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    Mutations in the riboflavin biosynthesis pathway confer resistance to furazolidone and abolish the synergistic interaction between furazolidone and vancomycin in Escherichia coli.
    (Microbiology Society, England, 2025-02-11) Wykes H; Le VVH; Rakonjac J
    The combined application of furazolidone and vancomycin has previously been shown to be synergistic against Gram-negative pathogens, with great therapeutic promise. However, the emergence and mechanism of resistance to this antibiotic combination have not been characterized. To fill this gap, we here selected Escherichia coli progeny for growth on the furazolidone-vancomycin combination at the concentration where the parent was sensitive. We show that selected clones were associated with increased resistance to neither, only one drug, or both furazolidone and vancomycin, but in all cases were associated with a decrease in the growth inhibition synergy. Using whole-genome sequencing, we identified various gene mutations in the resistant mutants. We further investigated the mechanism behind the most frequently arising mutations, those in the riboflavin biosynthesis genes ribB and ribE, that represent novel mutations causing furazolidone resistance and diminished vancomycin-furazolidone synergy. It was found that these ribB/ribE mutations act predominantly by decreasing the activity of the NfsA and NfsB nitroreductases. The emergence of the ribB/ribE mutations imposes a significant fitness cost on bacterial growth. Surprisingly, supplementing the medium with riboflavin, which compensates for the affected riboflavin biosynthesis pathway, could restore the normal growth of the ribB/ribE mutants while having no effects on the furazolidone resistance phenotype. Searching the ribB/ribE mutations in the public sequencing database detects the presence of the furazolidone-resistance-conferring ribE mutations (TKAG131-134 deletion or duplication) in clinical isolates from different countries. Hypotheses explaining why these ribE mutations were found in clinical isolates despite having poor fitness were further discussed.
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    When less is more: shortening the Lpp protein leads to increased vancomycin resistance in Escherichia coli.
    (Springer Nature Limited, 2023-12-01) Wykes H; Le VVH; Olivera C; Rakonjac J
    Vancomycin is a naturally occurring cell-wall-targeting glycopeptide antibiotic. Due to the low potency of this antibiotic against Gram-negative pathogens, such as Escherichia coli, there is a limited knowledge about interactions between vancomycin and this group of bacteria. Here, we show that an in-frame 63 bp deletion of the lpp gene caused a fourfold increase in vancomycin resistance in E. coli. The resulting protein, LppΔ21, is 21 amino acids shorter than the wild-type Lpp, a helical structural lipoprotein that controls the width of the periplasmic space through its length. The mutant remains susceptible to synergistic growth inhibition by combination of furazolidone and vancomycin; with furazolidone decreasing the vancomycin MIC by eightfold. These findings have clinical relevance, given that the vancomycin concentration required to select the lpp mutation is reachable during typical vancomycin oral administration for treating Clostridioides difficile infections. Combination therapy with furazolidone, however, is likely to prevent emergence and outgrowth of the lpp-mutated Gram-negative coliforms, avoiding exacerbation of the patient's condition during the treatment.
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    In vitro synergy between sodium deoxycholate and furazolidone against enterobacteria
    (BioMed Central Ltd, 2020-01-06) Le VVH; Olivera C; Spagnuolo J; Davies IG; Rakonjac J
    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.
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    Characterisation of the synergistic vancomycin-furazolidone action against Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Masters in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2017) Weerasinghe, Raveen Marlon
    The use of antibiotic combinations is garnering increased interest in the recent years due to the spread of antibiotic-resistant bacteria. The shortage of antibacterial therapy options is particularly severe for infections caused by Gram-negative bacteria, due to the formidable barrier to molecules > 600 Da imposed by the outer membrane. Vancomycin is a large glycopeptide antibiotic to which the outer membrane is poorly permeable, hence the minimal inhibitory concentration of this antibiotic for Escherichia coli is very high (~500 mg/L). Due to the resistance of E. coli and other Gram-negative pathogens to an increasing number of < 600 Da antibiotics including beta lactams, aminoglycosides and quinolones, enabling vancomycin use on Gram-negative bacteria would be valuable. Furazolidone was reported to increase sensitivity of E. coli to vancomycin, and this interaction has been investigated in this thesis in order to explore the potential of the vancomycin-furazolidone combination for clinical applications. The initial analysis of the vancomycin-furazolidone synergy demonstrated that their interaction is synergistic rather than merely additive. Furthermore, effectiveness of this combination for growth inhibition and eradication of E. coli biofilm was investigated. However, despite the synergy between vancomycin and furazolidone, the concentration of vancomycin in combinations required for growth inhibition and killing of E. coli in a planktonic mode and as a biofilm was above the nephrotoxicity (toxicity in the kidneys) threshold and therefore too high to treat infections with this organism systemically. However, by adding deoxycholic acid to the combination, the bactericidal vancomycin concentration was decreased below the nephrotoxicity threshold. The mechanism of synergy in the planktonic mode of growth was investigated through the analysis of E. coli gene-knock-out mutants and it was observed that TolC, the outer membrane channel common to a number of efflux systems (exporting enterobactin, xenobiotics and metabolites) is likely to be involved in vancomycin-furazolidone synergy. However, it was not possible to reliably pinpoint any particular efflux pump or enterobactin accumulation as factors in synergy. Using the genetic approach, it was found that DNA excision repair endonuclease UvrABC was ruled out as a factor involved in synergy. Overall this study characterised the synergy between vancomycin and furazolidone, initiated the enquiry into the mechanisms of interaction between these two antibiotics and examined its effectiveness against biofilms.