https://orcid.org/0000-0001-5553-2512
Figures
Abstract
Introduction
The ST-4821 complex (cc4821) is a leading cause of serogroup C and serogroup B invasive meningococcal disease in China where diverse strains in two phylogenetic groups (groups 1 and 2) have acquired fluoroquinolone resistance. cc4821 was recently prevalent among carriage isolates in men who have sex with men in New York City (USA). Genome-level population studies have thus far been limited to Chinese isolates. The aim of the present study was to build upon these with an extended panel of international cc4821 isolates.
Methods
Genomes of isolates from Asia (1972 to 2017), Europe (2011 to 2018), North America (2007), and South America (2014) were sequenced or obtained from the PubMLST Neisseria database. Core genome comparisons were performed in PubMLST.
Results
Four lineages were identified. Western isolates formed a distinct, mainly serogroup B sublineage with alleles associated with fluoroquinolone susceptibility (MIC <0.03 mg/L) and reduced penicillin susceptibility (MIC 0.094 to 1 mg/L). A third of these were from anogenital sites in men who have sex with men and had unique denitrification gene alleles. Generally 4CMenB vaccine strain coverage was reliant on strain-specific NHBA peptides.
Discussion
The previously identified cc4821 group 2 was resolved into three separate lineages. Clustering of western isolates was surprising given the overall diversity of cc4821. Possible association of this cluster with the anogenital niche is worthy of monitoring given concerns surrounding antibiotic resistance and potential subcapsular vaccine escape.
Citation: Lucidarme J, Zhu B, Xu L, Bai X, Gao Y, González-López JJ, et al. (2020) Genomic analysis of the meningococcal ST-4821 complex–Western clade, potential sexual transmission and predicted antibiotic susceptibility and vaccine coverage. PLoS ONE 15(12): e0243426. https://doi.org/10.1371/journal.pone.0243426
Editor: William M. Shafer, Emory University School of Medicine, UNITED STATES
Received: October 8, 2020; Accepted: November 23, 2020; Published: December 10, 2020
Copyright: © 2020 Lucidarme et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All draft genomes are available from the https://pubmlst.org/organisms/neisseria-spp database (accession number(s) are listed in the S1 Table).
Funding: This work was supported by grants (ZS) from the National Key Program for Infectious Disease of China (contract no. 2013ZX10004221 and 2018ZX10714002-001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: JL, XB and RB perform contract research on behalf of Public Health England for GlaxoSmithKline, Pfizer, and Sanofi Pasteur. GT has performed contract research on behalf of the School of Public Health, University of West Attica for GlaxoSmithKline and Pfizer. This does not alter our adherence to PLOS ONE policies on sharing data and materials. BZ, JJG, YG, RM, KJS, AS, PS, BS, PT, LX, ZS declare no conflicts of interest.
Introduction
Neisseria meningitidis, the meningococcus, is a leading cause of meningitis and sepsis. Six serogroups (A, B, C, W, X and Y) cause the majority of invasive meningococcal disease (IMD) globally. Prior to 2003/4, meningococcal serogroup A (MenA) was the most common cause of IMD in China with periodic epidemics [1]. In 1982 the country introduced MenA polysaccharide vaccination after which IMD stabilised at 0.2 to 1.0 per 100,000 [2].
In 2003/4 and 2004/5, a number of serogroup C (MenC) outbreaks occurred in Anhui province due to a novel strain—the Sequence Type (ST)-4821 complex (cc4821) [2]. A national survey of case, case-contact, and carrier isolates in 2004/5 revealed the existence of MenC cc4821 in disease and/or carriage in 11 other provinces [2]. Subsequent analysis of geo-temporally diverse Chinese MenC isolates identified several cc4821 case and carrier isolates dating back as far as 1978 [3]. In 2005 the serogroup A polysaccharide vaccine was replaced with a MenA and MenC polysaccharide vaccine [4]. In recent years MenC cc4821 has become a leading and widespread cause of IMD in China. cc4821 has also emerged as a leading cause of serogroup B (MenB) disease which has tended to be more sporadic in nature [4]. cc4821 isolates express diverse PorA subtypes, however, the majority of the MenC cases, particularly those associated with the outbreaks, possessed PorA P1.7–2,14 [4].
High-resolution phylogenetic analyses of small numbers (n = 22 to 32) of Chinese cc4821 case and carrier isolates have found that cc4821 comprises two distinct groups—group 1 (or group I) and group 2 (or group II) [5, 6]. The group 1 isolates were very closely related to one-another and included a subcluster of isolates with PorA P1.7–2,14 (the epidemic clone [6]). The group 2 isolates were relatively diverse and had a lower proportion of invasive isolates. In the study by Guo et al. (2018) [6] the most common DNA gyrase subunit A (gyrA) allele among the group 2 isolates, as well as the majority (22/29) of a distinct panel of group 2 isolates from Shanghai, was a wild type allele, allele 12, which is associated with susceptibility (MIC ≤0.03 mg/L; EUCAST v10.0) to ciprofloxacin among 137/138 PubMLST Neisseria isolates (accessed 10th September 2020). Ciprofloxacin is commonly used for prophylaxis against IMD. The remaining group 2 isolates possessed a variety of gyrA alleles with resistance-associated mutations (MICS >0.03 mg/L; EUCAST v10.0) that affect amino acid residue 91 [7]. All of the group 1 isolates possessed gyrA allele 71, containing the resistance-associated mutation T91I.
Concerns about antibiotic resistance are confounded by the existence of multiple MenB strains in both groups 1 and 2 [5, 6] as a result of multiple capsular switch events [6]. No MenB vaccine is currently licensed in China. Two subcapsular vaccines that are licensed elsewhere afford strain coverage dependant on the presence and expression of cross-reactive peptide variants among the prevailing meningococcal population [8, 9]. The 4CMenB vaccine potentially covers isolates sufficiently expressing PorA P1.4, and/or a sufficiently cross-reactive factor H-binding protein (fHbp) variant 1 peptide, and/or a Neisseria adhesin A (NadA) variant NadA-1 or NadA-2/3 peptide, and/or a cross-reactive neisserial heparin–binding antigen (NHBA) peptide. Coverage by MenB-fHbp is dependent on sufficient expression of fHbp. Potential coverage of individual isolates is determined using the ELISA-based Meningococcal Antigen Typing System (MATS; 4CMenB) [10] or the flow-cytometry based Meningococcal Antigen Surface Expression (MeASurE; MenB-fHbp) [11] assays. A genetic version of MATS (gMATS) has also been developed based on a large international MATS dataset [12]. The emergence of a serogroup W cc4821 strain in both carriage and disease is also a cause for concern [13], although highly effective serogroup W conjugate vaccines are available.
Although invasive disease due to cc4821 is rare outside of Asia, Guo et al. (2018) previously noted the expansion of non-Chinese cc4821 isolates within the PubMLST Neisseria database [6]. In a recent meningococcal carriage study in 706 men who have sex with men (MSM) in New York City (USA), cc4821 was the most prevalent clonal complex identified accounting for 14.3% (24/168) isolates [14]. Members of another strain, ST-11 complex lineage 11.2, have caused multiple outbreaks of IMD in MSM in Europe and the USA and a large multistate outbreak of urethritis among predominantly heterosexual men in the USA [15]. The latter strain (US_NmUC) has recently been detected in proctitis in MSM in the United Kingdom [16]. The strains responsible have shown several adaptations to the anogenital niche including the acquisition of active/efficient denitrification genes (aniA and norB) to enable growth in an anaerobic environment [17, 18]. A recent study in gonococci found an association between loss of function (LOF) mutation in the efflux pump component, MtrC, and cervical infection. This extended to US_NmUC in which 8.7% of isolates possessed LOF mutations versus 0.62% in a large invasive disease collection [19].
In the present study, we expanded upon earlier work by sequencing additional Chinese genomes and performing high resolution phylogenetic analyses on these and an extended panel of all cc4821 genomes from multiple countries on several continents that are currently available on the PubMLST Neisseria database (accessed 12th September 2019). We also charted the distribution of ciprofloxacin resistance-associated gyrA mutations and penA mutations associated with reduced susceptibility to penicillin; the nitrite reductase gene aniA; and potential cc4821 strain coverage by subcapsular (MenB) vaccines. Predicted functionality of MtrC was assessed in a set of anogenital isolates.
Methods
Genome sequencing and database searches
New genome sequences were obtained for 138 Chinese cc4821 isolates collected between 1978 to 2017, inclusive (S1 Table). DNA was extracted from overnight cultures using the Wizard genomic DNA purification kit (Promega, WI, USA) according to the manufacturer’s instructions. Paired end libraries were generated with average insert lengths of 350 bp or 500 bp. Sequencing was performed on Illumina HiSeq 2000, 2500 or 4000 platforms (Illumina, CA, USA).Genome assembly was performed using SOAPdenovo (release 1.04; http://soap.genomics.org.cn/soapdenovo.html).
Other cc4821 genomes were obtained from the PubMLST Neisseria isolate database (https://pubmlst.org/neisseria/) using the ‘search or browse database’ function (accessed 12/09/2019). Isolates with >4 ST-4821 alleles among clonal complex-unassigned isolates (incomplete MLST profiles or unassigned STs) were also included.
Associated metadata and genotypic data were exported using the ‘export dataset’ function. Stated capsular groups are from the ‘capsule_group’ field which is determined automatically from the contents of the serogroup and/or genogroup fields in PubMLST.
Phylogenetic analyses and identification of putative recombination events
Genomes were compared using the PubMLST genome comparator tool [20] in conjunction with the meningococcal core genome MLST scheme (N. meningitidis cgMLST v1.0) [21] using an ST-41/44 complex genome (PubMLST ID 19266; M01-240149) as an outgroup. The resulting distance matrixes were visualised and annotated using SplitsTree4 (version 4.12.8) [22].
Putative recombinations were identified by comparing genomes in term of all NEIS loci and identifying blocks of differing genes between closely related isolates.
BLAST searches and nucleotide/amino acid sequence alignments
Allelic sequence data were obtained from the PubMLST Neisseria database using the ‘sequence attribute search’ tool. BLAST searches were performed using the PubMLST BLAST tool. Nucleotide and amino acid sequence alignments were performed using BioEdit (version 7.0.9.0) [23].
Classification of antibiotic susceptibility
Minimum inhibitory concentration (MIC) breakpoints for antibiotic sensitivities differ according to publication and by the organisations that define them, including occasional revisions over time. Current EuCAST (v10.0) breakpoints for intravenous administration of penicillin are susceptible ≤0.06 mg/L and resistant >0.25 mg/L. Studies in animal models, however, suggest an upper breakpoint of >1 mg/L for resistance [24]. Current EuCAST (v10.0) breakpoints for ciprofloxacin for use in the prophylaxis of meningococcal disease are susceptible ≤0.03 mg/L and resistant >0.03 mg/L.
For the present study, isolates possessing up to five mutations (F504L, A510V, I515V, H541N and I566V) within the deduced amino acid sequence for a 402 bp fragment (penA) of the penicillin binding protein 2 gene (PubMLST locus NEIS1753) were predicted to have reduced susceptibility to penicillin (0.094 to 1 mg/L) in accordance with Taha et al. (2007) [25]. Isolates with alterations to residues T91 or D95 of the deduced amino acid sequence of the DNA gyrase gene (gyrA; PubMLST locus NEIS1320) were predicted to be resistant (MIC, ≥0.03 mg/L) to ciprofloxacin in accordance with Hong et al., 2013 [7]. Alterations to residues D86, S87, S88 or E91 of the deduced amino acid sequence of the DNA topoisomerase IV subunit A gene (parC; PubmLST locus NEIS1525) were also noted having previously been associated with incrementally higher ciprofloxacin MICs when observed in tandem with gyrA mutations in N. gonorrhoeae [26].
Results
Final genome panel
In addition to the 138 new genomes, the database search yielded 50 genomes including 49 cc4821 genomes and a single ST-unassigned genome (PubMLST ID 85386) that had four MLST alleles in common with both ST-4821 and ST-8. The latter genome was subsequently assigned to ST-14840 that, in accordance with the rules by which 7-locus STs are assigned (https://pubmlst.org/neisseria/info/complexes.shtml), was assigned to the ST-8 complex. Given the uncertainty regarding the isolate’s true descent it was included in subsequent analyses employing high resolution cgMLST. Collectively, the genomes were from carriage (n = 121), invasive disease (n = 57), anogenital sites (n = 6) and unspecified (n = 4). The isolates were isolated between 1972 and 2018 in Asia (1972 to 2017, including China, n = 170; Japan, n = 1); Europe (2011 to 2018, including UK, n = 6; Italy, n = 3; Ireland, n = 2; Greece, n = 1; Malta, n = 1; Spain. N = 1; Sweden, n = 1); North America (2007, USA, n = 1); and South America (2014, Brazil, n = 1) (Table 1).
Population structure
Figs 1 and 2 show the population structure of the genomes highlighting geographical origins, and serogroup and disease status, respectively. The cc4821 isolates were diverse and formed four distinct lineages among several relatively diffuse isolates. To maintain a linkage to the previous studies by Zhu et al. (2015) [5] and Guo et al. (2018) [6] these were designated lineage 1 (corresponding to Zhu/Guo group I/1) and lineages 2a, 2b and 2c (collectively corresponding to Zhu/Guo group II/2). The positions of isolates used in the earlier studies are shown in S1 Fig. The ST-14840 (cc8) isolate, a Spanish urethral swab isolate from 2018, belonged to lineage 2c confirming it was descended from cc4821 meningococci, rather than those of cc8 to which it is officially assigned. Each of the four lineages was represented by isolates from multiple Chinese provinces (S2 Fig). With the exception of a single diffuse isolate from China in 1980, PorA P1.7–2,14 was only observed within a discrete lineage 1 cluster that included the epidemic clone isolates as designated by Guo et al. (2018) [6] (S3 Fig). There were 55 sequence types among the study genomes. Among the predominant sequence types, ST-4821 was distributed between lineage 1 and several diffuse isolates from 1973 to 1985 (n = 5) and 2009 (n = 1). ST-5664 and ST-9454 were confined to lineage 2a. ST-8491 was confined to MenW isolates of lineage 2b. ST-3200 and ST-5798 were confined to lineage 2c (S4 Fig).
With the exception of a Japanese isolate in lineage 1, all of the non-Chinese isolates belonged to a discreet ‘Rest of the world’ (Row) cluster. The phylogeny was based on a core genome (1605 loci) comparison. The scale bar represents the number of different loci. UK = United Kingdom.
Group B and C isolates are interspersed throughout though lineage 1 is predominantly MenC while lineages 2a and 2c are mainly MenB. The MenW isolates formed a discreet cluster in lineage 2b. cnl = capsule null. NG = non-groupable.
The geo-temporal composition and disease status of the four lineages was as follows:
Lineage 1.
The majority of lineage 1 isolates (n = 51/52, 2004 to 2017) were from China including 16 carrier isolates belonging to MenC (n = 10; 2005 to 2014), MenB (n = 4; 2009–2016), and serogroup unknown (n = 2; 2007 and 2009); 34 invasive isolates belonging to MenB (n = 6, 2008 to 2014) and MenC (n = 28; 2004 to 2016); and a single MenB isolate with disease status unknown (2005). The remaining isolate was an invasive MenC isolate from Japan (2017).
Lineage 2a.
All lineage 2a isolates (n = 49; 2005 to 2017) were from China and most (n = 45/49) were MenB. The lineage included 44 carrier isolates (2005 to 2016; MenB, n = 40; MenC, n = 1; MenX, n = 1; capsule null (cnl), n = 1; and unknown, n = 1), and five invasive isolates (2011 to 2017; all MenB) (Fig 2). Four of the invasive isolates belonged to a distinct cluster of relatively closely related isolates.
Lineage 2b.
All of the lineage 2b isolates (n = 15; 1972 to 2017) were from China. The majority (n = 13/15) were carrier isolates including a single MenB (1980), two MenC (1972 and 2007), and ten MenW isolates (2009 to 2017). The two invasive isolates (2002 and 2005) belonged to MenC and formed a distinct sublineage.
Lineage 2c.
The majority (n = 40/57) of the lineage 2c isolates were from China including 29 carrier isolates belonging to MenB (n = 26; 1977 to 2016) and MenC (n = 3; 2016 to 2017), ten invasive isolates belonging to MenB (n = 9; 2005 to 2013) and MenC (n = 1; 2005) (Fig 2), and a single MenB isolate (2005) with unknown disease status.
The remaining lineage 2c isolates (n = 17/57) were from nine countries in Europe and North and South America and formed a distinct cluster within lineage 2c designated the ‘cc4821 lineage 2c RoW (rest of world) cluster’ (Fig 1). These included carrier MenB isolates from meningococcal carriage studies in the USA (n = 1 isolate; 2007; high school children) [27]; Brazil (n = 1 isolate; 2014; adolescents) [28]; Italy (n = 2 isolates; 2016; young adults); Ireland (n = 2 isolates; 2017; Universities); Greece (n = 1 isolate; 2018; adolescents), Sweden (n = 1 isolate; 2018; universities) and Malta (n = 1 isolate; 2018; university); and invasive MenB isolates from Italy (n = 1; 2017) and the UK (n = 1; 2014) (Fig 2). The remaining six lineage 2c isolates were all from anogenital sites among men who have sex with men (MSM). These included three UK MenB isolates (2011, 2012 and 2013) that were incidentally isolated from rectal swabs during routine gonococcal screening [29]. Two of these were from dual meningococcal/gonococcal coinfections. A further UK MenB rectal swab isolate (2017; age 20 to 30 years) was from a meningococcal/gonococcal coinfected asymptomatic contact of a gonococcal urethritis patient. Another MenB isolate was from a urethral swab from a meningococcal/Chlamydia trachomatis coinfected sex worker with symptoms of urethritis in Spain (2018; 20 to 30 years). The final isolate belonged to MenC and was a urethral swab isolate from a UK bisexual male practicing protected insertive sex and displaying symptoms of urethritis (2018; 30 to 40 years). Coinfections were ruled out for C. trachomatis, Mycoplasma genitalium and gonococcus.
Diffuse isolates.
The diffuse isolates that were not assigned to particular lineages (n = 15; 1973 to 2011) were all from China and comprised ten carrier isolates including MenB (n = 2; 2009 to 2011) and MenC (n = 8; 1973 to 2008) isolates; three invasive MenB isolates (1985 to 2011); and two MenB isolates with unspecified disease status (1980 to 1985).
Antibiotic resistance determinant genotyping
The different lineages/sublineages contained varying distributions of isolates with fluoroquinolone resistance-associated (MICs ≥ 0.03 mg/L) gyrA and parC alleles and penA alleles associated with reduced penicillin susceptibility (0.094 to 1 mg/L) (Fig 3). Detailed lineage breakdowns of genotypic antibiotic resistance determinants are depicted in S5 Fig and as follows:
gyrA alleles were considered to be associated with fluoroquinolone resistance (MIC ≥ 0.03 mg/L) if they possessed mutations affecting residues T91 or D95 of the corresponding peptide. penA alleles were considered to be associated with reduced penicillin susceptibility (0.094 to 1 mg/L) if they encoded the following mutations: F504L, A510V, I515V, H541N and I566V.
Lineage 1.
All 52 of the lineage 1 isolates possessed mutant (resistance-associated) gyrA alleles (51x allele 71 and 1x allele 326). Among these, 45 isolates (including 18/20 epidemic clone isolates) possessed wild type (WT; susceptibility-associated) penA alleles. One of these, a MenB carrier isolate (2009), also possessed a mutant parC allele (mutation S88P). Three isolates, including an invasive MenB (2014) and MenC (2012) isolate and a carrier MenB isolate (2016), possessed mutant penA alleles (5x reduced susceptibility-associated alleles). A further three carrier isolates (including 2/20 epidemic clone) and one invasive isolate did not have full length penA sequence data. The single Japanese epidemic clone isolate possessed mutant gyrA and parC (mutation S87I) alleles and WT penA alleles.
Lineage 2a.
Among the lineage 2a carrier isolates, 8/40 MenB isolates (2011 to 2016) and 1/1 MenX isolates (2009) possessed mutant gyrA and penA alleles. Of these, two MenB isolates (2014 to 2016) also possessed mutant parC alleles (mutation E91K). A further 22/40 MenB isolates (2006 to 2016), and a single cnl (2009) and serogroup-unknown (2016) isolates possessed mutant gyrA and WT penA alleles. Of these, a single MenB isolate (2013) also possessed a mutant parC allele (mutation S87I). The remaining 10/40 MenB (2005 to 2014) and 1/1 MenC (2005) carrier isolates possessed WT gyrA and penA alleles.
Among the five MenB lineage 2a invasive isolates, one (2017) possessed mutant gyrA and penA alleles, two (2011 to 2013) possessed mutant gyrA alleles and WT penA alleles, and two (both 2014) possessed WT gyrA and mutant penA alleles.
Lineage 2b.
Among the lineage 2b carrier isolates, 1/1 MenB (1980) and 1/2 MenC (1972) possessed WT gyrA and penA alleles. The other MenC isolate (2007) and 8/10 MenW isolates (2009 to 2017) possessed mutant gyrA and WT penA alleles. The remaining 2/10 (2014 and 2016) MenW isolates possessed mutant gyrA and penA alleles. The two invasive MenC isolates (2002 and 2005) possessed WT gyrA and penA alleles.
Lineage 2c.
Among the Chinese lineage 2c MenB carrier isolates, 9/26 isolates (1977 to 2011) possessed WT gyrA and penA alleles, 12/26 (2005 to 2016) possessed mutant gyrA and WT penA alleles, including one isolate with a mutant (S87I) parC allele, 4/26 (2012 to 2014) possessed WT gyrA and mutant penA alleles, and 1/26 (2013) possessed mutant alleles for both gyrA and penA. All three Chinese MenC carrier isolates (2016 to 2017) possessed mutant alleles for both gyrA and penA. Among the Chinese invasive MenB lineage 2c isolates, 4/9 possessed WT gyrA and penA alleles, 1/9 possessed WT gyrA and mutant penA alleles, 2/9 possessed mutant gyrA and WT penA alleles, and 2/9 possessed mutant alleles for both gyrA and penA. The single Chinese invasive MenC isolate possessed mutant gyrA and WT penA alleles. A further MenB isolate with unknown disease status possessed WT gyrA and penA alleles.
All 17 lineage 2c RoW isolates possessed WT gyrA alleles and mutant penA alleles.
Diffuse isolates.
Among the diffuse carrier isolates 2/2 MenB (2009 and 2011) and 3/8 MenC isolates (1973 to 1978) possessed WT gyrA and penA alleles. A further 4/8 MenC isolates (2005 to 2008) possessed mutant gyrA and WT penA alleles, while 1/8 MenC isolates (1980) possessed WT gyrA and incomplete penA alleles.
Among the diffuse invasive isolates, 2/3 MenB isolates (1985 and 2011) possessed WT alleles for both gyrA and penA. The remaining isolate (2009) possessed mutant gyrA and penA.
The two diffuse isolates with unknown disease status (1980 and 1985) possessed WT gyrA and penA alleles.
Subcapsular vaccine antigen genes
None of the cc4821/ST-14840 isolates possessed alleles for PorA P1.4 or NadA. Two isolates had incomplete nhba sequences and one, a UK invasive rectal swab isolate from 2017, had a frameshifted nhba allele. The remaining 185 isolates possessed alleles for one of 32 intact NHBA peptides, 22 of which were unique to China, and 27 of which were only, or predominantly, observed in cc4821 on the PubMLST Neisseria database (accessed 24/06/20). 4CMenB strain coverage of all but one of the encoded NHBA peptides was unpredictable according to gMATS. The remaining peptide, peptide 10 was predicted by gMATS to be covered and occurred in a single lineage 2a MenB carrier isolate from 2013 (this isolate possessed an allele for a non-covered variant 2 fHbp peptide). The majority of isolates 144/188 possessed fHbp variant 2 or 3 alleles and a further two isolates, a Chinese invasive MenC lineage 1 (non-epidemic clone) isolate from 2011 and a Chinese lineage 2c MenC carrier isolate from 2017, possessed a truncated allele (nonsense mutation) and a complete deletion of fhbp, respectively. The remaining 42 isolates possessed alleles for one of 13 fHbp variant 1 peptides, eight of which were only, or predominantly, found in cc4821, and six of which were unique to China on the PubMLST Neisseria database (accessed 24/06/20). All 13 peptides afforded unpredictable 4CMenB strain coverage according to gMATS. Given the general absence of gMATS-predictable strain coverage, potential strain coverage was estimated based on the most conservative criteria that any NHBA peptide and any fHbp variant 1 peptide is potentially covered. Potential antigen-specific strain coverage of cc4821 lineages/sublineages by 4CMenB is shown in Fig 4. A detailed breakdown of lineage-specific potential 4CMenb strain coverage is as follows:
Potential strain coverage was estimated based on the most conservative criteria that any NHBA peptide and any fHbp variant 1 peptide is potentially covered.
Lineage 1.
At least 50 of the 52 lineage 1 isolates were potentially covered by NHBA though this was heavily reliant on peptide 503 (n = 43) being covered. The two remaining isolates had incomplete nhba sequences. Interestingly, only 3/16 carrier isolates, including 1/5 epidemic clone carrier isolates (1/4 MenB and 2/10 MenC) were also potentially covered by fHbp. By contrast, 28/35 of the invasive isolates (3/6 MenB and 25/29 MenC), including 13/15 invasive epidemic clone isolates, were potentially covered by fHbp. One invasive MenC (2011) isolate possessed a truncated fhbp allele with a nonsense mutation.
Lineage 2a.
All 49 lineage 2a isolates were potentially covered by NHBA though this was heavily reliant on peptides 910 and 688 being covered since these accounted for a half and a quarter of the isolates, respectively. Among the invasive isolates, 2/5 were also potentially covered by virtue of one of two different fHbp peptides.
Lineage 2b.
All of the lineage 2b isolates were potentially covered by NHBA, though this was reliant on peptide 697 being covered. Additionally, 1/13 carrier isolates (MenW, 2014) and the two invasive MenC isolates were potentially covered by virtue of fHbp.
Lineage 2c.
All of the 40 Chinese lineage 2c isolates were potentially covered by NHBA though this was mainly reliant on coverage of peptides 669 (n = 22) and 697 (n = 9). In addition, 4/25 carrier isolates and 2/10 invasive isolates (MenB, 2011 and MenC, 2005) were potentially covered by fHbp. An fhbp gene was lacking in one MenC carrier isolate (2017) and was replaced by N. lactamica sequence as previously reported among proposed ST-286 complex isolates [30].
Among the lineage 2c RoW isolates, 16/17 were potentially covered by virtue of NHBA, in particular peptide 669 (n = 15). One MenB isolate from a rectal swab from an invasive disease patient possessed a frameshifted nhba allele. None of the RoW isolates were covered by fHbp.
Distribution of the nitrite reductase (aniA) gene previously associated with adaptation to the anogenital niche
The acquisition of a working nitrite reductase (aniA) gene has previously been implicated in the adaptation of meningococci to the anogenital niche [17, 18]. The distribution of aniA alleles within the cc4821 population structure is shown in S6 Fig. A number of RoW cluster and lineage 2c isolates possessed aniA (NEIS1549) allele 8 that was unique to these and a small number of other ST-3200 (cc4821) related isolates on the PubMLST Neisseria database (accessed 21/07/20). A genome comparison of all lineage 2c isolates in terms of all NEIS loci showed that isolates with aniA allele 8 also possessed a unique nitric oxide reductase (norB; NEIS1548) allele–allele 379. Most other lineage 2c isolates possessed aniA allele 287 and norB allele 838 in common with diverse isolates from lineages 2a and 2b. The most commonly observed alleles of the loci immediately upstream and downstream of aniA and norB did not differ between the two sets of isolates suggesting a possible recombination event. A nucleotide alignment of aniA, norB and flanking genes between isolates representative of the putative ancestral (aniA allele 287 and norB allele 838) and putative recombinant (aniA allele 8 and norB allele 379) states identified a potentially horizontally acquired region of 2022 bp covering 82% of the 5’ end of aniA, 30% of the 5’end of norB, and the 381 bp intergenic region (S7 Fig). The extent of the change in the respective peptides was relatively modest comprising P23Q, A43T, T45A and the addition of A27, P28, A29 and E30 for AniA (S8A Fig), and I133V, V146A, D206G, T208V for NorB (S8B Fig).
A BLAST search of all genomes (>2Mb) on the PubMLST Neisseria database (n = 30,044; accessed 1st September 2020) for the putatively acquired 2022 bp sequence identified n = 103 exact, full-length matches, all of which were meningococcal. Thirty eight of these belonged to, or were closely related to, cc4821, including n = 29 cc4821 lineage 2c isolates from the current study with aniA allele 8 (n = 28) or aniA allele 733 (n = 1); five cc4821 carriage study isolates from the UK and Sweden that were not included in the current study (all aniA allele 8); and four clonal complex-unassigned isolates with <4 MLST alleles in common with ST-4821 (all aniA allele 8). The latter four isolates each had 4 to 5 loci in common with ST-3200. The cc8 isolate descended of cc4821 (aniA allele 8) was also identified. The remaining BLAST hits were for MenA ST-4 complex (n = 14; 1915 to 1972) and MenA ST-5 complex (n = 48; 1966 to 2014) isolates and two isolates with incomplete MLST profiles but matching cc5 at six loci.
Discussion
High-resolution phylogenetic analyses have revolutionised our understanding of meningococcal population structures and our ability to identify and track existing and emerging strains and outbreaks on a global level [31, 32]. Such analyses benefit from the inclusion of geo-temporally diverse isolates to provide the broadest possible view of the corresponding diversity and to put the constituent strains in context. To this end, our study included all currently available cc4821 genomes on the PubMLST database (accessed 12/09/19).
A key finding of our study is the existence of four well defined lineages, versus the two that were previously identified [5, 6]. Lineage 1 corresponds to the previously identified group I/1 which includes the predominantly MenC epidemic clone with PorA P1.7–2,14 [6]. Lineages 2a, 2b, and 2c, and a number of diffuse isolates/singletons, collectively correspond to the previously designated group II/2. It was previously noted that group 2 was relatively diverse but the inclusion of more than five times as many genomes in the present study has helped to resolve the constituent lineages. The star-like arrangement of the four lineages along with the non-cc4821 outgroup on the phylogeny suggests that the four lineages have diverged concurrently from an early common ancestor, however, the relative closeness of lineages 2a, 2b and 2c to one-another and the benefits of maintaining continuity with previous publications justifies the chosen lineage designations (2a, 2b and 2c).
Interestingly, with the exception of a lineage 1 epidemic clone MenC case isolate from Japan in 2017, all of the remaining 17 non-Chinese isolates, from nine countries on three continents (2007 to 2018), exclusively belonged to a distinct cluster (the RoW cluster) within lineage 2c. Furthermore, whilst PorA subtype P1.17–6,23 and closely-related subtypes were only associated with the RoW cluster among our genome panel, they also accounted for 14/27 PorA subtypes among non-Chinese, non-genomic isolate submissions on the PubMLST Neisseria database (accessed 13th July 2020; S2 Table). The lineage-specific distribution of several predominant Sequence Types suggests that the previously reported ‘ST-3200 group’ of invasive MenB isolates in Taiwan from 1996 to 2002 [33] would also form part of the broader lineage 2c. The global expansion of this particular sublineage may be due to an unknown feature of the strain making it more transmissible in these populations or may simply be opportune e.g. due to an international mass gathering [34].
More than a third (6/17) of the lineage 2c RoW cluster isolates were from an anogenital site among MSM. Although two groups of isolates (a group of three and a group of two) were from patients attending sexual health services that utilised common microbiology laboratories (at opposite ends of the UK), the corresponding isolates were distributed over time (2011, 2012 and 2013; and 2017 and 2018, respectively) and within the population structure of the corresponding cluster. Furthermore, the second group also differed from one-another by serogroup. This, along with the finding of the recent msm carriage study in New York (USA) [14], may be indicative of a propensity for this cluster to infect the anogenital site as has previously been observed with ST-11 complex lineage 11.2 strains [15, 16, 18]. One factor in the emergence of anogenital-associated cc11 strains was the acquisition of an active nitrite reductase (aniA) gene [15, 18]. Among the cc4821 lineages, only lineage 1 lacked aniA functionality, however, 14/17 lineage 2c RoW isolates, including the anogenital isolates, and 15/40 Chinese lineage 2c isolates shared a putative ancestral recombination involving partial sequences of the divergent aniA and norB genes along with the intergenic/promoter regions. Although the corresponding sequence was exclusively meningococcal on the PubMLST database, its initial origin was likely in MenA-associated lineages dating back to 1915. It is possible that the newly generated alleles or the newly acquired intergenic region (containing promoter regions) where many of the changes occurred, are more efficient than the existing ones. This, however, requires further investigation. Formal bioinformatic analyses are also required to confirm the origin of the putatively horizontally acquired sequence. Another possible explanation for this particular cluster alone being observed in anogenital sites is that Chinese anogenital meningococci from other lineage 2c clusters or the other cc4821 lineages may simply not be collected, sequenced, posted or identified as such on PubMLST. Interestingly, lineage 2c also included a rare isolate lacking an fhbp gene and therefore unable to express fHbp which has also previously been implicated in adaptation to the anogenital niche [18]. None of the anogenital isolates possessed LOF mutations in mtrC that have been associated with cervical infection in gonococci and urogenitally adapted meningococci from uretheritis outbreaks among heterosexual males [19]. One possible explanation for this is that cc4821 isolates were all isolated from MSM and the cervix may not have been encountered in recent ancestral isolates.
The absence of the lineage 1 epidemic clone outside of Asia is surprising given that it has caused most cases of cc4821 disease within China. This may, in part, be due to a lack of corresponding genomes. Canada, for example, has reported IMD due to a P1.7–2,14:ST-4821 isolate [31, 35] but no genome is currently available. On our phylogeny, with the exception of a single diffuse isolate from China in 1980, PorA P1.7–2,14 was only observed within the discrete cluster representing the epidemic clone. This supports PorA P1.7–2,14 as a specific marker for the clone among cc4821 isolates. In addition, PorA VR1 P1.7–2 alone was a strong marker for the epidemic clone cluster accounting for 22/23 (96%) isolates. Three of the epidemic clone isolates possessed a different VR2. In light of this, the lack of the epidemic clone outside of China is further supported by available PorA data among non-Chinese, non-genomic, cc4821 isolate submissions on the PubMLST Neisseria database (accessed 13th July 2020). None of the isolates from Europe (France, n = 2, 2009 and 2011; Spain, n = 2, 2018; Czech Republic, n = 1, 2007; Italy, n = 1, 2020), North America (USA, n = 14, 2015 to 2019), South America (Brazil, n = 1, 2014), Australasia (Australia, n = 2, 2012 and 2014; New Zealand, n = 1, 2017) or Asia (Taiwan, n = 2, 2004 and 2005; Vietnam, n = 1, 2012) possessed the epidemic clone-associated PorA (P1.7–2,14) (S2 Table).
The widespread distribution of fluoroquinolone resistance in cc4821 is well documented [36]. The present study confirms the distribution of diverse resistance-associated (MICs > 0.03 mg/L) mutant gyrA alleles throughout the four cc4821 lineages and, with the exception of lineage 1, these were interspersed with isolates possessing susceptibility-associated (MICs ≤0.03 mg/L) WT alleles. A small number of well distributed isolates also possessed mutant parC alleles that may further increase fluoroquinolone MICs. Notably, the RoW cluster does not possess a mutant gyrA, however, it may be susceptible to stable acquisition of fluoroquinolone resistance given the situation in the broader clonal complex and the gradual emergence of fluoroquinolone resistance in Western countries [35, 37–39]. Interestingly, all of the RoW isolates possessed a mutant penA allele associated with reduced penicillin susceptibility (0.094 to 1 mg/L). Although diverse mutant penA alleles were found among Chinese isolates in each of the four lineages, they were relatively rare. As the present study constituted a genomic survey, we focussed on antibiotic resistance/reduced susceptibility determinants commonly associated with cc4821, rather than phenotypic MICs. Further work may include consideration of other potential resistance determinants such as multidrug efflux systems or beta lactamase, the latter of which has recently been reported among meningococci in North America and Europe [37, 40, 41].
Potential 4CMenB strain coverage of lineages 2a, 2b and 2c, including the RoW cluster was heavily reliant on NHBA. As the corresponding peptides are rare among existing MATS datasets there is a critical need for MATS analysis of isolates representing each of the main peptides. This may then be used to extend the current list of covered, not covered, and unpredictable isolates as defined by gMATS [12]. Perhaps reassuringly, more than half of the lineage 1 isolates, including 85% (28/33) of invasive isolates were also potentially covered by fHbp. This vaccine may, therefore, be an option if an fHbp lineage 1-possessing MenB strain should emerge to cause a large outbreak or epidemic. The majority of isolates without a variant 1 fhbp possessed a variant 2 or 3 gene and so the majority of isolates are potentially covered by Men-fHbp, though this would need confirming with the MeASurE assay [11]. Of concern, two isolates lacked a functional fhbp and one isolate lacked a function nhba. Such isolates and their corresponding strains have infrequently been reported before [30, 34, 42] and should be monitored due to the potential for escape of subcapsular vaccines. They should also be considered when devising new vaccine formulations.
In conclusion, cc4821 can now be considered in terms of four distinct lineages and the phylogeny herein can form a template with which to analyse genomes from prevailing cases. A small and distinct cluster of lineage 2c is responsible for the majority of cases outside of China and may have an affinity for anogenital sites. In light of emerging fluoroquinolone and penicillin resistance, uncertain strain coverage by subcapsular vaccines, and the repeated emergence of MenB cc4821 strains through capsule switching, there is an urgent need to test isolates representative of each lineage in the MATS and MeASurE assays.
Supporting information
S1 Fig. Distribution of previously designated group I/1 and group II/2 isolates within cc4821 population structure.
https://doi.org/10.1371/journal.pone.0243426.s001
(DOCX)
S2 Fig. Distribution of Chinese provinces represented within the cc4821 population structure.
https://doi.org/10.1371/journal.pone.0243426.s002
(DOCX)
S3 Fig. Distribution of genotypic PorA subtypes in cc4821 lineage 1.
https://doi.org/10.1371/journal.pone.0243426.s003
(DOCX)
S4 Fig. Distribution of sequence types within cc4821 population structure.
https://doi.org/10.1371/journal.pone.0243426.s004
(DOCX)
S5 Fig. Distribution of fluoroquinolone resistance-associated gyrA and parC alleles and penA alleles associated with reduced penicillin susceptibility within the cc4821 population structure.
https://doi.org/10.1371/journal.pone.0243426.s005
(DOCX)
S6 Fig. Distribution of nitrite reductase (aniA) alleles within the cc4821 population structure.
https://doi.org/10.1371/journal.pone.0243426.s006
(DOCX)
S7 Fig. Alignment of aniA, norB and flanking genes between cc4821 lineage 2c isolates representative of the ancestral and novel recombinant states.
https://doi.org/10.1371/journal.pone.0243426.s007
(DOCX)
S8 Fig. Alignment of AniA and NorB from putative ancestral and recombinant cc4821 lineage 2 isolates.
https://doi.org/10.1371/journal.pone.0243426.s008
(DOCX)
S1 Table. Isolate/genome listing, genotypic data and accession numbers.
https://doi.org/10.1371/journal.pone.0243426.s009
(XLSX)
S2 Table. PorA subtypes among non-Chinese, non-genomic PubMLST submissions highlighting P1.17–6,23 and closely-related subtypes that were only associated with the RoW cluster among the genome panel.
https://doi.org/10.1371/journal.pone.0243426.s010
(DOCX)
Acknowledgments
This publication made use of the Neisseria Multi Locus Sequence Typing website (https://pubmlst.org/neisseria/) sited at the University of Oxford [43]. The development of this site has been funded by the Wellcome Trust and European Union.
The authors would like to thank Dr Desiree Bennett and Dr Robert Cunney of the Irish Meningitis and Sepsis Reference Laboratory, Children’s Health Ireland at Temple Street, Dublin, Ireland, and Dr. Judit Serra-Pladevall from the Clinical Microbiology Department of Hospital Universitari Vall d'Hebron. Thank you also to Dr David Pace—Department of Child and Adolescent Health, Mater Dei Hospital, Malta, and Ms Marlene Farrugia, Ms Sabrina Goodlip, Ms Julie Haider, Dr Graziella Zahra—Department of Pathology, Mater Dei Hospital, Malta for their instrumental contribution to the Maltese meningococcal carriage study referred to in this research. Thank you to Roisin Ure of the Bacterial Respiratory Infection Service, Scottish Microbiology Reference Laboratory, Glasgow Royal Infirmary, Glasgow, United Kingdom and Dr Andrew Winter, Consultant in Sexual Health & HIV Medicine & Joint Clinical Lead for e-Health, NHS Greater Glasgow and Clyde. Thank you to Dr Paola Vacca of the Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy, and Andrea Ciammaruconi, Antonella Fortunato, Silvia Fillo, and Florigio Lista of the Scientific Department, Army Medical Center, Rome, Italy. We would also like to thank Aiswarya Lekshmi and the staff of the Public Health England Meningococcal Reference Unit.
References
- 1. Zhu P, Hu X, Xu L. Typing Neisseria meningitidis by analysis of restriction fragment length polymorphisms in the gene encoding the class 1 outer membrane protein: application to assessment of epidemics throughout the last 4 decades in China. J Clin Microbiol. 1995 Feb;33(2):458–62. pmid:7714207.
- 2. Shao Z, Li W, Ren J, Liang X, Xu L, Diao B, et al. Identification of a new Neisseria meningitidis serogroup C clone from Anhui province, China. Lancet. 2006 Feb 4;367(9508):419–23. pmid:16458767.
- 3. Zhang X, Shao Z, Yang E, Xu L, Xu X, Li M, et al. Molecular characterization of serogroup C Neisseria meningitidis isolated in China. J Med Microbiol. 2007 Sep;56(Pt 9):1224–9. pmid:17761487.
- 4. Zhou H, Shan X, Sun X, Xu L, Gao Y, Li M, et al. Clonal characteristics of invasive Neisseria meningitidis following initiation of an A + C vaccination program in China, 2005–2012. J Infect. 2015 Jan;70(1):37–43. pmid:25107632.
- 5. Zhu B, Xu Z, Du P, Xu L, Sun X, Gao Y, et al. Sequence Type 4821 Clonal Complex Serogroup B Neisseria meningitidis in China, 1978–2013. Emerg Infect Dis. 2015 Jun;21(6):925–32. pmid:25989189.
- 6. Guo Q, Mustapha MM, Chen M, Qu D, Zhang X, Chen M, et al. Evolution of Sequence Type 4821 Clonal Complex Meningococcal Strains in China from Prequinolone to Quinolone Era, 1972–2013. Emerg Infect Dis. 2018 Apr;24(4):683–90. pmid:29553310.
- 7. Hong E, Thulin Hedberg S, Abad R, Fazio C, Enriquez R, Deghmane AE, et al. Target gene sequencing to define the susceptibility of Neisseria meningitidis to ciprofloxacin. Antimicrob Agents Chemother. 2013 Apr;57(4):1961–4. pmid:23357770.
- 8. Serruto D, Bottomley MJ, Ram S, Giuliani MM, Rappuoli R. The new multicomponent vaccine against meningococcal serogroup B, 4CMenB: immunological, functional and structural characterization of the antigens. Vaccine. 2012 May 30;30 Suppl 2:B87–97. pmid:22607904.
- 9. Jiang HQ, Hoiseth SK, Harris SL, McNeil LK, Zhu D, Tan C, et al. Broad vaccine coverage predicted for a bivalent recombinant factor H binding protein based vaccine to prevent serogroup B meningococcal disease. Vaccine. 2010 Aug 23;28(37):6086–93. pmid:20619376.
- 10. Donnelly J, Medini D, Boccadifuoco G, Biolchi A, Ward J, Frasch C, et al. Qualitative and quantitative assessment of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines. Proc Natl Acad Sci U S A. 2010 Nov 9;107(45):19490–5. pmid:20962280.
- 11. McNeil LK, Donald RGK, Gribenko A, French R, Lambert N, Harris SL, et al. Predicting the Susceptibility of Meningococcal Serogroup B Isolates to Bactericidal Antibodies Elicited by Bivalent rLP2086, a Novel Prophylactic Vaccine. mBio. 2018 Mar 13;9(2). pmid:29535195.
- 12. Muzzi A, Brozzi A, Serino L, Bodini M, Abad R, Caugant D, et al. Genetic Meningococcal Antigen Typing System (gMATS): A genotyping tool that predicts 4CMenB strain coverage worldwide. Vaccine. 2019 Feb 8;37(7):991–1000. pmid:30661831.
- 13. He B, Jia Z, Zhou H, Wang Y, Jiang X, Ma H, et al. CC4821 serogroup W meningococcal disease in China. Int J Infect Dis. 2014 Dec;29:113–4. pmid:25461240.
- 14. Ngai S, Weiss D, Bell JA, Majrud D, Zayas G, Crawley A, et al. Carriage of Neisseria meningitidis in Men Who Have Sex With Men Presenting to Public Sexual Health Clinics, New York City. Sex Transm Dis. 2020 Aug;47(8):541–8. pmid:32520884.
- 15. Retchless AC, Kretz CB, Chang HY, Bazan JA, Abrams AJ, Norris Turner A, et al. Expansion of a urethritis-associated Neisseria meningitidis clade in the United States with concurrent acquisition of N. gonorrhoeae alleles. BMC Genomics. 2018 Mar 2;19(1):176. pmid:29499642.
- 16. Brooks A, Lucidarme J, Campbell H, Campbell L, Fifer H, Gray S, et al. Detection of the United States Neisseria meningitidis urethritis clade in the United Kingdom, August and December 2019—emergence of multiple antibiotic resistance calls for vigilance. Euro Surveill. 2020 Apr;25(15). pmid:32317054.
- 17. Tzeng YL, Bazan JA, Turner AN, Wang X, Retchless AC, Read TD, et al. Emergence of a new Neisseria meningitidis clonal complex 11 lineage 11.2 clade as an effective urogenital pathogen. Proc Natl Acad Sci U S A. 2017 Apr 18;114(16):4237–42. pmid:28373547.
- 18. Taha MK, Claus H, Lappann M, Veyrier FJ, Otto A, Becher D, et al. Evolutionary Events Associated with an Outbreak of Meningococcal Disease in Men Who Have Sex with Men. PLoS One. 2016;11(5):e0154047. pmid:27167067.
- 19. Ma KC, Mortimer TD, Hicks AL, Wheeler NE, Sanchez-Buso L, Golparian D, et al. Adaptation to the cervical environment is associated with increased antibiotic susceptibility in Neisseria gonorrhoeae. Nat Commun. 2020 Aug 17;11(1):4126. pmid:32807804.
- 20. Jolley KA, Maiden MC. BIGSdb: Scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics. 2010 Dec 10;11:595. pmid:21143983.
- 21. Bratcher HB, Corton C, Jolley KA, Parkhill J, Maiden MC. A gene-by-gene population genomics platform: de novo assembly, annotation and genealogical analysis of 108 representative Neisseria meningitidis genomes. BMC Genomics. 2014 Dec 18;15:1138. pmid:25523208.
- 22. Huson DH. SplitsTree: analyzing and visualizing evolutionary data. Bioinformatics. 1998;14(1):68–73. pmid:9520503.
- 23. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser. 1999;41:95–8.
- 24. Belkacem N, Hong E, Antunes A, Terrade A, Deghmane AE, Taha MK. Use of Animal Models To Support Revising Meningococcal Breakpoints of beta-Lactams. Antimicrob Agents Chemother. 2016 Jul;60(7):4023–7. pmid:27090179.
- 25. Taha MK, Vazquez JA, Hong E, Bennett DE, Bertrand S, Bukovski S, et al. Target gene sequencing to characterize the penicillin G susceptibility of Neisseria meningitidis. Antimicrob Agents Chemother. 2007 Aug;51(8):2784–92. pmid:17517841.
- 26. Shultz TR, Tapsall JW, White PA. Correlation of in vitro susceptibilities to newer quinolones of naturally occurring quinolone-resistant Neisseria gonorrhoeae strains with changes in GyrA and ParC. Antimicrob Agents Chemother. 2001 Mar;45(3):734–8. pmid:11181352.
- 27. Harrison LH, Shutt KA, Arnold KE, Stern EJ, Pondo T, Kiehlbauch JA, et al. Meningococcal carriage among Georgia and Maryland high school students. J Infect Dis. 2015 Jun 1;211(11):1761–8. pmid:25505298.
- 28. Moura A, Kretz CB, Ferreira IE, Nunes A, de Moraes JC, Reis MG, et al. Molecular characterization of Neisseria meningitidis isolates recovered from 11-19-year-old meningococcal carriers in Salvador, Brazil. PLoS One. 2017;12(9):e0185038. pmid:28931058.
- 29. Harrison OB, Cole K, Peters J, Cresswell F, Dean G, Eyre DW, et al. Genomic analysis of urogenital and rectal Neisseria meningitidis isolates reveals encapsulated hyperinvasive meningococci and coincident multidrug-resistant gonococci. Sex Transm Infect. 2017 Sep;93(6):445–51. pmid:28137933.
- 30. Lucidarme J, Tan L, Exley RM, Findlow J, Borrow R, Tang CM. Characterization of Neisseria meningitidis isolates that do not express the virulence factor and vaccine antigen factor H binding protein. Clin Vaccine Immunol. 2011 Jun;18(6):1002–14. pmid:21508163.
- 31. Lucidarme J, Hill DM, Bratcher HB, Gray SJ, du Plessis M, Tsang RS, et al. Genomic resolution of an aggressive, widespread, diverse and expanding meningococcal serogroup B, C and W lineage. J Infect. 2015 Nov;71(5):544–52. pmid:26226598.
- 32. Lucidarme J, Scott KJ, Ure R, Smith A, Lindsay D, Stenmark B, et al. An international invasive meningococcal disease outbreak due to a novel and rapidly expanding serogroup W strain, Scotland and Sweden, July to August 2015. Euro Surveill. 2016 Nov 10;21(45). pmid:27918265.
- 33. Chiou CS, Liao JC, Liao TL, Li CC, Chou CY, Chang HL, et al. Molecular epidemiology and emergence of worldwide epidemic clones of Neisseria meningitidis in Taiwan. BMC Infect Dis. 2006 Feb 15;6:25. pmid:16478548.
- 34. Lucidarme J, Lekshmi A, Parikh SR, Bray JE, Hill DM, Bratcher HB, et al. Frequent capsule switching in 'ultra-virulent' meningococci—Are we ready for a serogroup B ST-11 complex outbreak? J Infect. 2017 Aug;75(2):95–103. pmid:28579305.
- 35. Tsang RS, Law DK, Deng S, Hoang L. Ciprofloxacin-resistant Neisseria meningitidis in Canada: likely imported strains. Can J Microbiol. 2017 Mar;63(3):265–8. pmid:28140652.
- 36. Zhao P, Xu L, Zhang A, Zhu B, Shao Z. Evolutionary analysis of gyrA gene from Neisseria meningitidis bacterial strains of clonal complex 4821 collected in China between 1978 and 2016. BMC Microbiol. 2020 Mar 30;20(1):71. pmid:32228482.
- 37. McNamara LA, Potts C, Blain AE, Retchless AC, Reese N, Swint S, et al. Detection of Ciprofloxacin-Resistant, beta-Lactamase-Producing Neisseria meningitidis Serogroup Y Isolates—United States, 2019–2020. MMWR Morb Mortal Wkly Rep. 2020 Jun 19;69(24):735–9. pmid:32555137.
- 38. Tzanakaki G, Georgakopoulou T, Xirogianni A, Papandreou A, Deghmane AE, Magaziotou I, et al. First report of meningococcal ciprofloxacin resistance in Greece due to invasive isolates of the sequence type ST-3129. Eur J Clin Microbiol Infect Dis. 2020 Jul 3. pmid:32621148.
- 39. Vacca P, Vocale C, Fazio C, Ambrosio L, Luzi K, Neri A, et al. Invasive meningococcal disease due to ciprofloxacin-resistant Neisseria meningitidis Sequence Type 7926: the first case in Italy, likely imported. J Glob Antimicrob Resist. 2019 Sep;18:177–8. pmid:31325619.
- 40. Hong E, Deghmane AE, Taha MK. Acquisition of Beta-Lactamase by Neisseria meningitidis through Possible Horizontal Gene Transfer. Antimicrob Agents Chemother. 2018 Sep;62(9). pmid:29941641.
- 41. Tsang RSW, Ahmad T, Jamieson FB, Tyrrell GJ. WGS analysis of a penicillin-resistant Neisseria meningitidis strain containing a chromosomal ROB-1 beta-lactamase gene. J Antimicrob Chemother. 2019 Jan 1;74(1):22–8. pmid:30295754.
- 42. Wang X, Cohn A, Comanducci M, Andrew L, Zhao X, MacNeil JR, et al. Prevalence and genetic diversity of candidate vaccine antigens among invasive Neisseria meningitidis isolates in the United States. Vaccine. 2011 Jun 24;29(29–30):4739–44. pmid:21571026.
- 43. Jolley KA, Bray JE, Maiden MCJ. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res. 2018;3:124. pmid:30345391.