Effect of EGTA, a Calcium Chelator, on Streptococcus mutans Biofilm

Streptococcus mutans, the primary causative agent of human dental caries has the ability to form biofilm on tooth surface. Previous study showed that calcium is required for biofilm formation process in S. mutans. Here, we investigated the effect of EGTA, a calcium chelator, on S. mutans biofilm formation and S. mutans' integration into pre-existing biofilm.

Methods:

Overnight culture of Spectinomycin-resistant, UA140 S. mutans derivative strain was diluted 500 fold in fresh BHI medium supplemented with 0.5% sucrose, and used for seeding two sets of 48-well plates: one without, and one with pre-formed biofilm of wild type UA140 S. mutans strain. Briefly, 1 ml of diluted culture was seeded into each well, followed by EGTA at various concentrations of 0 to 10 mM. After overnight incubation at 37ªC under anaerobic condition, supernatant and biofilm samples within each well of the two sets were serially diluted and plated onto BHI and BHI/spectinomycin agar plates. Plates were incubated at 37ªC under anaerobic condition for 2 days before colonies were counted. The integration of S. mutans into preformed biofilm can be revealed by the viable count of SpeR colonies within the preformed biofilm samples.

Results:

EGTA has no significant effect on S. mutans' biofilm formation when added at a concentration lower than 0.35 mM; while higher concentrations showed drastic inhibitory effect on both cellular viability and ability to form biofilm. Surprisingly, in presence of pre-existing biofilm, EGTA, even at 10 mM concentration, did not have significant effect on viability of S. mutans in supernatant or within the biofilm, and neither did it show significant effect on S. mutans' ability to integrate into pre-existing biofilm.

Conclusion:

The results indicate that pre-formed S. mutans biofilm might employ a mechanism to titrate out EGTA, thus rendering it ineffective.

This research was supported by NIH grant GM54666.