Chromosomal genes modulating fosfomycin susceptibility in uropathogenic Escherichia coli: a genome-wide analysis

Escherichia coli acquires fosfomycin resistance through chromosomal mutations that reduce drug uptake and by drug-inactivating enzymes. However, the complete resistance mechanisms remain to be elucidated. The aim of this study was to elucidate the genetic mechanisms regulating fosfomycin susceptibility in uropathogenic E. coli (UPEC). We constructed a highly saturated transposon mutant library containing >340,000 unique Tn5 insertions in a clinical UPEC strain. We conducted transposon-directed insertion site sequencing (TraDIS) to screen for chromosomal genes whose mutations are beneficial for bacterial growth and survival in the presence of fosfomycin at 4 and 32 µg/mL. TraDIS analysis identified 67 genes including known resistance determinants (n = 13) as well as a set of novel genes modulating fosfomycin susceptibility (n = 54). These genes are involved in pyruvate metabolism, pentose phosphate pathway, nucleotide biosynthesis, DNA repair, protein translation, cellular iron homeostasis, and biotin biosynthesis. Deletion of 16 selected genes in the wild-type strain resulted in growth advantages and decreased susceptibility when exposed to fosfomycin. Notably, deletion of DNA repair genes (i.e., mutL and mutS) and purine synthesis genes (i.e., purB and its upstream gene hflD) led to the most significant advantages in competitive and non-competitive growth in the presence of fosfomycin, as well as the highest increase of fosfomycin MIC (8- to 16-fold). These findings provide a genome-wide overview of genes required for maintaining fosfomycin susceptibility in E. coli, highlighting new mutations and functional pathways that may be used by UPEC to develop clinical resistance.