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

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.

Identification of Streptococcus pneumoniae and other Mitis streptococci: importance of molecular methods

“…This situation is associated with the common genetic ancestry of the Mitis group, relatively frequent HGT among streptococcal species, facilitated by their natural competence, together with a gene loss, resulting in populations with highly diverse gene content [89,90]. In consequence, the core genome (genes common for each genome of a species) of S. pneumoniae is much smaller than the pangenome (a complete set of genes of a species) and many of these genes are shared with other Mitis streptococci, including determinants of factors playing the role in interactions with the host allowing a successful survival [91][92][93][94]. The recent genomic analyses of the Mitis group streptococci are discussed in more detail below (see Genomic studies in the identification of Mitis streptococci).…”

“…Four of loci characteristics for S. pneumoniae , including the piaABCD operon, the locus containing SP2020 and two single genes, encoding a putative ABC transporter and a hypothetical protein, were common with the set of 49 genes from the previously mentioned study [ 92 ]. Additional analyses of six selected genes, longer than 500 bp, considered specific for S. pneumoniae and proposed as PCR targets revealed the presence of four of them in a few genomes of S. pseudopneumoniae consistent with the need to use more than a single marker for identification purposes [ 94 ].…”

“…Of these 224 genes, 49 ones, which were totally absent from S. mitis , S. pseudopneumoniae and S. oralis , included genes encoding surface-located proteins PspA, CbpA and “Xisco”, the iron uptake operon piaABCD , genes involved in carbohydrate metabolism and others. Pangenomic analysis of 32 genomes of S. pneumoniae , 36 genomes of S. mitis and 13 genomes of S. pseudopneumoniae followed by a validation of results on larger panels of streptococcal genomes revealed ten genes, residing in five loci, specific solely for S. pneumoniae and nine genes unique for S. pseudopneumoniae [ 94 ]. Four of loci characteristics for S. pneumoniae , including the piaABCD operon, the locus containing SP2020 and two single genes, encoding a putative ABC transporter and a hypothetical protein, were common with the set of 49 genes from the previously mentioned study [ 92 ].…”

Bacterial pathogen detection by conventional culture‐based and recent alternative (polymerase chain reaction, isothermal amplification, enzyme linked immunosorbent assay, bacteriophage amplification, and gold nanoparticle aggregation) methods in food samples: A review

“…In addition, bioinformatics, such as pan‐genome analysis, can also be applied to develop new primer/probe (Kim, Gu, Kim, & Lee, 2020; Wilson et al, 2009). For example, Gonzales‐Siles et al (2020) used pan genome approach to find unique gene markers such as yfmC , fepD , yfhA , yusV , gmu R and ybhF , gdpE , ypdB , kpdD for identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae , respectively. Furthermore, multiplex PCR has been developed to detect and differentiate pathogens in one step, which has economic value as it reduces cost and time.…”

Comparative Genomic and Functional Evaluations of Bacillus subtilis Newly Isolated from Korean Traditional Fermented Foods

“…Competence pheromone SRCM103571, SRCM103689 Pangenome analysis compares the entire gene set of all strains of a species, enabling assessment of the genomic diversity of entire repertoires of genes and identification of the core genomic elements [62]. To analyze the genomic differences among B. subtilis isolated from fermented products in East Asian countries, we next performed pangenome analysis using 61 genomes of different B. subtilis species, including 29, 3, and 24 B. subtilis strains isolated from China, Japan, and Korea, respectively, as well as the 5 strains isolated in this study.…”