Nitrofurantoin has been used for decades for the treatment of
urinary tract infections (UTIs), but clinically significant resistance in Escherichia coli is uncommon.
Nitrofurantoin concentrations in the gastrointestinal tract tend to be low, which might facilitate selection of
nitrofurantoin-resistant (NIT-R) strains in the gut flora. We subjected two
nitrofurantoin-susceptible intestinal E. coli strains (ST540-p and ST2747-p) to increasing
nitrofurantoin concentrations under aerobic and anaerobic conditions. Whole-genome sequencing was performed for both susceptible isolates and selected mutants that exhibited the highest
nitrofurantoin resistance levels aerobically (ST540-a and ST2747-a) and anaerobically (ST540-an and ST2747-an). ST540-a/ST540-an and ST2747-a (aerobic MICs of >64 μg/ml) harbored mutations in the known
nitrofurantoin resistance determinants nfsA and/or nfsB, which encode
oxygen-insensitive
nitroreductases. ST2747-an showed reduced
nitrofurantoin susceptibility (aerobic MIC of 32 μg/ml) and exhibited remarkable growth deficits but did not harbor nfsA/nfsB mutations. We identified a 12-nucleotide deletion in ribE, encoding
lumazine synthase, an essential
enzyme involved in the biosynthesis of
flavin mononucleotide (
FMN), which is an important cofactor for NfsA and NfsB. Complementing ST2747-an with a functional wild-type
lumazine synthase restored
nitrofurantoin susceptibility. Six NIT-R E. coli isolates (NRCI-1 to NRCI-6) from stools of UTI patients treated with
nitrofurantoin,
cefuroxime, or a
fluoroquinolone harbored mutations in nfsA and/or nfsB but not ribE. Sequencing of the ribE gene in six intestinal and three urinary E. coli strains showing reduced
nitrofurantoin susceptibility (MICs of 16 to 48 μg/ml) also did not identify any relevant mutations. NRCI-1, NRCI-2, and NRCI-5 exhibited up to 4-fold higher anaerobic MICs, compared to the mutants generated in vitro, presumably because of additional mutations in
oxygen-sensitive
nitroreductases.