Evaluation of combined ceftriaxone and dexamethasone therapy in experimental cephalosporin-resistant pneumococcal meningitis

C. Cabellosa,*, J. Martínez-Lacasaa, F. Tubaub, A. Fernándeza, P. F. Viladricha, J. Liñaresb and F. Gudiola

a Laboratory of Experimental Infection, Infectious Diseases Service; b Microbiology Service, Ciutat Sanitària i Universitària de Bellvitge, C/Feixa Llarga s/n, 08907 L'Hospitalet, Barcelona, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The treatment of meningitis caused by strains of Streptococcus pneumoniae with decreased susceptibility to third-generation cephalosporins is an increasingly frequent and difficult problem. In this study a rabbit model of meningitis was used to determine the efficacy of ceftriaxone at different dosages, and to establish the effect of the addition of dexamethasone to the chemotherapeutic regimen. Groups of eight rabbits were inoculated with 106 cfu/mL of a cephalosporin- resistant strain of S. pneumoniae (MIC of cefotaxime/ceftriaxone 2 mg/L). Eighteen hours after inoculation, ceftriaxone (50 or 100 mg/kg/day) with or without dexamethasone (0.25 mg/kg/ day) was administered for a period of 48 h. The ceftriaxone dose of 50 mg/kg/day was not fully effective in this model (therapeutic failure rate 28%). With a dose of 100 mg/kg/day there were no therapeutic failures and all CSF cultures were below the level of detection at 48 h. CSF ceftriaxone concentrations, area under the time–concentration curve and time above the MIC were not significantly different with or without dexamethasone. However, concomitant use of dexamethasone resulted in higher CSF bacterial counts and a higher number of therapeutic failures (57% with the 50 mg/kg/day dose and 28% with the 100 mg/kg/day dose). Increasing doses of ceftriaxone might be an effective mode of therapy for meningitis caused by S. pneumoniae with MIC <= 2 mg/L. However, in contrast to cephalosporin-sensitive cases, in cases caused by ceftriaxone-resistant strains, concomitant use of dexamethasone was associated with a higher failure rate even when a higher dosage of ceftriaxone was used.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The increasing prevalence of penicillin- and cephalosporin- resistant Streptococcus pneumoniae worldwide has changed the empirical therapy of pneumococcal meningitis. Since the MICs of cefotaxime and ceftriaxone for S. pneumoniae were 2–4 times lower than that of penicillin G, these drugs were considered to be the therapy of choice for meningitis caused by organisms with intermediate penicillin resistance.1–4 Efficacy against pneumococcal strains with higher levels of resistance is controversial. The use of ceftriaxone combined with vancomycin or rifampicin in adults and children has been suggested as the best alternative,5 but no clinical series has been published to date. Increasing the dosage of cephalosporins in order to achieve higher CSF antibiotic concentrations is an alternative strategy, and a previous study described several cases that were cured with high doses of cefotaxime.6 Dexamethasone has been shown to reduce inflammatory activity in experimental bacterial meningitis and to reduce neurological sequelae in paediatric cases of Haemophilus influenzae and pneumococcal meningitis,7–10 but its routine use as adjunctive therapy is not yet widely accepted.10 In experimental models it has been demonstrated that use of dexamethasone reduces CSF concentrations of vancomycin significantly, and also ceftriaxone (although not to a significant degree).11,12 The aim of this study was to determine the efficacy of increased doses of ceftriaxone in the therapy of meningitis caused by S. pneumoniae with MICs <= 2 mg/L and to establish the effect of dexamethasone on ceftriaxone therapy.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The design of the rabbit meningitis model followed an established protocol.13 Four groups of eight rabbits were used, as well as a control group which was inoculated but not treated. Female New Zealand white rabbits weighing 2 kg were anaesthetized intramuscularly with 35 mg/kg ketamine (Ketolar, Parke-Davis, El Prat de Llobregat, Spain) and 5 mg/kg xylazine (Rompun, Bayer AG, Leverkusen, Germany), and a dental acrylic helmet was affixed to each rabbit's calvaria. Twenty-four hours later the animals were anaesthetized again and placed in a stereotaxic frame. A spinal needle was introduced into the cisterna magna, 200 µL of CSF was withdrawn and 200 µL of 106 cfu/mL of a strain of S. pneumoniae belonging to serotype 23 F (MIC of penicillin 4 mg/L; MIC of ceftriaxone 2 mg/L) was instilled into the subarachnoid space. Eighteen hours later the rabbits were anaesthetized again with urethane (Sigma Chemical Company, St Louis, MO, USA) 1.75 g/kg subcutaneously and thiopental (Penthotal sodico, Abbott Laboratories, Madrid, Spain) 5 mg/kg iv and a baseline CSF sample was taken. Then an iv dose of 0.25 mg of dexamethasone (Fortecortin, Merck, Mollet del Vallés, Barcelona, Spain) or saline (Suero fisiológico Braun, Braun SA Rubí, Barcelona, Spain) was administered, and 10 min later an iv dose of either 50 or 100 mg/kg of ceftriaxone (Roche, Madrid, Spain) was administered (serum ceftriaxone concentrations at 30 min after a 75 mg/kg dose are c. 200 mg/L12 and serum ceftriaxone concentrations at 60–90 min after a 125 mg/kg dose are also c. 200 mg/L5). Dexamethasone was administered every 12 h over a 48 h period (four doses) and ceftriaxone was administered every 24 h (two doses). Serial CSF samples were taken at 2 (peak), 6, 24 (trough), 26 (peak) and 48 h (trough). WBC counts, lactic acid concentration, direct and quantitative bacterial cultures, ceftriaxone concentrations in CSF and CSF bactericidal activities at trough and peak time points were determined for each sample of CSF. WBC counts were performed by optical microscopy with a Neubauer chamber after RBC had been lysed with Turk solution (made in house; 0.2% acetic acid and methylene blue). CSF lactic acid concentrations were determined by Lactate PAP (bioMérieux SA, Marcy l'Etoile, France) and read by spectrophotometer.

Serial ten-fold dilutions were performed to determine bacterial counts at each time point (the detection limit with this method was 102 cfu/mL, and a value of 0 (log 10 = 1) was assigned to the first and subsequent sterile cultures).

MICs of penicillin and ceftriaxone were determined by a microdilution method with cation-adjusted Mueller–Hinton broth (Difco Laboratories, Detroit, MI, USA) supplemented with 3% lysed horse blood, with an inoculum of approximately 5 x 105 cfu/mL as recommended by the NCCLS.14 The MIC was defined as the lowest concentration of antibiotic for which no visible turbidity was apparent.

CSF ceftriaxone concentrations were determined by an agar disc diffusion method15 in antibiotic medium 2 (Difco Laboratories), using Bacillus subtillis ATCC 12432 as the indicator strain. Standard antibiotic solutions were prepared in saline. To avoid inter-day variability the concentrations of drugs from which a standard curve was determined and all CSF samples were assayed in duplicate in a single day. The assay variability for individual samples was <=10%. The level of detection was 0.06 mg/L.

CSF bactericidal activities were performed by a microdilution method16 with cation-adjusted Mueller–Hinton broth supplemented with 2–5% lysed horse blood. Serial two-fold dilutions (range 1:2 to 1:4096) of CSF were performed, and a concentration of 5 x 105 cfu/mL of the same strain as the meningitis model was used for the inoculum. Bacteriostatic activity was defined as the highest dilution without visible turbidity and bactericidal activity was defined as the highest dilution capable of killing 99.9% of the initial inoculum.

To minimize the effect of carry over of antibiotic agent, an entire agar plate was used for each sample. The sample was placed on the plate in a single streak down the centre, allowed to be absorbed into the agar until the plate surface appeared dry, and then the inoculum was spread over the plate.17

Therapeutic failure was defined as an increase in bacterial concentration compared with a previous count, and the death of the rabbit.

Since slight but not statistically significant differences in CSF ceftriaxone concentrations were found at certain time points, it was decided to perform a further set of experiments in order to determine the area under the time–concentration curve (AUC; by the linear trapezoidal rule), which might better demonstrate such differences. The rabbit model was set up as before and 13 rabbits were treated with ceftriaxone 100 mg/kg/day and 13 rabbits with ceftriaxone 100 mg/kg/day plus dexamethasone. Several samples of 50 µL of CSF were withdrawn at 1, 2, 4, 6, 8, 10, 12, 24, 26, 28, 30, 32, 34, 36 and 48 h. Only bacterial concentrations and CSF ceftriaxone concentrations were determined for these samples.

Statistical analysis

Fisher's exact test was used, when appropriate, to determine categorical variables. Student's t test was used to determine continuous variables. The N-par Mann–Whitney U Wilcoxon rank sum test was used to compare median bactericidal titres.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial concentrations

Ceftriaxone at 50 mg/kg/day reduced bacterial counts by >3 log cfu/mL at 24 h, with a mean log cfu/mL of 1.69 at 24 h versus 5.91 at 0 h (FigureGo). However, in two animals bacterial counts were above the level of detection at 24 h, and in one animal bacteria were detected at 48 h.



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Figure. Mean bacterial concentrations in CSF during treatment of experimental S. pneumoniae meningitis. Symbols: ——, control; {diamondsuit}, ceftriaxone 50 mg/kg/day; {blacksquare}, ceftriaxone 50 mg/kg/day plus dexamethasone; •, ceftriaxone 100 mg/kg/day; +, ceftriaxone 100 mg/kg/day plus dexamethasone.

 
Concomitant use of dexamethasone resulted in statistically significant higher bacterial counts at 24 h, with a mean log cfu/mL of 1.69 in ceftriaxone-treated animals versus 4.61 in animals treated with ceftriaxone plus dexamethasone (P < 0.05).

Ceftriaxone at 100 mg/kg/day decreased bacterial counts by >3 log cfu/mL at 6 h, with a mean log cfu/mL 1.68 versus 5.6 at 0 h. With this regimen, at 24 h 5 of 8 animals had bacterial counts under the level of detection and at 48 h bacterial counts in all animals were under the level of detection. A higher bacterial count was found with concomitant use of dexamethasone, although the difference was not statistically significant: the mean log cfu/mL was 1.18 in ceftriaxone- treated animals versus 2.18 in ceftriaxone plus dexamethasone-treated animals at 24 h, and 0 in ceftriaxone-treated animals versus 1.2 mean log cfu/mL in ceftriaxone plus dexamethasone-treated animals at 48 h (FigureGo).

Comparisons between bacterial counts with the different regimens at a range of time points are shown in the Figure.Go Bacterial counts at 24 h in animals treated with ceftriaxone 100 mg/kg/day versus animals treated with ceftriaxone 50 mg/kg/day plus dexamethasone also showed statistically significant differences (P < 0.05).

CSF ceftriaxone concentrations

Mean CSF ceftriaxone concentrations at peak and trough on the first and second days of therapy are shown in Table IGo. For the 50 mg/kg/day dose these were lower when concomitant dexamethasone was used, but this difference was not statistically significant. Differences were more evident at 2 h (peak of the first day): 3.57 mg/L in the ceftriaxone group versus 1.75 mg/L in the ceftriaxone plus dexamethasone group (P = 0.06). Mean trough CSF ceftriaxone concentrations were below the MIC for the infecting strain at 24 and 48 h; the variations between groups were unremarkable.


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Table I. Mean CSF ceftriaxone concentrations (mg/L) and bactericidal activity in rabbits with pneumococcal meningitis
 
The effects of increasing the dose to 100 mg/kg/day were variable (Table IGo). The most noteworthy finding was a higher peak concentration with the 100 mg/kg/day dose than with the 50 mg/kg/day dose, although the difference was disproportionate. Some differences were observed with the use of dexamethasone, but these were not statistically significant: at 26 h (peak of the second day) concentrations were 6.24 mg/L in the ceftriaxone group versus 4.43 mg/L in the ceftriaxone plus dexamethasone group.

Area under the time–concentration curve

No statistically significant differences were observed in CSF ceftriaxone concentrations between the time points during the first 24 h (Table IIGo). Nor were statistically significant differences observed in the AUC of the two therapy groups: 25.81 mg•h/L in the ceftriaxone group versus 23.61 mg•h/L in the ceftriaxone plus dexamethasone group. After 24 h there was a higher mortality in the ceftriaxone plus dexamethasone group, and so comparisons of CSF levels and AUC calculations were not possible.


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Table II. Mean CSF ceftriaxone concentrations (mg/L) in rabbits with pneumococcal meningitis included in the AUC study
 
Percentage of time above MIC

This set of experiments also allowed the time above the MIC to be determined: on the first day this was 4–6 h (20%) in the ceftriaxone (with or without dexamethasone) groups and after the second dose it ranged 6–8 h (29%) in both groups.

CSF bactericidal activity

Median CSF bactericidal activities are shown in Table IGo. There were no statistically significant differences between groups, and all bacterial activities were low (<=1:2). With the higher dose of ceftriaxone, bactericidal activities were also low, but were one dilution higher.

Therapeutic failures

The dose of 50 mg/kg/day resulted in therapeutic failures in 2/7 subjects (28%). Concomitant use of dexamethasone resulted in a higher failure rate: 4/7 (57%). The only group in which there were no therapeutic failures (0/8) was for those given ceftriaxone 100 mg/kg/day alone, but in the group given 100 mg/kg/day plus dexamethasone there were 2/7 (28%) therapeutic failures. The difference in therapeutic efficacy between the ceftriaxone 100 mg/kg/day and ceftriaxone 50 mg/kg/day plus dexamethasone groups was statistically significant (P < 0.05).

Inflammatory activity

Concomitant use of dexamethasone resulted in differences in CSF inflammatory activity. With the dose of 50 mg/kg/ day the mean CSF WBC at 24 h was 11,900 cells/µL in the ceftriaxone group versus 4780 cells/µL in the ceftriaxone plus dexamethasone group (P < 0.05). With ceftriaxone 100 mg/kg/day no differences were observed in the mean WBC between the groups treated with or without dexamethasone, but there were statistically significant differences in the mean CSF lactic acid concentration at 48 h: 6.71 mg/L in the ceftriaxone group versus 3.18 mg/L in the ceftriaxone plus dexamethasone group (P < 0.05).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, doses of ceftriaxone of 50 mg/kg/day failed to eradicate a cephalosporin-resistant strain of S. pneumoniae (MIC of ceftriaxone 2 mg/L). Increasing the dose to 100 mg/ kg/day improved bacterial killing, with sterilization of CSF at 48 h, and no therapeutic failures occurred. In addition CSF ceftriaxone levels were higher and bactericidal activities better when a dose of 100 mg/kg/day was used, showing that increased dosing concentrations may be effective in the therapy of cephalosporin-resistant pneumococcal meningitis. Overall, bactericidal activities were low, as predicted by the MIC for the infecting strain. In a recent report, Klugman et al.1 showed that the CSF of children receiving ceftriaxone at a range of doses, with dexamethasone, was unable to kill cephalosporin-intermediate or fully -resistant strains when the CSF ceftriaxone concentration was <5 mg/L, but at higher concentrations bactericidal activity was present. In another study, the percentage of time above the MBC was the variable that independently predicted bacterial killing rate.18

Adult patients with pneumococcal meningitis cannot usually be treated with >=100 mg/kg/day of ceftriaxone, because of the limitation in total daily dose (4 g) recommended by the manufacturers. It is possible to increase the dose of other similar broad-spectrum cephalosporins, such as cefotaxime, for which a total daily dose limitation is not given. Our group has reported the successful use of cefotaxime 300 mg/kg/day concomitantly with dexamethasone in ten cases of pneumococcal meningitis caused by strains with decreased susceptibility to third-generation cephalosporins.6 Increased dosage of third-generation cephalosporins might be an effective therapy for cephalosporin-resistant pneumococcal meningitis, at least for MICs <= 2 mg/L. However, in our study, with the use of dexamethasone the results were unacceptable even at the higher recommended dose. We have previously shown11 that dexamethasone can be used safely with ceftriaxone to treat penicillin-sensitive strains. In this study, although small differences were detected, there were no statistically significant differences in ceftriaxone levels in the CSF, bactericidal activities or therapeutic failures. Paris et al.12 found that ceftriaxone concentrations were lower at each time point in animals given dexamethasone in a meningitis model using two cephalosporin-resistant pneumococcal strains, although again the differences were not statistically significant. These small, though not statistically significant differences might be of biological relevance in cases owing to cephalosporin-resistant pneumococci. In the present study, AUC data showed that dexamethasone did not decrease ceftriaxone levels in the CSF. However, concomitant use of dexamethasone worsened results in terms of bacterial killing and bactericidal activity, with a higher number of therapeutic failures for both dose regimens. The mechanism of these failures remains unclear. Although possible differences in CSF ceftriaxone concentrations may appear the best explanation, this is not supported by our data. Nevertheless, percentage of time above MICs of the dosing intervals was lower than accepted for achieving therapeutic success in most cases (therapeutic failures with percentage of time above the MIC values in the range of 30% might be expected).19 The concomitant use of dexamethasone may accentuate this therapeutic limitation. Our data indicate that increasing doses of ceftriaxone may be an effective mode of therapy for cephalosporin-resistant cases, but concomitant use of dexamethasone makes this approach unreliable.


    Acknowledgments
 
This work was supported in part by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISss) 92/277 and 95/385 of Spain's National Health Service. F. Tubau, J. Martínez-Lacasa and A. Fernández were supported by grants from the Fundació August Pi i Sunyer. This paper was presented in part at the Thirty-Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, American Society for Microbiology, in San Francisco, California, September 1995 (abstract no. B25). The study was approved by the Ethical Committee for Animal Experiments at the University of Barcelona (Campus de Bellvitge).


    Notes
 
* Corresponding author. Tel: +34-93-335-7011 ext. 2487; Fax: +34-93-260-7637; E-mail: ccabellos{at}csub.scs.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Klugman, K. P., Friedland, I. R. & Bradley, J. S. (1995). Bactericidal activity against cephalosporin-resistant Streptococcus pneumoniae in cerebrospinal fluid of children with acute bacterial meningitis. Antimicrobial Agents and Chemotherapy 39, 1988–92.[Abstract]

2 . Liñares, J., Alonso, T., Perez, J. L., Ayats, J., Dominguez, M. A., Pallares, R. et al. (1992). Decreased susceptibility of penicillinresistant pneumococci to twenty-four ß-lactam antibiotics. Journal of Antimicrobial Chemotherapy 30, 279–88.[Abstract]

3 . Spangler, S. K., Jacobs, M. R. & Appelbaum, P. C. (1994). Susceptibilities of 177 penicillin-susceptible and -resistant pneumococci to FK 037, cefpirome, cefepime, ceftriaxone, cefotaxime, ceftazidime, imipenem, biapenem, meropenem and vancomycin. Antimicrobial Agents and Chemotherapy 38, 898–900.[Abstract]

4 . Viladrich, P. F., Gudiol, F., Liñares, J., Rufí, G., Ariza, J. & Pallarés, R. (1988). Characteristics and antibiotic therapy of adult meningitis due to penicillin-resistant pneumococci. American Journal of Medicine 84, 839–46.[ISI][Medline]

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6 . Viladrich, P. F., Cabellos, C., Pallares, R., Tubau, F., Martínez-Lacasa, J., Liñares, J. et al. (1996). High dose of cefotaxime in treatment of adult meningitis due to Streptococcus pneumoniae with decreased susceptibility to broad spectrum cephalosporins. Antimicrobial Agents and Chemotherapy 40, 218–20.[Abstract]

7 . Girgis, N. I., Farid, Z., Mikhail, I. A., Farrag, I., Sulstan, Y. & Kilpatrick, M. E. (1989). Dexamethasone treatment for bacterial meningitis in children and adults. Pediatric Infectious Diseases Journal 8, 848–51.[ISI][Medline]

8 . Lebel, M. H., Freij, B. J., Syrogianopoulos, G. A., Chrane, D. F., Hoyt, M. J., Stewart, S. M. et al. (1988). Dexamethasone therapy for bacterial meningitis: results of two double blind, placebo controlled trials. New England Journal of Medicine 319, 964–71.[Abstract]

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12 . Paris, M. M., Hickey, S. M., Uscher, M. I., Shelton, S., Olsen, K. D., McCracken, G. H., Jr (1994). Effect of dexamethasone on therapy of experimental penicillin- and cephalosporin-resistant pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 38, 1320–4.[Abstract]

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15 . Chapin-Robertson, K. & Edberg, S. C. (1991). Measurements of antibiotics in human body fluids: techniques and significance. In Antibiotics in Laboratory Medicine, 3rd edn, (Lorian, V., Ed.), pp. 295–366. Williams & Wilkins, Baltimore.

16 . Griffin, J. (1992). Serum inhibitory and bactericidal titers. In Clinical Microbiology Procedures Handbook, (Isenberg, H. D., Ed.), pp. 1–18. American Society for Microbiology, Washington, DC.

17 . Knapp, C. & Moody, J. A. (1992). Test to assess bactericidal activity. In Clinical Microbiology Procedures Handbook, (Isenberg, H. D., Ed.), p. 12. American Society for Microbiology, Washington, DC.

18 . Lutsar, I., Ahmed, A., Friedland, I. R., Trujillo, M., Wubbel, L., Olsen, K. et al. (1997). Pharmacodynamics and bactericidal activity of ceftriaxone therapy in experimental cephalosporin-resistant pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 41, 2414–7.[Abstract]

19 . Vogelman, B., Gudmundsson, S., Legett, J., Turnidge, J., Ebert, S. & Craig, W. A. (1988). Correlation of antimicrobial pharmacokinetic parameters with therapeutic efficacy in an animal model. Journal of Infectious Diseases 158, 831–47.[ISI][Medline]

Received 25 May 1999; returned 9 August 1999; revised 24 September 1999; accepted 12 November 1999