Identification of species of Abiotrophia, Enterococcus, Granulicatella and Streptococcus by sequence analysis of the ribosomal 16S-23S intergenic spacer region.

The feasibility of sequence analysis of the ribosomal 16S-23S intergenic spacer region (ITS) was evaluated for identification of 24 species of Streptococcus, one species of Abiotrophia, 18 species of Enterococcus and three species of Granulicatella. As GenBank currently lacks ITS sequence entries for many species of these four genera, the ITS sequences of 38 type strains were first sequenced and submitted to GenBank to facilitate species identification of these genera. Subsequently, the ITS sequences of 217 strains (84 reference strains and 133 clinical isolates) were determined and species identification was made by blast search for homologous sequences in public databases. Species other than Streptococcus contained multiple ITS fragments and only the shortest fragment was analysed. A total of 25 isolates (11.5 %) produced discrepant identification by ITS sequencing. The 25 discordant strains were analysed further by sequencing of the 16S rRNA gene for species clarification, and 21 were found to be identified correctly by ITS sequence analysis. The correct identification rate by ITS sequencing was 98.2 % (213/217). Several closely related enterococcal and streptococcal species/subspecies contained specific ITS signature sequences that were useful for differentiating these bacteria. In conclusion, ITS sequencing provides a useful approach towards identifying this group of pathogens on a molecular platform alongside 16S rRNA gene sequencing.