Kristin Stuertz

Organotypic hippocampal cultures. A model of brain tissue damage in Streptococcus pneumoniae meningitis.

Hippocampal slices of newborn rats were exposed to either heat-inactivated Streptococcus pneumoniae R6 (hiR6) equivalent to 10(6) and 10(8) CFU/ml, lipoteichoic acid (LTA) (0.3 microg/ml and 30 microg/ml), peptidoglycans (PG) (0.3, 30, 50 and 100 microg/ml), pneumococcal DNA (pDNA) (0.3 and 30 microg/ml) or medium only (control). Cell injury was examined by Nissl staining, Annexin V and NeuN immunohistochemistry, and quantified by propidium iodide (PI) uptake and by determining neuron-specific enolase (NSE) concentration in the culture medium. Necrotic and apoptotic cell damage occurred in all treatment groups. Overall damage (Nissl and PI staining) was most prominent after hiR6 (10(8) CFU/ml), followed by LTA (30 microg/ml), pDNA (30 microg/ml), and not detectable after PG (30 microg/ml) exposure. PG (100 microg/ml) induced severe damage. Apoptotic cells were most frequent after exposure to LTA and hiR6. Damage in the neuronal cell layers (NeuN, NSE) was most severe after treatment with hiR6 (10(8) CFU/ml), followed by PG (100 microg/ml), pDNA (30 microg/ml), and LTA (30 microg/ml).

Lower Lipoteichoic and Teichoic Acid CSF Concentrations During Treatment of Pneumococcal Meningitis with Non-bacteriolytic Antibiotics than with Ceftriaxone

In the rabbit model of Streptococcus pneumoniae meningitis, treatment with rifabutin, quinupristin-dalfopristin, moxifloxacin and trovafloxacin led to smaller increases of the CSF concentrations of the pro-inflammatory cell wall components lipoteichoic and teichoic acids (LTA and TA) than did treatment with ceftriaxone. Low doses of moxifloxacin were associated with higher LTA and TA concentrations in CSF than were high doses.

Rifabutin for Experimental Pneumococcal Meningitis

Rifabutin is a lipophilic antibacterial with high in vitro activity against many pathogens involved in bacterial meningitis including pneumococci. Resistance to beta-lactam antibiotics in pneumococci is not associated with a decreased sensitivity to rifabutin (30 strains from Germany with intermediate penicillin resistance; MIC range of penicillin: 0.125-1 mg/l, MIC of rifabutin: < 0.008-0.015 mg/l). Rifabutin at doses of 0.625, 1.25, 2.5, 5 and 10 mg/kg/h i.v. was investigated in a rabbit model of meningitis using a Streptococcus pneumoniae type 3 (MIC/MBC of rifabutin: 0.015/0.06 mg/l). The bacterial density in CSF at the onset of treatment was 7.3 +/- 0.6 log CFU/ml (mean +/- SD). Rifabutin decreased bacterial CSF titers in a dose-dependent manner [delta log CFU/ml/h (slope of the regression line log CFU/ml vs. time) at a dose of 0.625 mg/kg/h: -0.16 +/- 0.06 (n = 3), at 1.25 mg/kg/h: -0.20 +/- 0.12 (n = 4), at 2.5 mg/kg/h: -0.24 +/- 0.04 (n = 4), at 5 mg/kg/h: -0.31 +/- 0.10 (n = 8), and at 10 mg/kg/h: -0.29 +/- 0.10 (n = 5)]. At high doses rifabutin was as active as ceftriaxone at 10 mg/kg/h (delta log CFU/ml/h: -0.29 +/- 0.10, n = 10). Two and 5 h after initiation of therapy, CSF TNF-alpha activities were lower with rifabutin 5 mg/kg/h than with ceftriaxone (medians 2 vs. 141 U/ml, p = 0.005 at 2 h; median 51 vs. 120 U/ml 5 h after initiation of therapy, p = 0.04). This did not result, however, in a decrease of indicators of neuronal damage. In conclusion, intravenous rifabutin was bactericidal in experimental pneumococcal meningitis. Provided that a well-tolerated i.v. formulation will be available it may qualify as a reserve antibiotic for pneumococcal meningitis, in particular when strains with a reduced sensitivity to beta-lactam antibiotics are the causative pathogens.

Reduction of meningeal macrophages does not decrease migration of granulocytes into the CSF and brain parenchyma in experimental pneumococcal meningitis

Leukocyte infiltration of the CSF and brain parenchyma and other parameters of inflammation during pneumococcal meningitis were investigated after reduction of meningeal macrophages in rabbits by intracisternal injection of dichloromethylene-diphosphonate (Cl2MDP)-containing liposomes. Macrophages in the meninges were reduced, in median, by approximately 77% after three intrathecal injections of 100 microl of liposomes containing Cl2MDP at 12 h intervals. Production of the cytokines interleukin-1 and tumor necrosis factor-alpha as well as infiltration of the CSF and nervous tissue by leukocytes was not significantly altered in infected animals after treatment with Cl2MDP-containing liposomes. The median CSF concentration of neuron specific enolase (NSE) as a parameter of neuronal damage was higher in infected Cl2MDP-treated animals (median [median (25th/75th percentiles): 44.7 (33.2/54.3) microg/l vs. 13.9 (10.4/23.9) microg/l; P = 0.01]). Therefore, the reduction of meningeal macrophages does not appear to attenuate inflammation in the subarachnoid space in experimental pneumococcal meningitis. Meningeal macrophages seem, however, to be important for the protection of neuronal tissue in bacterial meningitis.

Glycerol does not reduce neuronal damage in experimental Streptococcus pneumoniae meningitis in rabbits.

To study the effect of high-dose glycerol therapy on inflammation and neuronal destruction in a model of experimental pneumococcal meningitis, 14 New Zealand White rabbits were infected intracisternally with Streptococcus pneumoniae type 3. Sixteen hours after infection, 7 animals received intravenous glycerol therapy (1 g kg−1 bolus and 0.5 g kg 1h−1 maintenance dose) and 7 animals served as untreated controls.After 8 h of therapy, the glycerol CSF:serum ratio exceeded the previously observed values in rabbits with an intact blood-CSF barrier (0.72±0.25 vs. 0.35), i.e. glycerol crossed the blood-CSF barrier more readily in animals altered by meningitis than in healthy animals. In contrast, the brain tissue:serum ratio of glycerol (grey matter 0.33±0.29, white matter 0.30±0.31) was substantially lower than the CSF:serum ratio (p=0.03 and p=0.047). There was no significant effect of glycerol on intracranial pressure, brain water content and neuron-specific enolase release into the CSF. Glycerol significantly increased the density of neuronal apoptosis in the dentate gyrus of the hippocampal formation. Therefore, glycerol does not appear to be beneficial in experimental pneumococcal meningitis.

Release of Teichoic and Lipoteichoic Acids from 30 Different Strains of Streptococcus pneumoniae during Exposure to Ceftriaxone, Meropenem, Quinupristin/Dalfopristin, Rifampicin and Trovafloxacin

SummaryThe release of teichoic acids (TA) and lipoteichoic acids (LTA) from 30 different strains of Streptococcus pneumoniae during exposure to ceftriaxone, meropenem, quinupristin/dalfopristin, rifampicin and trovafloxacin at concentrations of 10 μg/ml and of the respective MIC was determined by an enzyme immunoassay. At 10 μg/ml the most rapid and intense release was detected during treatment with the β-lactam antibiotics ceftriaxone and meropenem, the lowest release was seen with rifampicin and quinupristin/dalfopristin. Trovafloxacin delayed the release of TA/LTA. The maximum concentrations of TA/LTA, however, during trovafloxacin treatment were almost as high as those during exposure to ceftriaxone and meropenem. During exposure to the MIC, ceftriaxone, meropenem, rifampicin and trovafloxacin released significantly higher amounts of TA/LTA than during exposure to 10 μg/ml (p < 0.01). Only quinupristin/dalfopristin released small amounts of TA/LTA at the low and high concentration. In conclusion, at high concentrations antibiotics that do not affect the bacterial cell wall released less pro-inflammatory compounds from S. pneumoniae than ceftriaxone and meropenem. This may be of value in the treatment of meningitis and sepsis.

S100B in the cerebrospinal fluid--a marker for glial damage in the rabbit model of pneumococcal meningitis.

The rabbit model provides an important experimental setting for the evaluation of antibiotic agents against pneumococcal meningitis. One of the primary targets of this model is the study of neuronal and glial cell damage in bacterial meningitis. The aim of this investigation was to evaluate whether a significant increase of S100B in the cerebrospinal fluid (CSF) as an indicator of white matter damage could be observed in this meningitis model. Seven rabbits were infected intracisternally with S. pneumoniae, and CSF S100B concentrations were examined serially before infection, at 12h, 14h, 17h, 20h, and at 24h after infection. The course of CSF S100B increase and its relation to other parameters of brain tissue destruction and CSF inflammation were measured. Axonal damage was visualized by amyloid precursor protein (APP) immunostaining and demyelination by Luxol Fast Blue/Periodic Acid Schiff (LFB-PAS) stain. In each animal, we observed a distinct rise in S100B concentration in the CSF due to pneumococcal meningitis. We conclude that the CSF concentration of the glial S100B protein can be used as an additional parameter for future interventional studies focusing on glial cell damage in the rabbit model of bacterial meningitis.

Intravenous granulocyte colony-stimulating factor increases the release of tumour necrosis factor and interleukin-1beta into the cerebrospinal fluid, but does not inhibit the growth of Streptococcus pneumoniae in experimental meningitis.

Granulocyte colony‐stimulating factor (G‐CSF) possesses an antimicrobial effect in several animal models of infection. To evaluate a possible effect of G‐CSF on the course of pneumococcal meningitis, rabbits infected intracisternally (i.c.) with Streptococcus pneumoniae type 3 (n = 7) received 50 μg/kg of rhG‐CSF intravenously (i.v.) 1 h prior to infection. Seven infected animals served as controls. Uninfected rabbits received 10 μg of G‐CSF (n = 3), 2 μg G‐CSF (n = 3) or saline (n = 3) i.c. G‐CSF injected i.c. did not produce cerebrospinal fluid (CSF) leucocytosis. Compared with the control group, i.v. G‐CSF given prior to i.c. infection increased the percentage of granulocytes in blood 6 h and 12 h after infection. Twelve hours after infection, CSF tumour necrosis factor (TNF) activity and CSF interleukin (IL)‐1β concentrations were significantly higher in G‐CSF‐treated animals. G‐CSF did not decrease bacterial growth in the subarachnoid space and the CSF leucocyte densities were not influenced. At 24 h after infection, G‐CSF did not reduce the CSF concentrations of neurone‐specific enolase and the density of apoptotic neurones in the dentate gyrus of the hippocampus. In conclusion, i.v. G‐CSF increased the concentration of pro‐inflammatory cytokines in the CSF but did not decrease the growth of Streptococcus pneumoniae in the subarachnoid space.

Streptococcal meningitis: effect of CSF filtration on inflammation and neuronal damage

Abstract The effect of CSF filtration on inflammation and neuronal damage was studied in experimental Streptococcus pneumoniae meningitis. New Zealand white rabbits received either antibiotic therapy alone (ceftriaxone i.v., 20 mg/kg bolus, 10 mg/kg maintenance dose; n = 10) or ceftriaxone plus CSF filtration (n = 11) 12 h after intracisternal infection. Immediately after the onset of antibiotic therapy 300 μl cisternal CSF was removed, passed through a miniaturized CSF-1 filter at a constant flow of 20 μl/min, and then reinjected. This procedure was repeated six times at intervals of 20 min. Antibiosis plus CSF filtration caused a transient reduction in CSF bacterial titers and leukocyte counts compared with antibiosis alone (P = 0.04 and 0.02 5 h after initiation of therapy). CSF lipoteichoic acid concentrations were not reduced. The concentration of neuron-specific enolase in CSF and the density of apoptotic neurons in the dentate gyrus were almost equal 12 h after the onset of treatment. Adjuvant CSF filtration accelerated the elimination of viable bacteria from CSF in comparison to antibiotic treatment alone. Parameters of neuronal destruction, however, were not reduced.