Daptomycin produces an enhanced bactericidal activity compared to ceftriaxone, measured by [3H]choline release in the cerebrospinal fluid, in experimental meningitis due to a penicillin-resistant pneumococcal strain without lysing its cell wall

Daptomycin monotherapy was superior to ceftriaxone monotherapy and was highly efficacious in experimental pneumococcal meningitis, sterilizing the cerebrospinal fluid (CSF) of three of three rabbits after 4 to 6 h. With daptomycin therapy only a negligible release of [(3)H]choline as marker of cell wall lysis was detectable in the CSF, peaking around 250 cpm/min after 4 h, compared to a peak of around 2,400 cpm/min after 4 to 6 h for the ceftriaxone-treated rabbits.

Clindamycin is neuroprotective in experimental Streptococcus pneumoniae meningitis compared with ceftriaxone

In animal models of Streptococcus pneumoniae meningitis, rifampin is neuroprotective in comparison to ceftriaxone. So far it is not clear whether this can be generalized for other protein synthesis-inhibiting antimicrobial agents. We examined the effects of the bactericidal protein synthesis-inhibiting clindamycin (n = 12) on the release of proinflammatory bacterial components, the formation of neurotoxic compounds and neuronal injury compared with the standard therapy with ceftriaxone (n = 12) in a rabbit model of pneumococcal meningitis. Analysis of the CSF and histological evaluation were combined with microdialysis from the hippocampal formation and the neocortex. Compared with ceftriaxone, clindamycin reduced the release of lipoteichoic acids from the bacteria (p = 0.004) into the CSF and the CSF leucocyte count (p = 0.011). This led to lower extracellular concentrations of hydroxyl radicals (p = 0.034) and glutamate (p = 0.016) in the hippocampal formation and a subsequent reduction of extracellular glycerol levels (p = 0.018) and neuronal apoptosis in the dentate gyrus (p = 0.008). The present data document beneficial effects of clindamycin compared with ceftriaxone on various parameters linked with the pathophysiology of pneumococcal meningitis and development of neuronal injury. This study suggests neuroprotection to be a group effect of bactericidal protein synthesis-inhibiting antimicrobial agents compared with the standard therapy with beta-lactam antibiotics in meningitis.

Matrix metalloproteinase-9 in pneumococcal meningitis: activation via an oxidative pathway

In experimental bacterial meningitis, matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) contribute to brain damage. MMP-9 increases in cerebrospinal fluid (CSF) during bacterial meningitis and is associated with the brain damage that is a consequence of the disease. This study assesses the origin of MMP-9 in bacterial meningitis and how ROS modulate its activity. Rat brain-slice cultures and rat polymorphonuclear cells (PMNs) that had been challenged with capsule-deficient heat-inactivated Streptococcus pneumoniae R6 (hiR6) released MMP-9. Coincubation with either catalase, with the myeloperoxidase inhibitor azide, or with the hypochlorous acid scavenger methionine almost completely prevented activation, but not the release, of MMP-9, in supernatants of human PMNs stimulated with hiR6. Thus, in bacterial meningitis, both brain-resident cells and invading PMNs may act as sources of MMP-9, and stimulated PMNs may activate MMP-9 via an ROS-dependent pathway. MMP-9 activation by ROS may represent a target for therapeutic intervention in bacterial meningitis.

Cerebral vasculature is the major target of oxidative protein alterations in bacterial meningitis

We have previously shown that antioxidants such as a-phenyl-tert-butyl nitrone or N-acetylcysteine attenuate cortical neuronal injury in infant rats with bacterial meningitis, suggesting that oxidative alterations play an important role in this disease. However, the precise mechanism(s) by which antioxidants inhibit this injury remain(s) unclear. We therefore studied the extent and location of protein oxidation in the brain using various biochemical and immunochemical methods. In cortical parenchyma, a trend for increased protein carbonyls was not evident until 21 hours after infection and the activity of glutamine synthetase (another index of protein oxidation) remained unchanged. Consistent with these results, there was no evidence for oxidative alterations in the cortex by various immunohistochemical methods even in cortical lesions. In contrast, there was a marked increase in carbonyls, 4-hydroxynonenal protein adducts and manganese superoxide dismutase in the cerebral vasculature. Elevated lipid peroxidation was also observed in cerebrospinal fluid and occasionally in the hippocampus. All of these oxidative alterations were inhibited by treatment of infected animals with N-acetylcysteine or alpha-phenyl-tert-butyl nitrone. Because N-acetylcysteine does not readily cross the blood-brain barrier and has no effect on the loss of endogenous brain antioxidants, its neuroprotective effect is likely based on extraparenchymal action such as inhibition of vascular oxidative alterations.

Current concepts in the pathogenesis of meningitis caused by Streptococcus pneumoniae

In spite of improved antimicrobial therapy, bacterial meningitis still results in brain damage leading to significant long-term neurological sequelae in a substantial number of survivors, as confirmed by several recent studies. Meningitis caused by Streptococcus pneumoniae is associated with a particularly severe outcome. Experimental studies over the past few years have increased our understanding of the molecular mechanisms underlying the events that ultimately lead to brain damage during meningitis. Necrotic damage to the cerebral cortex is at least partly mediated by ischemia and oxygen radicals and therefore offers a promising target for adjunctive therapeutic intervention. Neuronal apoptosis in the hippocampus may represent the major pathological process responsible for cognitive impairment and learning disabilities in survivors. However, the mechanisms involved in causing this damage remain largely unknown. Anti-inflammatory treatment with corticosteroids aggravates hippocampal damage, thus underlining the potential shortcomings of current adjuvant strategies. In contrast, the combined inhibition of matrix metalloproteinase and tumour necrosis factor-alpha converting enzyme protected both the cortex and hippocampus in experimental meningitis, and may represent a promising new approach to adjunctive therapy. It is the hope that a more refined molecular understanding of the pathogenesis of brain damage during bacterial meningitis will lead to new adjunctive therapies.

Oxidative stress in brain during experimental bacterial meningitis: differential effects of alpha-phenyl-tert-butyl nitrone and N-acetylcysteine treatment

Antioxidant treatment has previously been shown to be neuroprotective in experimental bacterial meningitis. To obtain quantitative evidence for oxidative stress in this disease, we measured the major brain antioxidants ascorbate and reduced glutathione, and the lipid peroxidation endproduct malondialdehyde in the cortex of infant rats infected with Streptococcus pneumoniae. Cortical levels of the two antioxidants were markedly decreased 22 h after infection, when animals were severely ill. Total pyridine nucleotide levels in the cortex were unaltered, suggesting that the loss of the two antioxidants was not due to cell necrosis. Bacterial meningitis was accompanied by a moderate, significant increase in cortical malondialdehyde. While treatment with either of the antioxidants alpha-phenyl-tert-butyl nitrone or N-acetylcysteine significantly inhibited this increase, only the former attenuated the loss of endogenous antioxidants. Cerebrospinal fluid bacterial titer, nitrite and nitrate levels, and myeloperoxidase activity at 18 h after infection were unaffected by antioxidant treatment, suggesting that they acted by mechanisms other than modulation of inflammation. The results demonstrate that bacterial meningitis is accompanied by oxidative stress in the brain parenchyma. Furthermore, increased cortical lipid peroxidation does not appear to be the result of parenchymal oxidative stress, because it was prevented by NAC, which had no effect on the loss of brain antioxidants.

Marked elevation in cortical urate and xanthine oxidoreductase activity in experimental bacterial meningitis

Experimental bacterial meningitis due to Streptococcus pneumoniae in infant rats was associated with a time-dependent increase in CSF and cortical urate that was approximately 30-fold elevated at 22 h after infection compared to baseline. This increase was mirrored by a 20-fold rise in cortical xanthine oxidoreductase activity. The relative proportion of the oxidant-producing xanthine oxidase to total activity did not increase, however. Blood plasma levels of urate also increased during infection, but part of this was as a consequence of dehydration, as reflected by elevated ascorbate concentrations in the plasma. Administration of the radical scavenger alpha-phenyl-tert-butyl nitrone, previously shown to be neuroprotective in the present model, did not significantly affect either xanthine dehydrogenase or xanthine oxidase activity, and increased even further cortical accumulation of urate. Treatment with the xanthine oxidoreductase inhibitor allopurinol inhibited CSF urate levels earlier than those in blood plasma, supporting the notion that urate was produced within the brain. However, this treatment did not prevent the loss of ascorbate and reduced glutathione in the cortex and CSF. Together with data from the literature, the results strongly suggest that xanthine oxidase is not a major cause of oxidative stress in bacterial meningitis and that urate formation due to induction of xanthine oxidoreductase in the brain may in fact represent a protective response.

Endothelin inhibition improves cerebral blood flow and is neuroprotective in pneumococcal meningitis

By using an infant rat model of pneumococcal meningitis, we determined whether endothelins contribute to neuronal damage in this disease. Cerebrospinal fluid analysis demonstrated a significant increase of endothelin-1 in infected animals compared with uninfected controls. Histopathological examination 24 hours after infection showed brain damage in animals treated with ceftriaxone alone (median, 9.2% of cortex; range, 0-49.1%). In infected animals treated intraperitoneally with the endothelin antagonist bosentan (30 mg/kg, every 12 hours) also, injury was reduced to 0.5% (range, 0-8.6%) of cortex. Cerebral blood flow was reduced in infected animals (6.5 +/- 4.0 ml/min/100 g of brain vs 14.9 +/- 9.1 ml/min/100 g in controls. Treatment with bosentan restored cerebral blood flow to levels similar to controls (12.8 +/- 5.3 ml/min/100 g). Improved blood flow was not mediated by nitric oxide production, because bosentan had no effect on cerebrospinal fluid or plasma nitrite/nitrate concentrations at 6, 12, or 18 hours. These data indicate that endothelins contribute to neuronal injury in this model of pneumococcal meningitis by causing cerebral ischemia.

An infant mouse model of brain damage in pneumococcal meningitis

Bacterial meningitis due to Streptococcus pneumoniae is associated with an significant mortality rate and persisting neurologic sequelae including sensory-motor deficits, seizures, and impairments of learning and memory. The histomorphological correlate of these sequelae is a pattern of brain damage characterized by necrotic tissue damage in the cerebral cortex and apoptosis of neurons in the hippocampal dentate gyrus. Different animal models of pneumococcal meningitis have been developed to study the pathogenesis of the disease. To date, the infant rat model is unique in mimicking both forms of brain damage documented in the human disease. In the present study, we established an infant mouse model of pneumococcal meningitis. Eleven-days-old C57BL/6 (n = 299), CD1 (n = 42) and BALB/c (n = 14) mice were infected by intracisternal injection of live Streptococcus pneumoniae. Sixteen hours after infection, all mice developed meningitis as documented by positive bacterial cultures of the cerebrospinal fluid. Sixty percent of infected C57BL/6 mice survived more than 40 h after infection (50% of CD1, 0% of BALB/c). Histological evaluations of brain sections revealed apoptosis in the dentate gyrus of the hippocampus in 27% of infected C57BL/6 and in 5% of infected CD1 mice. Apoptosis was confirmed by immunoassaying for active caspase-3 and by TUNEL staining. Other forms of brain damage were found exclusively in C57BL/6, i.e. caspase-3 independent (pyknotic) cell death in the dentate gyrus in 2% and cortical damage in 11% of infected mice. This model may prove useful for studies on the pathogenesis of brain injury in childhood bacterial meningitis.

Rapid diagnosis of experimental meningitis by bacterial heat production in cerebrospinal fluid

BACKGROUND: Calorimetry is a nonspecific technique which allows direct measurement of heat generated by biological processes in the living cell. We evaluated the potential of calorimetry for rapid detection of bacterial growth in cerebrospinal fluid (CSF) in a rat model of bacterial meningitis. METHODS: Infant rats were infected on postnatal day 11 by direct intracisternal injection with either Streptococcus pneumoniae, Neisseria meningitidis or Listeria monocytogenes. Control animals were injected with sterile saline or heat-inactivated S. pneumoniae. CSF was obtained at 18 hours after infection for quantitative cultures and heat flow measurement. For calorimetry, 10 microl and 1 microl CSF were inoculated in calorimetry ampoules containing 3 ml trypticase soy broth (TSB). RESULTS: The mean bacterial titer (+/- SD) in CSF was 1.5 +/- 0.6 x 108 for S. pneumoniae, 1.3 +/- 0.3 x 106 for N. meningitidis and 3.5 +/- 2.2 x 104 for L. monocytogenes. Calorimetric detection time was defined as the time until heat flow signal exceeded 10 microW. Heat signal was detected in 10-microl CSF samples from all infected animals with a mean (+/- SD) detection time of 1.5 +/- 0.2 hours for S. pneumoniae, 3.9 +/- 0.7 hours for N. meningitidis and 9.1 +/- 0.5 hours for L. monocytogenes. CSF samples from non-infected animals generated no increasing heat flow (<10 microW). The total heat was the highest in S. pneumoniae ranging from 6.7 to 7.5 Joules, followed by L. monocytogenes (5.6 to 6.1 Joules) and N. meningitidis (3.5 to 4.4 Joules). The lowest detectable bacterial titer by calorimetry was 2 cfu for S. pneumoniae, 4 cfu for N. meningitidis and 7 cfu for L. monocytogenes. CONCLUSION: By means of calorimetry, detection times of <4 hours for S. pneumoniae and N. meningitidis and <10 hours for Listeria monocytogenes using as little as 10 microl CSF were achieved. Calorimetry is a new diagnostic method allowing rapid and accurate diagnosis of bacterial meningitis from a small volume of CSF.

Gene and protein expression of galectin-3 and galectin-9 in experimental pneumococcal meningitis

Inflammation of the subarachnoid and ventricular space contributes to the development of brain damage i.e. cortical necrosis and hippocampal apoptosis in pneumococcal meningitis (PM). Galectin-3 and -9 are known pro-inflammatory mediators and regulators of apoptosis. Here, the gene and protein expression profile for both galectins was assessed in the disease progression of PM. The mRNA of Lgals3 and Lgals9 increased continuously in the cortex and in the hippocampus from 22 h to 44 h after infection. At 44 h after infection, mRNA levels of Lgals9 in the hippocampus were 7-fold and those of Lgals3 were 30-fold higher than in uninfected controls (P<0.01). Galectin-9 protein did not change, but galectin-3 significantly increased in cortex and hippocampus with the duration of PM. Galectin-3 was localized to polymorphonuclear neutrophils, microglia, monocytes and macrophages, suggesting an involvement of galectin-3 in the neuroinflammatory processes leading to brain damage in PM.

Limited efficacy of adjuvant therapy with dexamethasone in preventing hearing loss due to experimental pneumococcal meningitis in the infant rat

Sensorineural hearing loss (SNHL) is the most common sequel of bacterial meningitis (BM) and is observed in up to 30% of survivors when the disease is caused by Streptococcus pneumoniae. BM is the single most important origin of acquired SNHL in childhood. Anti-inflammatory dexamethasone holds promises as potential adjuvant therapy to prevent SNHL associated with BM. However, in infant rats, pneumococcal meningitis (PM) increased auditory brainstem response (ABR) thresholds [mean difference = 54 decibels sound pressure level (dB SPL)], measured 3 wk after infection, irrespective to treatment with ceftriaxone plus dexamethasone or ceftriaxone plus saline (p < 0.005 compared with mock-infected controls). Moreover, dexamethasone did not attenuate short- and long-term histomorphologic correlates of SNHL. At 24 h after infection, blood-labyrinth barrier (BLB) permeability was significantly increased in infected animals of both treatment groups compared with controls. Three weeks after the infection, the averaged number of type I neurons per square millimeter of the Rosenthal's canal dropped from 0.3019 +/- 0.0252 in controls to 0.2227 +/- 0.0635 in infected animals receiving saline (p < 0.0005). Dexamethasone was not more effective than saline in preventing neuron loss (0.2462 +/- 0.0399; p > 0.05). These results suggest that more efficient adjuvant therapies are needed to prevent SNHL associated with pediatric PM.

Adjuvant TACE inhibitor treatment improves the outcome of TLR2-/- mice with experimental pneumococcal meningitis

BACKGROUND: Streptococcus (S.) pneumoniae meningitis has a high lethality despite antibiotic treatment. Inflammation is a major pathogenetic factor, which is unresponsive to antibiotics. Therefore adjunctive therapies with antiinflammatory compounds have been developed. TNF484 is a TNF-alpha converting enzyme (TACE) inhibitor and has been found efficacious in experimental meningitis. Toll-like receptor 2 (TLR2) contributes to host response in pneumococcal meningitis by enhancing bacterial clearing and downmodulating inflammation. In this study, TNF484 was applied in mice, which lacked TLR2 and exhibited a strong meningeal inflammation. METHODS: 103 CFU S. pneumoniae serotype 3 was inoculated subarachnoidally into C57BL/6 wild type (wt) mice or TLR2-/-, CD14-/- and CD14-/-/TLR2-/- mice. Severity of disease and survival was followed over 9 days. Response to antibiotics (80 mg/kg ceftriaxone i.p. for 5 days) and/or TACE inhibitor treatment (1 mg/kg s.c. twice daily for 4 days) was evaluated. Animals were sacrificed after 12, 24, and 48 h for analysis of bacterial load in cerebrospinal fluid (CSF) and brain and for TNF and leukocyte measurements in CSF. RESULTS: TLR2-/- mice were significantly sicker than the other mouse strains 24 h after infection. All knockout mice showed higher disease severity after 48 h and died earlier than wt mice. TNF release into CSF was significantly more elevated in TLR2-/- than in the other strains after 24 h. Brain bacterial numbers were significantly higher in all knockout than wt mice after 24 h. Modulation of outcome by antibiotic and TACE inhibitor treatment was evaluated. With antibiotic therapy all wt, CD14-/- and TLR2-/-/CD14-/- mice, but only 79% of TLR2-/- mice, were rescued. TACE inhibitor treatment alone did not rescue, but prolonged survival in wt mice, and in TLR2-/- and CD14-/- mice to the values observed in untreated wt mice. By combined antibiotic and TACE inhibitor treatment 95% of TLR2-/- mice were rescued. CONCLUSION: During pneumococcal meningitis strong inflammation in TLR2-deficiency was associated with incomplete responsiveness to antibiotics and complete response to combined antibiotic and TACE inhibitor treatment. TACE inhibitor treatment offers a promising adjuvant therapeutic strategy in pneumococcal meningitis.

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.