Polymicrobial Aggregates in Human Saliva Build the Oral Biofilm

Abstract

Biofilm community development has been established as a sequential process starting from the attachment of single cells on a surface. However, microor ganisms 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 spatio temporal attachment and biofilm community development. Using multiscale imag ing, 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 addi tion to a few single-cell forms. Phylogenetic analysis reveals a mixture of complex consortia of aerobes and anaerobes in which bacteria traditionally considered early and late colonizers are found mixed together. When individually tracked during colo nization and biofilm initiation, aggregates rapidly proliferate and expand tridimen sionally, modulating population growth, spatial organization, and community scaf folding. In contrast, most single cells remain static or are incorporated by actively growing aggregates. These results suggest an alternative biofilm development pro cess 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.