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Journal of Clinical Microbiology, April 2009, p. 1281-1282, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.02022-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Identification of Acinetobacter Genomic Species 13TU by Sequence Analysis of the 16S-23S rRNA Gene Spacer Region

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The genus Acinetobacter currently contains up to 32 described named and unnamed (genomic) species (3). Acinetobacter baumannii, genomic species 3, and 13TU, three of the most clinically relevant species, are genetically and phenotypically very similar to an environmental species, Acinetobacter calcoaceticus, and are therefore grouped together into the so-called A. calcoaceticus-A. baumannii (Acb) complex (3). Because phenotypic identification of Acinetobacter isolates to the species level has proven to be insufficient, several genotypic methods have been developed for genomic species identification (1, 3, 4).

In the last 3 years, our laboratory has routinely used the 16S-23S rRNA gene intergenic spacer (ITS) sequence-based method described by Chang et al. (1) to delineate isolates belonging to the Acb complex to the genomic species level. We found the procedure accurate and easy to perform. However, we have recently encountered a discrepancy during the characterization of Acinetobacter strain 74510 isolated originally from a clinical source at the Prince of Wales Hospital, Hong Kong, in 1995 (2). Strain 74510 was previously identified as genomic species 13TU by restriction analysis of amplified ribosomal DNA (2, 4). However, examination of the single PCR amplicon of the ITS region of strain 74510 (GenBank accession no. FJ360743) revealed an ITS sequence length of 607 bp, as opposed to the 615 bp reported by Chang et al. for the genomic species 13TU reference strain BCRC 15417 (1). On the other hand, DNA sequence analysis of this 607-bp ITS of strain 74510 showed an identity of 0.9561 to that of strain BCRC 15417 (Table 1). Upon further analysis of nucleotide sequences in GenBank, the strain 74510 ITS sequence was also found to share increasing identity (0.9950 to 0.9967) with three other genomic species 13TU strains, namely, v104-2, 00574, and DR25612/96 (Table 1). Interestingly, the ITS length (607 bp) of these three genomic species 13TU strains was also 607 bp, identical to that of strain 74510 (Table 1). In addition, the ITS sequences of these genomic species 13TU strains 74510, v104-2, 00574, and DR25612/96 appeared to be more closely related to the A. baumannii (genomic species 2) strain BCRC 10591^T than to the 13TU reference strain BCRC 15417 (0.9538 to 0.9588 versus 0.9171 identity) (Table 1), which reinforces the notion that genetic similarity among A. baumannii and genomic species 13TU is high (3) and perhaps suggests that BCRC 15417 may not be as representative for genomic species 13TU.

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TABLE 1. ITS sequence similarity between A. baumannii and genomic species 13TUa

Acinetobacter genomic species 13TU is the second most clinically relevant species in the Acb complex (3, 5, 6). Therefore, we believe that it is important to extend the number of Acinetobacter genomic species 13TU ITS sequences in the ITS sequence database for more-accurate genomic species identification. Also, we would suggest that the reference ITS sequence length for genomic species 13TU be 607 bp long and that strain 74510 could be considered as the "type" strain for genomic species 13TU.

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1
1. Chang, H. C., Y. F. Wei, L. Dijkshoorn, M. Vaneechoutte, C. T. Tang, and T. C. Chang. 2005. Species-level identification of isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex by sequence analysis of the 16S-23S rRNA gene spacer region. J. Clin. Microbiol. 43:1632-1639.[Abstract/Free Full Text]
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2. Chu, Y.-W., M. Afzal-Shah, E. T. S. Houang, M.-F. Palepou, D. J. Lyon, N. Woodford, and D. M. Livermore. 2001. IMP-4, a novel metallo-ß-lactamase from nosocomial Acinetobacter spp. collected in Hong Kong between 1994 and 1998. Antimicrob. Agents Chemother. 45:710-714.[Abstract/Free Full Text]
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3. Dijkshoorn, L., A. Nemec, and H. Seifert. 2007. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat. Rev. Microbiol. 5:939-951.[CrossRef][Medline]
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4. Dijkshoorn, L., B. van Harsselaar, I. Tjernberg, P. J. M. Bouvet, and M. Vaneechoutte. 1998. Evaluation of amplified ribosomal DNA restriction analysis for identification of Acinetobacter genomic species. Syst. Appl. Microbiol. 21:33-39.[Medline]
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5. Spence, R. P., K. J. Towner, C. J. Henwood, D. James, N. Woodford, and D. M. Livermore. 2002. Population structure and antibiotic resistance of Acinetobacter DNA group 2 and 13TU isolates from hospitals in the United Kingdom. J. Med. Microbiol. 51:1107-1112.[Abstract/Free Full Text]
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6. van Dessel, H., T. E. Kamp-Homans, A. C. Fluyt, S. Brisse, A. M. de Smet, L. Dijkshoorn, A. Troelsta, J. Verhoef, and E. M. Mascini. 2002. Outbreak of a susceptible strain of Acinetobacter species 13 (sensu Tjernberg and Ursing) in an adult neurosurgical intensive care unit. J. Hosp. Infect. 51:89-95.[CrossRef][Medline]

						Raffaele Zarrilli* Maria Giannouli Anna Di Popolo Federica Tomasone Dipartimento di Scienze Mediche Preventive Università di Napoli Federico II Ceinge, Biotecnologie Avanzate Naples, Italy Yiu-Wai Chu Public Health Laboratory Services Branch Centre for Health Protection The Government of the Hong Kong SAR, China
						* Phone: 39-081-7463026 Fax: 39-081-7463352 E-mail: rafzarri{at}unina.it

Authors' Reply

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Acinetobacter calcoaceticus, Acinetobacter baumannii, Acinetobacter genomic species (gen. sp.) 3, and Acinetobacter gen. sp. 13TU, which are included in the Acinetobacter calcoaceticus-A. baumannii complex, are difficult to distinguish by phenotypic methods (1). Members of the complex are the most common acinetobacters among clinical isolates, except for A. calcoaceticus, which is a soil organism and is rarely isolated from clinical samples. Strains of the complex have become an increasing cause of concern in hospitals worldwide because they can give rise to outbreaks. However, different (genomic) species in the complex display significant variability in antimicrobial susceptibility (2). From this, it follows that this differentiation of species within the complex is important from a clinical/epidemiological point of view.

The usefulness of the 16S-23S rRNA gene ITS sequence for the differentiation of (genomic) species in the A. calcoaceticus-A. baumannii complex was demonstrated in our previous study (1) and was confirmed by restriction analysis of amplified ribosomal DNA, amplified fragment length polymorphism analysis of genomic DNA, and the foregoing letter commenting on our study. For sequence-based identification, it is important that accurate reference sequences are available in the public databases. In our previous study (1), the ITS sequence of Acinetobacter gen. sp. 13TU strain BCRC 15417 (identical to strains LMG 993 and ATCC 17903) was used as the reference sequence for identification of other clinical isolates of this species, since strain BCRC 15417 is the only strain of the species deposited in public culture collections. In addition to the reference strain, we deposited the ITS sequences of two clinical isolates (strain no. 00574 [accession no. AY510070] and v104-2 [accession no. AY510071]) of Acinetobacter gen. sp. 13TU in GenBank.

It is interesting to note that indeed strains 00574, v104-2, and 74510 (GenBank accession no. FJ360743) have an ITS length of 607 bp, instead of 615 bp for Acinetobacter gen. sp. 13TU strain BCRC 15417. The length of the ITS fragment seems to be an important inherent characteristic of species in the A. calcoaceticus-A. baumannii complex and could be used as a primary guide for identification of species in the complex. For example, the ITS length is 637 to 638 bp for A. calcoaceticus, 607 bp for A. baumannii, and 619 bp for Acinetobacter gen. sp. 3 (1).

The commentators found that the sequence similarity (0.9538) between Acinetobacter gen. sp. 13TU strain 74510 and A. baumannii BCRC 10591^T was almost identical to that (0.9561) between strain 74510 and Acinetobacter gen. sp. 13TU strain BCRC 15417. Moreover, the ITS sequence similarities between Acinetobacter gen. sp. 13TU strain 74510 and several Acinetobacter gen. sp. 13TU clinical isolates (v104-2, 00574, and DR25612/96) ranged from 0.9950 to 0.9967. Based on the ITS lengths and the sequence similarities among these characterized isolates of Acinetobacter gen. sp. 13TU, it seems that Acinetobacter gen. sp. 13TU strain BCRC 15417 is not a "typical" representative of the species.

We agree with the viewpoint of the commentators that the ITS sequence (GenBank accession no. AY601830) of Acinetobacter gen. sp. 13TU strain BCRC 15417 is not a good representative of the species. However, there would be no problem for identification of clinical isolates in the A. calcoaceticus-A. baumannii complex by BLAST search, since several "typical" sequences of Acinetobacter gen. sp. 13TU are already available in the GenBank database.

We also agree with the suggestion of the commentators that it is important to extend the number of Acinetobacter genomic species 13TU ITS sequences in the ITS sequence database for more-accurate identification of the species. In addition, we agree that the reference ITS sequence length for Acinetobacter gen. sp. 13TU is 607 bp and not 615 bp, as is the case for strain BCRC 15417, and that strain 74510 could be recognized as the type strain of Acinetobacter gen. sp. 13TU.

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1
1. Chang, H. C., Y. F. Wei, L. Dijkshoorn, M. Vaneechoutte, C. T. Tang, and T. C. Chang. 2005. Identification of Acinetobacter isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex by sequence analysis of the 16S-23S rRNA gene spacer region. J. Clin. Microbiol. 43:1632-1639.[Abstract/Free Full Text]
2
2. Dijkshoorn, L., A. Nemec, and H. Seifert. 2007. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat. Rev. Microbiol. 5:939-951.[CrossRef][Medline]

						Mario Vaneechoutte Laboratory Bacteriology Research Department of Clinical Chemistry, Microbiology, and Immunology Ghent University Hospital Ghent, Belgium Lenie Dijkshoorn Department of Infectious Diseases Leiden University Medical Center Leiden, The Netherlands Tsung Chain Chang* Department of Medical Laboratory Science and Biotechnology College of Medicine National Cheng Kung University Tainan, Taiwan
						* Phone: 886-6-2353535, ext. 5790 Fax: 886-6-2363956 E-mail: tsungcha{at}mail.ncku.edu.tw

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Journal of Clinical Microbiology, April 2009, p. 1281-1282, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.02022-08
Copyright © 2009, 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 Zarrilli, R. Articles by Chang, T. C. Search for Related Content PubMed PubMed Citation Articles by Zarrilli, R. Articles by Chang, T. C.