Experimental study of teicoplanin, alone and in combination, in the therapy of cephalosporin-resistant pneumococcal meningitis

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1 Journal of Antimicrobial Chemotherapy (2005) 55, doi: /jac/dkh496 Advance Access publication 16 November 2004 JAC Experimental study of teicoplanin, alone and in combination, in the therapy of cephalosporin-resistant pneumococcal meningitis A. Fernández, C. Cabellos*, F. Tubau, J. M. Maiques, A. Doménech, S. Ribes, J. Liñares, P. F. Viladrich and F. Gudiol Laboratory of Experimental Infection, Infectious Diseases Service and Microbiology Service, IDIBELL, Hospital Universitari de Bellvitge, Barcelona, Spain Received 30 June 2004; returned 23 August 2004; revised 1 October 2004; accepted 1 October 2004 Objectives: The aim of the study was to determine the efficacy of teicoplanin, alone and in combination with ceftriaxone, in a rabbit model of cephalosporin-resistant pneumococcal meningitis, and to assess the effect of concomitant therapy with dexamethasone. Methods: In vitro killing curves of teicoplanin, with and without ceftriaxone, were performed. Groups of eight animals per treatment were inoculated with a cephalosporin-resistant pneumococcal strain (penicillin MIC, 4 mg/l; ceftriaxone MIC, 2 mg/l; teicoplanin MIC, 0.03 mg/l) and treated over a 26 h period. Teicoplanin was administered at a dose of 15 mg/kg, alone and in combination with ceftriaxone at 100 mg/kg with or without dexamethasone at 0.25 mg/kg. CSF samples were collected at different time-points, and bacterial titres, white blood cell counts, lactate and protein concentrations and bacteriostatic/bactericidal titres were determined. Blood and CSF teicoplanin pharmacokinetic and pharmacodynamic parameters were determined. Results: Teicoplanin alone promoted a decrease in bacterial counts at 6 h of log cfu/ml and was bactericidal at 24 h, without therapeutic failures. Similar good results were obtained when dexamethasone was used simultaneously, in spite of the penetration of teicoplanin into the CSF being significantly reduced, from 2.31% to 0.71%. Teicoplanin and ceftriaxone combinations were synergic in vitro, but not in the meningitis model. Conclusions: Teicoplanin alone was very effective in this model of cephalosporin-resistant pneumococcal meningitis. The use of concomitant dexamethasone resulted in lower CSF teicoplanin levels, but not in therapeutic failures. The combination of teicoplanin plus ceftriaxone and dexamethasone might be a good alternative for the empirical therapy of pneumococcal meningitis. Additional data should confirm our experiments, in advance of clinical trials to assess efficacy in humans. Keywords: glycopeptides, bacterial meningitis, dexamethasone, Streptococcus pneumoniae Introduction The global increase in penicillin- and cephalosporin-resistant Streptococcus pneumoniae has led to changes in the therapy of pneumococcal meningitis. 1 6 High doses of cefotaxime have been successfully used to treat infections caused by organisms with intermediate resistance to cephalosporins, but sporadic failures have occurred. 7 9 Also, the possibility of emergence of very high-level cephalosporin resistance, as occurred in Tennessee 23F strain infections, should be taken into account. Glycopeptides retain their activity against drug-resistant pneumococci and may be useful in this setting. However, monotherapy with systemic vancomycin in adults has been associated with clinical and bacteriological failures, better explained by the erratic and borderline vancomycin CSF levels, which decrease when dexamethasone is administered concomitantly. 10,11 Some experimental studies have suggested that vancomycin plus ceftriaxone would be synergic against pneumococci, and most authorities recommend the administration of this combination for pneumococcal meningitis caused by resistant strains as well as for initial empirical therapy. 5,12,13 Oritavancin, a semi-synthetic glycopeptide, has also proved to be effective in the rabbit model of both susceptible and resistant pneumococcal meningitis, but clinical experience is lacking *Corresponding author. Tel: ; Fax: ; ccabellos@csub.scs.es JAC vol.55 no.1 q The British Society for Antimicrobial Chemotherapy 2004; all rights reserved.

2 Teicoplanin in pneumococcal meningitis Teicoplanin is a glycopeptide antibiotic, obtained in 1978 by fermentation from Actinoplanes teichomyceticus. It is less active than vancomycin against staphylococci, but equal or more active against streptococci; specifically, it presents excellent in vitro activity against S. pneumoniae strains, usually with MICs four or five dilutions under those of vancomycin and similar to those of oritavancin. The pharmacokinetics of teicoplanin allow its administration in a single daily dose, and the drug exhibits a lower propensity than vancomycin to cause side effects. Clinical experience in the treatment of meningeal infections with teicoplanin is very limited, and non-existent in the particular case of pneumococcal meningitis. In this work, we sought to evaluate the efficacy of teicoplanin, alone and in combination with ceftriaxone, in a rabbit model of cephalosporin-resistant pneumococcal meningitis, and to assess the effects of dexamethasone on the CSF antibiotic levels and inflammatory parameters. Materials and methods Bacterial strain The S. pneumoniae strain HUB2349, which had been isolated from a patient with meningitis, was used in the study. MICs and MBCs of penicillin, cefotaxime, ceftriaxone, vancomycin and teicoplanin were determined by the macrodilution method in cation-adjusted Mueller Hinton broth supplemented with 3% 5% of horse lysed blood according to NCCLS guidelines. 23 MICs/MBCs were as follows: penicillin, 4/4 mg/l; cefotaxime and ceftriaxone, 2/4 mg/l; vancomycin, 0.25/0.50 mg/l; and teicoplanin, 0.03/0.25 mg/l. In vitro killing curves Killing curves were performed with glass tubes containing a final volume of 10 ml of cation-adjusted Mueller Hinton broth with 5% of horse lysed blood. The final bacterial inoculum was cfu/ml. Concentrations of 32 MIC, 8 MIC, 2 MIC, 1 MIC and 1/2 MIC of teicoplanin, concentrations of 2 MIC, 1 MIC, 1/2 MIC and 1/4 MIC of ceftriaxone, and several associations of both antibiotics with one of them at subinhibitory concentration, were studied. Bacterial titres were determined at 0, 6 and 24 h of incubation by serial dilution of samples that were plated on agar containing 5% sheep blood. No carryover effect was observed. The detection limit was 1 log 10 cfu/ml. Synergy was defined as the effect of a drug combination >2-log killing over the most active drug alone with one of the drugs at subinhibitory concentration. The bactericidal effect was defined as a decrease >_ 3 log cfu/ml of the initial inoculum. Inoculum preparation The microorganism was grown overnight on blood-agar plates (BAP) and bacterial suspensions were prepared. The inoculum was made by suspending the colonies in brain heart infusion broth, adjusting the turbidity to the equivalent of a 0.5 McFarland standard (10 8 cfu/ml) and making 10-fold dilutions in saline in order to obtain a concentration of 10 6 cfu/ml. For each experiment, the inoculum size was determined by plating 0.1 ml on BAP, with subsequent counting of the colonies after h of incubation at 358C. Pharmacokinetics and pharmacodynamics Several pharmacokinetic (PK) and pharmacodynamic (PD) parameters of teicoplanin and teicoplanin + dexamethasone were determined in serum and CSF: maximal achievable concentration, area under the curve calculated by linear trapezoidal rule, percentage of CSF penetration, time above the MIC and C max /MIC. Two groups of rabbits (n = 10 each) were intracisternally inoculated with the study strain. Eighteen hours later, during a period of 24 h, the rabbits were treated with 15 mg/kg/day of intravenous (iv) teicoplanin or teicoplanin + dexamethasone (0.25 mg/24 h, divided in two doses). CSF and serum samples were taken at 1, 2, 5, 8, 10, 12 and 24 h of therapy, and were used to determine teicoplanin concentrations by fluorescence polarized immunoassay using a TDX analyzer calibrated to achieve a minimal detectable concentration of 0.03 mg/l. CSF cultures were also performed to determine bacterial concentration at each time-point. PK and PD parameters were obtained by a computer-assisted method (PK functions for Microsoft Excel; J. I. Usansky, A. Desai and D. Tang-Liu, Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, CA 92606, USA) after determination of antibiotic concentration at the different time points. Meningitis rabbit model The rabbit model of meningitis was performed according to an established protocol 24 and was approved by the Ethical Committee for Animal Experiments at the University of Barcelona (Campus de Bellvitge). For all experiments, rabbits (2 kg female New Zealand white) were challenged in groups of eight animals per group. On the first day of the experiment, they were anaesthetized intramuscularly with ketamine 35 mg/kg (Ketolar; Parke-Davis, El Prat de Llobregat, Spain) and xylazine 5 mg/kg (Rompun; AG Bayer, Leverkusen, Germany). A dental acrylic helmet was fixed to the calvaria and the animals were returned to their cages. Twenty-four hours later, the rabbits were anaesthetized again by the same method and placed in a stereotaxic frame. A 25 gauge spinal needle was introduced in the cisterna magna and 200 ml of CSF was withdrawn as a sterility control. Meningitis was then induced by instillation of 200 ml of a bacterial suspension adjusted to 10 6 cfu/ml of the studied strain into the subarachnoid space (see inoculum preparation above). Eighteen hours later, the rabbits were anaesthetized subcutaneously with urethane 1.75 g/kg (ethyl-carbamate; Sigma Chemical Company, St Louis, MI, USA) and phenobarbital 5 mg/kg (Penthotal sodico; Abbott Laboratories, Madrid, Spain). Blood cultures were taken at this time to detect secondary bacteraemia; 0.1 ml of blood collected from the central ear vein was suspended in 5 ml of trypticase soy broth and incubated overnight at 378C. A baseline 200 ml 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 S.A. Rubí, Barcelona, Spain) was administered, and 10 min later, antibiotic therapy was started with one of the following therapy schedules: teicoplanin, teicoplanin + dexamethasone, teicoplanin + ceftriaxone, teicoplanin + ceftriaxone + dexamethasone. There was also a control group infected but not treated. Antibiotic doses were as follows: teicoplanin 15 mg/kg/day (Targocid; Aventis Pharma, Alcorcon, Madrid, Spain) and ceftriaxone 100 mg/kg/day (Rocefalin; Roche, Madrid, Spain), all in a single dose. The total dose of dexamethasone was 0.5 g/24 h, divided every 12 h over a 48 h period (four doses). The teicoplanin dosage of 15 mg/kg/day was chosen in order to have good serum levels based on previous experience with rabbit models. 25 Ceftriaxone and dexamethasone doses were the same as used in previous experiments. 26 CSF samples were taken at 0, 2, 6, 24, 26 and 48 h of therapy. Brain oedema, expressed as g of water/100 g of dry weight, was determined after sacrifice of the rabbits with an overdose of phenobarbital at 48 h. Therapeutic failure was defined as an increase in bacterial concentration of at least 1 log cfu/ml compared with 79

3 A. Fernández et al. a previous count. A therapy was considered bactericidal when a reduction of 3 log cfu/ml was achieved. Sample processing CSF bacterial titres, white blood cells (WBC), protein and lactate concentrations, and bacteriostatic/cidal activity were determined. For colony counts, direct cultures and serial 10-fold dilutions were performed (the detection limit by this method was 10 2 cfu/ml and a value of 1.9 log cfu/ml was assigned to the first sterile culture and of 0 to the subsequent ones). To avoid carryover antimicrobial agent interference, the sample was placed onto 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. 27 For WBC counts, 10 ml of each sample was diluted 1:1 with Turk solution (acetic acid and Methylene Blue prepared in-house) and read with a Neubauer camera. After centrifugation, CSF was stored at 808C in order to determine the rest of the parameters. CSF protein concentration was determined by the Bradford method (Bio-Rad Protein Assay), and lactate CSF concentration by Lactate PAP (biomérieux, France). CSF bactericidal activities were performed by a microdilution method 28 with cation-adjusted Mueller Hinton broth (Difco Laboratories, Detroit, MI, USA) with 2% 5% lysed horse blood. Statistical analysis Comparisons among different therapy groups were, respectively, performed by ANOVA multiple-comparison test or Kruskall Wallis test (inflammatory activity). A P value of <0.05 was considered significant. Results In vitro killing curves Teicoplanin alone was bactericidal at 24 h at a concentration of 8 MIC and higher, equivalent to 0.25 mg/l. Ceftriaxone was also bactericidal at 24 h at a concentration of 1 MIC, equivalent to >_2 mg/l. A combination of 1/2 MIC of both (subinhibitory concentrations) showed a synergic effect at 6 h and a bactericidal effect at 24 h. Combinations of teicoplanin 1 MIC + ceftriaxone 1/2 MIC, ceftriaxone 1/2 MIC + teicoplanin 2 MIC and ceftriaxone 1/4 MIC + teicoplanin 2 MIC were also synergic at 6 h and bactericidal at 24 h. Variations in log cfu/ml at 6 and 24 h are shown in Table 1. Pharmacokinetic and pharmacodynamic experiments The different parameters are shown in Table 2. Peak and trough CSF teicoplanin concentration with the concomitant use of dexamethasone was statistically significantly lower than with teicoplanin alone. Accordingly, CSF penetration decreased from 2.31% to 0.71%. PD parameters such as t > MIC and C max /MIC were also lower when using concomitant dexamethasone. Blood cultures Eighteen hours after the inoculation, 100% of the animals presented positive blood cultures, suggesting that this is a highly invasive pneumococcal strain. Table 1. In vitro activities of ceftriaxone and teicoplanin against S. pneumoniae at 6 h and 24 h Drugs (fold MIC) Difference log cfu/ml at 6 h 24 h TEI (8) TEI (2) TEI (1) TEI (1/2) CRO (2) CRO (1) CRO (1/2) CRO (1/4) CRO (1) + TEI (2) CRO (1) + TEI (1) CRO (1) + TEI (1/2) CRO (1/2) + TEI (2) CRO (1/2) + TEI (1) CRO (1/2) + TEI (1/2) CRO (1/4) + TEI (2) CRO (1/4) + TEI (1) CRO (1/4) + TEI (1/2) TEI, teicoplanin; CRO, ceftriaxone. In vivo activity of the antimicrobial regimens Table 3 shows the in vivo bacterial reduction in CSF at 6 and 24 h with the different therapeutic schedules. All therapies were bactericidal and all CSF samples under the level of detection at 24 h of the experiment. No therapeutic failures were observed in any of the combinations. Teicoplanin alone showed good behaviour, indifferent to the concomitant administration of dexamethasone ( 2.66 log cfu/ml for teicoplanin versus 2.75 log cfu/ml for teicoplanin + dexamethasone at 6 h; not significant). The addition of ceftriaxone did not significantly improve the activity of teicoplanin with or without dexamethasone ( 2.05 log cfu/ml for teicoplanin + ceftriaxone and 2.14 log cfu/ml for teicoplanin + ceftriaxone + dexamethasone at 6 h; not significant). CSF bactericidal activity (bacteriostatic and bactericidal titres) Bacteriostatic and bactericidal titres are shown in Table 4. Peak bactericidal activity (2 and 26 h) ranged between 1:4 (teicoplanin, teicoplanin + dexamethasone) and 1:32 (teicoplanin + ceftriaxone). Trough bactericidal activity (24 and 48 h) ranged between <1:2 (teicoplanin, teicoplanin + dexamethasone, teicoplanin + ceftriaxone + dexamethasone) and 1:8 (teicoplanin + ceftriaxone). Overall, the combination of teicoplanin + ceftriaxone gave higher bacteriostatic/bactericidal activities, whereas the addition of dexamethasone produced a decrease in the bacteriostatic/bactericidal titres. Inflammatory activity in CSF The median of CSF WBC and the arithmetic means of lactate and protein concentrations at 2, 6 and 24 h are shown in Table 5. No statistically significant differences were observed in the cytochemical activity promoted by the different antibiotic therapies. 80

4 Teicoplanin in pneumococcal meningitis Table 2. Pharmacokinetics and pharmacodynamics parameters of teicoplanin in serum and CSF with and without dexamethasone (DEX) C mas (mg/l) C min (mg/l) AUC (mg h/l) t > MIC (%) Penetration (%) AUC/MIC C max /MIC (IQ) C max /MBC Serum ± ± Serum (+ DEX) ± ± CSF 1.09 ± 0.93 a 0.25 ± 0.17 a CSF (+ DEX) 0.32 ± ± C max and C min values are given as means ± S.D.. a P < 0.05 versus + DEX. Table 3. Decrease in log cfu/ml (means ± S.D.) in experimental pneumococcal meningitis with the different therapeutic schedules Initial titres (cfu/ml) Dlog cfu/ml 6 h Dlog cfu/ml 24 h Control 5.45 ± TEI 5.11 ± ± ± 0.55 TEI+DEX 5.75 ± ± ± 0.69 TEI+CRO 5.00 ± ± ± 0.60 TEI+CRO+DEX 5.85 ± ± ± 0.42 TEI, teicoplanin; DEX, dexamethasone; CRO, ceftriaxone. Table 4. Median CSF bacteriostatic/bactericidal activity with different antibiotic schedules 2 h 24 h 26 h 48 h TEI 1:16/1:4 1:4 1:8/1:2 1:16/1:8 1:4/<1:2 TEI + DEX 1:16/1:8 1:4/<1:2 1:8/1:4 1:2 1:4/<1:2 TEI + CRO 1:32/1:32 1:8/1:4 1:64/1:32 1:64 1:8/1:8 TEI + CRO + DEX 1:8/1:8 1:2/1:2 1:8/1:8 1:4/<1:2 TEI, teicoplanin; DEX, dexamethasone; CRO, ceftriaxone. The effect of dexamethasone was studied, bearing in mind the percentages of variation of the different parameters at a determined time-point compared with 0 h. The teicoplanin ± dexamethasone group did not present significant differences in terms of variation of WBC, lactate concentration or protein concentration in CSF. However, the combination of teicoplanin + ceftriaxone presented a higher inflammatory activity than the combination plus dexamethasone, regarding WBC concentration at 2 h and protein concentration at 24 h. Brain oedema was in the range g of water/100 g of dry weight, without significant differences between groups. Discussion Teicoplanin alone, administered at a dose of 15 mg/kg, was effective in this rabbit model of cephalosporin-resistant pneumococcal meningitis, promoting a decrease in bacterial counts at 6 h of 2.66 log cfu/ml, and being bactericidal at 24 h, without therapeutic failures. Similar good results were obtained when dexamethasone was used simultaneously, in spite of the penetration of teicoplanin into the CSF being significantly reduced, Table 5. Percentage of variation with respect to 0 h in inflammatory activity in experimental pneumococcal meningitis TEI TEI ± DEX TEI ± CRO TEI ± CRO ± DEX WBC 2h a 6h h Protein concentration 2 h h h a Lactate concentration 2h h h Brain oedema (g of water/100 g of dry weight) 379 ± ± ± ± 8 TEI, teicoplanin; DEX, dexamethasone; CRO, ceftriaxone. a P < 0.05 versus same combination without dexamethasone. 81

5 A. Fernández et al. from 2.31% to 0.71%. It has been shown previously that the antiinflammatory activity of dexamethasone may reduce the penetration of the glycopeptides vancomycin 11,26,29,30 and oritavancin 14 through the blood brain barrier, resulting in lower levels in CSF. According to the present results, this phenomenon appears to be a universal effect involving all glycopeptides. In our experience, the administration of both vancomycin and oritavancin with dexamethasone, using the same meningitis model and the same pneumococcal strain, was associated with therapeutic failures, 14,26 whereas the use of teicoplanin was not. Although we do not have a complete explanation for this observation, we think that the absence of failures is, to a large extent, in accordance with the PD parameters observed. In fact, peak CSF levels of teicoplanin were several times above the MIC in all cases, the C max /MIC ratio was 10 and the percentage of time above the MIC between doses (t > MIC) was 35% at worst. It has been suggested that the ratio C max /MBC in the CSF should be equal or superior to 4 in order to avoid failures as much as possible. 12 Since the ratio C max /MBC in our teicoplanin plus dexamethasone group was lower than 4 (Table 2), our results should be confirmed by additional data. In our previous experience with oritavancin, 14 despite very low MIC/MBC values (0.008/0.008 mg/l) therapeutic failures occurred. However, in that study, oritavancin CSF levels were below the level of detection when dexamethasone was used, making the ratio C max /MIC and C max /MBC impossible to determine but was close to zero. The conserved efficacy of teicoplanin in the presence of dexamethasone might be of special interest in the clinical setting. If additional studies confirm our results, the possibility of using teicoplanin instead of vancomycin as empirical therapy could be considered and assessed in clinical trials. Currently, more experience is needed before recommending the concomitant use of dexamethasone and teicoplanin alone to treat patients. The addition of ceftriaxone to teicoplanin did not represent a significant improvement in bacterial decrease, despite the synergy shown in the in vitro studies at most concentrations tested. A synergic effect of ceftriaxone and vancomycin against resistant strains has been found in an experimental model of pneumococcal meningitis, 12 but we have been unable to demonstrate this effect in any of the experiments performed with the different glycopeptides. 14,26 However, the combination produced higher bactericidal titres (1:32) than teicoplanin alone (1:4). As with oritavancin, it is possible that the rapid decrease produced by teicoplanin in the bacterial concentration prevented the manifestation of a synergic effect. The use of ceftriaxone evoked some degree of enhanced inflammatory activity (increase in protein concentration) that was down-modulated by dexamethasone. The possibility of meningitis cases resulting from S. pneumoniae, with very high resistance to third-generation cephalosporins, is of special interest following the promising results with this glycopeptide: empirical therapy with the combination cephalosporin glycopeptide would result, in fact, in monotherapy. The experience with teicoplanin in bacterial meningitis is very limited. The administration of 400 mg to four patients resulted in a CSF concentration of 1 mg/l in one case and of less than 0.5 mg/l in the other three cases. With a dose of 1200 mg/day, the CSF levels achieved were mg/l. 31 The dose of 15 mg/kg used in our experiments achieved a serum teicoplanin concentration mg/l in the rabbits, similar to that achieved with the usual dose of 400 mg in humans; 32 however, to treat severe infections higher doses (around 800 mg/day) have been used in humans and are known to be safe. A loading dose of teicoplanin would be of interest to treat pneumococcal meningitis in humans, in order to increase levels enhancing C max /CMB ratios in the first hours of therapy. The high degree of protein binding of teicoplanin, 90% in humans and rabbits, seems not to be a problem in terms of efficacy, as occurs with other antibiotics used in meningitis, such as ceftriaxone. In conclusion, teicoplanin alone was very effective in this model of penicillin and cephalosporin pneumococcal meningitis. The use of concomitant dexamethasone resulted in lower CSF teicoplanin levels but not in therapeutic failures. Combinations of teicoplanin and ceftriaxone were synergic in vitro, but did not produce any significant improvement in bacterial decrease. In the clinical setting, the combination of teicoplanin plus ceftriaxone and dexamethasone might be a good alternative for the empirical therapy of pneumococcal meningitis. Additional data should confirm our experiments before performing clinical trials to assess this efficacy in humans. Acknowledgements This work was supported in part by a grant FIS 97/507 (Fondo de Investigaciones Sanitarias, Spain). A.F., A.D. and S.R. were also supported by the same organization and J.M. by CIRIT (Generalitat de Catalunya). References 1. Baquero, F., García-Rodríguez, J. A., García de Lomas, J. et al. (1999). Antimicrobial resistance of 1113 Streptococcus pneumoniae isolates from patients with respiratory tract infections in Spain: results of a 1-year ( ) multicentre surveillance study. Antimicrobial Agents and Chemotherapy 43, Doern, G. V., Heilmann, K. 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