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 threefold 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 Gramnegative 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.

Introduction. There is an urgent need for effective therapies against bacterial infections, especially those caused by antibiotic-resistant Gram-negative pathogens. Hypothesis. Synergistic combinations of existing antimicrobials show promise due to their enhanced efficacies and reduced dosages which can mitigate adverse effects, and therefore can be used as potential antibacterial therapy. Aim. In this study, we sought to characterize the in vitro interaction of 5-nitrofurans, vancomycin and sodium deoxycholate (NVD) against pathogenic bacteria. Methodology. The synergy of the NVD combination was investigated in terms of growth inhibition and bacterial killing using checkerboard and time-kill assays, respectively. Results. Using a three-dimensional checkerboard assay, we showed that 5-nitrofurans, sodium deoxycholate and vancomycin interact synergistically in the growth inhibition of 15 out of 20 Gram-negative strains tested, including clinically significant pathogens such as carbapenemase-producing Escherichia coli , Klebsiella pneumoniae and Acinetobacter baumannii , and interact indifferently against the Gram-positive strains tested. The time-kill assay further confirmed that the triple combination was bactericidal in a synergistic manner. Conclusion. This study demonstrates the synergistic effect of 5-nitrofurans, sodium deoxycholate and vancomycin against Gram-negative pathogens and highlights the potential of the combination as a treatment for Gram-negative and Gram-positive infections.

We report the complete genome of a multidrug-resistant Escherichia coli strain isolated from a New Zealand patient with a history of hospitalization in India. The strain, carrying eight plasmids, harbors chromosome-encoded nfsA and nfsB mutations, which cause nitrofuran resistance, and class C β-lactamase (blaEC) and plasmid-encoded blaNDM-1, blaCTX-M-15, and blaCMY-6, as well as other antibiotic resistance genes.

Synergistic antibiotic combinations are a promising alternative strategy for developing effective therapies for multidrug-resistant bacterial infections. The synergistic combination of the existing antibiotics nitrofurans and vancomycin with sodium deoxycholate shows promise in inhibiting and killing multidrug-resistant Gram-negative bacteria.

21Antimicrobial combinations have been proven to be a promising approach in the 22 confrontation with multi-drug resistant bacterial pathogens, owing to enhancement of 23 antibacterial efficacy, deceleration of resistance development rate and mitigation of side 24 effects by lowering the doses of two drugs. In the present study, we report that combination 25 of furazolidone (FZ) and other nitrofurans with a secondary bile salt, Sodium Deoxycholate 26 (DOC), generates a profound synergistic effect on growth inhibition and lethality in 27 enterobacteria, including Escherichia coli, Salmonella, Citrobacter gillenii and Klebsiella 28pneumoniae. Taking E. coli as the model organism to study the mechanism of DOC-FZ 29 synergy, we found that the synergistic effect involves FZ-mediated inhibition of efflux pumps 30 that normally remove DOC from bacterial cells. We further show that the FZ-mediated nitric 31 oxide production contributes to the synergistic effect. This is to our knowledge the first report 32 of nitrofuran-DOC synergy against Gram-negative bacteria. 33

The hamigerans are diterpenoid secondary metabolites isolated from the New Zealand marine sponge Hamigera tarangaensis. Of all the hamigerans that have been isolated and characterised at Victoria University of Wellington, hamigeran G showed the most potent anti-proliferative activity against a mammalian cancer cell line. We previously reported that it might be targeting the Golgi network of cells based on a chemical genomic screen on yeast (Saccharomyces cerevisiae). Here, we investigated the effects of hamigeran G on the Golgi network of mammalian cells and showed that it did not have a significant effect on Golgi apparatus morphology or Golgi network functions such as protein secretion and endocytosis. Results of this study, therefore, conclude that the Golgi network is unlikely to be the primary target of hamigeran G's anti-proliferative activity. Further work is needed to fully elucidate the mechanism of action and target(s) of hamigeran G.

Effective therapeutic options are urgently needed to tackle antibiotic resistance. Furazolidone (FZ), vancomycin (VAN), and sodium deoxycholate (DOC) show promise as their combination can synergistically inhibit the growth of, and kill, multidrug-resistant Gram-negative bacteria that are classified as critical priority by the World Health Organization. Here, we investigated the mechanisms of action and synergy of this drug combination using a transcriptomics approach in the model bacterium Escherichia coli. We show that FZ and DOC elicit highly similar gene perturbations indicative of iron starvation, decreased respiration and metabolism, and translational stress. In contrast, VAN induced envelope stress responses, in agreement with its known role in peptidoglycan synthesis inhibition. FZ induced the SOS response consistent with its DNA damaging effects, but we demonstrate that using FZ in combination with the other two compounds enables use of lower dosages and largely mitigates its mutagenic effects. Based on the gene expression changes identified, we propose a synergy mechanism where the combined effects of FZ, VAN, and DOC amplify damage to Gram-negative bacteria while simultaneously suppressing antibiotic resistance mechanisms.