Alterations in chromosomal genes nfsA, nfsB, and ribE are associated with nitrofurantoin resistance in Escherichia coli from the United Kingdom

Abstract

Antimicrobial resistance in enteric or urinary Escherichia coli is a risk factor for invasive E. coli infections. Due to widespread trimethoprim resistance amongst urinary E. coli and increased bacteraemia incidence, a national recommendation to prescribe nitrofurantoin for uncomplicated urinary tract infection was made in 2014. Nitrofurantoin resistance is reported in <6% urinary E. coli isolates in the UK, however, mechanisms underpinning nitrofurantoin resistance in these isolates remain unknown. This study aimed to identify the genetic basis of nitrofurantoin resistance in urinary E. coli isolates collected from north west London and then elucidate resistance-associated genetic alterations in available UK E. coli genomes. As a result, an algorithm was developed to predict nitrofurantoin susceptibility. Deleterious mutations and gene-inactivating insertion sequences in chromosomal nitroreductase genes nfsA and/or nfsB were identified in genomes of nine confirmed nitrofurantoin-resistant urinary E. coli isolates and additional 11 E. coli isolates that were highlighted by the prediction algorithm and subsequently validated to be nitrofurantoin-resistant. Eight categories of allelic changes in nfsA, nfsB, and the associated gene ribE were detected in 12412 E. coli genomes from the UK. Evolutionary analysis of these three genes revealed homoplasic mutations and explained the previously reported order of stepwise mutations. The mobile gene complex oqxAB, which is associated with reduced nitrofurantoin susceptibility, was identified in only one of the 12412 genomes. In conclusion, mutations and insertion sequences in nfsA and nfsB were leading causes of nitrofurantoin resistance in UK E. coli . As nitrofurantoin exposure increases in human populations, the prevalence of nitrofurantoin resistance in carriage E. coli isolates and those from urinary and bloodstream infections should be monitored.

Exploring the in-situ evolution of Nitrofurantoin resistance in clinically derived Uropathogenic Escherichia coli isolates

“…Consistent with the literature, the predominant mutations leading to Nit R were deletions, although 1646 BASE to 1646 6 evolved Nit R via the acquisition of a point mutation in nfsA: T37M. Growth and MIC data provided strong evidence that T37M, like other previously identified point mutations 10,12 , was functioning as an inactivating mutation in nfsA. Data also suggested that the method by which nfs genes were inactivated (DnfsA, nfsAT37M, DnfsB, DnfsB30) did not significantly alter nitrofurantoin resistance.…”

“…While genome surveillance studies support Nit R being linked to point mutations and deletions of the nfsA/nfsB genes 10,12 , they are unable to explain the factors that drive resistance phenotype selection. Understanding these factors are key to informing prescribing guidelines that underpin UTI treatments and future urology antibiotic stewardship programmes.…”

“…In E. coli , for example, the inactivation, via deletion or point mutation, of the chromosomally located genes, nfsA and nfsB , which account for 70% and 30 % of nitroreductase activity respectively, appear a common mechanism of gaining Nit R 9 . Other pathways leading to Nit R include the horizonal acquisition of the efflux system oqxAB or chromosomal mutations in ribE 10 .…”

“…The inactivation of nfsA and nfsB follows a two-step evolutionary pathway: nfsA before nfsB 11 . Supporting this evolutionary pathway, genetic surveillance studies of Nit R uncovered nfsA - nfsB + isolates, but seldom the reciprocal nfsA + nfsB - combination 10,12 . In vitro experiments have also demonstrated that Δ nfsA single mutants exhibit intermediate levels of nitrofurantoin resistance (MIC < 64 mg/L) compared to their parental isolates 12 .…”

Exploring the in situ evolution of nitrofurantoin resistance in clinically derived uropathogenic Escherichia coli isolates

“… 7 Other pathways leading to Nit R include the horizonal acquisition of the efflux system oqxAB or chromosomal mutations in ribE. 8 …”