Identification of 43StreptococcusSpecies by Pyrosequencing Analysis of thernpBGene

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Abstract
Pyrosequencing technology was evaluated for identification of species within theStreptococcusgenus. Two variable regions in thernpBgene, 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 pairsStreptococcus anginosus/S. constellatusandS. infantis/S. peroris. A total of 113 blood culture isolates were identified by pyrosequencing analysis of partialrnpBsequences. All but eight isolates could be unambiguously assigned to a specific species when the first 30 nucleotides of the two regions were compared to anrnpBdatabase 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 entirernpBDNA sequence analysis that confirmed the identifications by pyrosequencing. In conclusion, pyrosequencing analysis of thernpBgene can reliably identifyStreptococcusspecies with high resolution.

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