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Species Identification and Phylogenetic Analysis of Borrelia burgdorferi Sensu Lato Using Molecular Biological Methods

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Borrelia burgdorferi

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1690))

Abstract

Bacterial species identification is required in different disciplines and—depending on the purpose—levels of specificity or resolution of typing may vary. Nowadays, molecular methods are the mainstay for bacterial identification and sequence-based analyses are of ever-growing importance. For diagnostics, immediate results are needed and often real-time PCR of one or two loci is the method of choice while for epidemiological or evolutionary studies sequence data of several loci improve phylogenetic resolution to required levels. Multilocus sequence typing (MLST) and multilocus sequence analyses (MLSA) utilize sequences information of several housekeeping loci (eight for Borrelia) to distinguish between species. This method has been widely used for bacterial species and strain identification and will be described in this chapter.

As more and more diversity is being detected in the Borrelia burgdorferi sensu lato species complex, the importance of accurate species and strain typing has come to the fore. This is particularly significant with a view of differentiating human pathogenic and non-pathogenic strains or species and understanding the epidemiology, ecology, population structure, and evolution of species.

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References

  1. Aguero-Rosenfeld ME, Wang G, Schwartz I, Wormser GP (2005) Diagnosis of lyme borreliosis. Clin Microbiol Rev 18:484–509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Lebech AM, Hansen K, Brandrup F, Clemmensen O, Halkier-Sorensen L (2000) Diagnostic value of PCR for detection of Borrelia burgdorferi DNA in clinical specimens from patients with erythema migrans and Lyme neuroborreliosis. Mol Diagn 5:139–150

    Article  CAS  PubMed  Google Scholar 

  3. Stanek G, Fingerle V, Hunfeld KP, Jaulhac B, Kaiser R, Krause A et al (2010) Lyme borreliosis: clinical case definitions for diagnosis and management in Europe. Clin Microbiol Infect 17:69–79

    Article  Google Scholar 

  4. Fukunaga M, Koreki Y (1995) The flagellin gene of Borrelia miyamotoi sp. nov. and its phylogenetic relationship among Borrelia species. FEMS Microbiol Lett 134:255–258

    Article  CAS  PubMed  Google Scholar 

  5. Fukunaga M, Takahashi Y, Tsuruta Y, Matsushita O, Ralph D, McClelland M et al (1995) Genetic and phenotypic analysis of Borrelia miyamotoi sp. nov., isolated from the ixodid tick Ixodes persulcatus, the vector for Lyme disease in Japan. Int J Syst Bacteriol 45:804–810

    Article  CAS  PubMed  Google Scholar 

  6. Richter D, Schlee DB, Matuschka FR (2003) Relapsing fever-like spirochetes infecting European vector tick of Lyme disease agent. Emerg Infect Dis 9:697–701

    Article  PubMed  PubMed Central  Google Scholar 

  7. Scoles GA, Papero M, Beati L, Fish D (2001) A relapsing fever group spirochete transmitted by Ixodes scapularis ticks. Vector Borne Zoonotic Dis 1:21–34

    Article  CAS  PubMed  Google Scholar 

  8. Hovius JW, de Wever B, Sohne M, Brouwer MC, Coumou J, Wagemakers A et al (2013) A case of meningoencephalitis by the relapsing fever spirochaete Borrelia miyamotoi in Europe. Lancet 382:658

    Article  PubMed  PubMed Central  Google Scholar 

  9. Krause PJ, Narsimhan S, Wormser GP, Rollend L, Fikrig E, Lepore T et al (2013) Human Borrelia miyamotoi infection in the United States. N Engl J Med 368:290–291

    Article  Google Scholar 

  10. Platonov AE, Karan LS, Kolyasnikova NM, Makhneva NA, Toporkova MG, Maleev VV et al (2011) Humans infected with relapsing fever spirochete Borrelia miyamotoi, Russia. Emerg Infect Dis 17:1816–1823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sato K, Takano A, Konnai S, Nakao M, Ito T, Koyama K et al (2014) Human infections with Borrelia miyamotoi, Japan. Emerg Infect Dis 20:1391–1393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Boden K, Lobenstein S, Hermann B, Margos G, Fingerle V (2016) Neuroborreliosis in the immunocompromised caused by the relapsing fever spirochete Borrelia miyamotoi. Emerg Infect Dis 22(9):1617–1620. doi: 10.3201/eid2209.152034

  13. Venczel R, Knoke L, Pavlovic M, Dzaferovic E, Vaculova T, Silaghi C et al (2016) A novel duplex real-time PCR permits simultaneous detection and differentiation of Borrelia miyamotoi and Borrelia burgdorferi sensu lato. Infection 44(1):47–55

    Article  CAS  PubMed  Google Scholar 

  14. Enright MC, Spratt BG (1999) Multilocus sequence typing. Trends Microbiol 7:482–487

    Article  CAS  PubMed  Google Scholar 

  15. Urwin R, Maiden MC (2003) Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol 11:479–487

    Article  CAS  PubMed  Google Scholar 

  16. Bishop CJ, Aanensen DM, Jordan GE, Kilian M, Hanage WP, Spratt BG (2009) Assigning strains to bacterial species via the internet. BMC Biol 7:3

    Article  PubMed  PubMed Central  Google Scholar 

  17. Feil EJ, Enright MC, Spratt BG (2000) Estimating the relative contributions of mutation and recombination to clonal diversification: a comparison between Neisseria meningitidis and Streptococcus pneumoniae. Res Microbiol 151:465–469

    Article  CAS  PubMed  Google Scholar 

  18. Jolley KA (2009) Internet-based sequence-typing databases for bacterial molecular epidemiology. Methods Mol Biol 551:305–312

    Article  CAS  PubMed  Google Scholar 

  19. Maiden MC (2006) Multilocus sequence typing of bacteria. Annu Rev Microbiol 60:561–588

    Article  CAS  PubMed  Google Scholar 

  20. Maiden MC, Jansen van Rensburg MJ, Bray JE, Earle SG, Ford SA, Jolley KA et al (2013) MLST revisited: the gene-by-gene approach to bacterial genomics. Nat Rev Microbiol 11:728–736

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Margos G, Vollmer SA, Ogden NH, Fish D (2011) Population genetics, taxonomy, phylogeny and evolution of Borrelia burgdorferi sensu lato. Infect Genet Evol 11:1545–1563

    Article  PubMed  PubMed Central  Google Scholar 

  22. Margos G, Binder K, Dzaferovic E, Hizo-Teufel C, Sing A, Wildner M et al (2015) PubMLST.org—the new home for the Borrelia MLSA database. Ticks Tick Borne Dis 6:869–871

    Article  PubMed  Google Scholar 

  23. Margos G, Chu CY, Takano A, Jiang BG, Liu W, Kurtenbach K et al (2015) Borrelia yangtzensis sp. nov. a rodent associated species in Asia is related to B. valaisiana. Int J Syst Evol Microbiol. doi:10.1099/ijsem.0.000491

  24. Margos G, Piesman J, Lane RS, Ogden NH, Sing A, Straubinger RK et al (2014) Borrelia kurtenbachii sp. nov., a widely distributed member of the Borrelia burgdorferi sensu lato species complex in North America. Int J Syst Evol Microbiol 64:128–130

    Article  PubMed  Google Scholar 

  25. Margos G, Wilske B, Sing A, Hizo-Teufel C, Cao WC, Chu C et al (2013) Borrelia bavariensis sp. nov. is widely distributed in Europe and Asia. Int J Syst Evol Microbiol 63:4284–4288

    Article  PubMed  Google Scholar 

  26. Chu CY, Liu W, Jiang BG, Wang DM, Jiang WJ, Zhao QM et al (2008) Novel genospecies of Borrelia burgdorferi sensu lato from rodents and ticks in southwestern China. J Clin Microbiol 46:3130–3133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Postic D, Garnier M, Baranton G (2007) Multilocus sequence analysis of atypical Borrelia burgdorferi sensu lato isolates – description of Borrelia californiensis sp. nov., and genomospecies 1 and 2. Int J Med Microbiol 297:263–271

    Article  CAS  PubMed  Google Scholar 

  28. Richter D, Postic D, Sertour N, Livey I, Matuschka FR, Baranton G (2006) Delineation of Borrelia burgdorferi sensu lato species by multilocus sequence analysis and confirmation of the delineation of Borreliaspielmanii sp. nov. Int J Syst Evol Microbiol 56:873–881

    Article  CAS  PubMed  Google Scholar 

  29. James MC, Gilbert L, Bowman AS, Forbes KJ (2014) The heterogeneity, distribution, and environmental associations of Borrelia burgdorferi Sensu Lato, the agent of Lyme Borreliosis, in Scotland. Front Public Health 2:129

    Article  PubMed  PubMed Central  Google Scholar 

  30. Cerar T, Strle F, Stupica D, Ruzic-Sabljic E, McHugh G, Steere AC et al (2016) Differences in genotype, clinical features, and inflammatory potential of Borrelia burgdorferi sensu stricto strains from Europe and the United States. Emerg Infect Dis 22:818–827

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hanincova K, Mukherjee P, Ogden NH, Margos G, Wormser GP, Reed KD et al (2013) Multilocus sequence typing of Borrelia burgdorferi suggests existence of lineages with differential pathogenic properties in humans. PLoS One 8:e73066

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hoen AG, Margos G, Bent SJ, Kurtenbach K, Fish D (2009) Phylogeography of Borrelia burgdorferi in the eastern United States reflects multiple independent Lyme disease emergence events. Proc Natl Acad Sci U S A 106:15013–15018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ogden NH, Bouchard C, Kurtenbach K, Margos G, Lindsay LR, Trudel L et al (2010) Active and passive surveillance and phylogenetic analysis of Borrelia burgdorferi elucidate the process of Lyme disease risk emergence in Canada. Environ Health Perspect 118:909–914

    Article  PubMed  PubMed Central  Google Scholar 

  34. Ogden NH, Margos G, Aanensen DM, Drebot MA, Feil EJ, Hanincová K et al (2011) Investigation of genotypes of Borrelia burgdorferi in Ixodes scapularis ticks collected in surveillance in Canada. Appl Environ Microbiol 77:3244–3254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Fingerle V, Schulte-Spechtel UC, Ruzic-Sabljic E, Leonhard S, Hofmann H, Weber K et al (2008) Epidemiological aspects and molecular characterization of Borrelia burgdorferi s.l. from southern Germany with special respect to the new species Borrelia spielmanii sp. nov. Int J Med Microbiol 298:279–290

    Article  CAS  PubMed  Google Scholar 

  36. Pritt BS, Mead PS, Johnson DK, Neitzel DF, Respicio-Kingry LB, Davis JP et al (2016) Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study. Lancet Infect Dis 16(5):556–564

    Google Scholar 

  37. Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E, Davis JP (1982) Lyme disease-a tick-borne spirochetosis? Science 216:1317–1319

    Article  CAS  PubMed  Google Scholar 

  38. Johnson RC, Schmidt GP, Hyde FW, Steigerwalt AG, Brenner DJ (1984) Borrelia burgdorferi sp. nov.: etiological agent of Lyme disease. Int J Syst Bacteriol 34:496–497

    Article  Google Scholar 

  39. Fukunaga M, Okada K, Nakao M, Konishi T, Sato Y (1996) Phylogenetic analysis of Borrelia species based on flagellin gene sequences and its application for molecular typing of Lyme disease borreliae. Int J Syst Bacteriol 46:898–905

    Article  CAS  PubMed  Google Scholar 

  40. Fukunaga M, Sohnaka M (1992) Tandem repeat of the 23S and 5S ribosomal RNA genes in Borrelia burgdorferi, the etiological agent of Lyme disease. Biochem Biophys Res Commun 183:952–957

    Article  CAS  PubMed  Google Scholar 

  41. Postic D, Assous MV, Grimont PA, Baranton G (1994) Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons. Int J Syst Bacteriol 44:743–752

    Article  CAS  PubMed  Google Scholar 

  42. Postic D, Ras NM, Lane RS, Hendson M, Baranton G (1998) Expanded diversity among Californian borrelia isolates and description of Borrelia bissettii sp. nov. (formerly Borrelia group DN127). J Clin Microbiol 36:3497–3504

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Schwartz JJ, Gazumyan A, Schwartz I (1992) rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi. J Bacteriol 174:3757–3765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wang G, van Dam AP, Schwartz I, Dankert J (1999b) Molecular typing of Borrelia burgdorferi sensu lato: taxonomic, epidemiological, and clinical implications. Clin Microbiol Rev 12:633–653

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Michel H, Wilske B, Hettche G, Gottner G, Heimerl C, Reischl U et al (2004) An ospA-polymerase chain reaction/restriction fragment length polymorphism-based method for sensitive detection and reliable differentiation of all European Borrelia burgdorferi sensu lato species and OspA types. Med Microbiol Immunol 193:219–226

    Article  CAS  PubMed  Google Scholar 

  46. Nolte O (2012) Nucleic acid amplification based diagnostic of Lyme (neuro-)borreliosis – lost in the jungle of methods, targets, and assays? Open Neurol J 6:129–139

    Article  PubMed  PubMed Central  Google Scholar 

  47. Wang G, Liveris D, Mukherjee P, Jungnick S, Margos G, Schwartz I (2014) Molecular typing of Borrelia burgdorferi. Curr Protoc Microbiol 34:12C.5.1–12C.531

    Article  Google Scholar 

  48. Crowder CD, Matthews HE, Schutzer S, Rounds MA, Luft BJ, Nolte O et al (2010) Genotypic variation and mixtures of Lyme Borrelia in Ixodes ticks from North America and Europe. PLoS One 5:e10650

    Article  PubMed  PubMed Central  Google Scholar 

  49. Margos G, Gatewood AG, Aanensen DM, Hanincova K, Terekhova D, Vollmer SA et al (2008) MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi. Proc Natl Acad Sci U S A 105:8730–8735

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Qiu WG, Bruno JF, McCaig WD, Xu Y, Livey I, Schriefer ME et al (2008) Wide distribution of a high-virulence Borrelia burgdorferi clone in Europe and North America. Emerg Infect Dis 14:1097–1104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Rudenko N, Golovchenko M, Grubhoffer L, Oliver JH Jr (2011) Borrelia carolinensis sp. nov., a novel species of the Borrelia burgdorferi sensu lato complex isolated from rodents and a tick from the south-eastern USA. Int J Syst Evol Microbiol 61:381–383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452

    Article  CAS  PubMed  Google Scholar 

  55. Green MR, Sambrook J (2012) Molecular cloning, vol 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  56. Jacquot M, Gonnet M, Ferquel E, Abrial D, Claude A, Gasqui P et al (2014) Comparative population genomics of the Borrelia burgdorferi species complex reveals high degree of genetic isolation among species and underscores benefits and constraints to studying intra-specific epidemiological processes. PLoS One 9:e94384

    Article  PubMed  PubMed Central  Google Scholar 

  57. Huelsenbeck JP, Ronquist F (2005) Bayesian analysis of molecular evolution using MrBayes. In: Nielsen R (ed) Statistical methods in molecular evolution. Springer, New York, pp 183–232

    Chapter  Google Scholar 

  58. Guindon S, Lethiec F, Duroux P, Gascuel O (2005) PHYML online—a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res 33:W557–W559

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Nylander JAA (2004) MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University, Uppsala

    Google Scholar 

  60. Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG (2004) eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol 186:1518–1530

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Francisco AP, Bugalho M, Ramirez M, Carrico JA (2009) Global optimal eBURST analysis of multilocus typing data using a graphic matroid approach. BMC Bioinformatics 10:152

    Article  PubMed  PubMed Central  Google Scholar 

  62. Francisco AP, Vaz C, Monteiro PT, Melo-Cristino J, Ramirez M, Carrico JA (2012) PHYLOViZ: phylogenetic inference and data visualization for sequence based typing methods. BMC Bioinformatics 13:87

    Article  PubMed  PubMed Central  Google Scholar 

  63. Ribeiro-Goncalves B, Francisco AP, Vaz C, Ramirez M, Carrico JA (2016) PHYLOViZ online: web-based tool for visualization, phylogenetic inference, analysis and sharing of minimum spanning trees. Nucleic Acids Res 44(W1):W246–W251

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Bavarian Health and Food Safety Authority, National Reference Center for Borrelia, Veterinärstr. 2, 85764, Oberschleissheim, Germany

    Gabriele Margos, Isabell Notter & Volker Fingerle

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  1. Gabriele Margos
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  2. Isabell Notter
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  3. Volker Fingerle
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Correspondence to Gabriele Margos .

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Editors and Affiliations

  1. Department of Veterinary Medicine, University of Maryland, College Park, Maryland, USA

    Utpal Pal

  2. Department of Microbiology, Ondokuz Mayis University, Samsun, Turkey

    Ozlem Buyuktanir

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Margos, G., Notter, I., Fingerle, V. (2018). Species Identification and Phylogenetic Analysis of Borrelia burgdorferi Sensu Lato Using Molecular Biological Methods. In: Pal, U., Buyuktanir, O. (eds) Borrelia burgdorferi. Methods in Molecular Biology, vol 1690. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7383-5_2

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  • DOI: https://doi.org/10.1007/978-1-4939-7383-5_2

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