Mutators can increase both the rate of occurrence and specificity of nitrofurantoin resistance mutations

Abstract Antimicrobial resistance is a significant global health crisis, with many antibiotics losing effectiveness within a decade of their introduction. The antibiotic nitrofurantoin, however, counters this trend, as it has sustained low resistance rates despite prolonged and widespread use. A key factor behind nitrofurantoin’s success is that resistance requires two independent inactivating mutations in separate genes, nfsA and nfsB . However, this inherent safeguard may be undermined by elevated mutation rates, a risk that remains unquantified for this antibiotic. Here, we investigated how mutation rates influence nitrofurantoin resistance in Escherichia coli using experimental evolution with both low-level and high-level mutator strains, alongside genomic analyses of uropathogenic nitrofurantoin-resistant clinical isolates. Under experimental conditions, increasing mutation rates increased the development of nitrofurantoin resistnace. Moreover, changes in the mutations that occurred also shifted the spectrum of resistance mutations from broad-impact frameshifts and indels to specific amino acid substitutions in the proteins’ active sites. Of the nitrofurantoin-resistant clinical isolates we analysed, nearly 40% harboured disruptive variants in DNA replication fidelity and repair genes — a prevalence at the higher end of what is typically observed in uropathogenic E. coli . These findings indicate that elevated mutation rates pose a risk to the continued efficacy of nitrofurantoin, highlighting the need for increased monitoring of mutator-driven resistance.