Genotyping of Yersinia pestis

  • Yanjun Li
  • Yujun Cui
Part of the Springer Protocols Handbooks book series (SPH)


Variation occurs on an individual bacterial genome and then becomes fixed in the population by evolutionary force, including both natural selection and neutral drift, subsequently shaping polymorphism of the species. Genome variations include single-nucleotide mutation, small fragment insertions and deletions (indels), structure variation [also known as SV, including large genome rearrangement (gain and loss)], and copy number variation. By determining these variations, we can identify bacteria strains of different populations and designate their genotypes or even distinguish isolates of different clinical samples during the same epidemic, which provides clues for source tracing in epidemic investigations and supports development of infectious disease control strategies. By comparison with Yersinia pestis reference genome sequences, different types of variations have been detected, and corresponding genotyping methods were developed. These methods included single-nucleotide polymorphism typing that targets single-nucleotide mutations, different region typing that targets the presence/absence of large genome fragments, multiple-locus variable number tandem repeat analysis that targets multiple tandem repeat loci, and clustered regularly interspaced short palindromic repeat (CRISPR) methods that target the composition of repeat-spacer arrays in three CRISPR loci. Here, we summarize these protocols including their experimental operation, data recording, and data analysis.

Key words

Yersinia pestis Genotyping SNPs VNTRs CRISPRs 


  1. 1.
    Morelli G, Song Y, Mazzoni CJ, Eppinger M, Roumagnac P, Wagner DM, Feldkamp M, Kusecek B, Vogler AJ, Li Y et al (2010) Yersinia pestis genome sequencing identifies patterns of global phylogenetic diversity. Nat Genet 42(12):1140–1143CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Vogler AJ, Chan F, Nottingham R, Andersen G, Drees K, Beckstrom-Sternberg SM, Wagner DM, Chanteau S, Keim P (2013) A decade of plague in Mahajanga, Madagascar: insights into the global maritime spread of pandemic plague. MBio 4(1):e00623–e00612CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Yan Y, Wang H, Li D, Yang X, Wang Z, Qi Z, Zhang Q, Cui B, Guo Z, Yu C et al (2014) Two-step source tracing strategy of Yersinia pestis and its historical epidemiology in a specific region. PLoS One 9(1):e85374CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Rasmussen S, Allentoft ME, Nielsen K, Orlando L, Sikora M, Sjogren KG, Pedersen AG, Schubert M, Van Dam A, Kapel CM et al (2015) Early divergent strains of Yersinia pestis in Eurasia 5,000 years ago. Cell 163(3):571–582CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Achtman M, Morelli G, Zhu P, Wirth T, Diehl I, Kusecek B, Vogler AJ, Wagner DM, Allender CJ, Easterday WR et al (2004) Microevolution and history of the plague bacillus, Yersinia pestis. Proc Natl Acad Sci U S A 101(51):17837–17842CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Cui Y, Yu C, Yan Y, Li D, Li Y, Jombart T, Weinert LA, Wang Z, Guo Z, Xu L et al (2013) Historical variations in mutation rate in an epidemic pathogen, Yersinia pestis. Proc Natl Acad Sci U S A 110(2):577–582CrossRefPubMedGoogle Scholar
  7. 7.
    Radnedge L, Agron PG, Worsham PL, Andersen GL (2002) Genome plasticity in Yersinia pestis. Microbiology 148(Pt 6):1687–1698CrossRefPubMedGoogle Scholar
  8. 8.
    Zhou D, Han Y, Song Y, Tong Z, Wang J, Guo Z, Pei D, Pang X, Zhai J, Li M et al (2004) DNA microarray analysis of genome dynamics in Yersinia pestis: insights into bacterial genome microevolution and niche adaptation. J Bacteriol 186(15):5138–5146CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Dai E, Tong Z, Wang X, Li M, Cui B, Dai R, Zhou D, Pei D, Song Y, Zhang J et al (2005) Identification of different regions among strains of Yersinia pestis by suppression subtractive hybridization. Res Microbiol 156(7):785–789CrossRefPubMedGoogle Scholar
  10. 10.
    Li Y, Dai E, Cui Y, Li M, Zhang Y, Wu M, Zhou D, Guo Z, Dai X, Cui B et al (2008) Different region analysis for genotyping Yersinia pestis isolates from China. PLoS One 3(5):e2166CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Vogler AJ, Birdsell D, Wagner DM, Keim P (2009) An optimized, multiplexed multi-locus variable-number tandem repeat analysis system for genotyping Francisella tularensis. Lett Appl Microbiol 48(1):140–144CrossRefPubMedGoogle Scholar
  12. 12.
    Le Fleche P, Jacques I, Grayon M, Al Dahouk S, Bouchon P, Denoeud F, Nockler K, Neubauer H, Guilloteau LA, Vergnaud G (2006) Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay. BMC Microbiol 6:9CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ciammaruconi A, Grassi S, De Santis R, Faggioni G, Pittiglio V, D’Amelio R, Carattoli A, Cassone A, Vergnaud G, Lista F (2008) Fieldable genotyping of Bacillus anthracis and Yersinia pestis based on 25-loci multi locus VNTR analysis. BMC Microbiol 8:21CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Song Y, Tong Z, Wang J, Wang L, Guo Z, Han Y, Zhang J, Pei D, Zhou D, Qin H et al (2004) Complete genome sequence of Yersinia pestis strain 91001, an isolate avirulent to humans. DNA Res 11(3):179–197CrossRefPubMedGoogle Scholar
  15. 15.
    Deng W, Burland V, Plunkett G 3rd, Boutin A, Mayhew GF, Liss P, Perna NT, Rose DJ, Mau B, Zhou S et al (2002) Genome sequence of Yersinia pestis KIM. J Bacteriol 184(16):4601–4611CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Parkhill J, Wren BW, Thomson NR, Titball RW, Holden MT, Prentice MB, Sebaihia M, James KD, Churcher C, Mungall KL et al (2001) Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413(6855):523–527CrossRefPubMedGoogle Scholar
  17. 17.
    Chain PS, Hu P, Malfatti SA, Radnedge L, Larimer F, Vergez LM, Worsham P, Chu MC, Andersen GL (2006) Complete genome sequence of Yersinia pestis strains Antiqua and Nepal516: evidence of gene reduction in an emerging pathogen. J Bacteriol 188(12):4453–4463CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Le Fleche P, Hauck Y, Onteniente L, Prieur A, Denoeud F, Ramisse V, Sylvestre P, Benson G, Ramisse F, Vergnaud G (2001) A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis. BMC Microbiol 1:2CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Pourcel C, Andre-Mazeaud F, Neubauer H, Ramisse F, Vergnaud G (2004) Tandem repeats analysis for the high resolution phylogenetic analysis of Yersinia pestis. BMC Microbiol 4:22CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Klevytska AM, Price LB, Schupp JM, Worsham PL, Wong J, Keim P (2001) Identification and characterization of variable-number tandem repeats in the Yersinia pestis genome. J Clin Microbiol 39(9):3179–3185CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Girard JM, Wagner DM, Vogler AJ, Keys C, Allender CJ, Drickamer LC, Keim P (2004) Differential plague-transmission dynamics determine Yersinia pestis population genetic structure on local, regional, and global scales. Proc Natl Acad Sci U S A 101(22):8408–8413CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Li Y, Cui Y, Cui B, Yan Y, Yang X, Wang H, Qi Z, Zhang Q, Xiao X, Guo Z et al (2013) Features of variable number of tandem repeats in Yersinia pestis and the development of a hierarchical genotyping scheme. PLoS One 8(6):e66567CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Li Y, Cui Y, Hauck Y, Platonov ME, Dai E, Song Y, Guo Z, Pourcel C, Dentovskaya SV, Anisimov AP et al (2009) Genotyping and phylogenetic analysis of Yersinia pestis by MLVA: insights into the worldwide expansion of Central Asia plague foci. PLoS One 4(6):e6000CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Sorek R, Kunin V, Hugenholtz P (2008) CRISPR – a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol 6(3):181–186CrossRefPubMedGoogle Scholar
  25. 25.
    Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819):1709–1712CrossRefPubMedGoogle Scholar
  26. 26.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Pourcel C, Salvignol G, Vergnaud G (2005) CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151(Pt 3):653–663PubMedGoogle Scholar
  28. 28.
    Vergnaud G, Li Y, Gorge O, Cui Y, Song Y, Zhou D, Grissa I, Dentovskaya SV, Platonov ME, Rakin A et al (2007) Analysis of the three Yersinia pestis CRISPR loci provides new tools for phylogenetic studies and possibly for the investigation of ancient DNA. Adv Exp Med Biol 603:327–338CrossRefPubMedGoogle Scholar
  29. 29.
    Cui Y, Li Y, Gorge O, Platonov ME, Yan Y, Guo Z, Pourcel C, Dentovskaya SV, Balakhonov SV, Wang X et al (2008) Insight into microevolution of Yersinia pestis by clustered regularly interspaced short palindromic repeats. PLoS One 3(7):e2652CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Riehm JM, Vergnaud G, Kiefer D, Damdindorj T, Dashdavaa O, Khurelsukh T, Zoller L, Wolfel R, Le Fleche P, Scholz HC (2012) Yersinia pestis lineages in Mongolia. PLoS One 7(2):e30624CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Grissa I, Bouchon P, Pourcel C, Vergnaud G (2007) On-line resources for bacterial micro-evolution studies using MLVA or CRISPR typing. Biochimie.

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Yanjun Li
    • 1
  • Yujun Cui
    • 2
  1. 1.PLA Navy General HospitalBeijingChina
  2. 2.Beijing Institute of Microbiology and EpidemiologyBeijingChina

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