Characteristics of Neisseria meningitidis strains isolated from patients with symptoms of meningococcal meningitis in Poland in 1995-2000

Phenotype and genotype identification of 179 Neisseria meningitidis strains isolated from cerebrospinal fluid or blood of patients with meningococcal infection, hospitalized in Poland, was performed. This is the first analysis of that type conducted in Poland. We analyzed strains collected in 1995-2000 from laboratories located all over the country. Phenotype Neisseria meningitidis B:22:P1.14 was the predominant among analyzed invasive strains in Poland. Type 22 is characteristic to most of the strains isolated in our country. No strain from analyzed group belonged to known epidemic clusters. One penicillin resistant strain (MIC = 2 mg/l) and about 27% strains with decreased susceptibility to penicillin (0.1 = < MIC < 1.0 mg/l) were present among 166 N. meningitidis tested. All strains were susceptible to ciprofloxacin and rifampicin.     

Serotypes and subtypes of Neisseria meningitidis serogroup B strains associated with meningococcal disease in Canada, 1977-1989

Typing of Neisseria meningitidis serogroup B disease isolates was carried out using a panel of serotype-and subtype-specific monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assays (ELISA). Three hundred and sixty-two strains isolated from 1977 to 1986 were typed using five serotyping and seven subtyping reagents and outer membrane vesicles as antigens. Serotype 2b accounted for 30% of the disease isolates. The most common subtype was P1.2, which occurred on 18.5% of all strains or 48.6% of the serotype 2b strains. Of the 362 strains typed, 135 (37.3%) were serotyped and 122 (33.7%) were subtyped. Overall, 185 (51.1%) of the strains could be assigned a serotype and (or) subtype. Strains (221) isolated during the years 1987-1989 were typed using a panel of 6 serotyping and 12 subtyping reagents by whole-cell ELISA. Strains of serotypes 4 (21.7%) and 15 (20.8%) were the most common and carried a wide variety of subtypes. The most common subtypes were P1.2 (11.8%) and P1.16 (9.5%). Of the 221 strains analyzed, 132 (59.7%) were assigned a serotype and 123 (55.7%) a subtype and with all 18 MAbs, 192 (86.9%) of the strains were serotyped and (or) subtyped. Two different MAbs to the four epitopes 2a, 15, P1.2, and P1.16 gave discordant reactions of 0.3, 6.6, 2.6, and 2.2%, respectively, when used to analyze over 300 strains of N. meningitidis.     

Polysaccharide-protein complexes isolated from different strains of Neisseria meningitidis serogroup B

Fractionation of the biomass of 3 serogroup B N. meningitidis strains, obtained from solid serum-free and liquid synthetic media, by increasing concentrations of cetavlone revealed that the formation of natural polysaccharide-protein complexes with the ratio of their components approaching 1:1 was possible under the conditions ensuring the intensive synthesis of capsular polysaccharide. Two strains, 125 and 1642, grown on a solid amino peptide-containing medium regularly produced two polysaccharide-protein complexes with the protein/polysaccharide ratio approaching 1:1. One of these complexes passed easily into the supernatant fluid and could be precipitated with 0.1% cetavlone. The second complex was more firmly bound to the outer membrane of the cell and could be precipitated with 1% cetavlone. In most experiments an additional fraction with high protein content in relation to sialic acid was isolated from the biomass.     

Sequence variation in class 1 outer membrane protein in Neisseria meningitidis isolated from patients with meningococcal infection and close household contacts

Abstract Aspergillus oryzae , which is widely used for Japanese traditional fermentation, produced at least two lipolytic enzymes (L1 and L2). Southern hybridization analysis of restriction enzyme-digested genomic DNA fragments of Aspergillus oryzae with 23-mer oligonucleotides synthesized according to the amino acid sequence of the enzyme L1 as probes suggested that there is single copy of the L1 gene in the genome. DNA fragments containing the L1 gene were cloned in Escherichia coli . Nucleotide sequencing of the DNA fragments revealed an open reading frame consisting of 213 amino acid residues. It had three putative introns whose sizes were 52 bp, 48 bp and 53 bp, respectively. Putative CAAT and TATA boxes were found at positions −147 and −100 from A (+1) of the translational initiation codon, and a polyadenylation site at 158 bp downstream of the stop codon. The deduced amino acid sequence of the L1 gene was highly similar to those of cutinases from phytopathogenic fungi. Thus, it is interesting to note that the non-phytopathogenic fungus, A. oryzae , produces cutinase, which seems to play an important role in flavor formation.     

Characterization of Neisseria meningitidis strains isolated from invasive meningococcal disease cases in Canada in 2001

With the recent introduction of polysaccharide-protein conjugated vaccines for the control of serogroup C meningococcal disease and the emergence of different variants of serogroup C meningococci, it is likely the epidemiology of meningococcal disease in many countries may be affected. We have therefore analysed and reported the characteristics of Neisseria meningitidis strains collected in 2001 from the Canadian surveillance program on invasive meningococcal disease. Only strains collected from normally sterile clinical sites of patients were studied. Of the 289 isolates obtained from individual patients, 173 (59.9%) were serogroup C, 76 (26.3%) were serogroup B, 30 (10.4%) were serogroup Y, and 10 (3.5%) were serogroup W135. Ninety-six percent of the serogroup C isolates belonged to the ET-15 clone, with an additional 2.3% belonging to other electrophoretic types within the ET-37 clonal complex. Different antigenic variants of the endemic serogroup C ET-15 clone were responsible for localized outbreaks in different parts of the country. One novel variant with the antigenic composition of C:2a:P1.1,7 was reported in two provinces, Quebec and Ontario. Eighteen percent of the meningococci isolated from patients in Ontario belonged to serogroup Y, compared with only 8% in the rest of Canada. The current data highlight the importance of strain characterization by serogroup, serotype, and serosubtype antigens in providing useful information for the surveillance of meningococcal disease in Canada.     

A novel monoclonal antibody to Neisseria meningitidis serogroup X capsular polysaccharide and its potential use in quantitation of meningococcal vaccines

A novel murine hybridoma monoclonal antibody (MAb) was produced against the capsular polysaccharide (CP) of Neisseria meningitidis serogroup X (MenX) in order to develop a sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of the meningococcal polysaccharide. The MAb only reacted with the CP from MenX and did not react with CPs from N. meningitidis serogroups A, C, Y and W (MenA, MenC, MenY, MenW). The affinity constant (Ka) of the MAb measured by non-competitive ELISA was 7.25 × 10⁷ M⁻¹. The application of this MAb in a sandwich ELISA was demonstrated by its ability to properly quantitate three lots of an experimental meningococcal CP-based vaccine. The MAb obtained in this work could be a valuable reagent for the detection and quantitation of future meningococcal vaccines containing MenX CP.     

Pulse electrophoresis of serogroup A streptocci isolated in Moscow

Pulse electrophoresis of group A streptococci (GAS) isolated in 1998 - 2004 in Moscow from patients during outbreaks of tonsillitis in child institutions, military unit and also from sporadic cases in children and adults was performed. During analysis of 48 strains 16 pulse electrotypes were recognized. These data allow to consider that in different child institutions (and other groups) accumulation and spreading of most adapted to such environment variants of GAS are take place and population structure of GAS in such institutions is unique on molecular and other characteristics. This study showed that complex typing of GAS (use of pulse electrophoresis and PCR for detection of erythrogenic toxins A and C) will help to improve molecular-epidemiologic surveillance for streptococcal group A infection, development of recommendations on reduction of morbidity from this infection and also to decrease risk of its severe forms, complications and mortality.     

Genetic structure of Neisseria meningitidis populations in relation to serogroup, serotype, and outer membrane protein pattern.

The genetic structure of populations of Neisseria meningitidis was examined by an analysis of electrophoretically demonstrable allelic variation at 15 genes encoding enzymes in 650 isolates of eight serogroups (A, B, C, W135, X, Y, Z, and 29E) and 38 nonserogroupable isolates. A total of 331 distinctive multilocus genotypes (electrophoretic types, ETs) was identified, among which mean genetic diversity per locus (H = 0.547) was greater than in Escherichia coli and other bacterial species thus far studied. The intercontinental distribution of some ETs and the recovery of organisms of identical genotype over periods of many years strongly suggest that the genetic structure of N. meningitidis is basically clonal as a consequence of low rates of recombination of chromosomal genes. Variation among strains in serogroup, serotype, and the electrophoretic pattern of the major outer membrane proteins has little relationship to the complex structure of populations revealed by enzyme electrophoresis, which involves 14 major lineages of clones diverging from one another at genetic distances greater than 0.50. Genetic diversity among ETs of isolates of the same serogroup was, on average, 84% of that in the total sample. Clones of serogroup A were unusual in being genotypically less heterogeneous than those of other serogroups and in forming a single phylogenetic group. Isolates of the same serotype or outer membrane protein pattern were also highly heterogeneous; on average, 87 and 97%, respectively, of the total species diversity was represented by ETs of the same serotype or outer membrane protein.     

Immunogenicity and evolutionary variability of epitopes within IgA1 protease from serogroup A Neisseria meningitidis

Five murine epitopes were defined and mapped within IgA1 protease produced by Neisseria meningitidis. Epitopes 1 and 2 were present in IgA1 protease from all strains, and from Neisseria gonorrhoeae. Epitopes 3 through to 5 varied between subgroups of serogroup A meningococci. but have remained constant over decades within the subgroups, except for epitope 4, which changed between 1983 and 1987 during the spread of subgroup III meningococci from Asia to Africa. Binding of monoclonal antibodies to epitopes 1, 4 and 5 neutralized enzymatic function. Human sera containing antibodies to lgA1 protease as a result of natural infection inhibited binding of monoclonal antibodies to epitope 4 but not to the other epitopes.     

Typing and susceptibility to penicillin of Neisseria meningitidis isolated from patients in Cuba (1993-1999)

The susceptibility to penicillin of 111 Neisseria meningitidis strains was assessed by the agar-dilution procedure and serosubtypes were determined by a whole-cell enzyme-linked immunoassay using monoclonal antibodies reagents. Thirty-five isolates showed reduced sensitivity to penicillin (MIC > or = 0.1 mg/l and < or = 1 mg/l) and no resistant strains were detected. The most common phenotype was B:4:P1.15 (77.5%) and a rising trend of non-typeable and non-subtypeable strains was detected. The increase in levels of minimal inhibitory concentrations of meningococci to penicillin gives cause for concern and the increase in non-typeable and non-subtypeable isolation demand the use of molecular biology techniques for their typing.     

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.     

Novel identification of expressed genes and functional classification of hypothetical proteins from Neisseria meningitidis serogroup A

To implement the 2-DE database of serogroup A Neisseria meningitidis (MenA) and improve its potential of investigation in bacterial biology, cell extracts were separated by tricine-SDS-PAGE and 131 novel proteins were identified by microLC-ESI-IT-MS/MS. These identifications extended to 404, the number of MenA gene expression products characterized at the proteome level, approximately covering 20% of the total ORFs predicted from genome sequence. This technical approach was particularly useful in ascertaining expression of ribosomal as well as hypothetical proteins. Particular attention was paid to functional characterization of hypothetical proteins by means of software analyses and database searches.     

Effect of a booster dose of serogroup B meningococcal vaccine on antibody response to Neisseria meningitidis in mice vaccinated with different immunization schedules

The generation and maintenance of memory antibody response by different primary immunization schedules with the Cuban-produced outer membrane protein based vaccine was investigated in a murine model. We analyzed the duration of the antibody response (IgG-ELISA and bactericidal titer) and the effect of a booster dose on the antibody response. The IgG avidity index was determined in an attempt to find a marker for memory development. This study also included an analysis of IgG subclasses induced by primary and booster immunization. The specificity of bactericidal antibodies was investigated using local strains of the same serotype/serosubtype (4,7:P1.19,15) as the vaccine strain and mutant strains lacking major outer membrane proteins. A significant recall response was induced by a booster dose given 7 months after a primary series of 2, 3 or 4 doses of vaccine. The primary antibody response showed a positive dose-effect. In contrast, a negative dose-effect was found on the booster bactericidal antibody response. There was a significant increase in IgG1 levels after the fourth and booster doses. Three doses of vaccine were required to induce a significant increase in IgG avidity. Two injections of vaccine induced a significant antibody response to PorA protein, while 4 injections induced a larger range of specificities.     

A群奈瑟氏脑膜炎球菌荚膜多糖-破伤风类毒素结合疫苗的制备及其免疫原性研究

采用溴化氰(CNBr)活化多糖,以无水己二酸二肼(ADH)作为连接剂,1乙基13(3二甲基氨基丙基)碳化二亚胺(EDAC)为偶联剂制备A群奈瑟氏脑膜炎球菌荚膜多糖(GAMP)与破伤风类毒素(TT)的结合物,经皮下免疫NIH小鼠,用ELISA检测小鼠血清中抗GAMP及抗载体蛋白的IgG抗体水平。用补体介导的体外杀菌试验检测血清中GAMP抗体的杀菌活性。结果显示,实验中制备的多糖衍生物和多糖蛋白质结合物都具有GAMP抗原特异活性。结合物免疫小鼠后可诱生比多糖单独免疫更高水平的GAMP血清IgG抗体,并能形成免疫记忆,产生再次应答。结合物免疫小鼠所诱生的血清GAMP抗体较之多糖组具有更强的体外杀菌活性。表明此方法制备的结合物可获得优于多糖的、稳定的特异免疫原性。     

Genotypic emm-typing of streptococcal cultures, group A, isolated from patients with different clinical manifestations of streptococcal infection in Moscow

The results of the intraspecific typing of group A streptococci (SGA), isolated from patients with different manifestations of group A streptococcal infection, carried out in Russia for the first time, are presented. The genotypic method of emm-typing, based on sequencing the DNA area coding the variable part of the molecule of SGA M-protein was used. The data obtained in this study made it possible to follow changes in the pattern of SGA M-types in Moscow and the specific features of SGA in comparison with the analogous data on other territories. New emm-types of SGA circulating on the territory of Russia were detected and described.     

The protective activity of the detoxified lipopolysaccharide of Neisseria meningitidis serogroup A in in vivo experiments

The immunogenic potency, toxicity, homologous and heterologous protective activity of lipopolysaccharide preparations obtained from serogroup A N. meningitidis (LPS A) were studied in animal experiments. These preparations were shown to possess very high protective activity. The alkaline treatment of native LPS A decreased the toxicity of the preparation almost 20 times and did not affect its immunogenic potency. Detoxified LPS A was capable of protecting mice from fatal meningococcemia resulting from infection with N. meningitidis strains, serogroups A, B and C; the adsorption of the preparation on aluminium hydroxide did not affect its protective activity. In view of the properties of detoxified LPS A revealed in this investigation, it may be regarded as a possible vaccinal preparation.     

Multilocus genotypes determined by enzyme electrophoresis of Neisseria meningitidis isolated from patients with systemic disease and from healthy carriers

Variation in nine enzymes in 152 isolates of Neisseria meningitidis from Norway (118 from blood or cerebrospinal fluid of patients with systemic disease and 34 from the pharynx of healthy carriers) was analysed by starch-gel electrophoresis. All nine enzymes were polymorphic and the number of allozymes (electromorphs) identified per locus ranged from 3 to 12, with a mean of 6.1. Among the 152 isolates, 55 unique combinations of electromorphs (electrophoretic types, ETs) were distinguished. Twenty ETs were represented among the carrier isolates and 37 among the systemic isolates; hence, only two ETs were found in both groups of isolates. ET-5 was identified 67 times among the 118 systemic isolates (58%), indicating an association of this ET with invasiveness; ET-5 was also the most common type among the carrier isolates (18%). Genetic similarity between ETs was analysed by pairwise comparison of all 55 ETs with respect to the number of electromorphs by which they differed. No evidence of a general genetic difference between carrier and case isolates was found. Two well-defined clusters of ETs were observed, each including one of the two most common ETs identified among the systemic isolates (ET-5 and ET-37), together with isolates differing from them only at one or two loci. All isolates of ET-5 and ET-37, as well as their closely related variants defined by the similarity matrix, were resistant to sulphonamide, independent of their antigenic characteristics and isolation site. The extensive allozyme variation among isolates of the same serogroup demonstrated the limited value of serogrouping as an epidemiological tool. All but one isolate of serotype 15:P1.16 were electrophoretically similar, as were all the 2a:P1.2 isolates. The 15:P1.15 isolates, however, were genetically heterogeneous. The distribution of alleles in genotypes identified among the systemic isolates indicated that genetic recombination may occur in natural populations of N. meningitidis.     

Genetic diversity of Neisseria meningitidis strains isolated in Rio de Janeiro, Brazil, evaluated by multilocus enzyme electrophoresis

AIMS: To analyse Neisseria meningitidis isolates from meningococcal meningitis cases in Rio de Janeiro (Brazil) from 1990 to 1993 and 1999-2002, to determine the genetic and relatedness with hypervirulent and epidemic strains. METHODS AND RESULTS: The isolates were analysed by multilocus enzyme electrophoresis (MEE) clustering into 83 electrophoretic types (ET). All isolates from 1999 to 2002, formed a cluster which included one strain of the ET-5 complex worldwide associated with epidemics. CONCLUSIONS: The overall results suggested a panmictic structure probably because of recombination events. The observation of a separated cluster including isolates from 1999 to 2002 and an ET-5 complex strain, also suggested the introduction of strains genetically related with this hypervirulent complex in the State of Rio de Janeiro (Brazil) over the last 5 years. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of strains related to the ET-5 complex in several states of Brazil was already described elsewhere, but this is the first time it was reported in the State of Rio de Janeiro. Our findings reinforce the necessity to genetically determine the clones which should be considered to produce a national vaccine against meningococcal meningitis.