Our findings show that the corncob microhabitat is selective for a subset of the Streptococcus species in dental plaque. The most abundant streptococci in dental plaque are members of the S. mitis/oralis/infantis cluster and S. sanguinis, which together make up about 90% of plaque streptococci. The species S. cristatus is a minor component of the genus in plaque as a whole, making up less than 4% of supragingival plaque Streptococcus in Human Microbiome Project samples across 148 individuals [7]. Nonetheless, S. cristatus was the most abundant Streptococcus species in corncobs. Thus, the site-specificity displayed by S. cristatus is for a well-defined microhabitat within dental plaque: adhered to Corynebacterium filaments as corncobs. The association is not exclusive, however, in that a different Streptococcus species, identified by FISH as a member of the S. mitis/oralis/infantis group, was almost as abundant as S. cristatus in corncobs. Detailed comparison of staining from the Streptococcus genus vs. species probes indicated that S. cristatus and the S. mitis/oralis/infantis group together comprised almost all the Streptococcus cells in corncobs; other unidentified Streptococcus were rarely present. In particular, S. gordonii, a species with overall abundance in supragingival plaque similar to that of S. cristatus, was detected in surrounding plaque but not in corncobs. Thus, the corncob represents an interaction between Corynebacterium and a limited subset of the pool of plaque Streptococcus species.
Members of other genera present in corncobs likewise were adjacent to multiple partners but not all potential partners in plaque. Cells hybridizing with the Porphyromonas probe were present in corncobs, either as the only cocci surrounding a filament or sharing a central filament with S. mitis/oralis/infantis, S. cristatus, or both. The additional outer layer of Pasteurellaceae was found adjacent to cells of both S. mitis/oralis/infantis and S. cristatus, but not Porphyromonas. This distribution indicates that the Pasteurellaceae-Streptococcus relationship in corncobs, like the Corynebacterium-Streptococcus relationship, is a selective interaction. Although Pasteurellaceae spp. associated with two different Streptococcus spp. partners, it did not associate with Porphyromonas spp. Further study will be needed to determine the mechanistic underpinnings of this spatial selectivity: whether it results from differential binding or differential reproductive success of Pasteurellaceae spp. when bound to Streptococcus spp. rather than Porphyromonas spp., or both.
Although corncob-like structures have been reported to form around other taxa, several lines of evidence suggest that in supragingival plaque the corncob filament is generally Corynebacterium spp. In vitro studies [30] have shown that Fusobacterium nucleatum, when mixed with Streptococcus, can form the central filament of corncob-like structures. However, we have not seen an association of cocci with Fusobacterium spp. in natural dental plaque. Our previous results with a probe set targeting different filamentous bacteria in plaque, including Fusobacterium, indicated that the corncob association was highly specific to Corynebacterium spp. [12]. Although staining of Corynebacterium was variable in intensity, the central filament of corncobs, when staining was evident, was always Corynebacterium. Other filamentous or elongated taxa such as Fusobacterium, Leptotrichia, and Capnocytophaga were not detected as the central filament even when they were detected in the immediate surroundings of the corncob. A previous study [34] showed associations of streptococci with hyphae of Candida albicans in natural plaque. However, Candida generally has low abundance in the healthy mouth. The quantification results in this study apply to the full population of corncob cocci that we visualized in healthy subjects, whether or not the identity of the central filament could be confirmed.
Our finding of complex but limited taxon composition in corncobs bears on an important question in microbial ecology, namely the question of how a stable, healthy interaction is maintained between a host and its microbiome [35]. Theoretical work predicts that mutualistic interactions tend to fall apart over time, for example because the loss of one of the partners results in the loss of the other, or because one partner ceases to behave as a mutualist and instead becomes a parasite [36]. Such a shift from mutualism to parasitism is more likely if the interaction is highly specific, so that an organism is dependent on a single partner [37]. Bacteria within the densely packed dental plaque biofilm depend on one another for metabolites and signals [19, 38, 39], but the composition of oral microbial communities is characterized by wide fluctuations in the relative abundance of taxa even as the overall community membership remains stable, a pattern known as stationary dynamics [40, 41]. We observed flexibility in the taxon relationships involved in corncobs, in the sense that several partners were capable of interacting with the central Corynebacterium filament and several streptococci could interact with the outer layer of Pasteurellaceae. The interactions we observe in corncobs suggest that each of the microbial participants is capable of interacting with multiple, albeit limited, potential partners, a feature that may encourage the long-term stability of the community.
A related open question in microbial ecology is whether microbial communities assemble with a consistent species composition or, alternatively, with a consistent set of functional genes that can be contributed by a range of different species [42, 43]. It has been proposed, for example, that under conditions common in the mouth (horizontal gene transfer and migration), species identity can be insignificant because genes, rather than species, inhabit niches [44]. Despite the flexibility we observed in the composition of individual corncobs, however, both S. cristatus and S. mitis/oralis/infantis were observed in corncobs in every donor. At the scale of individual corncobs or corncob segments, these distinct Streptococcus species were apparently interchangeable in their ability to bind to the central Corynebacterium filament and the exterior shell of Pasteurellaceae, yet both types persisted in the plaque community. This persistence suggests that the different taxa possess distinct ecological roles, or that mechanisms exist that stabilize the continued persistence of multiple, functionally redundant taxa within the same microbiome ecosystem. Our data thus indicate that in the corncob microhabitat within the dental plaque biofilm, species composition remains consistent from mouth to mouth.
The heterogeneity of corncob structures has important implications for mechanistic studies such as in vitro co-culture or multi-taxon metabolic modeling of plaque bacteria as a model microbial community. In addition to the Corynebacterium-S. cristatus relationship, our results show numerous pairs of taxa directly adjacent to one another in corncobs, including all combinations of S. cristatus, S. mitis/oralis/infantis, Porphyromonas spp., and Corynebacterium spp. as well as Pasteurellaceae with both S. mitis/oralis/infantis and S. cristatus. Thus, a number of potentially significant taxon-taxon relationships have been identified in this study, and our results suggest that a natural corncob may be modeled not only as a two-taxon relationship but also as a relationship containing three, four, or five partners. Although the species visualized here in corncobs have not yet been the subject of extensive in vitro investigation, other oral species within these same genera have been investigated and their taxon-taxon interactions have been shown to change the gene expression and biology of the partners [14, 45, 46]. Our results enable selection of taxa for in vitro co-culture studies that are grounded in the frequently adjacent taxa of natural plaque; these are the taxa that are likely to engage in metabolic interactions with physiologically relevant consequences.
Because of their location towards the outside of plaque, corncob taxa may represent the first organisms that a microbe would encounter when landing on the tooth biofilm. S. cristatus has been shown to inhibit biofilm formation of the periodontal pathogen P. gingivalis by repression of virulence genes [47, 48]. This finding suggests that corncobs composed of S. cristatus might therefore inhibit colonization of the mouth by this potential pathogen. On the other hand, some mitis group streptococci potentiate the virulence of C. albicans [49, 50]. Under what circumstances the species that participate in corncob structures inhibit or enhance the colonization and virulence of pathogens, and the mechanisms by which they do so, is an important question for further research. In studies of the development of plaque on epoxy resin crowns worn by volunteers, the initial plaque was coccus-rich, corncobs were first observed after three days, and filament-rich plaque did not occur until approximately 1 week of incubation [25, 26]. These observations might suggest that hedgehog structures and corncobs would be rare in people engaging in daily dental hygiene. In the present study, however, we detected corncobs in all donors and hedgehog structures in most, even though donors were instructed to refrain from oral hygiene for only 24 hours and no donor reported going longer than 26 hours without tooth brushing. We conclude that hedgehogs and corncobs can be formed in less than 24 hours in the dental plaque of healthy individuals, perhaps growing from already-established patches of filament-rich plaque, and that corncob structure and function may play an important role in normal oral microbial ecology.