Skip to main content
SpringerLink
Log in

Genotyping Methods

  • Protocol
  • First Online:
Pseudomonas Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1149))

Abstract

Genotyping allows for the identification of bacterial isolates to the strain level and provides basic information about the evolutionary biology, population biology, taxonomy, ecology, and genetics of bacteria. Depending on the underlying question and available resources, Pseudomonas aeruginosa strains may be typed by anonymous fingerprinting techniques or electronically portable sequence-based typing methods such as multiple locus variable number tandem repeat (VNTR) analysis (MLVA), multilocus sequence typing, or oligonucleotide microarray.

Macrorestriction fragment pattern analysis is a genotyping method that is globally applicable to all bacteria and hence has been and still is the reference method for strain typing in bacteriology. Agarose-embedded chromosomal DNA is cleaved with a rare-cutting restriction endonuclease and the generated 20–70 fragments are then separated by pulsed-field gel electrophoresis. The chapter provides a detailed step-by-step manual for SpeI genome fingerprinting of Pseudomonas chromosomes that has been optimized for SpeI fragment pattern analysis of P. aeruginosa.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Dabrowski W et al (2003) Optimisation of AP-PCR fingerprinting discriminatory power for clinical isolates of Pseudomonas aeruginosa. FEMS Microbiol Lett 218:51–57

    Article  CAS  Google Scholar 

  2. Mahenthiralingam E et al (1996) Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis. J Clin Microbiol 34:1129–1135

    Article  CAS  Google Scholar 

  3. Waters V et al (2012) Comparison of three typing methods for Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Eur J Clin Microbiol Infect Dis 31:3341–3350

    Google Scholar 

  4. Römling U et al (1994) Bacterial genome analysis by pulsed field gel eletrophoresis techniques. In: Chrambach A, Dunn MJ, Radola BJ (eds) Advances in electrophoresis, vol 7. Weinheim, VCH Verlagsgesellschaft, pp 353–406

    Google Scholar 

  5. Römling U, Greipel J, Tümmler B (1995) Gradient of genomic diversity in the Pseudomonas aeruginosa chromosome. Mol Microbiol 17:323–332

    Article  Google Scholar 

  6. Römling U, Schmidt KD, Tümmler B (1997) Large genome rearrangements discovered by the detailed analysis of 21 Pseudomonas aeruginosa clone C isolates found in environment and disease habitats. J Mol Biol 271:386–404

    Article  Google Scholar 

  7. Klockgether J et al (2011) Pseudomonas aeruginosa genomic structure and diversity. Front Microbiol 2:150

    Article  CAS  Google Scholar 

  8. Vu-Thien H et al (2007) Multiple-locus variable-number tandem-repeat analysis for longitudinal survey of sources of Pseudomonas aeruginosa infection in cystic fibrosis patients. J Clin Microbiol 45:3175–3183

    Article  CAS  Google Scholar 

  9. Gironde S, Manceau C (2012) Housekeeping gene sequencing and multilocus variable-number tandem-repeat analysis to identify subpopulations within Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. tomato that correlate with host specificity. Appl Environ Microbiol 78:3266–3279

    Article  CAS  Google Scholar 

  10. Sobral D et al (2012) A new highly discriminatory multiplex capillary-based MLVA assay as a tool for the epidemiological survey of Pseudomonas aeruginosa in cystic fibrosis patients. Eur J Clin Microbiol Infect Dis 31:2247–2256

    Article  CAS  Google Scholar 

  11. Maiden MCJ et al (1998) Multilocus sequence tying: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 95:3140–3145

    Article  CAS  Google Scholar 

  12. Boers SA, van der Reijden WA, Jansen R (2012) High-throughput multilocus sequence typing: bringing molecular typing to the next level. PLoS One 7:e39630

    Article  CAS  Google Scholar 

  13. Sarkar SF, Guttman DS (2004) Evolution of the core genome of Pseudomonas syringae, a highly clonal, endemic plant pathogen. Appl Environ Microbiol 70:1999–2012

    Article  CAS  Google Scholar 

  14. Bull CT et al (2011) Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley. Phyto Pathol 101:847–858

    Article  CAS  Google Scholar 

  15. Curran B et al (2004) Development of a multilocus sequence typing scheme for the opportunistic pathogen Pseudomonas aeruginosa. J Clin Microbiol 42:5644–5649

    Article  CAS  Google Scholar 

  16. Johnson JK et al (2007) Multilocus sequence typing compared to pulsed-field gel electrophoresis for molecular typing of Pseudomonas aeruginosa. J Clin Microbiol 45:3707–3712

    Article  CAS  Google Scholar 

  17. Kidd TJ et al (2011) Comparison of three molecular techniques for typing Pseudomonas aeruginosa isolates in sputum samples from patients with cystic fibrosis. J Clin Microbiol 49:263–268

    Article  Google Scholar 

  18. Wiehlmann L et al (2007) Population structure of Pseudomonas aeruginosa. Proc Natl Acad Sci USA 104:8101–8106

    Article  CAS  Google Scholar 

  19. Cramer N et al (2011) Microevolution of the major common Pseudomonas aeruginosa clones C and PA14 in cystic fibrosis lungs. Environ Microbiol 13:1690–1704

    Article  CAS  Google Scholar 

  20. Klockgether J et al (2013) Intraclonal diversity of the Pseudomonas aeruginosa cystic fibrosis airway isolates TBCF10839 and TBCF121838: distinct signatures of transcriptome, proteome, metabolome, adherence and pathogenicity despite an almost identical genome sequence. Environ Microbiol 15:191–210

    Google Scholar 

  21. Liu L et al (2012) Comparison of next-generation sequencing systems. J Biomed Biotechnol 2012:251364

    PubMed  PubMed Central  Google Scholar 

  22. Stover CK et al (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964

    Article  CAS  Google Scholar 

  23. Lee DG et al (2006) Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial. Genome Biol 7:R90

    Article  Google Scholar 

  24. Grothues D, Tümmler B (1991) New approaches in genome analysis by pulsed-field gel electrophoresis: application to the analysis of Pseudomonas species. Mol Microbiol 5:2763–2776

    Article  CAS  Google Scholar 

  25. Römling U, Fislage R, Tümmler B (1996) Macrorestriction mapping and analysis of bacterial genomes. In: Birren B, Lai E (eds) Nonmammalian genome analysis—a practical guide. Academic Press, San Diego, pp 165–195, http://dx.doi.org/10.1016/B978-012101285-4/50007-2

    Chapter  Google Scholar 

  26. Römling U, Tümmler B (2000) Achieving 100 % typeability of Pseudomonas aeruginosa by pulsed-field gel electrophoresis. J Clin Microbiol 38:464–465

    Article  Google Scholar 

Download references

Acknowledgments

PFGE equipment and protocols have initially been set up by Dietmar Grothues, Wilfried Bautsch, Joachim Greipel, Uta Koopmann, and Ute Römling. The protocols were subsequently modified, evaluated, and adapted to the needs of individual applications by (in chronological order) Ute Römling, Rainer Fislage, Thomas Heuer, Jutta Ulrich, Christiane Bürger, Karen Larbig, Stefanie Breitenstein, Peter Gudowius, Christian Weinel, and Jens Klockgether. The protocol for genotyping of P. aeruginosa by oligonucleotide microarray has been developed by Lutz Wiehlmann in collaboration with a team at CLONDIAG Chip Technologies GmbH.

Author information

Authors and Affiliations

  1. Clinical Research Group, Clinic for Pediatric Pneumology, Allergology and Neonatology, Hanover Medical School, Hanover, Germany

    Burkhard Tümmler

Authors
  1. Burkhard Tümmler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Burkhard Tümmler .

Editor information

Editors and Affiliations

  1. MRC Centre for Molecular Bacteriology and Infection (CMBI) Department of Life Sciences, Imperial College, London, UK

    Alain Filloux

  2. Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain

    Juan-Luis Ramos

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Tümmler, B. (2014). Genotyping Methods. In: Filloux, A., Ramos, JL. (eds) Pseudomonas Methods and Protocols. Methods in Molecular Biology, vol 1149. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-0473-0_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-0473-0_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-0472-3

  • Online ISBN: 978-1-4939-0473-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation