In vitro selection of resistance in Escherichia coli and Klebsiella spp. at in vivo fluoroquinolone concentrations.
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.
Drago
Lorenzo
L
Laboratory of Microbiology, Dept Preclinical Sciences LITA Vialba, University of Milan, Via GB Grassi 74, Milan 20157, Italy. lorenzo.drago@unimi.it
Nicola
Lucia
L
Mattina
Roberto
R
De Vecchi
Elena
E
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D002939:Ciprofloxacin / Q000494:pharmacology; D027081:DNA Gyrase / Q000235:genetics; D004252:DNA Mutational Analysis; D027101:DNA Topoisomerase IV / Q000235:genetics; D004148:Dioxolanes / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000382:microbiology; D024841:Fluoroquinolones / Q000494:pharmacology*; D006801:Humans; D007709:Klebsiella / Q000187:drug effects* / Q000302:isolation & purification; D007710:Klebsiella Infections / Q000382:microbiology; D064704:Levofloxacin; D008826:Microbial Sensitivity Tests; D009154:Mutation; D015242:Ofloxacin / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D012141:Respiratory Tract Infections / Q000382:microbiology; D012641:Selection, Genetic; D014552:Urinary Tract Infections / Q000382:microbiology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004148:Dioxolanes; D024841:Fluoroquinolones; D010879:Piperazines; D002939:Ciprofloxacin; D064704:Levofloxacin; D015242:Ofloxacin; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase; C074191:prulifloxacin
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