Genome-Wide Association Studies Identify an Association of Transferrin Binding Protein B Variation and Invasive Serogroup Y Meningococcal Disease in Older Adults.

BACKGROUND: Neisseria meningitidis serogroup Y, especially ST-23 clonal complex (Y:cc23), represents a larger proportion of invasive meningococcal disease (IMD) in older adults compared to younger individuals. This study explored the meningococcal genetic variation underlying this association. METHODS: Maximum-likelihood phylogenies and the pangenome were analyzed using whole-genome sequence (WGS) data from 200 Y:cc23 isolates in the Neisseria PubMLST database. Genome-wide association studies (GWAS) were performed on WGS data from 250 Y:cc23 isolates from individuals with IMD aged ≥65 years versus < 65 years. RESULTS: Y:cc23 meningococcal variants did not cluster by age group or disease phenotype in phylogenetic analyses. Pangenome comparisons found no differences in presence or absence of genes in IMD isolates from the different age groups. GWAS identified differences in nucleotide polymorphisms within the transferrin-binding protein B (tbpB) gene in isolates from individuals ≥65 years of age. TbpB structure modelling suggests these may impact binding of human transferrin. CONCLUSIONS: These data suggest differential iron scavenging capacity amongst Y:cc23 meningococci isolated from older compared to younger patients. Iron acquisition is essential for many bacterial pathogens including the meningococcus. These polymorphisms may facilitate colonization, thereby increasing the risk of disease in vulnerable older people with altered nasopharyngeal microbiomes and nutritional status.

Acquisition of Ciprofloxacin Resistance Among an Expanding Clade of ß-Lactamase-Positive, Serogroup Y Neisseria meningitidis in the United States.

BACKGROUND: Penicillin and ciprofloxacin are important for invasive meningococcal disease (IMD) management and prevention. IMD cases caused by penicillin- and ciprofloxacin-resistant Neisseria meningitidis containing a ROB-1 ß-lactamase gene (blaROB-1) and a mutated DNA gyrase gene (gyrA) have been recently reported in the United States. METHODS: We examined 2097 meningococcal genomes collected through US population-based surveillance from January 2011 to February 2020 to identify IMD cases caused by strains with blaROB-1- or gyrA-mediated resistance. Antimicrobial resistance was confirmed phenotypically. The US isolate genomes were compared to non-US isolate genomes containing blaROB-1. Interspecies transfer of ciprofloxacin resistance was assessed by comparing gyrA among Neisseria species. RESULTS: Eleven penicillin- and ciprofloxacin-resistant isolates were identified after December 2018; all were serogroup Y, sequence type 3587, clonal complex (CC) 23, and contained blaROB-1 and a T91I-containing gyrA allele. An additional 22 penicillin-resistant, blaROB-1- containing US isolates with wild-type gyrA were identified from 2013 to 2020. All 33 blaROB-1-containing isolates formed a single clade, along with 12 blaROB-1-containing isolates from 6 other countries. Two-thirds of blaROB-1-containing US isolates were from Hispanic individuals. Twelve additional ciprofloxacin-resistant isolates with gyrA T91 mutations were identified. Ciprofloxacin-resistant isolates belonged to 6 CCs and contained 10 unique gyrA alleles; 7 were similar or identical to alleles from Neisseria lactamica or Neisseria gonorrhoeae. CONCLUSIONS: Recent IMD cases caused by a dual resistant serogroup Y suggest changing antimicrobial resistance patterns in the United States. The emerging dual resistance is due to acquisition of ciprofloxacin resistance by ß-lactamase-containing N. meningitidis. Routine antimicrobial resistance surveillance will effectively monitor resistance changes and spread.

Genomic Analysis of Serogroup Y Neisseria meningitidis Isolates Reveals Extensive Similarities Between Carriage-Associated and Disease-Associated Organisms.

BACKGROUND: Neisseria meningitidis is a frequent colonizer of the human nasopharynx, with asymptomatic carriage providing the reservoir for invasive, disease-causing strains. Serogroup Y (MenY) strains are a major cause of meningococcal disease. High-resolution genetic analyses of carriage and disease isolates can establish epidemiological relationships and identify potential virulence factors. METHODS: Whole-genome sequence data were obtained for 99 MenY carriage isolates recovered in the United Kingdom during 1997-2010. Sequences were compared to those of 73 MenY invasive isolates recovered during 2010-2011, using a gene-by-gene approach. RESULTS: Comparisons across 1605 core genes resolved 91% of isolates into one of 8 clusters containing closely related disease and carriage isolates. Six clusters contained carried meningococci isolated during 1997-2001, suggesting temporal stability. One cluster of isolates, predominately sharing the designation Y: P1.5-1,10-1: F4-1: ST-1655 (cc23), was resolved into one subcluster with 86% carriage isolates and a second with 90% invasive isolates. These subclusters were defined by specific allelic differences in 5 core genes encoding glycerate kinase (glxK), valine-pyruvate transaminase (avtA), superoxide dismutase (sodB), and 2 hypothetical proteins. CONCLUSIONS: High-resolution genetic analyses detected long-term temporal stability and temporally overlapping carriage and disease populations for MenY clones but also evidence of a disease-associated clone.

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.

Persistent occurrence of serogroup Y/sequence type (ST)-23 complex invasive meningococcal disease among patients aged five to 14 years, Italy, 2007 to 2013.

In Italy, the incidence of invasive meningococcal disease (IMD) has remained stable since 2007 (around 0.3 cases/100,000 inhabitants). However, as reported for other European countries, an increase of serogroup Y Neisseria meningitidis has been observed. In this study we report IMD cases from 2007 to 2013 in Italy and investigate the clinical and epidemiological features of cases affected by serogroup Y. Molecular characteristics of serogroup Y strains are also described. During the study period, the proportion of IMD cases due to serogroup Y increased, ranging from 2% in 2007 to 17% in 2013 (odds ratio (OR): 8.8), whereby the five to 14 years age group was mostly affected (p < 0.001). Overall 81 serogroup Y IMD cases were identified, with a median age of 18 years, ranging from three months to 84 years. Of the 81 respective patient samples, 56 were further subject to molecular typing. The sequence type (ST)-23 complex (clonal complex (cc)23) was predominant among serogroup Y meningococci (54/56 samples), and included nine different STs. Presumably, ST-23 was the founding genotype, with all the other STs presenting as single-locus variants. All cc23 isolates analysed harboured mutations in the lpxL1 gene; however, no associations among lpxL1 mutations, ST and age group were identified. Overall, these findings generate scientific evidence for the use of the quadrivalent meningococcal conjugate vaccine in the five to 14 years age group.

Temporal variations in the serogroup distribution of invasive meningococcal disease in Quebec, Canada, due to emerging unique clade of serogroup Y strain belonging to the Sequence Type-23 clonal complex.

OBJECTIVE: To identify recent trends in invasive meningococcal diseases (IMD) in Quebec, Canada, with a focus on MenY cases and MenY strains. METHODS: IMD cases and MenY strains from January 1, 2015 to August 11, 2023 were analyzed for clonal analysis and prediction of susceptibility to MenB vaccines. MenY strains of ST-23 CC from Quebec were analyzed with global MenY strains by core-genomic multi-locus sequence typing (cg-MLST). RESULTS: Since 2015 the serogroup distribution of IMD in Quebec has shifted from predominantly MenB to mainly MenY, with most (80.9 %) of the latter belonging to ST-23 CC. The median age of MenY cases due to ST-23 CC were statistically younger than MenY cases due to non-ST-23 CC. MenY of ST-23 CC showed genetic diversity and the major genetic cluster were similar to the Swedish Y1 strain. The increase in invasive MenY disease in Quebec was due to a sub-clade of Lineage 23.1 which caused an elevated proportion of severe disease in young adults. CONCLUSION: The increase in invasive MenY disease in Quebec, Canada was driven by the expansion of a sub-clade of Lineage 23.1 in young adults. Currently available quadrivalent A,C,W,Y-conjugate meningococcal vaccines were predicted to provide protection against these strains.

An evaluation of the role of properdin in alternative pathway activation on Neisseria meningitidis and Neisseria gonorrhoeae.

Properdin, a positive regulator of the alternative pathway (AP) of complement is important in innate immune defenses against invasive neisserial infections. Recently, commercially available unfractionated properdin was shown to bind to certain biological surfaces, including Neisseria gonorrhoeae, which facilitated C3 deposition. Unfractionated properdin contains aggregates or high-order oligomers, in addition to its physiological "native" (dimeric, trimeric, and tetrameric) forms. We examined the role of properdin in AP activation on diverse strains of Neisseria meningitidis and N. gonorrhoeae specifically using native versus unfractionated properdin. C3 deposition on Neisseria decreased markedly when properdin function was blocked using an anti-properdin mAb or when properdin was depleted from serum. Maximal AP-mediated C3 deposition on Neisseriae even at high (80%) serum concentrations required properdin. Consistent with prior observations, preincubation of bacteria with unfractionated properdin, followed by the addition of properdin-depleted serum resulted in higher C3 deposition than when bacteria were incubated with properdin-depleted serum alone. Unexpectedly, none of 10 Neisserial strains tested bound native properdin. Consistent with its inability to bind to Neisseriae, preincubating bacteria with native properdin followed by the addition of properdin-depleted serum did not cause detectable increases in C3 deposition. However, reconstituting properdin-depleted serum with native properdin a priori enhanced C3 deposition on all strains of Neisseria tested. In conclusion, the physiological forms of properdin do not bind directly to either N. meningitidis or N. gonorrhoeae but play a crucial role in augmenting AP-dependent C3 deposition on the bacteria through the "conventional" mechanism of stabilizing AP C3 convertases.

Population structure and capsular switching of invasive Neisseria meningitidis isolates in the pre-meningococcal conjugate vaccine era--United States, 2000-2005.

BACKGROUND: A quadrivalent meningococcal conjugate vaccine (MCV4) was licensed in the United States in 2005; no serogroup B vaccine is available. Neisseria meningitidis changes its capsular phenotype through capsular switching, which has implications for vaccines that do not protect against all serogroups. METHODS: Meningococcal isolates from 10 Active Bacterial Core surveillance sites from 2000 through 2005 were analyzed to identify changes occurring after MCV4 licensure. Isolates were characterized by multilocus sequence typing (MLST) and outer membrane protein gene sequencing. Isolates expressing capsular polysaccharide different from that associated with the MLST lineage were considered to demonstrate capsular switching. RESULTS: Among 1160 isolates, the most common genetic lineages were the sequence type (ST)-23, ST-32, ST-11, and ST-41/44 clonal complexes. Of serogroup B and Y isolates, 8 (1.5%) and 3 (0.9%), respectively, demonstrated capsular switching, compared with 36 (12.9%) for serogroup C (P < .001); most serogroup C switches were from virulent serogroup B and/or serogroup Y lineages. CONCLUSIONS: A limited number of genetic lineages caused the majority of invasive meningococcal infections. A substantial proportion of isolates had evidence of capsular switching. The high prevalence of capsular switching requires surveillance to detect changes in the meningococcal population structure that may affect the effectiveness of meningococcal vaccines.

Amino acid 310 determines the donor substrate specificity of serogroup W-135 and Y capsule polymerases of Neisseria meningitidis.

The capsular polysaccharides of serogroup W-135 and Y meningococci are sialic acid-containing heteropolymers, with either galactose or glucose as the second sugar residue. As shown previously, sequences of the predicted enzymes that catalyse capsule polymerization, i.e. SiaD(W-135) and SiaD(Y), differ in only a few amino acids. By in vitro assays with purified recombinant proteins, SiaD(W-135) and SiaD(Y) were now confirmed to be the capsule polymerases harbouring both hexosyltransferase and sialyltransferase activity. In order to identify amino acids crucial for substrate specificity of the capsule polymerases, polymorphic sites were narrowed down by DNA sequence comparisons and subsequent site-directed mutagenesis. Serogroup-specific amino acids were restricted to the N-terminal part of the proteins. Exclusively amino acid 310, located within the nucleotide recognition domain of the enzymes' predicted hexosyltransferase moiety, accounted for substrate specificity as shown by immunochemistry and in vitro activity assay. Pro-310 determined galactosyltransferase activity that resulted in a serogroup W-135 capsule and Gly-310 determined glucosyltransferase activity that resulted in a serogroup Y capsule. In silico analysis revealed a similar amino acid-based association in other members of the same glycosyltransferase family irrespective of the bacterial species.

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.

Genotypic comparison of invasive Neisseria meningitidis serogroup Y isolates from the United States, South Africa, and Israel, isolated from 1999 through 2002.

The proportion of meningococcal disease in the United States, South Africa, and Israel caused by Neisseria meningitidis serogroup Y (NmY) was greater than the worldwide average during the period 1999-2002. Genotypic characterization of 300 NmY isolates by multilocus sequence typing, 16S rRNA gene sequencing, and PorA variable region typing was conducted to determine the relationships of the isolates from these three countries. Seventy different genotypes were found. Two groups of ST-23 clonal complex isolates accounted for 88% of the U.S. isolates, 12% of the South African isolates, and 96% of the isolates from Israel. The single common clone (ST-23/16S-19/P1.5-2,10-1) represented 57, 5, and 35% of the NmY isolates from the United States, South Africa, and Israel. The predominant clone in South Africa (ST-175/16S-21/P1.5-1,2-2), and 11 other closely related clones made up 77% of the South African study isolates and were not found among the isolates from the United States or Israel. ST-175 was the predicted founder of the ST-175 clonal complex, and isolates of ST-175 and related sequence types have been described previously in other African countries. Continued active surveillance and genetic characterization of NmY isolates causing disease in the United States, South Africa, and Israel will provide valuable data for local and global epidemiology and allow monitoring for any expansion of existing clonal complexes and detection of the emergence of new virulent clones in the population.

Serological and genetic characterization of meningococcal isolates in Korea.

Meningococcal disease has been regarded as a very rare infection in Korea. Until now, there have been no reports on the serological or genetic characterization of Neisseria meningitidis isolates in Korea. This study was the first report of the serogroup, PorA VR subtype, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and antimicrobial susceptibility of N. meningitidis isolates collected from 2002 to 2003. Of 11 meningococcal isolates, serogroup Y was found to be the most frequent (nine isolates). In addition, one isolate was from serogroup B and one was from serogroup 29E. Four isolates showed a reduced sensitivity to penicillin G. However, all strains tested were susceptible to chloramphenicol, cefotaxime, ciprofloxacin, and rifampin. Among the 11 isolates, seven PorA types were identified. P1.5-1, 2-2 was the most prevalent PorA type, accounting for 55.6% of the serogroup Y isolates. In terms of PFGE patterns, nine isolates of serogroup Y were divided into three clusters, but the isolates shared a high level of PFGE pattern similarity. The serogroup Y isolates were characterized as ST-1625 (five strains) and ST-23 (four). They belonged to the ST-23 complex/Cluster A3. In this study, the ST-23 complex/Cluster A3 was prevalent, with the PorA type P1.5-1, 2-2 accounting for 55.6% of the nine serogroup Y strains. Also, we identified the hypervirulent lineage strain such as ST-6667 of ST-41/44 complex/Lineage 3 in Korea. The results of this study show the need for comprehensive epidemiological surveillance to monitor any changes in the meningococcal disease situation so that prompt intervention can be initiated.

Characterization of invasive serogroup Y meningococci in Italy: prevalence of ST-23 Complex/Cluster A3.

The percentage of Neisseria meningitidis serogroup Y isolated from patients with invasive meningococcal disease (IMD) in Italy has increased from 1998 to 2006. In this study, phenotypic features and genetic relatedness have been investigated in all serogroup Y meningococci isolated during that period. Multilocus sequence typing (MLST) identified the ST-23 complex/Cluster A3 as the major clonal complex in 88.8% of the strains. That complex included all strains belonging to the sequence type (ST) 23 isolated from 1998 to 2004, whereas the ST-3171 was prevalent among strains in the years 2005 and 2006. The STs 23 and 3171 differ for only one nucleotide in the phosphoglucomutase (pgm) housekeeping gene. Over 80% of serogroup Y ST-23 complex/Cluster A3 strains showed phenotype Y:14:NST and 85% of the latter resulted indistinguishable by pulsed-field gel electrophoresis analysis. In 2005, serogroup Y meningococci with decreased susceptibility to penicillin were isolated for the first time in Italy. In the following year, three of the seven strains showed this phenotype. The results of this study allow us to draw a profile of the molecular characteristics of invasive serogroup Y in Italy and will be helpful to monitor the spread of this serogroup in the next years.

First description of a rifampicin-resistant Neisseria meningitidis serogroup Y strain causing recurrent invasive meningococcal disease in Hungary.

A Hungarian soldier previously immunized against Neisseria meningitidis by quadrivalent polysaccharide vaccine was twice infected with meningococci within six weeks. The patient was treated with ceftriaxone during both episodes and he successfully recovered. His close contacts received rifampicin prophylaxis. An investigation was performed to characterize the genetic background of the pathogens to ascertain if the recurrent invasive meningococcal disease was caused by the same strain and to find out the reason for reinfection. Both meningococci belonged to the fine type Y:P1.5-2,10-1:F4-1:ST-23. This is the first description of the Europe-wide prevalent N. meningitidis serogroup Y in Hungary. In the first episode, we found wild-type rpoB allele in the non-culturable sample implying the susceptibility to rifampicin. The culturable isolate from the second episode proved resistant to rifampicin and had a point mutation in the rpoB gene. The rifampicin resistance might have evolved during the prophylactic treatment of contacts. Previous immunization of the patient with polysaccharide vaccine was ineffective due to his immunodeficiency, thus immunization with conjugate vaccine was proposed. We have proposed the implementation of centralized rifampicin susceptibility testing of N. meningitidis strains within a defined time frame to intervene and administer appropriate prophylaxis to close contacts.

Meningococcal serogroup Y lpxL1 variants from South Africa are associated with clonal complex 23 among young adults.

OBJECTIVES: To determine the genotypes of serogroup Y meningococcus (MenY), and to determine the prevalence of and identify factors associated with MenY lpxL1 variants. METHODS: Isolates, collected from 2003 to 2007 through national surveillance for invasive meningococcal disease, were characterized by multilocus sequence typing and screened for interleukin-6 induction. LpxL1 genes were sequenced from low IL-6 inducers. RESULTS: MenY represented 13% (n = 219/1702) of meningococcal disease. Clonal complex (cc) 175, ST-23/Cluster A3 (cc23), cc11 and cc167 accounted for 82% (176/214), 11% (24/214), 3% (6/214) and 3% (7/214) respectively. Low cytokine induction was evident in 15% (32/218). Cc23 isolates (24/24) had an lpxL1 mutation, while among the remaining isolates the proportion of lpxL1 variants was 4% (8/189, p < 0.001), and these were all cc175. Compared to wild type isolates, lpxL1 variants were associated with patients aged 5-14 years [unadjusted OR (95% CI): 4.3 (1.5-12)] or 15-24 years [unadjusted OR (95% CI): 9.1 (2.8-29)] compared to children <5 years; and were more likely have been isolated from CSF than blood [unadjusted OR (95% CI): 3.5 (1-11.9)]. On multivariable analysis, age remained significant [adjusted OR (95% CI), 5-14 years: 4.2 (1.5-12); 15-24 years: 8.9 (2.7-29)]. CONCLUSION: LpxL1 variants were associated with cc23 among young adults.

Whole genome sequencing to investigate the emergence of clonal complex 23 Neisseria meningitidis serogroup Y disease in the United States.

In the United States, serogroup Y, ST-23 clonal complex Neisseria meningitidis was responsible for an increase in meningococcal disease incidence during the 1990s. This increase was accompanied by antigenic shift of three outer membrane proteins, with a decrease in the population that predominated in the early 1990s as a different population emerged later in that decade. To understand factors that may have been responsible for the emergence of serogroup Y disease, we used whole genome pyrosequencing to investigate genetic differences between isolates from early and late N. meningitidis populations, obtained from meningococcal disease cases in Maryland in the 1990s. The genomes of isolates from the early and late populations were highly similar, with 1231 of 1776 shared genes exhibiting 100% amino acid identity and an average π(N)  =  0.0033 and average π(S)  =  0.0216. However, differences were found in predicted proteins that affect pilin structure and antigen profile and in predicted proteins involved in iron acquisition and uptake. The observed changes are consistent with acquisition of new alleles through horizontal gene transfer. Changes in antigen profile due to the genetic differences found in this study likely allowed the late population to emerge due to escape from population immunity. These findings may predict which antigenic factors are important in the cyclic epidemiology of meningococcal disease.