Prevention of brain injury by the nonbacteriolytic antibiotic daptomycin in experimental pneumococcal meningitis

Bacteriolytic antibiotics cause the release of bacterial components that augment the host inflammatory response, which in turn contributes to the pathophysiology of brain injury in bacterial meningitis. In the present study, antibiotic therapy with nonbacteriolytic daptomycin was compared with that of bacteriolytic ceftriaxone in experimental pneumococcal meningitis, and the treatments were evaluated for their effects on inflammation and brain injury. Eleven-day-old rats were injected intracisternally with 1.3 x 10(4) +/- 0.5 x 10(4) CFU of Streptococcus pneumoniae serotype 3 and randomized to therapy with ceftriaxone (100 mg/kg of body weight subcutaneously [s.c.]; n = 55) or daptomycin (50 mg/kg s.c.; n = 56) starting at 18 h after infection. The cerebrospinal fluid (CSF) was assessed for bacterial counts, matrix metalloproteinase-9 levels, and tumor necrosis factor alpha levels at different time intervals after infection. Cortical brain damage was evaluated at 40 h after infection. Daptomycin cleared the bacteria more efficiently from the CSF than ceftriaxone within 2 h after the initiation of therapy (log(10) 3.6 +/- 1.0 and log(10) 6.3 +/- 1.4 CFU/ml, respectively; P < 0.02); reduced the inflammatory host reaction, as assessed by the matrix metalloproteinase-9 concentration in CSF 40 h after infection (P < 0.005); and prevented the development of cortical injury (cortical injury present in 0/30 and 7/28 animals, respectively; P < 0.004). Compared to ceftriaxone, daptomycin cleared the bacteria from the CSF more rapidly and caused less CSF inflammation. This combined effect provides an explanation for the observation that daptomycin prevented the development of cortical brain injury in experimental pneumococcal meningitis. Further research is needed to investigate whether nonbacteriolytic antibiotic therapy with daptomycin represents an advantageous alternative over current bacteriolytic antibiotic therapies for the treatment of pneumococcal meningitis.

Pneumococcal meningitis induces apoptosis in recently postmitotic immature neurons in the dentate gyrus of neonatal rats

Bacterial meningitis is associated with high rates of morbidity and mortality, despite advances in antibiotic therapy. Meningitis caused by Streptococcus pneumoniae is associated with a particularly high incidence of neurological sequelae including deficits resulting from damage to the hippocampus. Previous studies have documented that in neonatal rats with experimental pneumococcal meningitis, cells in the subgranular layer of the dentate gyrus undergo apoptosis. The aim of the present study was to define in more detail the nature of the dying cells in the dentate gyrus. Using bromodeoxyuridine labeling at different times before infection combined with immunocytochemistry, we identified the vulnerable cells as those which underwent mitosis 6-10 days before infection. A majority of these cells are of neuronal lineage. Thus, immature neuronal cells several days after the last cell division are preferentially triggered into apoptosis during pneumococcal meningitis. The loss of these cells may contribute to the long-lasting impairment of hippocampal function identified in animal models and in humans after bacterial meningitis.

Essential role of choline for pneumococcal virulence in an experimental model of meningitis

Objectives: The goal of the present study was to elucidate the contribution of the newly recognized virulence factor choline to the pathogenesis of Streptococcus pneumoniae in an animal model of meningitis. Results: The choline containing strain D39Cho(-) and its isogenic choline-free derivative D39Cho(-)licA64 -each expressing the capsule polysaccharide 2 - were introduced intracisternally at an inoculum size of 10(3) CFU into 11 days old Wistar rats. During the first 8 h post infection both strains multiplied and stimulated a similar immune response that involved expression of high levels of proinflammatory cytokines, the matrix metalloproteinase 9 (MMP-9), IL-10, and the influx of white blood cells into the CSF. Virtually identical immune response was also elicited by intracisternal inoculation of 10(7) CFU equivalents of either choline-containing or choline-free cell walls. At sampling times past 8 h strain D39Cho(-) continued to replicate accompanied by an intense inflammatory response and strong granulocytic pleiocytosis. Animals infected with D39Cho(-) died within 20 h and histopathology revealed brain damage in the cerebral cortex and hippocampus. In contrast, the initial immune response generated by the choline-free strain D39Cho(-)licA64 began to decline after the first 8 h accompanied by elimination of the bacteria from the CSF in parallel with a strong WBC response peaking at 8 h after infection. All animals survived and there was no evidence for brain damage. Conclusion: Choline in the cell wall is essential for pneumococci to remain highly virulent and survive within the host and establish pneumococcal meningitis.

Expression of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta in the brain during experimental group B streptococcal meningitis

We performed mRNA in situ hybridization for TNF-alpha and IL-1beta from infant rats with group B streptococcal meningitis. Induction of both cytokines was seen in the ependyma and the meninges at 4 h. Both cytokines were expressed in the brain parenchyma at 12 h. Induction of IL-1beta mRNA was seen in vessels within the brain cortex. Neutrophilic infiltrate at all time points examined was minimal and could not account for the observed cytokine expression.

New therapies for pneumococcal meningitis

The treatment of pneumococcal meningitis remains a major challenge, as reflected by the continued high morbidity and case fatality of the disease. The worldwide increase of penicillin-resistant pneumococci and more recently cephalosporin- and vancomycin-tolerant pneumococci has jeopardised the efficacy of standard treatments based on extended spectrum cephalosporins alone or in combination with vancomycin. This review provides a summary of newly developed antibiotics tested in the rabbit meningitis model. In particular, newer beta-lactam monotherapies (cefepime, meropenem, ertapenem), recently developed quinolones (garenoxacin, gemifloxacin, gatifloxacin, moxifloxacin) and a lipopeptide antibiotic (daptomycin) are discussed. A special emphasis is placed on the potential role of combination treatments with some of the new compounds, which are of interest based on the background of increasing resistance problems due to their often synergistic activity in the rabbit model of pneumococcal meningitis.

Blockade of NMDA receptor subtype NR2B prevents seizures but not apoptosis of dentate gyrus neurons in bacterial meningitis in infant rats

BACKGROUND: Excitotoxic neuronal injury by action of the glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype have been implicated in the pathogenesis of brain damage as a consequence of bacterial meningitis. The most potent and selective blocker of NMDA receptors containing the NR2B subunit is (R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol (RO 25-6981). Here we evaluated the effect of RO 25-6981 on hippocampal neuronal apoptosis in an infant rat model of meningitis due to Streptococcus pneumoniae. Animals were randomized for treatment with RO 25-6981 at a dosage of either 0.375 mg (15 mg/kg; n = 28) or 3.75 mg (150 mg/kg; n = 15) every 3 h or an equal volume of sterile saline (250 microl; n = 40) starting at 12 h after infection. Eighteen hours after infection, animals were assessed clinically and seizures were observed for a period of 2 h. At 24 h after infection animals were sacrificed and brains were examined for apoptotic injury to the dentate granule cell layer of the hippocampus. RESULTS: Treatment with RO 25-6981 had no effect on clinical scores, but the incidence of seizures was reduced (P < 0.05 for all RO 25-6981 treated animals combined). The extent of apoptosis was not affected by low or high doses of RO 25-6981. Number of apoptotic cells (median [range]) was 12.76 [3.16-25.3] in animals treated with low dose RO 25-6981 (control animals 13.8 [2.60-31.8]; (P = NS) and 9.8 [1.7-27.3] (controls: 10.5 [2.4-21.75]) in animals treated with high dose RO 25-6981 (P = NS). CONCLUSIONS: Treatment with a highly selective blocker of NMDA receptors containing the NR2B subunit failed to protect hippocampal neurons from injury in this model of pneumococcal meningitis, while it had some beneficial effect on the incidence of seizures.

The stereochemistry of the amino acid side chain influences the inflammatory potential of muramyl dipeptide in experimental meningitis

Intrathecal injections of 50 to 100 micro g of (N-acetylmuramyl-L-alanyl-D-isoglutamine) muramyl dipeptide (MDP)/rabbit dose-dependently triggered tumor necrosis factor alpha (TNF-alpha) secretion (12 to 40,000 pg/ml) preceding the influx of leukocytes in the subarachnoid space of rabbits. Intrathecal instillation of heat-killed unencapsulated R6 pneumococci produced a comparable leukocyte influx but only a minimal level of preceding TNF-alpha secretion. The stereochemistry of the first amino acid (L-alanine) of the MDP played a crucial role with regard to its inflammatory potential. Isomers harboring D-alanine in first position did not induce TNF-alpha secretion and influx of leukocytes. This stereospecificity of MDPs was also confirmed by measuring TNF-alpha release from human peripheral mononuclear blood cells stimulated in vitro. These data show that the inflammatory potential of MDPs depends on the stereochemistry of the first amino acid of the peptide side chain and suggest that intact pneumococci and MDPs induce inflammation by different pathways.

Activities of ertapenem, a new long-acting carbapenem, against penicillin-sensitive or -resistant pneumococci in experimental meningitis

The penetration of ertapenem, a new carbapenem with a long half-life, reached 7.1 and 2.4% into inflamed and noninflamed meninges, respectively. Ertapenem had excellent antibacterial activity in the treatment of experimental meningitis due to penicillin-sensitive and -resistant pneumococci, leading to a decrease of 0.69 +/- 0.17 and 0.59 +/- 0.22 log(10) CFU/ml x h, respectively, in the viable cell counts in the cerebrospinal fluid. The efficacy of ertapenem was comparable to that of standard regimens (ceftriaxone monotherapy against the penicillin-sensitive strain and ceftriaxone combined with vancomycin against the penicillin-resistant strain). In vitro, ertapenem in concentrations above the MIC was highly bactericidal against both strains. Even against a penicillin- and quinolone-resistant mutant, ertapenem had similar bactericidal activity in vitro.

Modeling of transfer kinetics at the serum-cerebrospinal fluid barrier in rabbits with experimental meningitis: application to grepafloxacin

The goals of the present study were to model the population kinetics of in vivo influx and efflux processes of grepafloxacin at the serum-cerebrospinal fluid (CSF) barrier and to propose a simulation-based approach to optimize the design of dose-finding trials in the meningitis rabbit model. Twenty-nine rabbits with pneumococcal meningitis receiving grepafloxacin at 15 mg/kg of body weight (intravenous administration at 0 h), 30 mg/kg (at 0 h), or 50 mg/kg twice (at 0 and 4 h) were studied. A three-compartment population pharmacokinetic model was fit to the data with the program NONMEM (Nonlinear Mixed Effects Modeling). Passive diffusion clearance (CL(diff)) and active efflux clearance (CL(active)) are transfer kinetic modeling parameters. Influx clearance is assumed to be equal to CL(diff), and efflux clearance is the sum of CL(diff), CL(active), and bulk flow clearance (CL(bulk)). The average influx clearance for the population was 0.0055 ml/min (interindividual variability, 17%). Passive diffusion clearance was greater in rabbits receiving grepafloxacin at 15 mg/kg than in those treated with higher doses (0.0088 versus 0.0034 ml/min). Assuming a CL(bulk) of 0.01 ml/min, CL(active) was estimated to be 0.017 ml/min (11%), and clearance by total efflux was estimated to be 0.032 ml/min. The population kinetic model allows not only to quantify in vivo efflux and influx mechanisms at the serum-CSF barrier but also to analyze the effects of different dose regimens on transfer kinetic parameters in the rabbit meningitis model. The modeling-based approach also provides a tool for the simulation and prediction of various outcomes in which researchers might be interested, which is of great potential in designing dose-finding trials.

Gemifloxacin is efficacious against penicillin-resistant and quinolone-resistant pneumococci in experimental meningitis

In experimental rabbit meningitis, gemifloxacin penetrated inflamed meninges well (22 to 33%) and produced excellent bactericidal activity (change in log(10) [Deltalog(10)] CFU/ml/h, -0.68 +/- 0.30 [mean and standard deviation]), even superior to that of the standard regimen of ceftriaxone plus vancomycin (-0.49 +/- 0.09 deltalog(10) CFU/ml/h), in the treatment of meningitis due to a penicillin-resistant pneumococcal strain (MIC, 4 mg/liter). Even against a penicillin- and quinolone-resistant strain, gemifloxacin showed good bactericidal activity (-0.48 +/- 0.16 deltalog(10) CFU/ml/h). The excellent antibacterial activity of gemifloxacin was also confirmed by time-kill assays over 8 h in vitro.

Cefepime is efficacious against penicillin- and quinolone-resistant pneumococci in experimental meningitis

In experimental rabbit meningitis, cefepime given at a dose of 100 mg/kg was associated with concentrations in the cerebrospinal fluid of between 5.3 and 10 mg/L and a bactericidal activity of -0.61 +/- 0.24 Delta log(10) cfu/mL x h, similar to the standard regimen of ceftriaxone combined with vancomycin (-0.58 +/- 0.14 Delta log(10) cfu/mL x h) in the treatment of meningitis due to a penicillin- and quinolone-resistant pneumococcal mutant strain (MIC 4 mg/L). Compared with the penicillin-resistant parental strain, the penicillin- and quinolone-resistant mutant was killed more slowly by cefepime and ceftriaxone in time-killing assays in vitro over 8 h.

Gentamicin increases the efficacy of vancomycin against penicillin-resistant pneumococci in the rabbit meningitis model

In experimental meningitis a single dose of gentamicin (10 mg/kg of body weight) led to gentamicin levels in around cerebrospinal fluid (CSF) of 4 mg/liter for 4 h, decreasing slowly to 2 mg/liter 4 h later. The CSF penetration of gentamicin ranged around 27%, calculated by comparison of areas under the curve (AUC in serum/AUC in CSF). Gentamicin monotherapy (-1.24 log(10) CFU/ml) was inferior to vancomycin monotherapy (-2.54 log(10) CFU/ml) over 8 h against penicillin-resistant pneumococci. However, the combination of vancomycin with gentamicin was significantly superior (-4.48 log(10) CFU/ml) compared to either monotherapy alone. The synergistic activity of vancomycin combined with gentamicin was also demonstrated in vitro in time-kill assays.

Efficacies of BMS 284756 against penicillin-sensitive, penicillin-resistant, and quinolone-resistant pneumococci in experimental meningitis

BMS 284756 penetrated well into inflamed meninges (44% +/- 11%) and produced good bactericidal activity (-0.82 +/- 0.22 Delta log(10) CFU/ml. h) in the treatment of experimental meningitis in rabbits due to a penicillin-sensitive strain. BMS 284756 monotherapy had a greater potency than the standard regimen of ceftriaxone and vancomycin (-0.49 +/- 0.08 Delta log(10) CFU/ml. h) against a penicillin-resistant strain (MIC, 4 mg/liter). Even against a penicillin- and quinolone-resistant strain, BMS 284756 showed good bactericidal activity (-0.52 +/- 0.12 Delta log(10) CFU/ml. h). The antibacterial activity of BMS 284756 was confirmed by time-killing assays over 8 h in vitro.

Inhibition of matrix metalloproteinases and tumour necrosis factor alpha converting enzyme as adjuvant therapy in pneumococcal meningitis

Matrix metalloproteinases (MMPs) and tumour necrosis factor alpha (TNF-alpha) converting enzyme (TACE) contribute synergistically to the pathophysiology of bacterial meningitis. TACE proteolytically releases several cell-surface proteins, including the proinflammatory cytokine TNF-alpha and its receptors. TNF-alpha in turn stimulates cells to produce active MMPs, which facilitate leucocyte extravasation and brain oedema by degradation of extracellular matrix components. In the present time-course studies of pneumococcal meningitis in infant rats, MMP-8 and -9 were 100- to 1000-fold transcriptionally upregulated, both in CSF cells and in brain tissue. Concentrations of TNF-alpha and MMP-9 in CSF peaked 12 h after infection and were closely correlated. Treatment with BB-1101 (15 mg/kg subcutaneously, twice daily), a hydroxamic acid-based inhibitor of MMP and TACE, downregulated the CSF concentration of TNF-alpha and decreased the incidences of seizures and mortality. Therapy with BB-1101, together with antibiotics, attenuated neuronal necrosis in the cortex and apoptosis in the hippocampus when given as a pretreatment at the time of infection and also when administration was started 18 h after infection. Functionally, the neuroprotective effect of BB-1101 preserved learning performance of rats assessed 3 weeks after the disease had been cured. Thus, combined inhibition of MMP and TACE offers a novel therapeutic strategy to prevent brain injury and neurological sequelae in bacterial meningitis.