Molecular identification of streptococcus mutans from patients with dental caries using PCR and gene sequencing

Abstract

Dental caries is the most prevalent oral disease in the world. Even in Sri Lanka, its prevalence is considerably high with a huge negative impact on our society and economy. Streptococcus mutans is the major bacterial etiological agent of dental caries. Precise identification of S. mutans from saliva or dental biofilms is important in the preparation of clinical isolates for testing the efficacy of oral healthcare formulas against dental caries and its risk assessment in vulnerable populations based on their counts in saliva. Since oral microbiome contains numerous species of Streptococci with subtle changes in morphological and biochemical characteristics, precise identification of S. mutans based on these characteristics is a tedious task. This study aims to precisely identify S. mutans from dental biofilms using PCR and gene sequencing. DNA was extracted using an optimized chemical method. Using species-specific Sm479F/R primers, a segment of the HtrA gene of S. mutans was amplified. Agarose gel electrophoresis of the PCR product confirmed the presence of a 479-bp PCR amplicon. Sequencing of the PCR product and alignment of it with sequences available in the NCBI database showed that it has 97.8% identity to the S. mutans reference sequence. Among the 32 Streptococcus bacteria isolated from clinical samples on Mitis Salivarius agar, 23 were confirmed to be S. mutans. In conclusion, PCR with species-specific primers Sm479F/R is an effective method for precise identification of S. mutans among multitude of Streptococci in the oral cavity. The S. mutans clinical isolates confirmed in this study can be used to test the efficacy of oral healthcare formulas. Further, this PCR can be improved to quantify S. mutans in saliva and utilized in population screening for risk assessment of dental caries, which may help early interventions to prevent the occurrence of dental caries in high-risk individuals.