Synergistic inhibitory activity of Glycyrrhizae Radix and Rubi Fructus extracts on biofilm formation of Streptococcus mutans.

BACKGROUND: Streptococcus mutans is a bacterium that causes oral diseases. Plaque, a biofilm produced by S. mutans and other bacteria, makes it difficult to remove cariogenic oral microorganisms, including biofilm producers. Glucan synthesis by glucosyltransferase is one of the mechanisms underlying plaque formation. This study demonstrates the effectiveness of inhibiting biofilm formation by interfering with the glucosyltransferase activity of S. mutans using edible herbal medicines. METHODS: This study investigated the inhibitory activity of Glycyrrhizae Radix extract, Rubi Fructus extract, glycyrrhizin from Glycyrrhizae Radix, and ellagic acid from Rubi Fructus against glucosyltransferase activity of S. mutans. Enzyme kinetic analysis identified the mechanism by which glycyrrhizin and ellagic acid inhibit enzyme activity. RESULTS: The conditions for synergistically inhibiting biofilm formation by combining Glycyrrhizae Radix and Rubi Fructus extracts were identified. Biofilm formation was also synergistically inhibited by mixing their respective active constituents, glycyrrhizin and ellagic acid. Glycyrrhizin and ellagic acid inhibited glucosyltransferase via noncompetitive and uncompetitive mechanisms, respectively, indicating that they inhibit it via distinct mechanisms. CONCLUSIONS: This study presents an effective oral hygiene method using the synergistic activity of two natural plant extracts to inhibit biofilm formation through different inhibitory mechanisms against glucosyltransferase of S. mutans.

Inhibitory Effect of Phenolic Acids in Rubus coreanus on Glucosyltransferase of Streptococcus mutans.

Streptococcus mutans, a typical dental caries-causing oral pathogen, forms dental biofilm by attaching to the surface of teeth. Its glucosyltransferase (GTFase) is responsible for synthesizing water-insoluble glucan from sucrose, which is an important factor for biofilm formation and for providing microbial resistance against stresses. Therefore, inhibiting the activity of GTFase is an effective approach to prevent the formation of dental biofilm even without killing S. mutans. In this study, we found that 2.5 mg/mL of methanol extracts of Camellia sinensis leaf, Diospyros kaki leaf, Nelumbo nucifera seed, Rubus coreanus fruit, and Ulmus davidiana rhizodermis inhibits both GTFase activity and biofilm formation of S. mutans without affecting cell growth. Phenolic acids such as ferulic acid, salicylic acid, and vanillic acid in R. coreanus fruit extract inhibited GTFase activity. Enzyme kinetic analysis shows that ferulic acid, salicylic acid, and vanillic acid are competitive, noncompetitive, and uncompetitive inhibitors, respectively. These results suggest that R. coreanus fruit extracts containing phenolic acids may control and prevent dental caries without killing S. mutans by inhibiting GTFase activity.

Nitrogen Sources Inhibit Biofilm Formation by Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, which results in severe economic damage to rice farms. Xoo produces biofilms for pathogenesis and survival both inside and outside the host. Biofilms, which are important virulence factors, play a key role in causing the symptoms of Xoo infection. In the present study, we investigated the nutritional conditions for biofilm formation by Xoo. Although Xoo biofilm formation may be initiated by interactions with the host, Xoo biofilm cannot mature without the support of favorable nutritional conditions. Nitrogen sources inhibited Xoo biofilm formation by overwhelming the positive effect that cell growth has on it. However, limited nutrients with low amino acid concentration supported biofilm formation by Xoo in the xylem sap rather than in the phloem sap of rice.

Inhibitory activity of monoacylglycerols on biofilm formation in Aeromonas hydrophila, Streptococcus mutans, Xanthomonas oryzae, and Yersinia enterocolitica.

Biofilm provides a bacterial hiding place by forming a physical barrier and causing physiological changes in cells. The elimination of biofilm is the main goal of hygiene. Chemicals that are inhibitory to biofilm formation have been developed for use in food, personal hygiene products, and medical instruments. Monoacylglycerols are recognized as safe and are used in food as emulsifiers. In this study, the inhibitory activity of monoacylglycerols on bacterial biofilm formation was evaluated systematically with four bacterial strains, Aeromonas hydrophila, Streptococcus mutans, Xanthomonas oryzae, and Yersinia enterocolitica. Monoacylglycerols with two specific lengths of fatty acid moiety, monolaurin and monobehenin, were found to have strong inhibitory activity toward bacterial biofilm formation of S. mutans, X. oryzae, and Y. enterocolitica in a strain specific manner. First, this result suggested that biofilm formation was not inhibited by the detergent characteristics of monoacylglycerols. This suggestion was supported by the inhibitory action of monolaurin on biofilm development but not on the initial cell attachment of Y. enterocolitica in flow cytometric observation. Second, it was also suggested that two distinct response mechanisms to monoacylglycerols existed in bacteria. The existence of these two inhibitory response mechanisms was bacterial strain specific.

Sanitizing Effect of Ethanol Against Biofilms Formed by Three Gram-Negative Pathogenic Bacteria.

Sanitizing effect of ethanol on a Yersinia enterocolitica biofilm was evaluated in terms of biomass removal and bactericidal activity. We found that 40 % ethanol was most effective for biofilm biomass removal; however, no significant difference was observed in bactericidal activity between treatment with 40 and 70 % ethanol. This unexpected low ethanol concentration requirement for biomass removal was confirmed using biofilms of two additional pathogenic bacteria, Aeromonas hydrophila and Xanthomonas oryzae. Although only three pathogenic Gram-negative bacteria were tested and the biofilm in nature was different from the biofilm in this study, the results in this study suggested the possible re-evaluation of the effective sanitizing ethanol concentration 70 %, which is the concentration commonly employed for sanitization, on bacteria in a biofilm.