Cationic Lipid Content in Liposome-Encapsulated Nisin Improves Sustainable Bactericidal Activity against Streptococcus mutans.

An oral infectious disease, dental caries, is caused by the cariogenic streptococci Streptococcus mutans. The expected preventive efficiency for prophylactics against dental caries is not yet completely observed. Nisin, a bacteriocin, has been demonstrated to be microbicidal against S. mutans, and liposome-encapsulated nisin improves preventive features that may be exploited for human oral health. Here we examined the bactericidal effect of charged lipids on nisin-loaded liposomes against S. mutans and inhibitory efficiency for insoluble glucan synthesis by the streptococci for prevention of dental caries. Cationic liposome, nisin-loaded dipalmitoylphosphatidylcholine/phytosphingosine, exhibited higher bactericidal activities than those of electroneutral liposome and anionic liposome. Bactericidal efficiency of the cationic liposome revealed that the vesicles exhibited sustained inhibition of glucan synthesis and the lowest rate of release of nisin from the vesicles. The optimizing ability of cationic liposome-encapsulated nisin that exploit the sustained preventive features of an anti-streptococcal strategy may improve prevention of dental caries.

Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation.

Water-insoluble glucan (WIG) produced by mutans streptococci, an important cariogenic pathogen, plays an important role in the formation of dental biofilm and adhesion of biofilm to tooth surfaces. Glucanohydrolases, such as mutanase (α-1,3-glucanase) and dextranase (α-1,6-glucanase), are able to hydrolyze WIG. The purposes of this study were to construct bi-functional chimeric glucanase, composed of mutanase and dextranase, and to examine the effects of this chimeric glucanase on the formation and decomposition of biofilm. The mutanase gene from Paenibacillus humicus NA1123 and the dextranase gene from Streptococcus mutans ATCC 25175 were cloned and ligated into a pE-SUMOstar Amp plasmid vector. The resultant his-tagged fusion chimeric glucanase was expressed in Escherichia coli BL21 (DE3) and partially purified. The effects of chimeric glucanase on the formation and decomposition of biofilm formed on a glass surface by Streptococcus sobrinus 6715 glucosyltransferases were then examined. This biofilm was fractionated into firmly adherent, loosely adherent, and non-adherent WIG fractions. Amounts of WIG in each fraction were determined by a phenol-sulfuric acid method, and reducing sugars were quantified by the Somogyi-Nelson method. Chimeric glucanase reduced the formation of the total amount of WIG in a dose-dependent manner, and significant reductions of WIG in the adherent fraction were observed. Moreover, the chimeric glucanase was able to decompose biofilm, being 4.1 times more effective at glucan inhibition of biofilm formation than a mixture of dextranase and mutanase. These results suggest that the chimeric glucanase is useful for prevention of dental biofilm formation.

Sustainable inhibition efficacy of liposome-encapsulated nisin on insoluble glucan-biofilm synthesis by Streptococcus mutans.

CONTEXT: Dental caries are an infectious oral bacterial disease caused by cariogenic streptococci. These streptococci inhabit dental biofilms which comprise insoluble glucans. OBJECTIVE: To prevent dental caries, nisin, a suitable agent active against Gram-positive bacteria, was examined in vitro for its ability to suppress insoluble glucan-biofilm synthesis by cariogenic streptococci. MATERIALS AND METHODS: To investigate glucan-biofilm synthesis by a typical cariogenic streptococcus, Streptococcus mutans 10449, the naked form of nisin was loaded onto a 96-well microplate in vitro model. To prolong the efficacy of nisin as a preventive agent, liposome-encapsulated nisin (nisin-liposome) was examined for its ability to inhibit the synthesis of glucan-biofilms on microplates. RESULTS: Naked nisin (100 pmol) completely suppressed insoluble glucan-biofilm synthesis by S. mutans 10449 following 1 h cultivation in 96-well microplates. The concentration of nisin-liposome required for the efficacious inhibition of glucan-biofilm synthesis was four times lower than that of naked nisin following 2 h cultivation. In particular, nisin-liposome (30 pmol nisin equivalent) prolonged the inhibitory activity of nisin against glucan-biofilm synthesis by S. mutans 10449 for up to 6 h, while naked nisin (30 pmol) gradually lost this inhibitory activity over the same period. In vitro release assay of nisin from the liposome showed that 76% nisin was released within 6 h. DISCUSSION AND CONCLUSION: The findings indicate the usefulness of nisin-liposome for the sustained release of nisin. Thus, nisin-liposome could play a potential role in preventive medicine as an inhibitor of the glucan-biofilm synthesis.