Identification of Acinetobacter genomic species by means of

amplified rDNA restriction analysis (ARDRA)

 

Current status of Acinetobacter taxonomy (February 2009)

Phenotypic differentiation between Acinetobacter genomic species is not always straightforward. Therefore several genotypic identification methods have been developed.

One of these consists of amplification of the 16S rRNA-gene followed by separate restriction digestions with different restriction enzymes. This yields restriction patterns which in combination result in ARDRA profiles which enable differentiation between most species. E.g., the restriction patterns obtained with respectively CfoI, AluI, MboI, RsaI and MspI and numbered respectively 1,1,1, 2 and 3 yield in combination the ARDRA profile 11123 which is characteristic for A. baumannii.

The whole procedure of DNA-extraction (starting from pure culture), amplification of the 16S rDNA, restriction digestion and agarose electrophoresis takes one day (for up to 30 strains). See Mycobacterium.

 

Figure 1. Restriction patterns obtained after restriction digestion with CfoI, AluI, MboI, RsaI and MspI for amplified 16S rDNA of different Acinetobacter species

 

Image12.gif

 

Table 1. ARDRA profiles of 202 Acinetobacter strains previously identified to genomic species by the use of DNA-DNA hybridizationa,b. Patterns are shown in Figure 1.

 

Genomic species

 

Pattern with enzyme

Strains

Reference strain

CfoI

AluI

MboI

RsaI

MspI

(N)

1 (A. calcoaceticus)

 

     2

2

1

1

3

7

ATCC 23055T

 

 

 3

2

1

1

3

1

RUH 583

2 (A. baumannii)

 

 1

1

1

2

3

27

ATCC 17904

 

  

 1

1

1

2

1

33

ATCC 19606T

 

  

1

1

1

2

1+3

4

LMD 82.54

3

 

 2

1

3

1

3

24

ATCC 19004

 

  

2+3

1

3

1

3

2

RUH 1163

4 (A. haemolyticus)

u

1

4

1

2

2

7

ATCC 17906T

5 (A. junii)

l

1

2

1

2

3

6

ATCC 17908T

6

 

 1

1

2

2

2

2

ATCC 17979

7 (A. johnsonii)

u

1

4

1

2

2

8

ATCC 17909T

 

 n

1

4

1

2

2+3

1

RUH 2859

8/9 (A. lwoffii)

 

3

3

2

1

2

19

NCTC 5866T

10

D

4

2

1

2

3

3

ATCC 17924

11

D

4

2

1

2

3

7

ATCC 11171

12 (A. radioresistens)

 

 7

3

2

2

1

3

SEIP 12.81

 

  

 7

3

2

2

3

3

IAM 13186T

 

  

 7

3

2

2

1+3

2

RUH 2862

13 BJ/14 TU

u

1

4

1

2

2

3

SEIP 5.84

 

  

 1

4

1

2

3

6

114 c, RUH 2866

 

 n

1

4

1

2

2+3

6

ATCC 17905

14 BJ

 

5

5

1

2

3

5

CCUG 14816

15 BJ

 

 1

2

1

2

2

2

79 d , RUH 1729

16

 

 1

2

1

4

2

3

ATCC 17988

 

  

 1

2

1

2+4

2

1

LUH 1734

17

l

1

2

1

2

3

2

SEIP 2/87

13TU

 

 2

1

1

1

1

2

ATCC 17903

 

  

 2

1

1

1

3

9

100 d

 

  

 2

1

1

1

1+3

2

RUH 2624

15 TU

 

6

2

1

1

3

2

151a c, RUH 1090

a T, type strain; ATCC, American Type Culture Collection, Rockville, Md, USA; LUH, RUH, collection Leiden University Hospital, Leiden, The Netherlands; LMD, LMD Culture Collection, University of Technology, Delft, The Netherlands; NCTC, National Collection of Type Cultures, London, United Kingdom; SEIP, Service des Entérobactéries de l’Institut Pasteur, Paris, France; IAM, Institute of Applied Microbiology, The University of Tokyo, Tokyo, Japan; CCUG, Culture Collection, University of Göteborg, Göteborg, Sweden.

b (N), Number of strains tested. Markings (u, l, n,D) denote profiles occurring in different genomic species. Most of these species can be further differentiated by restriction digestion with BfaI and BsmaI (Fig. 2 and Table 2).

c Strain and designation used by Tjernberg and Ursing, 1989.

d Strain and designation used by Bouvet and Jeanjean, 1989.

 

Figure 2. Further differentiation is possible with the enzymes BfaI and BsmaI

 Image13.gif

 

 

Table 2. Further discrimination between genomic species or strains with common ARDRA patterns by the use of additional restriction enzymes BfaI and BsmAIa. Restriction patterns are shown in Fig. 2.

Genomic species

ARDRA profile

ARDRA pattern with enzyme

Reference strain

 

 

BfaI

BsmAI

 

4 (A. haemolyticus)

14122

1

2

ATCC 17906T

7 (A. johnsonii)

14122

2

2

ATCC 17909T

13 BJ/14TU

14122

1

2

SEIP Ac 88.239

13BJ

14122

1+2

2

SEIP 5.84

13 BJ

14122+3

5

2

376b

14TU

14122+3

6

2

ATCC 17905

5 (A. junii)

12123

3

3

ATCC 17908T

17

12123

4

2

SEIP 2/87c

Unclassified

12123

4

2

640b, c, SEIP 1/87

Unclassified

12122

9

2

631b, CCUG 14818

Unclassified

12122

10

2

RUH 203

15BJ

12122

8

2

79b

10

42123

7

1

ATCC 17924

11

42123

7

2

ATCC 11171

 

a All strains of A. haemolyticus, A. johnsonii, DNA group 13BJ/14TU, and of A. junii, genomic species 17 were tested with enzyme BfaI. All strains of A. junii, and of DNA group 10, 11 and 17 were tested with BsmAI. In other cases single strains representatitve for a given ARDRA profile were tested.

b Bouvet and Jeanjean, 1989.

c Differentiation between SEIP 2/87 and SEIP 1/87 was made by restriction enzyme StyI (data not shown).

 

 

More ARDRA patterns and profiles have been described, some additional to known groups, some indicating the existence of other Acinetobacter genomic groups (Nemec et al. 2001, Nemec et al. 2003, Chu et al. 1999). See Table 3.

 

 Table 3. ARDRA profiles in strains not yet classifiable by DNA-DNA hybridizationa

Strain with designation as received/ other designation

Specimen

 

Restriction pattern with enzyme

ARDRA pattern identical to DNA group

 

 

 

CfoI

AluI

MboI

RsaI

MspI

 

A. ursingii/ A. phenon 1

 

 

1

4

3

5

3

 

A. schindleri/ A. phenon 2

 

 

1+5

2+4

1

2

2

 

A. venetianus

Sea

 

1

3

2

2

3

 

Ungrouped: 631b, CCUG 14818

Ear

g

1

2

1

2

2

15BJ

A. schindleri: RUH 203

Liquor

g

1

2

1

2

2

15BJ

Ungrouped: 640b, SEIP 1/87

Wound

 

1

2

1

2

3

5, 17

A. phenon 3: 80b, SEIP 14.83

Blood

4

1

4

1

4

3

-

A. phenon 3: 930b, SEIP 87.302

Trachea

4

1

4

1

4

3

-

A. phenon 3: 944b, SEIP Ac87.316

Wound

4

1

4

1

4

3

-

A. phenon 3: 1240b, RUH 65

Clinical

4

1

4

1

4

3

-

A. phenon 10: 1271b , RUH 422

Urine

 

1

5

1

2

3

-

A. ct13TU: MGH 99613, 10095c

Abscess

h

3

5

1

1

3

-

A. ct13TU: MGH 99614, 10169c

Sputum

h

3

5

1

1

3

-

A. ct13TU: MGH 99896, 5804c

Blood

 

3

1

3

1

3

-

A. ct3TU: MGH 99685, 10090c

Ulcer

 

3

1

1+3

1

3

-

Ungrouped: Isolate patient Kd

Blood

 

1

4

3

4

3

-

A. phenon 5: ATCC 13809

Unknown

 

2

5

1

1

3

-

Ungrouped: RUH 175

Blood

 

1

2

1

4

3

-

Ungrouped: RUH 581

Soil

 

3

3

1

1

2

-

A. ct13TU: RUH 1139

Pharynx

 

3

1

1

1

3

-

 

a           Markings g, 4 ,h denote profiles found in multiple unclassified strains.

b           Bouvet and Jeanjean, 1989.

c           Gerner-Smidt and Tjernberg, 1993.

d           Horrevorts et al., 1995.

 

References

Chu et al., 1999. Skin carriage of acinetobacters in Hong Kong. J. Clin. Microbiol. 37: 2962-2967.

Bouvet, P. J. M., and S. Jeanjean. 1989. Delineation of new proteolytic genomic species in the genus Acinetobacter. Res. Microbiol. 140: 291-299.

Dijkshoorn L., B. van Harsselaar, I. Tjernberg, P.J.M. Bouvet, & M. Vaneechoutte. 1998. Evaluation of amplified ribosomal DNA restriction analysis for identification of Acinetobacter genomic species. System. Appl. Microbiol. 21: 33-39.

Gerner-Smidt P. & I. Tjernberg. 1993. Acinetobacter in Denmark: II. Molecular studies of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. APMIS 101: 826-832.

Horrevorts, A., K. Bergman, L. Kollee, I. Breuker, I. Tjernberg, and L. Dijkshoorn. 1995. Clinical and epidemiological investigations of Acinetobacter genomospecies 3 in a neonatal intensive care unit. J. Clin. Microbiol. 33: 1567-1572.

Seifert H., L. Dijkshoorn, P. Gerner-Smidt, N. Pelzer, I. Tjernberg, & M. Vaneechoutte. 1997. Distribution of Acinetobacter species on human skin: comparison of phenotypic and genotypic identification methods. J. Clin. Microbiol. 35: 2819-2825

Vaneechoutte M., L. Dijkshoorn, I. Tjernberg, A. Elaichouni, P. De Vos, G. Claeys, & G. Verschraegen. 1995. Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. J. Clin. Microbiol. 33: 11-15.

Vaneechoutte, M., I. Tjernberg, F. Baldi, M. Pepi, R. Fani, E.R. Sullivan, J. van der Toorn, & L. Dijkshoorn. 1999. The oil-degrading Acinetobacter strain RAG-1 and the strains described as 'Acinetobacter venetianus sp. nov.' belong to the same genomic species. Res. Microbiol. 150: 69-73.

 Further reading

Nemec, A., T. De Baere, I. Tjernberg, M. Vaneechoutte, T.J.K. van der Reijden, and L. Dijkshoorn. 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., L. Dijkshoorn, I. Cleenwerck, T. De Baere, D. Janssens, T. J. Van Der Reijden, P. Jezek, and M. Vaneechoutte. 2003. Acinetobacter parvus sp. nov., a small colony forming species isolated from human specimens. Int. J. Syst. Evol. Microbiol. 53: 1563-1567.

 

For further details, please contact

Lenie Dijkshoorn: Dijkshoorn@rullf2.MedFac.LeidenUniv.nl

Alexandr Nemec: anemec@szu.cz or

Mario Vaneechoutte: Mario.Vaneechoutte@ugent.be

 

HOME