Pumps

2019 ◽  
Author(s):  
Vuong Van Hung Le ◽  
Catrina Olivera ◽  
Julian Spagnuolo ◽  
Ieuan Davies ◽  
Jasna Rakonjac
Keyword(s):  
Synergistic Effect ◽  
Model Organism ◽  
Sodium Deoxycholate ◽  
Efflux Pumps ◽  
Bacterial Cells ◽  
Multidrug Efflux ◽  
Resistance Development ◽  
E Coli ◽  
Multidrug Efflux Pumps

AbstractAntimicrobial combinations have been proven to be a promising approach in the confrontation with multi-drug resistant bacterial pathogens, owing to enhancement of antibacterial efficacy, deceleration of resistance development rate and mitigation of side effects by lowering the doses of two drugs. In the present study, we report that combination of furazolidone (FZ) and other nitrofurans with a secondary bile salt, Sodium Deoxycholate (DOC), generates a profound synergistic effect on growth inhibition and lethality in enterobacteria, including Escherichia coli, Salmonella, Citrobacter gillenii and Klebsiella pneumoniae. Taking E. coli as the model organism to study the mechanism of DOC-FZ synergy, we found that the synergistic effect involves FZ-mediated inhibition of efflux pumps that normally remove DOC from bacterial cells. We further show that the FZ–mediated nitric oxide production contributes to the synergistic effect. This is to our knowledge the first report of nitrofuran-DOC synergy against Gram-negative bacteria.

scholarly journals In vitro synergy between sodium deoxycholate and furazolidone against enterobacteria

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Vuong Van Hung Le ◽  
Catrina Olivera ◽  
Julian Spagnuolo ◽  
Ieuan G. Davies ◽  
Jasna Rakonjac
Keyword(s):  
Nitric Oxide ◽  
Growth Inhibition ◽  
Fold Increase ◽  
Sodium Deoxycholate ◽  
Efflux Pumps ◽  
Bacterial Cells ◽  
Bacterial Killing ◽  
E Coli

Abstract Background Antimicrobial combinations have been proven as a promising approach in the confrontation with multi-drug resistant bacterial pathogens. In the present study, we identify and characterize a synergistic interaction of broad-spectrum nitroreductase-activated prodrugs 5-nitrofurans, with a secondary bile salt, sodium deoxycholate (DOC) in growth inhibition and killing of enterobacteria. Results Using checkerboard assay, we show that combination of nitrofuran furazolidone (FZ) and DOC generates a profound synergistic effect on growth inhibition in several enterobacterial species including Escherichia coli, Salmonella enterica, Citrobacter gillenii and Klebsiella pneumoniae. The Fractional Inhibitory Concentration Index (FICI) for DOC-FZ synergy ranges from 0.125 to 0.35 that remains unchanged in an ampicillin-resistant E. coli strain containing a β-lactamase-producing plasmid. Findings from the time-kill assay further highlight the synergy with respect to bacterial killing in E. coli and Salmonella. We further characterize the mechanism of synergy in E. coli K12, showing that disruption of the tolC or acrA genes that encode components of multidrug efflux pumps causes, respectively, a complete or partial loss, of the DOC-FZ synergy. This finding indicates the key role of TolC-associated efflux pumps in the DOC-FZ synergy. Overexpression of nitric oxide-detoxifying enzyme Hmp results in a three-fold increase in FICI for DOC-FZ interaction, suggesting a role of nitric oxide in the synergy. We further demonstrate that DOC-FZ synergy is largely independent of NfsA and NfsB, the two major activation enzymes of the nitrofuran prodrugs. Conclusions This study is to our knowledge the first report of nitrofuran-deoxycholate synergy against Gram-negative bacteria, offering potential applications in antimicrobial therapeutics. The mechanism of DOC-FZ synergy involves FZ-mediated inhibition of TolC-associated efflux pumps that normally remove DOC from bacterial cells. One possible route contributing to that effect is via FZ-mediated nitric oxide production.

scholarly journals Expression of Pseudomonas aeruginosa Multidrug Efflux Pumps MexA-MexB-OprM and MexC-MexD-OprJ in a Multidrug-Sensitive Escherichia coli Strain

1998 ◽  
Vol 42 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Ramakrishnan Srikumar ◽  
Tatiana Kon ◽  
Naomasa Gotoh ◽  
Keith Poole
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Crystal Violet ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Efflux System ◽  
E Coli ◽  
Multidrug Efflux Pumps

ABSTRACT The mexCD-oprJ and mexAB-oprM operons encode components of two distinct multidrug efflux pumps inPseudomonas aeruginosa. To assess the contribution of individual components to antibiotic resistance and substrate specificity, these operons and their component genes were cloned and expressed in Escherichia coli. Western immunoblotting confirmed expression of the P. aeruginosa efflux pump components in E. coli strains expressing and deficient in the endogenous multidrug efflux system (AcrAB), although only the ΔacrAB strain, KZM120, demonstrated increased resistance to antibiotics in the presence of the P. aeruginosa efflux genes. E. coli KZM120 expressing MexAB-OprM showed increased resistance to quinolones, chloramphenicol, erythromycin, azithromycin, sodium dodecyl sulfate (SDS), crystal violet, novobiocin, and, significantly, several β-lactams, which is reminiscent of the operation of this pump in P. aeruginosa. This confirmed previous suggestions that MexAB-OprM provides a direct contribution to β-lactam resistance via the efflux of this group of antibiotics. An increase in antibiotic resistance, however, was not observed when MexAB or OprM alone was expressed in KZM120. Thus, despite the fact that β-lactams act within the periplasm, OprM alone is insufficient to provide resistance to these agents. E. coli KZM120 expressing MexCD-OprJ also showed increased resistance to quinolones, chloramphenicol, macrolides, SDS, and crystal violet, though not to most β-lactams or novobiocin, again somewhat reminiscent of the antibiotic resistance profile of MexCD-OprJ-expressing strains ofP. aeruginosa. Surprisingly, E. coli KZM120 expressing MexCD alone also showed an increase in resistance to these agents, while an OprJ-expressing KZM120 failed to demonstrate any increase in antibiotic resistance. MexCD-mediated resistance, however, was absent in a tolC mutant of KZM120, indicating that MexCD functions in KZM120 in conjunction with TolC, the previously identified outer membrane component of the AcrAB-TolC efflux system. These data confirm that a tripartite efflux pump is necessary for the efflux of all substrate antibiotics and that the P. aeruginosa multidrug efflux pumps are functional and retain their substrate specificity in E. coli.

scholarly journals Functional Cloning and Characterization of the Multidrug Efflux Pumps NorM from Neisseria gonorrhoeae and YdhE from Escherichia coli

2008 ◽  
Vol 52 (9) ◽  
pp. 3052-3060 ◽  
Author(s):  
Feng Long ◽  
Corinne Rouquette-Loughlin ◽  
William M. Shafer ◽  
Edward W. Yu
Keyword(s):  
Escherichia Coli ◽  
Neisseria Gonorrhoeae ◽  
Ethidium Bromide ◽  
Antimicrobial Agents ◽  
Dissociation Constants ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Active Efflux ◽  
E Coli ◽  
Multidrug Efflux Pumps

ABSTRACT Active efflux of antimicrobial agents is one of the most important adapted strategies that bacteria use to defend against antimicrobial factors that are present in their environment. The NorM protein of Neisseria gonorrhoeae and the YdhE protein of Escherichia coli have been proposed to be multidrug efflux pumps that belong to the multidrug and toxic compound extrusion (MATE) family. In order to determine their antimicrobial export capabilities, we cloned, expressed, and purified these two efflux proteins and characterized their functions both in vivo and in vitro. E. coli strains expressing norM or ydhE showed elevated (twofold or greater) resistance to several antimicrobial agents, including fluoroquinolones, ethidium bromide, rhodamine 6G, acriflavine, crystal violet, berberine, doxorubicin, novobiocin, enoxacin, and tetraphenylphosphonium chloride. When they were expressed in E. coli, both transporters reduced the levels of ethidium bromide and norfloxacin accumulation through a mechanism requiring the proton motive force, and direct measurements of efflux confirmed that NorM behaves as an Na+-dependent transporter. The capacities of NorM and YdhE to recognize structurally divergent compounds were confirmed by steady-state fluorescence polarization assays, and the results revealed that these transporters bind to antimicrobials with dissociation constants in the micromolar region.

scholarly journals Impaired Efflux of the Siderophore Enterobactin Induces Envelope Stress in Escherichia coli

2019 ◽  
Author(s):  
Randi L. Guest ◽  
Emily A. Court ◽  
Jayne L. Waldon ◽  
Kiersten A. Schock ◽  
Tracy L. Raivio
Keyword(s):  
Escherichia Coli ◽  
Nadh Dehydrogenase ◽  
Enteric Bacteria ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Gram Negative ◽  
E Coli ◽  
Multidrug Efflux Pumps ◽  
Pathway Activation ◽  
Envelope Stress

AbstractThe Cpx response is one of several envelope stress responses that monitor and maintain the integrity of the gram-negative bacterial envelope. While several conditions that are known or predicted to generate misfolded inner membrane proteins activate the Cpx response, the molecular nature of the Cpx inducing cue is not yet known. Studies have demonstrated that mutation of multidrug efflux pumps activates the Cpx response in many gram-negative bacteria. In Vibrio cholerae, pathway activation is due to accumulation of the catechol siderophore vibriobactin. However, the mechanism by which the Cpx response is activated by mutation of efflux pumps in Escherichia coli remains unknown. Here we show that inhibition of efflux by deletion of tolC, the outer membrane channel of several multidrug efflux pumps, activates the Cpx response in E. coli as a result of impaired efflux of the siderophore enterobactin. Enterobactin accumulation in the tolC mutant reduces activity of the NADH oxidation arm of the aerobic respiratory chain. However, NADH dehydrogenase I, NADH dehydrogenase II, and cytochrome bo3 do not contribute to Cpx pathway activation in the E. coli tolC mutant. We show that the Cpx response down-regulates transcription of the enterobactin biosynthesis operon. These results suggest that the Cpx response promotes adaptation to envelope stress in enteric bacteria that are exposed to iron-limited environments, which are rich in envelope-damaging compounds and conditions.

scholarly journals Function and Inhibitory Mechanisms of Multidrug Efflux Pumps

2021 ◽  
Vol 12 ◽  
Author(s):  
Kunihiko Nishino ◽  
Seiji Yamasaki ◽  
Ryosuke Nakashima ◽  
Martijn Zwama ◽  
Mitsuko Hayashi-Nishino
Keyword(s):  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Bacterial Cells ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Inhibitory Mechanisms ◽  
Multidrug Efflux Pumps ◽  
Multiple Antibiotics

Multidrug efflux pumps are inner membrane transporters that export multiple antibiotics from the inside to the outside of bacterial cells, contributing to bacterial multidrug resistance (MDR). Postgenomic analysis has demonstrated that numerous multidrug efflux pumps exist in bacteria. Also, the co-crystal structural analysis of multidrug efflux pumps revealed the drug recognition and export mechanisms, and the inhibitory mechanisms of the pumps. A single multidrug efflux pump can export multiple antibiotics; hence, developing efflux pump inhibitors is crucial in overcoming infectious diseases caused by multidrug-resistant bacteria. This review article describes the role of multidrug efflux pumps in MDR, and their physiological functions and inhibitory mechanisms.

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