Biofilm Formation of Streptococcus mutans

Streptococcus mutans strain UA159 (JM10:pJM1-ldh, luc+, Spc^R, luc under the control of the ldh promoter) was utilized as the model organism in the present study. [34 (link),35 (link),42 (link)] The selection of antibiotic-resistant colonies was performed on two passages of TH plates (Todd-Hewitt, BD Difco, New Jersey, NJ, USA), supplemented with 0.3% yeast extract (EMD Millipore Sigma, Burlington, MA, USA) and 800 µg/mL of spectinomycin (MP Biomedicals, Santa Ana, CA, USA). The plates were incubated under anaerobic conditions at 37 °C for 48 h. Planktonic cultures of S. mutans (JM10) were grown in THY culture medium at 37 °C for 16 h. Cultures having optical density (OD₆₀₀) levels equal to or higher than 0.900 (corresponding to 6.43 e⁺¹² CFU/mL) were used as inoculum to grow biofilms. Optimal biofilm growth parameters identified during a previous study from our group [1:50 dilution, 0.65× THY + 1% (w/v) sucrose, 1000 µL] [6 (link)] were then used to grow the biofilms. Aliquots (1.0 mL) of inoculated biofilm growth media were dispensed into the wells of sterile 24-well microtiter plates (Falcon, Corning, NY, USA), containing sterile specimens. Biofilms were grown for 24 h (static cultures, microaerophilic conditions, 37 °C). An additional set of specimens fabricated with OPTB was treated with 2% chlorhexidine gluconate (CHX) for 2 min and served as the control group.