JAC Advance Access originally published online on April 19, 2006
Journal of Antimicrobial Chemotherapy 2006 57(6):1116-1121; doi:10.1093/jac/dkl135
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Selection of ciprofloxacin resistance in Escherichia coli in an in vitro kinetic model: relation between drug exposure and mutant prevention concentration
1 Antibiotic Research Unit, Department of Medical Sciences, Clinical Bacteriology and Infectious Diseases, Uppsala University S-751 22 Uppsala, Sweden 2 Microbiology Programme, Department of Cell and Molecular Biology, Biomedical Center, Uppsala University S-751 24 Uppsala, Sweden
*Corresponding author. Tel: +46-18-611-5640; Fax: +46-18-611-5650; E-mail: otto.cars{at}smi.ki.se
Received 19 July 2005; returned 16 October 2005; revised 9 February 2006; accepted 21 March 2006
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Abstract |
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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 Cmax 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 Cmax. When resistance was selected, several subpopulations with different levels of susceptibility to ciprofloxacin emerged.
Conclusions: Neither T > MPC nor Cmax proved to be single correlates for preventing resistance development. For the two investigated wild-type strains, an AUC/MPC ratio of 
22 was the single pharmacodynamic index that predicted prevention of resistant mutant development.
Keywords: antibiotic resistance , MPC , PK/PD , E. coli
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Introduction |
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In clinical isolates of Escherichia coli, resistance to fluoroquinolones is associated with the accumulation of multiple genetic alterations, usually chromosomal mutations affecting the drug target or drug efflux,1 but in some cases a plasmid-borne gene, qnr, also contributes to the resistance.2 One possible way to prevent the selection of mutants with reduced susceptibility to fluoroquinolones is to adjust antibiotic dosing regimens upwards to the mutant prevention concentration (MPC). Resistant subpopulations are proposed to be selectively enriched in the mutant selection window (MSW), the concentration range between MIC and MPC.3 MPC is defined as the lowest antibiotic concentration that prevents growth of the least susceptible first-step resistant mutant among a large (1010 cfu) bacterial population.3,4 Thus, for bacteria to grow at the MPC, two or more resistance mutations would have to arise concurrently within one bacterium. Since the frequency of fluoroquinolone resistance mutations in E. coli is <10–7, in theory a bacterial population of >1014 cells would be needed for two concurrent resistance mutations to arise.5 Such large bacterial population sizes are considered unlikely in clinical infections.6
The factors that influence MPC are not well understood. A recent study has shown that MPC cannot be accurately predicted from MIC.7 Thus, to determine whether MPC can be clinically applied, it must be measured for relevant bacterial populations. To be useful clinically, the MPC should be within the concentration limits that can be safely reached in patients. Although the methodology of MPC has not been formally standardized, in most experimental designs bacterial populations of 1010 cells are applied to antibiotic-containing agar plates, and the MPC is then defined as the minimal drug concentration that prevents the growth of bacterial colonies.3,4,7–9 These studies use fixed antibiotic concentrations and the methodology has the advantage that it can be used to measure MPC on many different isolates or populations of bacteria. However, in clinical settings the concentrations of an antimicrobial agent will have a complex pattern over time and in different compartments, and clearance of a drug can make concentrations decrease rapidly between doses.10–12
To evaluate the significance and reliability of MPC for clinical application one should try to measure MPC under conditions that closely mimic the clinical environment. Dynamic in vitro models can be used to take account of some of these fluctuations and to simulate the pharmacokinetic profiles of humans.13–15 For the fluoroquinolones, studies integrating pharmacokinetics and pharmacodynamics (PK/PD) have shown that the area under the concentration-time curve (AUC)/MIC and the maximum concentraion of drug in serum (Cmax)/MIC are the most important pharmacodynamic indices predicting the efficacy of bacterial killing.13,16,17 In the present study an in vitro kinetic model was used to investigate and evaluate the MPC of ciprofloxacin for two susceptible E. coli strains and one strain carrying a first-step fluoroquinolone resistance mutation in the gyrA gene. The purpose was to determine MPC using a constant infusion of ciprofloxacin and then relate MPC to the PK/PD of the drug. To achieve the latter objective, the prevention of growth of resistant mutants was studied in relation to different dosing profiles.
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Bacterial strains
Two fluoroquinolone-susceptible E. coli strains were used: a clinical urinary tract infection (UTI) isolate, Nu14,18,19 and a reference strain, CCUG (Culture Collection University of Gothenburg) 17620 (=ATCC 25922).20 In addition, a strain with reduced susceptibility to fluoroquinolones, Nu118, was derived from Nu14 by selection on norfloxacin solid media at 2x MIC. Nu118 has a gyrA mutation causing the amino acid substitution S83A.
Media and growth conditions
Bacteria were grown as liquid cultures in Mueller–Hinton (MH) broth (Difco Laboratories, Detroit, MI, USA) and as colonies on MH agar (Difco Laboratories) plates. Separate broth cultures were prepared for each experiment. Incubations were carried out at 35°C.
Antibiotic
Ciprofloxacin (Bayer AG, Leverkusen, Germany) was obtained as a reference powder with known potency. The antibiotic was dissolved in 0.1 M NaOH at 10 mg/mL on the day of use and diluted in MH broth.
MIC determinations
MIC was determined using Etest on MH agar plates according to the instructions of the manufacturer (AB Biodisk, Solna, Sweden). The original strains were tested in triplicate on different occasions.
In vitro kinetic model
The in vitro kinetic model used in this study has been described previously.14,15,21 It consists of a spinner flask (110 mL) with an open bottom that is placed on a holder with an outlet connected to a pump (P-500; Pharmacia Biotech, Uppsala, Sweden). A filter membrane with a pore size of 0.45 µm is supported by a metal rack between the flask and the holder, impeding dilution of bacteria. In addition, a magnetic stirrer is attached to the flask to ensure a homogeneous mixing of the culture and to prevent membrane pore blockage. The flask has two side arms: one with a silicone membrane to facilitate repeated sampling and another connected to a plastic tube from a vessel containing fresh medium. From the culture flask, the medium is drawn through the filter at a given rate by the pump, and fresh medium is sucked into the flask at the same rate by the negative pressure built up inside. The antibiotic added to the flask is diluted according to the first-order kinetics C = Cmax e–kt, where Cmax is the initial antibiotic concentration, C is the antibiotic concentration at time t, k is the rate of elimination and t is the time that has elapsed since the addition of antibiotic. The apparatus was operated in a thermostatic room at 35°C.
Determination of mutant prevention concentration
To compare MPC values in our model using broth with previously published studies using cultures on antibiotic-containing agar,7,22 the in vitro kinetic model was used with constant ciprofloxacin concentrations. The flask was prepared with broth, and bacteria from 6 to 7 h broth cultures were added to achieve an inoculum of 106 cfu/mL. After incubation at 35°C for 3–4 h the flask contained 
1010 bacterial cells (108 cfu/mL), and ciprofloxacin was added to obtain the desired concentration. To maintain this concentration in the system throughout the experiment, ciprofloxacin was added to the fresh medium that was continuously supplemented to the culture. The experiments were run for 24 h and samples were withdrawn at various time points. Appropriate dilutions were made in phosphate-buffered saline, pH 7.4, and bacteria were cultured on MH agar plates. After 24 h at 35°C the colonies were counted. The limit of detection for viable counts was 10 cfu/mL. For Nu14, ciprofloxacin concentrations of 4x, 8x, 16x, 32x and 64x the MIC were used and for the other strains at least two concentrations were tested to identify the MPC. For Nu14, six experiments were performed at 4x, 8x and 16x MIC, and for the other concentrations and the remaining strains experiments were done in triplicate. MIC was measured on single colonies from the 24 h samples to detect any changes in susceptibility. MPC was defined as the minimal concentration needed to prevent regrowth of cells after 24 h. The lack of mutant growth was confirmed by the absence of bacteria with increased MIC at 24 h.
Determination of time above MPC needed to prevent emergence of resistance (T > MPCPR) in the pharmacokinetic model
The pharmacodynamic index T > MPCPR was defined as the minimal time required for a ciprofloxacin concentration to be above the MPC to prevent the selection of resistant bacteria. Bacterial cultures were prepared as described above. The experiments were started with Cmax set at MPC (MPC = 16x MIC for Nu14). After 2, 4, 6, 8, 10, 14 or 18 h of exposure to these static concentrations, the antibiotic was diluted, with an elimination half-life (t1/2) of 4 h for the remaining time (total time 24 h). At the end point of each experiment (24 h) a sample of the population (35 mL) was removed from the flask and washed twice by centrifugation (1400 g for 15 min) with re-suspension in MH broth to avoid antibiotic transfer, and appropriate dilutions were spread on agar plates containing 4x, 8x and 16x MIC ciprofloxacin to detect resistant bacteria (limit of detection 1 cfu/10 mL). The unexposed controls were washed similarly. Each experiment was performed twice.
The T > MPCPR was further studied for each of the strains in experiments where a t1/2 of 4 h was used from time zero, to simulate the kinetics of ciprofloxacin in humans. In this series of experiments Cmax of ciprofloxacin ranged from 2x to 1024x MIC, depending on the strain, and the experiments were performed at least twice. At the end of each experimental cycle the bacteria were washed with MH broth as described above, prior to spreading on plates with and without ciprofloxacin. For Nu118, 45 mL of the cell suspension was removed from the flask and cultured on agar plates containing four ciprofloxacin concentrations (2x, 4x, 8x and 16x MIC). For Nu14, experiments were also carried out with Cmax of 64x MIC and t1/2 of 2 h.
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Determination of MIC and MPC at static ciprofloxacin concentrations
The MICs of ciprofloxacin were 0.008 (Nu14 and CCUG 17620) and 0.047 mg/L (Nu118), Table 1. The in vitro kinetic model was used with constant antibiotic concentrations for 24 h to determine the MPC of ciprofloxacin. From such experiments we determined that both the susceptible strains had an MPC of 0.128 mg/L ciprofloxacin, 16x MIC (Table 1), a concentration at which no mutants were detected in any experiment. This MPC for Nu14 is in agreement with that previously determined on agar, 0.1 mg/L.7 At concentrations below MPC (4x and 8x MIC) mutant bacteria arising from the susceptible strains had MICs of 0.094–0.19 mg/L. For Nu14, at 4x MIC, mutants were selected in all experiments, and, at 8x MIC, mutants were selected in four out of six experiments. For CCUG 17620, a concentration of 8x MIC selected for mutants in one out of three experiments. The MPC of the gyrA mutant based on similar time–kill curves was considerably lower in relation to its MIC (Table 1). Thus, Nu118 had an MPC of 4x MIC (0.188 mg/L). For Nu118, at a concentration below MPC (2x MIC), mutants with an MIC of 0.125 mg/L were selected in one of three experiments.
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We noted that after 24 h at MPC, a surviving population of cfu remained for Nu14 and Nu118 (but not for CCUG 17620) that were unaltered in their MIC values. In experiments with Nu14 over an extended time (72 h) and at a higher drug concentration (256x MIC), the persisting bacteria were still not completely eradicated (data not shown). However, when similar experiments (Cmax = 64x MIC) were made with smaller populations of Nu14, all cfu were eliminated within 24 h (109) or 2 h (107 cells). We have previously noted survivors/persisters in MPC assays on solid media.7
Determination of T > MPCPR in the pharmacokinetic model
In a first series of kinetic experiments Nu14 was exposed to static concentrations of ciprofloxacin at MPC (0.128 mg/L; 16x MIC) for 2–18 h before initiation of the dilution phase (t1/2 of 4 h). These experiments showed that a T > MPC of 18 h was sufficient to prevent the growth of resistant bacteria (Figure 1a). In experiments where T > MPC was shorter (
14 h) resistant mutants were selected on ciprofloxacin-containing agar (2x, 8x and 16x MIC) in all experiments.
In a second series of kinetic experiments, Cmax was varied (2–1024x MIC for Nu14; 4–128x MIC for CCUG 17620; 2–32x MIC Nu118) and a constant rate of drug dilution (t1/2 of 4 h) was initiated from time zero. For both susceptible strains a Cmax of 64x MIC, corresponding to a T > MPC of 8 h (33% of the dosing interval), prevented resistance development. When Cmax was lower, between 4 and 32x MIC, and T > MPC was less than 8 h, resistant mutants were selected in all experiments except one experiment of a triplicate (Cmax = 32x MIC) for Nu14 and one experiment of a duplicate (Cmax = 16x MIC) for CCUG 17620. Results of Nu14 are shown in Figures 1(b) and 2. A Cmax of 2x MIC did not allow selective amplification of resistant mutants and represents the lower boundary of the selective window. Analysis of these resistant mutants in Nu14 suggests the presence of subpopulations with different levels of reduced susceptibility to ciprofloxacin (Figure 2). For example, when Cmax was 4x MIC the cfu/mL at 24 h, was >108 on antibiotic-free plates, 105 on 4x and 8x MIC, and 102 on 16x MIC (Figure 2). CCUG 17620 results were similar to those for Nu14 except that mutants resistant to 16x MIC were not found. Additional experiments were performed with Nu14, where the t1/2 was shortened to 2 h but initial dose was kept the same (64x MIC), resulting in a T > MPC of 4 h. In these experiments resistance development was not prevented.
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For the mutant strain Nu118, a Cmax of only 8x MIC prevented resistance development. This corresponds to a T > MPC of 4 h (17% of the dosing interval). There was a subpopulation of Nu118 with a small decrease in susceptibility (2x MIC) in all experiments that was not taken into account when determining the T > MPCPR. The mutants selected from Nu118 when Cmax was lower (4x MIC; one out of two experiments) were resistant to 4x and 8x MIC.
At the end point of these experiments (24 h) we assayed for cfu at the highest ciprofloxacin concentrations tested. For all strains we found that some cells had survived/persisted under these conditions without any change in their original MIC, as also noted in our study with static concentrations on solid media.7 The numbers of survivors decreased with higher ciprofloxacin concentrations, arguing that the ‘paradoxical effect’23 is not the explanation. The numbers present at the highest ciprofloxacin concentrations tested were Nu14, 104 cfu/mL (at 1024x MIC); CCUG 17620, 106 cfu/mL (at 128x MIC); and Nu118, 107 cfu/mL (at 32x MIC). The persistence of a subpopulation of bacteria in the presence of an antibiotic is an established phenomenon,24 but why different bacterial populations and treatments give rise to more or less persister cells remains to be explained. The possible clinical importance of these persisting cells is unclear.
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Clinical and experimental studies indicate that suboptimal antibiotic dosage regimens may be a significant risk factor for emergence of resistance.25–30 Hence, it is important to optimize dosages, not only with respect to achieving a therapeutic effect, but also with respect to minimizing resistance development. MPC is a pharmacodynamic parameter which may be a useful tool to guide the dosage of antibiotics in order to reduce the emergence of bacteria with decreased antibiotic susceptibility. PK/PD modelling of ciprofloxacin in urinary tract infections (UTIs) shows that current regimens may not always yield urinary concentrations exceeding the MPC of E. coli during the whole dosage interval.7,31 We have previously studied the MPC of ciprofloxacin for UTI E. coli isolates with different levels of susceptibility in relation to static drug concentrations.7 In the present study, in order to better mimic the changing drug concentrations in the body, we used an in vitro kinetic model to study the selection of E. coli resistance in relation to the MPC and drug pharmacodynamics.
We began by using the in vitro kinetic model to measure the static ciprofloxacin MPC for each of three E. coli strains, two susceptible and one first-step resistant mutant. We found that both susceptible strains had an MPC of 16x MIC, while the mutant strain Nu118 had an MPC of 4x MIC. We note that the static MPC for Nu14 measured in liquid media in this study agrees with that previously measured on solid media.7
Having determined that the MPC for Nu14 (16x MIC) was similar in both agar and broth, we made 24 h experiments in the in vitro kinetic model where ciprofloxacin was maintained at MPC for different time periods before dilution (t1/2 4 h) was begun. These experiments showed that a T > MPC of 18 h was sufficient to prevent the selective enrichment of mutants.
In the next series of experiments (made on all strains) in the in vitro kinetic model, we varied the initial ciprofloxacin concentration (Cmax) and began dilution (t1/2 4 h) at time zero. We found that for both susceptible strains a Cmax of 64x MIC, corresponding to a T > MPC of 8 h (33% of the dosing interval), was sufficient to block the amplification of pre-existing resistant subpopulations and to prevent the selective enrichment of mutants. For the gyrA mutant Nu118, a Cmax of only 8x MIC, corresponding to a T > MPC of 4 h (17% of the dosing interval), prevented resistance development. These findings suggest that neither T > MPC nor Cmax are single correlates for preventing resistance development (Figure 1).
In Table 2 the dosage regimens with the lowest Cmax or shortest T > MPC that prevented emergence of resistance in these strains are compared with the dosage regimens with the highest Cmax or longest T > MPC where resistant mutants appeared. The data suggests that the single pharmacodynamic index that shows the least variation and therefore best predicts the prevention of resistance emergence in these strains is AUC/MPC. Thus, for both susceptible strains (Nu14 and CCUG 17620), an AUC/MPC 22 correlated with prevention of resistance emergence. For the gyrA mutant Nu118, an AUC/MPC of 11 was efficient to prevent resistance. Such levels can easily be reached in urine. However in serum, when protein binding and interindividual pharmacokinetic variability is taken into account, not all patients will reach this target with current dosages of ciprofloxacin.32
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To our knowledge there are no previous studies in pharmacokinetic models where the in vitro pharmacokinetics of fluoroquinolones have been studied in relation to the MPC of E. coli. We note that several studies that have focused on antibiotic resistance have failed to find a relationship between the emergence of bacterial resistance and a single pharmacodynamic index.33–36 Our results are, however, in broad agreement with a study in which five different fluoroquinolones were tested on Staphylococcus aureus, where the AUC/MPC was also the only parameter to correlate with the prevention of resistance development.37 In contrast, other studies, using moxifloxacin against a strain of Streptococcus pneumoniae,38 or using four fluoroquinolones against a strain of S. aureus,39 have identified a time within the mutant selection window (TMSW) <20% as the pharmacodynamic index that predicted prevention of the emergence of resistant bacteria. Our results, and a study using ciprofloxacin and S. aureus,36 did not support a simple relationship between TMSW and the prevention of the emergence of resistance. We do not have a ready explanation for the contradictions between different studies, but they could include species-specific effects or be related to methodological differences in the design or execution of the experiments.
In the present study, we have shown for E. coli and ciprofloxacin that a certain AUC/MPC ratio can influence the propensity for selection of resistance. Further studies are warranted to verify the usefulness of this pharmacodynamic index for the design of dosing regimens.
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None to declare.
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Acknowledgements |
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We thank Anita Perols and Juliana Larsson for excellent technical assistance. This work was supported by the EU Fifth Framework Programme (QLK2-CT-2001-00873) and Bayer AG.
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References |
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Allen GP, Kaatz GW, Rybak MJ. (2003) Activities of mutant prevention concentration-targeted moxifloxacin and levofloxacin against Streptococcus pneumoniae in an in vitro pharmacodynamic model. Antimicrob Agents Chemother 47:2606–14.
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Zinner SH, Lubenko IY, Gilbert D, et al. (2003) Emergence of resistant Streptococcus pneumoniae in an in vitro dynamic model that simulates moxifloxacin concentrations inside and outside the mutant selection window: related changes in susceptibility, resistance frequency and bacterial killing. J Antimicrob Chemother 52:616–22.
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W. H. F. Goessens, J. W. Mouton, M. T. ten Kate, A. J. Bijl, A. Ott, and I. A. J. M. Bakker-Woudenberg Role of ceftazidime dose regimen on the selection of resistant Enterobacter cloacae in the intestinal flora of rats treated for an experimental pulmonary infection J. Antimicrob. Chemother., March 1, 2007; 59(3): 507 - 516. [Abstract] [Full Text] [PDF]
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A. A. Firsov, M. V. Smirnova, I. Yu. Lubenko, S. N. Vostrov, Y. A. Portnoy, and S. H. Zinner Testing the mutant selection window hypothesis with Staphylococcus aureus exposed to daptomycin and vancomycin in an in vitro dynamic model J. Antimicrob. Chemother., December 1, 2006; 58(6): 1185 - 1192. [Abstract] [Full Text] [PDF]
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