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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 &lt;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 Selection of Resistance to Clinafloxacin, Ciprofloxacin, and Trovafloxacin in Streptococcus pneumoniae