Identification and capsular serotype sequetyping ofStreptococcus pneumoniaestrains

Abstract

ABSTRACTCorrect identification ofStreptococcus pneumoniae(pneumococcus) and differentiation from the closely related species of the Mitis group of the genusStreptococcus, as well as serotype identification, is important for monitoring disease epidemiology and assessing the impacts of pneumococcal vaccines. In this study, we assessed the taxonomic identifications of 422 publicly available genome sequences ofS. pneumoniae, S. pseudopneumoniaeandS. mitis, using different methods. Identification ofS. pneumoniae, by comparative analysis of thegroELpartial sequence, was possible and accurate, whereasS. pseudopneumoniaeandS. mitiscould be misclassified asS. pneumoniae, suggesting thatgroELis unreliable as a biomarker for differentiatingS. pneumoniaefrom its closest related species. The genome sequences ofS. pneumoniaeandS. pseudopneumoniaefulfilled the suggested thresholds of average nucleotide identity (ANI), i.e., > 95% genome sequence similarity to the sequence of respective type strains for identification of species, whereas none of theS. mitisgenome sequences fulfilled this criterion. However, ANI analyses of all sequencesversusall sequences allowed discrimination of the different species by clustering, with respect to species type strains. Thein silicoDNA-DNA distance method was also inconclusive for identification ofS. mitisgenome sequences, whereas presence of the “Xisco” gene proved to be a reliable biomarker forS. pneumoniaeidentification. Furthermore, we present an improved sequetyping protocol including two newly-designed internal sequencing primers with two PCRs, as well as an improved workflow for differentiation of serogroup 6 types. The proposed sequetyping protocol generates a more specific product by generating the whole gene PCR-product for sequencing, which increases the resolution for identification of serotypes. Validations of both protocols were performed with publicly availableS. pneumoniaegenome sequences, reference strains at the Culture Collection University of Gothenburg (CCUG), as well as with clinical isolates. The results were compared with serotype identifications, using real-time Q-PCR analysis, as well as the Quellung reaction or antiserum panel gel-precipitation. Our protocols provide a reliable diagnostic tool for taxonomic identification as well as serotype identification ofS. pneumoniae.