Molecular epidemiology of recent belgian isolates of Neisseria meningitidis serogroup B

In Belgium an increase in the incidence of meningococcal disease has been noted since the early 1990s. Four hundred twenty clinical strains isolated during the period from 1990 to 1995, along with a set of 30 European reference strains, and 20 Dutch isolates were examined by random-primer and repetitive-motif-based PCR. A subset was investigated by multilocus enzyme electrophoresis and pulsed-field gel electrophoresis. The data were compared with results obtained by serotyping (M. Van Looveren, F. Carion, P. Vandamme, and H. Goossens, Clin. Microbiol. Infect. 4:224-228, 1998). Both phenotypic and molecular epidemiological data suggest that the lineage III of Neisseria meningitidis, first encountered in The Netherlands in about 1980, has been introduced in Belgium. The epidemic clone, as defined by oligonucleotide D8635-primed PCR, encompasses mainly phenotypes B:4:P1.4 and B:nontypeable:P1.4, but strains with several other phenotypes were also encountered. Therefore, serotyping alone would underestimate the prevalence of the epidemic clone.     

Characterization of Neisseria meningitidis isolates and clinical features of meningococcal conjunctivitis in ten patients

Genetic defects of the blood platelet membrane glycoproteins, GPIIb-IIIa (alpha IIb/beta 3; CD41/CD61) and GPIb-V-IX (CD42) are the origin of several rare bleeding disorders, the best known of which are Glanzmann's thrombasthenia, Bernard-Soulier syndrome, and platelet-type von Willebrand's disease. In Glanzmann's thrombasthenia, GPIIb-IIIa are missing or defective and platelet aggregation is lacking or reduced. Either gene can be affected and mutations leading to lack of expression or to expression of poorly functional forms have been described. In Bernard-Soulier syndrome, GPIb-V-IX are missing or defective, leading to poor platelet adhesion at high-shear stress to damaged vessel wall and reduced platelet response to thrombin. Mutations in both GPIb alpha (CD42b) and GPIX (CD42a) have been described. Mutations in GPIb alpha can also lead to platelet-type von Willebrand's disease in which GPIb-V-IX are expressed normally but bind von Willebrand's factor spontaneously, which leads to platelet aggregation and thrombocytopenia.     

Cloning and characterization of the ponA gene encoding penicillin-binding protein 1 from Neisseria gonorrhoeae and Neisseria meningitidis

The ponA gene encoding penicillin-binding protein 1 (PBP 1) from Neisseria gonorrhoeae was cloned by a reverse genetic approach. PBP 1 was purified from solubilized membranes of penicillin-susceptible strain FA19 by covalent ampicillin affinity chromatography and used to obtain an NH2-terminal amino acid sequence. A degenerate oligonucleotide based on this protein sequence and a highly degenerate oligonucleotide based on a conserved amino acid motif found in all class A high-molecular-mass PBPs were used to isolate the PBP 1 gene (ponA). The ponA gene encodes a protein containing all of the conserved sequence motifs found in class A PBPs, and expression of the gene in Escherichia coli resulted in the appearance of a new PBP that comigrated with PBP 1 purified from N. gonorrhoeae. A comparison of the gonococcal ponA gene to its homolog isolated from Neisseria meningitidis revealed a high degree of identity between the two gene products, with the greatest variability found at the carboxy terminus of the two deduced PBP 1 protein sequences.     

Neisseria meningitidis produces iron-regulated proteins related to the RTX family of exoproteins

A monoclonal antibody (A4.85) which reacted with Fe-regulated proteins of Neisseria meningitidis, was used to isolate a lambda gt11 clone from N. meningitidis FAM20. Chromosomal fragments flanking the fragment expressing the A4.85 epitope were cloned, and their DNA sequences revealed a 3,345-bp open reading frame predicting a 122-kDa protein. This gene was named frpA (Fe-regulated protein). A computer similarity search of GenBank revealed high levels of similarity to members of the RTX family of cytotoxins, especially in a region of tandem 9-amino-acid repeats. These repeats are found in all members of the RTX family; similar repeats were present 13 times in the predicted FrpA protein. Antigenic relatedness between the meningococcal proteins and the RTX proteins was demonstrated by the reactivity of A4.85 with Escherichia coli hemolysin (HlyA) and Bordetella pertussis adenylate cyclase-hemolysin (CyaA). Similarly, FrpA was recognized by 9D4, a monoclonal antibody directed against B. pertussis CyaA. In addition to the frpA gene, a second gene (frpC) produced a larger RTX-related protein. The frpA and frpC loci were mutagenized in strain FAM20, resulting in the loss of RTX-related proteins. A 120-kDa protein was expressed from the reconstructed frpA gene in E. coli. The biological function of FrpA is unknown, but its similarity to other RTX toxins suggests that it may play an important role in the pathogenesis of meningococcal infection.     

Changes in clinical and epidemiologic characteristics in Western Bohemia of invasive meningococcal disease associated with the occurrence of an invasive clone of Neisseria meningitidis

The authors analyzed the incidence of meningococcal diseases in the West Bohemian region in 1982-1996. The draw attention to changes of clinical and epidemiological characteristics of the disease which appeared in 1994 in conjunction with a new invasive clonus of Neisseria meningitidis C:2a:P1.2, P1.5, ET-15/37. While in 1982-1993 invasive meningococcal diseases had in 75% the course of meningitis with a relatively low fatality (4%), during the subsequent period a marked change occurred. Since 1994 the disease took in the West Bohemian region in 58% the course of sepsis with a fatality of 16%. 25% cases of meningococcal meningitis were diagnosed combined sepsis and meningitis in 17%. The disease lost its seasonal character and the authors confirmed the highest incidence of the disease in the age group from 15-19 years and 0-4 years. Neisseria meningitidis group C was detected in 1994-1996 in 73% and the invasive clone C:2a:P1.2, P1.5, ET-15/37 in 62%.     

Diversity of the transferrin-binding protein Tbp2 of Neisseria meningitidis

In order to investigate the genetic basis for the observed polymorphism amongst meningococcal transferrin-binding proteins, Tbp2, the corresponding genes of different Neisseria meningitidis strains were cloned and sequenced. Comparison of the deduced amino acid (aa) sequences indicated that the Tbp2 were 76.6 to 81.2% homologous. Several stretches of aa have been found repeated both in the N- and C-terminal halves of the molecule.     

Analysis of obstetric complications reported to the National Patient Insurance Association in Finland from 1987 to 1995

OBJECTIVE: To review comparative studies evaluating oral propafenone for restoring sinus rhythm in recent onset atrial fibrillation. DATA SOURCES: A MEDLINE search of the English-language literature (1966 to 1996) along with any referenced articles not identified by MEDLINE. STUDY SELECTION: Because intravenous propafenone is not marketed in Canada, only studies evaluating oral propafenone were included. Studies were selected if they compared oral propafenone with placebo or other antiarrhythmic agents for converting recent onset atrial fibrillation to normal sinus rhythm. DATA SYNTHESIS: Propafenone is often used as a first-line agent for pharmacological cardioversion of atrial fibrillation. In earlier studies, the efficacy of propafenone in restoring sinus rhythm was reported to be low with conversion rates of 6% to 62%. Many of these studies were noncomparative and often included patients with refractory, chronic atrial fibrillation or employed suboptimal doses of propafenone. More recently propafenone has been evaluated in the treatment of recent onset atrial fibrillation by using a single 600 mg oral loading dose. Success rates of 76% at 8 h and 83% at 12 h following the loading dose are reported. The incidence of atrial flutter during active treatment was similar to that with placebo, with the majority exhibiting 2:1 or greater atrioventricular conduction ratios and heart rates 150 beats/min or less. CONCLUSIONS: A single 600 mg oral dose of propafenone is highly effective at restoring sinus rhythm in patients with acute onset atrial fibrillation with few adverse effects. The small studies reviewed cannot lead to definitive conclusions about the safety of propafenone without prior administration of agents for rate control.     

Transport of intact porphyrin by HpuAB, the hemoglobin-haptoglobin utilization system of Neisseria meningitidis

The meningococcal hemA gene was cloned and used to construct a porphyrin biosynthesis mutant. An analysis of the hemA mutant indicated that meningococci can transport intact porphyrin from heme (Hm), hemoglobin (Hb), and Hb-haptoglobin (Hp). By constructing a HemA- HpuAB- double mutant, we demonstrated that HpuAB is required for the transport of porphyrin from Hb and Hb-Hp.     

Preparation and analysis of isogenic mutants in the transferrin receptor protein genes, tbpA and tbpB, from Neisseria meningitidis

Isogenic mutants were constructed in the tbpA and tbpB genes from Neisseria meningitidis strain B16B6, which code for the transferrin receptor proteins, Tbp1 and Tbp2. Insertion mutants of the tbpA and tbpB genes were obtained by shuttle mutagenesis and by in vitro cassette mutagenesis, respectively. The isogenic mutants were verified by Southern blot and Western blot analysis. Isogenic mutants deficient in Tbp1 or Tbp2 demonstrated a reduced transferrin binding activity in intact cells and total membranes but were incapable of utilizing transferrin iron for growth. Tbp1 could be isolated by affinity methods from the mutant lacking Tbp2 but isolation of Tbp2 from the mutant lacking Tbp1 required the presence of exogenous Tbp1.     

Rapid epidemiological characterization of Neisseria meningitidis using polymerization chain reaction from biological samplings

OBJECTIVES: Due to the spread of the meningococcal infections, a good epidemiological surveillance is needed. Prophylactic measures should be undertaken because of the high transmissibility of these bacteria. One problem which hinders the epidemiological characterization is that the responsible strain should be isolated. The aim of this work is to develop a rapid and non culture typing method of Neisseria meningitidis. METHODS: Six cerebrospinal fluids were obtained from 5 different patients with meningococcal meningitis. A specific locus, pil A, for N. meningitidis was amplified by polymerization chain reaction (PCR). The polymorphism of this locus was then analyzed by digesting the PCR products with one of three different restriction enzymes. RESULTS: The polymorphism of this locus allowed us to establish the clonal relationships between the meningococcal strains involved in the infection. Three CSF corresponded to epidemiological strains. CONCLUSION: This typing method allows a rapid and less expensive epidemiological characterization of meningococcal infections. Moreover, it is a non culture typing method.     

Trends in breast cancer screening in Missouri from 1987 to 1995, and predictions for the years 2000 and 2010

BACKGROUND: Breast cancer is a major cause of morbidity and mortality in the United States (U.S.) and Missouri. In 1992, 3,915 new breast cancer cases were diagnosed and in 1995, 1,006 deaths from breast cancer were reported in Missouri. Although breast cancer incidence has increased in Missouri in the past 20 years, there are indications that early detection has also increased during the same period. Knowledge about which segments of the population have experienced the greatest increase in mammography screening rates helps in planning and implementation of breast cancer control programs at the state level. OBJECTIVES: Examine the prevalence and trends of lifetime mammography and 2-year mammography compliance in Missouri by age, race, and education from 1987 to 1995 and make predictions for the years 2000 and 2010. METHODS: We used data from the Missouri Behavioral Risk Factor Surveillance System (BRFSS), 1987 to 1995, to estimate the prevalence of ever having had a mammogram and compliance with mammography screening guidelines within two years by race, age, and education status among Missouri women over age 18. Using linear models, we regressed breast cancer screening prevalence estimates on time to obtain trends and predictions. RESULTS: Overall, African-American women were more likely to have had a lifetime mammogram than white women. However, we found a steady increase in the prevalence of ever having had a mammogram for all groups of women defined by age and education status, except among African Americans. Increase in the prevalence of ever having had a mammogram was much higher in women age 50 and older and slightly higher among women with a high school education or less. The average prevalence of 2-year mammography screening compliance was about 60% for all groups, a rate which did not significantly change between 1987 and 1995. By the year 2000, white women will have mammography rates equal to or higher than African-American women, and the majority of all women age 50 and older (98.3% to 100%) will have had a lifetime mammogram. CONCLUSION: Missouri target populations are predicted to attain Year 2000 National Health Objectives concerning lifetime mammography. Current efforts should be continued in order to maintain levels of mammography, particularly among African-American women.     

Anti-Gal binds to pili of Neisseria meningitidis: the immunoglobulin A isotype blocks complement-mediated killing

alpha 1,3-Galactosyl antibodies (anti-Gal) are ubiquitous natural human serum and secretory polyclonal antibodies that bind to terminal galactose-alpha 1,3-galactose (alpha-galactosyl) residues. Serum immunoglobulin G (IgG) anti-Gal can block alternative complement pathway-mediated lysis of representative gram-negative enteric bacteria that bind it to lipopolysaccharide alpha-galactosyl structures, thereby promoting survival of such bacteria in the nonimmune host. We wanted to know whether anti-Gal also could bind to the lipooligosaccharides (LOS) of Neisseria meningitidis. To our surprise, we found that serum and secretory anti-Gal bound to pili but not to LOS of certain strains. This suggested the presence of an immunogenic pilus carbohydrate epitope. Mild periodate oxidation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated outer membrane preparations from strains that bound anti-Gal followed by labeling of the neoaldehyde groups resulted in the labeling of bands that corresponded to pilin and LOS, confirming that pilin contains carbohydrate structures. A Bandeiraea simplicifolia lectin that also binds terminal alpha 1,3-galactosyl residues also bound to pilin. Serum IgG, IgA, and IgM anti-Gal as well as colostral secretory IgA anti-Gal bound to pilin, as judged by immunoblotting, and to the pili of intact piliated organisms, as judged by immunoelectron microscopy. Total serum anti-Gal (IgG, IgA, and IgM) and purified serum IgA1 anti-Gal, but not its purified IgG isotype, blocked complement-mediated lysis of a piliated meningococcal strain that bound anti-Gal to its pili. Colostral anti-Gal secretory IgA blocked killing of the same strain. Thus, anti-Gal IgA may promote disease when it binds to the pili of N. meningitidis strains.     

Genetic heterogeneity of strains of Neisseria meningitidis belonging to serotype 22 isolated in the Czech Republic

Strains of Neisseria meningitidis of serogroup B isolated in the Czech Republic frequently belong to serotype 22. We analyzed the genetic relationships among strains of this serotype by using the multilocus enzyme electrophoresis technique and the polymorphism of the pilA gene. Our results indicate that these strains correspond to a highly heterogeneous population rather than to the expansion of a single clone.     

Usefulness of the disk diffusion method for evaluating the sensitivity of Neisseria meningitidis to penicillin and cefotaxime

BACKGROUND: Routine susceptibility testing of Neisseria meningitidis to penicillin and other beta lactams is recommended after the isolation of N. meningitidis of moderately resistant to penicillin (MRP). We have evaluated the disk-diffusion method to determine susceptibility of N. meningitidis to penicillin (using disks of either penicillin or oxacillin) and to cefotaxime. METHODS: Fifty-four strains of N. meningitidis isolated from clinical samples were studied. MICs of penicillin and cefotaxime were determined by microdilution. Disks of 2 U of penicillin, 1 microgram of oxacillin and 30 micrograms of cefotaxime and two culture media, Mueller-Hinton agar (MHA) and MHA supplemented with 5% sheep blood (MHS) were used in the disk-diffusion assay. RESULTS: For disk of 2 U of penicillin assayed in MHA, 86.4% of the susceptible strains and 20% of MRP strains were considered susceptible when a breakpoint of 28 mm was considered. None of the MRP strains was considered susceptible when using MHS, but only 38.6% of susceptible strains appeared as such on this medium. When a 1 microgram oxacillin disk was used all MRP strains presented an inhibition zone < or = 10 mm on both MHA and MHS, but 54.4 and 4.5% of susceptible strains presented an inhibition zone > or = 11 mm on MHA and MHS, respectively. All strains were susceptible to cefotaxime, showing inhibition zones around a 30 micrograms disk on MHA and MHS of > or = 35 mm and > or = 25 mm, respectively. CONCLUSION: Disk diffusion with cefotaxime (30 micrograms) allows to determine susceptibility of N. meningitidis to this antimicrobial agent. Discs of penicillin (2 U) and oxacillin (1 microgram) are not useful for screening of MRP N. meningitidis.     

Expression of the L8 lipopolysaccharide determinant increases the sensitivity of Neisseria meningitidis to serum bactericidal activity

The influence of lipopolysaccharide (LPS) expression on the sensitivity of Neisseria meningitidis to serum bactericidal activity was investigated by using a series of variants that expressed different LPS determinants. For each of two different strains, a series of three LPS variants that expressed L3,7, L8, or both were analyzed. LPS variants were identified and monitored by colony blotting with murine monoclonal antibodies specific for the L8 or the L3,7,9 immunotype determinants. Differences in LPS expression were verified by analysis of proteinase K lysates of whole cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then by silver staining or immunoblotting. The variants were used as test strains in bactericidal assays with human sera and with murine sera and monoclonal antibodies. Expression of the L8 LPS epitope was correlated with increased sensitivity to serum bactericidal activity. The geometric mean bactericidal titers of 12 to 16 sets of pre- and postvaccination sera were determined for each variant. Mean serum titers increased progressively with increased expression of L8 on the target strain. The bactericidal titers of anti-outer membrane protein monoclonal antibodies also increased with increased L8 expression.     

Allelic polymorphism and site-specific recombination in the opc locus of Neisseria meningitidis

The opc gene is widespread in epidemic and endemic Neisseria meningitidis, but most strains of certain epidemic clones (ET-37 complex, Cluster A4) and a few random endemic isolates lack an opc gene. Four percent of the 1148 bp that contain opc plus the surrounding intergenic region was polymorphic (18 alleles), and many of the alleles contained a 230 bp insertion at a fixed location in the intergenic region. The presence or absence of the insertion reflects site-specific recombination. The alleles are stably inherited within clonal groupings for up to at least 50 years, with rare cases of horizontal genetic exchange. Most statistical methods indicated significant intragenic recombination events within this dataset.     

Clonal heterogeneity, distribution, and pathogenicity of methicillin-resistant Staphylococcus aureus

Four thousand eighty-eight Staphylococcus aureus isolates obtained from patients hospitalised in a university clinic and four community hospitals over a period of one year were screened for methicillin resistance. A resistance rate of 5% was detected among initial isolates. Distribution of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus showed an increased prevalence of MRSA in clinically significant specimens such as blood, central venous catheter tips, bronchial secretions, and wound secretions. Typing of 110 MRSA strains (initial isolates) by macrorestriction analysis of chromosomal DNA revealed 26 different genotypes that could be divided into five epidemic and 21 sporadic strains. More than 50% of all isolates belonged to one type that was confirmed to be closely related to the "southern-German" epidemic strain. Production of virulence factors such as enterotoxin A-D and toxic shock syndrome-toxin 1 among MRSA strains (initial isolates) occurred in ten of 26 different MRSA types. A strong correlation between genotype and toxin production was demonstrated.     

Characterization of the structure, function, and conformational stability of PorB class 3 protein from Neisseria meningitidis. A porin with unusual physicochemical properties

PorB proteins constitute the vast majority of channels in neisserial outer membranes and can be subdivided within meningococcal strains into two distinct and mutually exclusive families that are designated as class 2 and class 3 proteins. We recently characterized the functional activity and conformational stability of a PorB class 2 protein from Neisseria meningitidis (Minetti, C. A. S. A., Tai, J. Y., Blake, M. S., Pullen, J. K., Liang, S. M., and Remeta, D. P. (1997) J. Biol. Chem. 272, 10710-10720). To evaluate the structure-function relatedness among the PorB proteins, we have employed a combination of electrophoretic and spectroscopic techniques to assess the conformational stability of zwittergent-solubilized class 3 trimers. The functional, physicochemical, and structural properties of the meningococcal class 2 and class 3 proteins are comparable with the notable exception that the latter exhibits a significantly higher susceptibility to SDS. The SDS-induced dissociation and partial unfolding of PorB class 3 is characterized by a single two-state transition with a midpoint at 0.35% SDS. The native trimeric assembly dissociates reversibly, forming partially folded monomers that retain the characteristic beta-sheet content of the transmembrane domain with a concomitant increase in random coil structure arising from unfolding the rigid surface loops. These results provide new insight into the elucidation of porin folding pathways and the factors that govern the overall structural stability of meningococcal proteins.