Skip to content
1887

Microbial Genomics logo Microbial Genomics

Volume 11, Issue 8

Direct whole-genome sequencing enables strain typing of unculturable from oropharyngeal carriage specimens Open Access

Abstract

Oropharyngeal carriage of (.) is a prerequisite for invasive meningococcal disease. As such, genomic surveillance of disease-causing carriage strains can inform targeted public health responses. However, whole-genome sequencing (WGS) from isolates is often precluded due to the high rates of culture failure for . samples collected in carriage studies. This study outlines an alternative method to sequence . directly from oropharyngeal specimens that enables high-resolution molecular fine typing.

We performed direct probe-capture enrichment WGS (dWGS) of . on oropharyngeal specimens from the ‘B part of it’ South Australian and ‘B part of it NT’ Northern Territory meningococcal carriage studies (NCT03089086 and NCT04398849). Sequences were analysed using currently available bioinformatic tools, including the characterization of genogroup, multi-locus sequence typing (MLST), Antigen Sequence Typing (BAST), and type.

Sensitivity of dWGS typing compared to WGS for genogroup, MLST, , and BAST schemes was 88.89%, 72.22%, 100%, 94.44% and 88.24%, respectively. Genogroup and type were more reliably characterized in unculturable samples compared to the other typing schemes assessed. Factors that influenced accurate fine typing included the amount and proportion of . sequences, and the proportion of other species in enriched sequencing libraries. An alternative phylogenetic method (phylotyping) correctly predicted the clonal complex for 93.46% of the samples assessed. These results demonstrate that dWGS enables high-resolution molecular fine typing and can be applied to unculturable samples in . carriage studies.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): typing , metagenomics , Neisseria meningitidis and whole-genome sequencing
Funding
This study was supported by the:
  • GlaxoSmithKline Biologicals
    • Principal Award Recipient: HelenMarshall
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.001464
2025-08-04
2025-12-14

Metrics

Loading full text...

Full text loading...

/deliver/fulltext/mgen/11/8/mgen001464.html?itemId=/content/journal/mgen/10.1099/mgen.0.001464&mimeType=html&fmt=ahah

References

  1. Archer BN, Chiu CK, Jayasinghe SH, Richmond PC, McVernon J et al. Epidemiology of invasive meningococcal B disease in Australia, 1999-2015: priority populations for vaccination. Med J Aust 2017; 207:382–387 [View Article] [PubMed]
     [Google Scholar]
  2. Marshall HS, Lally N, Flood L, Phillips P. First statewide meningococcal B vaccine program in infants, children and adolescents: evidence for implementation in South Australia. Med J Aust 2020; 212:89–93 [View Article] [PubMed]
     [Google Scholar]
  3. Caugant DA, Maiden MCJ. Meningococcal carriage and disease--population biology and evolution. Vaccine 2009; 27 Suppl 2:B64–70 [View Article] [PubMed]
     [Google Scholar]
  4. Christensen H, May M, Bowen L, Hickman M, Trotter CL. Meningococcal carriage by age: a systematic review and meta-analysis. Lancet Infect Dis 2010; 10:853–861 [View Article] [PubMed]
     [Google Scholar]
  5. Rouphael NG, Stephens DS. Neisseria meningitidis: biology, microbiology, and epidemiology. Methods Mol Biol Clifton NJ 2012; 799:1–20
     [Google Scholar]
  6. Leong LEX, Coldbeck-Shackley RC, McMillan M, Bratcher HB, Turra M et al. The genomic epidemiology of Neisseria meningitidis carriage from a randomised controlled trial of 4CMenB vaccination in an asymptomatic adolescent population. The Lancet Regional Health - Western Pacific 2024; 43:100966 [View Article]
     [Google Scholar]
  7. Dolan-Livengood JM, Miller YK, Martin LE, Urwin R, Stephens DS. Genetic basis for nongroupable Neisseria meningitidis. J Infect Dis 2003; 187:1616–1628 [View Article] [PubMed]
     [Google Scholar]
  8. Jolley KA, Brehony C, Maiden MCJ. Molecular typing of meningococci: recommendations for target choice and nomenclature. FEMS Microbiol Rev 2007; 31:89–96 [View Article] [PubMed]
     [Google Scholar]
  9. Beddek AJ, Li MS, Kroll JS, Jordan TW, Martin DR. Evidence for capsule switching between carried and disease-causing Neisseria meningitidis strains. Infect Immun 2009; 77:2989–2994 [View Article] [PubMed]
     [Google Scholar]
  10. Stein-Zamir C, Shoob H, Abramson N, Block C, Keller N et al. Invasive meningococcal disease epidemiology and characterization of Neisseria meningitidis serogroups, sequence types, and clones; implication for use of meningococcal vaccines. Hum Vaccin Immunother 2019; 15:242–248 [View Article] [PubMed]
     [Google Scholar]
  11. Moreno J, Alarcon Z, Parra E, Duarte C, Sanabria O et al. Molecular characterization of Neisseria meningitidis isolates recovered from patients with invasive meningococcal disease in Colombia from 2013 to 2016. PLoS One 2020; 15:e0234475 [View Article] [PubMed]
     [Google Scholar]
  12. Mowlaboccus S, Mullally CA, Richmond PC, Howden BP, Stevens K et al. Differences in the population structure of Neisseria meningitidis in two Australian states: Victoria and Western Australia. PLoS One 2017; 12:e0186839 [View Article] [PubMed]
     [Google Scholar]
  13. Lahra MM, Hogan TR. Australian gonococcal surveillance programme annual report, 2021. Commun Dis Intell (2018) 2018; 46: [View Article]
     [Google Scholar]
  14. Rodrigues CMC, Jolley KA, Smith A, Cameron JC, Feavers IM et al. Meningococcal deduced vaccine antigen reactivity (MenDeVAR) index: a rapid and accessible tool that exploits genomic data in public health and clinical microbiology applications. J Clin Microbiol 2020; 59:e02161-20 [View Article] [PubMed]
     [Google Scholar]
  15. Tzeng YL, Martin LE, Stephens DS. Environmental survival of Neisseria meningitidis. Epidemiol Infect 2014; 142:187–190 [View Article] [PubMed]
     [Google Scholar]
  16. Caugant DA, Tzanakaki G, Kriz P. Lessons from meningococcal carriage studies. FEMS Microbiol Rev 2007; 31:52–63 [View Article] [PubMed]
     [Google Scholar]
  17. Marshall HS, Andraweera PH, Ward J, Kaldor J, Andrews R et al. An Observational study to assess the effectiveness of 4CMenB against meningococcal disease and carriage and gonorrhea in adolescents in the Northern Territory, Australia-Study Protocol. Vaccines (Basel) 2022; 10:309 [View Article] [PubMed]
     [Google Scholar]
  18. Jordens JZ, Williams JN, Jones GR, Heckels JE. Detection of meningococcal carriage by culture and PCR of throat swabs and mouth gargles. J Clin Microbiol 2002; 40:75–79 [View Article] [PubMed]
     [Google Scholar]
  19. McMillan M, Walters L, Mark T, Lawrence A, Leong LEX et al. B Part of It study: a longitudinal study to assess carriage of Neisseria meningitidis in first year university students in South Australia. Hum Vaccin Immunother 2019; 15:987–994 [View Article] [PubMed]
     [Google Scholar]
  20. Anyansi C, Straub TJ, Manson AL, Earl AM, Abeel T. Computational methods for strain-level microbial detection in colony and metagenome sequencing data. Front Microbiol 2020; 11:1925 [View Article] [PubMed]
     [Google Scholar]
  21. Itsko M, Retchless AC, Joseph SJ, Norris Turner A, Bazan JA et al. Full molecular typing of Neisseria meningitidis directly from clinical specimens for outbreak investigation. J Clin Microbiol 2020; 58:e01780-20 [View Article] [PubMed]
     [Google Scholar]
  22. Clark SA, Doyle R, Lucidarme J, Borrow R, Breuer J. Targeted DNA enrichment and whole genome sequencing of Neisseria meningitidis directly from clinical specimens. Int J Med Microbiol 2018; 308:256–262 [View Article] [PubMed]
     [Google Scholar]
  23. Retchless AC, Kretz CB, Rodriguez-Rivera LD, Chen A, Soeters HM et al. Oropharyngeal microbiome of a college population following a meningococcal disease outbreak. Sci Rep 2020; 10:632 [View Article] [PubMed]
     [Google Scholar]
  24. Marshall HS, McMillan M, Koehler A, Lawrence A, MacLennan JM et al. B Part of It protocol: a cluster randomised controlled trial to assess the impact of 4CMenB vaccine on pharyngeal carriage of Neisseria meningitidis in adolescents. BMJ Open 2018; 8:e020988 [View Article] [PubMed]
     [Google Scholar]
  25. Leong LEX, Coldbeck-Shackley RC, McMillan M, Bratcher HB, Turra M et al. The genomic epidemiology of Neisseria meningitidis carriage from a randomised controlled trial of 4CMenB vaccination in an asymptomatic adolescent population. Lancet Reg Health West Pac 2024; 43:100966 [View Article] [PubMed]
     [Google Scholar]
  26. Diallo K, Coulibaly MD, Rebbetts LS, Harrison OB, Lucidarme J et al. Development of a PCR algorithm to detect and characterize Neisseria meningitidis carriage isolates in the African meningitis belt. PLoS One 2018; 13:e0206453 [View Article] [PubMed]
     [Google Scholar]
  27. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018; 34:i884–i890 [View Article]
     [Google Scholar]
  28. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012; 9:357–359 [View Article] [PubMed]
     [Google Scholar]
  29. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J et al. The sequence alignment/map format and SAMtools. Bioinformatics 2009; 25:2078–2079 [View Article]
     [Google Scholar]
  30. Wood DE, Lu J, Langmead B. Improved metagenomic analysis with Kraken 2. Genome Biol 2019; 20:257 [View Article] [PubMed]
     [Google Scholar]
  31. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for illumina sequence data. Bioinforma Oxf Engl 2014; 30:2114–2120
     [Google Scholar]
  32. tseeman shovill [Internet]. n.d https://github.com/tseemann/shovill
  33. tseeman nullarbor [Internet]. n.d https://github.com/tseemann/nullarbor/tree/master
  34. Ren J, Chaisson MJP. lra: A long read aligner for sequences and contigs. PLoS Comput Biol 2021; 17:e1009078 [View Article] [PubMed]
     [Google Scholar]
  35. tseeman mlst [Internet]. n.d https://github.com/tseemann/mlst
  36. andersgs meningotype [Internet]. n.d https://github.com/MDU-PHL/meningotype
  37. ntopaz characterise_neisseria_captule [Internet]. n.d https://github.com/ntopaz/characterize_neisseria_capsule
  38. tseeman snippy [Internet]. n.d https://github.com/tseemann/snippy
  39. Price MN, Dehal PS, Arkin AP. FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One 2010; 5:e9490 [View Article] [PubMed]
     [Google Scholar]
  40. Yu G, Smith DK, Zhu H, Guan Y, Lam TTY. Ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods Ecol Evol 2017; 8:28–36
     [Google Scholar]
  41. Bennett JS, Jolley KA, Earle SG, Corton C, Bentley SD et al. A genomic approach to bacterial taxonomy: an examination and proposed reclassification of species within the genus Neisseria. Microbiology 2012; 158:1570–1580 [View Article]
     [Google Scholar]
  42. Muzzi A, Mora M, Pizza M, Rappuoli R, Donati C. Conservation of meningococcal antigens in the genus Neisseria. mBio 2013; 4:e00163–00113 [View Article]
     [Google Scholar]
  43. Clemence MEA, Maiden MCJ, Harrison OB. Characterization of capsule genes in non-pathogenic Neisseria species. Microbial Genomics 2018; 4: [View Article]
     [Google Scholar]
  44. Yadav S, Pahil S, Kumar MB, Mohindra R, Mohan B et al. Neisseria mucosa: a not so benign culprit of urinary tract infection: a case report. Indian J Med Microbiol 2024; 47:100514 [View Article] [PubMed]
     [Google Scholar]
  45. Lahra MM, Latham NH, Templeton DJ, Read P, Carmody C et al. Investigation and response to an outbreak of Neisseria meningitidis serogroup Y ST-1466 urogenital infections, Australia. Commun Dis Intell 2024; 48: [View Article]
     [Google Scholar]
  46. Unemo M, Norlén O, Fredlund H. The porA pseudogene of Neisseria gonorrhoeae – low level of genetic polymorphism and a few, mainly identical, inactivating mutations. APMIS 2005; 113:410–419 [View Article]
     [Google Scholar]
  47. McMillan M, Walters L, Sullivan T, Leong LEX, Turra M et al. Impact of meningococcal B (4CMenB) vaccine on pharyngeal Neisseria meningitidis carriage density and persistence in adolescents. Clin Infect Dis 2021; 73:e99–e106 [View Article]
     [Google Scholar]
/content/journal/mgen/10.1099/mgen.0.001464
Loading
Direct whole-genome sequencing enables strain typing of unculturable Neisseria meningitidis from oropharyngeal carriage specimens
M Gen 11, 001464 (2025); https://doi.org/10.1099/mgen.0.001464
/content/journal/mgen/10.1099/mgen.0.001464
/content/journal/mgen/10.1099/mgen.0.001464
Loading

Data & Media loading...

Supplements

Supplementary material 1

EXCEL

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An error occurred
Approval was partially successful, following selected items could not be processed due to error