Single- and multi-step resistance selection study of gemifloxacin compared with trovafloxacin, ciprofloxacin, gatifloxacin and moxifloxacin in Streptococcus pneumoniae.

The ability of sequential subcultures in subinhibitory concentrations of gemifloxacin, trovafloxacin, ciprofloxacin, gatifloxacin and moxifloxacin to select resistant mutants was studied in 16 pneumococci [eight with ciprofloxacin MICs (mg/L) 0.25-1; four with 8-16; four with 16-32]. Subculturing was done 50 times, or until mutants with elevated MICs (> or = 4 x) to the selecting drug emerged. Subculturing in gemifloxacin selected six resistant mutants (gemifloxacin MICs 2 mg/L); trovafloxacin selected nine (trovafloxacin MICs 2-4 mg/L); ciprofloxacin selected 11 (ciprofloxacin MICs 8-128 mg/L); gatifloxacin selected 13; and moxifloxacin selected 12 (gatifloxacin or moxifloxacin MICs 2-16 mg/L). DNA sequencing showed that most mutants had mutations in ParC at Ser-79 or Asp-83 and in GyrA at Ser-81 or Glu-85; some mutants also had mutations in ParE or GyrB. Some new mutations were found in ParE or GyrB that have not yet been reported; GyrB mutation might be associated with moxifloxacin resistance. Both DNA gyrase and topoisomerase IV were thought to be the target of gemifloxacin; gemifloxacin also selected mutants with single modifications in gyrA, parC or parE alone among derived mutants by repeated exposure to subinhibitory concentrations of fluoroquinolones. In the presence of reserpine, most mutants had lower MICs of ciprofloxacin and gemifloxacin (4-32 x), and gatifloxacin (4-8 x), suggesting an efflux mechanism; none had lower trovafloxacin and moxifloxacin MICs. All quinolones tested selected for resistance; judicious use and proper dosing will be necessary to avoid resistance selection of newer broad-spectrum fluoroquinolones.

In vitro development of resistance to ceftriaxone, cefprozil and azithromycin in Streptococcus pneumoniae.

Approval of ceftriaxone for the treatment of otitis media has led to fear of selection of resistant mutants owing to widespread use. To test this, we examined the ability of sequential subcultures in sub-MICs of ceftriaxone, cefprozil and azithromycin to select resistant mutants in 12 pneumococci. Daily subculturing was performed 50 times or until mutants with raised ceftriaxone, cefprozil or azithromycin MICs were selected. Of eight ceftriaxone-susceptible parents, ceftriaxone did not select for any resistant mutants, while cefprozil selected for four mutants (MICs 2-4 mg/L after 21-50 subcultures). Among four ceftriaxone-resistant parents, subculturing in ceftriaxone selected for one stable mutant with raised ceftriaxone MIC (>16 mg/L after 21 subcultures) and subculturing in cefprozil selected for one mutant with raised cefprozil MIC (64 mg/L after 44 subcultures). Mutations were observed in pbp2x and pbp1a. Among six azithromycin-susceptible parents, subculturing in azithromycin selected for five resistant mutants (MIC 0.5-32 mg/L after 10-42 passages) and among six azithromycin-resistant strains, subculturing selected for mutants with raised azithromycin MICs in all six strains (MIC 16-32 mg/L after 4-18 passages). All azithromycin-resistant mutants derived from azithromycinsusceptible parents had mutations in domain V of 23S rRNA while all azithromycin-resistant parents and derived mutants had mefE. Single-step mutation rates among the 12 strains at the MIC ranged from 1.5 x 10(-6) to <6.2 x 10(-10) for ceftriaxone, >1.3 x 10(-5) to 8.9 x 10(-8) for cefprozil and >1.1 x 10(-6) to 6.7 x 10(-10) for azithromycin. Multi-step and single-step testing showed that ceftriaxone selected for resistant mutants less often than cefprozil and azithromycin.

In vitro selection of resistance to clinafloxacin, ciprofloxacin, and trovafloxacin in Streptococcus pneumoniae.

Ability of daily sequential subcultures in subinhibitory concentrations of clinafloxacin, ciprofloxacin, and trovafloxacin to select resistant mutants was studied in 10 pneumococci (ciprofloxacin MICs, 1 to 4 microg/ml, and clinafloxacin and trovafloxacin MICs, 0.06 to 0.125 microg/ml [n = 9]; ciprofloxacin, clinafloxacin, and trovafloxacin MICs, 32, 0.5, and 2 microg/ml, respectively [n = 1]). Subculturing was done 50 times, or until MICs increased fourfold or more. Mutants for which MICs were fourfold (or more) higher than those for parent strains were selected in five strains by clinafloxacin, in six strains by trovafloxacin, and nine strains by ciprofloxacin. Sequence analysis of type II topoisomerase showed that most mutants had mutations in ParC at Ser79 or Asp83 and in GyrA at Ser81, while a few mutants had mutations in ParE or GyrB. In the presence of reserpine, the MICs of ciprofloxacin and clinafloxacin for most mutants were lower (four to eight times lower), but for none of the mutants were trovafloxacin MICs lower, suggesting an efflux mechanism affecting the first two agents but not trovafloxacin. Single-step mutation rates were also determined for eight strains for which the MICs were as follows: 0.06 microg/ml (clinafloxacin), 0.06 to 0.125 microg/ml (trovafloxacin), and 1 microg/ml (ciprofloxacin). Single-step mutation rates with drugs at the MIC were 2.0x10(-9) to <1.1x10(-11), 5.0x10(-4) to 3.6x10(-9), and 4.8x10(-4) to 6.7x10(-9), respectively. For two strains with clinafloxacin MICs of 0.125 to 0.5 microg/ml trovafloxacin MICs of 0. 125 to 2 microg/ml, ciprofloxacin MICs of 4 to 32 microg/ml mutation rates with drugs at the MIC were 1.1x10(-8)-9.6x10(-8), 3.3x10(-6)-6. 7x10(-8), and 2.3x10(-5)-2.4x10(-7), respectively. Clinafloxacin was bactericidal at four times the MIC after 24 h against three parent and nine mutant strains by time-kill study. This study showed that single and multistep clinafloxacin exposure selected for resistant mutants less frequently than similar exposures to other drugs studied.

In vitro development of resistance to telithromycin (HMR 3647), four macrolides, clindamycin, and pristinamycin in Streptococcus pneumoniae.

The ability of 50 sequential subcultures in subinhibitory concentrations of telithromycin (HMR 3647), azithromycin, clarithromycin, erythromycin A, roxithromycin, clindamycin, and pristinamycin to select for resistance was studied in five macrolide-susceptible and six macrolide-resistant pneumococci containing mefE or ermB. Telithromycin selected for resistance less often than the other drugs.

In vitro development of resistance to five quinolones and amoxicillin-clavulanate in Streptococcus pneumoniae.

The ability of 50 sequential subcultures in subinhibitory concentrations of ciprofloxacin, levofloxacin, grepafloxacin, sparfloxacin, trovafloxacin, and amoxicillin-clavulanate to select for resistance was studied for six penicillin-susceptible and four penicillin-intermediate pneumococci. Subculturing in ciprofloxacin, grepafloxacin, levofloxacin, and sparfloxacin led to selection of mutants requiring increased MICs for all 10 strains, with MICs rising from (i) 0.5 to 4.0 to (ii) 4.0 to 32.0 microgram/ml after 7 to 12 passages for ciprofloxacin, from (i) 0.06 to 0.25 to (ii) 0.5 to 8.0 microgram/ml after 5 to 23 passages for grepafloxacin, from (i) 0.5 to 1.0 to (ii) 4.0 to 64 microgram/ml after 14 to 49 passages for levofloxacin, and from (i) 0.125 to 0.25 to (ii) 1.0 to 16.0 microgram/ml after 8 to 26 passages for sparfloxacin. Subculturing in trovafloxacin led to increased MICs for eight strains, with MICs rising from (i) 0.06 to 0.125 to (ii) 0.5 to 8.0 microgram/ml after 6 to 28 passages. Subculturing in amoxicillin-clavulanate led to raised MICs for only one strain, with the MIC rising from 0.015 to 0. 125 microgram/ml after 24 passages. Double mutations in both ParC and GyrA led to high-level quinolone resistance when ParC mutations were at S79. Trovafloxacin MICs were 1 to 2 microgram/ml in double mutants with ParC mutations at positions other than S79 (e.g., D83). Mutations in ParE (at D435, R447, and E474) and GyrB (at S405, D406, and D435) were found in four and six mutants, respectively. In the presence of reserpine, 29 mutants had lower ciprofloxacin MICs (2 to 16 times lower), 8 mutants had lower levofloxacin MICs (2 times), and one mutant had a lower trovafloxacin MIC (2 times), suggesting the involvement of an efflux mechanism. In contrast to the case for quinolones, subculturing in the presence of amoxicillin-clavulanate did not select for resistance to this drug.