Elucidation on the effect of Theobroma cacao aqueous extract (cocoa) on the susceptibility and biofilm-forming ability of Streptococcus mutans on different sizes and shapes of nickel-titanium and stainless-steel orthodontic archwires [EMBARGOED]

Abstract

The extract of Theobroma cacao beans was reported to possess antimicrobial activity against the dental caries pathogen. Meanwhile, Nickel-Titanium (NiTi) and Stainless-Steel (SS) orthodontics archwires are commonly used for aligning teeth in the initial stages of orthodontic treatment. However, the antimicrobial effect of T. cacao on the Streptococcus mutans on the archwires as well as its effect on the biofilm formation is not completely understood. This study aimed to determine the effects of T. cacao on the presence of S. mutans on orthodontic archwires with different materials and sizes. Corresponding 0.014”, 0.018”, 0.017x0.025, and 0.019x0.025” dimension NiTi and SS archwires were used. To determine the antimicrobial effects of T. cacao on S. mutans, sterile 10 mm archwire sections were soaked in 200 mg/mL of T. cacao aqueous extract for 30 sec, 1 min and 2 min. Later, the archwires were placed on a Mueller-Hinton agar pre-swabbed with S. mutans (Ingbritt), standardized at optical density OD620nm 0.5. Finally, the plate was incubated at 37?C for 24 h and the inhibition zone was measured. To determine the antibiofilm activity of T. cacao on the S. mutans, a 10 mm length sterile archwire was treated with 2 mL of 200 mg/mL of T. cacao for 1 min. Following that, the archwires were transferred into a 6-well plate containing 2 mL BHI broth. Then, 2 mL of S. mutans standardized to an OD620nm 0.5 was inoculated into the well and incubated at 37?C for 24 h. The archwires were transferred into a 2 mL tube containing 2 mL of PBS and sonicated for 1 min. Later, 100 ?L of the suspension was collected and the total colony-forming unit was enumerated using the serial dilution method on brain heart infusion agar. BHI and 0.12% chlorhexidine gluconate (CHX) were used as the positive and negative control, respectively. The experiment was conducted in three biological replicates. Scanning electron microscope (SEM) analysis was conducted to observe biofilm formation over orthodontic archwires. The present study showed that S. mutans on orthodontic archwires were resistant to treatment with T. cacao aqueous extract. Furthermore, the antibiofilm assay showed that T. cacao-treated archwire had a comparable antibiofilm effect with CHX for 0.017x0.025” NiTi and SS archwires, and 0.018” NiTi. However, they were not statistically significant (P>0.05). T. cacao treatment significantly reduced S. mutans biofilm on 0.014” SS archwire (P<0.05). In addition, S. mutans cell count was found to reduce on all archwires compared with untreated controls except for 0.014” NiTi archwires where treatment with both CHX and T. cacao showed no difference. The biofilm adhesion was found to be significantly higher on NiTi compared to SS archwires on both 0.018” and 0.019x0.025” sizes (P<0.05). Finally, SEM images showed that T. cacao prevented the dispersion of S. mutans biofilm. In conclusion, T. cacao does not inhibit the growth of S. mutans on archwires, however, it possesses antibiofilm properties against the bacterium.