The genomic epidemiology of Neisseria meningitidis carriage from a randomised controlled trial of 4CMenB vaccination in an asymptomatic adolescent population.

Background: Oropharyngeal carriage of Neisseria meningitidis is frequent during adolescence, representing a major source of invasive meningococcal disease. This study examined the impact of a serogroup B vaccination (Bexsero, GSK 4CMenB) programme on adolescent N. meningitidis carriage using genomic data. Methods: A total 34,489 oropharyngeal samples were collected as part of a state-wide cluster randomised-controlled trial in South Australia during 2017 and 2018 (NCT03089086). Samples were screened for the presence of N. meningitidis DNA by porA PCR prior to culture. Whole genome sequencing was performed on all 1772 N. meningitidis culture isolates and their genomes were analysed. Findings: Unencapsulated meningococci were predominant at baseline (36.3% of isolates), followed by MenB (31.0%), and MenY (20.5%). Most MenB were ST-6058 from hyperinvasive cc41/44, or ST-32 and ST-2870 from cc32. For MenY, ST-23 and ST-1655 from cc23 were prevalent. Meningococcal carriage was mostly unchanged due to the vaccination programme; however, a significant reduction in ST-53 capsule-null meningococci prevalence was observed in 2018 compared to 2017 (OR = 0.52; 95% CI: 0.30-0.87, p = 0.0106). This effect was larger in the vaccinated compared to the control group (OR = 0.37; 95% CI: 0.12-0.98, p = 0.0368). Interpretation: While deployment of the 4CMenB vaccination did not alter the carriage of hyperinvasive MenB in the vaccinated population, it altered the carriage of other N. meningitidis sequence types following the vaccination program. Our findings suggest 4CMenB vaccination is unlikely to reduce transmission of hyperinvasive N. meningitidis strains and therefore ongoing targeted vaccination is likely a more effective public health intervention. Funding: This work was funded by GlaxoSmithKline Biologicals SA.

Impact of meningococcal ACWY conjugate vaccines on pharyngeal carriage in adolescents: evidence for herd protection from the UK MenACWY programme.

OBJECTIVE: Serogroup W and Y invasive meningococcal disease increased globally from 2000 onwards. Responding to a rapid increase in serogroup W clonal complex 11 (W:cc11) invasive meningococcal disease, the UK replaced an adolescent booster dose of meningococcal C conjugate vaccine with quadrivalent MenACWY conjugate vaccine in 2015. By 2018, the vaccine coverage in the eligible school cohorts aged 14 to 19 years was 84%. We assessed the impact of the MenACWY vaccination programme on meningococcal carriage. METHODS: An observational study of culture-defined oropharyngeal meningococcal carriage prevalence before and after the start of the MenACWY vaccination programme in UK school students, aged 15 to 19 years, using two cross-sectional studies: 2014 to 2015 "UKMenCar4" and 2018 "Be on the TEAM" (ISRCTN75858406). RESULTS: A total of 10 625 participants preimplementation and 13 438 postimplementation were included. Carriage of genogroups C, W, and Y (combined) decreased from 2.03 to 0.71% (OR 0.34 [95% CI 0.27-0.44], p < 0.001). Carriage of genogroup B meningococci did not change (1.26% vs 1.23% [95% CI 0.77-1.22], p = 0.80) and genogroup C remained rare (n = 7/10 625 vs 17/13 438, p = 0.135). The proportion of serogroup positive isolates (i.e. those expressing capsule) decreased for genogroup W by 53.8% (95% CI -5.0 - 79.8, p = 0.016) and for genogroup Y by 30.1% (95% CI 8.946·3, p = 0.0025). DISCUSSION: The UK MenACWY vaccination programme reduced carriage acquisition of genogroup and serogroup Y and W meningococci and sustained low levels of genogroup C carriage. These data support the use of quadrivalent MenACWY conjugate vaccine for indirect (herd) protection.

Genome-wide association studies reveal the role of polymorphisms affecting factor H binding protein expression in host invasion by Neisseria meningitidis.

Many invasive bacterial diseases are caused by organisms that are ordinarily harmless components of the human microbiome. Effective interventions against these microbes require an understanding of the processes whereby symbiotic or commensal relationships transition into pathology. Here, we describe bacterial genome-wide association studies (GWAS) of Neisseria meningitidis, a common commensal of the human respiratory tract that is nevertheless a leading cause of meningitis and sepsis. An initial GWAS discovered bacterial genetic variants, including single nucleotide polymorphisms (SNPs), associated with invasive meningococcal disease (IMD) versus carriage in several loci across the meningococcal genome, encoding antigens and other extracellular components, confirming the polygenic nature of the invasive phenotype. In particular, there was a significant peak of association around the fHbp locus, encoding factor H binding protein (fHbp), which promotes bacterial immune evasion of human complement by recruiting complement factor H (CFH) to the meningococcal surface. The association around fHbp with IMD was confirmed by a validation GWAS, and we found that the SNPs identified in the validation affected the 5' region of fHbp mRNA, altering secondary RNA structures, thereby increasing fHbp expression and enhancing bacterial escape from complement-mediated killing. This finding is consistent with the known link between complement deficiencies and CFH variation with human susceptibility to IMD. These observations demonstrate the importance of human and bacterial genetic variation across the fHbp:CFH interface in determining IMD susceptibility, the transition from carriage to disease.

Evolution of Sequence Type 4821 Clonal Complex Hyperinvasive and Quinolone-Resistant Meningococci.

Expansion of quinolone-resistant Neisseria meningitidis clone ChinaCC4821-R1-C/B from sequence type (ST) 4821 clonal complex (CC4821) caused a serogroup shift from serogroup A to serogroup C invasive meningococcal disease (IMD) in China. To determine the relationship among globally distributed CC4821 meningococci, we analyzed whole-genome sequence data from 173 CC4821 meningococci isolated from 4 continents during 1972-2019. These meningococci clustered into 4 sublineages (1-4); sublineage 1 primarily comprised of IMD isolates (41/50, 82%). Most isolates from outside China (40/49, 81.6%) formed a distinct sublineage, the Europe-USA cluster, with the typical strain designation B:P1.17-6,23:F3-36:ST-3200(CC4821), harboring mutations in penicillin-binding protein 2. These data show that the quinolone-resistant clone ChinaCC4821-R1-C/B has expanded to other countries. The increasing distribution worldwide of serogroup B CC4821 raises the concern that CC4821 has the potential to cause a pandemic that would be challenging to control, despite indirect evidence that the Trumenba vaccine might afford some protection.

Meningococcal carriage in periods of high and low invasive meningococcal disease incidence in the UK: comparison of UKMenCar1-4 cross-sectional survey results.

BACKGROUND: The incidence of invasive meningococcal disease in the UK decreased by approximately four times from 1999 to 2014, with reductions in serogroup C and serogroup B disease. Lower serogroup C invasive meningococcal disease incidence was attributable to implementation of the meningococcal serogroup C conjugate vaccine in 1999, through direct and indirect protection, but no vaccine was implemented against serogroup B disease. UK Meningococcal Carriage surveys 1-3 (UKMenCar1-3), conducted in 1999, 2000, and 2001, were essential for understanding the impact of vaccination. To investigate the decline in invasive meningococcal disease incidence, we did a large oropharyngeal carriage survey in 2014-15, immediately before the changes to meningococcal vaccines in the UK national immunisation schedule. METHODS: UKMenCar4 was a cross-sectional survey in adolescents aged 15-19 years who were enrolled from schools and colleges geographically local to one of 11 UK sampling centres between Sept 1, 2014, and March 30, 2015. Participants provided an oropharyngeal swab sample and completed a questionnaire on risk factors for carriage, including social behaviours. Samples were cultured for putative Neisseria spp, which were characterised with serogrouping and whole-genome sequencing. Data from this study were compared with the results from the UKMenCar1-3 surveys (1999-2001). FINDINGS: From the 19 641 participants (11 332 female, 8242 male, 67 not stated) in UKMenCar4 with culturable swabs and completed risk-factor questionnaires, 1420 meningococci were isolated, with a carriage prevalence of 7·23% (95% CI 6·88-7·60). Carriage prevalence was substantially lower in UKMenCar4 than in the previous surveys: carriage prevalence was 16·6% (95% CI 15·89-17·22; 2306/13 901) in UKMenCar1 (1999), 17·6% (17·05-18·22; 2873/16 295) in UKMenCar2 (2000), and 18·7% (18·12-19·27; 3283/17 569) in UKMenCar3 (2001). Carriage prevalence was lower for all serogroups in UKMenCar4 than in UKMenCar1-3, except for serogroup Y, which was unchanged. The prevalence of carriage-promoting social behaviours decreased from 1999 to 2014-15, with individuals reporting regular cigarette smoking decreasing from 2932 (21·5%) of 13 650 to 2202 (11·2%) of 19 641, kissing in the past week from 6127 (44·8%) of 13 679 to 7320 (37·3%) of 19 641, and attendance at pubs and nightclubs in the past week from 8436 (62·1%) of 13 594 to 7662 (39·0%) of 19 641 (all p<0·0001). INTERPRETATION: We show that meningococcal carriage prevalence in adolescents sampled nationally during a low incidence period (2014-15) was less than half of that in an equivalent population during a high incidence period (1999-2001). Disease and carriage caused by serogroup C was well controlled by ongoing vaccination. The prevalence of behaviours associated with carriage declined, suggesting that public health policies aimed at influencing behaviour might have further reduced disease. FUNDING: Wellcome Trust, UK Department of Health, and National Institute for Health Research.

The global meningitis genome partnership.

Genomic surveillance of bacterial meningitis pathogens is essential for effective disease control globally, enabling identification of emerging and expanding strains and consequent public health interventions. While there has been a rise in the use of whole genome sequencing, this has been driven predominately by a subset of countries with adequate capacity and resources. Global capacity to participate in surveillance needs to be expanded, particularly in low and middle-income countries with high disease burdens. In light of this, the WHO-led collaboration, Defeating Meningitis by 2030 Global Roadmap, has called for the establishment of a Global Meningitis Genome Partnership that links resources for: N. meningitidis (Nm), S. pneumoniae (Sp), H. influenzae (Hi) and S. agalactiae (Sa) to improve worldwide co-ordination of strain identification and tracking. Existing platforms containing relevant genomes include: PubMLST: Nm (31,622), Sp (15,132), Hi (1935), Sa (9026); The Wellcome Sanger Institute: Nm (13,711), Sp (> 24,000), Sa (6200), Hi (1738); and BMGAP: Nm (8785), Hi (2030). A steering group is being established to coordinate the initiative and encourage high-quality data curation. Next steps include: developing guidelines on open-access sharing of genomic data; defining a core set of metadata; and facilitating development of user-friendly interfaces that represent publicly available data.

Complete genome and methylome analysis of Neisseria meningitidis associated with increased serogroup Y disease.

Invasive meningococcal disease (IMD) due to serogroup Y Neisseria meningitidis emerged in Europe during the 2000s. Draft genomes of serogroup Y isolates in Sweden revealed that although the population structure of these isolates was similar to other serogroup Y isolates internationally, a distinct strain (YI) and more specifically a sublineage (1) of this strain was responsible for the increase of serogroup Y IMD in Sweden. We performed single molecule real-time (SMRT) sequencing on eight serogroup Y isolates from different sublineages to unravel the genetic and epigenetic factors delineating them, in order to understand the serogroup Y emergence. Extensive comparisons between the serogroup Y sublineages of all coding sequences, complex genomic regions, intergenic regions, and methylation motifs revealed small point mutations in genes mainly encoding hypothetical and metabolic proteins, and non-synonymous variants in genes involved in adhesion, iron acquisition, and endotoxin production. The methylation motif CACNNNNNTAC was only found in isolates of sublineage 2. Only seven genes were putatively differentially expressed, and another two genes encoding hypothetical proteins were only present in sublineage 2. These data suggest that the serogroup Y IMD increase in Sweden was most probably due to small changes in genes important for colonization and transmission.

UKMenCar4: A cross-sectional survey of asymptomatic meningococcal carriage amongst UK adolescents at a period of low invasive meningococcal disease incidence.

Carriage of Neisseria meningitidis, the meningococcus, is a prerequisite for invasive meningococcal disease (IMD), a potentially devastating infection that disproportionately afflicts infants and children. Humans are the sole known reservoir for the meningococcus, and it is carried asymptomatically in the nasopharynx of ~10% of the population. Rates of carriage are dependent on age of the host and social and behavioural factors. In the UK, meningococcal carriage has been studied through large, multi-centre carriage surveys of adolescents in 1999, 2000, and 2001, demonstrating carriage can be affected by immunisation with the capsular group C meningococcal conjugate vaccine, inducing population immunity against carriage. Fifteen years after these surveys were carried out, invasive meningococcal disease incidence had declined from a peak in 1999.  The UKMenCar4 study was conducted in 2014/15 to investigate rates of carriage amongst the adolescent population during a period of low disease incidence. The protocols and methodology used to perform UKMenCar4, a large carriage survey, are described here.

cgMLST characterisation of invasive Neisseria meningitidis serogroup C and W strains associated with increasing disease incidence in the Republic of Ireland.

INTRODUCTION AND AIMS: Since 2013 MenC and MenW disease incidence and associated mortality rates have increased in the Republic of Ireland. From 2002/2003 to 2012/2013, the average annual MenC incidence was 0.08/100,000, which increased to 0.34/100,000 during 2013/2014 to 2017/18, peaking in 2016/17 (0.72/100,000) with an associated case fatality rate (CFR) of 14.7%. MenW disease incidence has increased each year from 0.02/100,000 in 2013/2014, to 0.29/100,000 in 2017/18, with an associated CFR of 28.6%. We aimed to characterise and relate recent MenC isolates to the previously prevalent MenC:cc11 ET-15 clones, and also characterise and relate recent MenW isolates to the novel 'Hajj' clones. METHODS: Using WGS we characterised invasive (n = 74, 1997/98 to 2016/17) and carried (n = 16, 2016/17) MenC isolates, and invasive (n = 18, 2010/11 to 2016/17) and carried (n = 15, 2016/17) MenW isolates. Genomes were assembled using VelvethOptimiser and stored on the PubMLST Neisseria Bacterial Isolate Genome Sequence Database. Isolates were compared using the cgMLST approach. RESULTS: Most MenC and MenW isolates identified were cc11. A single MenC:cc11 sub-lineage contained the majority (68%, n = 19/28) of recent MenC:cc11 disease isolates and all carried MenC:cc11 isolates, which were interspersed and distinct from the historically significant ET-15 clones. MenW:cc11 study isolates clustered among international examples of both the original UK 2009 MenW:cc11, and novel 2013 MenW:cc11clones. CONCLUSIONS: We have shown that the majority of recent MenC disease incidence was caused by strain types distinct from the MenC:cc11 ET-15 clone of the late 1990s, which still circulate but have caused only sporadic disease in recent years. We have identified that the same aggressive MenW clone now established in several other European countries, is endemic in the RoI and responsible for the recent MenW incidence increases. This data informed the National immunisation Advisory Committee, who are currently deliberating a vaccine policy change to protect teenagers.

Genome Sequencing and Interrogation of Genome Databases: A Guide to Neisseria meningitidis Genomics.

Whole genome sequencing (WGS) has revolutionized molecular microbiology, allowing the population biology of bacterial pathogens to be examined with greater accuracy and detail. The study of Neisseria meningitidis isolates, in particular, has benefitted from the availability of WGS data allowing outbreak cases, hyper-invasive lineages, molecular epidemiology, and vaccine coverage to be determined. Here, we describe a suite of protocols for the optimum recovery and analysis of WGS data, including a brief overview of methods for N. meningitidis DNA extraction, sequencing, and analysis. Downstream analysis tools are described including a step-by-step guide to the use of PubMLST.org/neisseria . This freely accessible website provides a resource for the Neisseria community allowing the diversity of the meningococcal population to be extracted and exploited.

Characterization of meningococcal carriage isolates from Greece by whole genome sequencing: Implications for 4CMenB vaccine implementation.

Herd protection, resulting from the interruption of transmission and asymptomatic carriage, is an important element of the effectiveness of vaccines against the meningococcus. Whilst this has been well established for conjugate polysaccharide vaccines directed against the meningococcal capsule, two uncertainties surround the potential herd protection provided by the novel protein-based vaccines that are used in place of serogroup B (MenB) polysaccharide vaccines (i) the strain coverage of such vaccines against carried meningococci, which are highly diverse; and (ii) the generation of a protective immune response in the mucosa. These considerations are essential for realistic estimates of cost-effectiveness of new MenB vaccines. Here the first of these questions is addressed by the whole genome sequence (WGS) analysis of meningococci isolated from healthy military recruits and university students in Greece. The study included a total of 71 MenB isolates obtained from 1420 oropharyngeal single swab samples collected from military recruits and university students on voluntary basis, aged 18-26 years. In addition to WGS analysis to identify genetic lineage and vaccine antigen genes, including the Bexsero Antigen Sequence Type (BAST), the isolates were examined with the serological Meningococcal antigen Typing System (MATS) assay. Comparison of these data demonstrated that the carried meningococcal population was highly diverse with 38% of the carriage isolates showed expression of antigens matching those included in the 4CMenB vaccine. Our data may suggest a limited potential herd immunity to be expected and be driven by an impact on a subset of carriage isolates.

Establishment of the European meningococcal strain collection genome library (EMSC-GL) for the 2011 to 2012 epidemiological year.

Invasive meningococcal disease surveillance in Europe combines isolate characterisation and epidemiological data to support public health intervention. A representative European Meningococcal Strain Collection (EMSC) of IMD isolates was obtained, and whole genome sequenced to characterise 799 EMSC isolates from the epidemiological year July 2011-June 2012. To establish a genome library (GL), the isolate information was deposited in the pubMLST.org/neisseria database. Genomes were curated and annotated at 2,429 meningococcal loci, including those defining clonal complex, capsule, antigens, and antimicrobial resistance. Most genomes contained genes encoding B (n = 525; 65.7%) or C (n = 163; 20.4%) capsules; isolates were genetically highly diverse, with >20 genomic lineages, five of which comprising 60.7% (n = 485) of isolates. There were >350 antigenic fine-types: 307 were present once, the most frequent (P1.7-2,4:F5-1) comprised 8% (n = 64) of isolates. Each genome was characterised for Bexsero Antigen Sequence Typing (BAST): 25.5% (n = 204) of isolates contained alleles encoding the fHbp and/or the PorA VR1 vaccine component, but most genomes (n = 513; 64.2%) did not contain the NadA component. EMSC-GL will support an integrated surveillance of disease-associated genotypes in Europe, enabling the monitoring of hyperinvasive lineages, outbreak identification, and supporting vaccine programme implementation.

Frequent capsule switching in 'ultra-virulent' meningococci - Are we ready for a serogroup B ST-11 complex outbreak?

The meningococcal ST-11 complex (cc11) causes large invasive disease outbreaks with high case fatality rates, such as serogroup C (MenC) epidemics in industrialised nations in the 1990s and the serogroup W epidemic currently expanding globally. Glycoconjugate vaccines are available for serogroups A, C, W and Y. Broad coverage protein-based vaccines have recently been licensed against serogroup B meningococci (MenB), however, these do not afford universal MenB protection. Capsular switching from MenC to MenB among cc11 organisms is concerning because a large MenB cc11 (B:cc11) outbreak has the potential to cause significant morbidity and mortality. This study aimed to assess the potential for licensed and developmental non-capsular meningococcal vaccines to protect against B:cc11. The population structure and vaccine antigen distribution was determined for a panel of >800 geo-temporally diverse, predominantly MenC cc11 and B:cc11 genomes. The two licensed vaccines potentially protect against many but not all B:cc11 meningococci. Furthermore, strain coverage by these vaccines is often due to a single vaccine antigen and both vaccines are highly susceptible to vaccine escape owing to the apparent dispensability of key proteins used as vaccine antigens. cc11 strains with MenB and MenC capsules warrant special consideration when formulating future non-capsular meningococcal vaccines.

Molecular characterization of invasive capsule null Neisseria meningitidis in South Africa.

BACKGROUND: The meningococcal capsule is an important virulence determinant. Unencapsulated meningococci lacking capsule biosynthesis genes and containing the capsule null locus (cnl) are predominantly non-pathogenic. Rare cases of invasive meningococcal disease caused by cnl isolates belonging to sequence types (ST) and clonal complexes (cc) ST-845 (cc845), ST-198 (cc198), ST-192 (cc192) and ST-53 (cc53) have been documented. The clinical significance of these isolates however remains unclear. We identified four invasive cnl meningococci through laboratory-based surveillance in South Africa from 2003 through 2013, which we aimed to characterize using whole genome data. RESULTS: One isolate [NG: P1.7-2,30: F1-2: ST-53 (cc53)] contained cnl allele 12, and caused empyema in an adult male with bronchiectasis from tuberculosis, diabetes mellitus and a smoking history. Three isolates were NG: P1.18-11,42-2: FΔ: ST-192 (cc192) and contained cnl allele 2. One patient was an adolescent male with meningitis. The remaining two isolates were from recurrent disease episodes (8 months apart) in a male child with deficiency of the sixth complement component, and with the exception of two single nucleotide polymorphisms, contained identical core genomes. The ST-53 (cc53) isolate possessed alleles for NHBA peptide 191 and fHbp variant 2; whilst the ST-192 (cc192) isolates contained NHBA peptide 704 and fHbp variant 3. All four isolates lacked nadA. Comparison of the South African genomes to 61 additional cnl genomes on the PubMLST Neisseria database ( http://pubmlst.org/neisseria/ ), determined that most putative virulence genes could be found in both invasive and carriage phenotypes. CONCLUSIONS: Although rare, invasive disease by cnl meningococci may be associated with host immunodeficiency and such patients may benefit from protein-based meningococcal vaccines.

Isolation and characterization of Neisseria musculi sp. nov., from the wild house mouse.

Members of the genus Neisseria have been isolated from or detected in a wide range of animals, from non-human primates and felids to a rodent, the guinea pig. By means of selective culture, biochemical testing, Gram staining and PCR screening for the Neisseria-specific internal transcribed spacer region of the rRNA operon, we isolated four strains of the genus Neisseria from the oral cavity of the wild house mouse, Mus musculus subsp. domesticus. The isolates are highly related and form a separate clade in the genus, as judged by tree analyses using either multi-locus sequence typing of ribosomal genes or core genes. One isolate, provisionally named Neisseria musculi sp. nov. (type strain AP2031T=DSM 101846T=CCUG 68283T=LMG 29261T), was studied further. Strain AP2031T/N. musculi grew well in vitro. It was naturally competent, taking up DNA in a DNA uptake sequence and pilT-dependent manner, and was amenable to genetic manipulation. These and other genomic attributes of N. musculi sp. nov. make it an ideal candidate for use in developing a mouse model for studying Neisseria-host interactions.

Genomic epidemiology of age-associated meningococcal lineages in national surveillance: an observational cohort study.

BACKGROUND: Invasive meningococcal disease (IMD) is a worldwide health issue that is potentially preventable with vaccination. In view of its sporadic nature and the high diversity of Neisseria meningitidis, epidemiological surveillance incorporating detailed isolate characterisation is crucial for effective control and understanding the evolving epidemiology of IMD. The Meningitis Research Foundation Meningococcus Genome Library (MRF-MGL) exploits whole-genome sequencing (WGS) for this purpose and presents data on a comprehensive and coherent IMD isolate collection from England and Wales via the internet. We assessed the contribution of these data to investigating IMD epidemiology. METHODS: WGS data were obtained for all 899 IMD isolates available for England and Wales in epidemiological years 2010-11 and 2011-12. The data had been annotated at 1720 loci, analysed, and disseminated online. Information was also available on meningococcal population structure and vaccine (Bexsero, GlaxoSmithKline, Brentford, Middlesex, UK) antigen variants, which enabled the investigation of IMD-associated genotypes over time and by patients' age groups. Population genomic analyses were done with a hierarchical gene-by-gene approach. FINDINGS: The methods used by MRF-MGL efficiently characterised IMD isolates and information was provided in plain language. At least 20 meningococcal lineages were identified, three of which (hyperinvasive clonal complexes 41/44 [lineage 3], 269 [lineage 2], and 23 [lineage 23]) were responsible for 528 (59%) of IMD isolates. Lineages were highly diverse and showed evidence of extensive recombination. Specific lineages were associated with IMD in particular age groups, with notable diversity in the youngest and oldest individuals. The increased incidence of IMD from 1984 to 2010 in England and Wales was due to successive and concurrent epidemics of different lineages. Genetically, 74% of isolates were characterised as encoding group B capsules: 16% group Y, 6% group W, and 3% group C. Exact peptide matches for individual Bexsero vaccine antigens were present in up to 26% of isolates. INTERPRETATION: The MRF-MGL represents an effective, broadly applicable model for the storage, analysis, and dissemination of WGS data that can facilitate real-time genomic pathogen surveillance. The data revealed information crucial to effective deployment and assessment of vaccines against N meningitidis. FUNDING: Meningitis Research Foundation, Wellcome Trust, Public Health England, European Union.

Genomic resolution of an aggressive, widespread, diverse and expanding meningococcal serogroup B, C and W lineage.

OBJECTIVES: Neisseria meningitidis is a leading cause of meningitis and septicaemia. The hyperinvasive ST-11 clonal complex (cc11) caused serogroup C (MenC) outbreaks in the US military in the 1960s and UK universities in the 1990s, a global Hajj-associated serogroup W (MenW) outbreak in 2000-2001, and subsequent MenW epidemics in sub-Saharan Africa. More recently, endemic MenW disease has expanded in South Africa, South America and the UK, and MenC cases have been reported among European and North American men who have sex with men (MSM). Routine typing schemes poorly resolve cc11 so we established the population structure at genomic resolution. METHODS: Representatives of these episodes and other geo-temporally diverse cc11 meningococci (n = 750) were compared across 1546 core genes and visualised on phylogenetic networks. RESULTS: MenW isolates were confined to a distal portion of one of two main lineages with MenB and MenC isolates interspersed elsewhere. An expanding South American/UK MenW strain was distinct from the 'Hajj outbreak' strain and a closely related endemic South African strain. Recent MenC isolates from MSM in France and the UK were closely related but distinct. CONCLUSIONS: High resolution 'genomic' multilocus sequence typing is necessary to resolve and monitor the spread of diverse cc11 lineages globally.

Genome-Based Characterization of Emergent Invasive Neisseria meningitidis Serogroup Y Isolates in Sweden from 1995 to 2012.

Invasive meningococcal disease (IMD) caused by Neisseria meningitidis serogroup Y has increased in Europe, especially in Scandinavia. In Sweden, serogroup Y is now the dominating serogroup, and in 2012, the serogroup Y disease incidence was 0.46/100,000 population. We previously showed that a strain type belonging to sequence type 23 was responsible for the increased prevalence of this serogroup in Sweden. The objective of this study was to investigate the serogroup Y emergence by whole-genome sequencing and compare the meningococcal population structure of Swedish invasive serogroup Y strains to those of other countries with different IMD incidence. Whole-genome sequencing was performed on invasive serogroup Y isolates from 1995 to 2012 in Sweden (n = 186). These isolates were compared to a collection of serogroup Y isolates from England, Wales, and Northern Ireland from 2010 to 2012 (n = 143), which had relatively low serogroup Y incidence, and two isolates obtained in 1999 in the United States, where serogroup Y remains one of the major causes of IMD. The meningococcal population structures were similar in the investigated regions; however, different strain types were prevalent in each geographic region. A number of genes known or hypothesized to have an impact on meningococcal virulence were shown to be associated with different strain types and subtypes. The reasons for the IMD increase are multifactorial and are influenced by increased virulence, host adaptive immunity, and transmission. Future genome-wide association studies are needed to reveal additional genes associated with serogroup Y meningococcal disease, and this work would benefit from a complete serogroup Y meningococcal reference genome.

Nasal Inoculation of the Commensal Neisseria lactamica Inhibits Carriage of Neisseria meningitidis by Young Adults: A Controlled Human Infection Study.

BACKGROUND: Herd protection by meningococcal vaccines is conferred by population-level reduction of meningococcal nasopharyngeal colonization. Given the inverse epidemiological association between colonization by commensal Neisseria lactamica and meningococcal disease, we investigated whether controlled infection of human volunteers with N. lactamica prevents colonization by Neisseria meningitidis. METHODS: In a block-randomized human challenge study, 310 university students were inoculated with 10(4) colony-forming units of N. lactamica or were sham-inoculated, and carriage was monitored for 26 weeks, after which all participants were reinoculated with N. lactamica and resampled 2 weeks later. RESULTS: At baseline, natural N. meningitidis carriage in the control group was 22.4% (36/161), which increased to 33.6% (48/143) by week 26. Two weeks after inoculation of N. lactamica, 33.6% (48/143) of the challenge group became colonized with N. lactamica. In this group, meningococcal carriage reduced from 24.2% (36/149) at inoculation to 14.7% (21/143) 2 weeks after inoculation (-9.5%; P = .006). The inhibition of meningococcal carriage was only observed in carriers of N. lactamica, was due both to displacement of existing meningococci and to inhibition of new acquisition, and persisted over at least 16 weeks. Crossover inoculation of controls with N. lactamica replicated the result. Genome sequencing showed that inhibition affected multiple meningococcal sequence types. CONCLUSIONS: The inhibition of meningococcal carriage by N. lactamica is even more potent than after glycoconjugate meningococcal vaccination. Neisseria lactamica or its components could be a novel bacterial medicine to suppress meningococcal outbreaks. This observation explains the epidemiological observation of natural immunity conferred by carriage of N. lactamica. CLINICAL TRIALS REGISTRATION: NCT02249598.

A gene-by-gene population genomics platform: de novo assembly, annotation and genealogical analysis of 108 representative Neisseria meningitidis genomes.

BACKGROUND: Highly parallel, 'second generation' sequencing technologies have rapidly expanded the number of bacterial whole genome sequences available for study, permitting the emergence of the discipline of population genomics. Most of these data are publically available as unassembled short-read sequence files that require extensive processing before they can be used for analysis. The provision of data in a uniform format, which can be easily assessed for quality, linked to provenance and phenotype and used for analysis, is therefore necessary. RESULTS: The performance of de novo short-read assembly followed by automatic annotation using the pubMLST.org Neisseria database was assessed and evaluated for 108 diverse, representative, and well-characterised Neisseria meningitidis isolates. High-quality sequences were obtained for >99% of known meningococcal genes among the de novo assembled genomes and four resequenced genomes and less than 1% of reassembled genes had sequence discrepancies or misassembled sequences. A core genome of 1600 loci, present in at least 95% of the population, was determined using the Genome Comparator tool. Genealogical relationships compatible with, but at a higher resolution than, those identified by multilocus sequence typing were obtained with core genome comparisons and ribosomal protein gene analysis which revealed a genomic structure for a number of previously described phenotypes. This unified system for cataloguing Neisseria genetic variation in the genome was implemented and used for multiple analyses and the data are publically available in the PubMLST Neisseria database. CONCLUSIONS: The de novo assembly, combined with automated gene-by-gene annotation, generates high quality draft genomes in which the majority of protein-encoding genes are present with high accuracy. The approach catalogues diversity efficiently, permits analyses of a single genome or multiple genome comparisons, and is a practical approach to interpreting WGS data for large bacterial population samples. The method generates novel insights into the biology of the meningococcus and improves our understanding of the whole population structure, not just disease causing lineages.