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

Abstract

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 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.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.

Insertions or Deletions (Indels) in the rrn 16S-23S rRNA Gene Internal Transcribed Spacer Region (ITS) Compromise the Typing and Identification of Strains within the Acinetobacter calcoaceticus-baumannii (Acb) Complex and Closely Related Members

“…The feasibility of applying ITS sequences to identify reliably members of the Acinetobacter Acb complex has been addressed here. Earlier work had claimed that the length of the ITS region was an inherent characteristic for each Acb species, and hence could be used potentially as a primary tool for their individual identification [25] . This proposal seems to hold true for A. baumannii, where ITS copies of all strains appear to share the same length (608 bp), and where the only sequence differences are single nucleotide polymorphisms.…”

“…However, while the majority of the publicly available ITS sequences of the A. nosocomialis strains are 607 bp, those of the type strain A. nosocomialis BCRC15417 T are 615 bp ( Table 1 ). Furthermore, the differences in sequence between the 607 bp and 615 bp A. nosocomialis ITSs must be substantial as such a small (8 bp) length difference between otherwise identical ITS >600 bp would give a similarity score of >98%, and not the reported 95% [18] . Whether this variance is the result of point mutations or recombination events has never been examined.…”

Dissemination of ceftazidime-resistant Acinetobacter baumannii clonal complex 92 in Korea

“…A total of 376 nonduplicate consecutive clinical isolates of A. baumannii were collected from 19 different hospitals in Korea in 2008. The isolates were identified using the ATB 32 GN system (bioMérieux, Marcy l’Etoile, France) and 16S‐23S rRNA intergenic spacer region sequencing (Zarrilli et al. 2009).…”

Species identification within Acinetobacter calcoaceticus–baumannii complex using MALDI-TOF MS

“…Molecular methods such as 16S rRNA and rpoB gene sequencing and have shown to be useful in differentiating members of the Acinetobacter genus (La Scola et al, 2006;Zarrilli et al, 2009;Wang et al, 2014). However, neither method is sufficient to differentiate species such as A. pittii and A. calcoaceticus (Higgins et al, 2010), and other molecular methods such as PCR amplification of intrinsic resistance genes or gyrB are used.…”