Identification of Acinetobacter genomic species 13TU by sequence analysis of the 16S-23S rRNA gene spacer region.

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 socalled 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 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. 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.