Value of a novel Neisseria meningitidis--specific polymerase chain reaction assay in skin biopsy specimens as a diagnostic tool in chronic meningococcemia

BACKGROUND: Chronic meningococcemia (CM) is a diagnostic challenge. Skin lesions are frequent but in most cases nonspecific. Polymerase chain reaction (PCR)-based diagnosis has been validated in blood and cerebrospinal fluid for acute Neisseria meningitidis infection, in patients in whom routine microbiologic tests have failed to isolate the bacteria. In 2 patients with CM, we established the diagnosis by a newly developed PCR-based approach performed on skin biopsy specimens. OBSERVATIONS: Two patients presented with fever together with systemic and cutaneous manifestations suggestive of CM. Although findings from blood cultures remained negative, we were able to identify N meningitidis in the skin lesions by a newly developed PCR assay. In 1 patient, an N meningitidis strain of the same serogroup was also isolated from a throat swab specimen. Both patients rapidly improved after appropriate antibiotherapy. CONCLUSIONS: To our knowledge, we report the first cases of CM diagnosed by PCR testing on skin biopsy specimens. It is noteworthy that, although N meningitidis-specific PCR is highly sensitive in blood and cerebrospinal fluid in acute infections, our observations underscore the usefulness of PCR performed on skin lesions for the diagnosis of chronic N meningitidis infections. Whenever possible, this approach should be systematically employed in patients for whom N meningitidis infection cannot be confirmed by routine microbiologic investigations.

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.

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.

Fluoroquinolones in the Treatment of Meningitis

The continuous increase of resistant pathogens causing meningitis has limited the efficacy of standard therapeutic regimens. Due to their excellent activity in vitro and their good penetration into the cerebrospinal fluid (CSF), fluoroquinolones appear promising for the treatment of meningitis caused by gram-negative microorganisms, ie, Neisseria meningitidis and nosocomial gram-negative bacilli. The newer fluoroquinolones (moxifloxacin, gemifloxacin, gatifloxacin, and garenoxacin) have excellent activity against gram-positive microorganisms. Studies in animal models and limited clinical data indicate that they may play a future role in the treatment of pneumococcal meningitis. Analysis of pharmacodynamic parameters suggests that CSF concentrations that produce a C(peak)/minimal bactericidal concentration (MBC) ratio of at least 5 and concentrations above the MBC during the entire dosing interval are a prerequisite for maximal bactericidal activity in meningitis. Of interest, newer fluoroquinolones act synergistically with vancomycin and beta-lactam antibiotics (ceftriaxone, cefotaxime, meropenem) against penicillin-resistant pneumococci in experimental rabbit meningitis, potentially providing a new therapeutic strategy. Clinical trials are needed to further explore the usefulness of quinolones as single agents or in combination with other drugs in the therapy of pneumococcal meningitis.

Characterization of apxIVA, a new RTX determinant of Actinobacillus pleuropneumoniae

A fourth type of RTX determinant was identified in Actinobacillus pleuropneumoniae and was designated apxIVA. When expressed in Escherichia coli, recombinant ApxIVA showed a weak haemolytic activity and co-haemolytic synergy with the sphingomyelinase (beta-toxin) of Staphylococcus aureus. These activities required the presence of an additional gene, ORF1, that is located immediately upstream of apxIVA. The apxIVA gene product could not be detected in A. pleuropneumoniae cultures grown under various conditions in vitro; however, pigs experimentally infected with A. pleuropneumoniae serotypes 1, 5 and 7 started to produce antibodies that reacted with recombinant ApxIVA 14 d post-infection, indicating that apxIVA is expressed in vivo. In addition, sera from pigs naturally and experimentally infected with any of the serotypes all reacted with recombinant ApxIVA. The apxIVA gene from the serotype 1 A. pleuropneumoniae type strain Shope 4074T encodes a protein with a predicted molecular mass of 202 kDa which has typical features of RTX proteins including hydrophobic domains in the N-terminal half and 24 glycine-rich nonapeptides in the C-terminal half that bind Ca2+. The glycine-rich nonapeptides are arranged in a modular structure and there is some variability in the number of modules in the ApxIVA proteins of different serotypes of A. pleuropneumoniae. The deduced amino acid sequences of the ApxIVA proteins have significant similarity with the Neisseria meningitidis iron-regulated RTX proteins FrpA and FrpC, and to a much lesser extent with other RTX proteins. The apxIVA gene could be detected in all A. pleuropneumoniae serotypes and seems to be species-specific. Although the precise role of this new RTX determinant in pathogenesis of porcine pleuropneumonia needs to be determined, apxIVA is the first in vivo induced toxin gene that has been described in A. pleuropneumoniae.

Cerebrospinal-fluid cytokine and chemokine profile in patients with pneumococcal and meningococcal meningitis

BACKGROUND Bacterial meningitis is characterized by an intense inflammatory reaction contributing to neuronal damage. The aim of this study was to obtain a comparative analysis of cytokines and chemokines in patients with pneumococcal (PM) and meningococcal meningitis (MM) considering that a clear difference between the immune response induced by these pathogens remains unclear. METHODS The cyto/chemokines, IL-1beta, IL-2, IL-6, TNF-alpha, IFN-gamma, IL-10, IL-1Ra, CXCL8/IL-8, CCL2/MCP-1, CLL3/MIP-1alpha, CCL4/MIP-1gamma and G-CSF, were measured in cerebrospinal fluid (CSF) samples from patients with PM and MM. Additionally, a literature review about the expression of cytokines in CSF samples of patients with MB was made. RESULTS Concerning cytokines levels, only IFN-gamma was significantly higher in patients with Streptococcus pneumoniae compared to those with Neisseria meningitidis, regardless of the time when the lumbar puncture (LP) was made. Furthermore, when samples were compared considering the timing of the LP, higher levels of TNF-alpha (P <0.05) were observed in MM patients whose LP was made within 48 h from the initial symptoms of disease. We also observed that the index of release of cyto/chemokines per cell was significantly higher in PM. From the literature review, it was observed that TNF-alpha, IL-1beta and IL-6 are the best studied cytokines, while reports describing the concentration of the cytokine IL-2, IL-1Ra, G-CSF and CCL4/MIP-1beta in CSF samples of patients with bacterial meningitis were not found. CONCLUSION The data obtained in this study and the previously published data show a similar profile of cytokine expression during PM and MM. Nevertheless, the high levels of IFN-gamma and the ability to release high levels of cytokines with a low number of cells are important factors to be considered in the pathogenesis of PM and thereby should be further investigated. Moreover, differences in the early response induced by the pathogens were observed. However, the differences observed are not sufficient to trigger changes in the current therapy of corticosteroids adopted in both the PM and MM.

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.

Inhibition of meningitis-associated neutrophil apoptosis by TNF-α depends on functional PI3-kinase in monocytes

In bacterial meningitis, neutrophils cope with bacterial infection but also lead to tissue damage. The balance of beneficial and harmful effects may depend on the lifespan of the neutrophils in the CNS. Here, we show that CSF of patients with meningococcal meningitis contains a neutrophil apoptosis-inhibiting capacity that correlates with TNF-α content. In vitro experiments show that Neisseria meningitidis as well as LPS derived from these bacteria regulated neutrophil apoptosis mainly by stimulating TNF-α production in monocytes. Whereas LPS-induced PI3K-dependent survival signals in monocytes are critical for neutrophil survival, PI3K signaling in granulocytes did not contribute to the increased lifespan of neutrophils. We conclude that LPS-driven PI3K signaling in monocytes regulates neutrophil apoptosis and thereby, may be crucial in the initiation of secondary brain damage in bacterial meningitis.

Inhibition of matrix metalloproteinases attenuates brain damage in experimental meningococcal meningitis.

BackgroundApproximately 7% of survivors from meningococcal meningitis (MM) suffer from neurological sequelae due to brain damage in the course of meningitis. The present study focuses on the role of matrix metalloproteinases (MMPs) in a novel mouse model of MM-induced brain damage.MethodsThe model is based on intracisternal infection of BALB/c mice with a serogroup C Neisseria meningitidis strain. Mice were infected with meningococci and randomised for treatment with the MMP inhibitor batimastat (BB-94) or vehicle. Animal survival, brain injury and host-response biomarkers were assessed 48 h after meningococcal challenge.ResultsMice that received BB-94 presented significantly diminished MMP-9 levels (p¿<¿0.01), intracerebral bleeding (p¿<¿0.01), and blood-brain barrier (BBB) breakdown (p¿<¿0.05) in comparison with untreated animals. In mice suffering from MM, the amount of MMP-9 measured by zymography significantly correlated with both intracerebral haemorrhage (p¿<¿0.01) and BBB disruption (p¿<¿0.05).ConclusionsMMPs significantly contribute to brain damage associated with experimental MM. Inhibition of MMPs reduces intracranial complications in mice suffering from MM, representing a potential adjuvant strategy in MM post-infection sequelae.

Characterization of Neisseria gonorrhoeae isolates detected in Switzerland (1998–2012): emergence of multidrug-resistant clones less susceptible to cephalosporins

Background: The spread of Neisseria gonorrhoeae (Ng) isolates resistant to the clinically implemented antibiotics is challenging the efficacy of treatments. Unfortunately, phenotypic and molecular data regarding Ng detected in Switzerland are scarce. Methods: We compared the characteristics of Ng detected during 1998–2001 (n = 26) to those detected during 2009–2012 (n = 34). MICs were obtained with the Etest and interpreted as non-susceptible (non-S) according to EUCAST criteria. Sequence type (ST) was achieved implementing the NG-MAST. BlaTEM, ponA, penA, mtrR, penB, tet (M), gyrA, parC, mefA, ermA/B/C/F, rplD, rplV, and 23S rRNA genes were analyzed. Results: The following susceptibility results were obtained (period: % of non-S, MIC90 in mg/L): penicillin (1998–2001: 42.3%, 3; 2009–2012: 85.3%, 16), cefixime (1998–2001: 0%, ≤0.016; 2009–2012: 8.8%, 0.125), ceftriaxone (1998–2001: 0%, 0.004; 2009–2012: 0%, 0.047), ciprofloxacin (1998–2001: 7.7%, 0.006; 2009–2012: 73.5%, ≥32), azithromycin (1998–2001: 11.5%, 0.25; 2009–2012: 23.6%, 0.38), tetracycline (1998–2001: 65.4%, 12; 2009–2012: 88.2%, 24), spectinomycin (1998–2001: 0%, 12; 2009–2012: 0%, 8). The prevalence of multidrug-resistant (MDR) isolates increased from 7.7% in 1998–2001 to 70.6% in 2009–2012. International STs and genogroups (G) emerged during 2009–2012 (G1407, 29.4%; G2992, 11.7%; G225, 8.8%). These isolates possessed distinctive mechanisms of resistance (e.g., G1407: PBP1 with L421, PBP2 pattern XXXIV, GyrA with S91F and D95G, ParC with S87R, PorB with G120K and A121N, mtrR promoter with A deletion). Conclusions: The prevalence of penicillin- ciprofloxacin- and tetracycline-resistant Ng has reached dramatic levels, whereas cefixime and ceftriaxone show MICs that tend to increase during time. International MDR clones less susceptible to cephalosporins are rapidly emerging indicating that the era of untreatable gonococcal infections is close.