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

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 (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 ( = 13) as well as a set of novel genes modulating fosfomycin susceptibility ( = 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. and ) and purine synthesis genes (i.e., and its upstream gene ) 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 , highlighting new mutations and functional pathways that may be used by UPEC to develop clinical resistance.