Heterogeneity among 16S-23S rRNA intergenic spacers of species within the Streptococcus milleri group

The 16S-23S rRNA intergenic spacer has been suggested as a suitable region of the bacterial genome from which to derive useful taxonomic information, particularly with regard to identification at the species level. To investigate this approach as an aid to the identification of the three species comprising the ‘Streptococcus milleri group’ (SMG), the spacers of isolates of Streptococcus intermedius, Streptococcus anginosus and Streptococcus constellatus were amplified by PCR and length polymorphisms determined by agarose gel electrophoresis. Phenotypically atypical isolates which had been identified presumptively as belonging to these three species were also included. Spacers from two representatives of each spacer length found within the three SMG species were sequenced. 16S-23S rRNA intergenic spacer length polymorphisms allowed discrimination between S. anginosus (350 bp or 450 bp amplification product) and S. constellatus (380 bp amplification product), species that are difficult to differentiate phenotypically. S. intermedius (330 bp or 450 bp amplification product) and S. anginosus (350 bp or 450 bp amplification product) were not reliably distinguished by this method but are phenotypically distinct. Sequencing data demonstrated that the spacers had a central region of highly variable length flanked by conserved regions which included a single tRNAAla gene. Polymorphism in the length of the 16S-23S spacer determined by PCR provides a rapid and useful adjunct to strain identification for S. anginosus and S. constellatus, which are not readily differentiated phenotypically.