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

Abstract

ABSTRACT 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 uhp T 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. 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, does not impair virulence in uropathogenic E. coli , the major cause of urinary tract infections, suggesting a re-evaluation of current susceptibility guidelines is warranted.