IS1-related large-scale deletion of chromosomal regions harbouring oxygen-insensitive nitroreductase genenfsBcauses nitrofurantoin heteroresistance inEscherichia coli

Abstract Nitrofurantoin is a broad-spectrum first-line antimicrobial used for managing uncomplicated urinary tract infection. Loss-of-function mutations in chromosomal genes nfsA, nfsB , and ribE of Escherichia coli are known to reduce nitrofurantoin susceptibility. Here, we report monoclonal nitrofurantoin heteroresistance in E. coli and a novel genetic mechanism associated with this phenomenon. Subpopulations with reduced nitrofurantoin susceptibility in cultures of two E. coli blood strains were identified using population analysis profiling. Four colonies of each strain growing on agar with 0.5×MIC nitrofurantoin were sub-cultured in broth with 0.5×MIC nitrofurantoin (n=2) or without nitrofurantoin (n=2). Moreover, one colony of each strain growing without nitrofurantoin exposure was selected as a reference for genomic comparison. Whole-genome sequencing of all isolates were conducted on Illumina and Nanopore MinION systems. Both strains had a nitrofurantoin MICs of 64 mg/L. The proportion of cells grown at 0.5×MIC was two and 99 per million, respectively, which is distinct to that of a homogeneously susceptible or resistant isolate. All isolates grown at 0.5×MIC had 11–66 kbp deletions in chromosomal regions harbouring nfsB , and all these deletions were immediately adjacent to IS 1 -family insertion sequences. Although this study is limited to E. coli and nitrofurantoin, our findings suggest IS 1 -associated genetic deletion represents a hitherto unrecognised mechanism of heteroresistance that could compromise infection management and impact conventional antimicrobial susceptibility testing. Impact statement Nitrofurantoin is widely used for treating and preventing urinary tract infection. Prevalence of nitrofurantoin resistance generally is low in E. coli . Here, we report nitrofurantoin heteroresistance in two E. coli blood strains and attribute this phenotype to IS 1 -associated deletion of chromosomal regions harbouring oxygen-insensitive nitroreductase gene nfsB . Our discoveries demonstrate a novel genetic mechanism of heteroresistance and suggest detecting nitrofurantoin heteroresistance in E. coli urinary isolates for improving prescribing. Data summary Whole-genome sequencing reads and genome assemblies generated in this study have been deposited under BioProject PRJEB58678 in the European Nucleotide Archive (ENA). Accession numbers are listed in Supplementary Table 1. Previously generated Illumina whole-genome sequencing reads of parental isolates EC0026B and EC0880B are available under ENA accessions ERR3142418 and ERR3142524, respectively.