FIG. 1.

Stepwise enrichment of ciprofloxacin-resistant H. influenzae mutants. (A) Effect of ciprofloxacin concentration on recovery of colonies. Wild-type H. influenzae ATCC 49247 (open circles) was applied to ciprofloxacin-containing agar at the indicated concentrations, and colonies were recovered. Arrows indicate ciprofloxacin concentrations used to obtain colonies for which nucleotide sequence information was obtained. Arrows a and b indicate recovery of Ser-84-to-Tyr GyrA variants. Strain KD2308 (arrow b) was used for second-round selection (solid circles). Cells from positions c and d contained an additional parC mutation that changed Ser-84 to Arg. Strain KD2322 (arrow d) was used for a third round (open triangles). Cells from positions e and f contained an additional gyrA mutation that changed Asp-88 to Asn. Strain KD2364 (arrow f) was used for a fourth round (solid triangles). Cells from positions g and h contained an additional parC mutation that changed Glu-88 to Lys. Dashed lines, drug concentrations at which no colony was recovered; dotted lines, guides for determining MIC₍₉₉₎ and MPC, as indicated. (B) Relationship of mutant selection window to serum drug concentration. Shaded areas represent mutant selection window defined by MIC₍₉₉₎ and MPC, which were obtained from data shown in panel A. (i) Wild-type cells; (ii) first-step mutant; (iii) second-step mutant; (iv) third-step mutant.