Identification of 43 Streptococcus Species by Pyrosequencing Analysis of the rnpB Gene

Abstract

Pyrosequencing technology was evaluated for identification of species within the Streptococcus genus. Two variable regions in the rnpB gene, which encodes the RNA subunit of endonuclease P, were sequenced in two reactions. Of 43 species, all could be identified to the species level except strains of the species pairs Streptococcus anginosus/S. constellatus and S. infantis/S. peroris. A total of 113 blood culture isolates were identified by pyrosequencing analysis of partial rnpB sequences. All but eight isolates could be unambiguously assigned to a specific species when the first 30 nucleotides of the two regions were compared to an rnpB database comprising 107 streptococcal strains. Principal coordinate analysis of sequence variation of strains from viridans group streptococci resulted in species-specific clusters for the mitis and the salivarius groups but not for the anginosus group. The identification capacity of pyrosequencing was compared to the biochemical test systems VITEK 2 and Rapid ID 32 Strep. The concordance between pyrosequencing and VITEK 2 was 75%, and for Rapid ID 32 Strep the corresponding figure was 77%. Isolates with discrepant identifications in the three methods were subjected to entire rnpB DNA sequence analysis that confirmed the identifications by pyrosequencing. In conclusion, pyrosequencing analysis of the rnpB gene can reliably identify Streptococcus species with high resolution.Species identification by molecular methods is emerging in the field of microbiological diagnostics and the demand for simple, sensitive, specific, and robust tests is increasing. Broad range identification methods are needed for genera with a large number of species. The genus Streptococcus is a diverse group of approximately 50 species, including many important pathogens for humans and domesticated animals. Current means of classification rely on manual or automated phenotypic test systems, but up to 25% of examined streptococcal strains are incorrectly or ambiguously identified (13, 24). The misidentifications occur because of phenotypic trait variability among strains within a given species (3) and inconsistency in biochemical traits of the same strain (41). Small alterations in manual procedures of the tests may also give false results.Several DNA-based methods have been evaluated for species identification of streptococci. DNA hybridization has been successfully used for detection of few species (4, 18) but has a limited use in broad range identification. Streptococcal species can be identified by measuring the tRNA intergenic lengths (8) and up to 31 species were distinguishable when the fragments were separated by capillary electrophoresis (2). Size separation can also be used for species identification when combined with amplification of selected targets (11, 23), arbitrarily primed PCR (33), and restriction of amplified rRNA genes (21, 37). The latter can discriminate a large number of species, but in all of these methods the identification is based on complex band patterns with limitations in resol...

Utility of Pyrosequencing in Identifying Bacteria Directly from Positive Blood Culture Bottles

“…variability within the 23S rRNA gene targets did not provide species-level identification for all bacteria, it did provide genus-level identification for most other organisms associated with bloodstream infection. Certain highly related bacteria, e.g., S. pneumoniae and S. mitis, or Enterobacter species and C. freundii were not differentiated using this target; DNA targets outside of the rRNA genes will most likely be needed for this purpose (3,9,10,20,21,25). The overall agreement between pyrosequencing and culturebased identification methods was high (97.8% [264/270]).…”

“…Turenne et al used single-stranded conformation polymorphism analysis of PCR amplicons to distinguish between organisms (23), while Peters et al used fluorescence in situ hybridization to identify pathogens out of positive blood cultures (19). Several investigators have used pyrosequencing (Biotage, Uppsala, Sweden) to identify numerous bacteria, yeasts, and fungi (9)(10)(11).…”

A Genome-Wide Profiling Strategy as an Aid for Searching Unique Identification Biomarkers for Streptococcus

“…However, it involves more inputs and selection of other genes out of a few thousand genes within the genome is not an easy task. In spite of the fact that around 23 genes have been used frequently in many studies carried out for identifying Streptococcus, no consensus gene has been identified [3,4,11,[21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. It has also not been realized that except recA, the rest of the 22 genes are not present in all the species of Streptococcus.…”

“…Although, multilocus sequence analysis (MLSA) has been relatively quite successful in the identification of a wide range of bacterial species [33], however, using sequences of 7 HKGs: guaA, map, pfl, ppaC, pyk, rpoB, and sodA, also did not prove suitable for rapid identification of Streptococcus species [21]. MLSA using gdh, ddl along with guaA, map, pfl, ppaC, pyk, rnpB, rpoB, sodA, and tuf have been useful in identifying Streptococcus species [32,34,35].…”

Association of oral streptococci community dynamics with severe early childhood caries as assessed by PCR-denaturing gradient gel electrophoresis targeting the rnpB gene

“…All sequences of the dominant bands could be identified to the species level except for the species pairs of S. mitis/pneumoniae and S. infantis/peroris. The inability of our assay to distinguish these two pairs of species may due to a high level of similarity between them, making identification of some genotypes of these species difficult solely on the basis of the rnpB gene (Innings et al, 2005;Täpp et al, 2003).…”