Novel mechanisms of TolC-independent decreased bile-salt susceptibility in Escherichia coli.

Bile salts, including sodium deoxycholate (DOC), are secreted into the intestine to aid fat digestion and contribute to antimicrobial protection. Gram-negative pathogens such as Escherichia coli, however, are highly resistant to DOC, using multiple mechanisms of which the multidrug efflux pump AcrAB-TolC is the dominant one. Given that TolC-mediated efflux masks the interaction of DOC with potential targets, we sought to identify those targets by identifying genes whose mutations cause an increase in the MIC to DOC relative to the ∆tolC parental strain, that lacks TolC-associated functional efflux pumps. Using a mutant screen, we isolated twenty independent spontaneous mutants that had a higher MICDOC than the E. coli parental ∆tolC strain. Whole genome sequencing of these mutants mapped most mutations to the ptsI or cyaA gene. Analysis of knock-out mutants and complementation showed that elimination of PtsI, a component of the carbohydrate phosphotransferase system, or one of the two key proteins involved in cAMP synthesis and signaling, adenylate cyclase (CyaA) or cAMP receptor protein (Crp) causes low-level increased resistance of a ∆tolC E. coli strain to DOC.

In vitro synergy between sodium deoxycholate and furazolidone against enterobacteria.

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 ß-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.

Novel 5-Nitrofuran-Activating Reductase in Escherichia coli.

The global spread of multidrug-resistant enterobacteria warrants new strategies to combat these pathogens. One possible approach is the reconsideration of "old" antimicrobials, which remain effective after decades of use. Synthetic 5-nitrofurans such as furazolidone, nitrofurantoin, and nitrofurazone are such a class of antimicrobial drugs. Recent epidemiological data showed a very low prevalence of resistance to this antimicrobial class among clinical Escherichia coli isolates in various parts of the world, forecasting the increasing importance of its uses to battle antibiotic-resistant enterobacteria. However, although they have had a long history of clinical use, a detailed understanding of the 5-nitrofurans' mechanisms of action remains limited. Nitrofurans are known as prodrugs that are activated in E. coli by reduction catalyzed by two redundant nitroreductases, NfsA and NfsB. Furazolidone, nevertheless, retains relatively significant antibacterial activity in the nitroreductase-deficient ΔnfsA ΔnfsBE. coli strain, indicating the presence of additional activating enzymes and/or antibacterial activity of the unreduced form. Using genome sequencing, genetic, biochemical, and bioinformatic approaches, we discovered a novel 5-nitrofuran-activating enzyme, AhpF, in E. coli The discovery of a new nitrofuran-reducing enzyme opens new avenues for overcoming 5-nitrofuran resistance, such as designing nitrofuran analogues with higher affinity for AhpF or screening for adjuvants that enhance AhpF expression.

Oxidation of anthracene on platinum macro- and micro-electrodes: Sonoelectrochemical, cryoelectrochemical and sonocryoelectrochemical studies.

The electro- and sonoelectro-chemical oxidation of anthracene in acetonitrile has been explored at room and low temperature (-40 degrees C) at a platinum micro- and macro-electrode, respectively. From both methodologies, the effective number of electrons, n(eff), in the oxidation process is evaluated at both room temperature and at the limit of the solvent-electrolyte freezing point. It is observed that n(eff) is close to 1 at low temperature while at room temperature it is higher, indicating sluggish follow up kinetics at lower temperature. The oxidative electrolysis of anthracene within an undivided cell, under various conditions is investigated with the product(s) obtained analysed and quantified by (1)H NMR. At room temperature under both 'silent' and 'sono' conditions the electrochemical oxidation produces 9,10-anthraquinone and bianthrone while at low temperature with acoustic streaming, anthracene-9,10-diol is additionally produced. Electrolyses with intentionally added water and also with oxygen saturation is investigated and its influence on the reaction pathway reported. In particular, the addition of water (1.4M) favours the formation of anthraquinone in a 3:1 ratio with bianthrone. Overall, the effect of lower temperature is to favour the formation of anthraquinone or its reduced form whilst the addition of insonation at low temperature is to substantially enhance the product yield per Coulomb of charge injected.

Glyco- and peptidomimetics from three-component Joullié-Ugi coupling show selective antiviral activity.

Chlorination-elimination chemistry coupled with three-component Joullié-Ugi reaction and facile deprotection allowed efficient access to an array of polyhydroxylated pyrrolidines through parallel synthesis that may be considered to be a library of imino (aza) sugars (glycomimetics) and/or dihydroxyprolyl peptides (peptidomimetics). The utility of generating such a library was illustrated by screening against 15 different targets that revealed potent and selective inhibition of the Gaucher's disease glycosyltransferase enzyme glucosylceramide synthase and of primary pathogen model for human hepatitis C virus (HCV) and bovine diarrhoeal virus (BVDV). An observed selectivity for this HCV model over hepatitis B virus and remarkably low toxicity suggest a novel mode of action.