Biological cost of single and multiple norfloxacin resistance mutations in Escherichia coli implicated in urinary tract infections

• Resistance to fluoroquinolones in urinary tract infection (UTIs)caused by Escherichia coli is associated with multiple mutations,typically those that alter DNA gyrase and DNA topoisomeraseIV and those that regulate AcrAB-TolC-mediated efflux. We askedwhether a fitness cost is associated with the accumulation ofthese multiple mutations. Mutants of the susceptible E. coliUTI isolate Nu14 were selected through three to five successivesteps with norfloxacin. Each selection was performed with theMIC of the selected strain. After each selection the MIC wasmeasured; and the regions of gyrA, gyrB, parC, and parE, previouslyassociated with resistance mutations, and all of marOR and acrRwere sequenced. The first selection step yielded mutations ingyrA, gyrB, and marOR. Subsequent selection steps yielded mutationsin gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associatedmutations were identified in almost all isolates after selectionsteps 1 and 2 but in less than 50% of isolates after subsequentselection steps. Selected strains were competed in vitro, inurine, and in a mouse UTI infection model against the startingstrain, Nu14. First-step mutations were not associated withsignificant fitness costs. However, the accumulation of threeor more resistance-associated mutations was usually associatedwith a large reduction in biological fitness, both in vitroand in vivo. Interestingly, in some lineages a partial restorationof fitness was associated with the accumulation of additionalmutations in late selection steps. We suggest that the relativebiological costs of multiple mutations may influence the evolutionof E. coli strains that develop resistance to fluoroquinolones.

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