Pharmacodynamic modeling of the evolution of levofloxacin resistance in Staphylococcus aureus

Abstract

Previously, we demonstrated the importance of low-level-resistant variants to the evolution of resistance in Staphylococcus aureus exposed to ciprofloxacin in an in vitro system and developed a pharmacodynamic model which predicted the emergence of resistance. Here, we examine and model the evolution of resistance to levofloxacin in S. aureus exposed to simulated levofloxacin pharmacokinetic profiles. Enrichment of subpopulations with mutations in grlA and low-level resistance varied with levofloxacin exposure. A regimen producing average steady-state concentrations (Cavg ss) just above the MIC selected grlA mutants with up to 16-fold increases in the MIC and often additional mutations in grlA/grlB and gyrA. A regimen providing Cavg ss between the MIC and the mutant prevention concentration (MPC) suppressed bacterial numbers to the limit of detection and prevented the appearance of bacteria with additional mutations or high-level resistance. Regimens producing Cavg ss above the MPC appeared to eradicate low-level-resistant variants in the cultures and prevent the emergence of resistance. There was no relationship between the time concentrations remained between the MIC and the MPC and the degree of resistance or the presence or type of mutations that appeared in grlA/B or gyrA. Our pharmacodynamic model described the growth and levofloxacin killing of the parent strains and the most resistant grlA mutants in the starting cultures and correctly predicted conditions that enrich subpopulations with low-level resistance. These findings suggest that the pharmacodynamic model has general applicability for describing fluoroquinolone resistance in S. aureus and further demonstrate the importance of low-level-resistant variants to the evolution of resistance.

FIG. 1.

Total MRSA 8043 (left) and MRSA 8282 (right) bacterial counts (levofloxacin, 0 μg/ml) and subpopulations resistant to levofloxacin at 0.5 to 16 μg/ml in samples collected from the in vitro system at the indicated times during growth control and simulated levofloxacin dosage regimen experiments. Asterisks above the subpopulation bars indicate that bacteria were recovered in numbers below the reliable limit of detection (100 CFU/ml); no symbol above the bars denotes that no bacteria were recovered at the indicated concentration. Broth microdilution MICs of the total bacterial population in the absence (presence) of reserpine are indicated within the population profiles. Results are expressed as means of results of two separate experiments. The key for all panels appears in the top left panel.

FIG. 2.

Measured central (filled symbols) and peripheral (open symbols) compartment concentrations for simulated levofloxacin dosage regimens. The fitted curves from the one-compartment pharmacokinetic model are superimposed on the symbols. The levofloxacin MICs for MRSA 8043 and MRSA 8282 are depicted by the lower dot-and-dash horizontal line. The levofloxacin MPCs for MRSA 8043 (solid line) and MRSA 8282 (dashed line) are also shown.

FIG. 3.

Observed viable counts of MRSA 8043 (filled symbols, top panel), MRSA 8043L0-1 (open symbols, top panel), MRSA 8282 (filled symbols, bottom panel), and MRSA 8282L0-1 (open symbols, bottom panel) during growth control experiments and following exposure to simulated levofloxacin pharmacokinetic profiles. Viable counts are plotted as the means and ranges from two separate experiments. The reliable limit of detection for these experiments was 70 CFU/ml. The pharmacodynamic model-predicted viable count-versus-time profiles are shown by the solid (MRSA 8043 and MRSA 8282) and dashed (MRSA 8043L0-1 and MRSA 8282L0-1) curves. The key for both panels appears in the top panel. The slopes of the killing and regrowth portions of the viable-cell count curves were similar for replicate dosage regimen simulations with a given strain (error bars).