Evaluation of cefepime alone and in combination with vancomycin against penicillin-resistant pneumococci in the rabbit meningitis model and in vitro

Cynthia M. Gerbera, Marianne Cottagnoudb, Klaus Neftelb, Martin G. Täuberc and Philippe Cottagnouda,*

a Department of Internal Medicine, Inselspital, 3010 Berne; b Department of Internal Medicine, Zieglerspital, 3007 Berne; c Institute of Medical Microbiology, University of Berne, Berne, Switzerland


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Cefepime, a broad-spectrum, fourth-generation cephalosporin, showed excellent CSF penetration with levels ranging between 10 and 16 mg/L after two intravenous injections (100 mg/kg). The bactericidal activity of cefepime (–0.60 ± 0.28 {Delta}log10 cfu/mL/h) was superior to that of ceftriaxone (–0.34 ± 0.23 {Delta}log10 cfu/mL/h, P < 0.05) and vancomycin (–0.39 ± 0.19 {Delta}log10 cfu/mL/h, P < 0.05) in the treatment of rabbits with meningitis caused by an isolate highly resistant to penicillin (MIC of penicillin G: 4 mg/L). The addition of vancomycin to both cephalosporins did not significantly increase the killing rate compared with monotherapies (P > 0.05). Similar results were obtained in time–killing experiments in vitro.


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The increasing spread of penicillin-resistant strains has recently complicated the treatment of pneumococcal infections. In Europe, the highest rates of penicillin-resistant pneumococci have been registered in Spain and Hungary (c. 50% of all isolates).1,2 The resistance mechanism in pneumococci is based on structural modifications of the targets of ß-lactam antibiotics, the penicillin-binding proteins (PBPs), leading to decreased binding affinity between PBPs and antibiotics.3 The genetic basis for PBP modifications is the integration of heterogeneous DNA, mainly of non-pneumococcal origin, leading to the formation of ‘mosaic’ PBP genes.4 Potential DNA donors are streptococci present in the mouth flora.5

ß-Lactam antibiotics remain the drugs of choice for pneumococcal diseases,6 except when penetration to the site of infection is severely restricted, as is the case in meningitis. Because of treatment failures with cephalosporin monotherapy,7,8 a combination of vancomycin and a cephalosporin is often used for meningitis caused by resistant strains.9,10,11 Reliably active monotherapy would represent a significant advantage to the empirical therapy of meningitis. Cefepime is a broad-spectrum cephalosporin with good activity against the majority of human bacterial pathogens, including penicillin-resistant pneumococci, and good penetration into the CSF.12,13 In the present study we tested cefepime alone and in combination with vancomycin in the rabbit meningitis model and in vitro. Ceftriaxone, alone and combined with vancomycin served as comparison regimens.


    Material and methods
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Rabbit meningitis model

The meningitis model, originally described by Dacey and Sande,14 was slightly modified. Briefly, young New Zealand white rabbits weighing 2–2.5 kg were anaesthetized by im injections of ketamine (30 mg/kg) and xylazine (15 mg/kg), and were immobilized in stereotactic frames for induction of meningitis and CSF samplings. An inoculum containing c. 1 x 105 cfu of penicillin-resistant pneumococci serotype 6 was injected directly into the cisterna magna. The MICs of the antibiotics for the infecting strain were as follows: penicillin G, 4; ceftriaxone, 0.5; vancomycin, 0.12–0.25; and cefepime, 0.5 mg/L, respectively.

A long-acting anaesthetic (ethylcarbamate = urethane; 3.5 g/rabbit) was injected sc and animals were returned to their cages. Fourteen hours later the cisterna magna was punctured again for periodic CSF sampling before and 0.75, 2.5, 4, 6 and 8 h after initiation of therapy. Antibiotics were administered through a peripheral ear vein as bolus injections at the following concentrations: cefepime, 100 mg/kg; ceftriaxone, 125 mg/kg; and vancomycin, 20 mg/kg. Ceftriaxone was injected once at 0 h and cefepime and vancomycin at 0 and 4 h according to Friedland et al.15 Untreated controls received saline. During the whole experimental period rabbits were kept anesthetized by repeated iv injections of nembutal. At the end of the experiment (8 h) euthanasia was induced by a lethal iv dose of nembutal.

Numbers of bacteria (cfu) were measured by 10-fold serial dilutions of CSF samples, plated on blood agar plates containing 5% sheep blood and incubated overnight at 37°C. In parallel, 20 µL of undiluted CSF were plated (limit of detectability, 50 cfu/mL). Comparisons between different dilutions of CSF were used to exclude significant carryover effects during therapy. The antimicrobial activity of the regimens during the 8 h treatment was calculated by linear regression analysis and expressed as decrease of log10 cfu/mL/h ({Delta}log10 cfu/mL/h). A value of 1.7 (log10 of the limit of detectability) was assigned to the first sterile CSF sample and a value of 0 to any following sterile sample. The results are expressed as means ± S.D. Statistical significance of differences was determined by the Newman– Keuls test.

Measurement of antibiotic levels in the CSF

Antibiotic concentrations in the CSF were determined by the agar diffusion method. Standard curves were performed in saline with 5% rabbit serum in order to mimic CSF protein concentration during meningitis.16 Escherichia coli (ATCC 29522) was used as test strain for ceftriaxone and cefepime13 and Bacillus subtilis (ATCC 6633) for vancomycin.17 The intra- and interday variability of this method was less than 10%. The limit of detection was 0.5, 1 and 1.5 mg/L for vancomycin, ceftriaxone and cefepime, respectively.

In vitro assays

The pneumococcal strain was grown in C + Y medium18 to optical density 0.3 at 590 nm and then diluted 40-fold to 106 cfu/mL, corresponding to the CSF bacterial titre in rabbits before initiation of therapy. Antibiotics were added in concentrations ranging from 0.5 to 5 mg/L corresponding to 1x, 2x, 5x and 10 x MIC of cefepime and ceftriaxone (MIC 0.5 mg/L). Combinations of vancomycin (0.12 mg/L) with cefepime (0.5 mg/L) or ceftriaxone (0.5 mg/L) were also tested. Bacterial numbers were determined at 0, 2, 4 and 6 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 were expressed as means ± S.D. Synergy was defined as bactericidal effect of a drug combination significantly exceeding the sum of the bactericidal effects of each agent alone.


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Cefepime concentration in the CSF after the first dose (100 mg/kg) peaked at a mean of 13 mg/L, declining slowly to 9.5 mg/L 2 h later (Figure 1Go). Following the second injection, the mean peak level increased to 16 mg/L before decreasing to 14 mg/L 4 h later. During the entire experiment, cefepime CSF levels remained far above the MIC (0.5 mg/L). Ceftriaxone, injected once at a dose of 125 mg/kg, led to CSF levels ranging between 4 and 6 mg/L during the whole treatment period (Figure 2Go). The CSF level/MIC ratio ranged between 22 and 32 for cefepime and between 8 and 12 for ceftriaxone during the treatment period. The killing rates of the different substances are summarized in the TableGo. In untreated controls, bacterial numbers remained stable (0.06 ± 0.10 {Delta}log10 cfu/mL/h) for 8 h. Vancomycin and ceftriaxone produced comparable bactericidal activity, whereas cefepime showed significantly higher activity than ceftriaxone (P < 0.05, TableGo). The combination of vancomycin with either cefepime or ceftriaxone improved the killing rates of both cephalosporins, although the differences were not statistically significant (ceftriaxone plus vancomycin versus ceftriaxone; cefepime plus vancomycin versus cefepime). On the other hand, the combination of cefepime plus vancomycin was significantly superior to ceftriaxone or vancomycin alone (P < 0.05). Interestingly, cefepime monotherapy was as effective as vancomycin plus ceftriaxone, a combination often used for the treatment of meningitis caused by resistant pneumococci.



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Figure 1. Cefepime concentrations in CSF for 8 h after iv injection of 100 mg/kg cefepime. The arrow represents the time point of the second cefepime injection. The concentration of cefepime remained above the MIC (0.5 mg/L) during the entire treatment period.

 


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Figure 2. Ceftriaxone concentrations in CSF for 8 h after iv injection of 125 mg/kg ceftriaxone. The concentration of ceftriaxone remained above the MIC (0.5 mg/L) during the entire treatment period.

 

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Table.Cefepime versus ceftriaxone alone and in combination in the experimental meningitis caused by penicillin-resistant S. pneumoniae
 
In vitro, both cephalosporins had a comparable bactericidal activity with concentrations above the MIC (2x, 5x and 10 x MIC) (Figures 3 and 4GoGo). Combination therapies with a cephalosporin plus vancomycin were also tested in vitro. For this purpose, antibiotic concentrations equal to the MIC were chosen, producing only a slow bactericidal effect as monotherapy. Combinations with higher concentrations of the cephalosporins were not tested because of the pronounced in vitro bactericidal effect of these drugs as monotherapy (Figures 3 and 4GoGo). Figure 5Go shows the killing activity of cefepime, vancomycin and the combination of the two drugs. Whereas single antibiotics produced only poor bactericidal activity after 6 h (vancomycin: –0.49 {Delta}log10 cfu/mL; cefepime: –1.95 {Delta}log10 cfu/mL), the combination clearly increased the killing rate (–4.47 {Delta}log10 cfu/mL; P < 0.05). These data document a synergy between cefepime and vancomycin in vitro, while the improved killing effect of the combination in the animal model did not reach an extent that qualifies as synergy. Figure 6Go shows the antibacterial activity of ceftriaxone and vancomycin, using the same experimental setting. Ceftriaxone, at a concentration equal to the MIC (0.5 mg/mL) had modest bactericidal activity (–1.66 {Delta}log10 cfu/mL after 6 h). Synergy was observed by addition of vancomycin (0.12 mg/mL) to ceftriaxone, considerably increasing the killing rate to –4.71 {Delta}log10 cfu/mL (P < 0.05).



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Figure 3. Killing rates of cefepime in vitro with concentrations ranging from 1 to 5 mg/L (2–10 x MIC): {blacktriangleup}, 2 x MIC; {blacksquare}, 5 x MIC; {diamondsuit}, 10 x MIC; {square}, control. Experiments were performed in triplicate in liquid cultures and killing rates were expressed as means ± S.D.

 


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Figure 4. Killing rates of ceftriaxone in vitro with concentrations ranging from 1 to 5 mg/L (2–10x MIC): {blacktriangleup}, 2 x MIC; {blacksquare}, 5 x MIC; {diamondsuit}, 10 x MIC; {square}, control. Experiments were performed in triplicate in liquid cultures and killing rates were expressed as means ± S.D.

 


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Figure 5. Killing rates of vancomycin (•), cefepime ({diamondsuit}) and cefepime combined with vancomycin ({blacktriangleup}), in vitro using concentrations equal to the MIC. Experiments were performed in triplicate in liquid cultures and killing rates were expressed as means ± S.D. (*P < 0.05 versus cefepime or vancomycin).

 


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Figure 6. Killing rates of vancomycin (•), ceftriaxone alone ({diamondsuit}) and ceftriaxone combined with vancomycin ({blacktriangleup}) in vitro using concentrations equal to the MIC. Experiments were performed in triplicate in liquid cultures and killing rates were expressed as means ± S.D. (*P < 0.05 versus ceftriaxone or vancomycin).

 

    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae is one of the major causative agents of community acquired infections (e.g. pneumonia, otitis media) and is responsible for 3000 cases of meningitis per year in the USA.19 Infections caused by penicillinresistant strains can be treated with high doses of ß-lactam antibiotics, provided that the penetration of the drugs into infected tissues is sufficient.6 Since concentrations of most antibiotics are considerably lower in the subarachnoid space than in serum, therapy with ß-lactams of meningitis caused by strains highly resistant to penicillin and cephalosporins (MIC > 1 mg/L) can be ineffective. Despite recent experimental data suggesting a possible role for newer quinolones against penicillin-resistant pneumococci,20 the standard treatment for these infections remains a combination of a cephalosporin (cefotaxime or ceftriaxone) with vancomycin.

Little is known about the effectiveness of cefepime in meningitis caused by resistant pneumococci. In the rabbit model of experimental meningitis, we compared cefepime alone and in combination with vancomycin with the standard therapy consisting of ceftriaxone combined with vancomycin.

The cefepime dose (100 mg/kg) was deliberately chosen in order to mimic the pharmacokinetics of high doses of cefepime in humans (2 g iv).21 Forty-five minutes after intravenous injection, blood levels in rabbits (110 mg/L) were comparable to those reported in humans (109 mg/L). Three hours after injection, the serum level in rabbits (30 mg/L) correlated closely with trough levels measured in humans (33 mg/L).21 This provided the rationale to administer a second dose of cefepime to rabbits at this time. With this dosing regimen, CSF concentrations of cefepime ranged from 10 to 16 mg/L, with ratios of CSF concentration/MIC above 20 during the entire treatment period (Figure 1Go). These results confirm previous reports of the excellent penetration of cefepime into the subarachnoid space during meningitis (20% versus 9% for ceftriaxone).22,23

The doses of vancomycin (2 x 20 mg/kg) and ceftriaxone (1 x 125 mg/kg) were standard doses that have been used in previous studies in the same model15 and produced serum and CSF concentrations corresponding to high-dose regimens in humans.24,25 In the present study, ceftriaxone CSF concentrations (4–6 mg/L) corresponded to concentrations described in previous studies.15 Similarly, mean peak CSF concentrations were 4.0 ± 1.7 mg/L after 2 x 20 mg of vancomycin, corresponding closely to the CSF concentration measured in humans.25,26

The reason for the superior killing effect of cefepime compared with ceftriaxone in vivo is not immediately clear, as they had similar antibacterial activity in vitro. The higher ratio of CSF concentration/MIC might conceivably have favoured cefepime over ceftriaxone, since we have previously shown that this pharmacodynamic parameter is a major determinant of bactericidal activity in this model.13

The addition of vancomycin improved the antimicrobial efficacy of both cephalosporins, however, without reaching an extent that qualified as synergy. A synergy between ceftriaxone and vancomycin in vivo and in vitro against resistant pneumococci has been described in previous studies using similar experimental systems to those of the present study.15

In an attempt to confirm in vitro the additive effect of cefepime and vancomycin observed in vivo, we selected concentrations that led to a marginal decrease of bacterial numbers with monotherapies (e.g. concentrations around the MIC). In this setting, the addition of vancomycin to either ceftriaxone or cefepime produced a synergic effect (Figures 5 and 6GoGo). To our knowledge, a synergy between cefepime and vancomycin has not been described previously.

The good penetration of cefepime into the inflamed meninges (c. 20%)23 and the excellent bactericidal activity against penicillin-resistant pneumococci qualify cefepime alone or in combination with vancomycin as a potential candidate for the treatment of pneumococcal meningitis caused by strains highly resistant to penicillin. This new combination could be particularly useful in cases where broad antibacterial activity is required in the initial empirical treatment of meningitis. Although our data are preliminary, cefepime with or without vancomycin deserves further clinical evaluation for the therapy of pneumococcal meningitis.


    Acknowledgments
 
This work was supported by a grant from Bristol–Myers Squibb Corporation.


    Notes
 
* Corresponding author. Tel: +41-31-632-2111; Fax: +41-31-632-3847; E-mail: pcottagn{at}insel.ch Back


    References
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
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Received 2 March 1999; returned 14 June 1999; revised 4 August 1999; accepted 31 August 1999