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Identification and capsular serotype sequetyping of Streptococcus pneumoniae strains

Journal article
Authors Lucia Gonzales-Siles
Franciso Salvà-Serra
Anna Degerman
Rickard Nordén
Magnus Lindh
Susann Skovbjerg
Edward R.B. Moore
Published in Journal of Medical Microbiology
Volume 68
Issue 8
Pages 1173-1188
ISSN 0022-2615
Publication year 2019
Published at Centre for antibiotic resistance research, CARe
Institute of Biomedicine, Department of Infectious Medicine
Pages 1173-1188
Language en
Keywords Streptococcus pneumoniae, serotype, sequetyping, Streptococcus mitis group, invasive pneumococcal disease, multiplex pcr, mitis group, s-mitis, vaccine, burden, assay, gene, differentiation, diagnosis, Microbiology, crobiology, v59, p2317, ates of america, v102, p2567
Subject categories Infectious Medicine


Purpose. Correct serotype identification of Streptococcus pneumoniae (pneumococcus) is important for monitoring disease epidemiology and assessing the impacts of pneumococcal vaccines. Furthermore, correct identification and differentiation of the pathogenic S. pneumoniae from closely related commensal species of the mitis group of the genus Streptococcus are essential for correct serotype identification. Methodology. A new protocol for determining the existing 98 serotypes of pneumococcus was developed, applying two PCR amplifications and amplicon sequencing, using newly designed internal primers. The new protocol was validated using S. pneumoniae genome sequences, reference strains with confirmed serotypes and clinical isolates, and comparing the results with those from the traditional Quellung reaction or antiserum panel gel precipitation, in addition to real-time PCR analysis. The taxonomic identifications of 422 publicly available (GenBank) genome sequences of S. pneumoniae, Streptococcus pseudopneumoniae and Streptococcus mitis were assessed by whole-genome sequence average nucleotide identity based on blast (ANIb) analysis. Results. The proposed sequetyping protocol generates a 1017 bp whole cpsB region sequence, increasing resolution for serotype identification in pneumococcus isolates. The identifications of all GenBank genome sequences of S. pneumoniae were confirmed, whereas most of the S. pseudopneumoniae and almost all of the S. mitis genome sequences did not fulfil the ANIb thresholds for species-level identification. The housekeeping biomarker gene, groEL, correctly identified S. pneumoniae but often misclassified S. pseudopneumoniae and S. mitis as S. pneumoniae. Conclusions. These studies affirm the importance of applying reliable identification protocols for S. pneumoniae before serotyping; our protocols provide reliable diagnostic tools, as well as an improved workflow, for serotype identification of pneumococcus and differentiation of serogroup 6 types.

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