AUTHOR=Gonzales-Siles Lucia , Karlsson Roger , Schmidt Patrik , Salvà-Serra Francisco , Jaén-Luchoro Daniel , Skovbjerg Susann , Moore Edward R. B. , Gomila Margarita 

TITLE=A Pangenome Approach for Discerning Species-Unique Gene Markers for Identifications of Streptococcus pneumoniae and Streptococcus pseudopneumoniae

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=10

YEAR=2020

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2020.00222

DOI=10.3389/fcimb.2020.00222

ISSN=2235-2988

ABSTRACT=<p>Correct identifications of isolates and strains of the Mitis-Group of the genus <italic>Streptococcus</italic> 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. <italic>Streptococcus pneumoniae</italic> and <italic>Streptococcus pseudopneumoniae</italic> are the most closely related species of the clade. In this study, publicly-available genome sequences for <italic>Streptococcus pneumoniae</italic> and <italic>S. pseudopneumoniae</italic> were analyzed, using a pangenomic approach, to find candidates for species-unique gene markers; ten species-unique genes for <italic>S. pneumoniae</italic> and nine for <italic>S. pseudopneumoniae</italic> were identified. These species-unique gene marker candidates were verified by PCR assays for identifying <italic>S. pneumoniae</italic> and <italic>S. pseudopneumoniae</italic> strains isolated from clinical samples. All determined species-level unique gene markers for <italic>S. pneumoniae</italic> were detected in all <italic>S. pneumoniae</italic> clinical isolates, whereas fewer of the unique <italic>S. pseudopneumoniae</italic> 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 <italic>S. pneumoniae</italic> gene markers and for three of the <italic>S. pseudopneumoniae</italic> gene markers. Application of multiple species-level unique biomarkers of <italic>S. pneumoniae</italic> and <italic>S. pseudopneumoniae</italic>, is proposed as a protocol for the routine clinical laboratory for improved, reliable differentiation, and identification of these pathogenic and commensal species.</p>