Polymicrobial Aggregates in Human Saliva Build the Oral Biofilm

Biofilm community development has been established as a sequential process starting from the attachment of single-cells on a surface. However, microorganisms are often found as aggregates in the environment and in biological fluids. Here, we conduct a comprehensive analysis of the native structure and composition of aggregated microbial assemblages in human saliva and investigate their spatiotemporal attachment and biofilm community development. Using multiscale imaging, cell-sorting, and computational approaches combined with sequencing analysis, a diverse mixture of aggregates varying in size, structure, and microbial composition including bacteria associated with host-epithelial cells can be found in saliva, in addition to few single-cell forms. Phylogenetic analysis reveals a mixture of complex consortia of aerobes and anaerobes in which bacteria traditionally considered as early and late colonizers are found mixed together. When individually-tracked during colonization and biofilm initiation, aggregates rapidly proliferate and expand tri-dimensionally modulating population growth, spatial organization, and community scaffolding. In contrast, most single-cells remain static or are incorporated by actively growing aggregates. These results suggest an alternative biofilm development process whereby aggregates containing different species or associated with human cells, collectively adhere to the surface as "growth-nuclei" to build the biofilm and shape polymicrobial communities at various spatial and taxonomic scales.