Insights into the evolutionary trajectories of fluoroquinolone resistance in Streptococcus pneumoniae

Objectives: Fluoroquinolone resistance in Streptococcus pneumoniae typically arises through specific site mutations, but dynamic variation of mutations in the resistance evolution and interaction among these muta-tions have not been clearly demonstrated. The objectives of this study were to investigate the dynamics of allele frequency in populations evolved under fluoroquinolone pressure and pervasive interactions among mutations present in the evolutionary trajectories. Methods: Thirty-three evolved populationswere obtained by serial passages in the presence of antibiotic pressure and these populations were sequenced by using the Paired-End Illumina method. Mutants that occurred in the evolutionary trajectories were constructed by transforming the parental strain with PCR fragments containing corresponding mutations. Results: The number of target mutations increased progressively, consistent with phenotypic adaptation to moxifloxacin and levofloxacin. However, more mutations are required for high-level resistance to moxifloxacin than levofloxacin. Pervasive interactions, including positive epistasis between mutations, play a role in the evo-lutionary trajectories of resistance to the two drugs. Twomutations (R447C and P454S) in gyrBwere identified to confer 2-fold increases in resistance to moxifloxacin and levofloxacin based on the background of the double mutant S81F/S79F in parC. Moreover, the dynamics of allele frequency in evolved populations was reveale