Hyperinvasive Lineages Research Articles

Meningococcal carriage in men who have sex with men presenting at a sexual health unit in Spain.

Neisseria meningitidis (Nm) is asymptomatically carried in the nasopharynx of 5-10% adults, although certain populations, such as men who have sex with men (MSM), exhibit a higher colonisation rate. Interest in Nm carriage has been renewed, owed to meningitis outbreaks within populations of MSM. The aim of this study was to characterise Nm isolates and risk factors for its carriage among MSM attending a sexual health unit. A retrospective cross-sectional study was undertaken between June 2018 and December 2021. We took anal, oropharyngeal, urethral, and blood samples as part of the sexually transmitted infection screening procedures routinely implemented. Nm isolates were subjected to antimicrobial susceptibility testing; the serogroup and genogroup were determined by multi-locus sequence typing. A total of 399 subjects were recruited, and the Nm oropharyngeal carriage rate was 29%, similar among both people living with HIV (PLWH) and uninfected individuals. Nm carriage was less common in vaccinated individuals, especially those who had received the tetravalent vaccine (2.6% vs. 10.6%, p = 0.008). The most frequent serogroups were B (40%) and non-groupable (45%). Most of the isolates were susceptible to ciprofloxacin (96%) and ceftriaxone (100%). However, we identified 21 strains (20%) belonging to hyperinvasive lineages (CC11, CC4821, CC32, CC41/44, CC213, and CC269), most of which belonged to serogroup B. Given that vaccination with MenACWY was associated with a low Nm carriage, we encourage routine vaccination of all MSM. Moreover, the administration of the meningitis B vaccine should also be assessed considering that several invasive lines included in serogroup B are circulating among MSM.

Genomic surveillance of Neisseria meningitidis serogroup B invasive strains: Diversity of vaccine antigen types, Brazil, 2016-2018.

BackgroundNeisseria meningitidis serogroup B remains a prominent cause of invasive meningococcal disease (IMD) in Brazil. Because two novel protein-based vaccines against serogroup B are available, the main purpose of this study was to provide data on the diversity and distribution of meningococcal vaccine antigen types circulating in Brazil.MethodologyGenetic lineages, vaccine antigen types, and allele types of antimicrobial-associated resistance genes based on whole-genome sequencing of a collection of 145 Neisseria meningitidis serogroup B invasive strains recovered in Brazil from 2016 to 2018 were collected.ResultsA total of 11 clonal complexes (ccs) were identified among the 145 isolates, four of which were predominant, namely, cc461, cc35, cc32, and cc213, accounting for 72.0% of isolates. The most prevalent fHbp peptides were 24 (subfamily A/variant 2), 47 (subfamily A/variant 3), 1 (subfamily B/variant 1) and 45 (subfamily A/variant 3), which were predominantly associated with cc35, cc461, cc32, and cc213, respectively. The NadA peptide was detected in only 26.2% of the isolates. The most frequent NadA peptide 1 was found almost exclusively in cc32. We found seven NHBA peptides that accounted for 74.5% of isolates, and the newly described peptide 1390 was the most prevalent peptide exclusively associated with cc461. Mutated penA alleles were detected in 56.5% of the isolates, whereas no rpoB and gyrA mutant alleles were found.ConclusionDuring the study period, changes in the clonal structure of circulating strains were observed, without a predominance of a single hyperinvasive lineage, indicating that an epidemiologic shift has occurred that led to a diversity of vaccine antigen types in recent years in Brazil.

Meningococcal strain evolution, dusty dry air and respiratory tract infections: An explosive relationship.

Meningococcal strain evolution, dusty dry air and respiratory tract infections: An explosive relationship.

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.

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.

Bioinformatic analysis of meningococcal Msf and Opc to inform vaccine antigen design.

Neisseria meningitidis is an antigenically and genetically variable Gram-negative bacterium and a causative agent of meningococcal meningitis and septicaemia. Meningococci encode many outer membrane proteins, including Opa, Opc, Msf, fHbp and NadA, identified as being involved in colonisation of the host and evasion of the immune response. Although vaccines are available for the prevention of some types of meningococcal disease, none currently offer universal protection. We have used sequences within the Neisseria PubMLST database to determine the variability of msf and opc in 6,500 isolates. In-silico analysis revealed that although opc is highly conserved, it is not present in all isolates, with most isolates in clonal complex ST-11 lacking a functional opc. In comparison, msf is found in all meningococcal isolates, and displays diversity in the N-terminal domain. We identified 20 distinct Msf sequence variants (Msf SV), associated with differences in number of residues within the putative Vn binding motifs. Moreover, we showed distinct correlations with certain Msf SVs and isolates associated with either hyperinvasive lineages or those clonal complexes associated with a carriage state. We have demonstrated differences in Vn binding between three Msf SVs and generated a cross reactive Msf polyclonal antibody. Our study has highlighted the importance of using large datasets to inform vaccine development and provide further information on the antigenic diversity exhibited by N. meningitidis.

Molecular characterization of Neisseria meningitidis isolates recovered from 11-19-year-old meningococcal carriers in Salvador, Brazil.

Characterization of meningococci isolated from the pharynx is essential towards understanding the dynamics of meningococcal carriage and disease. Meningococcal isolates, collected from adolescents resident in Salvador, Brazil during 2014, were characterized by multilocus sequence typing, genotyping or whole-genome sequencing. Most were nongroupable (61.0%), followed by genogroups B (11.9%) and Y (8.5%). We identified 34 different sequence types (STs), eight were new STs, distributed among 14 clonal complexes (cc), cc1136 represented 20.3% of the nongroupable isolates. The porA and fetA genotypes included P1.18,25–37 (11.9%), P1.18–1,3 (10.2%); F5-5 (23.7%), F4-66 (16.9%) and F1-7 (13.6%). The porB class 3 protein and the fHbp subfamily A (variants 2 and 3) genotypes were found in 93.0 and 71.0% of the isolates, respectively. NHBA was present in all isolates, and while most lacked NadA (94.9%), we detected the hyperinvasive lineages B:P1.19,15:F5-1:ST-639 (cc32); C:P1.22,14–6:F3-9:ST-3780 (cc103) and W:P1.5,2:F1-1:ST-11 (cc11). This is the first report on the genetic diversity and vaccine antigen prevalence among N. meningitidis carriage isolates in the Northeast of Brazil. This study highlights the need for ongoing characterization of meningococcal isolates following the introduction of vaccines and for determining public health intervention strategies.

Transcriptomic buffering of cryptic genetic variation contributes to meningococcal virulence

BackgroundCommensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence.ResultsDespite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region.ConclusionsOur data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis.

Emergence of a new Neisseria meningitidis clonal complex 11 lineage 11.2 clade as an effective urogenital pathogen

Neisseria meningitidis (Nm) clonal complex 11 (cc11) lineage is a hypervirulent pathogen responsible for outbreaks of invasive meningococcal disease, including among men who have sex with men, and is increasingly associated with urogenital infections. Recently, clusters of Nm urethritis have emerged primarily among heterosexual males in the United States. We determined that nonencapsulated meningococcal isolates from an ongoing Nm urethritis outbreak among epidemiologically unrelated men in Columbus, Ohio, are linked to increased Nm urethritis cases in multiple US cities, including Atlanta and Indianapolis, and that they form a unique clade (the US Nm urethritis clade, US_NmUC). The isolates belonged to the cc11 lineage 11.2/ET-15 with fine type of PorA P1.5-1, 10-8; FetA F3-6; PorB 2-2 and express a unique FHbp allele. A common molecular fingerprint of US_NmUC isolates was an IS1301 element in the intergenic region separating the capsule ctr-css operons and adjacent deletion of cssA/B/C and a part of csc, encoding the serogroup C capsule polymerase. This resulted in the loss of encapsulation and intrinsic lipooligosaccharide sialylation that may promote adherence to mucosal surfaces. Furthermore, we detected an IS1301-mediated inversion of an ∼20-kb sequence near the cps locus. Surprisingly, these isolates had acquired by gene conversion the complete gonococcal denitrification norB-aniA gene cassette, and strains grow well anaerobically. The cc11 US_NmUC isolates causing urethritis clusters in the United States may have adapted to a urogenital environment by loss of capsule and gene conversion of the Neisseria gonorrheae norB-aniA cassette promoting anaerobic growth.

Genomic analysis of urogenital and rectal Neisseria meningitidis isolates reveals encapsulated hyperinvasive meningococci and coincident multidrug-resistant gonococci

ObjectiveInvasive meningococcal disease (IMD) outbreaks in men who have sex with men (MSM) have been associated with meningococcal colonisation of the urethra and rectum, but little is known about this...

Metabolic shift in the emergence of hyperinvasive pandemic meningococcal lineages

Hyperinvasive lineages of Neisseria meningitidis, which persist despite extensive horizontal genetic exchange, are a major cause of meningitis and septicaemia worldwide. Over the past 50 years one such lineage of meningococci, known as serogroup A, clonal complex 5 (A:cc5), has caused three successive pandemics, including epidemics in sub-Saharan Africa. Although the principal antigens that invoke effective immunity have remained unchanged, distinct A:cc5 epidemic clones have nevertheless emerged. An analysis of whole genome sequence diversity among 153 A:cc5 isolates identified eleven genetic introgression events in the emergence of the epidemic clones, which primarily involved variants of core genes encoding metabolic processes. The acquired DNA was identical to that found over many years in other, unrelated, hyperinvasive meningococci, suggesting that the epidemic clones emerged by acquisition of pre-existing metabolic gene variants, rather than ‘virulence’ associated or antigen-encoding genes. This is consistent with mathematical models which predict the association of transmission fitness with the emergence and maintenance of virulence in recombining commensal organisms.

Distribution of the type III DNA methyltransferases modA, modB and modD among Neisseria meningitidis genotypes: implications for gene regulation and virulence.

Neisseria meningitidis is a human-specific bacterium that varies in invasive potential. All meningococci are carried in the nasopharynx, and most genotypes are very infrequently associated with invasive meningococcal disease; however, those belonging to the ‘hyperinvasive lineages’ are more frequently associated with sepsis or meningitis. Genome content is highly conserved between carriage and disease isolates, and differential gene expression has been proposed as a major determinant of the hyperinvasive phenotype. Three phase variable DNA methyltransferases (ModA, ModB and ModD), which mediate epigenetic regulation of distinct phase variable regulons (phasevarions), have been identified in N. meningitidis. Each mod gene has distinct alleles, defined by their Mod DNA recognition domain, and these target and methylate different DNA sequences, thereby regulating distinct gene sets. Here 211 meningococcal carriage and >1,400 disease isolates were surveyed for the distribution of meningococcal mod alleles. While modA11-12 and modB1-2 were found in most isolates, rarer alleles (e.g., modA15, modB4, modD1-6) were specific to particular genotypes as defined by clonal complex. This suggests that phase variable Mod proteins may be associated with distinct phenotypes and hence invasive potential of N. meningitidis strains.

An OMV Vaccine Derived from a Capsular Group B Meningococcus with Constitutive FetA Expression: Preclinical Evaluation of Immunogenicity and Toxicity.

Following the introduction of effective protein-polysaccharide conjugate vaccines against capsular group C meningococcal disease in Europe, meningococci of capsular group B remain a major cause of death and can result in debilitating sequelae. The outer membrane proteins PorA and FetA have previously been shown to induce bactericidal antibodies in humans. Despite considerable antigenic variation among PorA and FetA OMPs in meningococci, systematic molecular epidemiological studies revealed this variation is highly structured so that a limited repertoire of antigenic types is congruent with the hyperinvasive meningococcal lineages that have caused most of the meningococcal disease in Europe in recent decades. Here we describe the development of a prototype vaccine against capsular group B meningococcal infection based on a N. meningitidis isolate genetically engineered to have constitutive expression of the outer membrane protein FetA. Deoxycholate outer membrane vesicles (dOMVs) extracted from cells cultivated in modified Frantz medium contained 21.8% PorA protein, 7.7% FetA protein and 0.03 μg LPS per μg protein (3%). The antibody response to the vaccine was tested in three mouse strains and the toxicological profile of the vaccine was tested in New Zealand white rabbits. Administration of the vaccine, MenPF-1, when given by intramuscular injection on 4 occasions over a 9 week period, was well tolerated in rabbits up to 50 μg/dose, with no evidence of systemic toxicity. These data indicated that the MenPF-1 vaccine had a toxicological profile suitable for testing in a phase I clinical trial.

Sequence Type 4821 Clonal Complex Serogroup B Neisseria meningitidis in China, 1978-2013.

Serogroup B Neisseria meningitidis strains belonging to sequence type 4821 clonal complex (CC4821), a hyperinvasive lineage first identified for serogroup C in 2003, have been increasingly isolated in China. We characterized the outer membrane protein genes of 48 serogroup B and 214 serogroup C strains belonging to CC4821 and analyzed the genomic sequences of 22 strains. Four serogroup B strains had porin A (i.e., PorA), PorB, and ferric enterobactin transport (i.e., FetA) genotypes identical to those for serogroup C. Phylogenetic analysis of the genomic sequences showed that the 22 CC4821 strains from patients and healthy carriers were unevenly clustered into 2 closely related groups; each group contained serogroup B and C strains. Serogroup B strains appeared variable at the capsule locus, and several recombination events had occurred at uncertain breakpoints. These findings suggest that CC4821 serogroup C N. meningitidis is the probable origin of highly pathogenic CC4821 serogroup B strains.

Genomic Analysis of the Evolution and Global Spread of Hyper-invasive Meningococcal Lineage 5

BackgroundThe predominant model for bacterial pandemics is the emergence of a virulent variant that diversifies as it spreads in human populations. We investigated a 40-year meningococcal disease pandemic caused by the hyper-invasive ET-5/ST-32 complex. MethodsA global collection of Neisseria meningitidis isolates dating from 1969 to 2008 was whole genome sequenced (WGS) and analysed using a gene-by-gene approach at http://pubmlst.org/neisseria. FindingsAnalysis of WGS data identified a ‘Lineage 5 pan genome’ of 1940 genes, 1752 (92%) of which were present in all isolates (Lineage 5 ‘core genome’). Genetic diversity, which was mostly generated by horizontal gene transfer, was unevenly distributed in the genome; however, genealogical analysis of diverse and conserved core genes, accessory genes, and antigen encoding genes, robustly identified a star phylogeny with a number of sub-lineages. Most European and American isolates belonged to one of two closely related sub-lineages, which had diversified before the identification of the pandemic in the 1970s. A third, genetically more diverse sub-lineage, was associated with Asian isolates. Several isolates had acquired DNA from the related gonococcus. InterpretationThese data were inconsistent with a single point of origin followed by pandemic spread, rather suggesting that the sub-lineages had diversified and spread by asymptomatic transmission, with multiple distinct strains causing localised hyperendemic outbreaks.

Metabolism and virulence in Neisseria meningitidis.

A longstanding question in infection biology addresses the genetic basis for invasive behavior in commensal pathogens. A prime example for such a pathogen is Neisseria meningitidis. On the one hand it is a harmless commensal bacterium exquisitely adapted to humans, and on the other hand it sometimes behaves like a ferocious pathogen causing potentially lethal disease such as sepsis and acute bacterial meningitis. Despite the lack of a classical repertoire of virulence genes in N. meningitidis separating commensal from invasive strains, molecular epidemiology suggests that carriage and invasive strains belong to genetically distinct populations. In recent years, it has become increasingly clear that metabolic adaptation enables meningococci to exploit host resources, supporting the concept of nutritional virulence as a crucial determinant of invasive capability. Here, we discuss the contribution of core metabolic pathways in the context of colonization and invasion with special emphasis on results from genome-wide surveys. The metabolism of lactate, the oxidative stress response, and, in particular, glutathione metabolism as well as the denitrification pathway provide examples of how meningococcal metabolism is intimately linked to pathogenesis. We further discuss evidence from genome-wide approaches regarding potential metabolic differences between strains from hyperinvasive and carriage lineages and present new data assessing in vitro growth differences of strains from these two populations. We hypothesize that strains from carriage and hyperinvasive lineages differ in the expression of regulatory genes involved particularly in stress responses and amino acid metabolism under infection conditions.

Implications of Differential Age Distribution of Disease-Associated Meningococcal Lineages for Vaccine Development

New vaccines targeting meningococci expressing serogroup B polysaccharide have been developed, with some being licensed in Europe. Coverage depends on the distribution of disease-associated genotypes, which may vary by age. It is well established that a small number of hyperinvasive lineages account for most disease, and these lineages are associated with particular antigens, including vaccine candidates. A collection of 4,048 representative meningococcal disease isolates from 18 European countries, collected over a 3-year period, were characterized by multilocus sequence typing (MLST). Age data were available for 3,147 isolates. The proportions of hyperinvasive lineages, identified as particular clonal complexes (ccs) by MLST, differed among age groups. Subjects <1 year of age experienced lower risk of sequence type 11 (ST-11) cc, ST-32 cc, and ST-269 cc disease and higher risk of disease due to unassigned STs, 1- to 4-year-olds experienced lower risk of ST-11 cc and ST-32 cc disease, 5- to 14-year-olds were less likely to experience ST-11 cc and ST-269 cc disease, and ≥25-year-olds were more likely to experience disease due to less common ccs and unassigned STs. Younger and older subjects were vulnerable to a more diverse set of genotypes, indicating the more clonal nature of genotypes affecting adolescents and young adults. Knowledge of temporal and spatial diversity and the dynamics of meningococcal populations is essential for disease control by vaccines, as coverage is lineage specific. The nonrandom age distribution of hyperinvasive lineages has consequences for the design and implementation of vaccines, as different variants, or perhaps targets, may be required for different age groups.

Three Outbreak-causingNeisseria meningitidis SerogroupC Clones, Brazil1

During 2003–2012, 8 clusters of meningococcal disease were identified in Rio de Janeiro State, Brazil, all caused by serogroup C Neisseria meningitidis. The isolates were assigned to 3 clonal complexes (cc): cc11, cc32, and cc103. These hyperinvasive disease lineages were associated with endemic disease, outbreaks, and high case-fatality rates.

Editorial Commentary: The Endgame for Serogroup A Meningococcal Disease in Africa?

Editorial Commentary: The Endgame for Serogroup A Meningococcal Disease in Africa?

Evolutionary and genomic insights into meningococcal biology.

Epidemic disease caused by Neisseria meningitidis, the meningococcus, has been recognized for two centuries, but remains incompletely controlled and understood. There have been dramatic reductions in serogroup A and C meningococcal disease following the introduction of protein-polysaccharide conjugate vaccines, but there is currently no comprehensive vaccine against serogroup B meningococci. Genetic analyses of meningococcal populations have provided many insights into the biology, evolution and pathogenesis of this important pathogen. The meningococcus, and its close relative the gonococcus, are the only pathogenic members of the genus Neisseria, and the invasive propensity of meningococci varies widely, with approximately a dozen 'hyperinvasive lineages' responsible for most disease. Despite this, attempts to identify a 'pathogenome', a subset of genes associated with the invasive phenotypes, have failed; however, genome-wide studies of representative meningococcal isolates using high-throughput sequencing are beginning to provide details on the relationship of invasive phenotype and genotype in this fascinating organism and how this relationship has evolved.