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Whole genome typing of the recently emerged Canadian serogroup W Neisseria meningitidis sequence type 11 clonal complex isolates associated with invasive meningococcal disease

Open AccessPublished:February 04, 2018DOI:https://doi.org/10.1016/j.ijid.2018.01.019

      Abstract

      Objectives

      This study was performed to analyze the Canadian invasive serogroup W Neisseria meningitidis (MenW) sequence type 11 (ST-11) clonal complex (CC) isolates by whole genome typing and to compare Canadian isolates with similar isolates from elsewhere.

      Methods

      Whole genome typing of 30 MenW ST-11 CC, 20 meningococcal group C (MenC) ST-11 CC, and 31 MenW ST-22 CC isolates was performed on the Bacterial Isolate Genome Sequence database platform. Canadian MenW ST-11 CC isolates were compared with the 2000 MenW Hajj outbreak strain, as well as with MenW ST-11 CC from other countries.

      Results

      Whole genome typing showed that the Canadian MenW ST-11 CC isolates were distinct from the traditional MenW ST-22 CC; they were not capsule-switched contemporary MenC strains that incorporated MenW capsules. While some recent MenW disease cases in Canada were caused by MenW ST-11 CC isolates showing relatedness to the 2000 MenW Hajj strain, many were non-Hajj isolates similar to current MenW ST-11 isolates found globally. Geographical and temporal variations in genotypes and surface protein antigen genes were found among the MenW ST-11 CC isolates.

      Conclusions

      The current MenW ST-11 isolates did not arise by capsule switching from contemporary MenC ST-11 isolates. Both the Hajj-related and non-Hajj MenW ST-11 CC strains were associated with invasive meningococcal disease in Canada.

      Keywords

      Introduction

      Neisseria meningitidis, the causative agent of invasive meningococcal disease (IMD), normally resides as a commensal in the respiratory tract of healthy carriers. Strains that belong to some clonal complexes (CCs) and normally express capsular antigens of A, B, C, W, X, or Y are known to be commonly associated with invasive disease and may cause outbreaks or epidemics (
      • Caugant D.A.
      Population genetics and molecular epidemiology of Neisseria meningitidis.
      ,
      • Harrison L.H.
      • Trotter C.L.
      • Ramsay M.E.
      Global epidemiology of meningococcal disease.
      ). These hypervirulent strains are identified by multilocus enzyme electrophoresis or multilocus sequence typing (MLST) as electrophoretic type (ET) or sequence type (ST), respectively.
      One such hypervirulent CC is the ET-37 or ST-11 CC, which is a collection of closely related ETs or STs. ST-11 or ET-37 CC is a long established hypervirulent CC, and the earliest known ST-11 CC isolate is a serogroup B (MenB) strain from 1917 (
      • Caugant D.A.
      Population genetics and molecular epidemiology of Neisseria meningitidis.
      ). In North America and Europe, ST-11 CC in the 1960s and 1970s was associated with MenB (
      • Caugant D.A.
      Population genetics and molecular epidemiology of Neisseria meningitidis.
      ). However, in the mid-1980s, a serogroup C (MenC) strain designated as ET-15 (which is a genetic variant and member of the ST-11 CC) emerged to cause IMD that eventually spread globally (
      • Jelfs J.
      • Munro R.
      • Ashton F.E.
      • Caugant D.A.
      Genetic characterization of a new variant within the ET-37 complex of Neisseria meningitidis associated with outbreaks in various parts of the world.
      ). Sporadic and endemic serogroup W (MenW) IMD has been caused by strains typed as ST-22 CC (
      • Tsang R.S.W.
      • Deeks S.L.
      • Wong K.
      • Marchand-Austin A.
      • Jamieson F.B.
      Invasive serogroup W Neisseria meningitides (MenW) in Ontario, Canada shows potential clonal replacement during the period January 1, 2009 to June 30, 2016.
      ). The first significant outbreak caused by MenW was also due to a ST-11 strain in Saudi Arabia associated with the Hajj pilgrimage in 2000 (
      • Taha M.K.
      • Achtman M.
      • Alonso J.M.
      • Greenwood B.
      • Ramsay M.
      • Fox A.
      • et al.
      Serogroup W135 meningococcal disease in Hajj pilgrims.
      ). The MenW Hajj strain was globally disseminated as pilgrims returned home from Mecca (
      • Aguilera J.F.
      • Perrocheau A.
      • Meffre C.
      • Hahne S.
      • the W135 Working Group
      Outbreak of serogroup W135 meningococcal disease after the Hajj pilgrimage, Europe 2000.
      ,
      • Mayer L.W.
      • Reeves M.W.
      • Al-Hamdan N.
      • Sacchi C.T.
      • Taha M.K.
      • Ajelllo G.W.
      • et al.
      Outbreak of W135 meningococcal disease in 2000: not emergence of a new W135 strain but clonal expansion within the electrophoretic type -37 complex.
      ). Since the Hajj outbreak in 2000, increases in MenW disease have been observed in Africa, China, and South America (
      • du Chatelet P.I.
      • Traore Y.
      • Gessner B.D.
      • Antignac A.
      • Naccro B.
      • Njanpop-Lafourcade B.-M.
      • et al.
      Bacterial meningitis in Burkina Faso: surveillance using field-based polymerase chain reaction testing.
      ,
      • Hu S.
      • Zhang W.
      • Li F.
      • Hu Z.
      • Ma E.
      • Zheng T.
      • et al.
      Neisseria meningitidis serogroup W sequence type 11, Anhui Province, China, 2011-2013.
      ,
      • Weidlich L.
      • Baethgen L.F.
      • Mayer L.W.
      • Moraes C.
      • Klein C.C.
      • Nunes L.S.
      • et al.
      High prevalence of Neisseria meningitidis hypervirulent lineages and emergence of W135:P1.5, 2:ST-11 clone in Southern Brazil.
      ,
      • Efron A.M.
      • Salcedo C.
      • Regueira M.
      • Vazquez J.A.
      W135 invasive meningococcal strains spreading in South America: significant increase in incidence rate in Argentina.
      ). In England and Wales, the increase in ST-11 MenW disease was first noted in 2009–2010 and has since increased yearly, prompting the introduction of a vaccination program for 13–18-year-olds in 2015 (
      • Ladhani S.N.
      • Beebeejaun K.
      • Lucidarme J.
      • Canmpbell H.
      • Gray S.
      • Kaczmarski E.
      • et al.
      Increase in endemic Neisseria meningitidis capsular group W sequence type 11 complex associated with severe invasive disease in England and Wales.
      ,
      • Campbell H.
      • Saliba V.
      • Borrow R.
      • Ramsay R.
      • Ladhani S.N.
      Targeted vaccination of teenagers following continued rapid endemic expansion of a single meningococcal group W clone (sequence type 11 clonal complex), United Kingdom, 2015.
      ). A similar increase in MenW disease due to the same strain has been observed in Australia since 2013 and has also led to the introduction of a targeted vaccination program for 0–4 and 15–19-year-olds in a region of western Australia (
      • Martin N.V.
      • Ong K.S.
      • Howden B.P.
      • Lahra M.M.
      • Lambert S.B.
      • Beard F.H.
      • et al.
      Rise in serogroup W meningotoccal disease in Australia, 2013–2015.
      , ). In Canada, the increase in MenW disease due to the ST-11 CC was first noted in 2014, and by 2016, ST-11 MenW was responsible for 19% of all culture-confirmed IMD (
      • Tsang R.S.W.
      • Deeks S.L.
      • Wong K.
      • Marchand-Austin A.
      • Jamieson F.B.
      Invasive serogroup W Neisseria meningitides (MenW) in Ontario, Canada shows potential clonal replacement during the period January 1, 2009 to June 30, 2016.
      ;
      • Tsang R.S.W.
      • Hoang L.
      • Tyrrell G.J.
      • Horsman G.
      • Van Caeseele P.
      • Jamieson F.
      • et al.
      Increase in Neisseria meningitides serogroup W invasive disease in Canada, 2000–2016.
      ).
      Unlike the recent MenW ST-11 disease in the UK, which began in the older adults and spread to all age groups including adolescents and infants (
      • Ladhani S.N.
      • Beebeejaun K.
      • Lucidarme J.
      • Canmpbell H.
      • Gray S.
      • Kaczmarski E.
      • et al.
      Increase in endemic Neisseria meningitidis capsular group W sequence type 11 complex associated with severe invasive disease in England and Wales.
      ), the Canadian MenW ST-11 cases have mostly occurred in the adult age group, with 62% of the cases older than 41 years of age (
      • Tsang R.S.W.
      • Hoang L.
      • Tyrrell G.J.
      • Horsman G.
      • Van Caeseele P.
      • Jamieson F.
      • et al.
      Increase in Neisseria meningitides serogroup W invasive disease in Canada, 2000–2016.
      ). MenW ST-11 disease in university students has also been reported from France (
      • Bassi C.
      • Taha M.
      • Merle C.
      • Hong E.
      • Levy-Bruhl D.
      • Barret A.
      • Njoya I.M.
      A cluster of invasive meningococcal disease (IMD) caused by Neisseria meningitidis serogroup W among university students, France, February to May 2017.
      ). While no fatal MenW ST-11 cases were identified in one Canadian study (
      • Tsang R.S.W.
      • Deeks S.L.
      • Wong K.
      • Marchand-Austin A.
      • Jamieson F.B.
      Invasive serogroup W Neisseria meningitides (MenW) in Ontario, Canada shows potential clonal replacement during the period January 1, 2009 to June 30, 2016.
      ), both the UK and Australia have reported high case-fatality rates (of 12% and 10.7%, respectively) (
      • Efron A.M.
      • Salcedo C.
      • Regueira M.
      • Vazquez J.A.
      W135 invasive meningococcal strains spreading in South America: significant increase in incidence rate in Argentina.
      ,
      • Campbell H.
      • Parikh S.R.
      • Borrow R.
      • Kaczmarski E.
      • Ramsay M.E.
      • Ladhani S.N.
      Presentation with gastrointestinal symptoms and high case fatality associated with group W meningococcal disease (MenW) in teenagers, England, July 2015 to January 2016.
      ). Unusual clinical presentations of predominantly gastrointestinal symptoms, but also necrotizing fasciitis, have also been reported from Europe (
      • Campbell H.
      • Parikh S.R.
      • Borrow R.
      • Kaczmarski E.
      • Ramsay M.E.
      • Ladhani S.N.
      Presentation with gastrointestinal symptoms and high case fatality associated with group W meningococcal disease (MenW) in teenagers, England, July 2015 to January 2016.
      ,
      • Russcher A.
      • Fanoy E.
      • van Olden G.D.J.
      • Graafland A.D.
      • van der Ende A.
      • Knol M.J.
      Necrotising fasciitis as atypical presentation of infection with emerging Neisseria meningitidis serogroup W (MenW) clonal complex 11, the Netherlands, March 2015.
      ).
      Whole genome analysis subdivided meningococci of the ST-11 CC into two sub-lineages, 11.2 (corresponding to the ET-15 type, which contained the unique polymorphism in its fumC gene) and 11.1 (corresponding to ET-37) (
      • Lucidarme J.
      • Hill D.M.C.
      • Bratcher H.B.
      • Gray S.J.
      • du Plessis M.
      • Tsang R.S.W.
      • et al.
      Genomic resolution of an aggressive, widespread, diverse and expanding meningococcal serogroup B, C and W lineage.
      ). All invasive MenW of the ST-11 CC belonged to sub-lineage 11.1 and were thought to have arisen from MenC ET-37 by a capsular switching event that occurred before 1970 (
      • Mustapha M.M.
      • Marsh J.W.
      • Krauland M.G.
      • Fernandez J.O.
      • de Lemos A.P.
      • Dunning Hotopp J.C.
      • et al.
      Genomic epidemiology of hypervirulent serogroup W, ST-11 Neisseria meningitidis.
      ). Within the sub-lineage 11.1, MenW can be further distinguished into the Hajj strain (responsible for the 2000 outbreak among Hajj pilgrims) and the South American strain (emerged in Brazil in 2003) (
      • Lucidarme J.
      • Hill D.M.C.
      • Bratcher H.B.
      • Gray S.J.
      • du Plessis M.
      • Tsang R.S.W.
      • et al.
      Genomic resolution of an aggressive, widespread, diverse and expanding meningococcal serogroup B, C and W lineage.
      ,
      • Mustapha M.M.
      • Marsh J.W.
      • Harrison L.H.
      Global epidemiology of capsular group W meningococcal disease (1970-2015): multifocal emergence and persistence of hypervirulent sequence type (ST)-11 clonal complex.
      ). A descendant from the South American strain (termed the original UK strain) was responsible for the initial increase in invasive MenW disease in the UK. A further descendant (termed the 2013 strain) is now responsible for most of the current invasive MenW disease in the UK and other European countries (France, Netherlands, Sweden) (
      • Knol M.J.
      • Hahne S.J.M.
      • Lucidarme J.
      • Campbell H.
      • de Melker H.E.
      • Gray S.J.
      • et al.
      Temporal associations between national outbreaks of meningococcal serogroup W and C disease in the Netherlands and England: an observational cohart study.
      ,
      • Lucidarme J.
      • Scott K.J.
      • 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.
      ) as well as the unusual clinical symptoms mentioned above (
      • Campbell H.
      • Parikh S.R.
      • Borrow R.
      • Kaczmarski E.
      • Ramsay M.E.
      • Ladhani S.N.
      Presentation with gastrointestinal symptoms and high case fatality associated with group W meningococcal disease (MenW) in teenagers, England, July 2015 to January 2016.
      ,
      • Russcher A.
      • Fanoy E.
      • van Olden G.D.J.
      • Graafland A.D.
      • van der Ende A.
      • Knol M.J.
      Necrotising fasciitis as atypical presentation of infection with emerging Neisseria meningitidis serogroup W (MenW) clonal complex 11, the Netherlands, March 2015.
      ).
      In this study, whole genome analysis was used to study the MenW and MenC strains in Canada. The objective was to determine whether the abrupt emergence of MenW ST-11 disease in Canada was due to capsule-switched contemporary MenC strains that acquired MenW capsules, or the 2000 Hajj-strain, or the current emerging non-Hajj strain common in other countries, or a combination of these or some previously unidentified new strain(s). In addition, the surface protein antigens of the Canadian MenW ST-11 CC (targets for the newer MenB vaccines) were characterized to explore if the latest MenB vaccines may also offer protection against the emerging MenW, as was predicted for the UK outbreak (
      • Ladhani S.N.
      • Giuliani M.M.
      • Biolchi A.
      • Pizza M.
      • Beebeejaun K.
      • Lucidarme J.
      • et al.
      Effectiveness of meningococcal B vaccine against endemic hypervirulent Neissseria meningitidis W strain, England.
      ). The Canadian MenW ST-11 CC isolates were also compared to current global MenW ST-11 CC isolates to study their genetic relationship.

      Materials and methods

      Neisseria meningitidis isolates

      In Canada, IMD is a notifiable disease and the provincial public health laboratories routinely receive case isolates from hospitals and clinical diagnostic laboratories for identification and serogrouping. Isolates are also forwarded to the National Microbiology Laboratory (NML) for additional characterization. From January 1, 2000 to March 31, 2017, NML identified 29 IMD cases and one non-IMD case due to MenW ST-11 CC. Thirty-one MenW ST-22 CC and 20 MenC ST-11 CC isolates recovered from across Canada during the same period were also included for comparison (Supplementary material, Table S1).

      Routine laboratory typing of Neisseria meningitidis

      Serogrouping, serotyping, serosubtyping, PorA genotyping, and MLST were done using standard methods described previously (
      • Jamieson F.B.
      • Rawte P.
      • Deeks S.L.
      • Zhou J.
      • Law D.K.S.
      • Deng S.
      • et al.
      Genetic and antigenic characterization of invasive endemic serogroup B Neisseria meningitidis in Ontario, Canada, in 2001-2010.
      ,
      • Zhou J.
      • Lefebvre B.
      • Deng S.
      • Gilcia R.
      • Deceuninck G.
      • Law D.K.S.
      • et al.
      Invasive serogroup B Neisseria meningitidis in Quebec, Canada, 2003 to 2010: persistence of the ST-269 clone since it first emerged in 2003.
      ).

      Whole genome sequencing, data assembly, and analysis

      DNA samples were extracted from pure isolate cultures following standard protocols using the Epicentre Masterpure Complete DNA and RNA Extraction Kit (Mandel Scientific, Guelph, ON, Canada). Multiplexed libraries were created with TruSeq sample preparation kits (Illumina, San Diego, CA, USA), and paired-end, 300-bp indexed reads were generated on the Illumina MiSeq platform (Illumina, San Diego, CA, USA). Genome sequence data of these isolates were assembled using SPAdes (
      • Bankevich A.
      • Nurk S.
      • Antipov D.
      • Gurevich A.A.
      • Dvorkin M.
      • Kulikov A.S.
      • et al.
      SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing.
      ). The assembled genomes were uploaded to the PubMLST Neisseria database (https://pubmlst.org/neisseria/), which runs the Bacterial Isolate Genome Sequence Database (BIGSdb) platform (
      • Jolley K.A.
      • Maiden M.C.
      BIGSdb: scalable analysis of bacterial genome variation at the population level.
      ).
      The isolates were compared by (1) extended MLST (eMLST), (2) ribosomal MLST (rMLST), and (3) core genome MLST (cgMLST) using the BIGSdb Genome Comparator tool (
      • Bratcher H.B.
      • Corton C.
      • Jolley K.A.
      • Parkhill J.
      • Maiden M.C.
      A gene-by-gene population genomics platform: de novo assembly, annotation and genealogical analysis of 108 representative Neisseria meningitidis genomes.
      ). Canadian MenW ST-11 CC (n = 30) were compared by cgMLST with other MenW ST-11 CC genomes (n = 188, two genomes per year from each country, available open access in the PubMLST Neisseria database, accessed in July 2017 and described in the Supplementary material, Table S2; genomes from the Hajj strain M7124, the ‘original UK’ strain M14 240001, and a representative ‘2013 strain’ M14 240054 were also included). A distance matrix based on the number of variable alleles was generated by the genome comparator tool, and the neighbor-net phylogenetic networks were generated by SplitsTree4 (version 4.14.5) (
      • Bratcher H.B.
      • Corton C.
      • Jolley K.A.
      • Parkhill J.
      • Maiden M.C.
      A gene-by-gene population genomics platform: de novo assembly, annotation and genealogical analysis of 108 representative Neisseria meningitidis genomes.
      ,
      • Huson D.H.
      SplitsTree: analyzing and visualizing evolutionary data.
      ). The Hajj strain M7124 (GenBank accession number CP009419.1) was included as a reference genome.
      The surface protein antigen genes, porA, porB, fetA, nadA, nhba, and fHbp, which encode, respectively, for class 1 outer membrane porin protein (PorA), class 2/3 outer membrane porin protein (PorB), iron-regulated Neisseria outer membrane protein (FetA), Neisseria adhesin A (NadA), Neisseria heparin binding antigen (NHBA), and factor H binding protein (fHbp), were also analyzed using the BIGSdb.

      Results

      Emergence of MenW ST-11 CC in Canada

      Prior to 2016, MenW was only responsible for <10% of all culture-positive IMD cases (NML unpublished data). The majority (67–100%) of invasive MenW isolated in Canada from 2000 to 2013 belonged to the ST-22 CC, but beginning in 2014 and by March 31, 2017, 56–88% of MenW were typed as ST-11 CC per year (Table 1).
      Table 1Characterization of serogroup W Neisseria meningitidis (MenW) in Canada by multilocus sequence typing to determine their clonal complex (CC).
      YearTotal numberNumber belonging toOthers
      ST-22 CCST-11 CC
      2000–200895853
      Three ST-11 CC MenW were identified between 2000 and 2008, including one in 2000 in British Columbia, one in 2001 in Ontario, and one in 2003 in Ontario.
      7 (ST-174 CC, n = 4; ST-23 CC, n = 3)
      2009121200
      20106600
      201110811 (ST-167 CC)
      20123201 (ST-23 CC)
      20134400
      20144130
      20159450
      201616
      One isolate was from the eye.
      214
      One isolate was from the eye.
      0
      2017
      From January 1 to March 31, 2017.
      4040
      ST, sequence type; CC, clonal complex.
      a Three ST-11 CC MenW were identified between 2000 and 2008, including one in 2000 in British Columbia, one in 2001 in Ontario, and one in 2003 in Ontario.
      b One isolate was from the eye.
      c From January 1 to March 31, 2017.
      There were 30 MenW ST-11 CC during the study period; three were from 2000 to 2003, with the first isolate identified in the year 2000. From 2004 to 2013, there was only one additional MenW ST-11 CC isolate. Three isolates of MenW ST-11 CC from three provinces were identified in 2014, increasing to five isolates from four provinces in 2015. However, during the 15 months in 2016–2017, 18 isolates from five provinces were identified (Table 1, Table 2).
      Table 2Characterization of 29 invasive and one non-invasive Canadian Neisseria meningitidis serogroup W, sequence type 11 clonal complex isolates, 2010 to 2017, by their antigen gene alleles
      Alleles are shown for the following order of antigen genes: porA, porB, fetA, nadA, nhba, and fHbp.
      and the antigens or peptides they were predicted to encode.
      porA alleles 1, 1091, and New* were predicted to encode PorA VR1=5, VR2=2. porB alleles 1, 194, and 244 were predicted to encode serotype antigen 2a; allele New** did not encode serotype 2a due to mutations (confirmed by serotyping result). fetA alleles 13, 421, and 1254 were predicted to encode FetA VR peptide F1-1. nadA alleles 3, 5, and 185 were predicted to encode peptides 3, 6, and 145, respectively (all variant 2/3); allele 256 was predicted not to encode NadA peptide due to a frame shift mutation. nhba alleles 17 and 72 were predicted to encode NHBA peptides 29 and 96, respectively. fHbp alleles 9, 22, and 1327 were predicted to encode fHbp peptides 9 (Novartis variant 1, Pfizer subfamily B), 22 (Novartis variant 2, Pfizer subfamily A), and 8 (Novartis variant 1, Pfizer subfamily B), respectively. New*=new porA allele and closest match to allele 1 (1 nucleotide difference). New**=new porB allele but closest match to allele 244 (6 nucleotide differences).
      Year, Province, ST
      ST=sequence type obtained by the standard seven-gene multilocus sequence typing.
      Gene alleles
      Alleles are shown for the following order of antigen genes: porA, porB, fetA, nadA, nhba, and fHbp.
      porAporBfetAnadAnhbafHbp
      2000 Hajj ST-11 strain M712411135729
      Canadian Hajj-related strains
       2000 British Columbia ST-1111135729
       2001 Ontario ST-1111135729
       2016 Manitoba ST-1111135729
      2016 Quebec, n = 3
      n refers to the number of isolates with that particular order of gene alleles.
      ST-11
      109119413256
      nadA allele 256 contains a frame shift mutation leading to a premature stop codon and therefore, no peptide predicted.
      729
      Canadian non-Hajj strains
       2011 British Columbia ST-1112441351722
       2014 Alberta ST-1112441351722
       2014 Ontario ST-10826
      Sequence types 247, 10826, 11371, and 12818 are single locus variants of ST-11 (https://pubmlst.org/neisseria/).
      12441351722
       2015 British Columbia ST-11371
      Sequence types 247, 10826, 11371, and 12818 are single locus variants of ST-11 (https://pubmlst.org/neisseria/).
      12441351722
       2015 Alberta ST-1112441351722
       2015 Ontario, n = 2 ST-1112441351722
       2015 Quebec ST-1112441351722
       2016 British Columbia, n = 2 ST-1112441351722
       2016 Alberta ST-1112441351722
       2016 Manitoba ST-1112441351722
       2016 Ontario, n = 5 ST-1112441351722
       2017 Quebec ST-1112441351722
      2014 British Columbia ST-111244131851722
      2016 British Columbia ST-111244125451722
      2017 Alberta ST-12818
      Sequence types 247, 10826, 11371, and 12818 are single locus variants of ST-11 (https://pubmlst.org/neisseria/).
      New*2441351722
      2017 British Columbia ST-111244135171327
      2017 British Columbia ST-111New**1351722
      2003 Ontario ST-247
      Sequence types 247, 10826, 11371, and 12818 are single locus variants of ST-11 (https://pubmlst.org/neisseria/).
      1142131722
      a Alleles are shown for the following order of antigen genes: porA, porB, fetA, nadA, nhba, and fHbp.
      b porA alleles 1, 1091, and New* were predicted to encode PorA VR1 = 5, VR2 = 2. porB alleles 1, 194, and 244 were predicted to encode serotype antigen 2a; allele New** did not encode serotype 2a due to mutations (confirmed by serotyping result). fetA alleles 13, 421, and 1254 were predicted to encode FetA VR peptide F1-1. nadA alleles 3, 5, and 185 were predicted to encode peptides 3, 6, and 145, respectively (all variant 2/3); allele 256 was predicted not to encode NadA peptide due to a frame shift mutation. nhba alleles 17 and 72 were predicted to encode NHBA peptides 29 and 96, respectively. fHbp alleles 9, 22, and 1327 were predicted to encode fHbp peptides 9 (Novartis variant 1, Pfizer subfamily B), 22 (Novartis variant 2, Pfizer subfamily A), and 8 (Novartis variant 1, Pfizer subfamily B), respectively. New* = new porA allele and closest match to allele 1 (1 nucleotide difference). New** = new porB allele but closest match to allele 244 (6 nucleotide differences).
      c ST = sequence type obtained by the standard seven-gene multilocus sequence typing.
      d n refers to the number of isolates with that particular order of gene alleles.
      e nadA allele 256 contains a frame shift mutation leading to a premature stop codon and therefore, no peptide predicted.
      f Sequence types 247, 10826, 11371, and 12818 are single locus variants of ST-11 (https://pubmlst.org/neisseria/).

      Genetic relationship of the traditional MenW ST-22 CC with the emerging MenW ST-11 CC and the MenC ST-11 CC isolates in Canada

      cgMLST showed that MenW ST-22 CC isolates formed a distinctly different cluster from the MenW ST-11 CC and the MenC ST-11 CC isolates (Figure 1). MenC ST-11 CC was divided into two sub-populations of lineage 11.2 and lineage 11.1. The MenW ST-11 CC isolates belonged to lineage 11.1.
      Figure 1
      Figure 1Phylogenetic analysis to compare the genetic relationship of 30 Canadian Neisseria meningitidis serogroup W (MenW) sequence type (ST)-11 clonal complex (CC) isolates, with 20 Canadian N. meningitidis serogroup C ST-11 CC isolates and 31 Canadian MenW ST-22 CC isolates by core genome multilocus sequence typing. The result is presented as a neighbor-net phylogenetic network. The scale bar indicates the number of allelic differences.
      In addition to the capsular genes that separated the MenW ST-11 CC from the MenC ST-11 CC isolates (Supplementary material, Figure S1a), rMLST and eMLST, which analyzed non-capsular genes (Supplementary material, Figure S1b, c), also confirmed that the MenW ST-11 isolates were not capsule-switched contemporary MenC ST-11 isolates.

      Whole genome analysis of Canadian MenW of ST-11 CC

      Analysis by eMLST or rMLST or cgMLST (Figure 2a–c) all divided the 30 MenW ST-11 CC isolates into either the Hajj-related isolates (n = 6) or the non-Hajj isolates (n = 24).
      Figure 2
      Figure 2Neighbor-net phylogenetic network comparing 30 Canadian Neisseria meningitidis serogroup W (MenW) sequence type (ST)-11 clonal complex isolates with the 2000 Hajj MenW ST-11 reference strain M7124 by (a) extended multilocus sequence typing (eMLST), (b) ribosomal MLST (rMLST), and (c) core genome MLST (cgMLST). The scale bar denotes the number of allelic differences.
      All six Hajj-related isolates were typed as ST-11 and they were found to cluster together (Figure 2c), which included isolates from 2000, 2001, and 2016 (Table 2). The 24 non-Hajj isolates were genetically more diverse (Figure 2a–c): 20 were typed as ST-11 and four were typed as ST-247, ST-11371, ST-12818, and ST-10826 (Table 2).
      Seventeen of the 24 Canadian non-Hajj MenW ST-11 CC isolates from 2014 to 2017 were clustered together to form a major group, which in turn could be divided into two sub-populations of eight or nine isolates (Figure 2c). The remaining seven isolates were somewhat more diverse, including the single 2003 Ontario isolate (typed as ST-247), which was most distinct from the rest of the isolates (Figure 2c).

      Protein antigen genes of Canadian MenW ST-11 CC

      Three of the six Canadian Hajj-related MenW (one each in 2000, 2001, and 2016) were found to have porA, porB, fetA, nadA, nhba, and fHbp gene alleles of 1, 1, 13, 5, 72, and 9, respectively, while the three Québec Hajj-related isolates were found to have distinct porA, porB, and nadA gene alleles (Table 2). The gene alleles in 18 non-Hajj isolates were determined to be 1, 244, 13, 5, 17, and 22. Five other non-Hajj isolates (four from British Columbia and one from Alberta) were determined to differ by a variant allele in one of the six antigen genes involving the porA, porB, fetA, nadA, or fHbp gene (Table 2). The single ST-247 (2003 Ontario) isolate differed from the rest in two gene alleles (fetA and nadA). The PorA Variable Region genotype, the PorB serotype antigen, and the FetA Variable Region type, as well as the Bexsero vaccine (GSK) antigen (NadA, NHBA, and fHbp) peptide types (or IDs), predicted to be encoded by the various strains are described in Table 2.

      Relationship of Canadian ST-11 MenW with ST-11 MenW from other countries

      The core genomes of the 30 Canadian MenW ST-11 CC isolates were compared with 188 genomes of MenW ST-11 CC from other countries (Figure 3). The six Canadian Hajj-related isolates were grouped together with isolates that clustered around the 2000 Hajj reference strain. The majority (n = 17 or 71%) of the 24 Canadian non-Hajj MenW isolates appeared to cluster with a representative of the ‘2013 strain’, while four appeared to be more similar to the ‘original UK strain’, and two grouped with the ‘South American strain’. The single non-Hajj ST-247 isolate from Ontario belonged to the older sub-lineages that were common prior to year 2000. The Canadian ST-11 MenW isolates that were related to the ‘2013 strain’ were recovered during the period 2014 (one isolate) and 2015 (three isolates) to 2017 (four isolates), and 90% (9/10) of the ST-11 MenW isolates from 2016 were found to belong to this group of ‘2013 strain’. The four isolates related to the ‘original UK strain’ were recovered between 2014 and 2016.
      Figure 3
      Figure 3Comparison of 30 Canadian Neisseria meningitidis serogroup W (MenW) sequence type (ST)-11 clonal complex (CC) isolates with 188 geo-temporally diverse MenW ST-11 CC isolates from other countries by core genome multilocus sequence typing (cgMLST). The result is presented as a neighbor-net phylogenetic network, generated using the genome comparator tool of BIGSdb with 1605-locus cgMLST v1.0. Data of the isolates are available through their PubMLST IDs described in the Supplementary material, Tables S1 and S2. The scale bar denotes the number of allelic differences. Isolates were placed into the following groups: ‘South American strain sub-lineage’, ‘Older sub-lineages’, and ‘Hajj strain sub-lineage’.
      South American strain sub-lineage, countries (n, number of isolates, year of isolation): (1) South American strain = Brazil (n = 4, 1997–2014), Canada (n = 2, 2011, 2014), Chile (n = 2, 2008, 2012), Finland (n = 1, 2015), Greece (n = 1, 2016), Italy (n = 2, 2014, (year information missing)), UK (n = 1, 2010), and USA (n = 1, 2009). (2) Original UK strain = Canada (n = 4, 2014–16), France (n = 1, 2014), Ireland (n = 3, 2013–15), Italy (n = 1, 2015), South Africa (n = 1, (year information missing)), Spain (n = 1, 2016), Sweden (n = 1, 2016), Netherlands (n = 3, 2012–13), and UK (n = 13, 2009–17). (3) 2103 strain = Canada (n = 17, 2014–17), Finland (n = 3, 2015–16), France (n = 3, 2015–16), Russia (n = 1, 2016), Sweden (n = 6, 2013–17), Netherlands (n = 3, 2015–16), and UK (n = 6, 2014–17).
      Older sub-lineages, countries (n, number of isolates, year of isolation): Canada (n = 1, 2003), Chile (n = 1, 1999), Czech Republic (n = 2, 1994, 1996), South Africa (n = 7, 2003–13), Sweden (n = 4, 1979–98), and UK (n = 10, 1975–2007).
      Hajj-strain sub-lineage, countries (n, number of isolates, year of isolation): Algeria (n = 4, 2001–15), Burkina Faso (n = 9, 2001–12), Canada (n = 6, 2000–16), Chad (n = 1, 2001), Central Africa Republic (n = 1, 2001), Cameroon (n = 4, 2000–01), Djibouti (n = 1, 2004), France (n = 3, 2000–14), Italy (n = 6, 2013–16), Morocco (n = 1, 2016), Madagascar (n = 5, 2001–16), Mali (n = 5, 1994–2012), Mauritius (n = 1, 2001), Niger (n = 8, 2001–15), Russia (n = 2, 2015, 2016), Sweden (n = 3, 2000–17), Saudi Arabia (n = 2, 2000), South Africa (n = 14, 2004–13), Senegal (n = 4, 2000–2002), Turkey (n = 3, 2005–06), UK (n = 11, 2000–07), and USA (n = 1, 2000).
      Others, countries (n, number of isolates, year of isolation): Algeria (n = 1, 1999), Brazil (n = 1, 2001), Chile (n = 1, 2011), Chad (n = 1, 1996), Indonesia (n = 1, 1996), Italy (n = 1, 2013), Mali (n = 1, 1994), Malta (n = 2, 1999), South Africa (n = 1, 2003), UK (n = 5, 1996–2005), and USA (n = 2, 2005, 2008).

      Discussion

      It was found that MenW IMD due to ST-11 CC was rare in Canada prior to 2014. During the 2000 international MenW Hajj outbreak, two cases (one in 2000 and one in 2001) of MenW ST-11 were identified in Canada and demonstrated by cgMLST to be caused by the Hajj strain. From 2000 to 2013, the majority of MenW diseases were caused by strains belonging to the ST-22 CC. Beginning in 2014, a significant shift in the genetic makeup of invasive MenW strains was noted, from ST-22 CC to ST-11 CC (
      • Tsang R.S.W.
      • Deeks S.L.
      • Wong K.
      • Marchand-Austin A.
      • Jamieson F.B.
      Invasive serogroup W Neisseria meningitides (MenW) in Ontario, Canada shows potential clonal replacement during the period January 1, 2009 to June 30, 2016.
      ). Concomitant to this genetic shift in the MenW strain was the increase in the proportion of IMD due to MenW (
      • Tsang R.S.W.
      • Hoang L.
      • Tyrrell G.J.
      • Horsman G.
      • Van Caeseele P.
      • Jamieson F.
      • et al.
      Increase in Neisseria meningitides serogroup W invasive disease in Canada, 2000–2016.
      ). The MenW ST-11 CC isolates have been shown to be unrelated to the contemporary MenC ST-11 CC isolates; therefore, they did not arise from the contemporary MenC strains by capsule switching. Core genome MLST (Figure 3) showed that the two ET-15 MenW from the USA were distinctly different from the rest of the MenW ST-11 isolates, and they were probably capsule-switched from serogroup C meningococci. The increase in MenW disease in Canada since 2014 was mainly caused by the non-Hajj ST-11 CC isolates. The present study also demonstrated the co-existence of both the Hajj-related and the non-Hajj isolates in Canada in the last few years. The non-Hajj isolates were found in British Columbia, Alberta, Manitoba, Ontario, and Quebec, while the recent Hajj-related isolates were in Manitoba and Quebec only.
      The Canadian MenW ST-11 CC isolates appeared to be similar to the current MenW ST-11 strains circulating globally based on cgMLST analysis (
      • Mustapha M.M.
      • Marsh J.W.
      • Krauland M.G.
      • Fernandez J.O.
      • de Lemos A.P.
      • Dunning Hotopp J.C.
      • et al.
      Genomic epidemiology of hypervirulent serogroup W, ST-11 Neisseria meningitidis.
      ,
      • Mustapha M.M.
      • Marsh J.W.
      • Harrison L.H.
      Global epidemiology of capsular group W meningococcal disease (1970-2015): multifocal emergence and persistence of hypervirulent sequence type (ST)-11 clonal complex.
      ,
      • Knol M.J.
      • Hahne S.J.M.
      • Lucidarme J.
      • Campbell H.
      • de Melker H.E.
      • Gray S.J.
      • et al.
      Temporal associations between national outbreaks of meningococcal serogroup W and C disease in the Netherlands and England: an observational cohart study.
      ,
      • Lucidarme J.
      • Scott K.J.
      • 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.
      ). Over half (17 out of 30 isolates or 56.7%) were similar to the ‘2013 strain’, 20% (six isolates) belonged to or were similar to the Hajj strain, 13.3% (four isolates) were grouped with the ‘original UK strain’, while only one (3.3%) belonged to a group of older sub-lineages common prior to the emergence of the Hajj strain in 2000. The province where the ‘original UK’ related strains, the Hajj-related strain, and isolates related to the ‘2013 strain’ were found, as well as their phylogenetic positions in the neighbor-net network diagram (Figure 3) and the year of their isolations, all suggested potential multiple introduction of these strains into Canada.
      The genetic heterogeneity of the Canadian non-Hajj MenW ST-11 CC isolates was also similar to that found in the MenW ST-11 from other countries. Whole genome typing differentiated between isolates obtained from different provinces, in contrast to the conventional seven-gene MLST scheme which cannot. Furthermore whole genome typing could also distinguish between the MenW ST-11 CC isolates in British Columbia and Ontario by placing some of them into two separate sub-populations (Figure 2c). In addition, whole genome typing demonstrated higher recombination and mutation rates in the surface protein antigen genes of isolates from British Columbia, as well as in the most recent isolates obtained in 2017 (Table 2).
      Among the six Hajj-related MenW ST-11 isolates identified in this study, those recovered in 2000 and 2001 were shown to be most similar to the 2000 Hajj reference strain M7124 (Figure 2c). These two earlier isolates probably represented the original importation of the Hajj strain into Canada during the first major MenW outbreak associated with the 2000 Hajj (
      • Taha M.K.
      • Achtman M.
      • Alonso J.M.
      • Greenwood B.
      • Ramsay M.
      • Fox A.
      • et al.
      Serogroup W135 meningococcal disease in Hajj pilgrims.
      ). In contrast, the four 2016 isolates could be separated into two clusters, consisting of three highly related isolates found in Quebec and the single isolate from Manitoba (Figure 2c), which appeared to be more distantly related to the original 2000 Hajj strain. As no Hajj-related MenW ST-11 was isolated in Canada between 2002 and 2015, it is hypothesized that these four 2016 Hajj-related ST-11 MenW isolates were recently imported. It is interesting to note that in the African meningitis belt, the 2012 MenW epidemic in Burkina Faso was due to strains related to or descended from the Hajj outbreak strain, while isolates from an earlier epidemic (2001 and 2002) were less closely related to the Hajj outbreak strain (
      • Retchless A.C.
      • Hu F.
      • Ouedraogo A.S.
      • Diarra S.
      • Knipe K.
      • Sheth M.
      • et al.
      The establishment and diversification of epidemic-associated serogroup W meningococcus in the African meningitis belt, 1994-2012.
      ).
      It is important to note that all of the Canadian MenW ST-11 CC isolates were demonstrated to express serogroup W capsular antigens, and that all provinces (except two) and territories in Canada now have quadrivalent conjugate meningococcal vaccine programs for children and adolescents (
      • Public Health Agency of Canada
      Canada’s provincial and territorial routine (and catch-up) vaccination programs for infants and children.
      ). Most provinces offer the vaccine to school children attending grade 9 or around age 14 years (assuming children begin grade 1 at age 6 years), but one province offers the vaccine to children in grade 4 and a territory provides the vaccine to grade 12 students who will be attending post-secondary education. The earliest program began in 2006, and by the end of 2011, six provinces already had this vaccine program in place (
      • Public Health Agency of Canada
      An Advisory Committee (ACS) Statement, National Advisory Committee on Immunization (NACI): update on quadrivalent meningococcal vaccines available in Canada.
      ). The impact of the quadrivalent conjugate vaccine introduced into the Canadian immunization programs has not been evaluated fully, including any possible effect on the recent emergence of MenW ST-11.
      Although none of the MenW ST-11 CC isolates in Canada was predicted to express fHbp, NadA, NHBA, and PorA antigens that match exactly with the Bexsero or Trumenba vaccine (Pfizer) components, the Canadian Hajj-related MenW ST-11 isolates were found to have fHbp genes that predicted the synthesis of fHbp peptide 9, a subfamily B or variant 1 protein. Since the fHbp component of Bexsero is also a subfamily B or variant 1 protein, some coverage by Bexsero against the Hajj MenW isolates may be expected because of cross-protection between fHbp proteins of the same subfamily. Coverage by Bexsero may be further provided by the cross-protective NHBA and NadA components, although three of the Canadian Hajj-related isolates were not predicted to express NadA proteins due to a frame-shift mutation in their nadA genes of allele 256. However, these MenB vaccines have not been proven to be protective against MenW disease, nor were they licensed for this use.
      Only one of the 24 Canadian non-Hajj MenW ST-11 isolates was found to have the fHbp gene that predicted synthesis of a subfamily B or variant 1 fHbp peptide. The majority (23/24 isolates or 96%) were predicted to synthesize fHbp peptide 22, a subfamily A or variant 2 protein, which had been shown to lack cross-reactivity with the subfamily B Bexsero component. However, coverage by Bexsero may be offered through the NadA and NHBA components of the vaccine, since all of the non-Hajj MenW ST-11 isolates were predicted to produce NHBA peptide 29 and NadA peptides of variant 2/3 (peptides 3, 6, or 145). Indeed sera from infants immunized with the Bexsero vaccine were shown to have high serum bactericidal titers (1:32 to 1: >128) to the current MenW strain in the UK, thus raising the possibility that Bexsero may offer protection against the current MenW disease (
      • Ladhani S.N.
      • Giuliani M.M.
      • Biolchi A.
      • Pizza M.
      • Beebeejaun K.
      • Lucidarme J.
      • et al.
      Effectiveness of meningococcal B vaccine against endemic hypervirulent Neissseria meningitidis W strain, England.
      ). In theory, Trumenba, a bivalent fHbp MenB vaccine, may also be predicted to cover MenW strains as long as fHbp is expressed and accessible in adequate amounts to be recognized by the anti-fHbp immune vaccine sera (
      • Harris S.L.
      • Zhy D.
      • Murphy E.
      • McNeil L.K.
      • Wang X.
      • Mayer L.H.
      • et al.
      Preclinical evidence for the potential of a bivalent fHBP vaccine to prevent serogroup C disease.
      ).
      The limitations of this study include potential underestimation of the true magnitude of MenW ST-11 CC disease, since only culture-confirmed case isolates were included. Furthermore, immunization history and travel history were not collected, and clinical information was also not collected, so it was not possible to determine whether there had been any unusual clinical presentations among the Canadian MenW ST-11 cases. Furthermore, it was unknown whether any case isolates were imported.
      In conclusion, this study demonstrated the usefulness of whole genome analysis of meningococci to reveal the presence of both the Hajj-related and the non-Hajj MenW ST-11 CC in Canada, with the majority of the latter group made up of isolates related to the ‘2013 strain’ that is spreading globally. Temporal and geographical variations in the genotypes and surface protein antigen genes of the MenW ST-11 CC isolates were also shown. Whole genome sequence (WGS) analysis provided further discrimination of the current MenW ST-11 CC isolates. WGS typing as part of routine surveillance will potentially identify IMD strain types that may be associated with increased transmission, severity of disease, higher case fatality rates, or unusual clinical presentations.

      Acknowledgements

      The authors thank the Genomics Research Development Initiative (GRDI) of the Government of Canada for providing financial support to this study. Tauqeer Ahmad is a post-doctoral fellow funded by GRDI.
      We thank the technical contributions of Dennis Law, Jianwei Zhou, and Saul Deng.
      This study made use of genomic data available on the PubMLST Neisseria Multilocus Sequence Typing website (https://pubmlst.org/neisseria/), developed by Keith Jolley and sited at the University of Oxford (Jolley and Maiden, BMC Bioinformatics 2010;11:595). The development of this site has been funded by the Wellcome Trust and European Union .
      The authors also thank all of the contributors of genomic data available from the above website, which we made use of in this study. A list of the strains that were used for the comparison of genomic data can be found in the Supplementary material (Table S2).
      This publication made use of the Meningitis Research Foundation Meningococcus Genome Library (http://www.meningitis.org/research/genome) developed by Public Health England, the Wellcome Trust Sanger Institute, and the University of Oxford as a collaboration. The project is funded by the Meningitis Research Foundation .

      Funding statement

      This study was supported by a Government of Canada Genomic Research and Development Initiative (GRDI) grant.

      Role of the funding source

      The funding source had no role in the design of the study, the collection, analysis, and interpretation of the data, the writing of this manuscript, or the decision to publish.

      Conflict of interest

      Rodica Gilca (RG) received research grant from Sanofi Pasteur and Pfizer for studies not related to this research. All other authors have no competing interest to declare.

      Appendix A. Supplementary data

      The following are Supplementary data to this article:
      • Figure S1

        Neighbor-net phylogenetic network graphs comparing 30 Canadian Neisseria meningitidis serogroup W (MenW) sequence type (ST)-11 clonal complex (CC) isolates with 20 Canadian N. meningitidis serogroup C ST-11 CC isolates by (a) capsular genes that made up of the capsule locus regions A, B, C, and D; (b) extended multilocus sequence typing (eMLST); and (c) ribosomal MLST (rMLST). The scale bar denotes the number of allelic differences.

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