分子分型技术用于流脑疫情的同源性分析

目的 应用多位点序列分型（Multilocus sequence typing,MLST）技术和脉冲场凝胶电泳（pulsed-field gel electrophoresis,PFGE）技术对大连市2014年4月同一工地两起流脑疫情得到的脑膜炎奈瑟氏菌株进行分子分型图谱分析,了解各菌株之间的亲缘关系。方法 对病例的脑脊液标本进行脑膜炎奈瑟菌PCR分群,对另一病例和所有密切接触者分离菌株进行多位点序列分型（MLST）试验和脉冲场凝胶电泳（PFGE）鉴定实验。结果 疑似病例标本PCR结果为脑膜炎奈瑟氏菌A群阳性,另一病例和所有密切接触者分离到的菌株PFGE图谱基本相同,表明来自同一克隆系。多位点序列分析结果为ST7。结论 两起疫情分离到的菌株型别之间具有高度的同源性,该病原菌类型为ST7的A群脑膜炎奈瑟氏菌。     

The influence of mutation, recombination, population history, and selection on patterns of genetic diversity in Neisseria meningitidis

Patterns of genetic diversity within populations of human pathogens, shaped by the ecology of host-microbe interactions, contain important information about the epidemiological history of infectious disease. Exploiting this information, however, requires a systematic approach that distinguishes the genetic signal generated by epidemiological processes from the effects of other forces, such as recombination, mutation, and population history. Here, a variety of quantitative techniques were employed to investigate multilocus sequence information from isolate collections of Neisseria meningitidis, a major cause of meningitis and septicemia world wide. This allowed quantitative evaluation of alternative explanations for the observed population structure. A coalescent-based approach was employed to estimate the rate of mutation, the rate of recombination, and the size distribution of recombination fragments from samples from disease-associated and carried meningococci obtained in the Czech Republic in 1993 and a global collection of disease-associated isolates collected globally from 1937 to 1996. The parameter estimates were used to reject a model in which genetic structure arose by chance in small populations, and analysis of molecular variation showed that geographically restricted gene flow was unlikely to be the cause of the genetic structure. The genetic differentiation between disease and carriage isolate collections indicated that, whereas certain genotypes were overrepresented among the disease-isolate collections (the "hyperinvasive" lineages), disease-associated and carried meningococci exhibited remarkably little differentiation at the level of individual nucleotide polymorphisms. In combination, these results indicated the repeated action of natural selection on meningococcal populations, possibly arising from the coevolutionary dynamic of host-pathogen interactions.     

Neisseria meningitidis PorA variable regions: rapid detection of P1.7 and P1.19 variants by PCR

AIM: Rapid characterization of variable region (VR)1 variants of the porA gene among invasive strains is crucial for outbreak management and epidemiology studies. Recent sequence analysis studies in Brazil showed that the VR1 P1.7 and P1.19 variants are highly prevalent, accounting for 68%, of the total number of VR1 variants characterized. The aim of this work is to develop a rapid polymerase chain reaction (PCR)-based method for genosubtyping Neisseria meningitidis by detection of porA variable regions P1.7 and P1.19. METHODS AND RESULTS: PCR primers for the detection of porA VR1 P1.7 and P1.19 were designed and tested using 198 clinical N. meningitidis isolates that had been previously evaluated by porA sequencing. All 50 strains with VR1 P1.7 and all 65 strains with VR1 P1.19 were positively identified by the respective VR-specific PCR and no false-positive reactions occurred. CONCLUSIONS: VR-specific PCR amplification accurately identified VR P1.7 and P1.19 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: To overcome the disadvantages of serosubtyping and sequencing for typing the porA VR1 segment of N. meningitidis, we developed a PCR-based method to rapidly and accurately detect VR1 P1.7 and P1.19 variants. This approach is highly specific and sensitive; moreover it may allow for genotype determination of culture-negative samples.     

Molecular techniques for the investigation of meningococcal disease epidemiology

Meningococcal disease remains a major cause of childhood morbidity and mortality world wide and no comprehensive vaccine is available against the causative organism, Neisseria meningitidis. Molecular studies of the diversity of this bacterium have provided a number of key insights into its biology, which have implications for control of meningococcal disease. These have included the identification of hyperinvasive lineages and the correlation of genetic type with antigenic type and disease epidemiology. In practical terms, such studies have enabled the application of DNA-based technologies in the development of improved methods for diagnosis and epidemiological monitoring. These data are of especial importance with the current, and ongoing, development and introduction of new meningococcal vaccines.     

A novel neisserial shuttle plasmid: a useful new tool for meningococcal research

We report the identification and nucleotide sequence analysis of a cryptic plasmid pMIDG2830 from the Gram-negative bacterium Neisseria flavescens. The largest open reading frame encodes a protein similar to the replication protein, RepA, found in pAB49 from Acinetobacter baumannii and pNI10 from Pseudomonas. Modified by the incorporation of a kanamycin resistance cassette, the plasmid can be stably maintained in Escherichia coli and Neisseria meningitidis, and can be used as a shuttle plasmid in meningococcal research.     

Identification and characterisation of a novel conserved outer membrane protein from Neisseria meningitidis

We have identified a homologue of the adhesin AIDA-I of Escherichia coli in Neisseria meningitidis. This gene was designated nhhA (Neisseria hia homologue), as analysis of the complete coding sequence revealed that it is more closely related to the adhesins Hia and Hsf of Haemophilus influenzae. The sequence of nhhA was determined from 10 strains, and found to be highly conserved. Studies of the localisation by Western immunoblot analysis of total cell proteins and outer membrane complex preparations and by immunogold electron microscopy revealed that NhhA is located in the outer membrane. A strain survey showed that nhhA is present in 85/85 strains of N. meningitidis representative of all the major disease-associated serogroups, based on Southern blot analysis. It is expressed in the majority of strains tested by Western immunoblot.     

A surveillance network for meningococcal disease in Europe

Between 1999 and 2004, the European Union Invasive Bacterial Infections Surveillance Network (EU-IBIS) received c. 50,000 reports of meningococcal disease from 27 participating countries. Analysis has demonstrated a major decline in the incidence of invasive disease in those countries that have introduced routine vaccination against serogroup C infection. The establishment of rapid reporting of W135 and B2a/B2b strains has been able to provide early reassurance that these strains are not emerging as major public health problems in Europe. Between September 2001 and February 2005, the EU-MenNet project offered further opportunities for enhancing this data resource. Collaborative projects included: improving the EU-IBIS website; reviewing case ascertainment in Europe; reviewing cost-effectiveness studies for meningococcal serogroup C conjugate (MCC) vaccination; international comparisons of MCC vaccine efficacy; and mathematical modelling studies. In addition, linking of data from the European Meningococcal Multi-locus Sequence Type Centre to epidemiological data was performed. Particular clonal complexes were found to be preferentially associated with certain serogroups. Case fatality was also found to vary with clonal complex, suggesting that genotype can be a marker for hypervirulence. The importance of close collaboration between networks of epidemiologists, microbiologists, and the wider scientific and public health community is demonstrated.     

Evaluation of the chick embryo for the determination of relative virulence of Neisseria meningitidis

Abstract The chick embryo model was evaluated as a method to compare virulence between selected strains of Neisseria meningitidis . Inoculation of 13-day-chick embryos via the egg yolk distinguished strains having an LD₅₀ of 10³ colony forming units (CFU) or greater (low virulence) from those having an LD₅₀ of approximately 10¹ or less (high virulence). A strain of serogroup B and a spontaneous nonpiliated strain of group C were found to be of relatively high virulence while a strain of N. lactamica , a serogroup A carrier strain, and certain nongroupable strains were found to be of low virulence. Strains having an LD₅₀ of 10² were not differentiated from either of these. Alternatively, inoculation of the chorioallantoic membrane (CAM) of 9-day-old chick embryos statistically differentiated most strains of N. meningitidis although inoculation via this route was less sensitive.     

Quality assessed nonculture techniques for detection and typing of meningococci

PCR protocols are increasingly used in laboratories worldwide for the diagnosis and confirmation of invasive meningococcal infection. Protocols are now available for the identification of Neisseria meningitidis, for genogrouping, susceptibility to antibiotics and genotyping of the corresponding isolates. The implementation of quality assurance (QA) schemes and standardization of protocols are required. Diagnostic and confirmatory PCRs should perform consistently in clinical and reference microbiology laboratories. General QA schemes address the issues of sample preparation, PCR laboratory environment, equipment and validation of protocols. Moreover, external QA interlaboratory studies are essential. The European Monitoring Group on Meningococci has provided a good forum to conduct such studies through the development and distribution of samples and protocols for nonculture detection and typing of N. meningitidis.     

Evidence for the role of glycoprotein G of respiratory syncytial virus in binding of Neisseria meningitidis to HEp-2 cells

Abstract Viral glycoproteins G and F are expressed on the surface of cells infected with respiratory syncytial virus (RSV). We investigated the role of these proteins in the previously reported enhanced binding of Neisseria meningitidis to RSV-infected HEp-2 cells. Virus particles attached to bacteria were detected by immunofluorescence with flow cytometry. Binding of FITC-labelled bacteria to RSV-infected cells was significantly inhibited by monoclonal antibody against glycoprotein G. Unlabelled bacteria interfered with binding of the anti-G monoclonal antibody to these cells. These interactions were not found with a monoclonal antibody against glycoprotein F. We propose that glycoprotein G of RSV expressed on the surface of infected cells might act as an additional receptor for meningococci.     

Identification of a cation transport pathway in Neisseria meningitidis PorB

Neisseria meningitidis is the main causative agent of bacterial meningitis. In its outer membrane, the trimeric Neisserial porin PorB is responsible for the diffusive transport of essential hydrophilic solutes across the bilayer. Previous molecular dynamics simulations based on the recent crystal structure of PorB have suggested the presence of distinct solute translocation pathways through this channel. Although PorB has been electrophysiologically characterized as anion‐selective, cation translocation through nucleotide‐bound PorB during pathogenesis is thought to be instrumental for host cell death. As a result, we were particularly interested in further characterizing cation transport through the pore. We combined a structural approach with additional computational analysis. Here, we present two crystal structures of PorB at 2.1 and 2.65 Å resolution. The new structures display additional electron densities around the protruding loop 3 (L3) inside the pore. We show that these electron densities can be identified as monovalent cations, in our case Cs⁺, which are tightly bound to the inner channel. Molecular dynamics simulations reveal further ion interactions and the free energy landscape for ions inside PorB. Our results suggest that the crystallographically identified locations of Cs⁺ form a cation transport pathway inside the pore. This finding suggests how positively charged ions are translocated through PorB when the channel is inserted into mitochondrial membranes during Neisserial infection, a process which is considered to dissipate the mitochondrial transmembrane potential gradient and thereby induce apoptosis. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Identification of a hotspot for transformation of Neisseria meningitidis by shuttle mutagenesis using signature-tagged transposons

Shuttle mutagenesis using signature-tagged transposons was employed to generate a library of individually tagged mutants of the Neisseria meningitidis strain B1940, which belongs to serogroup B. The use of tagged transposons allowed us to monitor for enrichment for single mutants during the process of shuttle mutagenesis, by amplification of the tags and subsequent sequence determination. Enrichment of a single clone occurred during the transformation of the meningococci with transposon-containing plasmid DNA. Sequence determination around the site of transposon insertion revealed that the transposon had mutagenized a previously unknown locus, which was designated hrtA (high rate of transformation). hrtA-mediated transformation was independent of TnMax5 and tag sequences, and it most probably involved recombination events. The hrtA locus is restricted to meningococci and gonococci and is present in few apathogenic neisserial species. Chromosomal mapping of hrtA and six further hrt sites revealed a random distribution of highly transforming DNA fragments on the meningococcal chromosome. In conclusion, our data demonstrate that shuttle mutagenesis of naturally competent bacteria using signature-tagged transposons allows the isolation of chromosomal DNA fragments, which exhibit a high transformation efficiency, and which, therefore, are likely to be involved in horizontal gene transfer. Received: 12 January 1998 / Accepted: 30 April 1998     

Simultaneous single-tube PCR-based assay for the direct identification of the five most common meningococcal serogroups from clinical samples

This study presents a stepdown multiplex PCR assay for the simultaneous detection of the five most common Neisseria meningitidis serogroups (A, B, C, W-135 and Y) in 530 clinical samples obtained from 428 patients (271 blood and 259 cerebrospinal fluid). The sensitivity and the specificity was calculated to 100% [positive predictive value 100% (95%, CI 99.0-100%) and negative predictive value 100% (95% CI 99.0-100%)]. The overall effectiveness permits the rapid, accurate and inexpensive detection of the five most prevalent meningococcal serogroups in clinical samples. It is potentially a valuable tool for diagnosis and epidemiological monitoring of disease due to N. meningitidis.     

Multilocus sequence typing for global surveillance of meningococcal disease

The global surveillance of bacterial pathogens is particularly important for bacteria with diverse and dynamic populations that cause periodic epidemics or pandemics. The isolate characterization methods employed for surveillance should: (1) generate unambiguous data; (2) be readily implemented in a variety of scenarios and be reproducible among laboratories; (3) be scalable and preferably available in a high throughput format; and (4) be cost effective. Multilocus sequence typing (MLST) was designed to meet these criteria and has been implemented effectively for a wide range of microorganisms. The 'Impact of meningococcal epidemiology and population biology on public health in Europe (EU-MenNet)' project had amongst its objectives: (1) to disseminate meningococcal MLST and sequence-based typing throughout Europe by establishing a centre for training and data generation, and (2) to produce a comprehensive Europe-wide picture of meningococcal disease epidemiology for the first time. Data produced from the project have shown the distribution of a relatively small number of STs, clonal complexes and PorA types that account for a large proportion of the disease-associated isolates in Europe. The project demonstrates how molecular typing can be combined with epidemiological data via the Internet for global disease surveillance.     

B群脑膜炎球菌多组分疫苗研究进展

目前获准上市的流脑疫苗主要有A群、A+C群脑膜炎球菌疫苗及A,C,W-135及Y群的四价脑膜炎球菌疫苗,B群脑膜炎球菌疫苗尚未研制成功。近年来,研究人员以疫苗外膜蛋白为基础,应用反向疫苗学技术对B群脑膜炎球菌疫苗进行了大量研究,现重点对B群脑膜炎球菌多组分疫苗的研究进展予以综述。     

Neisseria proteomics for antigen discovery and vaccine development

Neisseria meningitidis (meningococcus) is a major causative organism of meningitis and sepsis and Neisseria gonorrhoeae (gonococcus) is the causative organism of the sexually transmitted disease gonorrhea. Infections caused by meningococci are vaccine-preventable, whereas gonococcal vaccine research and development has languished for decades and the correlates of protection are still largely unknown. In the past two decades, complementary ‘omic’ platforms have been developed to interrogate Neisseria genomes and gene products. Proteomic techniques applied to whole Neisseria bacteria, outer membranes and outer membrane vesicle vaccines have generated protein maps and also allowed the examination of environmental stresses on protein expression. In particular, immuno-proteomics has identified proteins whose expression is correlated with the development of human natural immunity to meningococcal infection and colonization and following vaccination. Neisseria proteomic techniques have produced a catalog of potential vaccine antigens and investigating the functional and biological properties of these proteins could finally provide ‘universal’ Neisseria vaccines.     

Employment of broad-range 16S rRNA PCR to detect aetiological agents of infection from clinical specimens in patients with acute meningitis--rapid separation of 16S rRNA PCR amplicons without the need for cloning

AIMS: The aim of this study was to develop a polyacrylamide gel electrophoresis (PAGE) method for the rapid separation of 16S rRNA PCR amplicons from aetiological agents of acute meningitis. METHODS AND RESULTS: Blood samples from 40 patients with suspected acute meningococcal meningitis were examined for the presence of causal agents, including Neisseria meningitidis employing two methods: (i) broad-range 16S rRNA PCR in conjunction with PAGE and automated sequencing and (ii) species-specific PCR employing ABI TaqMan technology for N. meningitidis. Analysis of clinical specimens employing 16S rRNA PCR yielded 33/40 (82.5%) positive for the presence of bacterial DNA. Species-specific PCR yielded 30/40 (75%) clinical specimens positive for N. meningitidis. Prior to separation by PAGE, only 6/33 (18.2%) amplicons were able to be identified by sequence analysis, the remaining amplicons (n=27) did not yield an identification due to the presence of mixed 16S rRNA PCR amplicons. Following separation, amplicons were re-amplified and sequenced, yielding 24/27 (88.9%) positive for N. meningitidis and three specimens positive for Acinetobacter sp., Staphylococcus aureus and Streptococcus pneumoniae. One specimen was positive for both N. meningitidis and Streptococcus spp. and another specimen was positive for N. meningitidis and Pseudomonas sp., by broad-range PCR. Seven clinical specimens were negative for N. meningitidis and other eubacteria using both detection techniques. CONCLUSIONS: Clinical specimens including blood and cerebrospinal fluid from patients with suspected acute bacterial meningitis, may become contaminated with commensal skin flora, resulting in difficulties in downstream sequencing of pathogen plus contaminant DNA. This study allows for the rapid separation of amplified pathogen from contaminant DNA. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrated the usefulness of the rapid separation of multiple 16S rRNA PCR amplicons using a combination of PAGE and automated sequencing, without the need of cloning. Adoption of this technique is therefore proposed when trying to rapidly identify pathogens in clinical specimens employing broad-range 16S rRNA PCR.     

Antibody studies in mice of outer membrane antigens for use in an improved meningococcal B and C vaccine

Abstract Since 1988, N. meningitidis , B:4:P1.15, ET-5 complex, has been responsible for an epidemic of meningococcal disease in Greater São Paulo, Brazil. Despite current trials to develop an effective vaccine against group B meningococci, children less than 2 years old have not been protected. It has been suggested that iron-regulated proteins (IRPs) should be considered as potential antigens for meningococcal vaccines. The vaccines under study consisted of outer-membrane vesicles depleted of lipooligosaccharide from three serogroup B strains and one serogroup C strain, IRPs, meningococcal group C polysaccharide and aluminum hydroxide. Four different protein and C polysaccharide concentrations were studied. The ELISA and bactericidal results showed a higher antibody response when 2 injections of 2.0 μg doses were administered. Despite higher IgG reactivity against antigen preparations containing IRPs seen in ELISA, the bactericidal activity was not increased if the target strain was grown in iron-restricted medium. The influence of addition of alkaline-detoxified lipooligosaccharide (dLOS) on immunogenicity of the vaccine was also investigated, and the dLOS provided for a more functionally specific antibody response.     

Evaluation of touch-down real-time PCR based on SYBR Green I fluorescent dye for the detection of Neisseria meningitidis in clinical samples

Antibiotic treatment prior to transport or admission of patients to hospital has reduced the proportion of patients with invasive meningococcal disease (IMD) from whom Neisseria meningitidis can be isolated by standard microbiological techniques. Assays to detect the crgA gene were used to detect meningococcal DNA by both conventional polymerase chain reaction (PCR) and real-time PCR (RTPCR) in relation to microbiological diagnosis of cases over two years between 2002 and 2003. The sensitivity of both PCR assays for culture-confirmed cases was 93% and the specificity was 98.6%. Agreement between the two PCR assays was 96.2%. The inter- and intra-assay variations and effects of different amounts of DNA on the melting temperatures were examined. The touch-down RTPCR based on SYBR Green I fluorescent dye detected and characterized N. meningitidis in clinical samples within one hour.