Drug Resistance without a cost? Common and uncommon routes to fosfomycin resistance in Uropathogenic Escherichia coli

Fosfomycin kills bacteria by blocking the binding of phosphoenolpyruvate (PEP) to the bacterial enzyme MurA and halting peptidoglycan synthesis. While its use has increased with the emergence of antibiotic resistance, the mechanisms leading to fosfomycin resistance remain relatively unexplored. In uropathogenic Escherichia coli (UPEC) that accounts for >75% of urinary tract infections (UTIs), fosfomycin enters the cell primarily through UhpT, which transports glucose-6-phosphate (G6P) glycolysis intermediate into the cell. Mutations in uhp T lead to fosfomycin resistance and have been identified during antimicrobial susceptibility testing (AST) in non-susceptible inner colonies that form within the zone of inhibition. However, EUCAST and CLSI guidelines differ in how to read fosfomycin AST when such resistant colonies arise. Work from our lab and others demonstrated that glycolysis is dispensable during acute UTI. Moreover, G6P is scarce in urine, prompting us to test the hypothesis that uhp mutations may not impart a fitness cost to the pathogen. We report that loss of uhp indeed does not impair UPEC pathogenesis and that clinical isolates exist that lack the uhp locus altogether. Analysis of non-susceptible inner colonies revealed a suite of novel genes involved in fosfomycin resistance. One of them is PykF that converts PEP to pyruvate during glycolysis. Single deletions of pykF or its anaerobic homolog pykA do not attenuate UPEC. Based on our data, we raise the alarm that multiple routes lead to fosfomycin resistance and do not affect pathogenesis and propose that the current EUCAST and CLSI guidelines unify into how they evaluate fosfomycin AST. IMPORTANCE While fosfomycin resistance is rare, the observation of non-susceptible subpopulations among clinical Escherichia coli isolates is a common phenomenon during antimicrobial susceptibility testing (AST) in American and European clinical labs. Previous evidence suggests that mutations eliciting this phenotype are of high biological cost to the pathogen during infection, leading to current recommendations of neglecting non-susceptible colonies during AST. Here we report that the most common route to fosfomycin resistance, as well as novel routes described in this work do not impair virulence in uropathogenic E. coli , the major cause of urinary tract infections, suggesting a re-evaluation of current susceptibility guidelines is warranted.