Hyperinvasive Neisseria meningitidis in China Originates from Commensals in Healthy Carriers

Hyperinvasive Neisseria meningitidis in China Originates from Commensals in Healthy Carriers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hyperinvasive Neisseria meningitidis in China Originates from Commensals in Healthy Carriers Zhujun Shao, Zhizhou Tan, Hairui Wang, Huijun Zhao, Xiaodong Qi, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7042271/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract In June 2024, a novel hyperinvasive strain of Neisseria meningitidis ( N. meningitidis ) caused the deaths of two middle school students in Qinghai Province, China. This is the first hyperinvasive strain to emerge in the country since 2003 and likely originated from commensal strains in healthy carriers. Whole-genome sequencing identified the strain as serogroup C ST-8491, a commensal lineage circulating in China for over 15 years. Among close contacts, all of whom were classmates, the carriage rate reached 31.4%. Carrier ST-8491 isolates shared 98.5–99.9% genomic identity with the fatal strain. Two genetic variations—a duplication in the opal gene and a capsule polysaccharide synthesis locus variation acquired from the commensal, nonserogroupable ST-18331 strain—were identified as potential virulence factors. These findings demonstrate that commensal strains can directly evolve into fatal forms and underscore the need to monitor N. meningitidis transmission in healthy populations to prevent future outbreaks. Biological sciences/Microbiology/Clinical microbiology Health sciences/Diseases/Infectious diseases/Bacterial infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Some formidable pathogens have originated from commensals of humans, including numerous pathogenic Escherichia coli 1 and methicillin-resistant Staphylococcus aureus 2 . These pathogens pose a significant threat to public health due to the unpredictability of their outbreaks and the need to revise pathogen monitoring strategies 3 , 4 . Currently, most public health surveillance systems focus on pathogens that have already demonstrated pathogenicity or epidemic potential 5 , 6 . As increasing evidence suggests that commensal strains can evolve into pathogenic strains, monitoring strategies may need to be expanded to include healthy carriers. Neisseria meningitidis ( N. meningitidis ), an obligate human bacterium, is both a commensal and a major pathogen responsible for invasive meningococcal disease (IMD) 7 – 9 . Genetically, IMD strains are typically associated with a limited number of clonal complexes (CCs) or sequence types (STs), referred to as hyperinvasive strains 7 . In contrast, commensal strains, often referred to as carrier strains, exhibit greater genetic diversity. Commensal strains may form clonal complexes, exist outside of any clonal complex, or appear as rare strains within a clonal complex with high internal genetic diversity 10 , 11 . Commensal strains are carried by approximately 10% of the general population as part of the normal respiratory flora 7 , 10 . Phenotypically, the majority of hyperinvasive strains express a polysaccharide capsule and belong to one of six serogroups (A, B, C, Y, W, and X) 12 . In comparison, commensal strains, which often lack or have a reduced polysaccharide capsule, are classified as nonserogroupable (NG) 7 . The virulence factors of N. meningitidis remain controversial, as genomic analyses have yet to clearly identify virulence-related genes in N. meningitidis 13 , 14 . However, because commensal and hyperinvasive strains belong to genetically distinct populations 15 , N. meningitidis surveillance mainly focuses on hyperinvasive strains and strains of specific serogroups 16 , 17 . Commensal strains in healthy individuals are not included in routine monitoring, despite the long-standing hypothesis that commensal carrier strains may serve as a source of hyperinvasive strains 18 . Emerging hyperinvasive strains of N. meningitidis pose a significant public health threat 7 . While IMD typically occurs endemically, emerging hyperinvasive strains can trigger large, unpredictable epidemics 7 , 19 . Globally, the most recently emerged hyperinvasive strain is serogroup C (NmC) ST-10217, which appeared in Nigeria in 2013 and caused large outbreaks in 2016, with more than 14,000 IMD cases 20 , 21 . In China, the most recent hyperinvasive strain is NmC ST-4821, which emerged in 2003 and caused a series of outbreaks from 2003 to 2006, with over 1,500 IMD cases reported annually 22 23 . Since then, no new hyperinvasive strains have been reported in China. There is currently a lack of direct evidence confirming that hyperinvasive strains originate from commensal strains, and it is also generally believed that pre-existing hyperinvasive strains play a significant role in the emergence of new hyperinvasive strains. NmC ST-10217 may have originated from a commensal NG ST-9367 strain, acquiring a full NmC capsule polysaccharide synthesis ( cps ) locus from a hyperinvasive NmC ST-11 strain and an MDAΦ prophage from an unknown strain 19 . The origin of NmC ST-4821 remains unclear, but it likely originated from serogroup B (NmB) CC4821 strains 24 . In June 2024, two fatal cases of IMD occurred at a middle school in Xining City, Qinghai Province, China. Isolates from these cases revealed a novel hyperinvasive strain of N. meningitidis . Importantly, this new hyperinvasive strain likely originated from commensal strains in healthy carriers. This study aims to characterize the epidemiological and molecular traits of this emerging strain and ascertain its origin. Results Fatal case ascertainment In June 2024, two fatal cases due to IMD were successively reported in Xining City, Qinghai Province, China. The cases were students aged 14 and 15, in the same classroom at Huangzhong First Middle School. The first patient died on June 6 after seeking medical treatment on June 4. The second patient died on June 26 after seeking treatment the previous day. Two isolates were cultured from these two cases (Fig. 1 ). Close contact survey and disease control To control the spread of IMD, the CDC screened close contacts (including students, school staff, and family members) of the two fatal cases. Fourteen close contacts tested positive for N. meningitidis through PCR screenings; all were students in the classroom with the fatal cases. There were 51 students in the classroom, including the two fatal cases, resulting in a N. meningitidis prevalence rate of 31.4% in the classroom. These fourteen PCR-positive close contacts were all healthy and were identified through four screenings conducted on June 9, June 27, July 5, and July 7 (Fig. 1 ). N. meningitidis isolates were cultured from 10 of the 14 close contacts. One individual yielded two isolates (samples collected on June 9 and July 7). In total, 11 isolates were obtained from the 10 close contacts. Notably, of these close contacts from whom isolates were obtained, only one individual tested positive only once during the four PCR screenings; the remaining close contacts tested positive in at least two screenings (Fig. 1 , Table 1 and Table S2 ). Table 1 Neisseria meningitidis isolates from the Qinghai outbreak Isolates Isolation source PCR * MLST Serogroup cps locus Grouping QH01 Fatal case 1 - ST-8491 C C (complete) group 1 QH02 Fatal case 2 - ST-8491 C C (complete) group 1 QH03 Close contact 5 PPPP ST-8491 C C (complete, 25 SNPs in galE , cssB , and cssA ) group 2 QH04 Close contact 2 NNNP ST-8491 NG W (partial galU ) group 3 QH05 Close contact 6 PNPP ST-8491 NG W (partial galU ) group 3 QH06 Close contact 7 PNPP ST-8491 NG W (partial galU ) group 3 QH07 Close contact 8 PNNP ST-8491 NG W (partial galU ) group 3 QH08 Close contact 10 PNPP ST-8491 NG W (partial galU and csw ) group 4 QH09 Close contact 4 PNPP ST-8491 NG W (partial galU and csw ) group 4 QH10 Close contact 1 PPPP ST-18331 NG C (partial cssA, ctrA , and crtC ) group 5 QH11 Close contact 1 - ST-18331 NG C (partial cssA, ctrA , and crtC ) group 5 QH12 Close contact 3 PPNN ST-18331 NG C (partial cssA, ctrA , and crtC ) group 5 QH13 Close contact 9 PPNP ST-18331 NG C (partial cssA, ctrA , and crtC ) group 5 * A total of four rounds of PCR screening of close contacts were conducted on June 9, June 27, July 5, and July 7. The results of each close contact's PCR screening are displayed in chronological order, with “P” indicating a positive result and “N” indicating a negative result. The Ct values of the PCR-positive samples ranged from 17.9 to 38.6, with a mean of 26.95 (95% CI: 25.41, 28.99). The detailed PCR screening results for close contacts are shown in Table S2. Abbreviation: cps , capsule polysaccharide synthesis; NG, nonserogroupable; SNPs, single nucleotide polymorphisms. The detailed gene structure of the cps locus for each group is shown in Fig. 4. On June 28, the CDC administered prophylactic antibiotics (mainly cefixime) to PCR-positive students and emergency vaccinations of the ACYW135 quadrivalent meningococcal vaccine to all staff and students at the school. By July 7, a total of 3,198 individuals had been vaccinated, successfully controlling the spread of IMD, with no further cases reported (Fig. 1 ). Identification of a new hyperinvasive strain in fatal cases and carrier strains in close contacts Based on MLST, serogrouping, and cps locus sequence analysis, the 13 N. meningitidis isolates—two isolates (QH01 and QH02) from the fatal cases and 11 isolates (QH03 to QH13) from close contacts—were classified into five groups, labeled as group 1 through group 5 (Table 1 ). Isolates within the same group shared identical sequence types and serogroups, with their cps locus sequences exhibiting 100% identity. The two isolates from the fatal cases belonged to group 1, which represents a new hyperinvasive strain, were both NmC ST-8491 with the complete cps locus of NmC. In contrast, the carrier strains from the close contacts were classified into four groups, groups 2–5. A singular isolate from a close contact (QH03) belonged to group 2 and was also NmC ST-8491. This isolate contained the complete cps locus of NmC; however, it exhibited 25 single nucleotide polymorphisms (SNPs) compared to isolates in group 1. These SNPs were distributed in the galE , cssB , and cssA genes. Groups 3 and 4 consisted of four and two isolates, respectively, all obtained from close contacts. These isolates belonged to NG ST-8491 and contained an incomplete serogroup W (NmW) cps locus with a partial galU gene. The cps locus of group 3 and group 4 differed by a single SNP within the csw gene. Group 3 encoded a complete csw gene, whereas the csw gene in group 4 had a premature stop codon introduced by the SNP, resulting in a partial coding sequence. The MLST of group 5 was distinct from that of groups 1–4, as group 5 belonged to ST-18331, a newly reported sequence type with no available genome data. This group comprised four isolates, all obtained from close contacts. The isolates in group 5 were NG ST-18331 with an incomplete NmC cps locus, characterized by partial coding sequences of the cssA , ctrA , and crtC genes. Whole-genome alignment and identity analysis showed that the ST-8491 strains in Qinghai, including the hyperinvasive strain from the fatal cases (group 1) and those isolated from close contacts (groups 2, 3, and 4), exhibited a high degree of whole-genome identity (98.5–99.9%), despite differences in their serogroup and cps locus sequences. In contrast, the ST-18331 isolates (group 5) substantially diverged from the ST-8491 strains, with a whole-genome identity of only 88.8–89.6% ( Table S3 ). Origin of N. meningitidis strains in Qinghai The cgMLST analysis revealed that all 13 isolates in Qinghai belonged to CC4821. The ST-8491 and ST-18331 isolates formed two distinct genetic clusters (Fig. 2 A). All nine ST-8491 isolates from Qinghai (two from fatal cases and seven from close contacts) demonstrated high clonality, forming a cohesive monophyletic group and clustering with previously collected ST-8491 isolates in China. The four ST-18331 isolates (all from close contacts) constituted a separate monophyletic group, with substantial differences in their core genome compared to the ST-8491 isolates. The cgMLST analysis also revealed that both the ST-8491 and ST-18331 isolates were genetically distant from the hyperinvasive lineage of CC8421 (mainly ST-4821), which caused the IMD outbreak in China between 2003 and 2004 23,24 , as well as the globally spreading lineage of CC4821 (mainly ST-3200), which is rarely associated with IMD 24 . ST-8491 is a rare ST within the CC4821, with only 13 isolates documented in the PubMLST database, all originating from carriers in China 24 . The records revealed that ST-8491 was first identified in Hebei Province in 2009, with sporadic reports of its occurrence from 2009 to 2022 in seven provinces, predominantly in northern China (Fig. 2 B and 2 C). A phylogenetic analysis showed that the Qinghai ST-8491 isolates formed an independent lineage. Distinct genetic differences were observed between the Qinghai ST-8491 isolates and the previously identified ST-8491 isolates. The Qinghai isolates showed the closest genetic relationship to ST-8491 isolates LN21 and LN22 collected from Shandong Province and to isolates 651708 and 651402 collected from Xinjiang Uygur Autonomous Region (Fig. 2 B). Evolutionary relationship of the ST-8491 strains in Qinghai Whole-genome alignment and genetic variation analyses were performed on all nine ST-8491 isolates from Qinghai. The genome lengths of the isolates ranged from 2,162,319 bp to 2,168,266 bp, with nucleotide differences between genomes ranging from 452 bp to 16,328 bp (Fig. 3 B). These differences corresponded to 52 distinct regions of genetic variation scattered across the genomes of the nine isolates (Fig. 3 A). These genetic variations were likely the result of different evolutionary mechanisms, including mutation, homologous recombination, horizontal gene transfer, phase variation, duplication, insertion, and deletion. The size of these regions of variation ranged from 1 bp to 7,466 bp. Of the 52 regions of variation, 41 were unique to individual genomes, while 11 were shared by two or more genomes. The genomes of the two isolates from the fatal cases (QH01, QH02) shared only two specific regions of variation: one in the opal gene, which encodes the opacity protein OpA54, and the other in the cps locus. Both regions are likely key virulence factors contributing to the emergence of the novel hyperinvasive strain (Fig. 3 A). The specific genetic variation in the opal gene sequence was 280 bp in length, and identical sequences at another location were found in eight out of nine ST-8491 genomes (excluding QH08) from Qinghai, suggesting that this variation may have arisen through gene duplication (Fig. 3 A). The specific genetic variation in the cps locus was 7,466 bp in size. The genome of the QH03 isolate from close contact also contained a variation in the cps locus with a partially identical sequence to QH01 and QH02 from the fatal cases, although the regions of variation in QH03 was shorter, measuring 5,869 bp. Neither the 7,466 bp nor the 5,869 bp variation in the cps locus were found in other ST-8491 strains, indicating that these variations were likely acquired through recombination from non-ST-8491 strains. The whole genomes of the two fatal case isolates (QH01, QH02) showed a 3,365-bp nucleotide difference across 16 regions of variation (Fig. 3 A and 3 B), with the closest genetic distance among the isolates from Qinghai, suggesting they may have originated from a common ancestor (Fig. 3 C). The isolate from close contact 5 (QH03) was genetically closest to the fatal case isolates but exhibited a different length of genetic variation in the cps locus and lacked the opal regions of variation. Two independent recombination events in the cps locus likely occurred in the ST-8491 strains during the Qinghai outbreak (Fig. 3 C): one recombination event resulted in the emergence of the hyperinvasive strain infecting the two fatal cases (QH01, QH02), while the other recombination event gave rise to the carrier strain from close contact 5 (QH03). Evolution of the cps locus in the novel hyperinvasive strain The cps loci of all publicly available ST-8491 isolates were analyzed, revealing a relatively conserved gene organization pattern over a 15-year evolutionary span (2009–2024) (Fig. 4 A). The genes were grouped into six regions and arranged in the order B-D-A-C-E-D'. This organization differed from the classical gene arrangement of the cps locus in N. meningitidis , which followed the order D-A-C-E-D'-B 12 . The majority of genetic variations in the cps locus of the ST-8491 isolates were observed in the genes of region A. Consequently, three distinct cps loci were identified in the ST-8491 isolates: the unknown serogroup cps locus, the NmW cps locus, and the NmC cps locus (Fig. 2 A and 2 B). ST-8491 underwent at least two serogroup switching events from 2009 to 2024, evolving from unknown (2009) to W (2013–2024) to C (2024) (Fig. 4 A). The earliest ST-8491 isolates from 2009 had an unknown serogroup with a cps locus in region A containing five genes: galU , crtG , cssC , cssB , and cssA . In 2013, ST-8491 underwent its first serogroup switch by acquiring the capsular synthesis genes cssF and csw in region A, becoming serogroup W. Prior to this, no other NmW strains had been reported within the clonal complex CC4821 (Fig. 2 A). Therefore, the cssF and csw genes likely originated from another clonal complex, most probably from NmW CC11 strains 39 . From 2013 until the Qinghai outbreak, the cps locus of the NmW ST-8491 strains remained conserved. Minor genetic differences were found between the Qinghai ST-8491 isolates (QH04) and the 2013 ST-8491 isolate (131312) (Fig. 4 A), including a few SNPs in galE , csw , and cssC and an insertion in galU . In the Qinghai outbreak, a second serogroup switch occurred, with the cps locus changing to that of serogroup C. The galU , crtG , cssF , and csw genes in region A were replaced by reverse direction crtG , cssE , and csc genes. Additionally, genetic variations were observed in the upstream ( galE ) and downstream ( cssC , cssB , and cssA ) regions. An analysis of the cps locus of the ST-18331 strain from close contacts revealed only a 50.3–59.5% nucleotide identity with the cps locus of the Qinghai ST-8491 strains, likely due to differences in gene organization, with the sequence D-A-C-E-D'-B in ST-18331 and B-D-A-C-E-D' in ST-8491 (Fig. 4 B). Despite this, multiple sequence alignment showed a 7,466-bp identical region shared between ST-18331 and the hyperinvasive NmC ST-8491 strains (QH01 and QH02), encompassing partial galE and cssA and full ctrG , cssE , csc , cssC , and cssB genes. These findings suggested that the genetic variation in the cps locus of the hyperinvasive NmC ST-8491 strains originated from ST-18331. The upstream and downstream recombination breakpoints were likely located in the galE and cssA genes, respectively (Fig. 4 D). The carriage strain QH03, which was from a close contact, likely resulted from a similar recombination event but involving a shorter region with different breakpoints. The recombination region was 5,869 bp, including parts of the galE , ctrG , cssE , and csc genes ( Fig.s 4C ). Discussion This study reports the origin and emergence of a novel hyperinvasive N. meningitidis strain, NmC ST-8491, which led to the deaths of two middle school students in China. This is the first report of a novel hyperinvasive strain in China since the discovery of NmC ST-4821 in 2003 27 . Unlike the severe outbreaks caused by NmC ST-4821, the NmC ST-8491 strain was effectively controlled at the early stages of the outbreak, with no evidence of widespread transmission to date. We confirmed that the fatal NmC ST-8491 strain originated through recombination. The recombination receptor was an ST-8491 strain with an incomplete NmW cps locus, and the recombination donor was an ST-18331 strain with an incomplete NmC cps locus, both carried by healthy close contacts. Multiple lines of evidence suggest that these carriage strains are commensals 3 : they are nonserogroupable strains, exhibit significant genetic divergence from known hyperinvasive strains, and show persistent carriage in the same individuals as indicated by repeated PCR detection. Furthermore, ST-8491 has been present in the healthy population for at least 15 years. The isolation of ST-18331 from the same close contact after a one-month interval further supports its commensal nature. The distribution and prevalence of these commensal strains in the population are unclear, raising the concern that hyperinvasive ST-8491 could re-emerge through recombination in populations carrying both strains. Given that commensal ST-8491 strains have been widespread in northern China, this possibility is likely. Moreover, the outbreak occurred at Huangzhong First Middle School, located near the renowned Tibetan gompa, Kumbum Monastery, which attracts 2 to 3 million tourists and pilgrims annually, including 60,000 to 150,000 international visitors. It is possible that the commensal strains could have spread through the movement of these visitors. To prevent a recurrence of the outbreak, it is crucial to monitor commensal strains carried by healthy individuals. Extensive studies have shown that adolescents have the highest N. meningitidis carriage rates 25 . Our research suggested that the high carriage rate in adolescents may be a key driver in the emergence of new hyperinvasive strains. Efforts to monitor healthy populations should therefore focus on adolescents. Establishing causal relationships between a pathogen's virulence factors and disease outbreaks remains a complex challenge 14 , 26 . This study is notable for identifying the clear origin and emergence of a novel hyperinvasive strain at an early stage of the outbreak. To our knowledge, this marks the first documented instance of such a well-defined process in N. meningitidis . We isolated carrier strains highly similar to the hyperinvasive strain, allowing us to accurately pinpoint the two potential virulence factors in the genome of the hyperinvasive strain: the opal gene and the cps locus. Because the genetic variation in the opal gene likely arose from gene duplication and was also present in carrier strains, we hypothesized that it was not a critical virulence factor. In contrast, the cps locus appeared to be the only key factor associated with virulence. Furthermore, we discovered that a carrier strain had an identical serogroup as the hyperinvasive strain and shared 99.9% genomic identity, differing by only 25 SNPs in the cps locus. This finding suggested that even minor nucleotide variations in this locus may significantly modulate the virulence of N. meningitidis . This study demonstrated that hyperinvasive strains can emerge through recombination between commensal carrier strains, and were not associated with pre-existing hyperinvasive strains 18 . Therefore, monitoring N. meningitidis in carriers, instead of focusing solely on invasive strains, is crucial for preventing the emergence of new hyperinvasive strains. Methods Case investigation Two fatal cases of IMD were reported from hospitals in Xining City of Qinghai Province to the National Notifiable Disease Reporting System in China Centers for Disease Control and Prevention (CDC) in June 2024. These fatal cases of IMD were further confirmed by the CDC of Huangzhong District, the CDC of Xining City, and the CDC of Qinghai Province. Blood samples were collected from both fatal cases at the hospital for N. meningitidis isolation. Close contact survey The close contacts of the fatal cases were screened using a real-time PCR assay for N. meningitidis 27 . PCR-positive throat swab samples from the close contacts were used to isolate N. meningitidis . Characterization of the isolates and DNA extraction The 13 N. meningitidis isolates collected in the present study were cultured on blood agar plates at 37°C in a 5% CO 2 atmosphere for 18–22 h. Gram staining and biochemical tests (API-NH, BioMerieux) were performed to confirm the isolates. Serogrouping was performed using slide agglutination (Remel Agglutinating Sera, Thermo Scientific). Pure cultures of N. meningitidis were eluted with saline solution, and the supernatant was discarded after centrifugation. Genomic DNA was extracted using a DNA purification kit (Genomic DNA Purification Kit, Promega) according to the manufacturer's instructions. The purity and integrity of the DNA were assessed and quantified using a 5400 Fragment Analyzer system (Agilent). Genomic DNA that met quality control standards underwent whole-genome sequencing. Whole-genome sequencing Genomic DNA libraries were prepared separately for Illumina and Oxford Nanopore sequencing. Illumina libraries were constructed by sonication (Covaris M220) and TruSeq DNA kit, sequenced on NovaSeq 6000 with 150-bp paired-end reads. Raw reads were quality-checked with FastQC v0.11.8 and trimmed by Trimmomatic v0.39. Nanopore libraries used the SQK-NBD114.96 ligation kit and R10 flow cells, sequenced for 24 hours on MinKNOW v2.0; base-calling was done with Guppy v3.3.0. Genome assembly The hybrid genome assembly was performed using Unicycler v0.4.7, which allows for the integration of both Illumina reads (short and accurate) and Nanopore reads (long and less accurate) in conservative mode. The highly accurate Illumina reads were used to align against the Nanopore reads as a reference, correcting random sequencing errors and producing a high-quality genome assembly. The assembled sequence was then polished by aligning the Illumina paired-end reads using the BWA-MEM algorithm in Pilon v1.2.3. Multiple rounds of polishing were conducted until no further errors could be corrected. Genome alignment and genetic variation analysis FastMLST software ( https://github.com/EnzoAndree/FastMLST ) was used for multilocus sequence typing (MLST) on each genome. Whole-genome alignments were performed using the progressiveMauve algorithm implemented in Geneious software v2022.1, which applies the ClustalW progressive global alignment algorithm to each locally collinear block (LCB). The LCBs were extracted to confirm their homology, underwent a secondary alignment using MAFFT v7.490, and then were concatenated to calculate the overall genomic identity. Whole-genome alignments of the ST-8491 and ST-188331 strains from Qinghai (13 genomes in total) revealed eight LCBs, while whole-genome alignments of the nine ST-8491 isolates from Qinghai identified only one LCB. The genetic variations in the nine Qinghai ST-8491 isolates were manually checked to confirm that they represented authentic variations. For the nine ST-8491 isolates from Qinghai, 52 genetic variation regions were identified through whole-genome sequence alignment. Based on these variations, a profile similar to the cgMLST data structure was generated, consisting of 52 variation loci. A neighbor joining tree of the profile was then visualized using GrapeTree software v2.1 28 . The capsule polysaccharide synthesis ( cps ) locus of each genome was analyzed using the function characterize_neisseria_capsule.py 29 . The sequences of the cps locus, including approximately 5,000 bp upstream and 3,000 bp downstream of the locus, were extracted for multiple sequence alignment using MAFFT v7.490. The genomic structure of the cps locus of each genome was annotated and visualized using Geneious software v2022.1. The recombination analysis of the cps locus was performed by Simplot v3.5.1 using the Kimura distance model with a 20-bp window size and 100-bp step size. cgMLST analysis A neighbor joining tree was constructed based on the cgMLST profile of 390 isolates of the CC4821 clade ( Table S1 ) and 13 isolates obtained from Qinghai in the present study. The CC4821 clade encompassed all known N. meningitidis isolates that shared a common ancestor with CC4821, and comprised all identified CC4821 isolates, 23 unassigned (UA) isolates, and one CC8 isolate 24 . The results were then visualized using GrapeTree software v2.1 28 .. The cgMLST scheme and profile generated in this study are available at Chewie Nomenclature Server website ( https://chewbbaca.online ) 30 . Phylogenetic analysis A phylogenetic analysis of ST-8491 was performed based on genomes of 13 ST-8491 isolates in the PubMLST database ( Table S1 ) and genomes of 10 ST-8491 isolates from Qinghai using Gubbins software v3.3.1 31 . Reference genome 130903 (PubMLST ID: 57876) was used for whole genome sequence alignment, recombination analysis, and maximum likelihood (ML) phylogeny construction. The data were analyzed using five iterations in Gubbins. The starting phylogenetic tree was constructed and subsequent iterations were performed using IQTREE v2.2.6 with the GTRGAMMA model. The branch supports of the final tree were assessed using a bootstrap analysis with 1000 replicates. The ML tree of ST-8491 was visualized and annotated by the R packages ggtree v3.6.2 32 . Declarations Ethics approval: This study was approved by the Ethics Committee of the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention (approval number: ICDC-2022007). Consent to participate: According to the established national surveillance protocol and under China’s Law on the Prevention and Control of Infectious Diseases, the bacterial isolates were collected as part of an urgent outbreak response. The need for individual informed consent was waived by the Ethics Committee. Declaration of interests The authors declare no competing interests. Acknowledgements This work was supported by the National Key Research and Development Program of China (2022YFC2305303), the Beijing Natural Science Foundation (L212011), the Operation of Public Health Emergency Response Mechanisms-Infectious Disease Control (102393220020020000029), and the Enhancement of Infectious Disease Surveillance and Control Technological Capabilities (102393240020020000003). Data availability The genomes of isolates that sequenced and described in present study were available in pubMLST website ( https://pubmlst.org/organisms/neisseria-spp ). The PubMLST IDs for isolates QH01 to QH13 are 162129 to 162141. The cgMLST scheme and profile used in this study are available at Chewie Nomenclature Server website ( https://chewbbaca.online ). References Denamur, E., Clermont, O., Bonacorsi, S. & Gordon, D. The population genetics of pathogenic Escherichia coli . Nature Reviews Microbiology 19 , 37-54, doi:10.1038/s41579-020-0416-x (2021). Turner, N. A. et al. 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Genomic analysis of the meningococcal ST-4821 complex–Western clade, potential sexual transmission and predicted antibiotic susceptibility and vaccine coverage. PloS one 15 , e0243426, doi:10.1371/journal.pone.0243426 (2020). Shao, Z. et al. Identification of a new Neisseria meningitidis serogroup C clone from Anhui province, China. Lancet 367 , 419-423, doi:10.1016/S0140-6736(06)68141-5 (2006). Tan, Z. et al. Origin of Neisseria meningitidis clonal complex 4821. Emerging microbes & infections 14 , 2515461, doi:10.1080/22221751.2025.2515461 (2025). Christensen, H., May, M., Bowen, L., Hickman, M. & Trotter, C. L. Meningococcal carriage by age: a systematic review and meta-analysis. The Lancet. Infectious diseases 10 , 853-861, doi:10.1016/s1473-3099(10)70251-6 (2010). Mikucki, A. & Kahler, C. M. Microevolution and its impact on hypervirulence, antimicrobial resistance, and vaccine escape in Neisseria meningitidis . Microorganisms 11 , 3005 (2023). Dolan Thomas, J. et al. sodC -based real-time PCR for detection of Neisseria meningitidis . PloS one 6 , e19361, doi:10.1371/journal.pone.0019361 (2011). Zhou, Z. et al. GrapeTree: visualization of core genomic relationships among 100,000 bacterial pathogens. Genome research 28 , 1395-1404, doi:10.1101/gr.232397.117 (2018). Marjuki, H. et al. Whole-genome sequencing for characterization of capsule locus and prediction of serogroup of invasive meningococcal isolates. Journal of Clinical Microbiology 57 , e01609-01618, doi:10.1128/jcm.01609-18 (2019). Mamede, R., Vila-Cerqueira, P., Silva, M., Carriço, J. A. & Ramirez, M. Chewie Nomenclature Server (chewie-NS): a deployable nomenclature server for easy sharing of core and whole genome MLST schemas. Nucleic acids research 49 , D660-d666, doi:10.1093/nar/gkaa889 (2021). Croucher, N. J. et al. Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. Nucleic acids research 43 , e15, doi:10.1093/nar/gku1196 (2015). Yu, G. Using ggtree to visualize data on tree-like structures. Current Protocols in Bioinformatics 69 , e96, doi:10.1002/cpbi.96 (2020). Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7042271","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":482295056,"identity":"de185b81-7e6d-4ed4-ae8f-dfe863170c00","order_by":0,"name":"Zhujun Shao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYDACCSBmbGBgZmBgPnCAgY00LWwJpGkBAh4DBqK0yM9ufvjw547D7Aa3ez4e+FFmx8A/uwG/FoM7x4wNJM8cZja4c3bDwZ5zyQwSdw4Q0CKRYCZh2AbUciN3w2HGtgMgEQIOm5H+TSIRrCXnAXFaGG7kmEkchGhhIE4LUGWxYWNbOrPkjTQDkF94JG4QdtjGhz/brJP5biQ//gAMMTn+GYQcBgXJMAYPceqBwI5olaNgFIyCUTDyAABITEc7dLhGuAAAAABJRU5ErkJggg==","orcid":"","institution":"Chinese Center for Disease Control and Prevention","correspondingAuthor":true,"prefix":"","firstName":"Zhujun","middleName":"","lastName":"Shao","suffix":""},{"id":482295057,"identity":"331b6eea-569a-4c29-996d-d2278b28d7b1","order_by":1,"name":"Zhizhou Tan","email":"","orcid":"https://orcid.org/0000-0002-2844-9673","institution":"Chinese Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Zhizhou","middleName":"","lastName":"Tan","suffix":""},{"id":482295058,"identity":"bc05bdfe-631c-4722-9e3f-8aa62533f4ae","order_by":2,"name":"Hairui Wang","email":"","orcid":"","institution":"Chinese Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Hairui","middleName":"","lastName":"Wang","suffix":""},{"id":482295059,"identity":"2eefa650-a185-44be-adfa-3d7d8388e83f","order_by":3,"name":"Huijun Zhao","email":"","orcid":"","institution":"Xining Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Huijun","middleName":"","lastName":"Zhao","suffix":""},{"id":482295060,"identity":"c65a20ce-0316-46cd-9907-b74218e1ff14","order_by":4,"name":"Xiaodong Qi","email":"","orcid":"","institution":"Qinghai Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Qi","suffix":""},{"id":482295061,"identity":"325312fa-ad80-4a76-9457-65d209a1bd73","order_by":5,"name":"Chunguang Ma","email":"","orcid":"","institution":"Xining Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Chunguang","middleName":"","lastName":"Ma","suffix":""},{"id":482295062,"identity":"2be12e03-a3ba-4f45-adc0-e0d97e865614","order_by":6,"name":"Yaxin Zhang","email":"","orcid":"","institution":"Xining Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Yaxin","middleName":"","lastName":"Zhang","suffix":""},{"id":482295063,"identity":"4e4f32f6-c058-4d67-a985-67859439a667","order_by":7,"name":"Juan Xu","email":"","orcid":"","institution":"Chinese Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Xu","suffix":""},{"id":482295064,"identity":"f48e1226-5b10-4847-bdec-b0e5ace5442e","order_by":8,"name":"Jie Che","email":"","orcid":"","institution":"Chinese Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Che","suffix":""},{"id":482295065,"identity":"ba674d76-1e7d-49e2-96df-9e299920e86f","order_by":9,"name":"Li Xu","email":"","orcid":"","institution":"Chinese Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Xu","suffix":""},{"id":482295066,"identity":"6da9bb15-9f8b-489a-841a-752f5e2d43ec","order_by":10,"name":"Xueping Liu","email":"","orcid":"","institution":"Chinese Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Xueping","middleName":"","lastName":"Liu","suffix":""},{"id":482295067,"identity":"ca4888a3-a4a0-4626-89f5-0ff162ffb7fb","order_by":11,"name":"Zhifeng An","email":"","orcid":"","institution":"Qinghai Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Zhifeng","middleName":"","lastName":"An","suffix":""},{"id":482295068,"identity":"795dcaa7-a325-4365-9882-189c315f4a06","order_by":12,"name":"Haijian Zhou","email":"","orcid":"","institution":"Chinese Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Haijian","middleName":"","lastName":"Zhou","suffix":""},{"id":482295069,"identity":"76e91729-5285-43e7-add5-c6d4e4d232fb","order_by":13,"name":"Maojun Zhang","email":"","orcid":"","institution":"Chinese Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Maojun","middleName":"","lastName":"Zhang","suffix":""},{"id":482295070,"identity":"7943f060-ef6a-4fb9-86ba-1a02e065b0a0","order_by":14,"name":"Biao Kan","email":"","orcid":"https://orcid.org/0000-0002-6141-4552","institution":"China CDC","correspondingAuthor":false,"prefix":"","firstName":"Biao","middleName":"","lastName":"Kan","suffix":""},{"id":482295071,"identity":"9b630701-ea76-4ea3-b685-e28a496a4cb0","order_by":15,"name":"Bike Zhang","email":"","orcid":"","institution":"Chinese Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Bike","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-07-04 02:25:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7042271/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7042271/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90785878,"identity":"843ff106-d348-44f3-a6f0-46aff37d6b63","added_by":"auto","created_at":"2025-09-08 07:04:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106214,"visible":true,"origin":"","legend":"\u003cp\u003eTimeline of the invasive meningococcal disease outbreak in Qinghai Province, China. \u003cem\u003eNeisseria meningitidis \u003c/em\u003ewas identified and isolated from both the fatal cases and their close contacts. The sequence type and serogroup of each isolate are presented. Abbreviation: NmC, Serogroup C \u003cem\u003eNeisseria meningitidis\u003c/em\u003e; NG, nonserogroupable.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7042271/v1/aaa428aeb93ad9478b06b75a.png"},{"id":90786551,"identity":"c4818b8f-6c4f-4d20-9c77-2e5b13ecd8a5","added_by":"auto","created_at":"2025-09-08 07:12:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":324238,"visible":true,"origin":"","legend":"\u003cp\u003ecgMLST and phylogenetic analysis of \u003cem\u003eNeisseria meningitidis \u003c/em\u003eisolates in Qinghai. (\u003cstrong\u003eA\u003c/strong\u003e) A neighbor-joining tree of clonal complex 4821 clade. The tree is constructed based on cgMLST profiles of 390 isolates of the clonal complex 4821 clade and 13 isolates obtained from Qinghai in present study. The tips of the tree are color-coded according to the capsule polysaccharide synthesis (\u003cem\u003ecps\u003c/em\u003e) locus of each isolate, with the corresponding numbers of isolates belonging to each serogroup of \u003cem\u003ecps\u003c/em\u003e locus displayed in parentheses. (\u003cstrong\u003eB\u003c/strong\u003e) A phylogenetic tree of ST-8491. A maximum likelihood tree is generated using the nonrecombinant regions of a whole-genome alignment consisting of thirteen ST-8491 isolates from PubMLST database nine ST-8491 isolates from the Qinghai outbreak. Isolates derived from fatal cases are shown in bold. The scale bar on the tree represents the number of point mutations observed along each branch. The branch supports are evaluated using bootstrap with 1000 replicates. Bootstrap values above 70% are marked with asterisks. Each isolate is color-coded based on the \u003cem\u003ecps\u003c/em\u003e locus of its serogroup. Notably, one isolate, named 651708, is identified with a \u003cem\u003ecps\u003c/em\u003e locus classified as serogroup Y. However, its phenotype is determined to be serogroup W (Table S1). This discrepancy suggests that the serogroup Y \u003cem\u003ecps\u003c/em\u003e locus is likely misclassified, possibly due to the close genetic similarity between the \u003cem\u003ecsy\u003c/em\u003e gene in the serogroup Y locus and the \u003cem\u003ecsw\u003c/em\u003e gene in the serogroup W locus. (\u003cstrong\u003eC\u003c/strong\u003e) A map illustrates the distribution of ST-8491 strains across the provincial administrative regions of China, with the location of Xining City in Qinghai Province, where the outbreak occurred, marked.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7042271/v1/98636fe682e7f6190157c194.png"},{"id":90785877,"identity":"958af2d8-99c8-42f1-8607-e4b5b1e4c0bb","added_by":"auto","created_at":"2025-09-08 07:04:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":135866,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic identity, genetic variation and evolutionary relationships of Qinghai ST-8491 isolates. (\u003cstrong\u003eA\u003c/strong\u003e) Distribution of genetic variation regions among genomes of Qinghai ST-8491 isolates. Blue short lines represent variation regions identified in a single isolate, while red short lines denote those found in two or more isolates. Whole-genome identity analysis of nine Qinghai ST-8491 isolates is shown, brown represents 100% identity, light blue represents 30–100% identity. The length of the bases after whole-genome alignment is displayed at the top.Arrows indicate the positions of 52 variation regions identified across the genomes of the nine isolates. Isolates derived from fatal cases are shown in bold. Dark red arrows and red boxes highlight two variation regions exclusively found in isolates from fatal cases. (\u003cstrong\u003eB\u003c/strong\u003e) Number of nucleotide differences among genomes of Qinghai ST-8491isolates. (\u003cstrong\u003eC\u003c/strong\u003e) A neighbor-joining tree of Qinghai ST-8491 isolates. The tree illustrates the genetic distances among Qinghai ST-8491 isolates, based on profiles of 52 variation regions identified across their genomes. The tips of the tree are color-coded according to the capsule polysaccharide synthesis (\u003cem\u003ecps\u003c/em\u003e) locus of each isolate, with the corresponding numbers of isolates belonging to each serogroup of \u003cem\u003ecps\u003c/em\u003elocus displayed in parentheses.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7042271/v1/ff5be158582d22d7b90749f3.png"},{"id":90785879,"identity":"9b3b89f0-0388-4281-b7cd-51fb4e575176","added_by":"auto","created_at":"2025-09-08 07:04:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":220386,"visible":true,"origin":"","legend":"\u003cp\u003eSequence identity, gene structure, and recombination analysis of the capsule polysaccharide synthesis (\u003cem\u003ecps\u003c/em\u003e) locus. The \u003cem\u003ecps\u003c/em\u003elocus sequences analyzed included all coding genes as well as sequences approximately 5,000bp upstream and 3,000bp downstream of the \u003cem\u003ecps\u003c/em\u003e locus. Genes and sequences are color-coded in red and green to indicate their distinct evolutionary origins. Red genes represent those originating from the NG ST-18331 carrier strains, while green genes correspond to those from the NG ST-8491 carrier strains. The regions of recombination are highlighted in translucent red. In the sequence identity analysis, brown represents 100% identity, light blue represents 30–100% identity, and dark red represents below 30% identity. The gray band illustrates identical sequences within each sequence alignment, while genetic variations are delineated by gaps and black stripes. The length of the bases after each multiple sequence alignment is displayed at the top of each panel. (\u003cstrong\u003eA\u003c/strong\u003e) The sequence identity and gene structure of the \u003cem\u003ecps\u003c/em\u003e locus in ST-8491 isolates collected prior to the Qinghai outbreak are compared with those from ST-8491 isolates identified during the outbreak. For isolates collected prior to the outbreak, the serogroup, collection year, and province are indicated. For isolates collected during the outbreak, the serogroup, source of isolation, and group classification are provided. Isolates derived from fatal cases are highlighted in bold. (\u003cstrong\u003eB\u003c/strong\u003e) Sequence identity and gene structure of the \u003cem\u003ecps\u003c/em\u003e locus of the strains harboring serogroup C \u003cem\u003ecps\u003c/em\u003e locus in the Qinghai outbreak. (\u003cstrong\u003eC\u003c/strong\u003e) Sequence identity and gene structure of the \u003cem\u003ecps\u003c/em\u003e locus of the strains underwent to recombination in the Qinghai outbreak. (\u003cstrong\u003eD\u003c/strong\u003e) Recombination analysis of the representative strains in the Qinghai outbreak is conducted using Simplot, revealing potential recombination breakpoints. The Simplot analysis is performed by using the Kimura distance model with a 20-bp window size and a 100-bp step size.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7042271/v1/07f108ca27cb3dbfcc115d36.png"},{"id":90786604,"identity":"164d79f6-68db-4d70-a93c-7de9820ccdb0","added_by":"auto","created_at":"2025-09-08 07:20:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1403909,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7042271/v1/a38cb466-f484-4ae5-a1f0-3ef51e9c2190.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Hyperinvasive Neisseria meningitidis in China Originates from Commensals in Healthy Carriers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSome formidable pathogens have originated from commensals of humans, including numerous pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These pathogens pose a significant threat to public health due to the unpredictability of their outbreaks and the need to revise pathogen monitoring strategies \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Currently, most public health surveillance systems focus on pathogens that have already demonstrated pathogenicity or epidemic potential \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. As increasing evidence suggests that commensal strains can evolve into pathogenic strains, monitoring strategies may need to be expanded to include healthy carriers.\u003c/p\u003e\u003cp\u003e\u003cem\u003eNeisseria meningitidis\u003c/em\u003e (\u003cem\u003eN. meningitidis\u003c/em\u003e), an obligate human bacterium, is both a commensal and a major pathogen responsible for invasive meningococcal disease (IMD) \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Genetically, IMD strains are typically associated with a limited number of clonal complexes (CCs) or sequence types (STs), referred to as hyperinvasive strains \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In contrast, commensal strains, often referred to as carrier strains, exhibit greater genetic diversity. Commensal strains may form clonal complexes, exist outside of any clonal complex, or appear as rare strains within a clonal complex with high internal genetic diversity \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Commensal strains are carried by approximately 10% of the general population as part of the normal respiratory flora \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Phenotypically, the majority of hyperinvasive strains express a polysaccharide capsule and belong to one of six serogroups (A, B, C, Y, W, and X) \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In comparison, commensal strains, which often lack or have a reduced polysaccharide capsule, are classified as nonserogroupable (NG) \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The virulence factors of \u003cem\u003eN. meningitidis\u003c/em\u003e remain controversial, as genomic analyses have yet to clearly identify virulence-related genes in \u003cem\u003eN. meningitidis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, because commensal and hyperinvasive strains belong to genetically distinct populations \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eN. meningitidis\u003c/em\u003e surveillance mainly focuses on hyperinvasive strains and strains of specific serogroups \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Commensal strains in healthy individuals are not included in routine monitoring, despite the long-standing hypothesis that commensal carrier strains may serve as a source of hyperinvasive strains \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEmerging hyperinvasive strains of \u003cem\u003eN. meningitidis\u003c/em\u003e pose a significant public health threat \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. While IMD typically occurs endemically, emerging hyperinvasive strains can trigger large, unpredictable epidemics \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Globally, the most recently emerged hyperinvasive strain is serogroup C (NmC) ST-10217, which appeared in Nigeria in 2013 and caused large outbreaks in 2016, with more than 14,000 IMD cases \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In China, the most recent hyperinvasive strain is NmC ST-4821, which emerged in 2003 and caused a series of outbreaks from 2003 to 2006, with over 1,500 IMD cases reported annually \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Since then, no new hyperinvasive strains have been reported in China.\u003c/p\u003e\u003cp\u003eThere is currently a lack of direct evidence confirming that hyperinvasive strains originate from commensal strains, and it is also generally believed that pre-existing hyperinvasive strains play a significant role in the emergence of new hyperinvasive strains. NmC ST-10217 may have originated from a commensal NG ST-9367 strain, acquiring a full NmC capsule polysaccharide synthesis (\u003cem\u003ecps\u003c/em\u003e) locus from a hyperinvasive NmC ST-11 strain and an MDAΦ prophage from an unknown strain \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The origin of NmC ST-4821 remains unclear, but it likely originated from serogroup B (NmB) CC4821 strains \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn June 2024, two fatal cases of IMD occurred at a middle school in Xining City, Qinghai Province, China. Isolates from these cases revealed a novel hyperinvasive strain of \u003cem\u003eN. meningitidis\u003c/em\u003e. Importantly, this new hyperinvasive strain likely originated from commensal strains in healthy carriers. This study aims to characterize the epidemiological and molecular traits of this emerging strain and ascertain its origin.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eFatal case ascertainment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn June 2024, two fatal cases due to IMD were successively reported in Xining City, Qinghai Province, China. The cases were students aged 14 and 15, in the same classroom at Huangzhong First Middle School. The first patient died on June 6 after seeking medical treatment on June 4. The second patient died on June 26 after seeking treatment the previous day. Two isolates were cultured from these two cases (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClose contact survey and disease control\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo control the spread of IMD, the CDC screened close contacts (including students, school staff, and family members) of the two fatal cases. Fourteen close contacts tested positive for \u003cem\u003eN. meningitidis\u003c/em\u003e through PCR screenings; all were students in the classroom with the fatal cases. There were 51 students in the classroom, including the two fatal cases, resulting in a \u003cem\u003eN. meningitidis\u003c/em\u003e prevalence rate of 31.4% in the classroom. These fourteen PCR-positive close contacts were all healthy and were identified through four screenings conducted on June 9, June 27, July 5, and July 7 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eN. meningitidis\u003c/em\u003e isolates were cultured from 10 of the 14 close contacts. One individual yielded two isolates (samples collected on June 9 and July 7). In total, 11 isolates were obtained from the 10 close contacts. Notably, of these close contacts from whom isolates were obtained, only one individual tested positive only once during the four PCR screenings; the remaining close contacts tested positive in at least two screenings (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cstrong\u003eand Table S2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cem\u003eNeisseria meningitidis\u003c/em\u003e isolates from the Qinghai outbreak\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsolates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsolation source\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePCR\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMLST\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSerogroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ecps\u003c/em\u003e locus\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGrouping\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFatal case 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-8491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC (complete)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFatal case 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-8491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC (complete)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClose contact 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPPP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-8491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC (complete, 25 SNPs in \u003cem\u003egalE\u003c/em\u003e, \u003cem\u003ecssB\u003c/em\u003e, and \u003cem\u003ecssA\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClose contact 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNNNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-8491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eW (partial \u003cem\u003egalU\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClose contact 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePNPP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-8491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eW (partial \u003cem\u003egalU\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClose contact 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePNPP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-8491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eW (partial \u003cem\u003egalU\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClose contact 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePNNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-8491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eW (partial \u003cem\u003egalU\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClose contact 10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePNPP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-8491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eW (partial \u003cem\u003egalU\u003c/em\u003e and \u003cem\u003ecsw\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClose contact 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePNPP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-8491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eW (partial \u003cem\u003egalU\u003c/em\u003e and \u003cem\u003ecsw\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClose contact 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPPP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-18331\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC (partial \u003cem\u003ecssA, ctrA\u003c/em\u003e, and \u003cem\u003ecrtC\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClose contact 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-18331\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC (partial \u003cem\u003ecssA, ctrA\u003c/em\u003e, and \u003cem\u003ecrtC\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClose contact 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-18331\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC (partial \u003cem\u003ecssA, ctrA\u003c/em\u003e, and \u003cem\u003ecrtC\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQH13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClose contact 9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eST-18331\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC (partial \u003cem\u003ecssA, ctrA\u003c/em\u003e, and \u003cem\u003ecrtC\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egroup 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e*\u003c/sup\u003eA total of four rounds of PCR screening of close contacts were conducted on June 9, June 27, July 5, and July 7. The results of each close contact\u0026apos;s PCR screening are displayed in chronological order, with \u0026ldquo;P\u0026rdquo; indicating a positive result and \u0026ldquo;N\u0026rdquo; indicating a negative result. The Ct values of the PCR-positive samples ranged from 17.9 to 38.6, with a mean of 26.95 (95% CI: 25.41, 28.99). The detailed PCR screening results for close contacts are shown in Table S2.\u003cbr\u003eAbbreviation: \u003cem\u003ecps\u003c/em\u003e, capsule polysaccharide synthesis; NG, nonserogroupable; SNPs, single nucleotide polymorphisms. The detailed gene structure of the\u0026nbsp;\u003cem\u003ecps\u003c/em\u003e locus for each group is shown in Fig. 4.\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eOn June 28, the CDC administered prophylactic antibiotics (mainly cefixime) to PCR-positive students and emergency vaccinations of the ACYW135 quadrivalent meningococcal vaccine to all staff and students at the school. By July 7, a total of 3,198 individuals had been vaccinated, successfully controlling the spread of IMD, with no further cases reported (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of a new hyperinvasive strain in fatal cases and carrier strains in close contacts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on MLST, serogrouping, and \u003cem\u003ecps\u003c/em\u003e locus sequence analysis, the 13 \u003cem\u003eN. meningitidis\u003c/em\u003e isolates\u0026mdash;two isolates (QH01 and QH02) from the fatal cases and 11 isolates (QH03 to QH13) from close contacts\u0026mdash;were classified into five groups, labeled as group 1 through group 5 (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Isolates within the same group shared identical sequence types and serogroups, with their \u003cem\u003ecps\u003c/em\u003e locus sequences exhibiting 100% identity. The two isolates from the fatal cases belonged to group 1, which represents a new hyperinvasive strain, were both NmC ST-8491 with the complete \u003cem\u003ecps\u003c/em\u003e locus of NmC. In contrast, the carrier strains from the close contacts were classified into four groups, groups 2\u0026ndash;5. A singular isolate from a close contact (QH03) belonged to group 2 and was also NmC ST-8491. This isolate contained the complete \u003cem\u003ecps\u003c/em\u003e locus of NmC; however, it exhibited 25 single nucleotide polymorphisms (SNPs) compared to isolates in group 1. These SNPs were distributed in the \u003cem\u003egalE\u003c/em\u003e, \u003cem\u003ecssB\u003c/em\u003e, and \u003cem\u003ecssA\u003c/em\u003e genes. Groups 3 and 4 consisted of four and two isolates, respectively, all obtained from close contacts. These isolates belonged to NG ST-8491 and contained an incomplete serogroup W (NmW) \u003cem\u003ecps\u003c/em\u003e locus with a partial \u003cem\u003egalU\u003c/em\u003e gene. The \u003cem\u003ecps\u003c/em\u003e locus of group 3 and group 4 differed by a single SNP within the \u003cem\u003ecsw\u003c/em\u003e gene. Group 3 encoded a complete \u003cem\u003ecsw\u003c/em\u003e gene, whereas the \u003cem\u003ecsw\u003c/em\u003e gene in group 4 had a premature stop codon introduced by the SNP, resulting in a partial coding sequence. The MLST of group 5 was distinct from that of groups 1\u0026ndash;4, as group 5 belonged to ST-18331, a newly reported sequence type with no available genome data. This group comprised four isolates, all obtained from close contacts. The isolates in group 5 were NG ST-18331 with an incomplete NmC \u003cem\u003ecps\u003c/em\u003e locus, characterized by partial coding sequences of the \u003cem\u003ecssA\u003c/em\u003e, \u003cem\u003ectrA\u003c/em\u003e, and \u003cem\u003ecrtC\u003c/em\u003e genes.\u003c/p\u003e\n\u003cp\u003eWhole-genome alignment and identity analysis showed that the ST-8491 strains in Qinghai, including the hyperinvasive strain from the fatal cases (group 1) and those isolated from close contacts (groups 2, 3, and 4), exhibited a high degree of whole-genome identity (98.5\u0026ndash;99.9%), despite differences in their serogroup and \u003cem\u003ecps\u003c/em\u003e locus sequences. In contrast, the ST-18331 isolates (group 5) substantially diverged from the ST-8491 strains, with a whole-genome identity of only 88.8\u0026ndash;89.6% (\u003cstrong\u003eTable S3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOrigin of\u003c/strong\u003e \u003cstrong\u003eN. meningitidis\u003c/strong\u003e \u003cstrong\u003estrains in Qinghai\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cgMLST analysis revealed that all 13 isolates in Qinghai belonged to CC4821. The ST-8491 and ST-18331 isolates formed two distinct genetic clusters (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). All nine ST-8491 isolates from Qinghai (two from fatal cases and seven from close contacts) demonstrated high clonality, forming a cohesive monophyletic group and clustering with previously collected ST-8491 isolates in China. The four ST-18331 isolates (all from close contacts) constituted a separate monophyletic group, with substantial differences in their core genome compared to the ST-8491 isolates. The cgMLST analysis also revealed that both the ST-8491 and ST-18331 isolates were genetically distant from the hyperinvasive lineage of CC8421 (mainly ST-4821), which caused the IMD outbreak in China between 2003 and 2004 \u003csup\u003e23,24\u003c/sup\u003e, as well as the globally spreading lineage of CC4821 (mainly ST-3200), which is rarely associated with IMD \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eST-8491 is a rare ST within the CC4821, with only 13 isolates documented in the PubMLST database, all originating from carriers in China \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The records revealed that ST-8491 was first identified in Hebei Province in 2009, with sporadic reports of its occurrence from 2009 to 2022 in seven provinces, predominantly in northern China (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). A phylogenetic analysis showed that the Qinghai ST-8491 isolates formed an independent lineage. Distinct genetic differences were observed between the Qinghai ST-8491 isolates and the previously identified ST-8491 isolates. The Qinghai isolates showed the closest genetic relationship to ST-8491 isolates LN21 and LN22 collected from Shandong Province and to isolates 651708 and 651402 collected from Xinjiang Uygur Autonomous Region (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvolutionary relationship of the ST-8491 strains in Qinghai\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-genome alignment and genetic variation analyses were performed on all nine ST-8491 isolates from Qinghai. The genome lengths of the isolates ranged from 2,162,319 bp to 2,168,266 bp, with nucleotide differences between genomes ranging from 452 bp to 16,328 bp (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). These differences corresponded to 52 distinct regions of genetic variation scattered across the genomes of the nine isolates (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). These genetic variations were likely the result of different evolutionary mechanisms, including mutation, homologous recombination, horizontal gene transfer, phase variation, duplication, insertion, and deletion. The size of these regions of variation ranged from 1 bp to 7,466 bp.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eOf the 52 regions of variation, 41 were unique to individual genomes, while 11 were shared by two or more genomes. The genomes of the two isolates from the fatal cases (QH01, QH02) shared only two specific regions of variation: one in the \u003cem\u003eopal\u003c/em\u003e gene, which encodes the opacity protein OpA54, and the other in the \u003cem\u003ecps\u003c/em\u003e locus. Both regions are likely key virulence factors contributing to the emergence of the novel hyperinvasive strain (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The specific genetic variation in the \u003cem\u003eopal\u003c/em\u003e gene sequence was 280 bp in length, and identical sequences at another location were found in eight out of nine ST-8491 genomes (excluding QH08) from Qinghai, suggesting that this variation may have arisen through gene duplication (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The specific genetic variation in the \u003cem\u003ecps\u003c/em\u003e locus was 7,466 bp in size. The genome of the QH03 isolate from close contact also contained a variation in the \u003cem\u003ecps\u003c/em\u003e locus with a partially identical sequence to QH01 and QH02 from the fatal cases, although the regions of variation in QH03 was shorter, measuring 5,869 bp. Neither the 7,466 bp nor the 5,869 bp variation in the \u003cem\u003ecps\u003c/em\u003e locus were found in other ST-8491 strains, indicating that these variations were likely acquired through recombination from non-ST-8491 strains.\u003c/p\u003e\n\u003cp\u003eThe whole genomes of the two fatal case isolates (QH01, QH02) showed a 3,365-bp nucleotide difference across 16 regions of variation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB), with the closest genetic distance among the isolates from Qinghai, suggesting they may have originated from a common ancestor (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). The isolate from close contact 5 (QH03) was genetically closest to the fatal case isolates but exhibited a different length of genetic variation in the \u003cem\u003ecps\u003c/em\u003e locus and lacked the \u003cem\u003eopal\u003c/em\u003e regions of variation. Two independent recombination events in the \u003cem\u003ecps\u003c/em\u003e locus likely occurred in the ST-8491 strains during the Qinghai outbreak (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC): one recombination event resulted in the emergence of the hyperinvasive strain infecting the two fatal cases (QH01, QH02), while the other recombination event gave rise to the carrier strain from close contact 5 (QH03).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvolution of the\u003c/strong\u003e \u003cstrong\u003ecps\u003c/strong\u003e \u003cstrong\u003elocus in the novel hyperinvasive strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ecps\u003c/em\u003e loci of all publicly available ST-8491 isolates were analyzed, revealing a relatively conserved gene organization pattern over a 15-year evolutionary span (2009\u0026ndash;2024) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). The genes were grouped into six regions and arranged in the order B-D-A-C-E-D\u0026apos;. This organization differed from the classical gene arrangement of the \u003cem\u003ecps\u003c/em\u003e locus in \u003cem\u003eN. meningitidis\u003c/em\u003e, which followed the order D-A-C-E-D\u0026apos;-B \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The majority of genetic variations in the \u003cem\u003ecps\u003c/em\u003e locus of the ST-8491 isolates were observed in the genes of region A. Consequently, three distinct \u003cem\u003ecps\u003c/em\u003e loci were identified in the ST-8491 isolates: the unknown serogroup \u003cem\u003ecps\u003c/em\u003e locus, the NmW \u003cem\u003ecps\u003c/em\u003e locus, and the NmC \u003cem\u003ecps\u003c/em\u003e locus (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eST-8491 underwent at least two serogroup switching events from 2009 to 2024, evolving from unknown (2009) to W (2013\u0026ndash;2024) to C (2024) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). The earliest ST-8491 isolates from 2009 had an unknown serogroup with a \u003cem\u003ecps\u003c/em\u003e locus in region A containing five genes: \u003cem\u003egalU\u003c/em\u003e, \u003cem\u003ecrtG\u003c/em\u003e, \u003cem\u003ecssC\u003c/em\u003e, \u003cem\u003ecssB\u003c/em\u003e, and \u003cem\u003ecssA\u003c/em\u003e. In 2013, ST-8491 underwent its first serogroup switch by acquiring the capsular synthesis genes \u003cem\u003ecssF\u003c/em\u003e and \u003cem\u003ecsw\u003c/em\u003e in region A, becoming serogroup W. Prior to this, no other NmW strains had been reported within the clonal complex CC4821 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Therefore, the \u003cem\u003ecssF\u003c/em\u003e and \u003cem\u003ecsw\u003c/em\u003e genes likely originated from another clonal complex, most probably from NmW CC11 strains \u003csup\u003e39\u003c/sup\u003e. From 2013 until the Qinghai outbreak, the \u003cem\u003ecps\u003c/em\u003e locus of the NmW ST-8491 strains remained conserved. Minor genetic differences were found between the Qinghai ST-8491 isolates (QH04) and the 2013 ST-8491 isolate (131312) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), including a few SNPs in \u003cem\u003egalE\u003c/em\u003e, \u003cem\u003ecsw\u003c/em\u003e, and \u003cem\u003ecssC\u003c/em\u003e and an insertion in \u003cem\u003egalU\u003c/em\u003e. In the Qinghai outbreak, a second serogroup switch occurred, with the \u003cem\u003ecps\u003c/em\u003e locus changing to that of serogroup C. The \u003cem\u003egalU\u003c/em\u003e, \u003cem\u003ecrtG\u003c/em\u003e, \u003cem\u003ecssF\u003c/em\u003e, and \u003cem\u003ecsw\u003c/em\u003e genes in region A were replaced by reverse direction \u003cem\u003ecrtG\u003c/em\u003e, \u003cem\u003ecssE\u003c/em\u003e, and \u003cem\u003ecsc\u003c/em\u003e genes. Additionally, genetic variations were observed in the upstream (\u003cem\u003egalE\u003c/em\u003e) and downstream (\u003cem\u003ecssC\u003c/em\u003e, \u003cem\u003ecssB\u003c/em\u003e, and \u003cem\u003ecssA\u003c/em\u003e) regions.\u003c/p\u003e\n\u003cp\u003eAn analysis of the \u003cem\u003ecps\u003c/em\u003e locus of the ST-18331 strain from close contacts revealed only a 50.3\u0026ndash;59.5% nucleotide identity with the \u003cem\u003ecps\u003c/em\u003e locus of the Qinghai ST-8491 strains, likely due to differences in gene organization, with the sequence D-A-C-E-D\u0026apos;-B in ST-18331 and B-D-A-C-E-D\u0026apos; in ST-8491 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Despite this, multiple sequence alignment showed a 7,466-bp identical region shared between ST-18331 and the hyperinvasive NmC ST-8491 strains (QH01 and QH02), encompassing partial \u003cem\u003egalE\u003c/em\u003e and \u003cem\u003ecssA\u003c/em\u003e and full \u003cem\u003ectrG\u003c/em\u003e, \u003cem\u003ecssE\u003c/em\u003e, \u003cem\u003ecsc\u003c/em\u003e, \u003cem\u003ecssC\u003c/em\u003e, and \u003cem\u003ecssB\u003c/em\u003e genes. These findings suggested that the genetic variation in the \u003cem\u003ecps\u003c/em\u003e locus of the hyperinvasive NmC ST-8491 strains originated from ST-18331. The upstream and downstream recombination breakpoints were likely located in the \u003cem\u003egalE\u003c/em\u003e and \u003cem\u003ecssA\u003c/em\u003e genes, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). The carriage strain QH03, which was from a close contact, likely resulted from a similar recombination event but involving a shorter region with different breakpoints. The recombination region was 5,869 bp, including parts of the \u003cem\u003egalE\u003c/em\u003e, \u003cem\u003ectrG\u003c/em\u003e, \u003cem\u003ecssE\u003c/em\u003e, and \u003cem\u003ecsc\u003c/em\u003e genes (\u003cstrong\u003eFig.s 4C\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study reports the origin and emergence of a novel hyperinvasive \u003cem\u003eN. meningitidis\u003c/em\u003e strain, NmC ST-8491, which led to the deaths of two middle school students in China. This is the first report of a novel hyperinvasive strain in China since the discovery of NmC ST-4821 in 2003 \u003csup\u003e27\u003c/sup\u003e. Unlike the severe outbreaks caused by NmC ST-4821, the NmC ST-8491 strain was effectively controlled at the early stages of the outbreak, with no evidence of widespread transmission to date.\u003c/p\u003e\u003cp\u003eWe confirmed that the fatal NmC ST-8491 strain originated through recombination. The recombination receptor was an ST-8491 strain with an incomplete NmW \u003cem\u003ecps\u003c/em\u003e locus, and the recombination donor was an ST-18331 strain with an incomplete NmC \u003cem\u003ecps\u003c/em\u003e locus, both carried by healthy close contacts. Multiple lines of evidence suggest that these carriage strains are commensals \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e: they are nonserogroupable strains, exhibit significant genetic divergence from known hyperinvasive strains, and show persistent carriage in the same individuals as indicated by repeated PCR detection. Furthermore, ST-8491 has been present in the healthy population for at least 15 years. The isolation of ST-18331 from the same close contact after a one-month interval further supports its commensal nature.\u003c/p\u003e\u003cp\u003eThe distribution and prevalence of these commensal strains in the population are unclear, raising the concern that hyperinvasive ST-8491 could re-emerge through recombination in populations carrying both strains. Given that commensal ST-8491 strains have been widespread in northern China, this possibility is likely. Moreover, the outbreak occurred at Huangzhong First Middle School, located near the renowned Tibetan gompa, Kumbum Monastery, which attracts 2 to 3\u0026nbsp;million tourists and pilgrims annually, including 60,000 to 150,000 international visitors. It is possible that the commensal strains could have spread through the movement of these visitors. To prevent a recurrence of the outbreak, it is crucial to monitor commensal strains carried by healthy individuals. Extensive studies have shown that adolescents have the highest \u003cem\u003eN. meningitidis\u003c/em\u003e carriage rates \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Our research suggested that the high carriage rate in adolescents may be a key driver in the emergence of new hyperinvasive strains. Efforts to monitor healthy populations should therefore focus on adolescents.\u003c/p\u003e\u003cp\u003eEstablishing causal relationships between a pathogen's virulence factors and disease outbreaks remains a complex challenge \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. This study is notable for identifying the clear origin and emergence of a novel hyperinvasive strain at an early stage of the outbreak. To our knowledge, this marks the first documented instance of such a well-defined process in \u003cem\u003eN. meningitidis\u003c/em\u003e. We isolated carrier strains highly similar to the hyperinvasive strain, allowing us to accurately pinpoint the two potential virulence factors in the genome of the hyperinvasive strain: the \u003cem\u003eopal\u003c/em\u003e gene and the \u003cem\u003ecps\u003c/em\u003e locus. Because the genetic variation in the \u003cem\u003eopal\u003c/em\u003e gene likely arose from gene duplication and was also present in carrier strains, we hypothesized that it was not a critical virulence factor. In contrast, the \u003cem\u003ecps\u003c/em\u003e locus appeared to be the only key factor associated with virulence. Furthermore, we discovered that a carrier strain had an identical serogroup as the hyperinvasive strain and shared 99.9% genomic identity, differing by only 25 SNPs in the \u003cem\u003ecps\u003c/em\u003e locus. This finding suggested that even minor nucleotide variations in this locus may significantly modulate the virulence of \u003cem\u003eN. meningitidis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThis study demonstrated that hyperinvasive strains can emerge through recombination between commensal carrier strains, and were not associated with pre-existing hyperinvasive strains \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Therefore, monitoring \u003cem\u003eN. meningitidis\u003c/em\u003e in carriers, instead of focusing solely on invasive strains, is crucial for preventing the emergence of new hyperinvasive strains.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eCase investigation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTwo fatal cases of IMD were reported from hospitals in Xining City of Qinghai Province to the National Notifiable Disease Reporting System in China Centers for Disease Control and Prevention (CDC) in June 2024. These fatal cases of IMD were further confirmed by the CDC of Huangzhong District, the CDC of Xining City, and the CDC of Qinghai Province. Blood samples were collected from both fatal cases at the hospital for \u003cem\u003eN. meningitidis\u003c/em\u003e isolation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eClose contact survey\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe close contacts of the fatal cases were screened using a real-time PCR assay for \u003cem\u003eN. meningitidis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. PCR-positive throat swab samples from the close contacts were used to isolate \u003cem\u003eN. meningitidis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCharacterization of the isolates and DNA extraction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe 13 \u003cem\u003eN. meningitidis\u003c/em\u003e isolates collected in the present study were cultured on blood agar plates at 37°C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere for 18–22 h. Gram staining and biochemical tests (API-NH, BioMerieux) were performed to confirm the isolates. Serogrouping was performed using slide agglutination (Remel Agglutinating Sera, Thermo Scientific).\u003c/p\u003e\u003cp\u003ePure cultures of \u003cem\u003eN. meningitidis\u003c/em\u003e were eluted with saline solution, and the supernatant was discarded after centrifugation. Genomic DNA was extracted using a DNA purification kit (Genomic DNA Purification Kit, Promega) according to the manufacturer's instructions. The purity and integrity of the DNA were assessed and quantified using a 5400 Fragment Analyzer system (Agilent). Genomic DNA that met quality control standards underwent whole-genome sequencing.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWhole-genome sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGenomic DNA libraries were prepared separately for Illumina and Oxford Nanopore sequencing. Illumina libraries were constructed by sonication (Covaris M220) and TruSeq DNA kit, sequenced on NovaSeq 6000 with 150-bp paired-end reads. Raw reads were quality-checked with FastQC v0.11.8 and trimmed by Trimmomatic v0.39. Nanopore libraries used the SQK-NBD114.96 ligation kit and R10 flow cells, sequenced for 24 hours on MinKNOW v2.0; base-calling was done with Guppy v3.3.0.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenome assembly\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe hybrid genome assembly was performed using Unicycler v0.4.7, which allows for the integration of both Illumina reads (short and accurate) and Nanopore reads (long and less accurate) in conservative mode. The highly accurate Illumina reads were used to align against the Nanopore reads as a reference, correcting random sequencing errors and producing a high-quality genome assembly. The assembled sequence was then polished by aligning the Illumina paired-end reads using the BWA-MEM algorithm in Pilon v1.2.3. Multiple rounds of polishing were conducted until no further errors could be corrected.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenome alignment and genetic variation analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFastMLST software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/EnzoAndree/FastMLST\u003c/span\u003e\u003cspan address=\"https://github.com/EnzoAndree/FastMLST\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for multilocus sequence typing (MLST) on each genome.\u003c/p\u003e\u003cp\u003e Whole-genome alignments were performed using the progressiveMauve algorithm implemented in Geneious software v2022.1, which applies the ClustalW progressive global alignment algorithm to each locally collinear block (LCB). The LCBs were extracted to confirm their homology, underwent a secondary alignment using MAFFT v7.490, and then were concatenated to calculate the overall genomic identity. Whole-genome alignments of the ST-8491 and ST-188331 strains from Qinghai (13 genomes in total) revealed eight LCBs, while whole-genome alignments of the nine ST-8491 isolates from Qinghai identified only one LCB. The genetic variations in the nine Qinghai ST-8491 isolates were manually checked to confirm that they represented authentic variations.\u003c/p\u003e\u003cp\u003eFor the nine ST-8491 isolates from Qinghai, 52 genetic variation regions were identified through whole-genome sequence alignment. Based on these variations, a profile similar to the cgMLST data structure was generated, consisting of 52 variation loci. A neighbor joining tree of the profile was then visualized using GrapeTree software v2.1\u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe capsule polysaccharide synthesis (\u003cem\u003ecps\u003c/em\u003e) locus of each genome was analyzed using the function characterize_neisseria_capsule.py \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The sequences of the \u003cem\u003ecps\u003c/em\u003e locus, including approximately 5,000 bp upstream and 3,000 bp downstream of the locus, were extracted for multiple sequence alignment using MAFFT v7.490. The genomic structure of the \u003cem\u003ecps\u003c/em\u003e locus of each genome was annotated and visualized using Geneious software v2022.1. The recombination analysis of the \u003cem\u003ecps\u003c/em\u003e locus was performed by Simplot v3.5.1 using the Kimura distance model with a 20-bp window size and 100-bp step size.\u003c/p\u003e\u003cp\u003e\u003cb\u003ecgMLST analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA neighbor joining tree was constructed based on the cgMLST profile of 390 isolates of the CC4821 clade (\u003cb\u003eTable S1\u003c/b\u003e) and 13 isolates obtained from Qinghai in the present study. The CC4821 clade encompassed all known \u003cem\u003eN. meningitidis\u003c/em\u003e isolates that shared a common ancestor with CC4821, and comprised all identified CC4821 isolates, 23 unassigned (UA) isolates, and one CC8 isolate \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The results were then visualized using GrapeTree software v2.1 \u003csup\u003e28\u003c/sup\u003e.. The cgMLST scheme and profile generated in this study are available at Chewie Nomenclature Server website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chewbbaca.online\u003c/span\u003e\u003cspan address=\"https://chewbbaca.online\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA phylogenetic analysis of ST-8491 was performed based on genomes of 13 ST-8491 isolates in the PubMLST database (\u003cb\u003eTable S1\u003c/b\u003e) and genomes of 10 ST-8491 isolates from Qinghai using Gubbins software v3.3.1 \u003csup\u003e31\u003c/sup\u003e. Reference genome 130903 (PubMLST ID: 57876) was used for whole genome sequence alignment, recombination analysis, and maximum likelihood (ML) phylogeny construction. The data were analyzed using five iterations in Gubbins. The starting phylogenetic tree was constructed and subsequent iterations were performed using IQTREE v2.2.6 with the GTRGAMMA model. The branch supports of the final tree were assessed using a bootstrap analysis with 1000 replicates. The ML tree of ST-8491 was visualized and annotated by the R packages ggtree v3.6.2 \u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval: This study was approved by the Ethics Committee of the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention (approval number: ICDC-2022007).\u003c/p\u003e\n\u003cp\u003eConsent to participate: According to the established national surveillance protocol and under China\u0026rsquo;s Law on the Prevention and Control of Infectious Diseases, the bacterial isolates were collected as part of an urgent outbreak response. The need for individual informed consent was waived by the Ethics Committee.\u003c/p\u003e\u003cp\u003e\u003ch2\u003eDeclaration of interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2022YFC2305303), the Beijing Natural Science Foundation (L212011), the Operation of Public Health Emergency Response Mechanisms-Infectious Disease Control (102393220020020000029), and the Enhancement of Infectious Disease Surveillance and Control Technological Capabilities (102393240020020000003).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe genomes of isolates that sequenced and described in present study were available in pubMLST website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmlst.org/organisms/neisseria-spp\u003c/span\u003e\u003cspan address=\"https://pubmlst.org/organisms/neisseria-spp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The PubMLST IDs for isolates QH01 to QH13 are 162129 to 162141.\u003c/p\u003e\u003cp\u003eThe cgMLST scheme and profile used in this study are available at Chewie Nomenclature Server website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chewbbaca.online\u003c/span\u003e\u003cspan address=\"https://chewbbaca.online\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDenamur, E., Clermont, O., Bonacorsi, S. \u0026amp; Gordon, D. The population genetics of pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e. \u003cem\u003eNature Reviews Microbiology\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 37-54, doi:10.1038/s41579-020-0416-x (2021).\u003c/li\u003e\n\u003cli\u003eTurner, N. 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Using ggtree to visualize data on tree-like structures. \u003cem\u003eCurrent Protocols in Bioinformatics\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, e96, doi:10.1002/cpbi.96 (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7042271/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7042271/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn June 2024, a novel hyperinvasive strain of \u003cem\u003eNeisseria meningitidis\u003c/em\u003e (\u003cem\u003eN. meningitidis\u003c/em\u003e) caused the deaths of two middle school students in Qinghai Province, China. This is the first hyperinvasive strain to emerge in the country since 2003 and likely originated from commensal strains in healthy carriers. Whole-genome sequencing identified the strain as serogroup C ST-8491, a commensal lineage circulating in China for over 15 years. Among close contacts, all of whom were classmates, the carriage rate reached 31.4%. Carrier ST-8491 isolates shared 98.5\u0026ndash;99.9% genomic identity with the fatal strain. Two genetic variations\u0026mdash;a duplication in the \u003cem\u003eopal\u003c/em\u003e gene and a capsule polysaccharide synthesis locus variation acquired from the commensal, nonserogroupable ST-18331 strain\u0026mdash;were identified as potential virulence factors. These findings demonstrate that commensal strains can directly evolve into fatal forms and underscore the need to monitor \u003cem\u003eN. meningitidis\u003c/em\u003e transmission in healthy populations to prevent future outbreaks.\u003c/p\u003e","manuscriptTitle":"Hyperinvasive Neisseria meningitidis in China Originates from Commensals in Healthy Carriers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-08 07:04:00","doi":"10.21203/rs.3.rs-7042271/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"280fa1cc-9b50-4ccc-93bd-26b08ad49432","owner":[],"postedDate":"September 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":51209012,"name":"Biological sciences/Microbiology/Clinical microbiology"},{"id":51209013,"name":"Health sciences/Diseases/Infectious diseases/Bacterial infection"}],"tags":[],"updatedAt":"2025-09-08T07:04:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-08 07:04:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7042271","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7042271","identity":"rs-7042271","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}