Microbial Aggregates in Human Saliva Build the Oral Biofilm

Biofilm community development has been established as a sequential process starting from attachment of single cells on a surface as exemplified by the ecological succession model of early and late colonizers. However, microorganisms are often found as aggregates in the environment and in biological fluids, which could influence the biofilm formation process. How naturally occurring microbial aggregates modulate biofilm initiation remains underexplored. Here, we conduct a comprehensive analysis of the structure and composition of microbial aggregates in human saliva and investigate their spatiotemporal attachment and biofilm community development in dental plaque samples and on a tooth-mimetic apatitic surface. 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 buccal cells can be found in addition to single-cell forms in saliva. Phylogenetic analysis reveals a mixture of Streptococcus, Haemophilus, Neisseria, Porphyromonas and Veillonella, indicating complex consortia of aerobic and anaerobic bacteria. Notably, microbial aggregates are capable of binding to the apatite surface in which bacteria traditionally considered early and late colonizers are found mixed together. The colonizing microbes are then individually tracked during biofilm growth, and only a subset of aggregates actively proliferates and expands tri-dimensionally into structured communities. In contrast, most single-cells remain static or are incorporated by actively growing aggregates. These results suggest that microbial aggregates play a crucial role governing the biofilm initiation and assembly, modulating population growth, spatial organization, and community scaffolding. Rather than an orderly succession process of single-cells attachment in a sequential fashion, we propose an alternative model whereby aggregates of varying sizes and structures containing different species or associated with human cells, collectively adhere to the surface as "growth nuclei" to build the biofilm and shape microbial communities at various spatial and taxonomic scales.