Browsing by Author "Le VVH"

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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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    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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    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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    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.