Propolis Fluoride Inhibits Streptococcus mutans and Veillonella parvula Dual- species Biofilm Growth and alters Gene Expression

Abstract

Background Propolis fluoride 10% (PPF) was developed to arrest and prevent caries, with inhibitory effects on a single species of bacteria. However, no study has been conducted on the effectiveness of PPF in inhibiting multispecies biofilms, particularly early colonizers. This study aimed to investigate the effects of PPF on Streptococcus mutans and Veillonella parvula (Sm-Vp) dual-species biofilm growth and gene expression. Methods Biofilms were prepared using the 96-well method with 1, 3, and 6 h incubation, and 30% silver diamine fluoride was used as the positive control. Crystal violet assay and total plate counting (TPC) were performed to analyze inhibitory effects. An inverted microscope was used to analyze the formed structure. RNA was extracted from the samples for cDNA synthesis and standardized using a Qubit fluorometer for reverse-transcription quantitative-polymerase chain reaction. The target genes used in this study were NRAMP and SloR, and 16 srRNA was used as an internal control. Consequently, alterations of gene expression were quantified using Livak’s method (2^-∆∆Ct). Statistical analyses were performed using the Statistical Package for the Social Sciences software. Statistical significance was set at P < 0.05. Results Inverted microscopic observation showed lower aggregate formation in the treated and positive control groups, whereas large aggregates were observed in the negative control groups. The TPC and crystal violet results showed a significant difference between the control and treatment groups in the monospecies group (P < 0.05), and the lower mean was statistically significant (P < 0.05). The NRAMP and SloR/Dlg gene expressions were downregulated in PPF-treated samples. Conclusion The PPF-treated Sm-Vp biofilms showed lower biofilm formation and altered virulence and symbiotic-related gene expression. Therefore, PPF effectively inhibits Sm-Vp biofilm growth and prevents caries.