Institute for Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany
Received 4 August 2003; returned 25 September 2003; revised 10 October 2003; accepted 23 October 2003
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Abstract |
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Materials and methods: MIC and MPC for E.coli ATCC 25922 and the clinical isolate were determined on agar containing ciprofloxacin or levofloxacin, and for the ATCC strain on agar supplemented with nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin or clinafloxacin.
Results: Results for the ATCC strain and the clinical strain for ciprofloxacin or levofloxacin were similar. The MPC values for E.coli ATCC 25922 were 2 x MIC (trovafloxacin), 4 x MIC (ciprofloxacin, norfloxacin, ofloxacin), 8 x MIC (clinafloxacin, levofloxacin), 16 x MIC (sparfloxacin) and 32 x MIC (nalidixic acid) at 37°C and under aerobic conditions. Generally, a 37°C aerobic atmosphere was associated with the highest MPC values. As an exception, both the MIC and the MPC of ciprofloxacin were higher under anaerobic versus aerobic conditions (MICan 8 x MIC; MPCan = 4 x MPC) for both E.coli isolates. Irrespective of the quinolone or growth conditions, the MIC for mutants was 1256 x wild-type MIC. Calculated from published serum half-lives and the MPC values from this study, a putative selection period, in which resistant mutants might be selected, was calculated to be 14 h for nalidixic acid, 16 h for norfloxacin and ciprofloxacin, 28 h for ofloxacin, 30 h for trovafloxacin, 35 h for levofloxacin, 40 h for clinafloxacin, and 120 h for sparfloxacin.
Conclusions: As calculated from our model in respect to the length of the selection period, long serum half-lives of recently developed compounds could not be compensated for by a more favourable activity in terms of MPC. Higher concentrations of ciprofloxacin may be required under an anaerobic atmosphere to prevent the emergence of resistant mutants among 1010 cfu.
Keywords: MPC, quinolones, resistance
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Introduction |
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In the patient, antibiotic drug concentrations are not stable (like in vitro), but have complex patterns over time in different compartments. At various occasions drug concentrations are low enough to allow for the growth of resistant mutants, and the respective time span is called the selection period.3,9 Combining the model of the selection period with our data for the MPC for E.coli ATCC 25922, we calculated a putative selection period for each quinolone.
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Materials and methods |
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A recent clinical isolate of E.coli with wild-type susceptibility to nalidixic acid (MIC nalidixic acid = 1 mg/L) was obtained from urine routinely sent to the Institute for Medical Microbiology, University of Regensburg, Regensburg, Germany. E.coli ATCC 25922 was obtained from the American Type Culture Collection. The strains were maintained on Columbia agar plates (catalogue no. 10455; Merck, Darmstadt, Germany). MuellerHinton agar (Oxoid, Wesel, Germany) supplemented with serial dilutions of antibiotics was used for selection experiments and susceptibility testing. Colonies growing on selection plates were confirmed to be E.coli by demonstration of growth on MacConkey agar (catalogue no. 5465; Merck), a negative citrate reaction (catalogue no. 2501; Merck), and growth in SIM medium (catalogue no. 5470; Merck) with a positive indole reaction. Mutant strains were kept at 20°C (Microbank; Mast, Reinfeld, Germany) until used for susceptibility testing.
Antibacterial agents and susceptibility testing
Antimicrobial agents were provided by the following manufacturers: ciprofloxacin (Bayer AG, Leverkusen, Germany), clinafloxacin (Parke-Davis Pharmaceutical Research, Freiburg, Germany), levofloxacin (Hoechst Marion Roussel, Frankfurt, Germany), norfloxacin (Merck Sharp & Dohme, Haar, Germany), ofloxacin (Hoechst Marion Roussel), sparfloxacin (Rhone-Poulenc-Rohrer, Cologne, Germany) and trovafloxacin (Pfizer, Karlsruhe, Germany). Nalidixic acid was purchased from Sigma (catalogue no. N8878; Sigma, Deisenhofen, Germany). The agar dilution method was carried out on MuellerHinton agar according to NCCLS guidelines,10 and also under anaerobic conditions. Etest was carried out according to the instructions of the manufacturer (AB BIODISK, Solna, Sweden), and also under anaerobic conditions. For both methods, MICs for E.coli ATCC 25922 under aerobic conditions were within acceptable quality control ranges (see Table 2). MIC testing of E.coli by the agar dilution method, selection of mutants, subculture of mutants, and MIC determination of mutants were carried out on the same lot of antibiotic-supplemented agar plates. For the comparison of MICs for the original strains and the selected mutants, MIC testing of ofloxacin was carried out by the agar dilution method and Etest in parallel. To adjust for the different potency of the drugs when comparing the different quinolones, all MICs are expressed as multiple folds above wild-type MIC.
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A fresh overnight colony of E.coli was grown in 3 mL of LB broth (catalogue no. 7213; Merck) for 4 h (37°C, non-shaking), and transferred to 200 mL of pre-warmed LB broth. After growth to stationary phase (12 h, 37°C, 220 rpm, aerobic), bacteria were concentrated (3000g for 10 min), washed twice with ice-cold NaCl, and suspended in 4 mL of NaCl. Aliquots of 100 µL were used for inoculation of antibiotic-supplemented agar plates, and for serial dilution (for cell counts). Plates were kept under different growth conditions [37, 20°C, aerobic, anaerobic (GENbag anaer, catalogue no. 45 534; bioMérieux, Marcy lÉtoile, France)], and growth was checked visually after 48 h and 7 days. Colonies were counted, and the number of colonies was divided by the inoculum to calculate the fraction of cells recovered. All selectants were sub-cultured at 37°C and under aerobic conditions on agar containing the selecting quinolone at the concentration used for selection. Up to a number of 15, all mutants of each selection plate were picked for MIC determinations at 37°C and under aerobic conditions. All experiments were carried out twice.
Statistical calculations
SPSS 9.0 for Windows was used for calculation of 2 test results.
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Results |
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Discussion |
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In the patient, antibiotic drug concentrations are not stable (like in vitro), but have complex patterns over time in different compartments. At various occasions, drug concentrations are low enough to allow for the growth of resistant mutants, and the respective time span is called the selection period.3,9 Combining the model of the selection period with our data for the MPC, we calculated a selection period for each quinolone, as illustrated in Figure 3. The beginning of the selection period was set one dilution step below the MPC. The end of the selection period was arbitrarily set at 0.25 x MIC, a concentration still slightly favourable for the growth of resistant mutants, compared with the wild-type (data not shown). The number of dilution steps between these concentrations, multiplied by the serum half-life of the quinolone compound7,20 [clinafloxacin: data according to the manufacturer (Protocol 960034); Parke-Davis] gives the selection period. The selection period thus calculated was 14 h for nalidixic acid, 16 h for norfloxacin and ciprofloxacin, 28 h for ofloxacin, 30 h for trovafloxacin, 35 h for levofloxacin, 40 h for clinafloxacin and 120 h for sparfloxacin. Obviously, the long serum half-lives of clinafloxacin, trovafloxacin, or sparfloxacin (Table 1), despite their low relative concentrations sufficient to suppress the growth of mutants, result in relatively long selection periods (Table 1). Nalidixic acid, norfloxacin or ciprofloxacin compared more favourably, because the short serum half-lives compensate for the relatively high concentrations needed to suppress mutants. From this view, newly developed highly active compounds with increased serum half-lives, although desirable for reasons of compliance and ease of dosage, might favour the emergence of fluoroquinolone resistance. Two recent studies using an in vitro dynamic model, testing quinolones and Streptococcus pneumoniae21 or Staphylococcus aureus,22 demonstrated that resistant mutants are selectively enriched when antibiotic concentrations fall inside the selection period. Data generated in vitro and in vivo suggest that an AUC024/MIC ratio > 100 may be a useful parameter to guide therapy with the goal of preventing the selection of resistance, and that this ratio might correspond to a Cmax/MIC ratio of approximately 5:1 for ciprofloxacin.2123 Interestingly in our model, in vitro the MPC of ciprofloxacin was quite similar, 4 x MIC, suggesting that the somewhat arbitrary set-point of 1010 cfu might represent a realistic experimental challenge. However, our data in vitro may be used to compare the relative but not the absolute potency of different quinolones in prevention of resistance.
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In conclusion, 2 x MIC (trovafloxacin), 4 x MIC (ciprofloxacin, norfloxacin, ofloxacin), 8 x MIC (clinafloxacin, levofloxacin), 16 x MIC (sparfloxacin) or 32 x MIC (nalidixic acid) were required to suppress the growth of resistant mutants among 1010 cfu of E.coli ATCC 25922 under aerobic conditions. Higher concentrations were required for ciprofloxacin under anaerobic growth conditions, an observation supported by a higher MICan value. Calculated from the serum half-life and the MPC, respectively, the selection period was calculated to be 14 h for nalidixic acid, 16 h for norfloxacin and ciprofloxacin, 24 h for ciprofloxacin under anaerobic conditions, 28 h for ofloxacin, 30 h for trovafloxacin, 35 h for levofloxacin, 40 h for clinafloxacin, and 120 h for sparfloxacin. As calculated from our model in respect to the length of the selection period, long serum half-lives of recently developed highly active compounds could not be compensated for by a more favourable activity in terms of MPC. Dynamic models with repeated dosing and experiments in vivo with clinical isolates will be necessary to corroborate the findings of this study concerning the impact of growth conditions and drug concentrations on the emergence of quinolone resistance.
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Acknowledgements |
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Footnotes |
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References |
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