Quinupristin/dalfopristin attenuates the inflammatory response and reduces the concentration of neuron-specific enolase in the cerebrospinal fluid of rabbits with experimental Streptococcus pneumoniae meningitis

F. Trostdorfa, R. R. Reinertb, H. Schmidta, T. Nichterleinc, K. Stuertza, M. Schmitz-Saluea, I. Sadowskia, W. Brückd and R. Naua,*

a Department of Neurology, University of Göttingen d Department of Neuropathology, University of Göttingen; b Institute of Medical Microbiology, National Reference Centre for Streptococci, University of Aachen; c Department of Medical Microbiology, Klinikum Mannheim, Germany


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
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The inflammatory response following initiation of antibiotic therapy and parameters of neuronal damage were compared during intravenous treatment with quinupristin/dalfopristin (100 mg/kg as either a short or a continuous infusion) and ceftriaxone (10 mg/kg/h) in a rabbit model of Streptococcus pneumoniae meningitis. With both modes of administration, quinupristin/dalfopristin was less bactericidal than ceftriaxone. However, the concentration of proinflammatory cell wall components (lipoteichoic acid (LTA) and teichoic acid (TA)) and the activity of tumour necrosis factor (TNF) in cerebrospinal fluid (CSF) were significantly lower in the two quinupristin/dalfopristin groups than in ceftriaxone-treated rabbits. The median LTA/TA concentrations (25th/75th percentiles) were as follows: (i) 14 h after infection: 133 (72/155) ng/mL for continuous infusion of quinupristin/dalfopristin and 193 (91/308) ng/mL for short duration infusion, compared with 455 (274/2042) ng/mL for ceftriaxone (P = 0.002 and 0.02 respectively); (ii) 17 h after infection: 116 (60/368) ng/mL for continuous infusion of quinupristin/dalfopristin and 117 (41/247) ng/mL for short duration infusion, compared with 694 (156/2173) ng/mL for ceftriaxone (P = 0.04 and 0.03 respectively). Fourteen hours after infection the median TNF activity (25th/75th percentiles) was 0.2 (0.1/1.9) U/mL for continuous infusion of quinupristin/dalfopristin and 0.1 (0.01/3.5) U/mL for short duration infusion, compared with 30 (4.6/180) U/mL for ceftriaxone (P = 0.02 for each comparison); 17 h after infection the TNF activity was 2.8 (0.2/11) U/mL (continuous infusion of quinupristin/ dalfopristin) and 0.1 (0.04/6.1) U/mL (short duration infusion), compared with 48.6 (18/169) U/mL for ceftriaxone (P = 0.002 and 0.001). The concentration of neuron-specific enolase (NSE) 24 h after infection was significantly lower in animals treated with quinupristin/ dalfopristin: 4.6 (3.3/5.7) µg/L (continuous infusion) and 3.6 (2.9/4.7) µg/L (short duration infusion) than in those treated with ceftriaxone (17.7 (8.8/78.2) µg/L) (P = 0.03 and 0.009 respectively). In conclusion, antibiotic treatment with quinupristin/dalfopristin attenuated the inflammatory response within the subarachnoid space after initiation of antibiotic therapy. The concentration of NSE in the CSF, taken as a measure of neuronal damage, was lower in quinupristin/dalfopristin-treated rabbits than in ceftriaxone-treated rabbits.


    Introduction
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 Abstract
 Introduction
 Materials and methods
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 References
 
Over the past two decades, the antibiotic treatment of meningitis caused by Streptococcus pneumoniae has become increasingly complicated by the emergence and world-wide spread of strains with a reduced sensitivity to penicillin.1,2,3,4 Since penicillin-resistance in pneumococci is often associated with an increase in the MIC of other ß-lactam antibiotics, including third-generation cephalosporins, there have been failures in recent years in the treatment of meningitis caused by penicillin-resistant S. pneumoniae with the use of cefotaxime and ceftriaxone. 5,6,7

Although true microbiological failures are rare, the mortality of meningitis, including S. pneumoniae meningitis, has not changed over the past three decades. 8 In approximately one-third of the patients dying from this disease, severe brain oedema is present at autopsy. Proinflammatory bacterial products can produce brain oedema, so the burst of meningeal inflammation caused by the release of bacterial cell wall components, which is known to occur after initiation of antibiotic therapy,9– 9,10,11 may contribute to mortality. In addition, pneumococcal cell wall components have also been shown to exert a direct toxic effect on microglia, astrocytes and neurons in vitro. 12,13

Quinupristin/dalfopristin is the first injectable streptogramin; it belongs to the macrolide– lincosamide– streptogramin (MLS) family of antibiotics. It consists of two pristinamycin derivatives, quinupristin (RP 57669) and dalfopristin (RP 54476), in a 30:70 (w/w) ratio. The two components act synergically and have bactericidal activity against streptococci and staphylococci.14,15,16,17,18

Quinupristin/dalfopristin has been shown to release less proinflammatory teichoic and lipoteichoic acids from S. pneumoniae than ß-lactam antibiotics at equal bactericidal rates in vitro, 19 so it appears promising for the therapy of meningitis caused by penicillin-sensitive and penicillin-resistant S. pneumoniae. It has already been shown to be an effective antibacterial agent in a rabbit model of pneumococcal meningitis. Antibacterial activity clearly depended on the time of administration, indicating the importance of an increased permeability of the blood–CSF barrier to ensure sufficient antibacterial activity of quinupristin/dalfopristin in meningitis. 18 In healthy animals, penetration of quinupristin/dalfopristin into the central nervous system has been shown to be very low. 20

In the present study we compared the bactericidal activity of quinupristin/dalfopristin and ceftriaxone, and the effect of both antibacterials on the inflammatory response in the subarachnoid space and on markers of neuronal damage in a rabbit model of early S. pneumoniae meningitis.


    Materials and methods
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 Abstract
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 Materials and methods
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Test organism

A type 3 strain of S. pneumoniae originally isolated from an adult with meningitis (gift of M. G. Täuber, University of Bern, Switzerland), with an MIC and minimal bactericidal concentration (MBC) of ceftrixone of 0.03 and 0.06 mg/L respectively, was used in the in-vivo and in-vitro experiments. After several passages in rabbits, infected CSF was cultured for 24 h on blood agar plates, and the resulting bacteria were suspended in sterile saline. Samples were kept at -80°C.

The MIC and MBC of quinupristin/dalfopristin for this strain were determined by a broth macrodilution method. The MIC of 93 clinical S. pneumoniae isolates susceptible or intermediately resistant to penicillin was measured by an agar dilution method according to the recommendations of the National Committee for Clinical Labaratory Standards (NCCLS).

Drugs

Quinupristin/dalfopristin for experimental use was kindly provided by Rhône– Poulenc Rorer (Cologne, Germany). For iv administration it was dissolved in an aqueous solution containing 5% glucose.

Rabbit model

After intramuscular induction of anaesthesia with ketamine (25 mg/kg) and xylazine (5 mg/kg), New Zealand White rabbits (c. 2.5 kg; Charles River, Sulzfeld, Germany) were anaesthetized by iv urethane and fixed in a stereotaxic frame; a 22 G spinal needle was inserted into the cisterna cerebellomedullaris for the whole duration of the experiment (24 h). All animals were infected intracisternally and drugs were administered via an ear vein. Blood was sampled from the contralateral ear artery. All animals received the same amount of fluid (160 mL/24 h).

Twelve hours after infection, quinupristin/dalfopristin was administered either as an initial bolus of 10 mg/kg followed by a continuous infusion of 7.5 mg/kg/h over 12 h (n = 10) or as a short duration infusion of 100 mg/kg over 1 h (n = 10). The control animals (n = 10) received ceftriaxone as an initial bolus of 20 mg/kg with a maintenance dose of 10 mg/kg/h over 12 h.

Rabbits were killed 24 h after infection by iv injection of 75 mg of thiopental (Trapanal, Byk Gulden, Konstanz, Germany), and brains were removed immediately for histological examination.

Sample processing

CSF leucocytes were counted in a Fuchs– Rosenthal haemocytometer. Pneumococcal CSF counts were determined by plating 10 mL of serial ten-fold dilutions on blood agar plates, which were then incubated overnight at 37°C with 5% CO2. Bacterial titres at 12, 14, 17, 20, 24 h after infection were used for log-linear regression analysis.

The remaining CSF was immediately centrifuged at 3000g for 5 min. The supernatants were stored at -70°C for the determination of CSF concentrations of ceftriaxone, protein, lactate, tumour necrosis factor alpha (TNF), lipoteichoic and teichoic acids (LTA/TA) and neuron-specific enolase (NSE). CSF lactate was determined enzymatically (Biosen, Dreieich, Germany) and protein photometrically (BCA-Protein-Test, Pierce, Rockford, IL, USA) 12 h and 24 h after infection. CSF samples for determination of quinupristin/dalfopristin concentrations were diluted in 0.25 N HCl (0.25 mL/mL CSF, final pH approximately 5.5). For the measurement of quinupristin/dalfopristin concentrations in serum, blood was diluted in 0.25 N HCl (0.25 mL/mL blood) and 3.8% sodium citrate (0.1 mL/mL blood). Thereafter, the samples were centrifuged at 3000g for 5 min, and the supernatants were immediately frozen at -80°C. Ceftriaxone concentrations were measured in untreated serum and CSF.

Antibiotic assays

The concentrations of the two components of quinupristin/dalfopristin were determined at 14 and 24 h with a disc diffusion bioassay using Staphylococcus aureus HBD 511 sensitive to quinupristin and resistant to dalfopristin, and Staphylococcus epidermidis HBD 523 sensitive to dalfopristin and resistant to quinupristin. Both strains were kindly provided by Rhône– Poulenc Rorer. Filter paper discs were loaded with the samples, air-dried and placed on agar plates containing the respective strain in the agar. The diameters of the zones of inhibition were determined after 18 h of incubation at 37°C. Concentrations were calculated from the regression line of a standard curve with the respective antibiotic.

For small sample volumes, a broth microdilution method was developed using the same bacterial strains and dilution of the samples in cation-adjusted Mueller– Hinton broth (Radiometer, Willich, Germany). This assay yielded similar results to the disc diffusion test.

The concentrations of ceftriaxone in serum and CSF were determined by the agar-well diffusion technique in Antibiotic Medium No. 2 (Oxoid) with 0.4% agar, using Escherichia coli (no. 108; collection of Prof. Dr H. Hof, Department of Medical Microbiology, University of Heidelberg– Mannheim, Germany).

For serum and CSF samples, different standard curves were constructed using undiluted and 20-fold diluted rabbit serum. 21

Enzyme immunoassay for quantifiying lipoteichoic and teichoic acids

A newly developed enzyme immunoassay was used to measure the release of LTA as a result of bacterial lysis during antibiotic treatment. 19 Briefly, purified LTA serving for immunization and the construction of the standard curve were prepared from the unencapsulated S. pneumoniae R6 strain. Polyclonal antibodies were raised in New Zealand White rabbits immunized subcutaneously with 500 µg of LTA mixed with an equal volume of incomplete Freund's adjuvant. The enzyme immunoassay developed to quantify LTA and TA release from bacterial cultures used the commercially available TEPC-15 monoclonal antibody (Sigma, Deisenhofen, Germany) as the capture antibody and the polyclonal rabbit antiserum raised against LTA as the detector antibody. Various concentrations of quinupristin/dalfopristin as well as ceftriaxone added to control samples did not interfere with the LTA/TA assay. LTA/TA concentrations were measured in CSF samples taken 12, 14, 17, 20 and 24 h after infection.

Cytokine assay and quantification of neuronal damage

TNF activity in the CSF was measured using a cytolytic assay with actinomycin D-treated L929 cells. The CSF samples (in two different concentrations) were assayed in triplicate, and the standard curve samples in quintuplicate. NSE concentrations in the CSF were measured with an immunoluminometric assay (LIA-mat NSE Prolifigen, Byk-Sangtec, Dietzenbach, Germany). The addition of quinupristin/dalfopristin or ceftriaxone to selected samples did not influence the results of the NSE assay. In previous experiments, the density of apoptotic neurons in the gyrus dentatus of the hippocampal formation after 24 h of meningitis had been established as an early marker of neuronal damage. 22 To quantify neuronal apoptosis, the right hemisphere of the brain was removed immediately after killing of the rabbits; it was then fixed in 4% paraformaldehyde for 24 h. The fixed tissue was embedded in paraffin, and the dorsal hippocampal area was cut in 1 mm sections. The sections were mounted on poly-L-lysine-coated glass slides. Dewaxed sections were stained with haematoxylin and eosin (HE), and the DNA frag mentation was visualized by in-situ tailing to determine apoptotic cell death. 22 Deparaffinized and hydrated sections were treated with 5 mg/mL proteinase K (Sigma) for 15 min at 37°C. The sections were incubated for 1 h at 37°C in a reaction mixture containing 10 mL of 53 tailing buffer, 1 mL digoxigenin DNA labelling mix, 2 mL cobalt chloride, 12 U terminal transferase and the necessary amount of distilled water to give a volume of 50 mL. After washing the sections were incubated with 10% fetal calf serum (FCS) for 15 min at room temperature and then washed again. A solution of alkaline phosphatase-labelled anti-digoxigenin antibody in 10% FCS was placed on the sections for 60 min at 37°C. The colour reaction was developed with 4-nitroblue-tetrazolium-chloride/5-bromine-4-chloride-3-indolyl-phosphate.

The sections were counterstained with nuclear fast red– aluminium hydroxide. All reagents were purchased from Boehringer Mannheim, Germany. To determine the density of apoptotic neurons, HE-stained sections were used to measure the granular cell layer area with a Contron Videoplan computer (Grundig, Nürnberg, Germany). The density of apoptotic neurons was expressed as the number of marked neurons per mm2 of hippocampal granular cell layer.

Statistics

Data were expressed as medians and 25th/75th percentile or, when there was a Gaussian distribution, as means 6 S.D. An unpaired two-tailed U-test or, when the data were normally distributed, an unpaired t-test was used for comparisons between groups, and P< 0.05 was considered statistically significant.


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Antibiotic susceptibility

The MICs of quinupristin/dalfopristin were 0.25 mg/L for the penicillin-sensitive strains (n = 32) and 0.5 mg/L for the penicillin-intermediate strains (n = 31). Erythromycin-resistant strains (n = 30) also had a MIC of 0.5 mg/L. The S. pneumoniae type 3 strain used for the in-vivo experiments was susceptible to quinupristin/dalfopristin (MIC = 0.5 mg/L; MBC = 0.5 mg/L) and ceftriaxone (MIC = 0.03 mg/L; MBC = 0.06 mg/L).

In-vivo experiments

The antibiotic concentrations measured in serum and CSF during continuous infusion of quinupristin/ dalfopristin and ceftriaxone and short-duration infusion of quinupristin/dalfopristin are presented in Table I. Continuous infusion of quinupristin/dalfopristin reduced the CSF bacterial density less rapidly than ceftriaxone (from 7.7 ± 0.8 and 7.4 ± 0.6 log cfu/mL at 12 h to 6.3 ± 0.9 and 2.7 ± 0.9 log cfu/mL at 24 h, respectively; the change in log cfu/mL/h was – 0.11 ± 0.10 vs – 0.44 ± 0.12)(Figure 1). As a single dose of 100 mg/kg/h, quinupristin/ dalfopristin reduced the CSF bacterial density from 7.6 ± 0.9 log cfu/mL at 12 h to 6.2 ± 1.2 log cfu/mL at 17 h (i.e., 5 h after initiation of antibiotic therapy). Subsequently, the bacterial concentration increased again to 6.9 ± 1.6 log cfu/mL at 24 h ( Figure 1).


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Table I. Concentrations of quinupristin/dalfopristin and ceftriazone in serum and CSF (medians (25th/75th percentiles)) (ten animals per group)
 


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Figure 1. Antibacterial activity of quinupristin/dalfopristin ({blacksquare}, continuous infusion; {blacklozenge}, short duration infusion) and ceftriaxone (•) in the rabbit model of pneumococcal meningitis.

 
The median CSF leucocyte density at the time of initiation of antibiotic therapy in rabbits receiving quinupristin/dalfopristin was 753 (318/2965)/mL for the animals treated with a continuous infusion and 1101 (121/3802)/mL for those treated with a short duration infusion. For the animals receiving ceftriaxone, the median leucocyte density at initiation of antibiotic therapy was 237 (96/12453)/mL (differences not significant). With quinupristin/dalfopristin, maximum CSF leucocyte density was observed 24 h after infection with continuous infusion (11,040 (9653/16,298)/mL) and with the short duration infusion (11,813 (1929/14,959)/mL. During treatment with ceftriaxone, the maximum leucocyte density in the CSF was observed 17 h after infection (11,477 (4367/17,578)/mL), i.e. the maximum of inflammation was observed earlier with ceftriaxone than with quinupristin/ dalfopristin.

The CSF concentrations of LTA/TA were lower in the quinupristin/dalfopristin-treated animals than in the animals receiving ceftriaxone: the differences were significant at 14 h (medians: 133 (72/155) ng/mL (continuous infusion) versus 455 (274/2042) ng/mL (P = 0.002) and 193 (91/308) ng/mL (short duration infusion) versus 455 (274/2042) ng/mL (P = 0.02)) and 17 h (medians: 116 (60/368) ng/mL (continuous infusion) versus 694 (156/2173) ng/mL (P = 0.04) and 117 (41/247) ng/mL (short duration infusion) versus 694 (156/2173) ng/mL (P = 0.03)). Concentrations at 12 h, 20 h and 24 h were not significantly different(Figure 2).



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Figure 2. Median CSF concentrations of lipoteichoic and teichoic acids during treatment with quinupristin/dalfopristin as a continuous infusion ({blacksquare}) or short duration infusion ({blacklozenge}) and ceftriaxone (•). All samples were taken 12, 14, 17, 20 and 24 h after infection. {blacktriangleup} and {blacktriangledown} mark the 25th and 75th percentiles, respectively. *, P = 0.002 versus continuous infusion; P = 0.02 versus short duration infusion; **, P = 0.04 versus continuous infusion; P = 0.03 versus short duration infusion.

 
After initiation of antibiotic therapy the TNF CSF concentrations were lower in both quinupristin/ dalfopristin-treated groups than in the ceftriaxone-treated group. The difference was significant at 14 h (medians: 0.2 (0.1/1.9) U/mL versus 30 (4.6/180) U/mL (P = 0.02) and 0.1 (0.01/3.5) U/mL versus 30 (4.6/180) U/mL (P = 0.02)) and at 17 h (medians: 2.8 (0.2/11) U/mL versus 48.6 (18/168.8) U/mL (P = 0.002) and 0.1 (0.04/6.1) U/mL versus 48.6 (18/169) U/mL (P = 0.001)). At 20 h the TNF concentration was still significantly lower in animals treated with the bolus dose of quinupristin/dalfopristin (0.4 (0.04/4.1) U/mL versus 11.4 (5/39) U/mL (P = 0.01)). Twenty-four hours after infection no significant differences in TNF concentrations were observed (Figure 3).



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Figure 3. Median CSF concentrations of TNF during treatment with quinupristin/dalfopristin as a continuous ({blacksquare}) or short duration infusion ({blacklozenge}) and ceftriaxone (•). All samples were taken 12, 14, 17, 20 and 24 h after infection. {blacktriangleup} and {blacktriangledown} mark the 25th and 75th percentiles, respectively. *, P = 0.02; **, P = 0.002 versus continuous infusion; P = 0.001 versus short infusion.

 
CSF lactate and CSF protein concentrations 12 h and 24 h after infection were similar in the three treatment groups(Table II). The density of apoptotic neurons in the granular cell layer of the dentate gyrus after ceftriaxone and quinupristin/dalfopristin therapy was not significantly different (Table II).


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Table II. Concentrations (median (25th/75th percentile)) of lactate and protein in CSF, and the extent of neuronal damage 24 h after infection (mean ± SD) (ten animals per group)
 
The concentrations of NSE in the CSF 24 h after infection (12 h after initiation of antibiotic therapy) were significantly lower in quinupristin/dalfopristin-treated animals (Fig. 4Go).



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Figure 4. Neuron-specific enolase concentrations in the CSF 12 h after initiation of antibiotic therapy were significantly lower in quinupristin/dalfopristin-treated animals (with continuous (a) or short duration (b) infusion) than in ceftriaxone-treated animals. *, P = 0.03; **, P = 0.009.

 

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Although the amount of dalfopristin administered was approximately twice that of quinupristin, the dalfopristin serum concentrations were substantially lower than the corresponding quinupristin levels (Table I). This is in agreement with the results of Entenza et al., 16 who found that the half-life of dalfopristin was shorter than that of quinupristin in the rat (the former was undetectable 2 h after administration, while the latter was still detectable after 6 h). Since bioassays are of limited accuracy, we did not determine the elimination half-lives of quinupristin and dalfopristin. However, a rough estimate using the median serum concentrations after short-duration infusion yields a serum t1/2 of 3.45 h for quinupristin and of 1.22 h for dalfopristin. These half-lives were slightly longer than those measured in humans, rats and monkeys by HPLC. 20 Possibly, the bioassay recognized microbiologically active metabolites not detected by HPLC. Quinupristin/ dalfopristin was very active against S. pneumoniae in vitro, with respect to the MICs (present study) and to time– kill curves. 19 As observed by others, 17,23 the in-vitro activity of quinupristin/dalfopristin was not reduced in strains with a decreased sensitivity to penicillin and erythromycin.

Despite this excellent in-vitro activity, quinupristin/ dalfopristin as a short duration infusion over 1 h (100 mg/kg) or as a continuous infusion over 12 h (7.5 mg/kg) was only slowly bactericidal and significantly less active than ceftriaxone (10 mg/kg) in our study in vivo. Conversely, Tarasi et al. 18 reported higher bactericidal activity of quinupristin/dalfopristin in the CSF of rabbits with pneumococcal meningitis after iv infusion of a single 50 mg/kg dose (reduction by 2 log cfu in 2 h) or of two 50 mg/kg doses delivered at 2 h intervals (reduction by 3 log cfu in 4 h). The high activity of quinupristin/ dalfopristin was observed in the presence of intense meningeal inflammation, whereas it was substantially less active in the absence of strong meningeal inflammation. Antibiotic CSF concentrations were not reported, so the reason for these divergent observations remains unclear. 18 In the present study, antibacterial therapy was started early in the course of the illness, when the impairment of the blood– CSF barrier was moderate: in this way the immunomodulatory properties of antibiotics can best be studied. 10,11 The median quinupristin concentrations in CSF were 1.1 mg/L at 17 h and 1.3 mg/L at 20 h in rabbits treated with 7.5 mg/kg/h by continuous infusion. The dalfopristin CSF concentrations, however, were below the lower limit of quantification (0.5 mg/L) in six out of ten rabbits. Since antibiotic concentrations of at least ten times the MIC are usually required for rapid bacterial killing in CSF, 24 and since the dalfopristin concentrations were particularly low, concentrations in the present study apparently were not high enough to sterilize the CSF rapidly. To reach effective CSF concentrations in the presence of a moderately abnormal blood–CSF barrier, the dose of quinupristin/dalfopristin would need to be increased.

Recently, we observed that quinupristin/dalfopristin was as rapidly bactericidal as ß-lactam antibiotics against S. pneumoniae, but— like other antibacterials not directly affecting cell wall synthesis—released less LTA and TA in vitro. 19 This compares well with reports on Gram-negative bacteria, where the choice of antibiotic therapy influenced the time course and the absolute amount of endotoxin released.25,26,27 In the present study we demonstrated that the brisk release of proinflammatory cell wall components into the CSF was minimal during treatment with quinupristin/dalfopristin. The reduced release of bacterial cell wall components during continuous infusion or after bolus application of quinupristin/dalfopristin was the probable reason for the low TNF concentrations in the CSF after initiation of therapy. Both treatment groups receiving quinupristin/dalfopristin had significantly lower CSF TNF activities at 2 and 5 h after the start of antibiotic infusion than rabbits treated with ceftriaxone. This corresponded to a delayed increase of the median CSF leucocyte density in rabbits treated with quinupristin/ dalfopristin (the difference between these and ceftriaxone-treated rabbits, however, failed to reach statistical significance).

The CSF TNF activity and leucocyte density indicate an attenuated inflammatory response after initiation of antibiotic therapy with quinupristin/dalfopristin compared with ceftriaxone. This has also been demonstrated for rifabutin, which—like quinupristin/dalfopristin—exerts its bactericidal effect by interfering with bacterial protein synthesis. 11 Conversely, trovafloxacin only delayed but did not inhibit the increase of TNF activity in CSF occurring after initiation of antibiotic therapy. 10

Theoretically, the reason for the attenuated inflammatory response after initiation of antibiotic therapy with quinupristin/dalfopristin could be the low bactericidal activity in this model. A low antibacterial activity, however, often leads to an increased release of bacterial products, probably because bacterial multiplication is not terminated. This has been shown for the release of lipoteichoic acids in vitro and of endotoxin in E. coli meningitis in vivo: 6 h after initiation of antibacterial therapy, CSF endotoxin concentrations were higher following administration of the bacteriostatic compound chloramphenicol than after treatment with the bactericidal antibiotics cefotaxime, cefpirome, meropenem and gentamicin. 28 This suggests that the reduction in the release of proinflammatory cell wall components is a feature of quinupristin/dalfopristin and is not necessarily observed with other antibiotics unable to sterilize the CSF rapidly.

In the present study, we were able to show for the first time that the choice of antibacterial therapy had an impact on a marker of neuronal destruction. The CSF concentrations of NSE, which were correlated with clinical outcome in children, 29 were significantly lower in animals treated with quinupristin/dalfopristin than in those receiving ceftriaxone. This gives rise to optimism that an attenuated inflammatory response in the subarachnoid space may reduce the extent of neurological sequelae or even the mortality from bacterial meningitis. The density of neuronal apoptoses in the granular cell layer of the dentate gyrus, however, was not significantly lower in animals treated with quinupristin/dalfopristin than in those treated with ceftriaxone.

In conclusion, at the doses and the state of the blood–CSF barrier investigated, the in-vivo activity of quinupristin/dalfopristin was moderate compared with that of ceftriaxone. However, antibiotic treatment with quinupristin/dalfopristin attenuated the inflammatory response within the subarachnoid space occuring after initiation of therapy. Consistently, the NSE CSF concentration, a marker of neuronal damage, was significantly lower in quinupristin/dalfopristin-treated rabbits than in ceftriaxone-treated rabbits. The clinical use of quinupristin/dalfopristin in meningitis critically depends on whether higher serum and CSF concentrations than those observed in this study are safely tolerated in humans.


    Acknowledgments
 
The animal experiments were approved by the Animal Ethics Committee of the Government of Lower Saxony. The study was supported by grants from Rhône-Poulenc Rorer and Deutsche Forschungsgemeinschaft (Na 165/2-2). These data were presented, in part, at the Thirty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 28 September–1 October, 1997.


    Notes
 
* Corresponding author. Department of Neurology, University of Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany. Tel: +49-551-398455 or 396684; Fax: +49-551-398405; E-mail: rnau{at}gwdg.de Back


    References
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
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Received 12 February 1998; returned 7 May 1998; revised 26 June 1998; accepted 27 July 1998