Habitat analysis of 12 other abundant plaque genera ( Fig. 1E ) showed large differences in their degree of specificity to plaque but only modest differences in their relative abundance in SUPP compared with SUBP. Genera with strong plaque specificity, in addition to Corynebacterium, included Capnocytophaga, which was 10-fold more abundant in plaque than at nonplaque sites, as well as Lautropia and Rothia. By contrast, genera such as Streptococcus occupied a broad range of habitats. Despite being the single most abundant genus in SUPP, Streptococcus was substantially more abundant at nonplaque sites than in plaque on average. This wide-ranging habitat preference likely reflects the capacity of Streptococcus to be an efficient colonizer of multiple oral surfaces. Additional genera with broad habitat range in the mouth include Haemophilus and Veillonella. Supragingival plaque is often characterized as being composed primarily of Gram-positive aerobes, whereas Gram-negative anaerobes come to dominate subgingival plaque, particularly in individuals affected by periodontitis ( 21 ). However, habitat analysis of genera shows that the similarities between the two plaques are more striking than their differences in the healthy individuals sampled by the HMP. Most of the abundant genera are enriched in SUPP compared with SUBP by a small and relatively constant factor of ∼1.3–1.6. Some genera (Actinomyces, Porphyromonas, and Veillonella) are equally abundant. A few predominantly anaerobic genera, notably Prevotella and Fusobacterium, are more abundant in SUBP but only by a factor of ∼2. Thus, the overall similarity of distribution suggests a close connection between these two spatially adjacent communities.

Taxa that are present primarily or exclusively in one site may provide clues to the distinctive features of the habitat and the role that those taxa contribute to the site. Habitat analysis of the oral microbiome suggested that one genus, Corynebacterium, in particular was strikingly specific to supragingival and subgingival plaque. This genus was present in only trace amounts in saliva and on six of eight oral surfaces sampled (the tongue, buccal mucosa, keratinized gingivae, hard palate, tonsils, and throat) but made up 8% of the bacterial community in SUBP and more than 12% in SUPP ( Fig. 1C ) ( 19 , 20 ). The HOMD recognizes six oral species within the genus Corynebacterium. However, of these six, only two, Corynebacterium matruchotii and Corynebacterium durum, were present at significant levels in plaque. Although C. matruchotii was the dominant species in most individuals, C. durum was dominant in some ( Fig. 1D ). Taking the two species together, the genus not only had a high mean abundance, but also, it was consistently abundant, with a relative abundance of 3% or more in 90% of the individuals. The abundance and prevalence of Corynebacterium suggest that it plays an important role in the plaque community, whereas its plaque specificity suggests that it occupies a niche that is dependent on properties of the tooth surface and/or the gingival crevice.

Metagenomic sequence analysis points to Corynebacterium as a key taxon in supragingival plaque. (A) Prevalence abundance plot for supragingival plaque. (B) Cumulative abundance of genera in both supra- and subgingival plaque. Genera with greater than 3% abundance in SUPP, mean across 148 subjects, are indicated by colored dots in A and bar segments in B; B also shows the abundance of these genera in SUBP. Data are from the HMP ( 18 ) V3–V5 region of 16S rRNA, analyzed by oligotyping ( 19 ), and grouped by genus. (C) Corynebacterium is far more abundant in plaque than in other oral sites. Mean abundances of C. matruchotii and C. durum are shown for each oral site analyzed by oligotyping ( 19 ). BM, buccal mucosa; HP, hard palate; KG, keratinized gingiva; PT, palatine tonsils; SV, saliva; TD, tongue dorsum; TH, throat. (D) Corynebacterium is a major component of most plaque samples. Relative abundance of Corynebacterium in the HMP SUPP samples from 148 individuals ( 19 ). C. matruchotii is usually more abundant, but C. durum dominates some samples. (E) Habitat analysis identifies genera that are strongly characteristic of SUPP. The plaque to nonplaque ratio measures the relative abundance of each genus in two plaque sites compared with seven nonplaque sites sampled by the HMP [calculated as (mean SUBP + mean SUPP)/(mean BM + mean KG + mean HP + mean SV + mean PT + mean TH + mean TD)]. This ratio identifies Corynebacterium and Capnocytophaga as the taxa most preferentially abundant in plaque. The SUPP to SUBP ratio identifies these genera as relatively more abundant in SUPP than in SUBP. Colors in E are the same as those in A and B.

As an initial basis for identifying key taxa in supragingival plaque, we assessed the abundance and prevalence of the oligotypes grouped by genus. This analysis readily identified a group of bacterial genera that were both abundant and prevalent ( Fig. 1A ). Of 57 genera detected in supragingival plaque (SUPP), most had both low abundance and low prevalence. In contrast, 13 genera had at least 3% mean abundance and were also highly prevalent, each being detected in more than 90% of SUPP samples. Collectively, these 13 genera accounted for 85% of the sequencing data from SUPP. The same 13 genera also accounted for more than 80% of the subgingival plaque (SUBP) data ( Fig. 1B ), indicating a close relationship between the two plaque sites. Because of their abundance and prevalence, these taxa are likely to form both the spatial and the metabolic framework of the healthy plaque microbiome.

The Human Oral Microbiome Database (HOMD) ( 17 ) contains 707 entries at the species level. This enormous diversity poses an enormous challenge for efforts to sort out the spatial and structural relationships of the taxa. In an attempt to reduce the complexity to manageable proportions, we sought guidance from the 16S rRNA gene sequencing data generated by the HMP ( 18 ). We previously applied an information theory approach to analysis of the oral microbiome at the single-nucleotide level, resulting in high-resolution sequence groups termed oligotypes ( 19 ). The oligotypes were assigned to HOMD species and analyzed for each of nine oral habitats defined by the HMP. This analysis showed that most species of oral bacteria are habitat specialists and that the complexity can be reduced simply by considering only the bacteria resident in the oral habitat of interest. In the following discussion, we consider plaque to mean specifically the biofilm that forms on teeth as opposed to other oral substrates, such as gums or tongue, and we focus on the microbiota resident in plaque above the gum line, supragingival plaque.

Plaque Microbiota Is Organized into Highly Structured, Multigenus Consortia.

Bacteria are micron-sized and live in a chemical and structural environment with micron-scale heterogeneity. Therefore, an understanding of the micron-scale spatial organization of bacterial communities is necessary for understanding how these communities function. A striking degree of spatial organization can become visible, even with simple procedures, when plaque samples are prepared gently to retain their structure. For example, when plaque was spread out on a slide, conventional FISH with two probes revealed clumps of Corynebacterium, with long filaments that were coated at their tips by brightly staining cocci (Fig. 2). Associations between cocci and filaments in plaque were documented over 40 years ago and have been called “corncobs” (22, 23). However, the arrangement in dissected plaque, which we visualized with FISH, is striking in that the filaments are clearly continuous from the base of the clump to the tips, but the cocci are restricted to the tips or distal ends of the filaments (Fig. 2). This spatial arrangement suggests a role for Corynebacterium as a foundational taxon that structures the environment in a way that creates a microenvironment favorable to the growth of the cocci. Why the cocci are restricted to the distal ends is a key question, the answer to which requires more complete information about the surrounding structure.

Fig. 2. Corncob structures formed by Corynebacterium and cocci in plaque. Corynebacterium cells (magenta) are visible as long filaments, with cocci (green) bound to the tips of the filaments. Partially disrupted plaque was hybridized with a probe for Corynebacterium and a universal bacterial probe. Image was acquired using a Zeiss AxioImager 63× Plan-Apochromat 1.4 N.A. objective and Apotome structured illumination. (Scale bar: 20 μm.)

We used two complementary methods designed to preserve and visualize the spatial structure of the plaque community: whole-mount preparations and methacrylate embedding. Whole mounts permitted the imaging of entire 3D structures, including long filaments, but at the expense of slight spatial distortion resulting from compression. Embedding and sectioning preserved micron-scale spatial relationships more accurately but at the expense of loss of 3D continuity. Regardless of the preparation method, we detected similar microbial consortia in all samples. For a systems-level analysis of the spatial organization of these samples, we used Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH) (16) to differentiate up to 15 taxa simultaneously. In our previous proof of concept of CLASI-FISH, we labeled plaque that was partially dispersed to single-cell thickness (16), so that spectral signatures created by binary combinations of fluorophores could be read unambiguously. For this study, we wished to analyze more intact 3D structures, in which multiple cells may lie on top of one another, even in a single optical plane of focus. In such samples, overlapping cells with different binary signals in the same pixel could generate ambiguity in taxon identification. To avoid this ambiguity, we used a simplified labeling strategy, in which a single fluorophore served as the spectral signature to identify each taxon, and as many as 10 distinct fluorophores were used simultaneously.

The combination of sequence analysis with imaging allowed an assessment of spatial organization that was taxonomically both wide-reaching and refined. We used FISH probes with broad coverage, using probes for four phyla, two classes, three families, and 15 genera (Table S1). More specificity was provided by HMP sequencing data, which showed that, for most plaque genera, a small number of species was dominant. Of the 13 most abundant genera, one genus was represented by only a single major plaque species (Lautropia mirabilis), and six were represented primarily by two or three major species or small clusters of species (Table S2). Collectively, the probes that we used targeted 96–98% of the cells in a healthy supragingival plaque microbiome as judged by rRNA tag sequencing data from the HMP (Table S2). Among these probes, 2 family- and 11 genus-level probes covered 88% of the sequencing data and are shown in Figs. 2–8 and Figs. S1–S4. When describing imaging results in the following section, we will use the taxon name as shorthand for cells in the image that are reactive with the taxon-specific probe, but it should be kept in mind that these organisms are likely to be members of the species shown in Table S2. The genera Haemophilus and Aggregatibacter are phylogenetically intertwined in the family Pasteurellaceae and targeted by probe Pas111, which we refer to as Haemophilus/Aggregatibacter. The genera Neisseria, Kingella, and Eikenella are likewise intertwined in the family Neisseriaceae and targeted by probe Nei1030, which we refer to as Neisseriaceae.

Fig. 3. A hedgehog structure in plaque showing spatial organization of the plaque microbiome. Plaque was hybridized with a set of 10 probes each labeled with a different fluorophore. Each panel shows the superposition of several of these individual fluorophore channels. A–D and F–H show a single focal plane near the center of the structure, with two to three fluorophore channels shown in each of A–C and all nine specific probes superimposed in D. (E) Maximum intensity projection of three planes, representing a total of ∼2 μm of thickness, to visualize the continuity of Corynebacterium filaments from the center toward the edge of the structure. F is a detailed view of corncob structures. G is a detailed view of mixed filaments. H shows the fluorophore channel corresponding to the universal bacterial probe, showing that the specific probes (D) identify most of the cells that hybridize to the universal probe. I–L show a second focal plane near the periphery of the structure. Fluorophore channels shown correspond to the following genera in the figure: (A, E, and I) Corynebacterium and Streptococcus; (B and J) Capnocytophaga, Porphyromonas, and Haemophilus/Aggregatibacter; (C and K) Fusobacterium, Leptotrichia, and Neisseriaceae; (D and L) all nine specific probes; (F) Corynebacterium, Streptococcus, Porphyromonas, and Haemophilus/Aggregatibacter; (G) Corynebacterium, Fusobacterium, Leptotrichia, and Capnocytophaga; and (H) Bacteria. The plaque sample was fixed in 2% (wt/vol) paraformaldehyde, stored in 50% (vol/vol) ethanol, and spread onto the slide in 50% (vol/vol) ethanol in preparation for FISH.

Fig. 4. Complex corncob structures in SUPP. (A and B) Clusters of corncobs at the perimeter of hedgehog structures. (A) Whole mount of plaque hybridized with probes for Corynebacterium, Fusobacterium, Streptococcus, Porphyromonas, and Haemophilus/Aggregatibacter. (B) Methacrylate-embedded section hybridized with probes for Corynebacterium, Streptococcus, Porphyromonas, and Haemophilus/Aggregatibacter. (C) Gallery of representative images showing types of corncobs frequently observed. (Scale bar: C, 5 μm.)

Fig. 5. Filaments and rods of several genera intermingle at micron scales in an annulus of the hedgehog structure. The two images shown are from methacrylate-embedded, sectioned plaque from two different donors. Both samples were hybridized with probes for Corynebacterium, Fusobacterium, Leptotrichia, Streptococcus, Porphyromonas, Haemophilus/Aggregatibacter, and Neisseriaceae; the probe set in Upper also included a probe for Capnocytophaga.

Fig. 6. Localization of Actinomyces within hedgehogs, in patches within the base region of hedgehogs, and adjacent to them.

Fig. 7. Nested probing for species-level identification of Corynebacterium. Methacrylate-embedded, sectioned plaque was hybridized with a nested probe set targeting cells at the taxonomic levels of phylum, genus, and species. (A) Low-magnification image shows the three major oral genera of phylum Actinobacteria: Corynebacterium, Actinomyces, and Rothia. High-magnification views show (B) all Actinobacteria, (C) the three genera, and (D) C. matruchotii.

Fig. 8. A cauliflower structure in plaque composed of Lautropia, Streptococcus, Haemophilus/Aggregatibacter, and Veillonella. Scattered cells of Prevotella, Rothia, and Capnocytophaga are also visible.

Table S1. Probes used in this study

Table S2. The supragingival plaque microbiota in health

Fig. S1. Probe set hybridizes as expected with pure cultures. The set of 10 probes, each labeled with a distinct fluorophore, was applied to pure cultures and subjected to imaging and linear unmixing under the same conditions used to image plaque samples. Each of nine taxon-specific probes hybridized with its target taxon and showed no significant hybridization to nontarget taxa. The near-universal probe Eub338 hybridized with all taxa, with variable intensity.

Fig. S2. Corncob structures form around Corynebacterium and do not form around nearby Fusobacterium, Leptotrichia, or Capnocytophaga. A–C show methacrylate-embedded sections from three different donors. Rectangles indicate location of Insets; ovals highlight representative corncobs, each of which has a Corynebacterium core.

Fig. S3. Nested probe set provides species-level identification of hedgehog Corynebacterium. Sample was hybridized with nine probes, each labeled with a different fluorophore, targeting cells at the level of kingdom, phylum, genus, and species. (Top) Two different probes targeting phylum Actinobacteria identify a consistent set of cells. Genus-level probes (Middle Left) identify these cells as the three genera Corynebacterium, Actinomyces, and Rothia and (Middle Right) are shown in the context of cells labeled with the universal probe Eub338 plus autofluorescence. (Bottom Left) A second genus-level probe validates the identity of Corynebacterium cells, and (Bottom Right) a species-level probe identifies them as Corynebacterium matruchotii.

Fig. S4. Tile scan of a hedgehog structure in plaque. Image is a composite of seven fields of view showing a plaque sample with three adjacent hedgehogs.

We detected in plaque a complex microbial consortium characterized by the presence of a mass of Corynebacterium filaments with Streptococcus at the periphery. We refer to this structure as a “hedgehog” because of its spiny, radially oriented filaments. We identified nine taxa as regular participants in hedgehog structures: Corynebacterium, Streptococcus, Porphyromonas, Haemophilus/Aggregatibacter, Neisseriaceae, Fusobacterium, Leptotrichia, Capnocytophaga, and Actinomyces. Other genera were detected rarely or inconsistently in the hedgehog structures. To visualize the regular constituents of the consortium simultaneously, we constructed a probe set consisting of 10 probes: the 9 probes targeting these taxa plus the universal probe Eub338 reactive with essentially all bacteria. Each of these 10 probes consisted of a unique oligonucleotide conjugated to a unique fluorophore (Table S3). To validate the probes for specificity, we applied the 10-probe set to pure cultures, which we hybridized and imaged under the same conditions as natural plaque samples. All probes reacted strongly with the target taxon and insignificantly with the nontarget taxa (Fig. S1).

Table S3. Probe sets used in this study

This 10-probe set revealed large, organized hedgehog structures with a generally consistent composition and spatial arrangement (Fig. 3). The fluorescence signal from each of the probes was acquired with a spectral, confocal microscope, was differentiated using a linear unmixing algorithm (Materials and Methods), and is presented in false color, with combinations of probes shown superimposed as detailed in Fig. 3. Fig. 3 A–D and F–H shows a single focal plane near the middle of the structure. Corynebacterium filaments radiate outward from near the center of the image. The coccoid Streptococcus cells are arranged around the distal tips of the Corynebacterium filaments (Fig. 3A). Also located at the periphery of the structure, in the same region as the Streptococcus, are cells of Haemophilus/Aggregatibacter and Porphyromonas (Fig. 3B). Capnocytophaga occupies a wide band just inside the periphery (Fig. 3B). Also occupying this band but forming a more complete ring or annulus between the periphery and the base are Fusobacterium and Leptotrichia (Fig. 3C). Neisseriaceae forms clusters in and near the periphery (Fig. 3C). Actinomyces, which was represented by only a small number of cells in this particular structure, tended to be located near the base. All taxa are shown superimposed in Fig. 3D.

The spatial arrangement of Corynebacterium relative to other taxa in the structure is detailed in Fig. 3 E–G. Long filaments that move in and out of the plane of focus can be only partially captured in a single optical section (∼1-µm thickness). To visualize the continuity of these filaments, we generated a maximum intensity projection of three adjacent optical sections (Fig. 3E), which shows single filaments that are continuous for more than 50 µm and reach from the core to the periphery of the structure. Some filaments remain visible after they enter the region that contains Streptococcus, whereas others apparently disappear when they enter this zone. A detail of the periphery (Fig. 3F) shows that the corncob structures are composed of a filamentous core (sometimes visualized as Corynebacterium but frequently not stained) surrounded primarily by Streptococcus but also by cells of two other taxa, Porphyromonas and Haemophilus/Aggregatibacter, both of which are in close contact with Streptococcus cells. On their way to this corncob region in the periphery, the Corynebacterium filaments traverse the annulus that is densely populated with elongated rods of Fusobacterium, Leptotrichia, and Capnocytophaga, with cells of all four taxa oriented in roughly the same direction (Fig. 3G).

Completing the overview of the structure, a comparison of the fluorescent signal from the universal probe (Fig. 3H) to the overlay of nine specific probes (Fig. 3D) shows that the taxon-specific probes identify nearly all of the cells in the structure. A second focal plane near the exterior of the structure (Fig. 3 I–L) shows a view of the outer shell composed primarily of corncobs. Toward the center of the image, the edge of the Fusobacterium–Leptotrichia annulus can be seen in end-on view (Fig. 3K). In summary, the plaque hedgehog is a radially organized, multigenus consortium with a framework composed primarily of Corynebacterium, a multitaxon filament-rich annulus, and a periphery of corncob structures.

Corncobs are defined morphologically as structures in which coccoid cells, “kernels,” surround a central filament. Our CLASI-FISH results revealed that the kernels were of different taxonomic types and could be either single or double layer (Fig. 4). Single-layer corncobs had kernels of either Streptococcus or Porphyromonas; double-layer corncobs consisted of a combination of Streptococcus as the inner layer and Haemophilus/Aggregatibacter as the outer layer. The most common corncob had a single layer of Streptococcus kernels surrounded by a partial or complete layer of Haemophilus/Aggregatibacter. Porphyromonas kernels could be colinear with Streptococcus around the same filament, or could form entire corncobs of their own, but in either case were always organized in a single layer. In contrast, cells of Haemophilus/Aggregatibacter were never observed to form their own corncobs with Corynebacterium filaments. When present, they were always found adjacent to Streptococcus cells. The Haemophilus/Aggregatibacter–Streptococcus association was evidently specific, because Haemophilus/Aggregatibacter was not found adjacent to cells of Porphyromonas or other taxa in the absence of Streptococcus. Overall, the close spatial proximity of multiple taxa in corncobs suggests the possibility of significant competitive, exploitative, or mutualistic interactions among these taxa.

In a substantial fraction of corncob structures, weak or no fluorescence signal was detected from the central filament in the region where the kernels were present. Lack of hybridization to the central filament was particularly frequent in whole-mount preparations (Fig. 4A). In embedded and sectioned preparations, the central filament was more consistently visualized (Fig. 4B). Higher magnification images of longitudinal and cross-section views (Fig. 4C) illustrate the visualization or lack thereof of the central filament. However, in all cases in which the central filament was clearly labeled, it hybridized with the Corynebacterium probe, even in cases in which cells of other taxa, including Fusobacterium, Leptotrichia, and Capnocytophaga, were present in abundance immediately adjacent to the corncobs (Fig. S2). This observation indicates that, rather than binding promiscuously to any available filamentous substrate, the cocci are engaged in a highly specific interaction with Corynebacterium.

The filaments or elongated rods inhabiting plaque hedgehogs were striking in both their density and their spatial organization. Both Fusobacterium and Leptotrichia showed elongated morphology and were dispersed thinly at the periphery of the hedgehog but reached very high densities in the region immediately proximal to the periphery (Fig. 3C), a region that we call the filament-rich annulus. Capnocytophaga, likewise, reached high densities in the annulus but also extended into regions of the consortium that were rich in Neisseriaceae (Fig. 3 B and D). In whole-mount preparations, many cells in the filament-rich annulus overlapped in images in which all taxon channels were superimposed (Fig. 3D). This overlap was likely caused, in part, by compression of the 3D structure in whole-mount preparations, so that the cells were more densely packed than would occur in uncompressed material. In plaque embedded in methacrylate and sectioned, the compression was eliminated, and the images showed cells that were tightly packed but clearly resolved and distinct from one another (Fig. 5). Notably, these images showed that bacteria do not form large single-taxon clusters within hedgehogs. Instead, cells of at least four different taxa were intermingled at micron scales. These images show that the local environment of a cell in hedgehog consortia includes cells of several other taxa, even when we define local to mean within a radius of as little as 5–10 μm.

By contrast, the localization of Actinomyces relative to Corynebacterium was characterized more by patchy clusters than by intermingling. Actinomyces cells were generally detected in clumps within the base of the hedgehog or adjacent to hedgehogs, as shown in Figs. 6 and 7. The presence of Actinomyces near the base of a hedgehog is suggestive of the possibility that Corynebacterium finds its attachment site in plaque not directly on the tooth but on a preexisting biofilm containing Actinomyces.

Application of a nested probe set allowed identification of the framework Corynebacterium taxon to the species level. As shown in Fig. 7 and Fig. S3, only three genera, Corynebacterium, Actinomyces, and Rothia, comprised virtually all of the Actinobacteria present in plaque, and the species C. matruchotii comprised nearly all of the Corynebacterium in the hedgehog structure.

Hedgehog structures showed near-universal prevalence among individuals, but the fraction of plaque consisting of hedgehogs was highly variable from sample to sample, even within a single individual. We detected hedgehogs in every individual who was sampled on multiple occasions and in ∼80% of individuals sampled only once. The most exposed surface sampled, the tooth surface on the buccal side, yielded hedgehogs; so did plaque from the gingival margin. Some samples contained multiple hedgehog structures adjacent to one another (Fig. S4). Other samples lacked hedgehogs but contained other consortia. For example, clusters of Lautropia formed the center of a structure that also contained Streptococcus, Haemophilus/Aggregatibacter, and Veillonella and was reminiscent of a cauliflower (Fig. 8). Most samples contained a mixture of hedgehogs and other consortia. Because of this extensive variability and the time-intensive nature of spectral imaging analysis, higher-throughput imaging methods will be required to conduct a comprehensive analysis of spatial, temporal, and individual variation in the abundance of hedgehogs and other consortia in plaque.

In summary, we have discovered distinctive, multigenus consortia in dental plaque, with each taxon localized in a precise and well-defined spatial zone. The precision and reproducibility of this spatial organization indicate that micron-scale organization reflects a finely tuned interaction among the cells comprising oral microbial communities.