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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.
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 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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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.
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ACKNOWLEDGMENTS |
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