Abstract
Correct identifications of isolates and strains of the Mitis-Group of the genus Streptococcus are particularly difficult, due to high genetic similarity, resulting from horizontal gene transfer and homologous recombination, and unreliable phenotypic and genotypic biomarkers for differentiating the species. Streptococcus pneumoniae and Streptococcus pseudopneumoniae are the most closely related species of the clade. In this study, publicly-available genome sequences for Streptococcus pneumoniae and S. pseudopneumoniae were analyzed, using a pangenomic approach, to find candidates for species-unique gene markers; ten species-unique genes for S. pneumoniae and nine for S. pseudopneumoniae were identified. These species-unique gene marker candidates were verified by PCR assays for identifying S. pneumoniae and S. pseudopneumoniae strains isolated from clinical samples. All determined species-level unique gene markers for S. pneumoniae were detected in all S. pneumoniae clinical isolates, whereas fewer of the unique S. pseudopneumoniae gene markers were present in more than 95% of the clinical isolates. In parallel, taxonomic identifications of the clinical isolates were confirmed, using conventional optochin sensitivity testing, targeted PCR-detection for the “Xisco” gene, as well as genomic ANIb similarity analyses for the genome sequences of selected strains. Using mass spectrometry-proteomics, species-specific peptide matches were observed for four of the S. pneumoniae gene markers and for three of the S. pseudopneumoniae gene markers. Application of multiple species-level unique biomarkers of S. pneumoniae and S. pseudopneumoniae, is proposed as a protocol for the routine clinical laboratory for improved, reliable differentiation, and identification of these pathogenic and commensal species.
Highlights
Streptococcus pneumoniae, known as “pneumococcus,” is an important human pathogen, causing severe infections, such as meningitis, pneumonia, and bacteremia and responsible for considerable mortality worldwide (O’Brien et al, 2009)
Taxonomic identifications of the 133 genomes included in the study, by average nucleotide identity based on BLAST (ANIb) similarity analyses, confirmed that all 32 S. pneumoniae genomes were correctly classified and were, included in the study
Only 13 of the 36 S. pseudopneumoniae genomes in GenBank were confirmed, by ANIb similarity analyses, as S. pseudopneumoniae; 15 of the S. “pseudopneumoniae” genomes were identified as S. mitis and the remaining eight were identified as S. oralis
Summary
Streptococcus pneumoniae, known as “pneumococcus,” is an important human pathogen, causing severe infections, such as meningitis, pneumonia, and bacteremia and responsible for considerable mortality worldwide (O’Brien et al, 2009). Arbique et al (2004) described some of these atypical pneumococci as a new species, Streptococcus pseudopneumoniae, which is characterized by being bile insoluble and optochin-resistant, when incubated in 5% CO2 atmosphere, but optochin–susceptible when incubated under ambient atmosphere. Streptococcus pseudopneumoniae is a recognized member of the Viridans-Group streptococci (VGS) and is closely related, phylogenetically and taxonomically to S. pneumoniae and S. mitis, sharing nearly 100% 16S rRNA gene sequence similarity (Kilian et al, 2008). Different studies have shown that these gene markers can be detected in closely related species, and are not always reliable for S. pneumoniae species-level identifications (Whatmore et al, 2000; Kilian et al, 2008; Johnston et al, 2010; Simões et al, 2010; Rolo et al, 2013)
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