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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 [email protected]

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Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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

[email protected]


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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).


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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!


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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)

fromA. 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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009

[A. grimontii] = A. junii


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

PCR

PCR

Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009

6. tRNA intergenic spacer length polymorphism analysis: tDNA-PCR


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009

6. tRNA intergenic spacer length polymorphism analysis: tDNA-PCR


6 tdna pcr size determination by fluorescent capillary electrophoresis digitized dna fingerprints

6. tDNA-PCRSize determination by fluorescent capillary electrophoresis: digitized DNA-fingerprints

Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009


6 tdna pcr tdna pcr based identification listeria ivanovii subsp ivanovii 4 vs subsp londoniensis 4

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.


6 tdna pcr tdna pcr fingerprint library for the genus listeria

6. tDNA-PCR tDNA-PCR fingerprint library for the genus Listeria

Acinetobacter Taxonomy. Boerhaave Course. Leiden. 16 January 2009

2/3


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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


7 amplified rdna restriction analysis ardra pcr rflp analysis of the 16s rrna gene

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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!


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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.


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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-


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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

[email protected]


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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)


Acinetobacter taxonomy identification mario vaneechoutte laboratory bacteriology research university of ghent flander

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

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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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