JAC Advance Access published online on July 16, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm265
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Dose-related selection of fluoroquinolone-resistant Escherichia coli
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 Centre, Uppsala University, S-751 24 Uppsala, Sweden
* Corresponding author. E-mail: sara.olofsson{at}medsci.uu.se
Received 11 April 2007; returned 21 May 2007; revised 25 June 2007; accepted 25 June 2007
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Abstract |
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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, 
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/MPCwild-type of 35 prevented selection of the single mutant, whereas an AUC/MPCsingle mutant of 14 (equivalent to an AUC/MPCwild-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.
Key Words: antibiotic resistance , MPC , PK/PD
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Introduction |
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Fluoroquinolones have a broad antibacterial spectrum and are used to treat a variety of bacterial infections including urinary tract infections (UTIs).1 To preserve their effectiveness in the face of an increasing prevalence of resistant strains, optimal dosing regimens need to be implemented with respect to both bactericidal effect and potential for the selection of resistant mutants. Most dosing regimens of antibiotics are based on the pharmacodynamics of susceptible cells and ignore resistant mutant subpopulations.2 Mutant subpopulations are often present,3–5 and their enrichment has been observed in patients.6 These subpopulations will be enriched if doses fall inside the mutant selection window (MSW),7–9 which has led to the concept that levels should be kept above the mutant prevention concentration (MPC) to restrict resistance selection.10,11 The MPC, as originally defined, is the lowest drug concentration that prevents the growth of the least susceptible first-step resistant mutant. However, according to current breakpoints, organisms that are first- or second-step resistant mutants are often considered susceptible to fluoroquinolones, and so we have extended the definition of MPC to the lowest drug concentration that prevents growth of the most resistant ‘next-step’ mutant. There is good evidence for the selective enrichment of mutants within the MSW with in vitro dynamic models7,8,12–14 and in animal models.9,15 In the previous work,16 we suggested that AUC/MPC could serve as a measure of the drug exposure that prevents the selection of drug-resistant mutants. A simple logic for the use of AUC/MPC has been made,11 which fits with earlier empirical relationships.
Antibiotic exposure of mixed cultures of wild-type and mutant strains can provide important information, but requires cultivation and replication on antibiotic-containing agar. Such methods are time-consuming and cannot separate strains with small differences in MIC. We have developed a method, in which isogenic Escherichia coli strains with different levels of fluoroquinolone susceptibility can be detected by their different colony colours on agar. A fitness-neutral mutation in the araB gene of one of the strains causes red colony colour on tetrazolium-arabinose (TA) agar, whereas strains without this mutation make pink colonies. This difference in colony colour allows for a simple and accurate identification of the strains in a mixed population. We mixed a fluoroquinolone-susceptible strain with subpopulations of isogenic mutant strains in the proportion 99:1 and followed the changes in their populations as a function of exposure to fluoroquinolones. This mimics an extreme but potential in vivo situation, in which, possibly after repeated exposure to fluoroquinolones, a patient has a 1% level of mutants. Thus, in an in vitro kinetic model, the bacterial mixtures were exposed to simulated serum concentrations of currently used doses of norfloxacin, ciprofloxacin and moxifloxacin over a 24 h period and the pharmacodynamic effects on pre-existing mutants were determined for the different doses.
In the present work, we found that particular approved dosing regimens, e.g. ciprofloxacin 750 mg administered twice daily, were significantly better than others, e.g. norfloxacin 200 mg administered twice daily, at eradicating resistant mutants. This kind of analysis allows compounds and dosing regimens to be compared for their ability to restrict the selection of resistance.
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Materials and methods |
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Bacterial strains
Three isogenic strains of E. coli with different fluoroquinolone susceptibilities were used in the kinetic experiments: LM347, wild-type susceptibility; LM378, a gyrA single mutant; and LM421, a gyrA/marR double mutant. The three strains were all derived from E. coli MG1655, a fully sequenced laboratory strain,17 and their genotypes are listed in Table 1. Throughout the text, LM347 is referred to as the wild-type strain, LM378 as the single mutant and LM421 as the double mutant. Previous studies of natural and evolved E. coli UTI isolates showed that the gyrA S83L mutation is common among those with single mutations, whereas a gyrA mutation together with an efflux-regulatory mutation affecting either marR or acrR is common in those with double mutations.18,19
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Growth conditions
Bacterial growth in suspension was achieved in Mueller–Hinton (MH) broth (Difco Laboratories, Detroit, MI, USA). Solid TA indicator agar20 was used to assay the colour of bacterial colonies on plates. TA agar contains a colour indicator causing bacteria able to metabolize L-arabinose (LM378 and LM421, single and double fluoroquinolone-resistant mutants) to form pink colonies, whereas bacteria lacking this ability (LM347, araB::FRT, fluoroquinolone-susceptible) form red colonies. This phenotype allows for the simple identification of each strain in a mixed culture. The araB::FRT mutation was determined experimentally to be neutral in growth competition with an isogenic ara+ strain (relative growth rate of 1.002 ± 0.005). Preceding each experiment, strains were grown in MH for 6 h at 35°C, resulting in a bacterial concentration of 
5 x 108 cfu/mL.
Norfloxacin and ciprofloxacin powders were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Moxifloxacin was kindly provided by Bayer HealthCare AG (Leverkusen, Germany). To obtain 10 mg/mL stock solutions, norfloxacin and ciprofloxacin were dissolved in 0.1 M NaOH, whereas moxifloxacin was dissolved in sterile distilled water. Stock solutions were further diluted to appropriate concentrations in MH broth prior to each experiment.
MIC was determined using the Etest on Iso-Sensitest agar (Oxoid Ltd, Basingstoke, UK), according to the instructions of the manufacturer (AB BIODISK, Solna, Sweden). Original strains were tested in triplicate on separate occasions. To detect changes in susceptibility occurring during the kinetic experiments, MIC was determined for five separate colonies (where survivors were found) at the endpoint of the experiment. A change in susceptibility was defined as significant only if it differed by at least two steps on the Etest concentration scale from the MIC for the strain with which it was being compared. Each two steps on the Etest scales for norfloxacin, ciprofloxacin and moxifloxacin represents a doubling of antibiotic concentration.
The MPC of ciprofloxacin for each strain was determined by spreading a total of 
1010 bacterial cells on agar plates (109 cfu/plate) containing different ciprofloxacin concentrations and following the growth of resistant colonies up to 96 h, as described previously.21 The ciprofloxacin concentration steps used to measure MPC for the three strains were in mg/L: 0.275, 0.3, 0.5, 1.0, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.05, 2.2, 2.5, 5.0 and 10.0. Each experiment was performed in triplicate. MPC was defined as the lowest drug concentration that prevented growth of resistant next-step mutants in a large bacterial population (1010 cfu).
An in vitro kinetic model described previously22,23 was used to simulate the serum drug concentration–time curve in humans. The model 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. A magnetic stirrer is attached to the flask, ensuring homogenous mixing of the culture and preventing membrane pore blockage. The flask has two side arms: one has a silicon membrane inserted to facilitate repeated sampling and another is connected to a bottle with fresh medium. 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.
Antibiotic added to the flask is diluted according to the first-order kinetics C = Cmax x e–kt, where C is the antibiotic concentration at time t, Cmax the initial antibiotic concentration, k the rate of elimination and t the time elapsed since addition of antibiotic. The apparatus was placed in a thermostatic room at 35°C during the experiments.
The antibiotic concentrations used in the kinetic experiments corresponded to clinical doses of norfloxacin, ciprofloxacin and moxifloxacin, taking into account serum protein binding.24–26 The doses and initial concentrations (Cmax) used are listed in Table 3. The elimination half-life used was 5.1 h for norfloxacin,25 4 h for ciprofloxacin26 and 12 h for moxifloxacin.24
Six-hour bacterial cultures of wild-type and mutant bacteria were mixed in the flask in the proportion of 99:1, resulting in an initial wild-type concentration of 105 cfu/mL and a mutant concentration of 103 cfu/mL. The total bacterial cfu at the start of each experiment, taking into account the 110 mL volume in the growth flask, was 107 cfu. The low total cfu number was chosen to reduce the possibility that new spontaneous mutants would influence the outcome of competition experiments. These experiments are competition experiments and measure the selection or eradication of pre-existing mutants as a function of different fluoroquinolone dosing regimens. The 99:1 ratio would reflect an extreme but potential in vivo situation, in which, possibly after repeated exposure to fluoroquinolones, a patient has a 1% level of mutants. Furthermore, on the reasonable assumption that each mutant cell responds independently to drug exposure, and with our detection limit of 10 cfu/mL, then each experiment with a 99:1 ratio is quantitatively equivalent to making 104 parallel competition/selection experiments with each mutant cell present at a frequency of approximately 10–7 relative to the susceptible wild-type, close to the spontaneous mutation frequency to resistance. Accordingly, the eradication of all mutant cells in an experiment is a strong indication of a very effective treatment regimen. Antibiotic was added at the starting point of each experiment to obtain the desired initial concentration. The second antibiotic dose (norfloxacin and ciprofloxacin) was given after 12 h.
Samples of 400 µL were taken immediately after the initial introduction of bacteria into the vessel (before and after addition of the drug) and then after 2, 4, 6, 8, 12 and 24 h. The cultures were diluted in phosphate-buffered saline, pH 7.4, and 10 and 100 µL aliquots were seeded onto TA indicator agar. The plates were incubated for 24 h at 35°C after which red and pink colonies were counted. The detection limit was 10 cfu/mL. Five colonies of each strain, if present at 24 h, were picked and tested for MIC. Each experiment was performed three times.
Determination of antibiotic concentrations
Validation of drug pharmacokinetics in the in vitro model was carried out during experiments with ciprofloxacin 100 and 250 mg twice daily, where samples taken at 0, 2, 6, 12 and 24 h after addition of the drug were stored at –20°C. These samples were analysed using a microbiological diffusion model in order to determine the actual antibiotic concentrations. Plates were prepared with 35 mL of Iso-Sensitest agar (Oxoid Ltd) seeded with a test strain, E. coli MB3804. Thirty microlitres of four standard concentrations (0.125, 0.25, 0.5 and 1 mg/L) of ciprofloxacin and samples from the kinetic experiment were added to agar wells. The plates were incubated at 35°C overnight. The inhibition zones were measured and the antibiotic concentrations of the samples were calculated by linear regression. All assays were performed in triplicate and the correlation coefficient for the standard curves was always 0.99. The sensitivity was 0.025 mg/L, and the between-day coefficient of variation was 8%.
Five mutants with increased MICs, two derived from single mutants and three from double mutants, were collected at the endpoint of the ciprofloxacin experiments. These were sequenced to determine whether they had acquired additional mutations in genes previously shown to be associated with resistance mutations. The conditions and primer sequences for PCR amplification and nucleotide sequencing of gyrA, gyrB, parC, parE, marR and acrR were as described previously.18
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MIC and MPC
Isogenic strains of susceptible E. coli were constructed (see the Materials and methods section) carrying single (gyrA S83L) or double (gyrA S83L, marR) fluoroquinolone-resistance mutations (Table 1). The MICs and MPCs for the susceptible wild-type, single-mutant and double-mutant strains are presented in Table 2. For ciprofloxacin, the single and double mutants had MICs 16- and 63-fold over the susceptible wild-type MICs, respectively. The MPC of ciprofloxacin for the susceptible wild-type strain was 0.3, corresponding to a 25-fold increase in comparison with the MIC. Ciprofloxacin MPCs for the single and double mutants were 2.2 and 10 mg/L, respectively (10-fold increases in comparison with the MICs for the respective strains and equivalent to 200-fold and 800-fold increases, respectively, of the MIC for the susceptible wild-type strain). These values are all within the range previously found for clinical E. coli isolates with similar resistance genotypes and phenotypes.21
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Kinetic experiments
Mixed cultures containing drug-susceptible wild-type and either single or double fluoroquinolone-resistant mutants were grown in the presence of fluoroquinolones in the in vitro kinetic model (see the Materials and methods section).
Norfloxacin. Norfloxacin added at a concentration corresponding to a dose of 200 mg administered twice daily eradicated the wild-type strain in all experiments (Table 3). In these experiments, the single mutant with an original MIC of 0.75 mg/L was not eradicated and developed further loss of susceptibility with MICs ranging from 1.5 to 3 mg/L. The growth of the double mutant was unaffected by norfloxacin, and at the endpoint of each experiment, its MIC was unchanged. Thus, norfloxacin at approved doses failed to suppress outgrowth of either mutant.
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Ciprofloxacin. Time–kill curves of the mixed strains exposed to various doses of ciprofloxacin in the kinetic model are shown in Figure 1. Ciprofloxacin simulating a dose of 100 mg administered twice daily was sufficient to eradicate the susceptible wild-type strain but selected the single mutant. At 24 h, these selected mutant isolates had developed increased loss of susceptibility with MIC values up to 0.75 mg/L (original MIC 0.19).
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A dose simulating 250 mg administered twice daily eradicated both the wild-type and the single mutant, but selected the double mutant, which showed regrowth at 12 and 24 h. At the end of these experiments, the derivatives of the double mutant had MICs up to 1.5 mg/L when compared with the original MIC of 0.75 mg/L.
When the ciprofloxacin dose was increased to 500 mg administered twice daily, the double-mutant strain was still selected and MICs for derivatives ranged up to 2 mg/L at 24 h. Finally, ciprofloxacin concentrations simulating the highest dose, 750 mg administered twice daily, eradicated all bacteria, including the double mutant (Figure 1).
As a control on the validity of these assays, actual ciprofloxacin concentrations present in the model were determined during the kinetic experiments (100 and 250 mg twice daily) and are presented in Table 4. These concentrations are in good agreement with the predicted values.
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Moxifloxacin. Moxifloxacin is administered at a dose of 400 mg once daily and in the in vitro kinetic model a dosing concentration mimicking this was sufficient to eradicate the wild-type and the single mutant. However, when the bacterial mixture contained the double mutant, only the wild-type strain was completely killed. The double mutant was detected at 24 h in one out of three experiments, but the MIC value for the strain was unchanged.
Previous work has shown that with individual patient isolates, one cannot reliably predict MPC from MIC,21,27,28 and that AUC/MPC is more predictive than AUC/MIC for the prevention of mutant growth.16 AUC/MPC values were calculated for each of the clinical doses of ciprofloxacin used in the in vitro kinetic experiments (Table 3). An AUC/MPC of 35 prevented growth of the single mutant, whereas the next lowest ratio tested, an AUC/MPC of 14, was not restrictive. An AUC/MPCwild-type of 105 prevented growth of the double mutant, whereas the next lowest ratio tested, an AUC/MPCwild-type of 55, was not restrictive (Table 3). However, it may be more appropriate to consider restriction of growth of the double mutant relative to the single mutant because MPC relates to the prevention of growth of the next-step mutation. Accordingly, an AUC/MPCsingle mutant of 14 (based on the MPC for the single mutant of 2.2) prevented growth of the double mutant, whereas the next lowest ratio tested, an AUC/MPCsingle mutant of 8, was not restrictive.
Five mutants with significantly increased MICs (at least two steps on the Etest scale, a doubling of MIC) were isolated at the endpoint of ciprofloxacin dosing experiments and DNA sequences were determined for gyrA, gyrB, parC, parE, marOR and acrR in each of the strains. In one strain, derived from the double mutant exposed to a ciprofloxacin dose of 500 mg administered twice daily, the new mutation parC G171C was identified. This parC mutation, which has not previously been identified, increases the MIC for the strain by two steps on the Etest scale, from 0.75 to 1.5 mg/L. No new mutation was found in the genes sequenced in the other four mutant strains. For these four strains, the small increase in MIC may be within the limits of reproducibility of the Etest, but the lack of identified mutations is also consistent with previous findings that many mutations causing small increases in fluoroquinolone resistance are not found in the classical resistance-associated genes.19
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Discussion |
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Fluoroquinolones are one of the most widely used antibiotic classes,29 but resistance among important bacterial pathogens is increasing.30,31 During antibiotic treatment, the selection of resistant mutants may take place both at the infectious site and in the commensal flora, e.g. gut, skin and respiratory tract. Using isogenic strains with different levels of fluoroquinolone susceptibility, and a discriminative arabinose marker, we could simulate a potential bacterial infection consisting of a major wild-type population mixed with a less-susceptible mutant population. Drug pharmacokinetics at these various sites will differ. In these experiments, we have simulated drug concentrations obtained in serum. These concentrations are similar to those found in the interstitial tissue fluid.32,33 We have used an in vitro kinetic model to assess the relative ability of six different approved dosing regimens of commonly used fluoroquinolones to prevent the growth and selection of pre-existing resistant mutants.
All six approved dosing regimens tested eradicated the wild-type susceptible strain. Four of the six dosing regimens prevented the selection and growth of the single mutant. These were ciprofloxacin, 250, 500 and 750 mg administered twice daily, and moxifloxacin, 400 mg administered once daily (Table 3). Only one dosing regimen prevented selection and growth of the double mutant. This was ciprofloxacin 750 mg administered twice daily (Table 3).
Clinical and microbiological success in eradicating bacteria has previously been shown to correlate with an AUC/MIC of fluoroquinolones of >100.34–36 We found that all six approved fluoroquinolone dosing regimens eradicated the drug-susceptible wild-type strain, and all have AUC/MIC values of >100 (Table 3). However, the parameter MIC refers to drug-susceptible populations and is not relevant to the prevention of growth of resistant mutants. Indeed, there is little or no correlation between the value of AUC/MIC and the probability of resistance development.12,35,37–40 This is not surprising because the level of resistance of the most resistant next-step mutant varies among strains and cannot accurately be predicted from the MIC.21,28
In a previous study, we determined MPC of ciprofloxacin for two different susceptible wild-type E. coli and one resistant single mutant.16 For those wild-type strains, an AUC/MPC ratio 22 was found to prevent resistance development.16 In the present study, we have used approved fluoroquinolone dosing regimens to treat mixed populations of susceptible wild-type and a drug-resistant single mutant or double mutant. We found that an AUC/MPC value of 35 was sufficient to prevent the growth of the resistant single mutant, whereas the next-lowest tested AUC/MPC value of 14 was insufficient. This is consistent with our previous result and conclusion on the prevention of resistance acquisition in wild-type strains.16 The only dosing regimen that was effective at preventing growth of the double mutant was ciprofloxacin 750 mg administered twice daily. This drug exposure corresponded to an AUC/MPC relative to the susceptible wild-type of 105. However, as we define MPC in terms of the next-step mutation, the more relevant comparison is to relate the outcome of the double mutant with the MPC of the single mutant. When the AUC/MPC is calculated relative to the MPC of the single mutant, the ratio that prevents the growth of the double mutant is 14, although 8 is non-restrictive. This agrees well with our previous data on the prevention of emergence of ‘second-step’ resistance in a resistant single-mutant strain, where we found that an AUC/MPC of 11 was restrictive, whereas an AUC/MPC of 6 was not restrictive.16 Thus, the present study with mixed populations of susceptible and resistant strains supports the conclusions drawn from the previous study on the emergence of spontaneous resistant strains16 and suggests that AUC/MPC ratios of 20 and 10 correlate with the restriction of growth of resistant single and double mutants, respectively, against ciprofloxacin in E. coli. We note that concentrations of drug sufficient to block the outgrowth of pre-existing mutants will also block the acquisition of new mutants that arise due to fluoroquinolone treatment (e.g. via induction of the SOS response).
Because of the increasing frequency of drug-resistant pathogens, both in the community and in hospital environments, it is important that dosing regimens should be chosen so as to minimize the emergence or selection of antibiotic-resistant variants. This study shows that several of the currently approved dosing regimens of norfloxacin, ciprofloxacin and moxifloxacin are not sufficient to prevent the selection of resistant mutants of E. coli when serum concentrations are simulated. The pharmacodynamic exposure required to prevent the selection of resistance may however be obtained from the urine where the drug is concentrated.41 Higher doses may be required to prevent selection at extra-urinary sites, for example, in the bladder epithelium42 and among the commensal flora.43 However, the concentrations at these sites are not well determined. Because of the high numbers of bacteria that exist in the commensal flora (e.g. the intestinal tract or the skin), selection among commensals is potentially a more important driver of resistance than selection at the infectious sites.44 Studies on the importance of different dosing regimens in selecting mutants in the gut flora are warranted.
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This work was supported by grants from the SSAC Foundation to O. C. and from the European Union FP6 (EAR project) and the Swedish Research Council (Vetenskapsrådet) to D. H.
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None to declare.
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Acknowledgements |
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We thank a project student Kaire Rääk for assistance with some MPC measurements.
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References |
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