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.
Highlights
Acinetobacter spp. are important nosocomial pathogens and epidemiological survey data from around the world suggest that multi-antibiotic resistant Acinetobacter baumannii, A. pittii and A. nosocomialis are the most important clinically [1,2,3]
As intragenomic Internal Transcribed Spacer Region (ITS) sequence variance had never been confirmed in member species of this group, they proposed that the direct sequencing of Acinetobacter calcoaceticus-baumannii (Acb) ITS sequences provided a promising method for their identification [9]
The ITS of type strains sequenced by Chang et al [9] were designated by them as A. calcoaceticus LMG1046T, A. baumannii BCRC10591T and A. pittii LMG10350T, while the same strains sequenced here are designated as A. calcoaceticus ATCC23055T, A. baumannii ATCC19606T and A. pittii ATCC19004T (Table 1)
Summary
Acinetobacter spp. are important nosocomial pathogens and epidemiological survey data from around the world suggest that multi-antibiotic resistant Acinetobacter baumannii (genomic species 2), A. pittii (genomic species 3) and A. nosocomialis (genomic species TU13) are the most important clinically [1,2,3] These three species were grouped with A. calcoaceticus (genomic species BG1), a soil organism, into the A. calcoaceticus-A. baumannii (Acb) complex because of their close genetic similarities [4,2]. Interest in using chemotaxonomic and molecular methods to distinguish between Acb complex members has increased, as phenotypic characters have generally proved to be unreliable for this task Many such methods have been described [7], and include exploiting the sequence variations in the 16S–23S rRNA gene internal transcribed spacer region (ITS sequences) in the rrn operons [8]. All Acinetobacter spp. are known to possess multiple ITS copies [10,11], ITS targeted probes have been designed to identify Acb members [12,13,14,15,16,17] on the premise that the extent of Acb intra-species ITS copy sequence variance is low
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