Method: A six-species supragingival plaque community model composed of Actinomyces naeslundii, Fusobacterium nucleatum, Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis and Veillonella parvula was cultured in a constant depth film fermenter (CDFF). The CDFF consisted of a rotating turntable containing 15 removable polytetrafluoroethylene pans, each holding three enamel plugs as substrata for the biofilm. The CDFF was maintained at 370C under anaerobic conditions and fed with Artificial Saliva Medium (ASM) at a flow rate of 30 mL/h. In order to mimic in vivo oral conditions with food intake and carbohydrate challenge, 1% (w/v) sucrose solution in ASM was pumped into the CDFF four times daily at four hourly intervals. Sampling was carried out on Days 6, 12 and 19 after inoculation. Bacterial biofilm species composition was determined by 16S rDNA genomic sequencing. Lesion development and mineral content of enamel substratum was determined after sectioning using transverse microradiography.
Result: Polymicrobial biofilms established rapidly on the enamel substrata. 16S rDNA sequencing analysis showed that by Day 19, four of the six bacterial species had abundances above 10% of the total bacteria. The proportion of A. naeslundii increased from < 1% to > 20% over the 19 days. L. casei doubled from 2% (Day 6) to 4% (Day 19). S. sanguinis decreased from 49% (Day 6) to 15% (Day 19). Enamel lesion depth increased from 15 ± 5 µm at Day 6 to 81 ± 9 µm at Day 19.
Conclusion: We have developed a robust in vitro model where a polymicrobial biofilm initiates subsurface enamel demineralization in a reproducible manner. This six-species polymicrobial biofilm model will be useful for screening of novel anti-cariogenic compounds.