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Immunoadsorbent chromatography is described as a method to purify from rabbit antisera antibodies specific for protein serotype antigens of meningococci, exemplified by antibodies to the serotype 15 and P1.16 proteins, respectively.
An increase in B:15:P1.12 meningococci among isolates from patients with Neisseria meningitidis infection in Norway in recent years led to further characterization of such strains. Between 1987 and 1992, B:15:P1.12 strains constituted 9.8% (24 strains) of B:15 isolates. The B:15:P1.12 strains belonged to the electrophoretic type 5 (ET-5) complex, but 17 (71%) strains were a new clone (ET-5c) not found elsewhere in the world. All but one strain of ET-5c were responsible for a localized outbreak of systemic meningococcal disease in western Norway. A novel monoclonal antibody (202,G-12), developed against the unknown variable region 2 on the class 1 protein of one of these strains, bound to 19 of the 15:P1.12 strains, 4 strains bound the subtype P1.13 reference monoclonal antibody MN24H10.75, and the remaining strain showed no reaction. Sequencing of porA genes demonstrated a series of nine threonine residues in the deduced variable region 2 of the latter strain, while four and five threonine residues were found in the corresponding regions of strains reacting with the monoclonal antibodies 202,G-12 and MN24H10.75, respectively. Epitope mapping with synthetic peptides showed that 202,G-12 bound to a sequence of 11 amino acids which included the four threonine residues specific for subtype P1.13a. Immunoglobulin G antibodies against the P1.7,16 subtype protein, induced in volunteers after vaccination with the Norwegian meningococcal vaccine, did not cross-react on immunoblots with the subtype protein of clone ET-5c. Thus, postvaccination class 1 protein antibodies, assumed to be protective, may not be effective against infection with the new clone.
Neisseria meningitidis produces proteins (FrpA and FrpC) related to the RTX cytotoxin family. In meningococcal strain FAM20 these proteins were both localized in the outer membrane and secreted into the extracellular medium. An Escherichia coli strain with wild-type hemolysin secretion genes hlyB and hlyD and containing a cloned frpA gene secreted FrpA, whereas an isogenic hlyBD mutant strain did not. Low-stringency DNA hybridization revealed hlyBD-like sequences in N. meningitidis FAM20, suggesting that a similar RTX secretion system exists in meningococci. Structural features found at the C termini of other RTX proteins and thought to be important for their secretion were also found at the C terminus of FrpA. The secretion of FrpA from E. coli by heterologous RTX transport proteins further demonstrates the relation of the FrpA protein to RTX toxins.
Opsonic activity in sera from 27 military recruits vaccinated with the Norwegian meningococcal serogroup B outer membrane vesicle vaccine was measured as respiratory burst with polymorphonuclear leukocytes as the effector cells and meningococci of the epidemic strain as the target. The results were compared with antimeningococcal IgG antibodies against an outer membrane vesicle coat in an enzyme-linked immunosorbent assay and with serum bactericidal activity. The vaccinees were immunized twice, with a 6-week interval between the two. The serum samples studied were collected at day zero, after 6 weeks, and after 12 weeks. Both serum bactericidal activity and respiratory burst were measured by adding external serum as the complement source. The results revealed a significant increase in specific IgG response, serum bactericidal activity, and respiratory burst after vaccination. We found a highly significant correlation between the responses in all three assays (P < 0.0001). The highest correlation was found between respiratory burst and antimeningococcal IgG response (r = 0.93). This result strongly indicates that respiratory burst is mediated almost exclusively by IgG antibodies. The correlation between antimeningococcal IgG response and serum bactericidal activity was slightly lower (r = 0.83). The correlation between respiratory burst and serum bactericidal activity was further reduced (r = 0.78), and some of the sera revealed a marked preference for only one of the activities. This result means that respiratory burst and serum bactericidal activity in part are induced by different mediators, and to obtain a more complete picture of the potential protective activity, both assays should be applied to survey a vaccine trial.
Quantitative studies were carried out on the in vitro phagocytosis of 14C-labeled Neisseria meningitidis by mouse polymorphonuclear leukocytes. Intact, "loaded" leukocytes were found to excrete radioactive bacterial products back into supernatant fluids. Morphological events associated with the exocytosis events revealed a fusion between the phagocytic vacuole and plasma membranes of the leukocyte followed by an emptying of the vacuole contents. Egested materials were free from whole meningococci and consisted mainly of membranous vesicles.
Neisseria meningitidis (the meningococcus) is a naturally competent bacterial species in which intra- and interspecific horizontal gene transfer is a major source of genetic diversity. In strains of the electrophoretic type 37 (ET-37) complex and of the A4 cluster, we identified genomic DNA coding for a novel restriction-modification system and for the tail of a previously unidentified prophage. Furthermore, a novel 7.2-kb DNA segment restricted to clones of the ET-37 complex and the A4 cluster was isolated and shown to occur both as a plasmid (pJS-B) and as a chromosomal integration. Neither the genomic loci nor pJS-B was present in ET-5 complex, lineage 3, or serogroup A meningococci. The differential distribution of the DNA segments described herein, as well as of opcA, porB, nmeAI, nmeBI, and nmeDI described previously, supports the concept of genetic isolation of hypervirulent lineages responsible for most cases of serogroup C disease worldwide.
During periods of endemic meningococcal disease, serogroup B Neisseria meningitidis is responsible for a significant percentage of invasive diseases, and no particular clone or strain predominates (F. E. Ashton and D. A. Caugant, Can. J. Microbiol. 47: 293-289, 2001), However, in the winter of 2004 to 2005, a cluster of serogroup B meningococcal disease occurred in one region in the province of Québec, Canada. The N. meningitidis strain responsible for this cluster of cases was identified as sequence type ST-269 with the antigenic formula B:17:P1.19. Retrospective analysis of isolates from 2000 onwards showed that this clone first emerged in the province of Québec in 2003. The emergence of this clone of serogroup B meningococci occurred after a mass vaccination against serogroup C N. meningitidis, suggesting possible capsule replacement.
Population and evolutionary analyses of pathogenic bacteria are frequently hindered by sampling strategies that concentrate on isolates from patients with invasive disease. This is especially so for the gram-negative diplococcus Neisseria meningitidis, a cause of septicemia and meningitis worldwide. Meningococcal isolate collections almost exclusively comprise organisms originating from patients with invasive meningococcal disease, although this bacterium is a commensal inhabitant of the human nasopharynx and very rarely causes pathological effects. In the present study, molecular biology-based techniques were used to establish the genetic relationships of 156 meningococci isolated from healthy young adults in the Czech Republic during 1993. None of the individuals sampled had known links to patients with invasive disease. Multilocus sequence typing (MLST) showed that the bacterial population was highly diverse, comprising 71 different sequence types (STs) which were assigned to 34 distinct complexes or lineages. Three previously identified hyperinvasive lineages were present: 26 isolates (17%) belonged to the ST-41 complex (lineage 3); 4 (2.6%) belonged to the ST-11 (electrophoretic type [ET-37]) complex, and 1 (0.6%) belonged to the ST-32 (ET-5) complex. The data were consistent with the view that most nucleotide sequence diversity resulted from the reassortment of alleles by horizontal genetic exchange.
An outbreak of serogroup W-135 meningococcal disease was reported among pilgrims returning from the annual hajj (pilgrimage to Mecca) in mid-March 2000. Molecular characterization was used to investigate the similarity of the hajj-associated W-135 strains with those isolated in Sweden during a 23-year period (1978 to 2000). The same hajj-associated genosubtype, genosubtype P1.5,2,36b, has been documented in Sweden since 1979, while pulsed-field gel electrophoresis and the sulfadiazine resistance of the W-135 isolates indicated that the outbreak was probably due to a new clone of W-135 meningococci.
Invasive meningococcal infections are usually diagnosed by culture. This approach is far from optimal due to, e.g., treatment with precollection antibiotics. Molecular-genetics methods are therefore recognized as important tools for optimal laboratory confirmation of meningococcal infections as well as characterization of meningococci (Mc). The aims of the present study were to develop real-time PCRs for identification and genogrouping (A, B, C, Y, and W-135) of Mc and porA amplification that further can be used for subsequent genosubtyping. In a first run Mc were identified. In a second run they were genogrouped and porA genes were amplified. All the Mc isolates (n = 71) but one and cerebrospinal fluid samples (n = 11) tested gave the expected positive results. The specificity, inter- and intra-assay variations, and effects of different amounts of DNA on the melting temperatures were also explored. The LightCycler PCR system was found to effectively detect and characterize Mc in a few hours. This testing, including the DNA sequencing of the porA gene to reveal the genosubtype, does not take more than a working day, and the results can be compared to those from culturing with phenotypic analysis, which takes at least a few days.
Standard and fluorescence-based PCR assays were developed for the identification of serogroup A meningococci by detection of the mynA gene. This assay was evaluated using bacterial cultures but provides the sensitivity required for the detection of the mynA gene from bodily fluids during meningococcal disease.
A multilocus restriction typing (MLRT) method was developed to reduce the number of sequencing reactions required to determine the clonal relationships among serogroup B meningococci causing an epidemic in New Zealand. MLRT was a rapid, simple, and inexpensive method, and the results had an excellent correlation with multilocus sequence typing results.
In 2003, in the Zinder and Maradi regions (Niger), epidemics were due to serogroup A:4:P1.9 meningococci belonging to sequence type 7 (ST-7). In Niamey, only sporadic cases were reported: 55% of the meningococcus strains were in serogroup A, and 38% were in serogroup W135 and could be placed in ST-11, identical to the 2002 Burkina Faso epidemic clone, and in ST-2881, a new ST.
Modifications in the antiserum agar method for serogroup identification of meningococci, which reduce the amount of group-specific antisera required and increase long-term storage of prepoured antiserum agar plates, are described.
From 1966 to 1971, 298 cultures of meningococci from clinical material (cerebrospinal fluid or blood) were examined. Eighty-nine cultures were from the Manchester area and the remainder from other parts of England or Northern Ireland. Five per cent of strains were group A, 57% group B, and 31% group C; 6% were untypable. Eighteen strains (6%) had an MIC of 6.4 mug/ml or more of sodium sulphadiazine and 10 of these (3.5%) an MIC of 50 to 100 mug/ml. The incidence of sulphonamide resistance was higher in group A strains than in group B or group C strains.
A convenient and reliable method for serogrouping meningococci, based on immunospecific precipitation haloes in antiserum-agar plates, is described. It gave concordant results with conventional slide-agglutination in 900 strains of groups A, B or C tested. The antiserum-agar can also be used as a primary isolation medium for detecting nasopharyngeal carriers of a certain serogroup if antibiotics are added.
Rocket-line immunoelectrophoresis (R-LIE) with antigen containing intermediate gel, and co-agglutination utilising protein A-containing staphylococci coated with specific antibodies, were adapted for serotyping the prototypes of group B meningococci. Both were found to have the same specificity as agar gel double diffusion (AGDD) but they were more sensitive and more rapid than AGDD. R-LIE required, like AGDD, the extraction of relatively large quantities of bacteria, while the co-agglutination method, performed as a slide agglutination tests with results within a few minutes and no need of special equipment, required only a small amount of heated whole meningococcal cells. Meningococcal strains of serogroups B, C, and A from patients with carriers were serotyped and the results with all three methods were in agreement.
Initial cerebrospinal fluid (CSF) samples obtained from patients with pneumococcal meningitis usually contain far greater numbers of bacteria than initial CSF samples obtained from patients with meningococcal meningitis. Normal CSF was found to have an inhibitory effect on the growth of group A meningococci but not on type 1 pneumococci. The inhibitory effect of normal CSF was abolished by dialysis, indicating that the inhibitory factor has a low molecular weight. Heating normal CSF to 62.5 degrees C for 30 min resulted in a considerable reduction in the inhibitory effect, indicating that the inhibitory factor is heat labile.