Mutant prevention concentration of nalidixic acid, ciprofloxacin, clinafloxacin, levofloxacin, norfloxacin, ofloxacin, sparfloxacin or trovafloxacin for Escherichia coli under different growth conditions

Hans-Jörg Linde* and Norbert Lehn

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: We used two different strains of Escherichia coli, E.coli ATCC 25922 and a recent urinary isolate from a clinical sample, to investigate in vitro how the MIC and mutant prevention concentration (MPC) are affected by different temperatures (37 or 20°C) or oxygen tension (aerobic or anaerobic atmosphere; MIC, MICan; MPC, MPCan).

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 1–256 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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
According to Drlica et al.,13 the mutant prevention concentration (MPC) is the concentration of an antimicrobial agent necessary to prevent the growth of resistant mutants among 1010 cfu in vitro, and can be employed to compare the power of different antimicrobial agents to prevent emergence of antimicrobial resistance. More knowledge about the emergence of quinolone resistance is needed to reverse the worldwide trend of increasing quinolone resistance in Escherichia coli.46 In Germany for the year of 2001, prevalence of resistance of E.coli to ciprofloxacin reached 14.7% (Paul-Ehrlich-Society, Germany, http://www.p-e-g.de). In man, E.coli colonizes different aerobic and anaerobic habitats with different temperatures including the gut, its main reservoir (anaerobic, 37°C), and the perineal skin region (aerobic, <37°C). Growth conditions in other sites of colonization or in the setting of infection might also vary considerably. Treatment with quinolones, because of their excellent penetration into almost all tissues and body compartments, causes relevant drug concentrations in all these habitats.7 Excretion of quinolones in sweat has been implicated in the selection of quinolone-resistant staphylococci on the skin of healthy volunteers.8 We investigated in vitro, how different temperatures (37, 20°C) under aerobic or anaerobic atmosphere affected the growth of spontaneous mutants exposed to different concentrations of quinolones. E.coli ATCC 25922 was exposed to increasing concentrations of clinafloxacin, ciprofloxacin, levofloxacin, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin or trovafloxacin, and we determined the selection rate of resistant mutants and MPC. In addition, a clinical urinary isolate of E.coli and the ATCC strain E.coli 25922 were compared using ciprofloxacin or levofloxacin.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

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). Mueller–Hinton 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 Mueller–Hinton 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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Table 2. Results of MIC-testing (Etest) with nalidixic acid (NAL), ciprofloxacin (CIP), ofloxacin (OFX) or trovafloxacin (TVA) under aerobic or anaerobic conditions for E.coli ATCC 25922 and a clinical urinary isolate
 
Selection of resistant strains

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 l’Étoile, 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 {chi}2 test results.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mean number of cfu applied to agar plates supplemented with quinolones was 2.6 x 1010 and ranged between 7 x 108 and 1011 cfu. Compared with 48 h, reading of the plates after 7 days did not yield additional resistant colonies (data not shown). In general, the fraction of cells recovered from quinolone-supplemented plates decreased with increasing quinolone concentrations, or if plates were kept at 20°C or under anaerobic conditions. In contrast, a larger fraction was recovered from plates supplemented with ciprofloxacin under anaerobic versus aerobic conditions (Figure 1). For ofloxacin and nalidixic acid, the same finding was less obvious. With an MPC of 2 x MIC, trovafloxacin was the most, and nalidixic acid requiring 32 x MIC the least potent agent, with 4 x MIC for ciprofloxacin or norfloxacin or ofloxacin, 8 x MIC for clinafloxacin or levofloxacin, and 16 x MIC for sparfloxacin required, respectively (Table 1). As a notable exception, ciprofloxacin required 16 x MIC in an anaerobic atmosphere as opposed to 4 x MIC in an aerobic atmosphere to suppress the growth of mutants (Table 1).



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Figure 1. Selection rate of quinolone-resistant mutants of E.coli ATCC 25922 at different concentrations of ciprofloxacin (CIP), clinafloxacin (CLX), levofloxacin (LVX), nalidixic acid (NAL), norfloxacin (NOR), ofloxacin (OFX), sparfloxacin (SPX) or trovafloxacin (TVA) under different culture conditions (mean ± span/2, n = 2; cfu: colony forming units).

 

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Table 1. MPC for E.coli ATCC 25922 at 37°C (aerobic and anaerobic atmosphere), maximum selectant MIC, serum half-life (t1/2, h)7,20 and putative selection period (h; see also Figure 3) of nalidixic acid (NAL), norfloxacin (NOR), ciprofloxacin (CIP), ofloxacin (OFX), levofloxacin (LVX), sparfloxacin (SPX), clinafloxacin (CLX) or trovafloxacin (TVA) (n = 2) 
 
All mutants recovered from the selection plates re-grew on agar plates containing the respective quinolone concentration. With increasing selective drug concentrations, the number of colonies dropped, while the distribution of mutant MIC values shifted to higher concentrations. Figure 2 illustrates a typical distribution of MIC values for mutants recovered from ciprofloxacin-supplemented selection plates under different drug concentrations and growth conditions.



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Figure 2. MIC values (37°C, aerobic conditions) for mutant E.coli ATCC 25922 selected at increasing concentrations of ciprofloxacin (CIP) and different growth conditions (boxes: 50% of values; solid lines, median values; whiskers, 25/75% of values; filled circles, outliers).

 
MIC and MPC determinations for selected quinolones under aerobic and anaerobic atmosphere were repeated for E.coli ATCC 25922 and a clinical urinary isolate. MICs determined under aerobic or anaerobic atmosphere differed significantly for ciprofloxacin and ofloxacin but not for nalidixic acid or trovafloxacin (Table 2). Using MICan values, recalculation of the respective MPCan/MICan ratio for ciprofloxacin or ofloxacin resulted in much lower values comparable to the ratios calculated for other quinolones (Table 3).


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Table 3. MPCana/MIC and MPCan/MICanb of ciprofloxacin (CIP) or ofloxacin (OFX) for E.coli ATCC 25922 and a clinical urinary isolate
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Data generated in vitro and in vivo indicate that quinolone resistance in E.coli is a stepwise process involving target mutations in topoisomerase genes,11,12 and active efflux,13 among putative other factors.14,15 Consequently, emergence of resistance might differ upon exposure to different quinolones, assuming that the compounds differ in their affinity to their respective targets, diffusion, efflux, or other unknown differences.16 Drlica introduced and applied the concept of ‘mutant prevention concentration’3 for quinolones and Staphylococcus aureus,2 Mycobacterium tuberculosis,2,17 and Streptococcus pneumoniae.18 We compared the selection of resistant mutants among ~1010 cfu of E.coli ATCC 25922 for various quinolones at 20 or 37°C and under aerobic or anaerobic conditions. The conditions were chosen to account for the different habitats of E.coli in man, like the (perineal) skin, the gut, or a site of infection. Our data about the selection of mutants by nalidixic acid are in agreement with the findings of Forsgren & Striby,16 who concluded that nalidixic acid selected for resistant mutants at a rate of 0.2–20 x 10–9 at nalidixic acid concentrations of 64 x MIC, a rate much higher than for fluoroquinolones. In this study, of all quinolones, nalidixic acid required the highest concentration relative to the wild-type MIC to suppress the growth of mutants (32 x MIC). In contrast, trovafloxacin at 2 x MIC was the most potent quinolone to suppress the growth of resistant mutants at 37°C and in an aerobic atmosphere, with ciprofloxacin, norfloxacin and ofloxacin (all 4 x MIC) close behind. Unexpectedly, ciprofloxacin and (less obviously) ofloxacin (but not levofloxacin), two widely used fluoroquinolones, differed from all other compounds: one dilution step below the MPC, a higher number of mutants was recovered under anaerobic conditions. In addition, ciprofloxacin under anaerobic conditions required an MPC four times higher compared with aerobic conditions. Interestingly, for ciprofloxacin and ofloxacin but not for nalidixic acid or trovafloxacin, MICs were several fold higher under anaerobic versus aerobic growth conditions. The lower values obtained by the calculation of the MPCan/MICan (Table 3) instead of the MPCan/MIC ratio are in agreement with our findings for the other quinolone compounds. Hsieh et al.19 demonstrated with E.coli in vitro, that long-term anaerobic growth increased negative supercoiling, involving topoisomerase II. Thus, increased negative supercoiling might affect the interaction of the quinolone drug with the topoisomerase II–DNA complex (as indicated by the change in MIC of ciprofloxacin under anaerobic conditions) or favour the selection of specific mutants. At present, we have no further explanation for this in vitro observation; however, if true, anaerobic conditions (as present in the mammalian gut) may play a special role as the site of emergence of resistant mutants under therapy with ciprofloxacin or ofloxacin.

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 960–034); 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 AUC0–24/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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Figure 3. Selection period and selective concentration, adapted with permission from Baquero & Negri.9

 
We did not analyse mutants for resistance mechanisms. Also, we did not observe the growth of additional colonies after prolonged incubation for 7 days, as reported by Riesenfeld et al.24 However, our protocol differed substantially in the inoculum size, the selective conditions and the way colonies were picked.

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.


    Acknowledgements
 
We would like to thank Biljana Denkov and Regine Birngruber for excellent technical assistance. This study was presented in part at the Twelfth European Congress of Clinical Microbiology and Infectious Diseases, Milan, Italy, 2002.


    Footnotes
 
* Corresponding author. Tel: +49-941-944-6414; Fax: +49-941-944-6402; E-mail: Hans-Joerg.Linde{at}klinik.uni-regensburg.de Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Drlica, K. (2001). A strategy for fighting antibiotic resistance. ASM News 67, 27–33.[ISI]

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