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Acinetobacter Taxonomy & Identification Mario Vaneechoutte Laboratory Bacteriology Research University of Ghent Fla

Acinetobacter Taxonomy & Identification Mario Vaneechoutte Laboratory Bacteriology Research University of Ghent Flanders, Belgium Boerhaave Course. January 16 th , 2009 Leiden, the Netherlands. Slides available at: http://users.ugent.be/~mvaneech/LBR.htm Mario.Vaneechoutte@UGent.be.

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Acinetobacter Taxonomy & Identification Mario Vaneechoutte Laboratory Bacteriology Research University of Ghent Fla

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  1. Acinetobacter Taxonomy & Identification Mario Vaneechoutte Laboratory Bacteriology Research University of Ghent Flanders, Belgium Boerhaave Course. January 16th, 2009 Leiden, the Netherlands Slides available at: http://users.ugent.be/~mvaneech/LBR.htm Mario.Vaneechoutte@UGent.be

  2. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 Taxonomic position of the genus Acinetobacter The genus Acinetobacter(Brisou & Prévot 1954) Gram-negative, non-motile (but: see slide 6!)and non-fermentative (cocco)bacilli family of the Moraxellaceae (Rossau et al. 1991) γ-subclass of the Proteobacteria. Currently: 21 named species and 11 genomic species Its representatives are commonly present in soil and water, but most isolates of many species are from clinical sources. The taxonomy of the genus Acinetobacter exemplifies the dynamics of modern taxonomy as it has becomen possible by the combination of genotypic and phenotypic methods. This is illustrated by a brief history of Acinetobacter taxonomy. Brisou J, Prévot AR. 1954. Studies on bacterial taxonomy. X. The revision of species under Achromobacter group. Ann Inst Pasteur 86: 722-728. Rossau, R., Van Landschoot, A., Gillis, M., and De Ley, J. 1991. Taxonomy of Moraxellaceae fam. nov., a new bacterial family to accomodate the genera Moraxella, Acinetobacter, and Psychrobacter and related organisms. Int J Syst Bacteriol 41: 310-319.

  3. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 From one species in 1968 to 32 in 2009 1968: Acinetobacter calcoaceticus: one species genus (Baumann et al. 1968) At present: The genus Acinetobacter currently comprises 32 species of which 21 have been named, i.e., A. baumannii, A. baylyi, A. bereziniae, A. beijerinckii, A. bouvetii, A. calcoaceticus, A. gerneri, A. guillouiae, A. gyllenbergii, A. haemolyticus, A. johnsonii, A. junii, A. lwoffii, A. parvus, A. radioresistens, A. schindleri, A. tjernbergiae, A. towneri, A. tandoii, A. ursingii and A. venetianus whereas 11 remain unnamed and are designated as genomic species, i.e., genomic species 3 between 1 and 3 6 10 11 13TU close to 13TU 13BJ/14TU 14BJ 15TU 15BJ 16 17

  4. From one species in 1968 to 32 in 2009 Update February 2012: 1968: Acinetobacter calcoaceticus: one species genus (Baumann et al. 1968) At present: The genus Acinetobacter currently comprises 32 species of which 23 have been named, i.e., A. baumannii, A. baylyi, A. bereziniae, A. beijerinckii, A. bouvetii, A. calcoaceticus, A. gerneri, A. guillouiae, A. gyllenbergii, A. haemolyticus, A. johnsonii, A. junii, A. lwoffii, A. parvus, A. radioresistens, A. schindleri, A. tjernbergiae, A. towneri, A. tandoii, A. ursingii and A. venetianus whereas 11 remain unnamed and are designated as genomic species, i.e., genomic species 3 A. pittii (see slide 45) between 1 and 3 6 10 11 13TU  A. nosocomialis (see slide 45) close to 13TU 13BJ/14TU 14BJ 15TU 15BJ 16 17

  5. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 Acinetobacter pre-history Martinus Willem Beijerinck, Dutch microbiologist founder of the Delft School of Microbiology described an organism that was isolated from soil by enrichment cultivation on a calciumacetate-mineral medium (Beijerinck 1911) as Micrococcus calco-aceticus. In the following decades similar organisms were described independently and assigned different genus and species names like (Henriksen 1973) Herellea vaginicola Mima polymorpha Bacterium anitratum Moraxella lwoffii Beijerinck MW. 1911. Pigmenten als oxydatieproducten gevormd door bacteriën. Versl Koninkl Akad Wetensch Amsterdam 19: 1092-1103. Henriksen SD. 1973. Moraxella, Acinetobacter, and the Mimeae. Bacteriol Rev 37: 522-561.

  6. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 a-kineto-bacter Baumann et al. (1968): All these ‘taxa’ represent a single genus Genus name: Acinetobacter meaning: ‘immotile rod’ (a-kineto-bacter) BUT: (Update February 2012): Acinetobacter is motile!: Mussi et al. (2010) No clear-cut criteria for subdivision of the genus into species could be given: Only one species, named Acinetobacter calcoaceticus. Baumann P, Doudoroff M, Stanier RY. 1968. A study of the Moraxella group. II. Oxidative-negative species (genus Acinetobacter). J Bacteriol 95: 1520-1541. Mussi MA, JA Gaddy,M Cabruja, BA Arivett, AM Viale, R Rasia, LA Actis. 2010. The opportunistic human pathogen Acinetobacter baumannii senses and responds to light. J Bacteriol 192: 6336–6345 (doi:10.1128/JB.00917-10).

  7. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 1. DNA-transformation assay: Acinetobacter yes or no An important breakthrough was realized at the same time by Elliot Juni. Juni & Janik (1969) discovered a hypertransformableAcinetobacter strain, i.e. a strain able to take up DNA from lysed bacterial cells and able to recombine this DNA into its genome, and designated this strain as ‘A. calcoaceticus’, since A. calcoaceticus was the only valid species name at that time. Juni (1972) went on to show that this strain could be transformed by DNA-extracts prepared from 265 bacterial strains, described to belong to eleven different genera, and that in fact these strains were close genetic relatives, all belonging to the genus Acinetobacter Until today the transformation assay is a basic tool in Acinetobacter taxonomy and the ability to transform ‘A. calcoaceticus’ strain BD413. is a prerequisite for a strain to belong to the genus Acinetobacter (Juni 1984). BD413 is still at present an important biotechnological tool. Juni E, Janik A. 1969. Transformation of Acinetobacter calco-aceticus (Bacterium anitratum). J Bacteriol 98: 281-288. Juni E. 1972. Interspecies transformation of Acinetobacter: genetic evidence for a ubiquitous genus. J Bacteriol 112:917-931. Juni E. 1984. Acinetobacter (Brisou & Prévot 1954). Pp. 303-307, In Bergey's Manual of Systematic Bacteriology, Krieg NR, Holt JG, Eds. Baltimore/London, UK: Williams & Wilkins.

  8. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 Bouvet & Grimont (1986): DNA-hybridization + phenotypic characteristics Further progress had to await another sixteen years, with the publication by Bouvet & Grimont (1986), which can be considered as the start of modern taxonomy for the genus Acinetobacter. 12 DNA-DNA hybridization groups = species Six named species: DNA group 1: A. calcoaceticus DNA group 2: A. baumannii DNA group 4: A. haemolyticus DNA group 5: A. junii DNA group 7: A. johnsonii DNA group 8: A. lwoffii Six genomic species: DNA groups 3, 6, 9, 10, 11 and 12 because no phenotypic characteristics could be established to identify these species. 6 named species vs 6 genomic species: 6 + 6 = 12 species Bouvet PJM, Grimont PAD. 1986. Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., and Acinetobacter junii sp. nov. and emended descriptions of Acinetobacter calcoaceticus and Acinetobacter lwoffii. Int J Syst Bacteriol 36: 228-240.

  9. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 2. DNA-DNA hybridization: Determination of total DNA-homology 75% DNA-homology: all Mammalia are one species!

  10. The species concept Sexually reproducing eukaryotes Number of individuals H. sapiens Pan Gorilla Diversity Asexually reproducing prokaryotes with horizontal gene transfer Number of individuals M. canis M. catarrhalis NCTC 4103 Jannes, G., M. Vaneechoutte, M. Lannoo, ... and R. Rossau. 1993. Polyphasic taxonomy leading to the proposal of Moraxella canis sp. nov. for Moraxella catarrhalis-like strains. Int. J. Syst. Bacteriol. 43: 438-449. Diversity

  11. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 3. Phenotypic characterization of Acinetobacter species Phenotypic characteristics which can be used for identification present --> species name not present --> numbered species Bernards AT, Dijkshoorn L, Van der Toorn J, Bochner BR, van Boven CPA. 1995. Phenotypic characterization of Acinetobacter strains of 13 DNA-DNA hybridization groups by means of the Biolog system. J Med Microbiol 42: 113-119. Bernards AT, Van der Toorn J, van Boven CPA, Dijkshoorn L. 1996. Evaluation of the ability of the API 20NE system to identify Acinetobacter genomic species. J Clin Microbiol Infect Dis 15: 303-308. Kämpfer P, Tjernberg I, Ursing J. 1993. Numerical classification and identification of Acinetobacter genomic species. J Appl Microbiol 75: 259-268.

  12. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 A. radioresistens (= DNA group 12) Nishimura et al. (1988): A. radioresistens: a strain from a cotton sample, isolated following gamma ray irradation, other radioresistant strains, isolated from soil. 6 -> 7 + 6 = 13 Tjernberg & Ursing (1989), by means of DNA-DNA hybridization: A. radioresistens is synonymous to genomic species 12 of Bouvet & Grimont (1986). 7 + 6 -> 5 = 12 Bouvet PJM, Grimont PAD. 1986. Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., and Acinetobacter junii sp. nov. and emended descriptions of Acinetobacter calcoaceticus and Acinetobacter lwoffii. Int J Syst Bacteriol 36: 228-240. NishimuraY, InoT, Iizuka H. 1988.Acinetobacter radioresistens sp. nov. isolated from cotton and soil. Int J Syst Bacteriol 38: 209-211. Tjernberg I, Ursing J. 1989. Clinical strains of Acinetobacter classified by DNA-DNA hybridization. APMIS 97: 595-605.

  13. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 A. lwoffii (DNA group 8) = DNA-group 9 Tjernberg & Ursing (1989): the reference strains of A. lwoffii (DNA group 8) and of genomic species 9 are highly related: genomic species 9 should be omitted. 7 + 5 -> 4 = 11 Janssen et al. (1997): AFLP confirms the synonymity of A. lwoffii and genomic species 9: 13 strains of A. lwoffii and 9 strains from genomic species 9 form a homogeneous group nearly identical AFLP patterns for the A. lwoffii type strain NCTC5866T and the genomic species 9 reference strain CIP A162. Tjernberg I, Ursing J. 1989. Clinical strains of Acinetobacter classified by DNA-DNA hybridization. APMIS 97: 595-605. Janssen P, Maquelin K, Coopman R, Tjernberg I, Bouvet P, Kersters K, Dijkshoorn L. 1997. Discrimination of Acinetobacter genomic species by AFLP fingerprinting. Int J Syst Bacteriol 47: 1179-1187.

  14. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 Polyphasic taxonomy, Identification and Typing Technique RAPD, AP-PCR Typing (Selective restriction fragment hybridization) Selective restriction fragment amplification (AFLP) T T TI T TI T TI TI I TI t Identification Taxonomy , ARDRA MLSA

  15. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 4. Selective restriction fragment amplification: AFLP Purified genomic DNA Restriction digestion RFLP gTCATTgC  CAgTAACgAACCTg gTCATTgC CAgTAACCAACCTg gTCATTgC CAgTAACTAACCTg gTCATTgC CAgTAACAAACCTg Adapter Adapter-ligation PCR with selective primers with 0, 1 or more nucleotides overhang 1 primer is fluorescently labeled

  16. fragment length (bp) Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 4. Selective restriction fragment amplification: AFLP PCR: only a limited number of restriction fragments is amplified Automated (capillary) electrophoresis Detection of fluorescence + automated sizing: a digital fingerprint 1/3

  17. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 4. Selective restriction fragment amplification: AFLP AFLPtm: Identification of Acinetobacter species + genotyping of strains within each species Acinetobacter baumannii isolates > 50% homology = same species Janssen P, Maquelin K, Coopman R, Tjernberg I, Bouvet P, Kersters K, Dijkshoorn L. 1997. Discrimination of Acinetobacter genomic species by AFLP fingerprinting. Int J Syst Bacteriol 47: 1179-1187.

  18. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 1989: Numbering of proteolytic and haemolytic Acinetobacter species 1989: Two simultaneous and independent DNA-DNA hybridization studies on haemolytic - proteolytic Acinetobacter species. Tjernberg & Ursing (1989): genomic species 13, 14, 15 Bouvet & Jeanjean (1989): genomic species 13, 14, 15, 16, 17 This adds 8 genomic species to the genus: 7 + 4 ->12 = 19 Dijkshoorn et al. (1990): ATCC 17905 is used by both teams and was designated 13 by B&J, but 14 by T&U. Vaneechoutte et al. (1995): add affix 'BJ' or 'TU' to the number to clarify confusion. 13BJ, 14BJ, 15BJ, 16, 17 13TU, 14TU, 15TU 7 + 12 -> 11 = 18 Tjernberg I, Ursing J. 1989. Clinical strains of Acinetobacter classified by DNA-DNA hybridization. APMIS 97: 595-605. Bouvet PJ, Jeanjean S. 1989. Delineation of new proteolytic genomic species in the genus Acinetobacter. Res Microbiol 140: 291-299. Dijkshoorn L, Tjernberg I, Pot B, Michel MF, Ursing J, Kersters K. 1990. Numerical analysis of cell envelope protein profiles of Acinetobacter strains classified by DNA-DNA hybridization. System Appl Microbiol 13: 338-344. Vaneechoutte M, Dijkshoorn L, Tjernberg I, Elaichouni A, De Vos P, Claeys G, Verschraegen G. 1995.Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. J Clin Microbiol 33: 11-15.

  19. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 5. SDS PAGE of envelope proteins Dijkshoorn L, Tjernberg I, Pot B, Michel MF, Ursing J, Kersters K. 1990. Numerical analysis of cell envelope protein profiles of Acinetobacter strains classified by DNA-DNA hybridization. System Appl Microbiol 13: 338-344.

  20. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 The Acinetobacter calcoaceticus-baumannii complex A. calcoaceticus (DNA group 1) A. baumannii (DNA group 2) genomic species 3 genomic species 13TU Closely related, on the basis of high DNA-homology. Differentiation on the basis of biochemical characters is largely impossible. Therefore this group of four species is sometimes referred to as the ACB complex. Gerner-Smidt & Tjernberg (1993) recognized two additional genomic species, closely related to these four DNA groups. These new genomic species were designated ‘between 1 and 3’ and ‘close to 13TU’. 7 + 11 -> 13 = 20 Gerner-Smidt P, Tjernberg I. 1993.Acinetobacter in Denmark. II. Molecular studies of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. APMIS 101: 826-832.

  21. 6. Sequence determination of the amplified 16S rRNA gene Vaneechoutte, M., and T. De Baere. 2007. Taxonomy of the genus Acinetobacter, based on 16S ribosomal RNA gene sequences. Pp. 35-60, In: Acinetobacter Molecular Biology (Gerischer, U., Ed.), Horizon Scientific Press/Caister Academic Press, Norfolk, UK. ISBN 978-904455-20-2.

  22. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 6. Sequence determination of the amplified 16S rRNA gene Vaneechoutte & De Baere (2007): 16S rRNA gene sequence: 1450 nucleotides Threshold for species delineation: 1, 2, 3 or 4 nt difference: same species More than 4 nt difference: different species Vaneechoutte, M., and T. De Baere. 2007. Taxonomy of the genus Acinetobacter, based on 16S ribosomal RNA gene sequences. Pp. 35-60, In: Acinetobacter Molecular Biology (Gerischer, U., Ed.), Horizon Scientific Press/Caister Academic Press, Norfolk, UK. ISBN 978-904455-20-2.

  23. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 Australian sludge yields seven new species: A. baylyi, A. bouvetii, [A. grimontii], A. tjernbergiae, A. towneri, A. tandoii and A. gerneri Carr et al. 2003: An Australian group described seven novel Acinetobacter species from activated sludge: A. baylyi A. bouvetii A. gerneri [A. grimontii] A. tandoii A. tjernbergiae A. towneri 7 -> 14 + 13 = 27 Carr EL, Kämpfer P, Patel BKC, Gürtler V, Seviour RJ. 2003. Seven novel species of Acinetobacter isolated from activated sludge. Int J Syst Evol Microbiol 53: 953-963.

  24. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 [A. grimontii] = A. junii Vaneechoutte et al. (2008): tDNA-PCR could not differentiate A. grimontii (Carr et al. 2003) from A. junii (Bouvet & Grimont 1986) Polyphasic taxonomy study confirmed that A. grimontii was synonymous to A. junii (Vaneechoutte et al. 2008): * 16S rDNA sequence is not so different from A. junii as published by Carr et al. (2003) * DNA-homology was borderline at 65%. Retesting (other method): 85% * Biochemically: L-arginine + and L-glutamate +: in contrast to original description; in accordance with the phenotypic properties of A. junii. * AFLP-pattern: falls within the A. junii cluster: > 50% homology. 14 -> 13 + 13 = 26 Bouvet PJM, Grimont PAD. 1986. Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., and Acinetobacter junii sp. nov. and emended descriptions of Acinetobacter calcoaceticus and Acinetobacter lwoffii. Int J Syst Bacteriol 36: 228-240. Carr EL, Kämpfer P, Patel BKC, Gürtler V, Seviour RJ. 2003. Seven novel species of Acinetobacter isolated from activated sludge. Int J Syst Evol Microbiol 53: 953-963 Vaneechoutte M, De Baere T, Nemec A, van der Reijden TJK, Dijkshoorn L. 2008. Reclassification of Acinetobacter grimontii Carr et al. 2003 as a later synonym of Acinetobacter junii Bouvet and Grimont 1986. Int J Syst Evol Microbiol 58: 937-940.

  25. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 [A. grimontii] = A. junii

  26. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 6. tRNA intergenic spacer length polymorphism analysis: tDNA-PCR tRNA-genes are organized in clusters, separated by short spacers.Intergenic spacers have different length Using outward consensus primers, the different tRNA spacers can be amplified simultaneously

  27. T5A T3B Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 6. tRNA intergenic spacer length polymorphism analysis: tDNA-PCR Welsh & McClelland (1991): Consensus regions at the edges of the tRNA genes AGGTCGCGGGTTCGAATCC CTCAGTT GGGTTAGAGCACCGGACT Welsh J, McClelland M. 1991. Genomic fingerprints produced by PCR with consensus tRNA gene primers. Nucl Acids Res 19: 861-866.

  28. PCR PCR Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 6. tRNA intergenic spacer length polymorphism analysis: tDNA-PCR

  29. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 6. tRNA intergenic spacer length polymorphism analysis: tDNA-PCR

  30. 6. tDNA-PCRSize determination by fluorescent capillary electrophoresis: digitized DNA-fingerprints Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009

  31. 6. tDNA-PCR tDNA-PCR based identification:Listeria ivanovii subsp. ivanovii (4) vs subsp. londoniensis (4) Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 Vaneechoutte, M., P. Boerlin, H.-V. Tichy, E. Bannerman, B. Jäger, and J. Bille. 1998. Comparison of PCR-based DNA fingerprinting techniques for the identification of Listeria species and their use for atypical Listeria isolates. Int. J. Syst. Bacteriol. 48: 127-139.

  32. 6. tDNA-PCR tDNA-PCR fingerprint library for the genus Listeria Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 2/3

  33. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 6. tDNA-PCR A universal approach for bacterial species identification * Wiedmann-Al-Ahmad M, Tichy H-V, Schön G. 1994. Characterization of Acinetobacter type strains and isolates obtained from wastewater treatment plants by PCR fingerprinting. Appl Environm Microbiol 60: 4066-4071. * Ehrenstein B, Bernards AT, Dijkshoorn L, Gerner-Smidt P, Towner KJ, Bouvet PJ, Daschner FD, Grundmann H. 1996. Acinetobacter species identification by using tRNA spacer fingerprinting. J Clin Microbiol 34: 2414-2420. * Maes N, De Gheldre Y, De Ryck R, Vaneechoutte M, Meugnier H, Etienne J, Struelens M. 1997. Rapid and accurate identification of Staphylococcus species by tRNA intergenic spacer length polymorphism analysis. J Clin Microbiol 35: 2477-2481. * Vaneechoutte M, Boerlin P, Tichy H-V , Bannerman E, Jäger B, Bille J. 1998. Comparison of PCR-based DNA fingerprinting techniques for the identification of Listeria species and their use for atypical Listeria isolates. Int J Syst Bacteriol 48: 127-139. * Baele M, Baele P, Vaneechoutte M, Storms V, Butaye P, Devriese LA, Verschraegen G, Gillis M, Haesebrouck F. 2000. Application of tDNA-PCR for the identification of Enterococcus species. J Clin Microbiol 38: 4201-4207. * Baele M, Storms V, Haesebrouck F, Devriese LA, Gillis M, Verschraegen G, De Baere T, Vaneechoutte M. 2001. Application and evaluation of the interlaboratory reproducibility of tRNA intergenic length polymorphism analysis (tDNA-PCR) for identification of species of the genus Streptococcus. J Clin Microbiol 39: 1436-1442. *Baele, M., M. Vaneechoutte, R. Verhelst, M. Vancanneyt, L. A. Devriese, and F. Haesebrouck. 2002. Identification of Lactobacillus species using tDNA-PCR. J Microbiol Methods 50: 263-271. Stakenborg, T., J. Vicca, R. Verhelst, P. Butaye, D. Maes, A. Naessens, G. Claeys, C. De Ganck, F. Haesebrouck, and M. Vaneechoutte. 2005. Evaluation of tRNA gene PCR for the identification of mollicutes. J. Clin. Microbiol. 43: 4558-4566. * Other publications on Enterobacteriaceae, Gram-negative nonfermenters, Moraxella, Pasteurella, Mannheimia

  34. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 Acinetobacter phenons become species During the last decade, the laboratories of on the basis of LUMC, Leiden, the Netherlands (Lenie Dijkshoorn, Tanny van der Reijden): ARDRA, AFLP NIPH, Prague, Czech Republic (Alexandr Nemec): ARDRA, extensive biochemical testing (Martin Musilék): rpoB-sequencing LBR, Gent, Belgium (Mario Vaneechoutte): ARDRA, tDNA-PCR (Thierry De Baere): ARDRA, 16S rDNA sequencing delineated phenons, i.e. phenetic groups on the basis of detailed phenotypic characteristics combined with DNA fingerprinting (ARDRA) and DNA sequence data. Vaneechoutte M, Dijkshoorn L, Tjernberg I, Elaichouni A, De Vos P, Claeys G, Verschraegen G. 1995.Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. J Clin Microbiol 33: 11-15. Dijkshoorn L, van Harsselaar B, Tjernberg I, Bouvet PJM, Vaneechoutte M. 1998. Evaluation of amplified ribosomal DNA restriction analysis for identification of Acinetobacter genomic species. System Appl Microbiol 21: 33-39.

  35. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 Five additional species of clinical importance Nemec A, De Baere T, Tjernberg I, Vaneechoutte M, van der Reijden TJK, Dijkshoorn L. 2001. Acinetobacter ursingii sp. nov. and Acinetobacter schindleri sp. nov., isolated from human clinical specimens. Int J Syst Evol Microbiol 51: 1891-1899. Nemec A, Dijkshoorn L, Janssens D, De Baere T, van der Reijden TJK , Jezek P, Vaneechoutte M. 2003. Acinetobacter parvus sp. nov., a small colony forming species isolated from human specimens. Int J Syst Evol Microbiol 53: 1563-1567. Nemec A, MusílekM, Maixnerová M, De Baere T, van der Reijden TJK, Vaneechoutte M, Dijkshoorn L. 2009. Acinetobacter beijerinckii sp. nov. and Acinetobacter gyllenbergii sp. nov., haemolytic organisms isolated from humans. Int J Syst Evol Microbiol. 59: 118-124. 13 -> 18 + 13 = 31

  36. DNA-extraction Amplification of 16S rDNA Restriction digestion Agarose Gel Electrophoresis 7. Amplified rDNA Restriction Analysis (ARDRA) PCR-RFLP-analysis of the 16S rRNA gene Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 Vaneechoutte M, Dijkshoorn L, Tjernberg I, Elaichouni A, De Vos P, Claeys G, Verschraegen G. 1995.Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. J Clin Microbiol 33: 11-15. Dijkshoorn L, van Harsselaar B, Tjernberg I, Bouvet PJM, Vaneechoutte M. 1998. Evaluation of amplified ribosomal DNA restriction analysis for identification of Acinetobacter genomic species. System Appl Microbiol 21: 33-39.

  37. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 7. Amplified rDNA Restriction Analysis (ARDRA) PCR-RFLP-analysis of the 16S rRNA gene Vaneechoutte M, Rossau R, De Vos P, Gillis M, Janssens D, Paepe N, De Rouck A, Fiers T, Claeys G, Kersters K. 1992. Rapid identification of bacteria of the Comamonadaceae with amplified ribosomal DNA-restriction analysis (ARDRA). FEMS Microbiol Lett. 72: 227-233.

  38. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 7. Amplified rDNA Restriction Analysis (ARDRA) PCR-RFLP-analysis of the 16S rRNA gene Vaneechoutte, De Baere, Nemec, Dijkshoorn: Acinetobacter ARDRA Website

  39. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 7. Amplified rDNA Restriction Analysis (ARDRA) PCR-RFLP-analysis of the 16S rRNA gene Vaneechoutte, De Baere, Nemec, Van der Reijden, Dijkshoorn: Acinetobacter ARDRA Website

  40. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 ARDRA: Acinetobacter venetianus = RAG-1 Di Cello et al. (1997): isolation of fuel-degrading Acinetobacter strains from the Venice Lagoon (Italy). assignation of the name “A. venetianus” on the basis of 16S rDNA sequence. Vaneechoutte et al. (1999): ARDRA: these strains have a pattern identical to that of strain RAG-1, itself isolated from an Israeli beach after oil based enrichment, industrially important: production of a potent bioemulsifier, emulsan. Vaneechoutte et al. (1999): Confirmed by DNA-hybridization Thus far, five strains are in the LUH collection of this species, with at least three of these isolated independently from sea water and described to degrade long chain carbohydrates. Vaneechoutte et al. (2009): Finally validly described 18 -> 19 + 13 = 32! Di Cello F, Pepi M, Baldi F, Fani R. 1997. Molecular characterization of an n-alkane-degrading bacterial community and identification of a new species, Acinetobacter venetianus. Res Microbiol 148: 237-249. Vaneechoutte M, Tjernberg I, Baldi F, Pepi M, Fani R, Sullivan ER, van der Toorn J, Dijkshoorn L. 1999. Oil-degrading Acinetobacter strain RAG-1 and strains described as `Acinetobacter venetianus sp. nov.' belong to the same genomic species. Res Microbiol150: 69-73. Vaneechoutte M, Nemec A, Musílek M, van der Reijden TJK, van den Barselaar M, Tjernberg I, Calame W, Fani R, De Baere T, Dijks­hoorn L. 2009. Emended description of “Acinetobacter venetianus” Di Cello et al. 1997. Int J Syst Evol Microbiol: In press.

  41. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 ARDRA of strain RAG-1 and A. venetianus type strain C3 Amplification of 16S rRNA gene (1500 bp) + digestion with resp. CfoI AluI MboI RsaI MspI BfaI MM M M M MM Vaneechoutte, M., Tjernberg, I., Baldi, F., Pepi, M., Fani, R., Sullivan, E.R., van der Toorn, J., and Dijkshoorn, L. 1999. Oil-degrading Acinetobacter strain RAG-1 and strains described as `Acinetobacter venetianus sp. nov.' belong to the same genomic species. Res. Microbiol.150: 69-73.

  42. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 tDNA-PCR: Acinetobacter strain BD413 = A. baylyi The hypertransformable Acinetobacter strain BD413 (ADP1) (Juni & Janik 1969) plays a central role in Acinetobacter taxonomy, is also renown as a biotechnological workhorse, both exactly because of the ease with which it is transformed and cultured. It has been named A. calcoaceticus and A. lwoffii, among others ... Carr et al. (2003): seven new species from Australian sludge, among others: A. baylyi Vaneechoutte et al. (2006): tDNA-PCR: BD413 = A. baylyi type strain Confirmed by all other taxonomic techniques that are used by our consortium. BD413 is A. baylyi, not A. calcoaceticus or A. lwoffii, as it has been named. Juni E, Janik A. 1969. Transformation of Acinetobacter calco-aceticus (Bacterium anitratum). J Bacteriol 98: 281-288. Carr EL, Kämpfer P, Patel BKC, Gürtler V, Seviour RJ. 2003. Seven novel species of Acinetobacter isolated from activated sludge. Int J Syst Evol Microbiol 53: 953-963. Vaneechoutte M, Young DM, Ornston N, De Baere T, Nemec A, van der Reijden TJK, Carr E, Tjernberg I, Dijkshoorn L. 2006. The naturally transformable Acinetobacter strain ADP1 belongs to the newly described species Acinetobacter baylyi. Appl Environm Microbiol 72: 932-936.

  43. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 A. baylyi hypertransformability: PUZZLING! A. baylyi is an intriguing species: the few strains known are all hypertransformable, although they come from different continents. * Why does constitutive hypertransformability exist? * Why it is so typical for one species, i.e. A. baylyi? * Why only strains of all the different species of the genus Acinetobacter but no strains of species of other genera can transform the A. baylyi strains? * It is mind-boggling that hypertransformable strains collected from very different geographical sites still can be recognized as belonging to the same species by very different approaches. One would expect that the genome of each of the strains of this species would look very different, as a consequence of continued DNA-uptake and recombination, made possible by constituent hypertransformability. Vaneechoutte, M., and T. De Baere. 2007. Taxonomy of the genus Acinetobacter, based on 16S ribosomal RNA gene sequences. Pp. 35-60, In: Acinetobacter Molecular Biology (Gerischer, U., Ed.), Horizon Scientific Press/Caister Academic Press, Norfolk, UK. ISBN 978-904455-20-2.

  44. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 Genomic species 10 -> A. bereziniae Genomic species 11 -> A. guillouiae In addition, our consortium of three laboratories is in the process of naming the closely related genomic species 10 and genomic species 11, as respectively A. bereziniae and A. guillouiae 19 -> 21 + 13 -> 11 = 32! [Nemec A, PJM Bouvet,P Grimont,M Musílek,O Šedo, T De Baere, M Maixnerová,TJK van der Reijden,Z Zdráhal,M Vaneechoutte, L Dijkshoorn. Acinetobacter bereziniae sp. nov.and Acinetobacter guillouiae sp. nov., to respectively accommodate Acinetobacter genomic species 10 and Acinetobacter genomic species 11. IJSEM. In preparation.] Update February 2012: Nemec A, M Musílek, O Šedo, T De Baere, M Maixnerová, TJK. van der Reijden, Z Zdráhal, M Vaneechoutte, L Dijkshoorn. 2010. Acinetobacter berezinae sp. nov. and Acinetobacter guillouiae sp. nov., to accommodate, respectively, Acinetobacter genomic species 10 and Acinetobacter genomic species 11. IJSEM 60: 896–903.

  45. Update February 2012: Nemec A, L Krizova, M Maixnerova, TJ van der Reijden, P Deschaght, V Passet, M Vaneechoutte, S Brisse, L Dijkshoorn. 2011. Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus-Acinetobacter baumanniicomplex with the proposal of Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialissp. nov. (formerly Acinetobacter genomic species 13TU). Res. Microbiol. 162: 393-404. 21-> 23 +11-> 9= 32!

  46. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 Acinetobacter septicus = A. ursingii Kilic et al. (2008): New species causing nosocomial outbreak: 16S rDNA and rpoB sequence analysis: A. ursingii. DNA-homology with A. ursingii: 64.7-68.7%. No utilisation of citrate: indeed a difference with the description of A. ursingii. 32 -> 33? Nemec et al. (2008): AFLP: 59% homology to A. ursingii (above 50% treshold) rpoB sequence: 97-98% homology to A. ursingii, 79-84% with other species. Kilic A, Li H, Mellmann A, Basustaoglu AC, Kul M, Senses Z, Aydogan H, Stratton CW, Harmsen D, Tang YW. 2008.Acinetobacter septicus sp. nov. association with a nosocomial outbreak of bacteremia in a neonatal intensive care unit. J Clin Microbiol. 46: 902-908. Nemec, A., M. Musilek, M. Vaneechoutte, E. Falsen, and L. Dijkshoorn. 2008. Lack of evidence for ‘Acinetobacter septicus’ as a species different from Acinetobacter ursingii? J. Clin. Microbiol. 46: 2826-2827.

  47. Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 The future of Acinetobacter taxonomy and identification? 1. From 21 named + 11 unnamed species tot 32 named + 0 unnamed species? 2. Additional description of phenons as named species? 3. Additional detection of environmental species? (sludge) 4. Further insights into the clinical importance of the different species? A. ursingii, A. parvus 5. Preferred method for delineation of new species: DNA-hybridization --> AFLP? 6. Preferred method for identification of new strains to established species: DNA-fingerprinting: ARDRA, tDNA-PCR, AFLP --> MLST (Bartual et al. 2005)? 7. Comparison of phylogeny based on 16S rRNA gene, rpoB, recA, gyrB 8. Whole genome sequencing: 7 A. baumannii + 1 A. baylyi already sequenced (Vallenet et al. 2008) Bartual SG, Seifert H, Hippler C, Luzon MA, Wisplinghoff H, Rodriguez-Valera F. 2005. Development of a multilocus sequence typing scheme for characterization of clinical isolates of Acinetobacter baumannii. J Clin Microbiol 43:4382-4390. Vallenet et al. 2008. Comparative analysis of Acinetobacters: three genomes for three lifestyles. PloS One 3: e1805-

  48. Thanks! Acinetobacter Taxonomy & Identification Mario Vaneechoutte Laboratory Bacteriology Research University of Ghent Flanders, Belgium Boerhaave Course. January 16th, 2009 Leiden, the Netherlands Slides available at: http://users.ugent.be/~mvaneech/LBR.htm Mario.Vaneechoutte@UGent.be

  49. Yamamoto & Harayama 1996 Yamamoto et al. 1999 Krawczyk et al. 2002 La Scola et al. 2006 ribosomal RNA internally transcribed spacer 2 (ITS2) region (Chang et al. 2005)

  50. M. gallisepticum M. pneumoniae M. glycophilum M. bovirhinis M. columborale M. neurolyticum M. flocculare M. dispar M. hyopneumoniae M. hyorhinis M. arginini M. hyosynoviae M. orale M. columbinasale M. agalactiae M. bovis M. lipofaciens M. gallinarum Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009 7. Amplified rDNA Restriction Analysis (ARDRA) PCR-RFLP-analysis of the 16S rRNA gene 50-bp Genereuler 50-bp Genereuler 1000 900 800 700 600 500 400 300 250 200 150 100 Stakenborg T, Vicca J, Butaye P, Maes D, De Baere T, Verhelst R, Peeters J, de Kruif A, Haesebrouck, F Vaneechoutte M. 2005. Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of Mycoplasma species. BMC Infect. Dis. 2005, 5: 46.

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