1 Department of Internal Medicine, Spital Bern-Ziegler, Bern; 2 Department of Infectious Diseases, CHUV, Lausanne; 3 Department of Internal Medicine, Inselspital, Bern, Switzerland
Received 13 April 2003; returned 27 October 2003, revised 15 November 2003; accepted 17 November 2003
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Abstract |
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Keywords: Streptococcus pneumoniae, quinolones, ß-lactam antibiotics
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Introduction |
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Methods |
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The two pneumococcal strains (WB4 and KR4) were originally isolated from two patients with pneumonia at the University Hospital of Bern, Switzerland. The MICs for WB4 were: penicillin 4 mg/L, ceftriaxone 0.5 mg/L, cefotaxime 1 mg/L, vancomycin 0.120.25 mg/L, levofloxacin 1 mg/L, gatifloxacin 0.120.25 mg/L, moxifloxacin 0.12 mg/L and garenoxacin 0.03 mg/L. The MICs for KR4 were: penicillin 4 mg/L, ceftriaxone 0.5 mg/L, cefotaxime 1 mg/L, vancomycin 0.120.25 mg/L, levofloxacin 1 mg/L, gatifloxacin 0.25 mg/L, moxifloxacin 0.12 mg/L and garenoxacin 0.015 mg/L.
MICs were determined by broth macrodilution. The MIC was defined as the lowest concentration that inhibited visible growth after 12 and 24 h of incubation at 37°C.
Rabbit meningitis model
The meningitis model described by Dacey & Sande7 was used in this study. The experimental protocol was accepted by the local ethics committee (Veterinäramt des Kantons Bern). Young New Zealand white rabbits weighing 22.5 kg were anaesthetized by intramuscular injection of ketamine (30 mg/kg) and xylazine (15 mg/kg) and were immobilized in stereotactic frames for induction of meningitis and cfu sampling. An inoculum containing 105 cfu of the penicillin-resistant strain WB4 was instilled in the cisterna magna. A long-acting anaesthetic drug [ethylcarbamate (urethane): 3.5 g/rabbit] was injected subcutaneously, and animals were returned to their cages. Fourteen hours later the cisterna magna was punctured again for periodic CSF sampling before and 1, 2, 4, 5, 6 and 8 h after initiation of therapy. Anaesthesia was performed by repeat injections of pentobarbital sodium (Nembutal). Antibiotics were administered via a peripheral ear vein at the following concentrations: ceftriaxone 125 mg/kg and levofloxacin 10 mg/kg of body weight. Ceftriaxone and levofloxacin were injected once at hour zero. All antibiotics and anaesthetic drugs were purchased commercially.
Bacterial titres were measured by 10-fold serial dilutions of CSF samples, then plated on blood agar plates containing 5% sheep blood, and incubated overnight at 37°C. In parallel, 20 µL aliquots of undiluted samples were plated (limit of detectability: 50 cfu/mL). Comparisons between dilutions of CSF were used to exclude significant carryover effects during therapy. The antimicrobial activity of the different regimens during the 8 h treatment was calculated by linear regression analysis and expressed as a change in log10 cfu/mL.h and as a change in viable count over 8 h. A value of 1.7 (log10 of the limit of detectability) was assigned to the first sterile CSF sample, and a value of zero was assigned to any subsequent sterile CSF sample. The results are expressed as means ± S.D. Statistical significance was determined by the NewmanKeuls test.
Determination of antibiotic levels in CSF
Antibiotic concentrations in the CSF were measured by agar diffusion. Standard curves were performed in saline with 5% rabbit serum in order to mimic CSF protein concentrations.8 Bacillus subtilis ATCC 6633 was used as a test strain for levofloxacin9 and Escherichia coli (ATCC 25922) for ceftriaxone.10 The inter- and intraday variability was in the range 5%10%. The limits of detection were 0.5 mg/L for ceftriaxone and 0.3 mg/L for levofloxacin.
In vitro killing assays
The two pneumococcal strains (WB4 and KR4) were grown in C+Y11 to an optical density of 0.3 at 590 nm and then diluted 40-fold to 106 cfu, corresponding approximately to the CSF bacterial titre in rabbits before initiation of therapy. Ceftriaxone was added at a sub-MIC concentration (1/2 MIC against KR4 and WB4) and levofloxacin at the concentration corresponding to the MIC. Bacterial titres were determined at 0, 2, 4, 6 and 8 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 ± S.D.
Determination of fractional inhibitory concentration (FIC) index
FIC indices were measured using the chequerboard method, as described previously.12 In brief, the two pneumococcal strains (WB4 and KR4) were grown in C+Y medium until the logarithmic growth phase (optical density of 0.3 at 590 nm) and were then diluted 1:40. Approximately 0.51 x 106 cfu were pipetted into microtitre trays containing concentrations of levofloxacin and ceftriaxone that ranged from 1/32 x MIC to 2 x MIC. Microtitre plates were incubated at 37°C for 24 h. After 6, 12 and 24 h the plates were read for detection of inhibition of bacterial growth. The experiments were performed in duplicate and repeated once. FIC indices were calculated by the method of Eliopoulos & Moellering.13 Synergy was defined as an FIC index of 0.5, indifference as an FIC index of >0.5 to
4 and antagonism as an FIC index of >4.
Selection of quinolone-resistant derivatives in vitro
Experiments were designed to test the tendency of levofloxacin to select resistant strains in liquid cultures. Large inocula (107108 cfu/mL) of either WB4 or KR4 were exposed to stepwise increasing concentrations of antibiotics.14 Series of tubes containing two-fold increasing concentrations of levofloxacin were incubated either with WB4 or KR4 (107108 cfu/mL), as for MIC determination. After 12 h of incubation, 0.1 mL samples from the tubes containing the highest antibiotic concentration and still showing turbidity were used to inoculate a new series of tubes containing antibiotic serial dilutions. The experiments were performed over 12 cycles. The MIC was determined after each cycle.
In parallel, the same experimental protocol was used but ceftriaxone was added at a low concentration (0.03 mg/L corresponding to 1/16 MIC for the two strains) to the tubes containing serial dilutions of levofloxacin. After 12 h of incubation, the MIC of levofloxacin was determined as described above in tubes containing only levofloxacin.
Preparation of chromosomal DNA, PCR amplification and DNA sequence analysis
Chromosomal pneumococcal DNA was prepared as described previously.15 PCR amplification of the parC, parE, gyrA and gyrB genes was performed according to a published method.16 PCR amplification was performed with a GeneAmp PCR system 9700 apparatus (Perkin-Elmer). After amplification, PCR products were purified using a QIAquick PCR purification kit (Qiagen AG, Basel, Switzerland). Nucleotide sequencing for the PCR amplicons was carried out with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit according to the protocol of the manufacturer (Perkin-Elmer). An ABI PRISM 377 DNA sequencer was used for sequencing. All testing was performed in duplicate.
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Results |
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The efficacy of the different regimens in rabbit meningitis are summarized in Table 1. In untreated controls, a slight increase in bacterial titres was observed over 8 h (+0.29 ± 0.10 log10 cfu/mL). Ceftriaxone produced only moderate bactericidal activity (0.30 ± 0.09
log10 cfu/mL.h) without sterilizing the CSF of rabbits after 8 h (0 out of 9). Levofloxacin monotherapy produced significantly (P = 0.0085) higher antibacterial activity than ceftriaxone monotherapy, but managed to sterilize the CSF of only one rabbit. The combination regimen (ceftriaxone combined with levofloxacin) produced highly bactericidal activity, significantly superior compared with the monotherapies (P < 0.01 versus either monotherapy) and sterilized the CSF of eight out of nine rabbits at the end of the treatment period. In timekilling assays in vitro, antibiotic concentrations were chosen (ceftriaxone: 1/2x MIC and levofloxacin: 1x MIC) to produce only marginal intrinsic bactericidal activity in order to demonstrate potential synergy between the two compounds. Synergy was defined as the bactericidal effect of a drug combination significantly exceeding the sum of the bactericidal effects of each monotherapy.13 Against WB4, ceftriaxone (1/2x MIC) produced a slight decrease in the viable cell count over 8 h (0.8 log10 cfu/mL) (Figure 1). Levofloxacin monotherapy was more bactericidal with a decrease in bacterial titres around 3.9 log10 cfu/mL. The combination was highly bactericidal and acted synergistically (6.9 log10 cfu/mL), sterilizing the cultures after 8 h. Against KR4, the synergistic activity of the combination was even more pronounced, sterilizing all cultures after 4 h (Figure 2). Ceftriaxone monotherapy was also more active against KR4 than against WB4 (2.4 versus 0.8 log10 cfu/mL, respectively).
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Based on a previous experimental setting,17 levofloxacin-resistant mutants were selected in both WB4 and KR4 by sequential exposure to different levofloxacin concentrations over 12 cycles. In WB4 (Figure 3a), the MIC of levofloxacin increased to 8 mg/L after four cycles; after seven cycles, it increased to 64 mg/L and remained stable until the end of the experiment. In contrast, the MIC increased to a lesser extent with the addition of a low concentration of ceftriaxone (1/16 MIC). This latter concentration of ceftriaxone was the highest that allowed bacterial growth of pneumococcal cultures incubated with different concentrations of levofloxacin. After 12 cycles, the MIC was 16 mg/L. The increase in the MIC in the different treatment groups was confirmed by gene mutations: sequential exposure of WB4 to levofloxacin led to mutations in the target genes encoding for topoisomerase IV and gyrase (Ser79Phe in parC and Glu85
Lys in gyrA). In the combination treatment, mutations in parE (Asp435
Asn) and gyrA (Ser81
Phe) were detected (see Table 2). In the second strain, KR4, the selection of levofloxacin-resistant mutants was comparable. In the monotherapy group, the MIC of levofloxacin increased to 8 mg/L after four cycles and to 64 mg/L after nine cycles (Figure 3b). Analagous to WB4, the addition of ceftriaxone (1/16 MIC) led to an MIC increase to 16 mg/L after 12 cycles. In the monotherapy group, two mutations in parC (Ser79
Phe and Asp83
Tyr) and one mutation in gyrA (Glu85
Lys) were detected. In the combination regimen, mutations in parE (Asp435
Asn) and gyrA (Ser81
Phe) were found. It is interesting to note that the addition of ceftriaxone at a low concentration (1/16 MIC) did not lower the MIC of levofloxacin. On the other hand, no cross-resistance between levofloxacin and ceftriaxone has been detected. The MIC of ceftriaxone was not affected by serial incubation of these two pneumococcal strains with levofloxacin (Table 3).
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Discussion |
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The doses of ceftriaxone and levofloxacin were standard, mimicking levels achieved in humans. One injection of ceftriaxone led to CSF levels in rabbits in the range 5.23 mg/L, corresponding to levels measured in humans with bacterial meningitis.18 CSF levels obtained with one injection of levofloxacin were slightly higher than those measured in humans after 500 mg twice daily (peak levels: 3.3 mg/L in rabbits versus 2.561.29 mg/L in humans).19
An interesting feature of this study was the efficacy of the combination regimen compared with the monotherapy in experimental meningitis (5.33 log10 cfu/mL.8 h for the combination regimen versus 2.20
log10 cfu/mL.8 h for ceftriaxone and 3.45
log10 cfu/mL.8 h for levofloxacin), sterilizing the majority of the CSF of rabbits (eight out of nine). Ceftriaxone monotherapy produced killing rates similar to those published in previous studies against the same strain.12,20,21 Compared with a previous study, levofloxacin monotherapy produced slightly higher killing rates, although the CSF peak levels were in the same range.22
In vitro, synergy was equal between ceftriaxone and levofloxacin in timekilling assays and with the chequerboard method with FIC indices of 0.25 for both strains.
An interesting aspect of this study was the effect of ceftriaxone, added at a low concentration (1/16 MIC), on the development of levofloxacin-induced resistance in vitro. The choice of the low concentration of ceftriaxone was based on results observed in the chequerboard experiments.
In cycling experiments with both strains, levofloxacin led to a stepwise increase of resistance until high-level resistance (MIC 64 mg/L) was reached at the end of the experimental period (12 cycles). The increase in resistance correlated with the sequential emergence of point mutations in genes encoding the two target enzymes, i.e. topoisomerase IV and gyrase. All mutations described in Table 2 have been mentioned in the literature.
In both strains, ceftriaxone prevented the emergence of mutations in parC but led to mutations in parE (Asp435Asn). This mutation, which has been described in other isolates, selected after cycling with levofloxacin,23 seemed to contribute to a lesser extent to the MIC increase. The effect of the ß-lactam antibiotic, i.e. ceftriaxone added at a low concentration, on levofloxacin-induced resistance is not understood completely. Two explanations are conceivable: (i) the synergy between the two antibiotics might cause the bacterial populations to decrease below a critical threshold for longer, i.e. below 106107 cfu/mL where mutations occur; (ii) the combined antibacterial effect of antibiotics interfering with different targets (PBPs or gyrase and topoisomerase IV) might impede the development of mutations in the microorganism. The last hypothesis is probably the more unlikely because the MIC of levofloxacin was not affected by the addition of ceftriaxone (see Table 3).
The observation that antibiotics interfering with the cell wall synthesis might influence quinolone-induced resistance in pneumococci is not new. In the same experimental setting, we have shown previously that the addition of vancomycin reduced ciprofloxacin- and trovafloxacin-induced resistance in the strain WB4.17
In summary, we have demonstrated that a combination of ceftriaxone and levofloxacin was very efficacious in experimental pneumococcal meningitis and reduced the risk of quinolone-induced resistance. This combination might be used in the future as empirical treatment in bacterial meningitis, as an alternative to the recommended regimen based on ceftriaxone and vancomycin.
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Acknowledgements |
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Footnotes |
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References |
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