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Indian Journal of Microbiology logoLink to Indian Journal of Microbiology
. 2007 Jul 8;47(2):98–108. doi: 10.1007/s12088-007-0022-x

Polyphasic approach of bacterial classification — An overview of recent advances

O Prakash 1, M Verma 1, P Sharma 1, M Kumar 1, K Kumari 1, A Singh 1, H Kumari 1, S Jit 1, S K Gupta 1, M Khanna 1, R Lal 2,
PMCID: PMC3450112  PMID: 23100651

Abstract

Classification of microorganisms on the basis of traditional microbiological methods (morphological, physiological and biochemical) creates a blurred image about their taxonomic status and thus needs further clarification. It should be based on a more pragmatic approach of deploying a number of methods for the complete characterization of microbes. Hence, the methods now employed for bacterial systematics include, the complete 16S rRNA gene sequencing and its comparative analysis by phylogenetic trees, DNA-DNA hybridization studies with related organisms, analyses of molecular markers and signature pattern(s), biochemical assays, physiological and morphological tests. Collectively these genotypic, chemotaxonomic and phenotypic methods for determining taxonomic position of microbes constitute what is known as the ‘polyphasic approach’ for bacterial systematics. This approach is currently the most popular choice for classifying bacteria and several microbes, which were previously placed under invalid taxa have now been resolved into new genera and species. This has been possible owing to rapid development in molecular biological techniques, automation of DNA sequencing coupled with advances in bioinformatic tools and access to sequence databases. Several DNA-based typing methods are known; these provide information for delineating bacteria into different genera and species and have the potential to resolve differences among the strains of a species. Therefore, newly isolated strains must be classified on the basis of the polyphasic approach. Also previously classified organisms, as and when required, can be reclassified on this ground in order to obtain information about their accurate position in the microbial world. Thus, current techniques enable microbiologists to decipher the natural phylogenetic relationships between microbes.

Key words: Polyphasic, Bacterial classification

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References

  • 1.Gevers D., Cohan F.M., Lawrence J.G., Spratt B.G., Coenye T., Feil E.J., Stackebrandt E., Peer Y., Vandamme P., Thompson F.L., Swings J. Opinion: Re-evaluating prokaryotic species. Nat Rev Microbiol. 2005;3:733–739. doi: 10.1038/nrmicro1236. [DOI] [PubMed] [Google Scholar]
  • 2.Coenye T., Gevers D., Peer Y., Vandamme P., Swings J. Towards a prokaryotic genomic taxonomy. FEMS Microbiol Rev. 2005;29:147–167. doi: 10.1016/j.femsre.2004.11.004. [DOI] [PubMed] [Google Scholar]
  • 3.Mora R.R., Amann R. The species concept for prokaryotes. FEMS Microbiol Rev. 2001;25:39–67. doi: 10.1111/j.1574-6976.2001.tb00571.x. [DOI] [PubMed] [Google Scholar]
  • 4.Vandamme P., Pot B., Gillis M., Vos P., Kersters K., Swings J. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev. 1996;60:407–438. doi: 10.1128/mr.60.2.407-438.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Amann R.I., Ludwig W., Schleifer K.H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 1995;59:143–169. doi: 10.1128/mr.59.1.143-169.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Mayr E and Ashlock PD (1991) Principles of Systematic Zoology 2nd ed. McGraw-Hill, Inc. pp 1–12
  • 7.Simpson G.G. Principles of Animal Taxonomy. New York: Columbia University Press; 1961. [DOI] [PubMed] [Google Scholar]
  • 8.Clarridge J.E. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev. 2004;17:840–862. doi: 10.1128/CMR.17.4.840-862.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Woese C.R. Bacterial evolution. Microbiol Rev. 1987;51:221–272. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Schildkraut C.L., Marmur J., Doty P. The formation of hybrid DNA molecules and their use in studies of DNA homologies. J Mol Biol. 1961;3:595–617. doi: 10.1016/S0022-2836(61)80024-7. [DOI] [PubMed] [Google Scholar]
  • 11.Colwell R.R. Polyphasic taxonomy of the genus Vibrio: numerical taxonomy of Vibrio cholerae, Vibrio parahaemolyticus and related Vibrio species. J Bacteriol. 1970;104:410–433. doi: 10.1128/jb.104.1.410-433.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Mayr E. Systematics and the origin of species. New York: Columbia University Press; 1942. [Google Scholar]
  • 13.Grimont F., Grimont P.A.D. DNA fingerprinting. In: Stackebrandt E., Goodfellow M., editors. Nucleic Acid Techniques in Bacterial Systematics. England: John Wiley and Sons Ltd West Sussex; 1991. [Google Scholar]
  • 14.Stackebrandt E., Goebel B.M. Taxonomic note: A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol. 1994;44:846–849. [Google Scholar]
  • 15.Tenover F.C., Arbeit R.D., Goering R.V., Mickelsen P.A., Murray B.E., Persing D.H., Swaminathan B. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. J Clin Microbiol. 1995;33:2233–2239. doi: 10.1128/jcm.33.9.2233-2239.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Williams J.K.G., Kubelik A.R., Livak K.J., Rafalsky J.A., Tynger S.V. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 1990;18:6531–6535. doi: 10.1093/nar/18.22.6531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Schwartz D.C., Cantor R.C. Separation of yeast chromosomeized DNAs by pulse field gradient gel electrophoresis. Cell. 1984;37:67–75. doi: 10.1016/0092-8674(84)90301-5. [DOI] [PubMed] [Google Scholar]
  • 18.Regnault B., Grimont F., Grimont P.A. Universal ribotyping method using a chemically labelled oligonucleotide probe mixture. Res Microbiol. 1997;148:649–659. doi: 10.1016/S0923-2508(99)80064-3. [DOI] [PubMed] [Google Scholar]
  • 19.Maslow J.N., Mulligan M.E., Arbeit R.D. Molecular epidemiology: application of contemporary techniques to the typing of microorganism. Clin Infect Disease. 1993;17:153–164. doi: 10.1093/clinids/17.2.153. [DOI] [PubMed] [Google Scholar]
  • 20.Olive D.M., Bean P. Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol. 1999;37:1661–1969. doi: 10.1128/jcm.37.6.1661-1669.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Czekajło K.U., Giedrys-Kalemba S., Mędrala D. Phenotypic and genotypic characteristic of Pseudomonas aeruginosa strains isolated from hospitals in the north-west region of Poland. Polish J Microbiol. 2006;55:103–112. [PubMed] [Google Scholar]
  • 22.Versalovic J., Schneider M., Bruijn F.J., Lupski J.R. Genomic fingerprinting of bacteria using repetitive sequence based PCR (rep-PCR) Meth Cell Mol Biol. 1994;5:25–40. [Google Scholar]
  • 23.Louws F.J., Fulbright D.W., Stephens C.T., Bruijn F.J. Differentiation of genomic structure by rep-PCR fingerprinting to rapidly classify Xanthomonas campestris pv. vesicatoria. Phytopath. 1995;85:528–836. doi: 10.1094/Phyto-85-528. [DOI] [Google Scholar]
  • 24.Lupski J.R., Weinstock G.M. Short, interspersed repetitive DNA sequences in prokaryotic genomes. J Bacteriol. 1992;174:4525–4529. doi: 10.1128/jb.174.14.4525-4529.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Stern M.J., Ames G.F.L., Smith N.H., Robinson E.C., Higgins C.F. Repetitive extragenic palindromic sequences: a major component of the bacterial genome. Cell. 1984;37:1015–1026. doi: 10.1016/0092-8674(84)90436-7. [DOI] [PubMed] [Google Scholar]
  • 26.Hulton C.S.J., Higgins C.F., Sharp P.M. ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enterobacteria. Mol Microbiol. 1991;5:825–762. doi: 10.1111/j.1365-2958.1991.tb00755.x. [DOI] [PubMed] [Google Scholar]
  • 27.Martin B., Humbert O., Camara M., Guenzi E., Walker J., Mitchell T., Andrew P., Prudhomme M., Alloing G., Hakenbeck R., Morrison D.A., Boulnois G.J., Claverys J.-P. A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae. Nucl Acids Res. 1992;20:479–3483. doi: 10.1093/nar/20.3.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Versalovic J., Koeuth T., Lupski J.R. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucl Acids Res. 1991;19:6823–6831. doi: 10.1093/nar/19.24.6823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Toth I.K., Avrova A.O., Hyman L.J. Rapid identification and differentiation of the soft rot Erwinias by 16S–23S Intergenic transcribed spacer-PCR and restriction fragment length polymorphism analyses. Appl Environ Microbiol. 2001;67:4070–4076. doi: 10.1128/AEM.67.9.4070-4076.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Woese C.R., Stackebrandt E., Macke T.J., Fox G.E. A phylogenetic definition of major eubacterial taxa. Syst Appl Microbiol. 1985;6:143–151. doi: 10.1016/s0723-2020(85)80047-3. [DOI] [PubMed] [Google Scholar]
  • 31.Dubnau D., Smith I., Morell P., Marmur J. Gene conservation in Bacillus species I. Conserved genetic and nucleic acid base sequence homologies. Proc Natl Acad Sci USA. 1965;54:491–498. doi: 10.1073/pnas.54.2.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Pal R., Bala S., Dadhwal M., Kumar M., Dhingra G., Prakash O., Prabagaran S.R., Shivaji S., Cullum J., Holliger C., Lal R. Hexachlorocyclohexane-degrading bacterial strains Sphingomonas paucimobilis B90A, UT26 and Sp+, having similar lin genes, represent three distinct species, Sphingobium indicum sp. nov., Sphingobium japonicum sp. nov. and Sphingobium francense sp. nov., and reclassification of [Sphingomonas] chungbukensis as Sphingobium chungbukense comb. nov. Int J Syst Evol Microbiol. 2005;55:1965–1972. doi: 10.1099/ijs.0.63201-0. [DOI] [PubMed] [Google Scholar]
  • 33.Yang Z. Phylogenetic analysis using parsimony and likelihood methods. J Mol Evol. 1996;42:294–307. doi: 10.1007/BF02198856. [DOI] [PubMed] [Google Scholar]
  • 34.Ludwig W., Schleifer K. H. Phylogeny of bacteria beyond the 16S rRNA standard. ASM News. 1999;65:752–757. [Google Scholar]
  • 35.Ludwig W., Strunk O., Klugbauer S., Klugbauer N., Weizenegger M., Neumaier J., Bachleitner M., Schleifer K.H. Bacterial phylogeny based on comparative sequence analysis. Electrophoresis. 1998;19:554–568. doi: 10.1002/elps.1150190416. [DOI] [PubMed] [Google Scholar]
  • 36.Stackebrandt E., Liesack W. Nucleic acid and classification. In: Goodfellow M., O’Donnell A.G., editors. Handbook of new bacterial systematics. London: Academic Press Ltd.; 1993. pp. 151–194. [Google Scholar]
  • 37.Wayne L.G., Brenner D.J., Colwell R.R., Grimont P.A.D., Kandler O., Krichevsky M. I., Moore L.H., Moore W.E.C., Murray R.G.E., Stackebrandt E., Starr M.P., Truper H.G. International committee on systematics bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Microbiol. 1987;37:463–464. doi: 10.1099/00207713-37-4-463. [DOI] [Google Scholar]
  • 38.Mora R.R. DNA-DNA reassociation methods applied to microbial taxonomy and their critical evaluation. In: Stackebrandt E., editor. Molecular Identification, Systematics, and Population Structure of Prokaryotes. Berlin Heidelberg: Springer-Verlag; 2006. pp. 23–49. [Google Scholar]
  • 39.Stackebrandt E. The richness of prokaryotic diversity: there must be a species somewhere. Food Technol Biotechnol. 2003;41:7–22. [Google Scholar]
  • 40.Broekhuijsen M., Larsson P., Johansson A., Byström M., Eriksson U., Larsson E., Prior R.G., Sjöstedt A., Titball R.W., Forsman M. Genome-wide DNA microarray analysis of Francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent F. tularensis subsp. Tularensis. J Clin Microbiol. 2003;41:2924–2931. doi: 10.1128/JCM.41.7.2924-2931.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Goodfellow M., O’Donnell A.G. Roots of bacterial systematics. In: Goodfellow M., O’Donnell A. G., editors. Handbook of new bacterial systematics. London: Academic Press Ltd.; 1993. pp. 3–54. [Google Scholar]
  • 42.Schleifer K.H., Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev. 1972;36:407–477. doi: 10.1128/br.36.4.407-477.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Suzuki K., Goodfellow M., O’Donnell A.G. Cell envelopes and classification. In: Goodfellow M., O’Donnell A.G., editors. Handbook of New Bacterial Systematics. London: Academic Press Ltd; 1993. pp. 195–250. [Google Scholar]
  • 44.Busse J., Auling G. Polyamine pattern as a chemotaxonomic marker within the proteobacteria. Syst Appl Microbiol. 1988;11:1–8. [Google Scholar]
  • 45.Bishop D.H.L., Pandya K.P., King H.K. Ubiquinone and vitamin K in bacteria. Biochem J. 1962;83:606–614. doi: 10.1042/bj0830606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Collins M.D., Dorothy J. Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications. Microbiol Reviews. 1981;45:316–354. doi: 10.1128/mr.45.2.316-354.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.On S.L.W., Holmes B. Reproducibility of tolerance tests those are useful in the identification of compylobacteria. J Clin Microbiol. 1991;29:1785–1788. doi: 10.1128/jcm.29.9.1785-1788.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.On S.L.W., Holmes B. Assessment of enzyme detection tests useful in identification of campylobacteria. 1992;30:746–749. doi: 10.1128/jcm.30.3.746-749.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Colwell R.R., Austin B. Numerical taxonomy. In: Gerhardt T. P., Murray R. G. E., Costilow R. N., Nester E. W., Wood W. A., Krieg N. R., Phillips G. B., editors. Manual of Methods for General Bacteriology. Washington, DC: American Society for Microbiology; 1981. pp. 444–449. [Google Scholar]
  • 50.Sneath P. Numerical taxonomy 1. In: Krieg N. R., Holt J. G., editors. Bergey’s manual of systematics bacteriology. Baltimore: The Williams & Wilkins Co; 1984. pp. 111–118. [Google Scholar]
  • 51.Bala S., Khanna R., Dhadwal M., Prabhagaran S.R., Shivaji S., Cullum J., Lal R. Reclassification of Amycolatopsis mediterranei 46095 as Amycolatopsis rifamycinica. Int J Syst Evol Microbiol. 2004;54:1145–1149. doi: 10.1099/ijs.0.02901-0. [DOI] [PubMed] [Google Scholar]
  • 52.Majumdar S., Lal R. Reclassification of Amycolatopsis orientalis DSM 43387 as Amycolatopsis benzoatilytica sp. nov. Int J Syst Evol Microbiol. 2006;56:199–204. doi: 10.1099/ijs.0.63766-0. [DOI] [PubMed] [Google Scholar]
  • 53.Prakash O., Lal R. Description of Sphingobium fuligis sp. nov. a phenanthrene-degrading bacterium from a fly ash site dumping site and reclassification of Sphingomonas cloacae as Sphingobium cloacae comb. nov. Int J Syst Evol Microbiol. 2006;56:2147–2152. doi: 10.1099/ijs.0.64080-0. [DOI] [PubMed] [Google Scholar]
  • 54.Prakash O., Kumari K., Lal R. Pseudomonas delhiensis sp. nov., from a fly ash dumping site of a thermal power plant. Int J Syst Evol Microbiol. 2007;57:527–531. doi: 10.1099/ijs.0.64456-0. [DOI] [PubMed] [Google Scholar]
  • 55.Fox G.E., Wisotzkey J.D., Jurtshuk P., Jr How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol. 1992;42:166–170. doi: 10.1099/00207713-42-1-166. [DOI] [PubMed] [Google Scholar]
  • 56.Vandamme P., Harrington C.S., Jalava K., On S.L.W. Missidentifying helicobacters: the Helicobacter cinaedi example. J Clin Microbiol. 2000;38:2261–2266. doi: 10.1128/jcm.38.6.2261-2266.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Bellemare G., Vigne R., Jordon B.R. Interaction between Escherichia coli ribosomal proteins and 5S RNA molecules: recognition of prokaryotic 5S RNAs and rejection of eukaryotic 5S RNAs. Biochimie. 1973;55:340–350. doi: 10.1016/S0300-9084(73)80233-0. [DOI] [PubMed] [Google Scholar]
  • 58.Nomura M., Traub P., Bechmann H. Hybrid 30S ribosomal particles reconstituted from components of different bacterial origins. Nature. 1968;219:793–799. doi: 10.1038/219793b0. [DOI] [PubMed] [Google Scholar]
  • 59.Wrede P., Erdmann V.A. Activities of B. stearothermophilus 50S ribosomes reconstituted with prokaryotic ad eukaryotic 5S RNA. FEBS Lett. 1973;33:315–319. doi: 10.1016/0014-5793(73)80219-4. [DOI] [PubMed] [Google Scholar]
  • 60.Daya-Grosjean L., Geisser M., Stoffler G., Garret R.A. Heterologous protein-RNA interactions in bacterial ribosomes. FEBS Lett. 1973;37:17–20. doi: 10.1016/0014-5793(73)80416-8. [DOI] [PubMed] [Google Scholar]
  • 61.Wang Y., Zhang Z., Ramanam N. The actinomycete Thermobispora bispora contains two distinct types of transcriptionally active 16S rRNA genes. J Bacteriol. 1997;179:3270–3276. doi: 10.1128/jb.179.10.3270-3276.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Asai T., Zaporojets D., Squires C., Squires C.L. An Escherichia coli strain with all chromosomal rRNA operons inactivated: complete exchange of rRNA genes between bacteria. Proc NatI Acad Sci USA. 1999;96:1971–1976. doi: 10.1073/pnas.96.5.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Yap W.H., Zhang Z., Wang Y. Distinct type of rRNA operons exist in the genome of the actinomycete Thermomonospora chromogena and evidence for horizontal transfer of an entire rRNA operon. J Bacteriol. 1999;181:5201–5209. doi: 10.1128/jb.181.17.5201-5209.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Mylvaganam S., Dennis P.P. Sequence heterogeneity between the two genes encoding 16S rRNA from the halophilic archaebacterium Haloarcula marismortui. Genetics. 1992;130:399–410. doi: 10.1093/genetics/130.3.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Dennis P.P., Ziesche S., Mylvaganam S. Transcription analysis of two disparate rRNA operons in the halophilic archaeon Haloarcula marismortui. J Bacteriol. 1998;180:4804–4813. doi: 10.1128/jb.180.18.4804-4813.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Binnewies T.T., Motro Y., Hallin P. F., Lund O., Dunn D., La T., Hampson D. J., Bellgard M., Wassenaar T. M., Ussery D. W. ten years of bacterial genome sequencing: comparative — genomics — based discoveries. Funct Integr Genomics. 2006;6:165–185. doi: 10.1007/s10142-006-0027-2. [DOI] [PubMed] [Google Scholar]
  • 67.Perna N.T., Plunkett G., III, Burland V., Mau B., Glasner J.D., Rose D.J., Mayhew G.F., Evans P.S., Gregor J., Kirkpatrick H.A., Posfal G., Hackett J., Klink S., Boutin A., Shao Y., Miller L., Grotbeck E.J., Davis N.W., Lim A., Dimalanta E.T., Potamousis K.D., Apodaca J., Anantharaman T.S., Lin J., Yen G., Schwartz D.C., Welch R.A., Blattner F.R. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature. 2001;409:529–533. doi: 10.1038/35054089. [DOI] [PubMed] [Google Scholar]
  • 68.Daubin V., Gouy M., Perrie’re G. A phylogenomic approach to bacterial phylogeny: Evidence of a core of genes sharing a common history. Genome Res. 2002;12:1080–1090. doi: 10.1101/gr.187002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Lerat E., Daubin V., Moran N.A. From gene trees to organismal phylogeny in prokaryotes: The case of the gamma-proteobacteria. PLoS Biol. 2003;1:101–109. doi: 10.1371/journal.pbio.0000019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Olendzenski L Zhaxybayeva O & Gogarten JP (2002) What’s in a tree? Does horizontal gene transfer determine microbial taxonomy? In: Seckbach J (ed) Symbiosis Dordrecht, The Netherlands Kluwer pp. 63–78
  • 71.Ochman H., Lawrence J.G., Groisman E.A. Lateral gene transfer and the nature of bacterial innovation. Nature. 2000;405:299–304. doi: 10.1038/35012500. [DOI] [PubMed] [Google Scholar]
  • 72.Kunin V., Goldovsky L., Darzentas N., Ouzounis C. A. The net of life: Reconstructing the microbial phylogenetic network. Genome Research. 2005;15:954–959. doi: 10.1101/gr.3666505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Edward P.R., Ewing W.H. Identification of Enterobacteriaceae. 4th ed. New York: Elsevier Science Publishing Co. Inc.; 1986. [Google Scholar]

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