Neisseria Meningitidis Strains Research Articles

Early development in healthy children of serum opsonins against nonpathogenic Neisseria meningitidis.

In an earlier study, with the use of chemiluminescence (CL) and phagocytic killing, we could show that in the presence of serum from healthy adults polymorphonuclear leukocytes (PMNL) efficiently handle nonpathogenic Neisseria meningitidis strains, in sharp contrast to those associated with clinical disease. The major part of this difference was dependent on serum factors. In the present study 84 serum samples from children 1-3, 4-6, 7-9, and 10-14 years old were studied by the CL technique according to their ability to opsonize meningococci. There was a highly significant difference (p less than 0.001) in all four age groups when the CL indexes obtained with the pathogenic meningococci of the serogroups A, B and C were compared with those of the nonpathogenic menigococci: serogroup 29E and nongroupable meningococci. These findings imply that the ability to opsonize so-called nonpathogenic meningococci is developed early in life and may explain why they are only occasionally able to cause disease.

Specificity of antibodies to O-acetyl-positive and O-acetyl-negative group C meningococcal polysaccharides in sera from vaccinees and carriers

Most group C Neisseria meningitidis strains produce an O-acetyl-positive polysaccharide, a homopolymer of alpha-2----9-linked N-acetylneuraminic acid with O-acetyl groups at the C-7 and C-8 of its sialic acid residues. The majority of disease isolates have been reported to contain this polysaccharide. Some strains produce group C polysaccharide lacking O-acetyl groups. The licensed vaccine contains the O-acetyl-positive polysaccharide. We have measured the antibody specificities to the two polysaccharides in sera from asymptomatic group C meningococcal carriers and vaccinated adults by a new enzyme-linked immunosorbent assay (ELISA) procedure using methylated human serum albumin for coating the group C polysaccharide onto microtiter plates. Inhibition of binding of serum antibodies to polysaccharide-coated plates was measured by ELISA after incubation with O-acetyl-positive and O-acetyl-negative group C polysaccharides. Greater inhibition of binding of carrier sera was observed with the homologous polysaccharide. There was substantial inhibition of binding of vaccinee sera to the O-acetyl-positive polysaccharide-coated plate following preincubation with O-acetyl-positive polysaccharide, but homologous inhibition on plates coated with the O-acetyl-negative polysaccharide required much higher concentrations of polysaccharide. Carrier sera may have a higher proportion of antibodies with greater specificity for the O-acetyl-negative polysaccharide, while vaccinee sera contain antibodies with greater affinity for the O-acetyl-positive polysaccharide. Studies with monoclonal antibodies specific for O-acetyl-positive and O-acetyl-negative polysaccharides reveal that the percentage of group C meningococcal disease caused by O-acetyl-negative strains remains about 15%, as found over 15 years ago.

Iron and outer membrane proteins in the susceptibility of Neisseria meningitidis to human serum

The proportion of carrier-isolated Neisseria meningitidis strains sensitive to human serum (37.2%) was found to be significantly higher than that of case-isolated ones (4.1%), although the difference is too low to consider serum-resistance responsible for invasion in this microorganism. Serum-susceptibility was not related to the existence of specific outer membrane proteins, as is the case of N. gonorrhoeae. Iron restriction induced iron-regulated outer membrane proteins in each strain (but not the same proteins in all strains) but without any detectable effect on serum-susceptibility. Iron excess was also unable to induce changes in the susceptibility of N. meningitidis to human serum.

Failure of adherence to buccal cells and surface hydrophobicity as virulence markers in Neisseria meningitidis

Surface hydrophobicity and adherence to buccal epithelial cells were studied in 33 carrier and 34 invasive Neisseria meningitidis strains. It was found that hydrophobicity is statistically similar in both groups (P = 0.0507) although it could be considered that carrier strains are slightly more hydrophobic than invasive ones. Adherence was similar in both groups although more homogeneous in the carrier strains. No correlation could be demonstrated between these two properties nor can they be considered relevant as markers of the carrier or invasive status of this bacterium, indicating that at least in N. meningitidis they are not good properties to discriminate virulent strains.

Epitopes of serogroup B Neisseria meningitidis analysed in vitro and directly from cerebrospinal fluid

Two type B15 P1.16 strains of Neisseria meningitidis were examined by immunogold electron microscopy for accessibility of two outer membrane protein (OMPs) to monoclonal antibodies. Both strains exhibited cell-to-cell variation of one epitope of the Class 3 OMP (P3.15) and one of the Class 1 OMPs (P1.16) when grown in vitro. One strain, a nasopharyngeal isolate revealed this variation to be growth-phase independent and double labelling of both epitopes showed independent variation. CSF containing N. meningitidis was stored in liquid nitrogen without laboratory processing at the time of isolation of the second strain. Direct analysis of the organisms showed no cell-to-cell variation and immunoglobulin G on the surface. However, while there were similar labelling densities of the Class 1 epitope in vivo compared with either strain grown in vitro, there was a lower labelling density of the Class 3 epitope in vivo that was not caused by freeze-thawing. This reduction may be due to decreased expression of this epitope in vivo which casts doubts on the use of the Class 2/3 OMP as a vaccine candidate.

Epitopes of serogroup BNeisseria meningitidisanalysed in vitro and directly from cerebrospinal fluid

Two type B15 P1.16 strains of Neisseria meningitidis were examined by immunogold electron microscopy for accessibility of two outer membrane protein (OMPs) to monoclonal antibodies. Both strains exhibited cell-to-cell variation of one epitope of the Class 3 OMP (P3.15) and one of the Class 1 OMPs (P1.16) when grown in vitro. One strain, a nasopharyngeal isolate revealed this variation to be growth-phase independent and double labelling of both epitopes showed independent variation. CSF containing N. meningitidis was stored in liquid nitrogen without laboratory processing at the time of isolation of the second strain. Direct analysis of the organisms showed no cell-to-cell variation and immunoglobulin G on the surface. However, while there were similar labelling densities of the Class 1 epitope in vivo compared with either strain grown in vitro, there was a lower labelling density of the Class 3 epitope in vivo that was not caused by freeze-thawing. This reduction may be due to decreased expression of this epitope in vivo which casts doubts on the use of the Class 2/3 OMP as a vaccine candidate.

Serum antibodies to cross‐reactive Neisseria outer membrane antigens in healthy persons and patients with meningococcal disease

Outer membranes (OMs) were prepared from the Neisseria meningitidis (Nm) strain 44/76, N. gonorrhoeae (Ng) NRL 8658, and N. lactamica (N1) ATCC 23970. Paired serum samples from 16 patients with serogroup B Nm disease and single samples from 30 blood donors were tested for IgG antibody levels against the three OMs in indirect ELISA, before and after absorption of the sera with N1 OM. Immunoblot analysis was used to identify OM target antigens for cross-reacting and strain-specific antibodies. Most of the Nm- and NG-antibodies in sera from healthy adults were directed against OM antigens shared by the three Neisseria strains. Nm disease induced antibody formation against common Neisseria antigens, identified as the H.8 antigen and LPS determinants, against LPS determinants shared only by the Nm and Ng strains and against a variety of Nm-specific OM antigens. Very low levels of Nm-specific antibodies characterized the Nm patients in the acute phase. Also, the results indicate that the OM ELISA which has been used for the diagnosis of Nm disease, would be more useful if antibodies against common Neisseria antigens were removed from the sera before testing.

Protective efficacy of mouse serum to the N-propionyl derivative of meningococcal group B polysaccharide

The protective properties of antibodies induced by immunization of mice with a conjugate of tetanus toxoid and the N-propionyl derivative of group B meningococcal polysaccharide ( N-Pr-GBMP-TT) have been investigated. Mice immunized with the conjugate produced antibodies which were bactericidal for Neisseria meningitidis strains B:2b:P1.Ham and B:15:P1.16. Passive protection studies indicated that the conjugate serum completely eliminated or reduced considerably levels of bacteremia by the same strains in mice. There was no bactericidal activity or passive protection against a strain of N. meningitidis C:2b:P1.2. Following absorption of the conjugate serum with GBMP the non-absorbed antibody, directed to N-Pr-GBMP, was bactericidal and protected mice against bacteremia with group B meningococci. Thus N-Pr-GBMP antibodies which do not bind to the GBMP are protective in vitro and in vivo.

Characterization of a silent pilin gene locus from Neisseria meningitidis strain FAM18

We cloned and characterized a silent pilin locus ( pilS) in the chromosome of Neisseria meningitidis strain FAM18. This locus represents the sole region of the FAM18 chromosome with strong homology to a gonococcal pilin gene. The FAM18 pilS locus encodes two tandem, in-frame, truncated pilin genes and shares many features with the previously described pilS locus of N. meningitidis strain C114. However, DNA sequence comparison shows that different information resides in the hypervariable region of one of the gene copies between the two strains. The conservation of reading frames within silent copies and the sequence diversity in hypervariable regions are reminiscent of gonococcal pilS loci and suggest that pilS loci may be of functional importance in the meningococcus.

A reference procedure to study chemiluminescence induced in polymorphonuclear leukocytes by Neisseria meningitidis.

Luminol-enhanced chemiluminescence (CL) was used to study the ability of various strains of Neisseria meningitidis (MC) to induce oxidative metabolism of polymorphonuclear leukocytes (PMNL); an indirect measure of phagocytic activity. To circumvent variations related to different PMNL donors, a MC serogroup X strain was used as a control for indexing the CL responses induced by other MC strains. This procedure, with pooled serum from healthy blood donors to standardize opsonising conditions, gave reproducible and comparable results, irrespective of PMNL donors. Under these conditions, there was a highly significant difference between pathogenic and non-pathogenic MC strains as regards their ability to induce CL responses (p less than 0.001). The results indicated that the differences were due partly to opsonizing antibodies, partly to other differences related to pathogenicity of tested MC strains. These differences in leukocyte/MC interaction were also confirmed by phagocytic-killing experiments. The index procedure of CL measurements may be a suitable method to study the appearance of natural immunity to MC disease, as well as the pathogenicity of particular MC strains.

Host range of the conjugative 25.2-megadalton tetracycline resistance plasmid from Neisseria gonorrhoeae and related species

High-level tetracycline resistance in strains of Neisseria gonorrhoeae, Neisseria meningitidis, Kingella denitrificans, and Eikenella corrodens has recently been described. The resistance in each species is due to the acquisition of 25.2-megadalton conjugative plasmids that carry the tetracycline resistance determinant TetM. We examined the ability of commensal Neisseria species to serve as recipients in conjugation for these new plasmids. Most of the recipients (n = 21) tested had detectable conjugation frequencies (greater than 10(-9] with one or more of the donor strains. Transfer was not detected in Branhamella catarrhalis. Transconjugants were able to maintain the plasmids and act as donors in subsequent matings.

Detection of relatively penicillin G-resistant Neisseria meningitidis by disk susceptibility testing

Beginning in 1985, relatively penicillin G-resistant (Penr) meningococci which did not produce beta-lactamase were isolated from the blood and cerebrospinal fluid of patients in Spain. We identified 16 Penr (mean MIC, 0.3 microgram/ml; range, 0.1 to 0.7 microgram/ml) and 12 penicillin-susceptible (Pens; mean MIC, less than or equal to 0.06 microgram/ml) strains of Neisseria meningitidis by the agar dilution technique using an inoculum of 10(4) CFU and questioned which disk susceptibility test would best differentiate these two populations. We compared the disk susceptibility of these strains using disks containing 2 (P2) and 10 (P10) U of penicillin G, 2 (Am2) and 10 (Am10) micrograms of ampicillin, and 1 microgram of oxacillin (OX1). We also investigated susceptibility with disks containing 30 micrograms of each of cephalothin (CF30), cefoxitin (FOX30), cefuroxime (CXM30), and cefotaxime (CTX30) and 75 micrograms of cefoperazone (CFP75) and determined by cluster analysis any correlation with the zone diameters obtained with P2 disks. Using the P2 and AM2 disks (in contrast to the P10 and AM10 disks), we correctly differentiated all the Penr from Pens isolates. In addition, the zone diameters with the P2 disk gave the best correlation with the penicillin G MIC determinations. All 16 Penr strains and 3 of 12 Pens strains showed zone diameters of 6 mm around OX1 disks, limiting the usefulness of OX1 disks. The zone diameters obtained with CF30, CXM30, and OX1 disks correlated with those obtained with the P2 disk, which suggests that these antibiotics have similar effects on these strains. In contrast, the data obtained with FOX30, CTX30, and CFP75 disks did not cluster with those obtained with the P2 disk, which suggests that there was a difference in the bacterial target or reflects their greater activity. We conclude that the P2 disk tests more readily identify Penr meningococci than do the standard P10 disk tests.

Endotoxin Release from Invasive Meningococci Related to Sulfonamide Resistance, Serogroup and Serotype

The relationship between endotoxin liberation, sulfonamide resistance, serogroups and serotypes was studied in 28 Neisseria meningitidis strains isolated from patients with meningococcal disease. Sulfonamide resistance was present in 15/28 strains. 22 strains belonged to serogroup B, and 5 to group C; 1 strain was non-groupable. Free endotoxin activity in growing cultures of meningococci with endotoxin titre of greater than or equal to 10(2) was found in 27/28 strains. A high endotoxin activity was present in both sulfonamide-sensitive and -resistant invasive meningococci. A high endotoxin release with titre greater than or equal to 10(3) seemed to be more associated with serogroup C than B, and more to the serotypes 2 and 15/16 than to the non-typable strains.

Evaluation of hydrophobicity and adherence of Neisseria Meningitidis strains and a study of their correlation by analysis of alterations induced by antibiotics

Surface hydrophobicity and adherence to human buccal epithelial cells were evaluated in four Neisseria meningitidis strains. Hydrophobicity was measured by partition into p-xylene in the presence or absence of ammonium sulphate, whereas radioactivity-labelled bacteria were used to estimate the ability to adhere to buccal cells by liquid scintillation techniques. Eight antibiotics were employed to induce modifications in the two parameters in order to estimate their possible relationship. The presence of ammonium sulphate enhanced the partition into p-xylene and hence the hydrophobicity values obtained. There was no correlation between adherence and hydrophobicity (no matter what the method employed to evaluate the latter). The effect of the antibiotics varied as a function of the strain, method and/or parameter tested.

Studies on the implication of surface hydrophobicity in the adherence of Neisseria meningitidis to buccal epithelial cells.

The study of surface hydrophobicity and adherence to human buccal epithelial cells of four Neisseria meningitidis strains and the correlation analysis of the data obtained for both parameters has demonstrated that hydrophobicity of meningococci is not a causative factor in adherence in normal conditions. Modification of the bacterial surfaces (by freezing, heating, ultraviolet irradiation, fixation with glutaraldehyde or sodium metaperiodate cleavage of external sugars) induced significant changes in one or both parameters, but these changes were not correlative in both properties. In contrast to other bacteria, the hydrophobicity levels shown by our meningococci are not relevant to their adherent processes.

Neisserial antigen H.8 is immunogenic in patients with disseminated gonococcal and meningococcal infections.

Antigenic diversity among and within strains of Neisseria gonorrhoeae and Neisseria meningitidis has complicated studies of the pathogenesis of these strains and obstructed vaccine development. We previously described a distinct surface antigen (H.8) common to pathogenic Neisseria. We have now demonstrated in vivo expression of the H.8 antigen by detecting antibody responses to the antigen in 13 patients with disseminated neisserial infections. Each serum sample from a convalescent patient blocked the binding between the infecting meningococcal or gonococcal strain and a monoclonal antibody directed to the H.8 antigen, as demonstrated by binding-inhibition studies in enzyme-linked immunosorbent assays (P less than .005). Testing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting demonstrated an IgG response in each convalescent serum to an antigen co-migrating with the H.8 antigen. Specificity of this antibody response was confirmed by probing recombinant bacteriophage that expressed the H.8 antigen. The commonality and the immunogenicity of the H.8 antigen indicate its possible role in the pathogenesis of, and its potential as a vaccine component for, gonococcal and meningococcal diseases.

Influence of nutrient limitation and low pH on serogroup B Neisseria meningitidis capsular polysaccharide levels: correlation with virulence for mice.

Neisseria meningitidis strain M986, which possesses a polyanionic sialic acid capsular polysaccharide, was resistant to the bactericidal effects of normal rabbit serum, but sensitive when immune serum and complement were present. An isogenic strain PRM102, deficient in the ability to produce capsular polysaccharide, was sensitive to normal serum. Strain M986, when grown under conditions of low pH or nutrient limitation, synthesized increased levels of capsular polysaccharide. This was accompanied by an increase in cell surface hydrophilicity and virulence for mice. Cells grown in low-pH, iron-limited medium synthesized the highest concentration of polysaccharide and exhibited the highest cell surface hydrophilicity and virulence among the cases examined. The increase in capsular polysaccharide was partly explained by a decrease in the specific activity of a membrane-bound cytidine monophosphate-N acetylneuraminic acid hydrolase. The results suggest that conditions of nutrient limitation and low pH exert profound effects on the physicochemical nature of the meningococcal cell surface which, in turn, cumulate in enhanced virulence of this organism for mice.

Differentiation of B15 strains of Neisseria meningitidis by DNA restriction endonuclease fingerprinting.

The restriction endonuclease fingerprinting technique was applied to meningococcal DNA in an attempt to identify individual strains of Neisseria meningitidis B15 (serogroup B, serotype 15), which causes approximately 90% of cases of meningococcal disease in northern Norway. Thirty representative strains (10 each from asymptomatic pharyngeal carriers, patients with septicemia, and patients with meningitis) were investigated with the restriction endonucleases Hind III and Eco RI. The 10 carrier strains showed a remarkable heterogeneity of fingerprints that rendered each strain easily distinguishable from the others. The 10 strains from the blood and the 10 from the cerebrospinal fluid showed similar but not identical restriction patterns. The results obtained with the two endonucleases were in perfect agreement. Our data suggest that a large number of different B15 clones are present in the population of northern Norway, but that only one single clone causes invasive meningococcal disease.

Superoxide dismutase, catalase and peroxidase in four strains of Neisseria meningitidis of different virulence

Superoxide dismutase, catalase and peroxidase in four strains of Neisseria meningitidis of different virulence

Monoclonal antibodies to serotype 2 and serotype 15 outer membrane proteins of Neisseria meningitidis and their use in serotyping.

A series of murine monoclonal antibodies to serotype 2 and serotype 15 strains of Neisseria meningitidis were produced which were specific for outer membrane proteins of classes 1, 2, 3, and 5. A panel of eight monoclonal antibodies that exhibited a high degree of serotype specificity when reacted with prototype strains of known serotype were selected for study. Each of the corresponding epitopes was localized on a specific outer membrane protein by means of immunoprecipitation, electroblotting, or both. The serotype 2a-, 2b-, and 2c-specific antibodies bound to the class 2 protein, the serotype 15-specific antibody bound to the class 3 protein, two antibodies (3-1-P1.2 and 3-1-P1.16) bound to class 1 proteins, and two antibodies (1-1-P5.1 and 3-1-P5.2) bound to class 5 proteins. Six of these monoclonal antibodies were used in a spot-blot procedure to survey 122 case isolates (groups B, C, Y, and W135) and 363 carrier isolates (all serogroups) for the presence of the 2a, 2b, 2c, 15, P1.2, and P1.16 epitopes. A total of 66% of the case isolates and 30% of the carrier isolates reacted with one or more of the monoclonal antibodies. The use of monoclonal antibodies for serotyping of meningococci appears to be feasible and easy and appears to have significant advantages over the use of polyclonal typing sera.