Mutant prevention concentrations of pradofloxacin for susceptible and mutant strains of Escherichia coli with reduced fluoroquinolone susceptibility.

Pharmacodynamic and mutant prevention properties of the fluoroquinolone pradofloxacin (PRA) were measured against a set of 17 Escherichia coli strains carrying no, one or two known mutations conferring reduced fluoroquinolone susceptibility. The strains included susceptible wild-types, isogenic constructed mutants, isogenic selected mutants and clinical isolates. The effectiveness of PRA was determined with regard to preventing the selection of resistant mutants, using static and changing concentrations of drug. Ciprofloxacin was used as a reference drug. Minimum inhibitory concentrations (MICs) and mutant prevention concentrations (MPCs) of PRA for the susceptible wild-type strains were in the range 0.012-0.016mg/L and 0.2-0.3mg/L, respectively, giving a mean±standard deviation mutant prevention index (MPI=MPC/MIC) of 17.7±1.1. The mean MPI PRA of the 14 mutant strains was 19.2±12, and the mean MPI across all 17 strains was 18.9±10.8. In an in vitro kinetic model in which PRA was diluted with a half-life of 7h to mimic in vivo conditions, an initial concentration of PRA of 1.6-2.4mg/L (8-10× MPC), giving a PRA AUC/MPC ratio of 73-92, and a T>MPC of 21-23h was sufficient to prevent the selection of resistant mutants from the three susceptible wild-type strains. Dosing to reduce selection for antibiotic resistance in veterinary therapy has a role in reducing the reservoir of resistant mutants. We conclude that a level of dosing that prevents the selection of resistant mutants during therapy should be achievable in vivo.

Optimizing drug exposure to minimize selection of antibiotic resistance.

The worldwide increase in antibiotic resistance is a concern for public health. The fact that the choice of dose and treatment duration can affect the selection of antibiotic-resistant mutants is becoming more evident, and an increased number of studies have used pharmacodynamic models to describe the drug exposure and pharmacodynamic breakpoints needed to minimize and predict the development of resistance. However, there remains a lack of sufficient data, and future work is needed to fully characterize these target drug concentrations. More knowledge is also needed of drug pharmacodynamics versus bacteria with different resistance mutations and susceptibility levels. The dosing regimens should exhibit high efficacy not only against susceptible wild-type bacteria but, preferably, also against mutated bacteria that may exist in low numbers in "susceptible" populations. Thus, to prolong the life span of existing and new antibiotics, it is important that dosing regimens be carefully selected on the basis of pharmacokinetic and pharmacodynamic properties that prevent emergence of preexisting and newly formed mutants.

Dose-related selection of fluoroquinolone-resistant Escherichia coli.

OBJECTIVES: To investigate the effects of clinically used doses of norfloxacin, ciprofloxacin and moxifloxacin on survival and selection in Escherichia coli populations containing fluoroquinolone-resistant subpopulations and to measure the value of the pharmacodynamic index AUC/mutant prevention concentration (MPC) that prevents the growth of pre-existing resistant mutants. METHODS: Mixed cultures of susceptible wild-type and isogenic single (gyrA S83L) or double (gyrA S83L, Delta marR) fluoroquinolone-resistant mutants were exposed to fluoroquinolones for 24 h in an in vitro kinetic model. Antibiotic concentrations modelled pharmacokinetics attained with clinical doses. RESULTS: All tested doses eradicated the susceptible wild-type strain. Norfloxacin 200 mg administered twice daily selected for both single and double mutants. Ciprofloxacin 250 mg administered twice daily eradicated the single mutant, but not the double mutant. For that, 750 mg administered twice daily was required. Moxifloxacin 400 mg once daily eliminated the single mutant, but did not completely remove the double mutant. The MPC of ciprofloxacin was determined and based on those dose simulations that eradicated mutant subpopulations, an AUC/MPC(wild-type) of 35 prevented selection of the single mutant, whereas an AUC/MPC(single mutant) of 14 (equivalent to an AUC/MPC(wild-type) of 105) prevented selection of the double mutant. CONCLUSIONS: All tested clinical dosing regimens were effective in eradicating susceptible bacteria, but ciprofloxacin 750 mg twice daily was the only dose that prevented the selection of single- and double-resistant E. coli mutants. Thus, among approved fluoroquinolone dosing regimens, some are significantly more effective than others in exceeding the mutant selection window and preventing the enrichment of resistant mutants.

Selection of ciprofloxacin resistance in Escherichia coli in an in vitro kinetic model: relation between drug exposure and mutant prevention concentration.

OBJECTIVES: To evaluate the mutant prevention concentrations (MPCs) of ciprofloxacin for two susceptible and one first-step gyrA resistant mutant Escherichia coli strains in an in vitro kinetic model and to identify the pharmacodynamic index that best predicts prevention of resistance emergence. METHODS: An in vitro kinetic model was used to measure MPC with static antibiotic concentrations and to test different dosing profiles to study pharmacokinetics/pharmacodynamics indices important to prevent the growth of resistant mutants. In one set of kinetic experiments the starting concentration was equal to the MPC and the T > MPC was varied before antibiotic dilution was begun. In a second set of kinetic experiments C(max) was varied and dilution of the antibiotic was started at time zero. RESULTS: From the static experiments we calculated MPC values of 0.128 mg/L for both the susceptible strains (16x MIC) and 0.188 mg/L (4x MIC) for the first-step resistant (gyrA) strain. The kinetic experiments showed that the T > MPC needed to prevent the growth of resistant bacteria was shorter with an increased C(max). When resistance was selected, several subpopulations with different levels of susceptibility to ciprofloxacin emerged. CONCLUSIONS: Neither T > MPC nor C(max) proved to be single correlates for preventing resistance development. For the two investigated wild-type strains, an AUC/MPC ratio of > or =22 was the single pharmacodynamic index that predicted prevention of resistant mutant development.

Pharmacodynamic model to describe the concentration-dependent selection of cefotaxime-resistant Escherichia coli.

Antibiotic dosing regimens may vary in their capacity to select mutants. Our hypothesis was that selection of a more resistant bacterial subpopulation would increase with the time within a selective window (SW), i.e., when drug concentrations fall between the MICs of two strains. An in vitro kinetic model was used to study the selection of two Escherichia coli strains with different susceptibilities to cefotaxime. The bacterial mixtures were exposed to cefotaxime for 24 h and SWs of 1, 2, 4, 8, and 12 h. A mathematical model was developed that described the selection of preexisting and newborn mutants and the post-MIC effect (PME) as functions of pharmacokinetic parameters. Our main conclusions were as follows: (i) the selection between preexisting mutants increased with the time within the SW; (ii) the emergence and selection of newborn mutants increased with the time within the SW (with a short time, only 4% of the preexisting mutants were replaced by newborn mutants, compared to the longest times, where 100% were replaced); and (iii) PME increased with the area under the concentration-time curve (AUC) and was slightly more pronounced with a long elimination half-life (T(1/2)) than with a short T(1/2) situation, when AUC is fixed. We showed that, in a dynamic competition between strains with different levels of resistance, the appearance of newborn high-level resistant mutants from the parental strains and the PME can strongly affect the outcome of the selection and that pharmacodynamic models can be used to predict the outcome of resistance development.

Mutant prevention concentrations of ciprofloxacin for urinary tract infection isolates of Escherichia coli.

OBJECTIVES: To measure the mutant prevention concentration (MPC) of ciprofloxacin for a set of urinary tract infection (UTI) Escherichia coli isolates with different levels of susceptibility and determine whether MPC can be predicted from MIC. METHODS: MPC was defined as the lowest ciprofloxacin concentration that prevented the growth of resistant colonies when 10(10) bacteria were spread on solid medium and incubated for 96 h at 37 degrees C. MIC was measured by Etest. Bacteria surviving (persisting) at MPC were isolated and quantified from agar plugs taken after 96 h. The genes hipA and hipB were amplified by PCR from persisters and sequenced. RESULTS: Isolates with MICs above the NCCLS breakpoint for ciprofloxacin resistance (4 mg/L) typically have MPCs greater than 32 mg/L. Isolates with MICs below the breakpoint for ciprofloxacin susceptibility (1 mg/L) have MPCs up to 5 mg/L. MPC/MIC is approximately 16 for most susceptible isolates but there are several notable exceptions (MPC/MIC > 100). Resistant colonies arising one dilution step below MPC often had MIC > MPC. In every case tested, a proportion of cells survived (persisted), but did not grow into colonies, at MPC, without any increase in MIC. CONCLUSIONS: MPCs were determined for all ciprofloxacin-susceptible isolates. MPC is not accurately predicted from MIC. Colonies selected below MPC frequently have MIC > MPC, suggesting multiple mutations. A small fraction of cells from all strains tested survived for 96 h at MPC, without any associated increase in MIC. These survivors/persisters are not hipAB mutants.