Genomic Diversity among Strains and Closely Related Species.

The aim of this study was to investigate and clarify the ambiguous taxonomy of and its closely related species using state-of-the-art high-throughput sequencing techniques, and, furthermore, to determine whether sub-clusters identified within and in a previous study by multi locus sequence typing (MLST) using concatenation of seven housekeeping genes should either be classified as subspecies or distinct species. The strains in this study were broadly classified under group as genospecies I and genospecies II. Based on MLST data analysis, these were further classified as and . The whole genome sequencing of selected strains of ( = 17) and ( = 19) was carried out using Illumina Genome Analyzer IIxe and Roche 454 allowing paired-end and single-reads sequencing, respectively. The sequences obtained were aligned using CLC Genomic workbench version 5.1 and annotated using RAST (Rapid Annotation using Subsystem Technology) release version 59 accessible online. Additionally, genomes of seven publicly available strains of (k20, MG1, c505, OT175, OT171, OT170, and ) were also included. Comparative genomic analysis (CGA) using Mauve, Progressive Mauve, gene-by-gene, Core, and Pan Genome, and finally Digital DNA-DNA homology (DDH) analysis was carried out. DDH values were obtained using in silico genome-genome comparison. Evolutionary analysis using ClonalFrame was also undertaken. The mutation and recombination events were compared using chi-square test among and isolates (analysis methods are not included in the study). CGA results were consistent with previous traditional classification using MLST. It was found that strains of k20, MG1, c505, and OT175 clustered in group of isolates, while OT171, OT170, and appeared as separate branches. Similar clustering to MLST was observed for other isolates. The mutation and recombination events were significantly higher in than , highlighting the diversity of strains in the oral cavity. These findings suggest that forms six distinct groups, whereas forms three. The correct designation of isolates will help in the identification of clinical isolates found in dental plaque. Easily accessible online genomic sequence data will also accelerate the investigation of the biochemical characterisation and pathogenesis of this important group of micro-organisms.