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

Abstract

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 uropathogenicEscherichia 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 inuhpT 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 thatuhpmutations may not impart a fitness cost to the pathogen. We report that loss ofuhpindeed does not impair UPEC pathogenesis and that clinical isolates exist that lack theuhplocus 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 ofpykFor its anaerobic homologpykAdo 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.