Identification of clinically relevant viridans group streptococci by sequence analysis of the 16S-23S ribosomal DNA spacer region

Abstract

The feasibility of sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (ITS) for the identification of clinically relevant viridans group streptococci (VS) was evaluated. The ITS regions of 29 reference strains (11 species) of VS were amplified by PCR and sequenced. These 11 species were Streptococcus anginosus, S. constellatus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis, and S. uberis. The ITS lengths (246 to 391 bp) and sequences were highly conserved among strains within a species. The intraspecies similarity scores for the ITS sequences ranged from 0.98 to 1.0, except for the score for S. gordonii strains. The interspecies similarity scores for the ITS sequences varied from 0.31 to 0.93. Phylogenetic analysis of the ITS regions revealed that evolution of the regions of some species of VS is not parallel to that of the 16S rRNA genes. One hundred six clinical isolates of VS were identified by the Rapid ID 32 STREP system (bioMérieux Vitek, Marcy l'Etoile, France) and by ITS sequencing, and the level of disagreement between the two methods was 18% (19 isolates). Most isolates producing discrepant results could be unambiguously assigned to a specific species by their ITS sequences. The accuracy of using ITS sequencing for identification of VS was verified by 16S rDNA sequencing for all strains except strains of S. oralis and S. mitis, which were difficult to differentiate by their 16S rDNA sequences. In conclusion, identification of species of VS by ITS sequencing is reliable and could be used as an alternative accurate method for identification of VS.

FIG. 1.

Amplification of VS type strains with primers 13BF and 6R and separation of the PCR products by 2% agarose gel electrophoresis. Lanes: 1, S. mitis; 2, S. oralis; 3, S. constellatus; 4, S. intermedius; 5, S. salivarius; 6, S. anginosus; 7, S. mutans; 8, S. sanguinis; 9, S. gordonii; 10, S. parasanguis; 11, S. uberis; M, 100-bp ladder.

FIG. 2.

Phylogenetic relationship of various species of VS, S. pneumoniae, and S. pyogenes on the basis of the ITS sequences. The phylogenetic tree was generated by the neighbor-joining method within the MEGA package. The numbers at the nodes are the percentages of occurrence in 1,000 bootstrapped resamplings.