1887

Abstract

Multilocus sequence typing (MLST) is one of the most commonly used methods for studying microbial lineage worldwide. However, the traditional MLST process using Sanger sequencing is time-consuming and expensive. We have designed a workflow that simultaneously sequenced seven full-length housekeeping genes of 96 meticillin-resistant isolates with dual-barcode multiplexing using just a single flow cell of an Oxford Nanopore Technologies MinION system, and then we performed bioinformatic analysis for strain typing. Fifty-one of the isolates comprising 34 sequence types had been characterized using Sanger sequencing. We demonstrate that the allele assignments obtained by our nanopore workflow (nanoMLST, available at https://github.com/jade-nhri/nanoMLST) were identical to those obtained by Sanger sequencing (359/359, with 100 % agreement rate). In addition, we estimate that our multiplex system is able to perform MLST for up to 1000 samples simultaneously; thus, providing a rapid and cost-effective solution for molecular typing.

Funding
This study was supported by the:
  • Yu-Chieh Liao , Ministry of Science and Technology, Taiwan , (Award MOST 106-2923-B-400-001-MY3)
  • Yu-Chieh Liao , National Health Research Institutes , (Award PH-107-PP-05)
  • Feng-Jui Chen , National Health Research Institutes , (Award IV-107-PP-07)
Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.000336
2020-02-17
2020-06-02
Loading full text...

Full text loading...

/deliver/fulltext/mgen/6/3/mgen000336.html?itemId=/content/journal/mgen/10.1099/mgen.0.000336&mimeType=html&fmt=ahah

References

  1. Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998; 339:520–532 [CrossRef]
    [Google Scholar]
  2. Tong SYC, Davis JS, Eichenberger E, Holland TL, Fowler VG. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 2015; 28:603–661 [CrossRef]
    [Google Scholar]
  3. Tenover FC, Arbeit R, Archer G, Biddle J, Byrne S et al. Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus . J Clin Microbiol 1994; 32:407–415 [CrossRef]
    [Google Scholar]
  4. Shopsin B, Gomez M, Montgomery SO, Smith DH, Waddington M et al. Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J Clin Microbiol 1999; 37:3556–3563 [CrossRef]
    [Google Scholar]
  5. Enright MC, Day NPJ, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus . J Clin Microbiol 2000; 38:1008–1015 [CrossRef]
    [Google Scholar]
  6. Murchan S, Kaufmann ME, Deplano A, de Ryck R, Struelens M et al. Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains. J Clin Microbiol 2003; 41:1574–1585 [CrossRef]
    [Google Scholar]
  7. Lee GH, Pang S, Coombs GW. Misidentification of Staphylococcus aureus by the Cepheid Xpert MRSA/SA BC assay due to deletions in the spa gene. J Clin Microbiol 2018; 56:e00530-18 [CrossRef]
    [Google Scholar]
  8. Baum C, Haslinger-Loffler B, Westh H, Boye K, Peters G et al. Non-spa-typeable clinical Staphylococcus aureus strains are naturally occurring protein A mutants. J Clin Microbiol 2009; 47:3624–3629 [CrossRef]
    [Google Scholar]
  9. Hudson LO, Murphy CR, Spratt BG, Enright MC, Terpstra L et al. Differences in methicillin-resistant Staphylococcus aureus strains isolated from pediatric and adult patients from hospitals in a large county in California. J Clin Microbiol 2012; 50:573–579 [CrossRef]
    [Google Scholar]
  10. Maiden MCJ, Bygraves JA, Feil E, Morelli G, Russell JE et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 1998; 95:3140–3145 [CrossRef]
    [Google Scholar]
  11. Muñoz M, Camargo M, Ramírez JD. Estimating the intra-taxa diversity, population genetic structure, and evolutionary pathways of Cryptococcus neoformans and Cryptococcus gattii . Front Genet 2018; 9:148 [CrossRef]
    [Google Scholar]
  12. 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 [CrossRef]
    [Google Scholar]
  13. Boers SA, van der Reijden WA, Jansen R. High-throughput multilocus sequence typing: bringing molecular typing to the next level. PLoS One 2012; 7:e39630 [CrossRef]
    [Google Scholar]
  14. Singh P, Foley SL, Nayak R, Kwon YM. Multilocus sequence typing of Salmonella strains by high-throughput sequencing of selectively amplified target genes. J Microbiol Methods 2012; 88:127–133 [CrossRef]
    [Google Scholar]
  15. Takahashi H, Iwakawa A, Ohshima C, Kyoui D, Kumano S et al. A rapid typing method for Listeria monocytogenes based on high-throughput multilocus sequence typing (Hi-MLST). Int J Food Microbiol 2017; 243:84–89 [CrossRef]
    [Google Scholar]
  16. Zhang N, Wheeler D, Truglio M, Lazzarini C, Upritchard J et al. Multi-locus next-generation sequence typing of DNA extracted from pooled colonies detects multiple unrelated Candida albicans strains in a significant proportion of patient samples. Front Microbiol 2018; 9:1179 [CrossRef]
    [Google Scholar]
  17. Chen Y, Frazzitta AE, Litvintseva AP, Fang C, Mitchell TG et al. Next generation multilocus sequence typing (NGMLST) and the analytical software program MLSTEZ enable efficient, cost-effective, high-throughput, multilocus sequencing typing. Fungal Genet Biol 2015; 75:64–71 [CrossRef]
    [Google Scholar]
  18. Heather JM, Chain B. The sequence of sequencers: the history of sequencing DNA. Genomics 2016; 107:1–8 [CrossRef]
    [Google Scholar]
  19. Pérez-Losada M, Arenas M, Castro-Nallar E. Microbial sequence typing in the genomic era. Infect Genet Evol 2018; 63:346–359 [CrossRef]
    [Google Scholar]
  20. Cao MD, Ganesamoorthy D, Elliott AG, Zhang H, Cooper MA et al. Streaming algorithms for identification of pathogens and antibiotic resistance potential from real-time MinION(TM) sequencing. Gigascience 2016; 5:32 [CrossRef]
    [Google Scholar]
  21. Tarumoto N, Sakai J, Sujino K, Yamaguchi T, Ohta M et al. Use of the Oxford Nanopore MinION sequencer for MLST genotyping of vancomycin-resistant enterococci. J Hosp Infect 2017; 96:296–298 [CrossRef]
    [Google Scholar]
  22. Ardui S, Ameur A, Vermeesch JR, Hestand MS. Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics. Nucleic Acids Res 2018; 46:2159–2168 [CrossRef]
    [Google Scholar]
  23. Ho M, McDonald LC, Lauderdale TL, Yeh LL, Chen PC et al. Surveillance of antibiotic resistance in Taiwan, 1998. J Microbiol Immunol Infect 1999; 32:239–249
    [Google Scholar]
  24. Chen FJ, Huang IW, Wang CH, Chen PC, Wang HY et al. mecA-positive Staphylococcus aureus with low-level oxacillin MIC in Taiwan. J Clin Microbiol 2012; 50:1679–1683 [CrossRef]
    [Google Scholar]
  25. Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 1988; 16:10881–10890 [CrossRef]
    [Google Scholar]
  26. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 2018; 34:3094–3100 [CrossRef]
    [Google Scholar]
  27. Vaser R, Sović I, Nagarajan N, Šikić M. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res 2017; 27:737–746 [CrossRef]
    [Google Scholar]
  28. Loman NJ, Quick J, Simpson JT. A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat Methods 2015; 12:733–735 [CrossRef]
    [Google Scholar]
  29. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403–410 [CrossRef]
    [Google Scholar]
  30. Liao Y-C, Cheng H-W, Wu H-C, Kuo S-C, Lauderdale T-LY et al. Completing circular bacterial genomes with assembly complexity by using a sampling strategy from a single MinION run with barcoding. Front Microbiol 2019; 10:2068 [CrossRef]
    [Google Scholar]
  31. Urwin R, Maiden MCJ. Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol 2003; 11:479–487 [CrossRef]
    [Google Scholar]
  32. Srivathsan A, Baloğlu B, Wang W, Tan WX, Bertrand D et al. A MinION™-based pipeline for fast and cost-effective DNA barcoding. Mol Ecol Resour 2018; 18:1035–1049 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/mgen/10.1099/mgen.0.000336
Loading
/content/journal/mgen/10.1099/mgen.0.000336
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

EXCEL

Most cited this month 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