Mutations in the riboflavin biosynthesis pathway confer resistance to furazolidone and abolish the synergistic interaction between furazolidone and vancomycin in .

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 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 and , that represent novel mutations causing furazolidone resistance and diminished vancomycin-furazolidone synergy. It was found that these mutations act predominantly by decreasing the activity of the NfsA and NfsB nitroreductases. The emergence of the / 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 / mutants while having no effects on the furazolidone resistance phenotype. Searching the mutations in the public sequencing database detects the presence of the furazolidone-resistance-conferring mutations (TKAG deletion or duplication) in clinical isolates from different countries. Hypotheses explaining why these mutations were found in clinical isolates despite having poor fitness were further discussed.