As an alternative approach to the sequencing of homogenized samples, the microbiota of structurally preserved supragingival plaque samples were visualized using a combinatorial labelling and spectral imaging fluorescence in situ hybridization (CLASI-FISH) technique that was recently developed by the authors, which enables up to 15 fluorescent probes to be imaged simultaneously. Nine taxa were identified in data from the Human Microbiome Project as prevalent and abundant in the supragingival niche, and unique CLASI-FISH probes were designed for each of these taxa and applied to samples from 22 healthy volunteers. Several types of consortium structure were observed, including a spiny radial structure — reminiscent to the authors of hedgehogs — that was present in many of the samples. These 'hedgehog' structures were defined by signature radial protrusions of filamentous spines that were connected to cocci tips at the periphery and clumps of cells at the base; additional cells were present at the periphery or in an annulus ring beneath the periphery. Each of the nine taxa reproducibly occupied a characteristic position: Corynebacterium spp. (usually Corynebacterium matruchotii) filaments formed the framework; cells of the Streptococcus, Porphyromonas, Neisseriaceae and Haemophilus−Aggregatibacter taxa formed the periphery; Fusobacterium spp., Leptotrichia spp. and Capnocytophaga spp. cells formed the annulus; and Actinomyces spp. and Corynebacterium spp. cells formed the base.
The authors propose that these 'hedgehog' consortia are seeded by the attachment of Corynebacterium spp. cells to a pre-existing biofilm coating the surface of a tooth. Once seeded, the Corynebacterium spp. filaments seem to have a central role in organizing the consortium, as they provide an attachment surface for Streptococcus spp. and Porphyromonas spp. cells at the periphery, where the crevicular fluid supplies nutrients. In turn, Streptococcus spp. cells provide an attachment surface for Haemophilus spp. and/or Aggregatibacter spp. cells and, through the aerobic metabolism of sugars, create an anoxic microenvironment in the annulus that favours the metabolisms of Fusobacterium spp., Leptotrichia spp. and Capnocytophaga spp. cells.
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