Identification and capsular serotype sequetyping of Streptococcus pneumoniae strains

Area of Science:

• Microbiology
• Genomics
• Molecular Diagnostics

Background:

• Accurate serotype identification of Streptococcus pneumoniae (pneumococcus) is vital for disease epidemiology and pneumococcal vaccine impact assessment.
• Distinguishing pathogenic S. pneumoniae from closely related commensal Streptococcus species (mitis group) is essential for reliable serotyping.

Purpose of the Study:

• To develop and validate a novel molecular protocol for precise pneumococcal serotype identification.
• To differentiate S. pneumoniae from closely related species within the Streptococcus mitis group.

Main Methods:

• Development of a new sequetyping protocol using PCR amplification and amplicon sequencing with novel internal primers.
• Validation of the protocol against genome sequences, reference strains, and clinical isolates, comparing with Quellung reaction and real-time PCR.
• Taxonomic identification of 422 publicly available genome sequences using whole-genome average nucleotide identity based on blast (ANIb).

Main Results:

• The sequetyping protocol analyzes the 1017 bp cpsB region, enhancing serotype resolution for pneumococcal isolates.
• Confirmed identification of S. pneumoniae genomes, while most S. pseudopneumoniae and S. mitis genomes failed species-level identification thresholds via ANIb.
• The housekeeping gene groEL accurately identified S. pneumoniae but frequently misclassified S. pseudopneumoniae and S. mitis.

Conclusions:

• Reliable identification protocols are critical for S. pneumoniae before serotyping.
• The developed protocols offer dependable diagnostic tools and an improved workflow for pneumococcal serotype identification.
• Enhanced differentiation of pneumococcal serogroup 6 types is achieved with the new methodology.