The causative pathogen determines the inflammatory profile in cerebrospinal fluid and outcome in patients with bacterial meningitis

BACKGROUND The brain's inflammatory response to the infecting pathogen determines the outcome of bacterial meningitis (BM), for example, the associated mortality and the extent of brain injury. The inflammatory cascade is initiated by the presence of bacteria in the cerebrospinal fluid (CSF) activating resident immune cells and leading to the influx of blood derived leukocytes. To elucidate the pathomechanisms behind the observed difference in outcome between different pathogens, we compared the inflammatory profile in the CSF of patients with BM caused by Streptococcus pneumonia (n = 14), Neisseria meningitidis (n = 22), and Haemophilus influenza (n = 9). METHODS CSF inflammatory parameters, including cytokines and chemokines, MMP-9, and nitric oxide synthase activity, were assessed in a cohort of patients with BM from Burkina Faso. RESULTS Pneumococcal meningitis was associated with significantly higher CSF concentrations of IFN-γ , MCP-1, and the matrix-metalloproteinase (MMP-) 9. In patients with a fatal outcome, levels of TNF-α, IL-1 β, IL-1RA, IL-6, and TGF-α were significantly higher. CONCLUSION The signature of pro- and anti-inflammatory mediators and the intensity of inflammatory processes in CSF are determined by the bacterial pathogen causing bacterial meningitis with pneumococcal meningitis being associated with a higher case fatality rate than meningitis caused by N. meningitidis or H. influenzae.

[In search of strategies for preventing brain damage as a sequela of bacterial meningitis]

Multiplication of bacteria within the central nervous system compartment triggers a host response with an overshooting inflammatory reaction which leads to brain parenchyma damage. Some of the inflammatory and neurotoxic mediators involved in the processes leading to neuronal injury during bacterial meningitis have been identified in recent years. As a result, the therapeutic approach to the disease has widened from eradication of the bacterial pathogen with antibiotics to attenuation of the detrimental effects of host defences. Corticosteroids represent an example of the adjuvant therapeutic strategies aimed at downmodulating excessive inflammation in the infected central nervous system. Pathophysiological concepts derived from an experimental rat model of bacterial meningitis revealed possible therapeutic strategies for prevention of brain damage. The insights gained led to the evaluation of new therapeutic modalities such as anticytokine agents, matrix metalloproteinase inhibitors, antioxidants, and antagonists of endothelin and glutamate. Bacterial meningitis is still associated with persistent neurological sequelae in approximately one third of surviving patients. Future research in the model will evaluate whether the neuroprotective agents identified so far have the potential to attenuate learning disabilities as a long-term consequence of bacterial meningitis.

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-alpha-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H(2)O(2) into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly(12)-Gly(13)-Gly(14)-Ile(15)-Ile(16)-Gly(17). Recombinant GlpO lacking these six amino acids (GlpODeltaFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpODeltaFAD, similarly to anti-GlpO antibodies, neutralised H(2)O(2) production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.

Estrogen Modulates Hepatic Gene Expression and Survival of Rainbow Trout Infected with Pathogenic Bacteria Yersinia ruckeri

In the aquatic environment, fish are exposed to various stimuli at once and have developed different response mechanisms to deal with these multiple stimuli. The current study assessed the combined impacts of estrogens and bacterial infection on the physiological status of fish. Juvenile rainbow trout were exposed to two different concentrations of 17 beta-estradiol (E2) (2 or 20 mg/kg feed) and then infected with three concentrations of Yersinia ruckeri, a bacterial pathogen causing massive losses in wild and farmed salmonid populations. Organism-level endpoints to assess the impact of the single and combined treatments included hepatic vitellogenin transcript expression to evaluate the E2 exposure efficiency and survival rate of pathogen-challenged fish. The two E2 doses increased vitellogenin levels within the physiological range. Infection with Y. ruckeri caused mortality of trout, and this effect was significantly enhanced by a simultaneous exposure to high E2 dose. The hormone reduced survival at intermediate and high (10(4) and 10(6) colony forming units, cfu) bacterial concentrations, but not for a low one (10(2) cfu). Analysis of hepatic gene expression profiles by a salmonid 2 k cDNA microarray chip revealed complex regulations of pathways involved in immune responses, stress responses, and detoxicification pathways. E2 markedly reduced the expression of several genes implicated in xenobiotic metabolism. The results suggest that the interaction between pathogen and E2 interfered with the fish's capability of clearing toxic compounds. The findings of the current study add to our understanding of multiple exposure responses in fish.

The Pneumococcal Polysaccharide Capsule and Pneumolysin Differentially Affect CXCL8 and IL-6 Release from Cells of the Upper and Lower Respiratory Tract

The polysaccharide capsule and pneumolysin toxin are major virulence factors of the human bacterial pathogen Streptococcus pneumoniae. Colonization of the nasopharynx is asymptomatic but invasion of the lungs can result in invasive pneumonia. Here we show that the capsule suppresses the release of the pro-inflammatory cytokines CXCL8 (IL-8) and IL-6 from the human pharyngeal epithelial cell line Detroit 562. Release of both cytokines was much less from human bronchial epithelial cells (iHBEC) but levels were also affected by capsule. Pneumolysin stimulates CXCL8 release from both cell lines. Suppression of CXCL8 homologue (CXCL2/MIP-2) release by the capsule was also observed in vivo during intranasal colonization of mice but was only discernable in the absence of pneumolysin. When pneumococci were administered intranasally to mice in a model of long term, stable nasopharyngeal carriage, encapsulated S. pneumoniae remained in the nasopharynx whereas the nonencapsulated pneumococci disseminated into the lungs. Pneumococcal capsule plays a role not only in protection from phagocytosis but also in modulation of the pro-inflammatory immune response in the respiratory tract.

[Meningitis (I)--differential diagnosis; aseptic and chronic meningitis].

Meningitis is the most common serious manifestation of infection of the central nervous system. Inflammatory involvement of the subarachnoid space with meningeal irritation leads to the classical triad of headache, fever, and meningism, and to a pleocytosis of the cerebrospinal fluid (CSF). Meningitis is clinically categorized into an acute and chronic disease based on the acuity of symptoms. Acute meningitis develops over hours to days, while in chronic meningitis symptoms evolve over days or even weeks. Aseptic meningitis, in which no bacterial pathogen can be isolated by routine cultures, can mimic bacterial meningitis, but the disease has a much more favorable prognosis. Many cases of aseptic meningitis are caused by viruses, primarily enteroviruses, but bacteria and noninfectious etiologies also cause meningitis with negative cultures. Symptoms of meningeal inflammation with CSF pleocytosis that persist for more than 4 weeks define the chronic meningitis syndrome. The diagnosis is based on the patient history, clinical evidence of meningitis, CSF examination, and often imaging studies. The differential diagnosis is broad, and the predominant CSF cell type can provide clues as to the underlying disease. Empiric therapy is primarily based on the age of the patient, with modifications if there are positive findings on CSF gram stain or if the patient presents with special risk factors. In patients with chronic meningitis, a definite diagnosis is often not available or delayed for days, in which case empiric therapy may have to be initiated. It is important to cover the treatable causes of meningitis, for which the outcome is poor if treatment is delayed.

Rapid qualitative urinary tract infection pathogen identification by SeptiFast real-time PCR

Background Urinary tract infections (UTI) are frequent in outpatients. Fast pathogen identification is mandatory for shortening the time of discomfort and preventing serious complications. Urine culture needs up to 48 hours until pathogen identification. Consequently, the initial antibiotic regimen is empirical. Aim To evaluate the feasibility of qualitative urine pathogen identification by a commercially available real-time PCR blood pathogen test (SeptiFast®) and to compare the results with dipslide and microbiological culture. Design of study Pilot study with prospectively collected urine samples. Setting University hospital. Methods 82 prospectively collected urine samples from 81 patients with suspected UTI were included. Dipslide urine culture was followed by microbiological pathogen identification in dipslide positive samples. In parallel, qualitative DNA based pathogen identification (SeptiFast®) was performed in all samples. Results 61 samples were SeptiFast® positive, whereas 67 samples were dipslide culture positive. The inter-methodological concordance of positive and negative findings in the gram+, gram- and fungi sector was 371/410 (90%), 477/492 (97%) and 238/246 (97%), respectively. Sensitivity and specificity of the SeptiFast® test for the detection of an infection was 0.82 and 0.60, respectively. SeptiFast® pathogen identifications were available at least 43 hours prior to culture results. Conclusion The SeptiFast® platform identified bacterial DNA in urine specimens considerably faster compared to conventional culture. For UTI diagnosis sensitivity and specificity is limited by its present qualitative setup which does not allow pathogen quantification. Future quantitative assays may hold promise for PCR based UTI pathogen identification as a supplementation of conventional culture methods.

Monomeric and dimeric IgG fractions show differential reactivity against pathogen-derived antigens

Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both 'acquired' and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype-antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype-antiidiotype Ab pairs and thus in a comparable F(ab')(2) binding site availability of the different IgG fractions. Previously, we identified an increased anti-self-reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen-derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen-associated polysaccharides, classically regarded as T-independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.

Bacterial meningitis: current therapy and possible future treatment options

Despite targeted therapy, case-fatality rates and neurologic sequelae of bacterial meningitis remain unacceptably high. The poor outcome is mainly due to secondary systemic and intracranial complications. These complications seem to be both a consequence of the inflammatory response to the invading pathogen and release of bacterial components by the pathogen itself. Therefore, within the last decades, research has focused on the mechanism underlying immune regulation and the inhibition of bacterial lysis in order to identify new targets for adjuvant therapy. The scope of this article is to give an overview on current treatment strategies of bacterial meningitis, to summarize new insights on the pathophysiology of bacterial meningitis, and to give an outlook on new treatment strategies derived from experimental models.

Moraxella catarrhalis - pathogen or commensal?

Moraxella catarrhalis is an exclusively human commensal and mucosal pathogen. Its role as a disease-causing organism has long been questioned. Today, it is recognized as one of the major causes of acute otitis media in children, and its relative frequency of isolation from both the nasopharynx and the middle ear cavity has increased since the introduction of the heptavalent pneumococcal conjugate vaccine, which is associated with a shift in the composition of the nasopharyngeal flora in infants and young children. Although otitis media caused by M. catarrhalis is generally believed to be mild in comparison with pneumococcal disease, numerous putative virulence factors have now been identified and it has been shown that several surface components of M. catarrhalis induce mucosal inflammation. In adults with chronic obstructive pulmonary disease (COPD), M. catarrhalis is now a well-established trigger of approximately 10% of acute inflammatory exacerbations.Although the so-called cold shock response is a well-described bacterial stress response in species such as Escherichia coli, Bacillus subtilis or - more recently - Staphylococcus aureus, M. catarrhalis is the only typical nasopharyngeal pathogen in which this response has been investigated. Indeed, a 3-h 26°C cold shock, which may occur physiologically, when humans inspire cold air for prolonged periods of time, increases epithelial cell adherence and enhances proinflammatory host responses and may thus contribute to the symptoms referred to as common cold, which typically are attributed to viral infections.

Neurotoxicity of glia activated by gram-positive bacterial products depends on nitric oxide production

The present study examined the mechanism by which bacterial cell walls from two gram-positive meningeal pathogens, Streptococcus pneumoniae and the group B streptococcus, induced neuronal injury in primary cultures of rat brain cells. Cell walls from both organisms produced cellular injury to similar degrees in pure astrocyte cultures but not in pure neuronal cultures. Cell walls also induced nitric oxide production in cultures of astrocytes or microglia. When neurons were cultured together with astrocytes or microglia, the cell walls of both organisms became toxic to neurons. L-NAME, a nitric oxide synthase inhibitor, protected neurons from cell wall-induced toxicity in mixed cultures with glia, as did dexamethasone. In contrast, an excitatory amino acid antagonist (MK801) had no effect. Low concentrations of cell walls from either gram-positive pathogen added together with the excitatory amino acid glutamate resulted in synergistic neurotoxicity that was inhibited by L-NAME. The induction of nitric oxide production and neurotoxicity by cell walls was independent of the presence of serum, whereas endotoxin exhibited these effects only in the presence of serum. We conclude that gram-positive cell walls can cause toxicity in neurons by inducing the production of nitric oxide in astrocytes and microglia.

A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples

Early detection of bloodstream infections (BSI) is crucial in the clinical setting. Blood culture remains the gold standard for diagnosing BSI. Molecular diagnostic tools can contribute to a more rapid diagnosis in septic patients. Here, a multiplex real-time PCR-based assay for rapid detection of 25 clinically important pathogens directly from whole blood in <6 h is presented. Minimal analytical sensitivity was determined by hit rate analysis from 20 independent experiments. At a concentration of 3 CFU/ml a hit rate of 50% was obtained for E. aerogenes and 100% for S. marcescens, E. coli, P. mirabilis, P. aeruginosa, and A. fumigatus. The hit rate for C. glabrata was 75% at 30 CFU/ml. Comparing PCR identification results with conventional microbiology for 1,548 clinical isolates yielded an overall specificity of 98.8%. The analytical specificity in 102 healthy blood donors was 100%. Although further evaluation is warranted, our assay holds promise for more rapid pathogen identification in clinical sepsis.

The COLOSS BEEBOOK Volume II: Standard methods for Apis mellifera pest and pathogen research

n recent years, declines of honey bee populations have received massive media attention worldwide, yet attempts to understand the causes have been hampered by a lack of standardisation of laboratory techniques. Published as a response to this, the COLOSS BEEBOOK is a unique collaborative venture involving 234 bee scientists from 34 countries, who have produced the definitive guide to how to carry out research on honey bees. It is hoped that these volumes will become the standards to be adopted by bee scientists worldwide. Volume II includes approximately 600 separate protocols dealing with the study of the pests and diseases of the honey bee, Apis mellifera. These cover epidemiology and surveying techniques, virus diseases, bacterial diseases such as European and American foulbrood, fungal and microsporidian diseases such as Nosema, mites such as Acarapis, Varroa and Tropilaelaps, and other pests such as the small hive beetle.

Timing of bacterial carriage sampling in vaccine trials: A modelling study.

BACKGROUND Pathogenic bacteria are often asymptomatically carried in the nasopharynx. Bacterial carriage can be reduced by vaccination and has been used as an alternative endpoint to clinical disease in randomised controlled trials (RCTs). Vaccine efficacy (VE) is usually calculated as 1 minus a measure of effect. Estimates of vaccine efficacy from cross-sectional carriage data collected in RCTs are usually based on prevalence odds ratios (PORs) and prevalence ratios (PRs), but it is unclear when these should be measured. METHODS We developed dynamic compartmental transmission models simulating RCTs of a vaccine against a carried pathogen to investigate how VE can best be estimated from cross-sectional carriage data, at which time carriage should optimally be assessed, and to which factors this timing is most sensitive. In the models, vaccine could change carriage acquisition and clearance rates (leaky vaccine); values for these effects were explicitly defined (facq, 1/fdur). POR and PR were calculated from model outputs. Models differed in infection source: other participants or external sources unaffected by the trial. Simulations using multiple vaccine doses were compared to empirical data. RESULTS The combined VE against acquisition and duration calculated using POR (VEˆacq.dur, (1-POR)×100) best estimates the true VE (VEacq.dur, (1-facq×fdur)×100) for leaky vaccines in most scenarios. The mean duration of carriage was the most important factor determining the time until VEˆacq.dur first approximates VEacq.dur: if the mean duration of carriage is 1-1.5 months, up to 4 months are needed; if the mean duration is 2-3 months, up to 8 months are needed. Minor differences were seen between models with different infection sources. In RCTs with shorter intervals between vaccine doses it takes longer after the last dose until VEˆacq.dur approximates VEacq.dur. CONCLUSION The timing of sample collection should be considered when interpreting vaccine efficacy against bacterial carriage measured in RCTs.