Skip to main content
SpringerLink
Log in

Bacterial Taxonomics

Finding the Wood Through the Phylogenetic Trees

  • Protocol
Genomics, Proteomics, and Clinical Bacteriology

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 266))

Abstract

Bacterial taxonomy comprises systematics (theory of classification), nomenclature (formal process of naming), and identification. There are two basic approaches to classification. Similarities may be derived between microorganisms by numerical taxonomic methods based on a range of present-day observable characteristics (phenetics), drawing in particular on conventional morphological and physiological test characters as well as chemotaxonomic markers such as whole-cell protein profiles, mol% G+C content, and DNA-DNA homologies. By contrast, phylogenetics, the process of reconstructing possible evolutionary relationships, uses nucleotide sequences from conserved genes that act as molecular chronometers. A combination of both phenetics and phylogenetics is referred to as polyphasic taxonomy, and is the recommended strategy in description of new species and genera. Numerical analysis of small-subunit ribosomal RNA genes (rDNA) leading to the construction of branching trees representing the distance of divergence from a common ancestor has provided the mainstay of microbial phylogenetics. The approach has some limitations, particularly in the discrimination of closely related taxa, and there is a growing interest in the use of alternative loci as molecular chronometers, such as gyrA and RNAase P sequences. Comparison of the degree of congruence between phylogenetic trees derived from different genes provides a valuable test of the extent they represent gene trees or species trees. Rapid expansion in genome sequences will provide a rich source of data for future taxonomic analysis that should take into account population structure of taxa and novel methods for analysis of nonclonal bacterial populations.

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 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Blaxter, M. (2003) Molecular systematics: counting angels with DNA. Nature 421, 122–124.

    Article  PubMed  CAS  Google Scholar 

  2. Murray, R. G. E. and Stackebrandt, E. (1994) Taxonomic notes: implementation of the provisional status Candidatus for incompletely described procaryotes. Int. J. Syst. Bacteriol. 45, 186–187.

    Article  Google Scholar 

  3. De Groote, D., van Doorn, L. J., Ducatelle, R., Verachuuren, A., Haesebrouch, F., Quint, W. G., et al. (1999) “Candidatus Helicobacter suis,” a gastric helicobacter from pigs, and its phylogenetic relatedness to other gastrospirilla. Int. J. Syst. Bacteriol. 49, 1769–1777.

    Article  PubMed  Google Scholar 

  4. De Groote, D., van Doorn, L. J., Ducatelle, R., Verschuuren, A., Tilmant, K., Quint, W. G., et al. (1999) Phylogenetic characterization of “Candidatus Helicobacter bovis,” a new gastric Helicobacter in cattle. Int. J. Syst. Bacteriol. 49, 1707–1715.

    Article  PubMed  Google Scholar 

  5. Lawson, A. J., Linton, D., and Stanley, J. (1998) 16S rRNA gene sequences of “Candidatus Campylobacter hominis,” a novel uncultivated species, are found in the gastrointestinal tract of healthy humans. Microbiol. 49, 2063–2071.

    Google Scholar 

  6. Lawson, A. J., On, S. L., Logan, J. M., and Stanley, J. (2001) Campylobacter hominis sp. nov., from the human gastrointestinal tract. Int. J. Syst. Evol. Microbiol. 51, 651–660.

    PubMed  CAS  Google Scholar 

  7. Cowan, S. T. (1978) A Dictionary of Microbial Taxonomy (Hill, L. R., ed.), Cambridge University Press, Cambridge.

    Google Scholar 

  8. Lapage, S. P., Sneath, P. H. A., Lessel, E. F., Skerman, V. B. D., Seeliger, H. P. R., and Clark W. A., eds. (1992) International Code of Nomenclature of Bacteria (1990 Revision). Bacteriological Code. American Society for Microbiology, Washington, DC.

    Google Scholar 

  9. Skerman, V. B. D., McGowan, V., and Sneath, P. H. A. (1980) Approved lists of bacterial names. Int. J. Syst. Bacteriol. 30, 225–420.

    Article  Google Scholar 

  10. Holmes, B. (1995) Why do bacterial names change? PHLS Microbiol. Digest 12, 195–197.

    Google Scholar 

  11. Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A. D., Kämpfer, P., Maiden, C. J., et al. (2002) Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int. J. Syst. Evol. Microbiol. 52, 1043–1047.

    Article  PubMed  CAS  Google Scholar 

  12. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., et al. (1987) International Committee on Systematic Bacteriology: report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol. 37, 463–464.

    Article  Google Scholar 

  13. Garrity, G. M. and Holt, J. G. (2001) The road map to the manual, in Bergey’s Manual of Systematic Bacteriology, 2nd ed. (Boone, D. R., Castenholz, R. W., and Garrity, G. M., eds.), Springer, New York, pp. 119–166.

    Google Scholar 

  14. Cowan, S. J., Steel, K. J., Barrow, G. I., and Feltham, R. K. A. (1993) Cowan and Steel’s Manual for the Identification of Medical Bacteria, 3rd ed. Cambridge University Press, Cambridge, UK.

    Google Scholar 

  15. Ursing, J. B., Lior, H., and Owen, R. J. (1994) Proposal of minimal standards for describing new species of the family Campylobacteraceae. Int. J. Syst. Bacteriol. 44, 842–845.

    Article  PubMed  CAS  Google Scholar 

  16. Dewhurst, F. E., Fox, J. G., and On, S. L. W. (2000) Recommended minimal standards for describing new species of the genus Helicobacter. IJSEM 50, 2231–2237.

    Google Scholar 

  17. Sokal, R. R. and Sneath, P. H. A. (1963) Principles of Numerical Taxonomy. W. H. Freeman, San Francisco, CA.

    Google Scholar 

  18. On, S. L. W. and Holmes, B. (1995) Classification and identification of campylobacters, helicobacters and allied taxa by numerical analysis of phenotypic characters. Syst. Appl. Microbiol. 18, 374–390.

    Google Scholar 

  19. Goodfellow, M. and Minnikin, D. E., eds. (1985) Chemical Methods in Bacterial Systematics. The Society for Applied Bacteriology, Technical Series No. 20. Academic Press, New York.

    Google Scholar 

  20. Kersters, K. (1985) Numerical methods in the classification of bacteria by protein electrophoresis, in Computer-Assisted Bacterial Systematics (Goodfellow, M., Jones, D., and Priest, F. G., eds.), American Press, London, pp. 337–365.

    Google Scholar 

  21. Pot, B., Vandamme, P., and Kersters, K. (1994) Analysis of electrophoretic whole organism protein fingerprints, in Chemical Methods in Prokaryotic Systematics (Goodfellow, M. and O’Donnell, A. G., eds.), John Wiley & Sons, Chichester, UK, pp. 493–521.

    Google Scholar 

  22. Costas, M., Pot, B., Vandamme, P., Kersters, K., Owen, R. J, and Hill, L. R. (1990) Interlaboratory comparative study of the numerical analysis of one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoretic protein patterns of Campylobacter strains. Electrophoresis 11, 467–474.

    Article  PubMed  CAS  Google Scholar 

  23. Stanley, J., Linton, D., Burnens, A. P., Dewhirst, E., On, S. L., Porter, A., et al. (1994) Helicobacter pullorum sp. nov.—genotype and phenotype of a new species isolated from poultry and from human patients with gastroenteritis. Microbiol. 140, 3441–3449.

    Article  CAS  Google Scholar 

  24. Goodacre, R. (1994) Characterization and quantification of microbial systems using pyrolysis mass spectrometry: introducing neural networks to analytical pyrolysis. Microbiol. Eur. 2, 16–22.

    Google Scholar 

  25. Naumann, D., Helm, D., and Schultz, C. (1994) Characterization and identification of microorganisms by FT-IR spectroscopy and FT-IR microscopy, in Bacterial Diversity and Systematics (Priest, F. G., Ramos Cormenzana, A., and Tindall, B. J., eds.), Plenum, New York, pp. 67–85.

    Google Scholar 

  26. Claydon, M., Davey, S. N., Edwards-Jones, V., and Gordon, D. B. (1996) The rapid identification of intact microorganisms using mass spectrometry. Nat. Biotechnol. 14, 1584–1586.

    Article  PubMed  CAS  Google Scholar 

  27. Conway, G. C., Smole, S. C., Sarracino, D. A., Arbeit, R. D., and Leopold, P. E. (2001) Phyloproteomics: species identification of Enterobacteriaceae using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J. Mol. Microbiol. Biotechnol. 3, 103–112.

    PubMed  CAS  Google Scholar 

  28. Marmur, J. (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Molec. Biol. 3, 208–218.

    Article  CAS  Google Scholar 

  29. Stackebrandt, E. and Goodfellow M., eds. (1991) Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons, Chichester, UK.

    Google Scholar 

  30. Owen, R. J. and Pitcher, D. G. (1985) Current methods for estimating DNA base composition and levels of DNA-DNA hybridization. Chemical Methods in Bacterial Systematics. Goodfellow, M. and Minnikin, D. E. Soc. Appl. Bacteriol.—Technical Series 20, pp.67–93.

    Google Scholar 

  31. Xu, H. X., Kawamura, Y., Li, N., Zhao, L., Li, T. M., Li, Z. Y., et al. (2000) A rapid method for determining the G+C content of bacterial chromosomes by monitoring fluorescence intensity during DNA denaturation in a capillary tube. Int. J. Syst. Evol. Microbiol. 50, 1463–1469.

    PubMed  CAS  Google Scholar 

  32. Stackebrandt, E. and Goebel, B. M. (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol. 44, 846–849.

    Article  CAS  Google Scholar 

  33. DeSmedt, J. and DeLey, J. (1977) Intra-and intergeneric similarities of Agrobacterium ribosomal ribonucleic acid cistrons. Int. J. Syst. Bacteriol. 27, 222–240.

    Article  CAS  Google Scholar 

  34. Owen, R. J. and Hernandez, J. (1993) Ribotyping and arbitrary-primer PCR fingerprinting of Campylobacters, in New Techniques in Food and Beverage Microbiology (Kroll, R. G., Gilmour, A., and Sussman, M.), Soc. Appl. Bacteriol. Technical Series 31, pp. 265–285.

    Google Scholar 

  35. Owen, R. J., Ferrus, M., and Gibson, J. (2001) Amplified fragment length polymorphism genotyping of metronidazole-resistant Helicobacter pylori infecting dyspeptics in England. Eur. J. Clin. Microbiol. Infect. Dis. 7, 244–253.

    Article  CAS  Google Scholar 

  36. Desai, M., Logan, J. M., Frost, J. A., and Stanley, J. (2001) Genome sequence-based fluorescent amplified fragment length polymorphism of Campylobacter jejuni, its relationship to serotyping, and its implications for epidemiological analysis. J. Clin. Microbiol. 39, 3823–3829.

    Article  PubMed  CAS  Google Scholar 

  37. Marshall, S. M., Melito, P. L., Woodward, D. L., Johnson, W. M., Rodgers, F. G., and Mulvey, M. R. (1999) Rapid identification of Campylobacter, Arcobacter and Helicobacter isolates by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene. J. Clin. Microbiol. 37, 4158–4160.

    PubMed  CAS  Google Scholar 

  38. Hurtado, A. and Owen, R. J. (1997) A rapid identification scheme for Helicobacter pylori and other species of Helicobacter based on 23S rRNA gene polymorphisms. Syst. Appl. Microbiol. 20, 222–231.

    Google Scholar 

  39. Clewley, J. P. (1998) A user’s guide to producing and interpreting tree diagrams in taxonomy and phylogenetics. Part 3: Using restriction fragment length polymorphism patterns of bacterial genomes to draw trees. Comm. Dis. Publ. Hlth. 1, 208–210.

    CAS  Google Scholar 

  40. Darwin, C. (1859) On the Origin of Species, John Murray, London.

    Google Scholar 

  41. Haeckel, E. (1866) Generelle Morphologie der Organismen: Allgemeine Grundzuge der organischen Formen-Wissenschaft, mechanisch begrundet durch die von Charles Darwin reformirte Descendenz-Theorie. Georg Riemer, Berlin.

    Google Scholar 

  42. Orla-Jensen, S. (1921) The main lines of the natural bacterial system. J. Bacteriol. 6, 263–273.

    PubMed  CAS  Google Scholar 

  43. Kluyver, A. J. and van Neil C. B. (1936) Prospects for a natural system of classification of bacteria. Zentrabl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. II 94, 369–403.

    Google Scholar 

  44. Zuckerkandl, E. and Pauling, L. (1965) Molecules as documents of evolutionary history. J. Theoret. Biol. 8, 357–366.

    Article  CAS  Google Scholar 

  45. Whelan, S., Liò, P., and Goldman, N. (2001) Molecular phylogenetics: state-of-the-art methods for looking into the past. Trends Genet. 17, 262–272.

    Article  PubMed  CAS  Google Scholar 

  46. Kimura, M. (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120.

    Article  PubMed  CAS  Google Scholar 

  47. Woese, C. R. (1987) Bacterial evolution. Microbiol. Rev. 51, 221–271.

    PubMed  CAS  Google Scholar 

  48. Young, J. M. (2001) Implications of alternative classifications and horizontal gene transfer for bacterial taxonomy. Int. J. System. Evol. Microbiol. 51, 945–953.

    CAS  Google Scholar 

  49. Maidak, B. L., Cole, J. R., Lilburn, T. G., Parker, C. T., Jr., Saxman, P. R., Farris, R. J., et al. (2001) The RDP-II (Ribosomal Database Project). Nucleic Acids Res. 29, 173–174.

    Article  PubMed  CAS  Google Scholar 

  50. Hall, B. G. (2001) Phylogenetic Trees Made Easy. A How-to Manual for Molecular Biologists. Sinauer Associates, Sunderland, Massachusetts.

    Google Scholar 

  51. Clewley, J. P. (1998) A user’s guide to producing and interpreting tree diagrams in taxonomy and phylogenetics. Part 2. The multiple alignment of DNA and protein sequences to determine their relationships. Comm. Dis. Publ. Hlth. 1, 132–134.

    CAS  Google Scholar 

  52. McCormack, G. P. and Clewley, J. P. (2002) The application of molecular phylogenetics to the analysis of viral genome diversity and evolution. Rev. Mod. Virol. 12, 221–238.

    Article  CAS  Google Scholar 

  53. Jukes, T. H. and Cantor, C. R. (1969) Evolution of protein molecules, in Mammalian Protein Metabolism (Munro, H. N., ed.), Academic Press, New York, pp. 21–132.

    Google Scholar 

  54. Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.

    PubMed  CAS  Google Scholar 

  55. Harrington, C. S. and On, S. L. W. (1999) Extensive 16S rRNA gene sequence diversity in Campylobacter hyointestinalis strains: taxonomic and applied implications. Int. J. System. Bacteriol. 49, 1171–1175.

    Article  CAS  Google Scholar 

  56. Clayton, R. A., Sutton, G., Hinkle, P. S., Jr., Bult, C., and Fields, C. (1995) Intraspecific variation in small-subunit rRNA sequences in GenBank: why single sequences may not adequately represent Prokaryotic taxa. Int. System. Bacteriol. 45, 595–599.

    Article  CAS  Google Scholar 

  57. Hurtado, A., Clewley, J. P., Linton, D., Owen, R. J., and Stanley, J. (1997) Sequence similarities between large subunit ribosomal RNA gene intervening sequences from different Helicobacter species. Gene 194, 69–75.

    Article  PubMed  CAS  Google Scholar 

  58. Suerbaum, S. and Achtman, M. (1999) Evolution of Helicobacter pylori: the role of recombination. Trends Microbiol. 7, 182–184.

    Article  PubMed  CAS  Google Scholar 

  59. Falush, D., Kraft, C., Taylor, N. S., Correa, P., Fox, J. G., Achtman, M., et al. (2001) Recombination and mutation during long-term gastric colonization by Helicobacter pylori: estimates of clock rates, recombination size and minimal age. PNAS 98, 15,056–15,061.

    Article  PubMed  CAS  Google Scholar 

  60. Sneath, P. H. (1993) Evidence from Aeromonas for genetic crossing-over in ribosomal sequences. Int. J. Syst. Bacteriol. 43, 626–629.

    Article  PubMed  CAS  Google Scholar 

  61. Young, J. M. (2001) Implications of alternative classifications and horizontal gene transfer for bacterial taxonomy. Int. J. Syst. Evol. Microbiol. 51, 945–953.

    PubMed  CAS  Google Scholar 

  62. Hugenholtz, P. and Huber, T. (2003) Chemeric 16S rDNA sequences of diverse origin are accumulating in the public databases. Int. J. Syst. Evol. Microbiol. 53, 289–293.

    Article  CAS  Google Scholar 

  63. Ludwig, W. and Schleifer, K. H. (1994) Bacterial phylogeny based on 16S and 23S rRNA sequence analysis. FEMS Microbiol. Rev. 15, 155–173.

    Article  PubMed  CAS  Google Scholar 

  64. Ludwig, W. and Schleifer, K. H. (1999) Phylogeny of Bacteria beyond the 16S rRNA Standard. ASM News 65, 752–757.

    Google Scholar 

  65. Gupta, R. S. (2002) Phylogeny of bacteria: are we now close to understanding it? ASM News 68, 284–291.

    Google Scholar 

  66. Yamamoto, S. and Harayama, S. (1996) Phylogenetic analysis of Acinetobacter strains based on the nucleotide sequences of gyrB genes and on the amino acid sequences of their products. Int. J. Syst. Bacteriol. 46, 506–511.

    Article  PubMed  CAS  Google Scholar 

  67. Dauga, C. (2002) Evolution of the gyrB gene and the molecular phylogeny of Enterobacteriaceae: a model molecule for molecular systematic studies. Int. J. Syst. Evol. Microbiol. 52, 531–547.

    PubMed  CAS  Google Scholar 

  68. Fukushima, M., Kakinuma, K., and Kawaguchi, R. (2002) Phylogenetic analysis of Salmonella, Shigella and Escherichia coli strains on the basis of gyrB gene sequence. J. Clin. Microbiol. 40, 2779–2885.

    Article  PubMed  CAS  Google Scholar 

  69. Satomi, M., Kimura, B., Hamada, T., Harayama, S., and Fujii, T. (2002) Phylogenetic study of the genus Oceanospirillum based on 16S rRNA and gyrB genes: emended description of the genus Ocenaospirillum, description of Pseudospirillum gen. nov., Oceanobacter gen. nov. and Terasakiella gen. nov. and transfer of Oceanospirillum jannaschii and Pseudomonas stanieri to Marinobacterium as Marinobacterium jannaschii comb. nov. and Marinobacterium stanieri comb. nov. Int. J. Syst. Evol. Microbiol. 52, 739–747.

    Article  PubMed  CAS  Google Scholar 

  70. Weeks, D. L., Eskandari, S., Scott, D. R., and Sachs, G. (2000) A H+-gated urea channel: the link between Helicobacter pylori urease and gastric colonization. Science 287, 482–485.

    Article  PubMed  CAS  Google Scholar 

  71. Owen, R. J., Xerry, J., and Chisholm, S. A. (2001) Sequence diversity within the Helicobacter pylori ureI locus and identification of homologues in other ureolytic and nonureolytic species of Helicobacter of human and animal origin. Gut 49(Suppl 11), A9.

    Google Scholar 

  72. Pace, N. R. and Brown, J. W. (1995) Evolutionary perspective on the structure and function of ribonuclease P, a ribozyme. J. Bacteriol. 177, 1919–1926.

    PubMed  CAS  Google Scholar 

  73. Haas, E. S., Banta, A. B., Harris, J. K., Pace, N. R., and Brown, J. W. (1996) Structure and evolution of ribonuclease P RNA in Gram-positive bacteria. Nucleic Acids Research 24, 4775–4782.

    Article  PubMed  CAS  Google Scholar 

  74. Schön, A., Fingerhut, C., and Hess, W. R (2002) Conserved and variable domains within divergent Rnase P RNA gene sequences of Prochlorococcus strains. J. Syst. Evol. Microbiol. 52, 1383–1389.

    Article  Google Scholar 

  75. Zeaiter, Z., Fournier, P. E., Ogata, H., and Raoult, D. (2002) Phylogenetic classification of Bartonella species by comparing groEL sequences. Int. J. Syst. Evol. Microbiol. 52, 165–171.

    PubMed  CAS  Google Scholar 

  76. Colwell, R. R. (1970) Polyphasic taxonomy of the genus Vibrio: numerical taxonomy of Vibrio cholerae, Vibrio parahaemolyticus, and related Vibrio species. J. Bacteriol. 104, 410–433.

    PubMed  CAS  Google Scholar 

  77. Vandamme, P., Pot, B, Gillis, P, De Vos, P, Kersters, K., and Swings, J. (1996) Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol. Rev. 60, 407–438.

    PubMed  CAS  Google Scholar 

  78. Rossello-Mora, R. and Amann, R. (2001) The species concept for prokaryotes. FEMS Microbiol. Rev. 25, 39–67.

    Article  PubMed  CAS  Google Scholar 

  79. Falush, D., Wirth, T., Linz, B., Pritchard J. K., Stephens, M., Kidd, M., et al. (2003) Traces of human migrations in Helicobacter pylori populations. Science 299, 1582–1585.

    Article  PubMed  CAS  Google Scholar 

  80. Lan, R. and Reeves, P. R. (2000) Intraspecies variation in bacterial genomes: the need for a species genome concept. Trends Microbiol. 8, 396–401.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Enteric Pathogens, Specialist and Reference Microbiology Division, Health Protection Agency-Colindale, London, UK

    Robert J. Owen

Authors
  1. Robert J. Owen
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Antibiotic Resistance Monitoring and Reference Laboratory, Specialist and Reference Microbiology Division, Health Protection Agency-Colindale, London, UK

    Neil Woodford  & Alan P. Johnson  & 

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc.

About this protocol

Cite this protocol

Owen, R.J. (2004). Bacterial Taxonomics. In: Woodford, N., Johnson, A.P. (eds) Genomics, Proteomics, and Clinical Bacteriology. Methods in Molecular Biology™, vol 266. Humana Press. https://doi.org/10.1385/1-59259-763-7:353

Download citation

  • DOI: https://doi.org/10.1385/1-59259-763-7:353

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-218-6

  • Online ISBN: 978-1-59259-763-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation