Evolutionary risk analysis of mutators for the development of nitrofurantoin resistance

The rising prevalence of antimicrobial resistance is a significant global health crisis. However, nitrofurantoin remains an outlier, with low resistance rates despite prolonged and sustained use. This durability may stem from the requirement for two independent mutations in the nfsA and nfsB genes arising independently to confer resistance. However, microbes with elevated mutation rates known as ‘mutators’, are often associated with antimicrobial-resistant infections and may facilitate the independent acquisition of the two mutations needed for full resistance. We examined the role of mutators in the evolution of nitrofurantoin resistance in Escherichia coli using both experimental evolution and by analysing genomes of nitrofurantoin-resistant clinical isolates. In experimental populations, we found that nitrofurantoin resistance evolution was dramatically increased in mutators. Elevated mutation rates also shifted the type of resistance mutations observed from broad-impact frameshifts and indels to specific amino acid substitutions at the active sites in NfsA and NfsB. Over a third of nitrofurantoin-resistant clinical isolates possessed potentially disruptive variants in DNA replication fidelity and repair genes, though the phenotypic effects of many of these variants remain uncharacterised. Our results suggest that mutators are a potential threat to the long-term effectiveness of nitrofurantoin, and highlight a need for increased surveillance to monitor the association between mutations conferring mutator and resistance phenotypes.