Robust demarcation of fourteen different species groups within the genus Streptococcus based on genome-based phylogenies and molecular signatures

The genus Streptococcus comprises many important pathogens that impact human and animal health. Despite their widespread relevance, accurate classification and methods to reliably distinguish its members remain in-adequate. Hence, we conducted comprehensive phylogenomics and comparative genomic analyses on 70 genome-sequenced members of this genus to delineate their interrelationships. Comprehensive phylogenetic trees were constructed based on multiple datasets of proteins, including 737 core proteins for the genus Streptococcus, 87 proteins conserved within the phylum Firmicutes, GyrA-GyrB, and RpoB-RpoC sequences. Based on the core proteins for Streptococcus species, genetic relatedness based on average amino acid identity was also determined. In parallel, comparative genomic analyses on protein sequences from this genus have identified 134 highly specific molecular markers in the form of conserved signature indels (CSIs) that are specifically shared by either the entire genus or its distinct clades/subclades. Based on the results from these multiple lines of investigations, a reliable picture of the overall evolutionary relationships within the genus Streptococcus has emerged. At the highest level, members of this genus form 2 main clades termed the Mitis-Suis and Pyogenes-Equinus-Mutans clades. Among these 2 major clades, 14 distinct subclades have been identified which include 4 previously identified species groups (viz. Pyogenes, Mutans, Salivarius and Equinus (or Bovis)); the Mitis clade, which is comprised of the Anginosus subclade and three novel subclades (viz. Pneumoniae, Gordonii and Parasanguinis), and six novel subclades termed the Suis, Sobrinus, Halotolerans, Porci, Entericus and Orisratti subclades. Members of the aforementioned subclades are reliably distinguished on the basis of their distinct branching in phylogenetic trees and multiple identified CSIs that are specifically shared by them. The molecular markers described here serve to dermarcate and clarify the interrelationships among different species groups of streptococci, and provide important means for developing novel diagnostic tests and functional studies on these bacteria which should enhance our understanding of them.