Multilocus Sequence Typing of Streptococcus mutans

Objectives: MLST is a tool for the determination of the population structure of bacterial species with many epidemiological applications. The aim of this work was to develop an MLST scheme for S. mutans. A total of 100 independent strains from caries-free and caries-active subjects from the UK, Turkey, Hong Kong, South Africa and the USA were tested. Methods: cells were cultured on Columbia blood agar and DNA was extracted by boiling for PCR and both strands were sequenced using specific primers and standard protocols. The identity of each isolate was confirmed by 16S rRNA gene sequencing and partial sequences for 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), recP (transketolase), sodA (superoxide dismutase), and tyrS (tyrosyl-tRNA synthetase)] and 2 extracellular virulence genes [gtfB (glucosyltransferase B) and spaP (surface protein antigen I/II)] were obtained. Results: the number of alleles found varied between 19 (gtfB) and 27 (recP). Overall, 89 sequence types (STs) were defined from the data. The percentage nucleotide variation ranged from 1.0% (spaP) to 3.0% (recP). Analysis of the MLST data was carried out using the START2, BURST and SplitsTree4 programs. No major clonal complex ST founder was identified and no statistically significant evidence for intragenic recombination was found except in sodA (phi value = 0.048) and tyrS (phi value = 0.029). Conclusions: the high number of STs and the remarkably low sequence variation make it difficult to evaluate the rate of inter-strain recombination. However, our understanding of the ecology and physiology of S. mutans, suggest that it may only recently have been subjected to significant environmental pressures or it may be a newly evolved species, perhaps due to the introduction of refined carbohydrates to the human diet. Supported in part by the Wellcome Trust (Grant number 076381).