Abstract
Biofilms are structured microbial communities adhered to surfaces that cause many human infections. The study of oral biofilms has revealed complex composition, spatial organization, and phenotypic/genotypic diversity of the resident microbiota at the various sites in the mouth. Yet, knowledge about the spatial arrangement, positioning, and function of the polymicrobial community across the intact biofilm architecture remains sparse. Using multiple length scale imaging and computational analysis, we discovered unique spatial designs comprising mixed interbacterial species and interkingdom communities within intact biofilms formed on teeth of toddlers with caries. Intriguing structural patterns ranging from intermixed communities with extensive coaggregation (including bacterial-fungal clustering) to spatially segregated species forming a multilayered architecture were found. Among them, a distinctive 3-dimensional structure exhibited densely clustered cariogenic pathogens that were surrounded by outer layers of mixed bacterial communities in juxtaposition, forming a highly ordered spatial organization. These findings are particularly relevant as we approach the postmicrobiome era whereby studying the spatial structure of the pathogen and commensal microbiota may be important for understanding the microbiome function at the infection site to coordinate the disease process in situ.