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

Abstract

To determine whether ITS sequences in the rrn operon are suitable for identifying individual Acinetobacter Acb complex members, we analysed length and sequence differences between multiple ITS copies within the genomes of individual strains. Length differences in ITS reported previously between A. nosocomialis BCRC15417T (615 bp) and other strains (607 bp) can be explained by presence of an insertion (indel 13i/1) in the longer ITS variant. The same Indel 13i/1 was also found in ITS sequences of ten strains of A. calcoaceticus, all 639 bp long, and the 628 bp ITS of Acinetobacter strain BENAB127. Four additional indels (13i/2–13i/5) were detected in Acinetobacter strain c/t13TU 10090 ITS length variants (608, 609, 620, 621 and 630 bp). These ITS variants appear to have resulted from horizontal gene transfer involving other Acinetobacter species or in some cases unrelated bacteria. Although some ITS copies in strain c/t13TU 10090 are of the same length (620 bp) as those in Acinetobacter strains b/n1&3, A. pittii (10 strains), A. calcoaceticus and A. oleivorans (not currently acknowledged as an Acb member), their individual ITS sequences differ. Thus ITS length by itself can not by itself be used to identify Acb complex strains. A shared indel in ITS copies in two separate Acinetobacter species compromises the specificity of ITS targeted probes, as shown with the Aun-3 probe designed to target the ITS in A. pitti. The presence of indel 13i/5 in the ITS of Acinetobacter strain c/t13TU means it too responded positively to this probe. Thus, neither ITS sequencing nor the currently available ITS targeted probes can distinguish reliably between Acb member species.

Unusual features of the sequences of copies of the 16S–23S rRNA internal transcribed spacer regions of Acinetobacter bereziniae, Acinetobacter guillouiae and Acinetobacter baylyi arise from horizontal gene transfer events

“…1 in their paper) has been constructed with single variant ITS sequences, seemingly those of the shortest ITS copy from each strain. Tree topology would change markedly if the long ITS of A. baylyi ADP1 or those from A. baylyi 93A2 had been used instead, The same comments apply to A. bereziniae and A. guillouiae, and all species including those Acb complex members that possess multiple variant ITS copies (Maslunka et al, 2014). Furthermore, Chen et al (2007) stated that analysing the sequences of either their long or short ITS copies would not result in misidentification of A. baylyi strains.…”

“…The prospect that horizontal gene transfer may be responsible for the variable length ITS of Acinetobacter species is not one that has been explored previously, except with Acb members (Maslunka et al, 2014). These results demonstrate that such events may affect both those species containing variable length ITS, and those for which only single ITS lengths have been reported (Table 1, Figs S1, S2 and S3).…”

“…indels, genomic fragments probably acquired by horizontal transfer events involving other Acinetobacter species and, in some cases, members of unrelated bacterial genera (Maslunka et al, 2014). With the exceptions of Acb members (Maslunka et al, 2014) and A. baylyi (Carr et al, 2004), explanations for why ITS length/sequence variations might occur in Acinetobacter species have never been reported.…”

“…With the exceptions of Acb members (Maslunka et al, 2014) and A. baylyi (Carr et al, 2004), explanations for why ITS length/sequence variations might occur in Acinetobacter species have never been reported. Unlike the situation with Acb members (Maslunka et al, 2014), Carr et al (2004) proposed that the A. baylyi long and short ITS copies probably resulted from intra-genomic recombination events, without considering that indels might have been responsible.…”

“…For each, ITStargeted oligonucleotide probes have been described, but were designed using the only ITS sequence (that of the shortest copy) then publicly available for each species (Lin et al, 2008). Consequently, it was decided to extend the earlier study carried out with Acb and closely related Acinetobacter members (Maslunka et al, 2014) to attempt to determine the reasons for these reported ITS length variations, and to revisit the earlier work with A. baylyi (Carr et al, 2004). Attempts were also made to establish whether having intra-genomic variable length ITS copies might compromise the validity of the currently available A. bereziniae and A. guillouiae ITS-targeted probes, as is the case with members of the Acb complex (Maslunka et al, 2014).…”