Point-Of-Use Generation of Hypohalites to Remodel Dental Plaque Microcosm Biofilms

Shifts in the microbial populations of dental plaque cause periodontitis and caries. The indiscriminant biocide hypochlorite from subgingival neutrophils, and more selective-action hypothiocyanite generated by salivary peroxidase are highly reactive and unstable components of the innate immune system. We hypothesize that they selectively modulate plaque microbial populations and their pathogenicity. Objectives: To test this hypothesis by direct application of measured concentrations of hypohalites to dental plaque microcosm biofilms. Methods: Plaque microcosms were cultured from plaque-enriched saliva in the ‘Multiplaque Artificial Mouth,' supplied with sucrose for 6 min every 8h and with alternating 15 min periods of artificial saliva (0.5xDMM) and hypohalite concentrations from 0.1 to1 mM. The hypohalites were applied initially at 24h or 48h of growth. Active hypohalites were generated just prior to application; hypochlorite by a pH-jump to neutrality and hyothiocyanite by enzymatic generation using the LPO/SCN/ H2O2 system. The microbiota were characterized by checkerboard (CKB) analysis 0f 40 species and PCR-DGGE. Results: Biofilm growth was progressively inhibited by increased hypochlorite and hypothiocyanite concentrations, with minor microbiota differences that were more pronounced in the 24h preformed biofilm. DGGE analysis detected bands with hypochlorite concentration-dependent changes in intensity, and one band that disappeared with simultaneous application of 1 mM hypochlorite with DMM – indicating antimicrobial effects of secondary metabolites. DGGE showed more specific hypothiocyanite -induced remodeling: in the 48h preformed plaque one band only changed, 3 changed in the 24h plaque. Most CKB species were relatively unaffected. However both S. mutans and Tannerella forsythia seemed to be sensitive to both hypohalites. Conclusion: Concentrations up to 1mM, of these highly reactive hypohalites can induce plaque remodelling. Plaque age or size may be important determinants, with effects potentially confined to the plaque surface layers. These finding also support the hypothesis that compared to hypochlorite, hypothiocyanite effects on plaque are more selective. 1R21-DE016889-01A2