Characterization of the effects of 'old' nitrofuran antibiotics on Gram-negative bacteria : Master of Science (Microbiology), Massey University, Manawatu, New Zealand

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Nitrofurans are “old” antibacterials that are regaining popularity over the past decade due to the low prevalence of resistance amongst formidable Gram-negative bacteria. According to reports by the WHO, Escherichia coli and Pseudomonas aeruginosa are on the list of critical pathogens for which antibiotics are urgently needed because of the emergence and widespread dissemination of antibiotic resistance. E. coli and P. aeruginosa are Gram-negative bacteria that cause various diseases, such as uncomplicated urinary tract infections and chronic infections. Furazolidone, nitrofurantoin, and nitrofurazone are three nitrofuran antibiotics currently being investigated for their utility in treating infections caused by these pathogens. Nitrofurans are prodrugs that require activation through reduction by bacterial enzymes. In E. coli, three oxidoreductases, NfsA, NfsB, and AhpF, were shown to activate nitrofurans. Nevertheless, in the absence of these three enzymes, nitrofurans can still kill E. coli, albeit at an increased concentration, suggesting that additional oxidoreductases were reducing furazolidone from a prodrug to its active form. Due to the failure to identify additional nitrofuran-activating enzymes using mutagenic screens, it was hypothesised that the putative oxidoreductases must be essential for E. coli growth. Using a bioinformatic approach, I identified 18 essential oxidoreductases as candidates for nitrofuran-activating enzymes. These were investigated via overexpression from a high-copy-number plasmid in the E. coli triple mutant ΔnfsA ΔnfsB ΔahpF. Among those enzymes, five oxidoreductases, FolD, CydA, HemA, HemG, and MurB, decreased nitrofurantoin MIC when overexpressed and are, therefore, candidates for the nitrofuran-activating factors in E. coli. Furazolidone and two other nitrofurans were also investigated for antivirulence activity in P. aeruginosa PAO1 to determine whether its potency was more pronounced than the previously studied nitrofurazone. Effect on swimming and swarming motilities, biofilm formation, and pyocyanin production were assessed. Despite being non-inhibitory to the P. aeruginosa PAO1 growth at concentrations that are lethal to E. coli, furazolidone was found to be more potent than nitrofurazone and was capable of limiting biofilm formation and pyocyanin production. Overall, the results show the value of reviving “old” antibiotics as they can be used to treat E. coli infections even in the “resistant” mutants lacking three known activation enzymes and prove effective as antivirulence compounds in P. aeruginosa.