Raising the alarm: fosfomycin resistance associated with non-susceptible inner colonies imparts no fitness cost to the primary bacterial uropathogen

Fosfomycin kills bacteria by blocking the binding of phosphoenolpyruvate (PEP) to the bacterial enzyme MurA and halting peptidoglycan synthesis. While fosfomycin use has increased, 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 uhpT decrease fosfomycin susceptibility 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. We 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 in a well-established murine model of UTI and that clinical isolates exist that lack uhp altogether. Analysis of non-susceptible inner colonies revealed a suite of novel genes involved in fosfomycin resistance. One of them, PykF, 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 that does not affect colonization and persistence in the host urinary tract. We propose that the current EUCAST and CLSI guidelines unify how they evaluate fosfomycin AST.