Journal Articles

Permanent URI for this collectionhttps://mro.massey.ac.nz/handle/10179/7915

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Author: search.filters.author.Rakonjac JSubject: search.filters.subject.Escherichia coli

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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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    Correlated transcriptional responses provide insights into the synergy mechanisms of the furazolidone, vancomycin and sodium deoxycholate triple combination in Escherichia coli
    (American Society for Microbiology, 2021-10-27) Olivera C; Cox MP; Rowlands GJ; Rakonjac J; Bradford PA
    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 induces the SOS response consistent with its DNA-damaging effects, but we demonstrate that using FZ in combination with the other two compounds enables 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. IMPORTANCE 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. We examined the mechanism of action and synergy of these three antibacterials and proposed a mechanistic basis for their synergy. Our results highlight much-needed mechanistic information necessary to advance this combination as a potential therapy.
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    A large chromosomal inversion affects antimicrobial sensitivity of Escherichia coli to sodium deoxycholate
    (Microbiology Society, 2022-08-12) Le VVH; León-Quezada RI; Biggs PJ; Rakonjac J
    Resistance to antimicrobials is normally caused by mutations in the drug targets or genes involved in antimicrobial activation or expulsion. Here we show that an Escherichia coli strain, named DOC14, selected for increased resistance to the bile salt sodium deoxycholate, has no mutations in any ORF, but instead has a 2.1 Mb chromosomal inversion. The breakpoints of the inversion are two inverted copies of an IS5 element. Besides lowering deoxycholate susceptibility, the IS5-mediated chromosomal inversion in the DOC14 mutant was found to increase bacterial survival upon exposure to ampicillin and vancomycin, and sensitize the cell to ciprofloxacin and meropenem, but does not affect bacterial growth or cell morphology in a rich medium in the absence of antibacterial molecules. Overall, our findings support the notion that a large chromosomal inversion can benefit bacterial cells under certain conditions, contributing to genetic variability available for selection during evolution. The DOC14 mutant paired with its isogenic parental strain form a useful model as bacterial ancestors in evolution experiments to study how a large chromosomal inversion influences the evolutionary trajectory in response to various environmental stressors.
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    Correlated Transcriptional Responses Provide Insights into the Synergy Mechanisms of the Furazolidone, Vancomycin, and Sodium Deoxycholate Triple Combination in Escherichia coli.
    (27/10/2021) Olivera C; Cox MP; Rowlands GJ; Rakonjac J
    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 induces the SOS response consistent with its DNA-damaging effects, but we demonstrate that using FZ in combination with the other two compounds enables 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. IMPORTANCE 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. We examined the mechanism of action and synergy of these three antibacterials and proposed a mechanistic basis for their synergy. Our results highlight much-needed mechanistic information necessary to advance this combination as a potential therapy.