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Journal of Clinical Microbiology, August 2008, p. 2826-2827, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.01003-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Lack of Evidence for "Acinetobacter septicus" as a Species Different from Acinetobacter ursingii?

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In their recent paper, Kilic et al. (2) reported on a hospital outbreak of bloodstream infections caused by a non-glucose-acidifying Acinetobacter strain. The authors showed that this strain (AK001) was phenotypically most similar to Acinetobacter ursingii but had two properties atypical of this species: i.e., the ability to hemolyse sheep blood and the inability to grow on citrate. Additional taxonomic methods to clarify the taxonomic status of AK001 included 16S rRNA and rpoB gene sequence analysis and DNA-DNA reassociation. Although the results of the sequence analyses supported the close relationship of the strain to A. ursingii, the value of DNA-DNA reassociation between AK001 and the A. ursingii type strain was found to be 64.7 to 68.7%, which is slightly less than 70%, the recommended threshold for species delineation (6). The authors concluded that the strain represents a new species, for which the name "Acinetobacter septicus" was proposed.

As, in our opinion, the data provided by Kilic et al. (2) do not convincingly substantiate the proposal of a novel species, we analyzed strain AK001 using methods that had previously been used to resolve Acinetobacter taxonomy (3). AK001 was obtained by the Culture Collection, University of Göteborg (CCUG) from Y.-W. Tang and was designated CCUG 56015. The strain was shown to have properties consistent with the original description of A. ursingii, except for the inability to utilize citrate (4). It is of note, however, that there are other citrate-negative A. ursingii strains in our current collection, which indicates the intraspecies variation of this property. Importantly, the hemolytic activity of AK001 reported by Kilic et al. (2) could not be confirmed, although sheep blood agar plates obtained from different suppliers were used at different cultivation temperatures and with several control hemolytic Acinetobacter strains.

In the study by Kilic et al. (2), the 16S rRNA gene sequences of AK001 were 99.5% similar to that of the A. ursingii type strain, which is the value that can be found in strains within one species (4). To obtain more comprehensive information on the rpoB-based position of AK001 within the genus Acinetobacter, we determined the rpoB sequence of AK001 and compared it to that of eight A. ursingii strains and all hitherto-described species. The similarity values between AK001 (GenBank no. EU742582) and the A. ursingii strains (EU477105 and EU742575 to EU742581) ranged from 96.9% to 98.0%, which partially overlaps with the range of intraspecies values found for Acinetobacter beijerinckii (3). In contrast, the rpoB similarity values between AK001 and the other species were between 78.8% and 84.5%.

The proposal of "A. septicus" was based mainly on the results of DNA-DNA reassociation. However, DNA-DNA reassociation values close to the proposed value of 70% have to be treated with caution as incongruent results for the same strains can be obtained in different laboratories and/or by different methods, which may have taxonomic implications (5). Concerning also other limitations of DNA-DNA reassociation (1), no conclusions based on these intermediate values should be made without further evidence of genetic isolation between strains. To address this problem, the relatedness of AK001 to hitherto-described species was investigated by AFLP (Keygene NV) analysis, a whole-genome fingerprinting method. Previous studies using this method have shown that strains of the same (genomic) species cluster together at a 50% similarity level (4). AFLP analysis showed that AK001 and 25 reference A. ursingii strains were linked in a distinct cluster at a 59% similarity level, while AK001 was separated from other species at 34% similarity, which strongly supports the assignment of AK001 to A. ursingii.

Based on the above arguments, strain AK2001 is considered as belonging to A. ursingii and not representing a novel species.

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1. Hartford, T., and P. H. Sneath. 1988. Distortion of taxonomic structure from DNA relationships due to different choices of reference strains. Syst. Appl. Microbiol. 10:241-250.
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2. Kilic, A., H. Li, A. Mellmann, A. C. Basustaoglu, M. Kul, Z. Senses, H. Aydogan, C. W. Stratton, D. Harmsen, and Y.-W. Tang. 2008. Acinetobacter septicus sp. nov. association with a nosocomial outbreak of bacteremia in a neonatal intensive care unit. J. Clin. Microbiol. 46:902-908.[Abstract/Free Full Text]
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3. Nemec, A., T. De Baere, I. Tjernberg, M. Vaneechoutte, T. J. 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.[Abstract]
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4. Nemec, A., M. Musilek, M. Maixnerova, T. De Baere, T. J. van der Reijden, M. Vaneechoutte, and L. Dijkshoorn. Acinetobacter beijerinckii sp. nov. and Acinetobacter gyllenbergii sp. nov., haemolytic organisms isolated from humans. Int. J. Syst. Evol. Microbiol., in press.
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5. Vaneechoutte, M., T. De Baere, A. Nemec, M. Musílek, T. J. van der Reijden, and L. Dijkshoorn. 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.[Abstract/Free Full Text]
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6. Wayne, L. G., D. J. Brenner, R. R. Colwell, P. A. D. Grimont, O. Kandler, M. I. Krichevsky, L. H. Moore, W. E. C. Moore, R. G. E. Murray, E. Stackebrandt, M. P. Starr, and H. G. Trüper. 1987. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol. 37:463-464.[Free Full Text]

						Alexandr Nemec* Martin Musílek Centre of Epidemiology and Microbiology National Institute of Public Health robárova 48 100 42 Prague 10, Czech Republic Mario Vaneechoute Department of Clinical Chemistry, Microbiology and Immunology University Hospital, Blok A B-9000 Gent, Belgium Enevold Falsen Culture Collection, Department of Clinical Bacteriology University of Göteborg SE-413 46 Göteborg, Sweden Lenie Dijkshoorn Department of Infectious Diseases Leiden University Medical Center C5-P, P.O. Box 9600 2300 RC Leiden, The Netherlands
						* Phone: 420 267082266 Fax: 420 267082538 E-mail: anemec{at}szu.cz

Authors' Reply

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We would like to respond to this letter by first pointing out that, "Taxonomy ... is the most subjective branch of any biological science, and in many ways is more of an art than a science" (3). Nevertheless, we appreciate Nemec and colleagues' comments regarding our findings, as well as their contribution to additional characterization of the type strain of Acinetobacter septicus (6).

The type strain AK001 was first deposited into the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DMSZ), Germany, as DSM 19415. The proposal of AK001 as a novel Acinetobacter species (A. septicus) was mainly based on the findings of DNA-DNA hybridization. While there is no perfect tool for strain reassociation analysis, DNA-DNA hybridization remains the first choice, especially in the field of Acinetobacter taxonomy (8, 9). The DNA-DNA hybridization of A. septicus (DSM 19415) against A. ursingii (DSM 16037) was performed twice at the DSMZ according to standard, well-recognized procedures (2, 4, 5), and the results were interpreted according to the recommendations of an ad hoc committee (10). It will be interesting to see DNA-DNA hybridization results of A. septicus against the 25 reference A. ursingii strains in L. Dijkshoorn's collection. Whether a high-resolution typing method based on amplified fragment length polymorphism analysis can be used as a reliable tool for species reassociation merits further investigation.

Key biochemical reactions of AK001 (DSM 19415), including citrate utilization and hemolysis in sheep blood, were further characterized by DMSZ according to protocols described previously (1). AK001 was deposited in the Culture Collection, University of Göteborg (CCUG), Sweden, as CCUG 56015 approximately 1 year later. During this period of time, several passages of the strain have occurred, which may explain the differences in hemolysis seen by Nemec et al.

In summary, while we consider what percentages of differences are important as well as which techniques should be considered as the standard for species reassociation and circumscription, it is also important to consider the pathological and ecologic niches of microorganisms (7). A. septicus was first recognized as an important human pathogen while causing a sepsis outbreak in neonates.

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1. Bouvet, P. J. M., and P. A. D. Grimont. 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.[Abstract/Free Full Text]
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2. Cashion, P., M. A. Holder-Franklin, J. McCully, and M. Franklin. 1977. A rapid method for the base ratio determination of bacterial DNA. Anal. Biochem. 81:461-466.[CrossRef][Medline]
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3. Cowan, S. T. 1971. Sense and nonsense in bacterial taxonomy. J. Gen. Microbiol. 67:1-8.[Abstract/Free Full Text]
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4. De Ley, J., H. Cattoir, and A. Reynaerts. 1970. The quantitative measurement of DNA hybridization from renaturation rates. Eur. J. Biochem. 12:133-142.[Medline]
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5. Huss, V. A., H. Festl, and K. H. Schleifer. 1983. Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst. Appl. Microbiol. 4:184-192.
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6. Kilic, A., H. Li, A. Mellmann, A. C. Basustaoglu, M. Kul, Z. Senses, H. Aydogan, C. W. Stratton, D. Harmsen, and Y.-W. Tang. 2008. Acinetobacter septicus sp. nov. association with a nosocomial outbreak of bacteremia in a neonatal intensive care unit. J. Clin. Microbiol. 46:902-908.[Abstract/Free Full Text]
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7. Rossello-Mora, R., and R. Amann. 2001. The species concept for prokaryotes. FEMS Microbiol. Rev. 25:39-67.[Medline]
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8. Tjernberg, I., and J. Ursing. 1989. Clinical studies of acinetobacter classified by DNA-DNA hybridization. APMIS 97:595-605.[Medline]
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9. Vaneechoutte, M., T. De Baere, A. Nemec, M. Musilek, T. J. van der Reijden, and L. Dijkshoorn. 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.[Abstract/Free Full Text]
10
10. Wayne, L. G., D. J. Brenner, R. R. Colwell, P. A. D. Grimont, O. Kandler, M. I. Krichevsky, L. H. Moore, W. E. C. Moore, R. G. E. Murray, E. Stackebrandt, M. P. Starr, and H. G. Trüper. 1987. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol. 37:463-464.[Free Full Text]

						Yi-Wei Tang* Charles W. Stratton Departments of Pathology and Medicine Vanderbilt University Medical Center Nashville, Tennessee 37232-5310 Alexander Mellmann Dag Harmsen Institute for Hygiene and Department of Periodontology University Hospital Münster Münster, Germany
						* Phone: (615) 322-2035 Fax: (615) 343-8420 E-mail: yiwei.tang{at}vanderbilt.edu

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Journal of Clinical Microbiology, August 2008, p. 2826-2827, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.01003-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nemec, A. Articles by Harmsen, D. Search for Related Content PubMed PubMed Citation Articles by Nemec, A. Articles by Harmsen, D.