Tracking Dynamic Interactions during Plaque Formation

The mouth is an ecologically complex environment and host to a diverse microbial community (18, 20). Many of the microbes amass and grow between the teeth and along the gum line, resulting in formation of dental plaque. High cell density is a key characteristic of this microbial community, and plaque development is influenced by both cooperative and competitive interactions (12). Interactions occur through physical contact and through sensing the concentrations of extracellular compounds and signaling molecules. Communication between species may include favorable cross-feeding, competition for essential nutrients, killing through secretion of antimicrobial compounds, the production of extracellular molecules whose sole role is to influence gene expression, and the release of end products that broadcast metabolic activities throughout the community at the functional level (8, 13). Moreover, some or all of the interactions may occur simultaneously. Dissecting the spatial relationships between bacteria within the community and determining why juxtaposition of certain species is favorable are two primary challenges in oral biofilm research. Although complexity has made it extremely difficult to discern the full extent of interactions, in vivo accessibility makes dental plaque amenable to manipulation. In this issue of the Journal of Bacteriology, Chalmers et al. (1a) describe a novel approach for tracking the complex microbial interactions within this biofilm community. They, for the first time, conclusively demonstrate a role for specific cell-cell contact in the initial stages of plaque formation.

A HIGHLY SELECTIVE ORGANIZATION

Development of dental plaque is a progressive process initiated by adherence of specific bacterial species to salivary proteins and glycoproteins adsorbed on the tooth enamel surface. Data indicate that these interactions are highly selective (5, 14). The initial colonizers are often referred to as pioneer species, and colonization has been shown to be reproducibly sequential; species of Streptococcus, Actinomyces, Prevotella, Veillonella, and Neisseria are the pioneers that lead the way (5). This is followed by attachment of secondary colonizers, such as species of Fusobacterium and Capnocytophaga (8). Streptococcal species account for the largest fraction (63%) of the biomass during the initial stages of biofilm development (14), indicating the propensity of these organisms to accumulate at the enamel-saliva interface. These species create the physical foundation of dental plaque.

Direct contact between genetically distinct species (coaggregation) has been shown to occur with various oral isolates and is likely the origin of many of the spatial relationships within the biofilm structure. The direct-contact interaction was first described over 30 years ago by Gibbons and Nygaard(6), and since then researchers have shown that coaggregation occurs with organisms belonging to the same or different genera and can be disrupted by the addition of certain sugars (for reviews, see references 9, 10, and 19). Data from these in vitro studies have been used to develop a model for bacterial interactions before and during the initial stages of plaque biofilm development, as well as during the recruitment of secondary colonizers into the biofilm. Besides the temporal considerations influencing interactions during the maturation of plaque, multiple environmental parameters clearly affect interactions in vivo. Bacteria that inhabit certain sites within the oral cavity tend to associate with other bacteria typically isolated from the same location (e.g., bacteria originating from the tongue coaggregate best with other tongue-localized bacteria [7], indicating that there is direct spatial organization in the formation of oral biofilms). A number of lines of evidence indicate that coaggregation is directed by species-specific interactions (3). For example, adherence of the oral anaerobe Porphyromonas gingivalis to Streptococcus gordonii involves a discrete region of the streptococcal surface polypeptide SspB, designated BAR (4). Interestingly, P. gingivalis does not adhere to Streptococcus mutans, even though this species has a putative homologue of SspB with high sequence similarity. Hence, slight differences in the sequence of the surface polypeptide can eliminate interactions, supporting the hypothesis that coaggregation has specificity. Also, specific interactions can be disrupted by addition of sugars (such as lactose or galactose), indicating that specific receptor-ligand interactions are involved (1, 11, 22).

NOVEL TECHNIQUES: THE CORNERSTONE OF NEW DISCOVERIES

Translating findings from basic in vitro research to in vivo studies can pose significant challenges in biomedical research. However, because of the accessibility of the oral cavity this is not necessarily the case for microbiological studies of this location. In fact, a colleague at the Forsyth Institute has admitted to swishing cultures of his favorite bacterium around in his mouth (with institutional review board approval) to see if flagellum mutants were competitive in colonization—all for the sake of research. Such enthusiastic dedication, not withstanding, other less “risky” strategies are being employed. New tools, such as a retrievable enamel chip which is placed in the oral cavity for a designated amount of time and then removed for analyses, have been used in a number of studies (17). This retrievable system combined with novel molecular techniques, such as fluorescence in situ hybridization and immunofluorescence, are helping to merge in vivo studies with in vitro analyses (15, 16, 21).

Quantum dots (QD), which are luminescent nanocrystals, are also being used to label probes (antibodies) in order to localize bacterial cells within biological samples. QD of different sizes emit discrete colors of light when they are exposed to broad continuous excitation, which allows excitation of multiple QD simultaneously. Combinations of different QD and fluorophore-conjugated antibodies can be used with biological samples. Just recently, QD-conjugated probes were used to localize bacterial cells within dental plaque (2), and now Chalmers et al. (1a) have taken these techniques one step further. Using micromanipulation, these workers were able to capture initial biofilm cells from the surface, tease apart the species through selective plating, and then examine biofilm formation by the isolates in vitro, thus determining whether in vivo associations are also formed in vitro under simulated growth conditions (i.e., in the presence of saliva as the sole nutrient source). In essence, the associations were deconstructed in order to see if they would naturally reform in vitro, hence bringing the study of cell-cell interactions full circle. This clever use of advanced technologies is pioneering the way to determining the critical building blocks of dental plaque. These new techniques will likely eliminate our reliance on cell morphology. Who knows? Maybe all those cocci are a melting pot of many different functions.