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Background  Fluoroquinolones are potent antimicrobial agents used for the treatment of a wide variety of community- and nosocomial- infections. However, resistance to fluoroquinolones in Enterobacteriaceae is increasingly reported. Studies assessing the ability of fluoroquinolones to select for resistance have often used antimicrobial concentrations quite different from those actually acquired at the site of infection. The present study compared the ability to select for resistance of levofloxacin, ciprofloxacin and prulifloxacin at concentrations observed  in vivo  in twenty strains of  Escherichia coli  and  Klebsiella  spp. isolated from patients with respiratory and urinary infections. The frequencies of spontaneous single-step mutations at plasma peak and trough antibiotic concentrations were calculated. Multi-step selection of resistance was evaluated by performing 10 serial cultures on agar plates containing a linear gradient from trough to peak antimicrobial concentrations, followed by 10 subcultures on antibiotic-free agar.  E. coli  resistant strains selected after multi-step selection were characterized for DNA mutations by sequencing  gyrA ,  gyrB ,  parC  and  parE  genes.    Results  Frequencies of mutations for levofloxacin and ciprofloxacin were less than 10 -11  at peak concentration, while for prulifloxacin they ranged from <10 -11  to 10 -5 . The lowest number of resistant mutants after multistep selection was selected by levofloxacin followed by ciprofloxacin and prulifloxacin. Both ciprofloxacin- and prulifloxacin-resistant mutants presented mutations in  gyrA  and  parC , while levofloxacin resistance was found associated only to mutations in  gyrA .    Conclusions  Among the tested fluoroquinolones, levofloxacin was the most capable of limiting the occurrence of resistance.

In vitro selection of resistance in Escherichia coli and Klebsiella spp. at in vivo fluoroquinolone concentrations