Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase

Abstract

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodA(int)) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus, S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus, S. canis, S. cricetus, S. downei, S. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus, S. equinus, S. gordonii, S. iniae, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis, S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis, S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodA(int) fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of their sodA(int) fragments on the phylogenetic tree of the sodA(int) fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.

FIG. 1

Amplification of streptococcal type strains with the primers d1 and d2 and separation of the amplicons by 1% agarose gel electrophoresis. Lanes: 1, 1-kb ladder (Gibco, BRL); 2, S. acidominimus; 3, S. agalactiae; 4, S. alactolyticus; 5, S. anginosus; 6, S. bovis; 7, S. constellatus; 8, S. cricetus; 9, S. downei; 10, S. dysgalactiae; 11, S. equi subsp. equi; 12, S. equi subsp. zooepidemicus; 13, S. equinus; 14, S. gordonii; 15, S. intermedius; 16, S. mitis; 17, S. mutans; 18, S. oralis; 19, S. parasanguis; 20, S. pneumoniae; 21, S. porcinus; 22, S. pyogenes; 23, S. salivarius; 24, S. sanguis; 25, S. sobrinus; 26, S. thermophilus; 27, S. suis; 28, S. vestibularis. Arrowheads, 480-bp amplicon.

FIG. 2

Phylogenetic unrooted tree showing relationships among the sodA_int fragments from various streptococcal type strains. The tree was established from an analysis of the sequences listed in Table 1 by using the neighbor-joining method (26). The value on each branch is the estimated confidence limit (expressed as a percentage) for the position of the branch as determined by bootstrap analysis. The scale bar (neighbor-joining [NJ] distance) represents 10% differences in nucleotide sequences.

FIG. 3

Electropherograms showing part of the nucleotide sequence of sodA_int from S. porcinus. Sequence reactions were carried out with a pUC18ΩsodA_int recombinant plasmid with the −21 dye primer sequencing kit (A) and sodA_int PCR product with the degenerate oligonucleotide d2 and the dRhodamine dye terminator sequencing kit (B).