title: In vitro selection of resistance in Escherichia coli and Klebsiella spp. at in vivo fluoroquinolone concentrations.
abstract: 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.
authors.affiliation: Laboratory of Microbiology, Dept Preclinical Sciences LITA Vialba, University of Milan, Via GB Grassi 74, Milan 20157, Italy. lorenzo.drago@unimi.it
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Drago
authors.forename: Lucia
authors.initials: L
authors.lastname: Nicola
authors.forename: Roberto
authors.initials: R
authors.lastname: Mattina
authors.forename: Elena
authors.initials: E
authors.lastname: De Vecchi
mesh_terms: 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
chemical_list: 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
references.citation: Ann Clin Microbiol Antimicrob. 2009 Oct 24;8:27
references.pmid: 19852849
references.citation: Int J Antimicrob Agents. 2005 Jun;25(6):550-1
references.pmid: 15878822
references.citation: Pulm Pharmacol Ther. 2006;19 Suppl 1:20-9
references.pmid: 16360331
references.citation: J Antimicrob Chemother. 2005 Nov;56(5):914-8
references.pmid: 16174685
references.citation: J Antimicrob Chemother. 2006 Nov;58(5):1054-7
references.pmid: 16984897
references.citation: Rev Esp Quimioter. 2007 Jun;20(2):206-10
references.pmid: 17893757
references.citation: J Antimicrob Chemother. 2003 Apr;51(4):1011-6
references.pmid: 12654771
references.citation: Drugs. 2004;64(19):2221-34; discussion 2235-6
references.pmid: 15456336
references.citation: J Chemother. 2007 Oct;19(5):488-94
references.pmid: 18073146
references.citation: J Antimicrob Chemother. 2009 Apr;63(4):721-7
references.pmid: 19218275
references.citation: J Antimicrob Chemother. 2003 May;51(5):1109-17
references.pmid: 12697644
references.citation: Enferm Infecc Microbiol Clin. 2005 Jan;23(1):4-9
references.pmid: 15701325
references.citation: Can J Infect Dis Med Microbiol. 2006 Nov;17(6):337-40
references.pmid: 18382648
references.citation: Ann Pharmacother. 2004 Jul-Aug;38(7-8):1148-52
references.pmid: 15150380
references.citation: Int J Antimicrob Agents. 2002 Aug;20(2):93-9
references.pmid: 12297357
references.citation: Int J Antimicrob Agents. 2006 Aug;28 Suppl 1:S10-3
references.pmid: 16829056
references.citation: Antimicrob Agents Chemother. 1995 Feb;39(2):520-4
references.pmid: 7726525
references.citation: J Antimicrob Chemother. 2006 Dec;58(6):1274-8
references.pmid: 17040923
references.citation: Int J Antimicrob Agents. 2007 May;29 Suppl 3:S9-S22
references.pmid: 17659212
references.citation: Eur J Clin Microbiol Infect Dis. 2002 Dec;21(12):849-55
references.pmid: 12525919
references.citation: Clin Microbiol Infect. 2008 Jun;14(6):608-11
references.pmid: 18397336
references.citation: Clin Microbiol Infect. 2005 Sep;11(9):750-8
references.pmid: 16104991
references.citation: Chest. 2001 Apr;119(4):1114-22
references.pmid: 11296178
references.citation: J Korean Med Sci. 2006 Oct;21(5):816-22
references.pmid: 17043412
references.citation: Antimicrob Agents Chemother. 2004 Apr;48(4):1295-9
references.pmid: 15047532
references.citation: Pathol Biol (Paris). 2005 Oct-Nov;53(8-9):470-5
references.pmid: 16176863
references.citation: J Chemother. 2005 Feb;17(1):46-53
references.pmid: 15828443
references.citation: Antimicrob Agents Chemother. 2007 Nov;51(11):4171-3
references.pmid: 17846136
references.citation: Arzneimittelforschung. 2003;53(3):201-5
references.pmid: 12705176
references.citation: J Microbiol Immunol Infect. 2008 Aug;41(4):325-31
references.pmid: 18787740
references.citation: Chest. 2004 Mar;125(3):965-73
references.pmid: 15006955
references.citation: APMIS. 2007 Dec;115(12):1376-82
references.pmid: 18184408
references.citation: Clin Microbiol Infect. 2004 Jan;10(1):72-5
references.pmid: 14706090
references.citation: J Food Prot. 1998 Nov;61(11):1567-70
references.pmid: 9829205
references.citation: J Infect Dis. 2006 Jul 1;194(1):79-85
references.pmid: 16741885