我国脑膜炎奈瑟菌孔蛋白PorA、PorB的多态性分析

目的以蛋白质组学研究为基础,分析2003—2005年我国流脑暴发流行期间C群脑膜炎奈瑟菌菌株特征,建立以致病性相关蛋白多态性为目标的新分型方法。方法利用双向电泳和MALDI-TOF质谱鉴定分析2003—2005年流脑流行期内分离的66株C群脑膜炎奈瑟菌菌株及2株参考菌株的蛋白表达,重点分析与致病性相关的蛋白多态性特征。结果根据孔蛋白PorA、PorB在双向电泳中的多态性,建立了12个特征菌型。安徽菌株的PorA和PorB蛋白电泳谱型显示出了高度的一致性,而其他地区的菌株则显示出高度的多态性。结论PorA和PorB蛋白2-DE电泳谱型可以作为一种新的菌株分型方法,可能在菌型变迁检测和流脑暴发预警中具有重要应用前景。     

Identification of Neisseria meningitidis sequence type 66 in Poland

Investigation of two cases of invasive meningococcal disease within a single family revealed the presence of isolates of Neisseria meningitidis phenotype C:2b:P1.2,P1.5 belonging to sequence type (ST) 66. The ST66 clone is a single-locus variant of the widely distributed ST8 complex, which has been observed previously in Spain, Belgium, Australia and New Zealand. This hypervariable meningococcal lineage has been responsible for local epidemics worldwide. This is the first report of ST66 meningococcal isolates of this phenotype from Poland.     

Hyperinvasive genotypes of Neisseria meningitidis in France

Clinical isolates of Neisseria meningitidis from cases of meningococcal disease, collected between January 2000 and December 2004, were identified and typed at the French National Reference Centre. A representative subset of 546 isolates from among 2882 isolates was further genotyped by multilocus sequence typing to determine their genetic lineages (clonal complexes) and the degree of diversification among different clonal complexes. Representative isolates of the main clonal complexes were tested for their virulence in mice and for proapoptotic effects on human epithelial cells. High genetic diversity in some genetic lineages (ST-32 and ST-41/44) was correlated with heterogeneity in virulence in mice and proapoptotic effects on human epithelial cells. In contrast, the homogeneous genetic structure of isolates of the ST-11 clonal complex, regardless of their serogroup, correlated positively with a fatal outcome of the infection, increased virulence in mice and increased proapoptotic effects on human epithelial cells.     

Outbreak in France of Neisseria meningitidis B:15:P1.12 belonging to sequence type 1403

This report describes a meningococcal outbreak in France caused by Neisseria meningitidis B:15:P1.12 of sequence type 1403, which affected eight young patients, between November 2000 and February 2002. Epidemiological typing confirmed that a single strain was responsible. Favourable outcome, sequelae or death resulted in similar proportions as in other cases of meningococcal disease in France during the same period, but purpura was observed in all eight cases. The patients were aged between 14 and 28 years, whereas the median age of patients affected by other meningococcal strains during this period in the same area was 60.4 years.     

脑膜炎奈瑟氏菌逃避人类固有免疫系统的机制

脑膜炎奈瑟氏菌（-Nm）引起的脑脊髓膜炎在世界范围内是一种具有严重危害性的传染性疾病。因此研究其侵袭机制显得非常必要。Nm表现出对人类免疫系统的高度适应,通过其菌体自身结构如荚膜、脂多糖及对宿主的分子模拟能有效地避开抗菌肽、补体系统、吞噬细胞等宿主固有免疫系统。目前对脑膜炎奈瑟氏菌逃避及抵抗人类固有免疫系统的机制研究已取得了许多进展。     

Molecular characterisation of group A Neisseria meningitidis isolated in Sudan 1985-2001

A total of 33 group A Neisseria meningitidis (Mc) isolates, collected in Sudan between 1985 and 2001, were studied in order to describe the changes over time in a country within the meningitis belt of Africa. The isolates were characterised by traditional phenotypic methods (serogrouping, serotyping, serosubtyping and antibiogram) and molecular techniques (genosubtyping, pulsed-field gel electrophoresis [PFGE] with restriction endonucleases SpeI and NheI, and multilocus sequence typing [MLST]). Three clones of group A Mc were identified: one before 1988 (sulphadiazine sensitive, serotype 4, genosubtype P1.7,13-1,35-1, sequence type 4 [ST-4]); another during and after the 1988 epidemic (sulphadiazine resistant, serotype 4, genosubtype P1.20,9,35-1, ST-5); and a third causing the 1999 epidemic (sulphadiazine resistant, serotype 4, genosubtype P1.20,9,35-1, ST-7). The first clone showed major differences compared to the other two. The second and third clones had many similarities with differences in only a single gene (pgm) in the MLST (47 of the 450 bp) but significant other differences according to the PFGE patterns. Within the clones, genosubtyping and MLST gave identical information (except one base substitution in the aroE gene in one isolate). However, the PFGE patterns showed changes over time within the clones, where SpeI revealed somewhat more diversity than NheI.     

Invasive infections caused by Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae among children in St Petersburg, Russia.

This study investigated the causes of invasive bacterial infections in children aged <15 years in St Petersburg, Russia, during 2001-2003, using culture and antigen detection methods (rapid antigen latex agglutination (RAL)) for normally sterile body fluids. A pathogen was detected in 90 cases (culture 50, RAL 40). Neisseria meningitidis was the most common pathogen (66%), followed by Haemophilus influenzae (19%) and Streptococcus pneumoniae (16%). Meningitis was the main clinical diagnosis (68/90, 76%), with N. meningitidis serogroup B, H. influenzae type b (Hib), and S. pneumoniae serogroup 1 being the most common isolates. Hib was less prevalent in St Petersburg than it was in industrialised countries before the introduction of Hib vaccinations.     

脑膜炎奈瑟菌ACPS-BOMPC偶联物的免疫原性、安全性和稳定性

目的：评价脑膜炎奈瑟菌（Nm)A群荚膜多糖和B 群外膜蛋白复合物（ACPS－BOMPC）偶联物的免疫原性，安全性和稳定性，方法：通过小鼠和家兔免疫试验，ELISA和杀菌力试验，测定了偶联物的免疫原性和稳定性，通过小鼠和豚鼠毒性试验及热原性试验测定了BOMPC与偶联物的安全性。结果：免疫后87．5％的小鼠抗偶联物的抗体滴度达到1：7240－1：20480；81．25％小鼠抗偶联物蝇的ACPS抗体滴度达1：320以上，偶联后抗ACPS的抗体滴度比未偶联的ACPS的抗体滴度提高了8－128倍，偶联物免疫家兔产生了较高的抗体滴度，5个月未见抗ACPS－BOMPC抗体滴度明显下降；动物实验初步证明偶联物对B群Nm菌株有一定的保护作用；偶联物具有较好的稳定性，对小鼠，豚鼠无毒性。结论：所制备的ACPS－BOMPC偶联物具有较好的免疫原性，动物实验安全且稳定，有可能成为一种有效的抗Ａ群和抗B群Nm感染的疫苗。     

制备疫苗用W135/Y群脑膜炎球菌菌种的免疫原性及遗传稳定性观察

目的 为保证生产四价流脑(A、C、W135、Y群)结合疫苗质量一致性和可控性,对分离自国内的W135、Y群脑膜炎球菌CMCC(B)29037株和CMCC(B)29028株进行免疫原性及遗传稳定性观察.方法 将W135/Y群脑膜炎球菌工作种子批菌种分别连续传代至30代,并收获3、5、10、15、20、25及30代次菌液,对各代次菌进行免疫原性、抗原性、生化反应、毒性、毒力测定,并将30代次菌发酵培养后提取荚膜多糖进行质量分析.结果 CMCC(B)29037株和CMCC(B)29028株工作种子批菌种诱导小鼠产生总IgG抗体分别为1∶1114和1∶2229,杀菌抗体水平与IgG抗体间差异无统计学意义;试管凝集效价均达到1∶320,生化检定两菌株均发酵葡萄糖、麦芽糖,不发酵果糖、蔗糖、甘露醇和乳糖;两菌株的LD50均＞109,30代次内各代次菌的免疫原性、抗原性、生化反应和毒性均无差异.30代次菌液脑腔毒性测定显示均无病理改变,用第30代次菌生产的W135/Y群脑膜炎球菌荚膜多糖各项检定指标均合格.结论 分离自国内的CMCC(B)29037株和CMCC(B)29028株为脑膜炎球菌W135/Y群菌株,免疫原性、抗原性好,生化反应合格、安全性良好,连续传至30代次仍保持较好的安全性和免疫原性,30代次菌纯化的W135/Y群脑膜炎球菌荚膜多糖质量符合质控要求,可以用作四价流脑结合疫苗生产株.     

Intracellular survival and replication of Neisseria meningitidis in human brain microvascular endothelial cells

To cause meningitis the extracellular pathogen Neisseria meningitidis has to traverse the blood–cerebrospinal fluid (B–CSF) barrier. Postulating a transcellular passage, meningococci (MC) have been shown to adhere to and enter B–CSF barrier forming human brain microvascular endothelial cells (HBMEC). Furthermore, electron microscopy studies demonstrated that intracellular MC reside within membrane-bound compartments, both solitary and in groups. To investigate the ability of MC to survive and replicate intracellularly, prolonged gentamicin protection assays were performed. Encapsulated bacteria were found to survive and, after an initial delay, to replicate within HBMEC, whereas the number of intracellular capsule-deficient mutants decreased continuously. This strongly suggests that the capsule plays a pivotal role in the intracellular survival of MC. Further investigations were initiated to characterise the membrane-bound compartment, the Neisseria-containing vacuole (NCV). Immunfluorescence microscopy studies showed that NCVs interact with the endocytic pathway acquiring the early endosomal marker protein, transferrin receptor (TfR), and the late endosomal/lysosomal marker protein Lamp-1.     

两株B群脑膜炎奈瑟氏菌脂寡糖减毒前后抗原性与毒性的检测

两株Ｂ群脑膜炎奈瑟氏菌脂寡糖减毒前后抗原性与毒性的检测孙银燕胡绪敬王君摘要脂寡糖（ＬＯＳ）是Ｂ群Ｎｍ的一种主要外膜抗原，用ＳｅｐｈａｃｒｙｌＳ－３００ＨＲ或ＳｅｐｈａｄｅｘＧ－７５凝胶层析方法从两株Ｂ群Ｎｍ（５４２８５２和３４０７）中提取获得了纯度较...     

Characterisation of Neisseria meningitidis C:2b:P1.2,5 isolates in Poland

This study aimed to characterise Neisseria meningitidis C:2b:P1.2,5 isolates from Poland, which have now become predominant among serogroup C isolates in this country. Overall, 44 isolates (25 invasive and 19 from contact carriers) were typed by whole-cell ELISA and pulsed-field gel electrophoresis. Additionally, the invasive isolates were analysed by multilocus sequence typing, which revealed that they all belonged to the ST-8 complex/cluster A4. The emergence of this clone in other countries has resulted in mass immunisation campaigns and has been associated with a higher level of decreased susceptibility to penicillin; however the present study detected only one isolate that was penicillin-non-susceptible.     

National case‐control study on risk of death and Neisseria meningitidis sequence types in Denmark, 2000–2007

In the past decades efforts to further diminish the case‐fatality rate from meningococcal disease have proven challenging due to the often rapid progression of the disease in patients. In this study our objective was to characterise a subset of Neisseria meningitidis isolates to establish which sequence types were associated with increased mortality in Denmark during the period 2000–2007. We designed a matched case control and performed serogrouping, serotyping, serosubtyping and multilocus sequence typing (MLST) on 100 isolates. The clonal complex ST‐32/ET‐5 was found in 36% of the isolates, followed by the ST‐11/ET‐37 complex (14%) and ST‐41/44 complex/Lineage 3 (14%). Eight new sequence types were found. None of the clonal complexes were significantly associated with increased mortality. Phenotype B:15:P1.7,16 tended to be a better predictor of death than ST‐32. Although the numbers were low, the present study indicates that phenotyping may be a better predictor of mortality than MLST, which suggests that each typing method has its advantages and disadvantages. If this notion can be confirmed by other studies, it may stimulate additional research regarding the pathogenesis of severe illness, for example, if certain surface molecules trigger a cytokine storm more than others.     

Living in a changing environment: insights into host adaptation in Neisseria meningitidis from comparative genomics

Neisseria meningitidis (the meningococcus) colonizes the human nasopharynx of about 10% of the human population. However, for reasons that are still mostly unknown meningococci occasionally enter the cerebrospinal fluid leading to often fatal bacterial meningitis especially in children and young adults. The genetic basis for the observed differences in the pathogenic potential of different strains has only partially been unravelled so far. With the advent of whole genome sequencing technologies, complete genome sequences from three pathogenic meningococcal strains have become available and allow for a comprehensive analysis of the genomic and genetic differences occurring within this species. In this review, the general properties of the meningococcal genomes so far sequenced is given with an emphasis on the chromosomal rearrangements that have occurred, and the genomic islands and prophages that have been identified. The concomitant development of microarray technology for comparative genome hybridization studies of a large set of different meningococcal isolates as well as strains from other Neisseria species has extended our understanding of meningococcal population genetics on a genome-wide scale thus bridging the gap between meningococcal epidemiology and genomics. Finally, we briefly discuss the potential impact of meningococcal life style on its genome architecture and how in turn this genomic make-up might lead to a virulent phenotype making N. meningitidis an accidental pathogen. The overall properties of the meningococcal genome are characterized by genomic variability and instability, resulting in increased functional flexibility within this species.     

Dynamics of PCR-based diagnosis in patients with invasive meningococcal disease

Invasive meningococcal disease continues to be a life-threatening condition and rapid diagnosis is important for the administration of appropriate treatment. This study focused on the use of PCR for the diagnosis of meningococcal aetiology and the dynamics of PCR-based diagnosis over time in various biological samples. Sixty cerebrospinal fluid (CSF) and 144 serum samples collected during the first week of hospitalisation from 37 patients with laboratory-confirmed invasive meningococcal disease were investigated. Overall, 91.9% of CSF samples and 45.9% of serum samples were PCR-positive, while culture of CSF and blood was positive for only 35% and 39% samples, respectively. Positive PCR results were obtained until day 7 with CSF and until day 5 with serum. It is therefore recommended that samples for molecular diagnosis should be collected early in the course of suspected invasive meningococcal disease.     

韶关市2009—2011年健康人群C群脑膜炎奈瑟菌血清杀菌力抗体检测结果分析

目的了解韶关市健康人群血清C群脑膜炎奈瑟菌杀菌力水平,以评价健康人群对C群脑膜炎奈瑟菌的保护水平。方法2009—2011年,每年随机抽取8个年龄组健康人群血清共计811份,采用血清杀菌力试验检测血清中杀菌力抗体水平。结果811份健康人群血清中C群脑膜炎奈瑟菌杀菌力抗体总阳性率为25．40％,总保护率为23．06％,GNT为l：5．13,95％置信区间1：4．57～1：5．89。结论韶关市健康人群对c群脑膜炎奈瑟菌的传染有易感性,年龄越低其对C群脑膜炎奈瑟菌的抵抗力越弱,应加大对A＋C群流脑疫苗宣传力度,普及适龄儿童A＋C群流脑疫苗的免疫接种。     

Neisseria meningitidis W-135 in the Basque Country, northern Spain

Neisseria meningitidis W-135 accounted for nine (1.6%) of 562 cases of invasive meningococcal disease and 17 (3.9%) of 430 meningococcal isolates from healthy carriers. There was no mortality associated with the invasive nine isolates, which belonged to subtype P1.6 and geno-subtype P1.18-1. All invasive isolates and 15 of the 17 isolates from healthy carriers belonged to sequence type 22 by multilocus sequence typing, and showed a similarity of > 85% by pulsed-field gel electrophoresis following digestion with NheI. These results demonstrate that W-135 isolates in the Basque region of northern Spain have a high degree of similarity and are almost clonal.     

Bacteriocins (meningocins) in Norwegian isolates of Neisseria meningitidis: possible role in the course of a meningococcal epidemic

In the Neisseria meningitidis strain MC58 (serogroup B; ET-5 complex) genome three putative islands of horizontally transferred DNA (IHTs) have been identified. IHT-A2 codes for eight hypothetical proteins and two disrupted open reading frames with similarity to a secretion protein (NMB0097) and an ABC transporter (NMB0098). The strains MC58 and 44/76 (shown here) are meningocin resistant/weakly sensitive. None of these strains are meningocin producers. However, NMB0097 and NMB0098 homologues with open reading frames are found in meningocin producers (N. meningitidis P241 (serogroup A; systemic isolate) and BT878 (serogroup B; carrier isolate), and also in strain FAM18 (serogroup C; ET-37 complex). Knocking out either of the two genes in the strain BT878 yielded mutants that did not secrete meningocin. A similarly disrupted tolC mutant in strain BT878 still released meningocin. Among systemic meningococcal isolates prior to and at the onset (mid-1973 to the end of 1974) of the epidemic peaking in 1975 in North Norway, 12 of 30 (40%) isolates of serogroup A were meningocin producers. However, the rate for serogroup B was 1 of 45 (2.2%). Serogroup B meningocin-resistant/weakly sensitive non-producers dominated in the region from mid-1975 and spread to the rest of the country from then on. No producers were found in selected pharyngeal isolates from healthy carriers collected in Svalbard in the early spring of 1975. Our results suggest that meningocinogeny has played a part in the change from serogroup A to serogroup B among isolates in North Norway during the first half of 1975.     

A、C群脑膜炎奈瑟球菌免疫原性及毒性和传代稳定性研究

目的 为保证疫苗质量的一致性和可控性,对A、C群脑膜炎奈瑟球菌结合疫苗生产用菌株CMCC(B)29201和CMCC(B)29205株进行毒性和抗原传代稳定性研究,并对30代次菌进行脑腔毒性、免疫原性和产糖质量分析.方法 将A、C群脑膜炎奈瑟球菌工作种子批菌种分别连续传代至30代次并收获3、5、10、15、20、25及30代次菌液,小鼠腹腔注射观察各代次毒性,试管凝集试验和间接ELISA测定各代次抗原性.其中30代次菌液进行小鼠脑腔攻击观察是否引起脑组织病理改变,小鼠皮下免疫观察免疫原性,同时进行发酵培养提取荚膜多糖的质量分析.结果CMCC(B)29201株和CMCC(B)29205株工作种子批菌种的半数致死量(LD50)均≥109个/ml,30代次内的LD50也均≥109个/ml,30代次菌液脑腔毒性测定显示均无病理改变;抗原性试管凝集效价均达1∶320,30代次内的试管凝集效价均达1∶320,ELISA几何平均滴度(GMT)分别为1∶4835和1∶3915,且30代次内的ELISA效价分别为1∶4315和1∶3752以上;30代次菌液的血清杀菌抗体均≥1∶32,用第30代次工作种子批菌种生产的A群和C群脑膜炎奈瑟球菌荚膜多糖各项检定指标均达到国家标准.结论 A、C群脑膜炎奈瑟球菌结合疫苗生产用菌株CMCC(B)29201和CMCC(B)29205株毒性低、抗原性和免疫原性良好,连续传代至30代次仍保持较低的毒性和较好的抗原性及免疫原性,纯化的A群和C群脑膜炎奈瑟球菌荚膜多糖质量符合质控要求.