Wykes, H.; Le, V. V. H.; Rakonjac, J.

Antibiotic combinations are a promising strategy to counteract the global problem of increasing antibiotic resistance. We have previously demonstrated furazolidone-vancomycin synergy against Gram-negative pathogens. Here, we selected Escherichia coli progeny for growth on the furazolidone-vancomycin combination to which the parent was sensitive. We show that selected clones were associated with increased resistance to neither, only one of, or both furazolidone and vancomycin, but in all cases were associated with a decrease in furazolidone-vancomycin synergy. Among a variety of gene mutations identified in this screen, we investigated the mechanism behind the most frequently arising mutations, those in the riboflavin biosynthesis genes ribB and ribE, and found them to act predominantly through decreasing the activity of the NfsA and NfsB nitroreductases, which have FMN (flavin mononucleotide) or FAD (flavin adenine dinucleotide) as a prosthetic group. We further show that the ribB/ribE mutants isolated in our screen are riboflavin semi-auxotrophs. Riboflavin supplementation restored the normal growth of the ribB/ribE mutants but not the furazolidone sensitivity.