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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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    Extensive bacteriocin gene shuffling in the Streptococcus bovis/Streptococcus equinus complex reveals gallocin D with activity against vancomycin resistant enterococci.
    (Springer Nature Limited, 2020-08-10) Hill D; O'Connor PM; Altermann E; Day L; Hill C; Stanton C; Ross RP
    Streptococcus gallolyticus LL009 produces gallocin D, a narrow spectrum two component bacteriocin with potent activity against vancomycin-resistant enterococci. Gallocin D is distinct from gallocin A, a separate two component bacteriocin produced by S. gallolyticus. Although the gene clusters encoding gallocin A and gallocin D have a high degree of gene synteny, the structural genes are highly variable and appear to have undergone gene shuffling with other streptococcal species. Gallocin D was analysed in laboratory-based experiments. The mature peptides are 3,343 ± 1 Da and 3,019 ± 1 Da and could be readily synthesized and display activity against a vancomycin resistant Enterococcus strain EC300 with a MIC value of 1.56 µM. Importantly, these bacteriocins could contribute to the ability of S. gallolyticus to colonize the colon where they have been associated with colorectal cancer.
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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 of 5-nitrofurans, vancomycin and sodium deoxycholate against Gram-negative pathogens
    (Microbiology Society, 2021-01-15) Olivera C; Le VVH; Davenport C; Rakonjac J
    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.
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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.