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Antimicrobial Agents and Chemotherapy, August 2005, p. 3517-3519, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3517-3519.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Preincubation of Pneumococci with ß-Lactams Alone or Combined with Levofloxacin Prevents Quinolone-Induced Resistance without Increasing Intracellular Levels of Levofloxacin

Philippe Cottagnoud,^1* Maggie Johnson,² Marianne Cottagnoud,³ and Laura Piddock²

Department of Internal Medicine, Inselspital Bern, Freiburgstrasse, 3010 Bern, Switzerland,¹ Antimicrobial Agents Research Group, Division of Immunity and Infection, University of Birmingham, United Kingdom,² Clinic of Internal Medicine, Spital Bern-Ziegler, Morillonstrasse 75-91, 3007 Bern, Switzerland³

Received 27 October 2004/ Returned for modification 16 February 2005/ Accepted 20 April 2005

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Preincubation of pneumococci with sub-MIC concentrations of ceftriaxone (1/16x MIC), cefotaxime (1/8x MIC), and meropenem (1/4x MIC) alone or combined with levofloxacin (1/8x MIC) over 6 h prevents the emergence of levofloxacin-resistant mutants after 96 h of incubation but does not affect the intracellular accumulation of levofloxacin in two penicillin-resistant pneumococcal strains, suggesting a link between the mechanism of action of ß-lactams and the emergence of quinolone-induced resistance in pneumococci.

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The treatment of infections due to penicillin-resistant pneumococci remains a major challenge worldwide. Based on a recent study in 242 centers across the United States, penicillin resistance reached 33.6% and erythromycin resistance 41% in children (1). More alarming is the emergence of invasive pneumococcal isolates with very-high-level resistance against penicillin (MIC > 8 mg/liter) in the United States (11). In general, the newer quinolones have good activity against pneumococci, but a recent report of treatment failure due to the emergence of resistance during treatment may jeopardize the use of this class of antibiotics (5). A bactericidal antipneumococcal regimen that does not lead to the emergence of resistance would be a major progress. We have recently shown that cephalosporins (i.e., ceftriaxone and cefotaxime) and a carbapenem (i.e., meropenem) act synergistically with levofloxacin in vitro and in experimental meningitis against penicillin-resistant pneumococci (2, 6, 8). In addition, these combination regimens are able to prevent the emergence of levofloxacin-induced resistance in vitro in pneumococci. The aim of this study is to investigate the underlying mechanism of this interaction, especially the effect of ß-lactam antibiotics on the intracellular penetration of levofloxacin into pneumococci.

Preincubation of pneumococcal cultures with sub-MIC concentrations of ß-lactam antibiotics before plating on blood agar plates containing 1x MIC of levofloxacin. The two pneumococcal strains WB4 and KR4 were grown in C+Y (9) and then resuspended in fresh medium containing sub-MIC concentrations of ß-lactam antibiotics (1/16x MIC for ceftriaxone, 1/8x MIC for cefotaxime, and 1/4x MIC for meropenem) alone for 6 h. Preincubation with levofloxacin (1/8x MIC) alone or combined with cefotaxime (1/8x MIC) has also been tested. The MICs for both strains were similar: the MIC of ceftriaxone was 0.5 mg/liter, the MIC of cefotaxime was 0.5 mg/liter, the MIC of meropenem was 0.5 mg/liter, and the MIC of levofloxacin was 1 mg/liter. The dose of levofloxacin was chosen based on previous experiments with the checkerboard method. At the end of the incubation period, the optical density, measured at 590 nm, ranged between 0.3 and 0.5. The antibiotic doses were identical to those used in previous works, preventing the emergence of levofloxacin-induced resistance (2, 6, 8). Control cultures did not contain any antibiotics. After 6 h, bacterial titers were determined. The cultures then were centrifuged, the supernatant discarded, and the cultures resuspended in 50 µl of fresh medium (without antibiotics) and plated on blood agar plates containing 1x MIC of levofloxacin. Plates were incubated at 37°C for 96 h. Plates were checked daily for colonies.

Time-killing assays over 12 h. The two pneumococcal strains (WB4 and KR4) were grown in C+Y to an optical density of 0.3 at 590 nm and then diluted fivefold, corresponding approximately to bacterial titers used in experiments in vitro for the selection of quinolone-resistant mutants. Antibiotics were added in sub-MIC concentrations, as used in previous cycling experiments in vitro (2, 6, 8) (1/16x MIC for ceftriaxone, 1/8x MIC for cefotaxime, and 1/4x MIC for meropenem), and levofloxacin in concentrations corresponding to the MIC. Bacterial titers were determined at 0, 2, 4, 6, 8, 10, and 12 h by serial dilution of samples, plated on agar plates containing 5% sheep blood and incubated at 37°C for 24 h. Experiments were performed in triplicate, and results are expressed as means ± standard deviations.

Determination of intracellular levofloxacin levels. The modified fluorescence method for pneumococci was used essentially as described by Piddock and Johnson (10). A starter culture was added to 200 ml of brain heart infusion broth (Oxoid) and grown in 5% CO₂ at 37°C for 4 h until an optical density of 0.7 at 660 nm was attained. After centrifugation, the cells were harvested, washed, and resuspended as described by Piddock and Johnson (10) in phosphate buffer and allowed to equilibrate for 10 min at 37°C. Subinhibitory concentrations of each ß-lactam antibiotic were added to achieve a final concentration of 1/2x, 1/4x, or 1/8x MIC. Levofloxacin was added to give a final concentration of 10 µg/ml.

The concentration of levofloxacin accumulated was measured by fluorescent spectrophotometer at 5-min intervals up to 20 min at an excitation wavelength of 290 nm and an emission wavelength of 500 nm.

We tested the effect of preincubation of pneumococcal cultures with sub-MIC concentrations of ß-lactams alone or combined with levofloxacin on the emergence of levofloxacin-resistant mutants. The results are summarized in Table 1. Compared to the case with untreated controls, the addition of ß-lactam antibiotics in sub-MIC concentrations (ceftriaxone, 1/16x MIC; cefotaxime, 1/8x MIC; and meropenem, 1/4x MIC) alone or combined with levofloxacin (1/8x MIC) did not affect the growth rates after ca. 6 h of incubation (9.63 ± 0.70 log₁₀ CFU/ml for untreated controls versus 8.63 ± 0.70 log₁₀ CFU/ml for the combination regimen, the lowest bacterial titer). After 96 h of incubation, 308 ± 82 levofloxacin-resistant CFU emerged in untreated controls and 201 ± 149 CFU in the levofloxacin-preincubated cultures. The MIC of levofloxacin for 10 randomly selected colonies in untreated controls and in levofloxacin-preincubated cultures were 8 mg/liter and 4 to 8 mg/liter, respectively. Interestingly, in all cultures preincubated with ß-lactam antibiotics and in the combination regimen, no levofloxacin-resistant CFU were detected.

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TABLE 1. Effects of preincubation of pneumococcal cultures with sub-MIC concentrations of ß-lactam antibiotics on the emergence of levofloxacin-induced resistance

In a next set of experiments, the intracellular levofloxacin accumulation in both penicillin-resistant strains (WB4 and KR4) in the presence of sub-MIC concentrations of ceftriaxone, cefotaxime, and meropenem was measured (Fig. 1A to C). The intracellular levofloxacin levels have been determined by the modified fluorescence method, a well-established method developed by Piddock and Johnson (10). The results are expressed as nanograms of levofloxacin per mg of dry-weight cells. In the WB4 strain, levofloxacin, as monotherapy, accumulated rapidly, and the intracellular level peaked around 10 ng/mg dry-weight cells after 5 min and remained stable for the next 20 min. The addition of ceftriaxone in sub-MIC concentrations ranging from 1/8x MIC to 1/2x MIC in the same experimental setting did not influence the intracellular accumulation of levofloxacin (Fig. 1A). In this study, 1/16x MIC ceftriaxone (as used in an earlier study) was not tested, but based on the present results, it seems unlikely that a lower ceftriaxone concentration would influence the intracellular levofloxacin accumulation. Cefotaxime and meropenem added to levofloxacin in similar sub-MIC concentrations (1/8x to 1/2x MIC) did not influence the intracellular kinetic of levofloxacin (Fig. 1B and C). The experiments performed with the second penicillin-resistant strain, KR4, showed similar results (data not shown).

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FIG. 1. Panels A through C show the effects of three ß-lactam antibiotics (ceftriaxone in panel A, cefotaxime in panel B, and meropenem in panel C) added at different sub-MIC concentrations, on the intracellular accumulation of levofloxacin in the penicillin-resistant pneumococcal strain WB4. lev, levofloxacin; cef, ceftriaxone; cefo, cefotaxime; mer, meropenem; dry wt, dry-weight.

In a last set of experiments, the potential synergy between levofloxacin and ß-lactam antibiotics was investigated in vitro against the pneumococcal strain WB4, using high bacterial inocula (10⁸ to 10⁹ CFU/ml) similar to those used during previous experiments for the selection of quinolone-resistant mutants in vitro (2, 6, 8). In time-kill experiments over 12 h, the addition of ß-lactam antibiotics in sub-MIC concentrations did not significantly increase the killing rates (less than 1 log₁₀ CFU/ml) compared to levofloxacin monotherapy (data not shown).

We have recently shown that several ß-lactam antibiotics act synergistically with levofloxacin in experimental pneumococcal meningitis, producing a highly bactericidal effect against penicillin-resistant pneumococci. The most interesting feature of these studies was the fact that the addition of ß-lactam antibiotics in sub-MIC concentrations prevented the emergence of quinolone-resistant mutants in vitro. The aim of this study was to elucidate the underlying mechanism of this phenomenon. Basically, two scenarios are conceivable.

First, due to the synergy between the two antibiotic classes, the bacterial titer decreased rapidly and for a longer period under the critical threshold, under which levofloxacin-induced mutations do not occur. Initially, this hypothesis seemed plausible, especially when a small bacterial inoculum was used (ca. 10⁵ to 10⁶ CFU/ml, as encountered in experimental meningitis). A pronounced synergy between the two antibiotic classes could be demonstrated in this experimental setting in vitro and in vivo (2, 6, 8). However, with higher bacterial inocula (ca. 10⁸ CFU/ml) as used in cycling experiments in vitro, this effect was less evident, in general, less than 1 log₁₀ CFU/ml, making this hypothesis more questionable.

An alternative hypothesis would be an increased intracellular penetration of levofloxacin, due to an altered permeability of the cell wall caused by ß-lactam antibiotics that leads to levels above the mutation prevention concentration. The data presented in this study, however, did not support this hypothesis. The intracellular accumulation of levofloxacin into the two penicillin-resistant strains (WB4 and KR4) correlated closely to intracellular levels observed in other pneumococcal strains, described by Piddock and Johnson in a previous study (10). These data are in accordance with the previous observation that the addition of these ß-lactams in low concentrations did not influence the MIC for levofloxacin (2, 6, 8). In contrast to these results, we have demonstrated in a similar experimental setting in vitro that the addition of subinhibitory concentrations of vancomycin dramatically increased the intracellular penetration of gentamicin into the same pneumococcal strain WB4 (3, 4). Probably due to the higher molecular weight and cationic properties of aminoglycosides, the penetration of gentamicin is more influenced by the altered permeability of the cell wall, whereas the passage through the peptidoglycan network of the smaller sized and hydrophilic levofloxacin is not affected.

The most striking feature of this study was the effect of preincubation of pneumococcal cultures with sub-MIC concentrations of ß-lactams on the selection of levofloxacin-resistant mutants in vitro. In untreated controls, using an inoculum around 10⁹ CFU, circa 300 levofloxacin-resistant colonies emerged after a few days of incubation, corresponding approximately to the frequency of resistance selection in pneumococci previously described by Gootz et al. (7). Thus, the different sub-MIC ß-lactam concentrations (1/4x to 1/16x MIC) did not affect the growth rate during the preincubation period (Table 1) but completely impeded the selection of levofloxacin-resistant mutants in the following incubation phase on agar. Also, preincubation with the combination regimen completely prevented the emergence of quinolone resistance, confirming previous findings using different experimental settings (2, 6, 8). One might speculate that ß-lactams in low doses interfere with a bacterial system or structure which is not essential for physiological growth of the microorganisms but whose integrity is essential to allow bacteria to make mutations responsible for the resistance against quinolones.

In conclusion, the combination of ß-lactams and quinolones seems to be one of the most promising regimens for the treatment of pneumococcal infections, based on the highly bactericidal activity and the potential to prevent the emergence of quinolone-resistant microorganisms.

 
This study was partially supported by a grant from Aventis Pharma, Switzerland.

 
* Corresponding author. Mailing address: Department of Internal Medicine, Inselspital, Freiburgstrasse, 3010 Bern, Switzerland. Phone: 41-31-632-34-72. Fax: 41-31-632-38-47. E-mail: pcottagn{at}insel.ch.

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1
1. Brown, S. D., and D. J. Farrell. 2004. Antibacterial susceptibility among Streptococcus pneumoniae isolated from paediatric and adult patients as part of the PROTEKT US study in 2001-2002. J. Antimicrob. Chemother. 54(Suppl. 1):I23-I29.[CrossRef][Medline]
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2. Cottagnoud, P., M. Cottagnoud, F. Acosta, L. Flatz, F. Kuhn, A. Stucki, and J. Entenza. 2003. Meropenem prevents levofloxacin-induced resistance in penicillin-resistant pneumococci and acts synergistically with levofloxacin in experimental meningitis. Eur. J. Clin. Microbiol. Infect. Dis. 22:656-662.[CrossRef][Medline]
3
3. Cottagnoud, P., M. Cottagnoud, and M. G. Tauber. 2003. Vancomycin acts synergistically with gentamicin against penicillin-resistant pneumococci by increasing the intracellular penetration of gentamicin. Antimicrob. Agents Chemother. 47:144-147.[Abstract/Free Full Text]
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4. Cottagnoud, P., C. M. Gerber, M. Cottagnoud, and M. G. Tauber. 2002. Gentamicin increases the efficacy of vancomycin against penicillin-resistant pneumococci in the rabbit meningitis model. Antimicrob. Agents Chemother. 46:188-190.[Abstract/Free Full Text]
5
5. Davidson, R., R. Cavalcanti, J. L. Brunton, D. J. Bast, J. C. de Azavedo, P. Kibsey, C. Fleming, and D. E. Low. 2002. Resistance to levofloxacin and failure of treatment of pneumococcal pneumonia. N. Engl. J. Med. 346:747-750.[Free Full Text]
6
6. Flatz, L., M. Cottagnoud, F. Kuhn, J. Entenza, A. Stucki, and P. Cottagnoud. 2004. Ceftriaxone acts synergistically with levofloxacin in experimental meningitis and reduces levofloxacin-induced resistance in penicillin-resistant pneumococci. J. Antimicrob. Chemother. 53:305-310.[Abstract/Free Full Text]
7
7. Gootz, T. D., R. Zaniewski, S. Haskell, B. Schmieder, J. Tankovic, D. Girard, P. Courvalin, and R. J. Polzer. 1996. Activity of the new fluoroquinolone trovafloxacin (CP-99,219) against DNA gyrase and topoisomerase IV mutants of Streptococcus pneumoniae selected in vitro. Antimicrob. Agents Chemother. 40:2691-2697.[Abstract]
8
8. Kuhn, F., M. Cottagnoud, F. Acosta, L. Flatz, J. Entenza, and P. Cottagnoud. 2003. Cefotaxime acts synergistically with levofloxacin in experimental meningitis due to penicillin-resistant pneumococci and prevents selection of levofloxacin-resistant mutants in vitro. Antimicrob. Agents Chemother. 47:2487-2491.[Abstract/Free Full Text]
9
9. Lacks, S., and R. D. Hotchkiss. 1960. A study of the genetic material determining an enzyme in Pneumococcus. Biochim. Biophys. Acta 39:508-518.[Medline]
10
10. Piddock, L. J., and M. M. Johnson. 2002. Accumulation of 10 fluoroquinolones by wild-type or efflux mutant Streptococcus pneumoniae. Antimicrob. Agents Chemother. 46:813-820.[Abstract/Free Full Text]
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11. Schrag, S. J., L. McGee, C. G. Whitney, B. Beall, A. S. Craig, M. E. Choate, J. H. Jorgensen, R. R. Facklam, and K. P. Klugman. 2004. Emergence of Streptococcus pneumoniae with very-high-level resistance to penicillin. Antimicrob. Agents Chemother. 48:3016-3023.[Abstract/Free Full Text]

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Antimicrobial Agents and Chemotherapy, August 2005, p. 3517-3519, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3517-3519.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cottagnoud, P. Articles by Piddock, L. Search for Related Content PubMed PubMed Citation Articles by Cottagnoud, P. Articles by Piddock, L.