Multispecies oral biofilms studied at the single community level as a model system for spatiotemporal development of biofilms and interspecies interactions

Abstract

Oral biofilms are multispecies communities that are important in the development of the two most prevalent oral diseases---dental caries and periodontal disease. The primary initial colonizers of human enamel are streptococci, veillonellae and actinomyces. Bacteria of these genera coaggregate (cell-cell interactions between genetically distinct bacteria). The streptococci can be classified into two groups: those with receptor polysaccharides (RPS) and those with adhesins that recognize RPS. RPS mediates streptococcal intrageneric coaggregation. Besides coaggregation, veillonellae interact with streptococci metabolically by using lactic acid produced by streptococci. The hypothesis tested in this study is that coaggregation mediated interactions between streptococci and veillonellae are important for the development of oral biofilms in vivo. To translate naturally occurring interactions from undisturbed dental plaque to multispecies in vitro communities, a simple community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by immunofluorescence with quantum-dot-conjugated antibodies and micromanipulated from the tooth surface. Characterization of the application of quantum-dot primary immunofluorescence was accomplished and was essential for the success of this approach. An antibody-unreactive streptococcus invisible during micromanipulation was also obtained. The streptococci were identified as Streptococcus oralis and Streptococcus gordonii. The veillonellae could not be cultured; however, immunoreactive veillonellae cells were present in the original mixture and a Veillonella 16S-rRNA gene sequence was amplified. This sequence was similar to Veillonella sp. PK1910. The two streptococci coaggregated by an RPS-dependent mechanism, and both coaggregated with Veillonella sp. PK1910. Veillonella sp. PK1910 was used as a surrogate in in vitro community reconstruction. In vitro the two streptococcal isolates and Veillonella sp. PK1910 grew on saliva as the sole nutritional source and formed interdigitated multispecies clusters. Veillonella sp. PK1910 grew only in biofilms where streptococci were present. To map the spatial relationship between these species in vivo we used a novel approach integrating immunofluorescence and fluorescence in situ hybridization. Reconstruction in vitro of a community composed of Veillonella sp. PK1910, S. oralis, and S. gordonii demonstrates the vital role of RPS-mediated coaggregation in initial colonization of multispecies communities. The integrated in vitro - in vivo approach serves as a new paradigm for the study of oral biofilms and their development.