Vol. 34, No. 10
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1990, p. 1880-1884
0066-4804/90/101880-05$02.00/0 Copyright © 1990, American Society for Microbiology
Comparative In Vitro Activity of a New Quinolone, Fleroxacin, against Respiratory Pathogens from Patients with Cystic Fibrosis JOSEPH C. AKANIRO,1 CAROLINA E. VIDAURRE,l HARRIS R. STUTMAN,l.2* AND MELVIN I. MARKS' 2 Department of Pediatrics, Memorial Miller Children's Hospital, Long Beach, California 90806,1 and College of Medicine, University of California at Irvine, Irvine, California 926642 Received 12 December 1989/Accepted 19 June 1990
We evaluated fleroxacin, a newer fluoroquinolone, against isolates from sputum from patients with cystic fibrosis. These isolates included rough and mucoid Pseudomonas aeruginosa, Pseudomonas cepacia, Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli. Selected isolates were tested by the broth microdilution method to examine the influence of various pHs, inoculum sizes, and biological fluids (serum or sputum from patients with cystic fibrosis). Fleroxacin MICs for 50 and 90% of isolates of P. aeruginosa were 2.0 and 4 ,ug/ml, those for P. cepacia were 2 and 16 ,ug/ml, those for S. aureus were 0.5 and 1 ,ug/ml, those for H. influenzae were 0.06 and 0.06 ,ug/ml, and those for E. coli were 0.01 and 0.03 ,ug/ml, respectively. Fleroxacin activity against mucoid P. aeruginosa was similar to the activities of enoxacin and ofloxacin but eightfold lower than that of ciprofloxacin. It was twofold more active than norfloxacin and enoxacin but was twofold less active than ciprofloxacin, ofloxacin, and nafcillin against S. aureus. Fleroxacin inhibitory activity against P. cepacia was two- to fourfold lower than that of ciprofloxacin but eightfold greater than those of the other quinolones tested. Alterations in pH, diluent, and inoculum size did not significantly affect fleroxacin activity. These results, combined with available pharmacokinetic and tissue distribution data, support the need for clinical evaluation of fleroxacin in pulmonary infections in patients with cystic fibrosis.
Fleroxacin, RO 23-6240, is a newly developed synthetic trifluorinated quinolone with antimicrobial activity against a variety of pathogens, including mycobacteria, mycoplasmas, chlamydiae, and legionellae (24). Like the other quinolones, its mechanism of action involves the inhibition of supercoiling activity of DNA gyrase (1, 7, 18, 29). Data from preliminary investigations (26, 27) include an encouraging pharmacokinetic profile with excellent concentrations in serum and tissue and a very prolonged elimination half-life. Concentrations in serum may be two to three times those found with similar quinolone agents. Based on these preliminary data, the current study was designed to examine the in vitro activity of fleroxacin against isolates from sputum from patients with cystic fibrosis (CF) pulmonary infections. These infections are often characterized by the emergence of multiply resistant strains following prolonged and repeated courses of antimicrobial therapy. In addition to inhibitory activity, representative isolates were tested for the influence of pH, inoculum size, and biological fluids on fleroxacin activity.
enzae (n = 26), Escherichia coli (n = 24), and Staphylococcus aureus (n = 30) were tested. These organisms were obtained from 27 CF centers in the United States that were participating in a multicenter study sponsored by the National Institutes of Health. Prior to use, all frozen isolates were thawed and subcultured at least twice onto appropriate solid medium. Pseudomonas species were subcultured onto mucoid maintenance agar (10), and working stock cultures were stored on mucoid maintenance agar slants at room temperature. H. influenzae was subcultured onto chocolate agar plates and then transferred to chocolate agar slants. Initial subcultures of E. coli and S. aureus were done on blood agar plates, and subsequent maintenance was on Trypticase agar slants (BBL Microbiology Systems, Cock-
eysville, Md.). Susceptibility testing. Determinations of antimicrobial susceptibilities were performed by a standard microdilution method in which pH-adjusted and cation-supplemented Mueller-Hinton broth was used (21, 25). For testing of H. influenzae, haemophilus test medium (13) was used. By using the MIC-2000 Plus System (Dynatech Laboratories, Inc., Chantilly, Va.), 0.1 ml of the medium-antimicrobial broth was dispensed into wells of microdilution trays, forming a concentration gradient for each antimicrobial agent. All filled microdilution trays were sealed and stored in plastic bags at -70°C until needed. The plates were used within 4 weeks from the date of preparation. At this temperature and duration of storage, the antimicrobial agents were stable, as demonstrated by the consistency in susceptibilities of the reference organisms from the American Type Culture Collection (Rockville, Md.). Overnight cultures of the bacterial isolates were diluted to yield a final inoculum of approximately 2.0 x 105 CFU of test organisms per ml. Verification of the desired inoculum size was done by performing standard colony counts on solid antibiotic-free medium. The MIC was the lowest concentration showing no visible
MATERIALS AND METHODS
Antibiotics. Fleroxacin powder was supplied by Hoffmann-La Roche, Inc., Nutley, N.J. Reagent-grade antimicrobial agents were obtained from their respective manufacturers. All antimicrobial agents were initially dissolved in appropriate solvents, as recommended by the manufacturers, and then diluted to a stock concentration of 2,560 ,ug/ml. All working antimicrobial solutions were stored at -70°C and used within 4 weeks of preparation. Bacteria. A total of 200 bacterial isolates from the sputa of patients with CF consisting of Pseudomonas aeruginosa (n = 106), Pseudomonas cepacia (n = 14), Haemophilus influ*
Corresponding author. 1880
COMPARATIVE IN VITRO ACTIVITY OF FLEROXACIN
VOL. 34, 1990
1881
TABLE 1. Comparison of in vitro activities of fleroxacin and other antimicrobial agents against isolates in sputum from patients with CF Organism (no. of isolates tested)
P. aeruginosa (106)
P. cepacia (14)
H. influenzae (24)
Range
50o
90%o
0.125-16.0 0.032-2.0 0.063->16.0 0.063->16.0 0.25-64.0 0.032->64.0 0.125-32.0 2.0-64.0 2.0->64.0 0.5-256.0
0.5-64.0
2.0 0.25 1.0 2.0 2.0 4.0 1.0 8.0 8.0 4.0 2.0
4.0 0.5 4.0 4.0 4.0 8.0 2.0 32.0 >64.0 32.0 8.0
2.0->16.0 0.25->16.0 2.0->16.0 1.0-2.0 4.0->16.0 8.0->128.0 4.0-64.0 5.0-16.0 1.0->128.0 0.125-64.0 0.063/1.19->16.0/304.0
2.0 1.0 16.0 4.0 16.0 16.0 32.0 8.0 32.0 16.0 1.0/19.0
16.0 4.0 >16.0 >16.0 >16.0 >128.0 64.0 16.0 >128.0 16.0
Fleroxacin Ciprofloxacin Enoxacin Ofloxacin Ceftazidine Aztreonam Imipenem Amikacin Gentamicin Piperacillin Tobramycin Fleroxacin Ciprofloxacin Enoxacin Ofloxacin Norfloxacin Chloramphenicol Gentamicin Piperacillin Ticarcillin Tobramycin
TMP-SMXb S. aureus (30)
MIC (ILg/ml)a
Antimicrobial agent
Fleroxacin Ciprofloxacin Enoxacin Norfloxacin Ofloxacin TMP-SMX Cefazolin Nafcillin Cefuroxime Gentamicin Chloramphenicol Tetracycline
0.25-1.0 0.125-0.5 0.25-2.0 0.25-2.0 0.125-0.5
0.5 0.5 1.0 1.0 0.5
'
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1990, p. 1880-1884
0066-4804/90/101880-05$02.00/0 Copyright © 1990, American Society for Microbiology
Comparative In Vitro Activity of a New Quinolone, Fleroxacin, against Respiratory Pathogens from Patients with Cystic Fibrosis JOSEPH C. AKANIRO,1 CAROLINA E. VIDAURRE,l HARRIS R. STUTMAN,l.2* AND MELVIN I. MARKS' 2 Department of Pediatrics, Memorial Miller Children's Hospital, Long Beach, California 90806,1 and College of Medicine, University of California at Irvine, Irvine, California 926642 Received 12 December 1989/Accepted 19 June 1990
We evaluated fleroxacin, a newer fluoroquinolone, against isolates from sputum from patients with cystic fibrosis. These isolates included rough and mucoid Pseudomonas aeruginosa, Pseudomonas cepacia, Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli. Selected isolates were tested by the broth microdilution method to examine the influence of various pHs, inoculum sizes, and biological fluids (serum or sputum from patients with cystic fibrosis). Fleroxacin MICs for 50 and 90% of isolates of P. aeruginosa were 2.0 and 4 ,ug/ml, those for P. cepacia were 2 and 16 ,ug/ml, those for S. aureus were 0.5 and 1 ,ug/ml, those for H. influenzae were 0.06 and 0.06 ,ug/ml, and those for E. coli were 0.01 and 0.03 ,ug/ml, respectively. Fleroxacin activity against mucoid P. aeruginosa was similar to the activities of enoxacin and ofloxacin but eightfold lower than that of ciprofloxacin. It was twofold more active than norfloxacin and enoxacin but was twofold less active than ciprofloxacin, ofloxacin, and nafcillin against S. aureus. Fleroxacin inhibitory activity against P. cepacia was two- to fourfold lower than that of ciprofloxacin but eightfold greater than those of the other quinolones tested. Alterations in pH, diluent, and inoculum size did not significantly affect fleroxacin activity. These results, combined with available pharmacokinetic and tissue distribution data, support the need for clinical evaluation of fleroxacin in pulmonary infections in patients with cystic fibrosis.
Fleroxacin, RO 23-6240, is a newly developed synthetic trifluorinated quinolone with antimicrobial activity against a variety of pathogens, including mycobacteria, mycoplasmas, chlamydiae, and legionellae (24). Like the other quinolones, its mechanism of action involves the inhibition of supercoiling activity of DNA gyrase (1, 7, 18, 29). Data from preliminary investigations (26, 27) include an encouraging pharmacokinetic profile with excellent concentrations in serum and tissue and a very prolonged elimination half-life. Concentrations in serum may be two to three times those found with similar quinolone agents. Based on these preliminary data, the current study was designed to examine the in vitro activity of fleroxacin against isolates from sputum from patients with cystic fibrosis (CF) pulmonary infections. These infections are often characterized by the emergence of multiply resistant strains following prolonged and repeated courses of antimicrobial therapy. In addition to inhibitory activity, representative isolates were tested for the influence of pH, inoculum size, and biological fluids on fleroxacin activity.
enzae (n = 26), Escherichia coli (n = 24), and Staphylococcus aureus (n = 30) were tested. These organisms were obtained from 27 CF centers in the United States that were participating in a multicenter study sponsored by the National Institutes of Health. Prior to use, all frozen isolates were thawed and subcultured at least twice onto appropriate solid medium. Pseudomonas species were subcultured onto mucoid maintenance agar (10), and working stock cultures were stored on mucoid maintenance agar slants at room temperature. H. influenzae was subcultured onto chocolate agar plates and then transferred to chocolate agar slants. Initial subcultures of E. coli and S. aureus were done on blood agar plates, and subsequent maintenance was on Trypticase agar slants (BBL Microbiology Systems, Cock-
eysville, Md.). Susceptibility testing. Determinations of antimicrobial susceptibilities were performed by a standard microdilution method in which pH-adjusted and cation-supplemented Mueller-Hinton broth was used (21, 25). For testing of H. influenzae, haemophilus test medium (13) was used. By using the MIC-2000 Plus System (Dynatech Laboratories, Inc., Chantilly, Va.), 0.1 ml of the medium-antimicrobial broth was dispensed into wells of microdilution trays, forming a concentration gradient for each antimicrobial agent. All filled microdilution trays were sealed and stored in plastic bags at -70°C until needed. The plates were used within 4 weeks from the date of preparation. At this temperature and duration of storage, the antimicrobial agents were stable, as demonstrated by the consistency in susceptibilities of the reference organisms from the American Type Culture Collection (Rockville, Md.). Overnight cultures of the bacterial isolates were diluted to yield a final inoculum of approximately 2.0 x 105 CFU of test organisms per ml. Verification of the desired inoculum size was done by performing standard colony counts on solid antibiotic-free medium. The MIC was the lowest concentration showing no visible
MATERIALS AND METHODS
Antibiotics. Fleroxacin powder was supplied by Hoffmann-La Roche, Inc., Nutley, N.J. Reagent-grade antimicrobial agents were obtained from their respective manufacturers. All antimicrobial agents were initially dissolved in appropriate solvents, as recommended by the manufacturers, and then diluted to a stock concentration of 2,560 ,ug/ml. All working antimicrobial solutions were stored at -70°C and used within 4 weeks of preparation. Bacteria. A total of 200 bacterial isolates from the sputa of patients with CF consisting of Pseudomonas aeruginosa (n = 106), Pseudomonas cepacia (n = 14), Haemophilus influ*
Corresponding author. 1880
COMPARATIVE IN VITRO ACTIVITY OF FLEROXACIN
VOL. 34, 1990
1881
TABLE 1. Comparison of in vitro activities of fleroxacin and other antimicrobial agents against isolates in sputum from patients with CF Organism (no. of isolates tested)
P. aeruginosa (106)
P. cepacia (14)
H. influenzae (24)
Range
50o
90%o
0.125-16.0 0.032-2.0 0.063->16.0 0.063->16.0 0.25-64.0 0.032->64.0 0.125-32.0 2.0-64.0 2.0->64.0 0.5-256.0
0.5-64.0
2.0 0.25 1.0 2.0 2.0 4.0 1.0 8.0 8.0 4.0 2.0
4.0 0.5 4.0 4.0 4.0 8.0 2.0 32.0 >64.0 32.0 8.0
2.0->16.0 0.25->16.0 2.0->16.0 1.0-2.0 4.0->16.0 8.0->128.0 4.0-64.0 5.0-16.0 1.0->128.0 0.125-64.0 0.063/1.19->16.0/304.0
2.0 1.0 16.0 4.0 16.0 16.0 32.0 8.0 32.0 16.0 1.0/19.0
16.0 4.0 >16.0 >16.0 >16.0 >128.0 64.0 16.0 >128.0 16.0
Fleroxacin Ciprofloxacin Enoxacin Ofloxacin Ceftazidine Aztreonam Imipenem Amikacin Gentamicin Piperacillin Tobramycin Fleroxacin Ciprofloxacin Enoxacin Ofloxacin Norfloxacin Chloramphenicol Gentamicin Piperacillin Ticarcillin Tobramycin
TMP-SMXb S. aureus (30)
MIC (ILg/ml)a
Antimicrobial agent
Fleroxacin Ciprofloxacin Enoxacin Norfloxacin Ofloxacin TMP-SMX Cefazolin Nafcillin Cefuroxime Gentamicin Chloramphenicol Tetracycline
0.25-1.0 0.125-0.5 0.25-2.0 0.25-2.0 0.125-0.5
0.5 0.5 1.0 1.0 0.5
'
