Human Oral Multi-species Biofilms: Bacterial Communities in Health and Disease

Possibly the first biofilm samples ever examined from a microbiological perspective were obtained from the oral cavity: Antonie van Leeuwenhoek’s tooth scrapings. Since that time, oral microbiologists have made major contributions to microbial taxonomy, physiology, and ecology. The oral cavity distinguishes itself from other environments by having over 700 phylotypes (taxonomic units), nearly half of which have culturable representatives. Aerobic, facultatively anaerobic, and obligately anaerobic physiologies are present. Members of the microbial kingdoms Archaea, Bacteria and Fungi are present. What generates and maintains this diversity? Why are these communities attractive targets for study? How does community analysis using modern molecular methods differ from that using classical bacteriological approaches? We strive to answer these questions in the following contribution and, as far as possible, we rely on knowledge obtained from studies of plaque in situ. The oral cavity as an environment for biofilm growth Sites for biofilm development in the human oral cavity can be segregated into tooth-associated sites and soft-tissue sites. Tooth surfaces are one of the few non-shedding surfaces in the human body and thus present unique opportunities for biofilm development. Each tooth varies somewhat from its neighbors with respect to flow rate of saliva across its surface, and with respect to wear through contact with the tongue or cheek. In contrast to lingual or buccal tooth surfaces, interproximal (between teeth) tooth surfaces are shielded from wear. Teeth thus present colonization sites not only along a gradient of nutrient quantity/quality that develops according to proximity to the different salivary glands and to gingival sulci (the source of gingival crevicular fluid) (Hannig, 1999), but also along a gradient of shear stress that develops according to salivary flow rate and abrasion (Dawes, et al., 1989). Softtissue surfaces likewise present sites along similar gradients, however these surfaces are continuously shed and thus must be constantly recolonized. Turnover time of oral epithelia ranges from roughly 6 days (tongue, cheek) to as much as 12 days (gingiva) (Itoiz and Carranza, 2002). Desquamated epithelial cells have been noted as components of tooth-surface biofilms (Nyvad, 1993) (Figure 10.1). Clearly, colonization of the desquamating epithelial cells is a different process compared to colonization of the non-shedding tooth surface. caister.com/biofilmsbooks Kolenbrander et al. 176 | Although saliva bathes both surfaces, biofilms on these two kinds of surfaces are certainly distinct. Saliva is the fluid that transports nutrients of dietary origin, in the form of partially dissolved carbohydrates and peptides, to oral biofilms. In addition, the same proteins and glycoproteins that make up the salivary secretions themselves can be substrates for bacterial growth (Homer, et al., 1996; Palmer, et al., 2001). The majority of salivary secretions originate from the three different major glands: parotid, submandibular, and sublingual. Each gland secretes saliva of a different composition: for example, the parotid gland is the source of amylase, whereas the sublingual and submandibular glands secrete the bulk of the mucins (Scannapieco, 1994). Thus, while the secretions of these glands combine within the oral cavity to create the mixture known as whole saliva, proximity of a particular bacterial colonization site to the ducts of the glands influences the composition and flow rate of the saliva across the site. The major component of whole saliva is the group of glycoproteins known as mucins. Mucins are also the major component in other secretions such as cervical mucous and sweat, but saliva differs dramatically from other secretions in the type and amount of other components. For example, amylase is the most prevalent protein component of saliva yet is found in only small amounts in other body secretions. Several minor Figure 10.1 An epithelial cell detected on the enamel surface is colonized with multi-species bacterial biofilm communities in 8-hour supragingival dental plaque. Communities are documented with FISH probes (eubacterial probe EUB338, blue; Streptococcus spp. probe STR405, red) in conjunction with general nucleic acid stain (acridine orange, green). The nucleus of the epithelial cell is stained with acridine orange (green). Streptococcus spp. cells (purple, colocalization of red + blue) are closely associated with non-Streptococcus spp. cells (blue) on the epithelial surface. (Bar 5 m; inset—same region at lower magnification). Reprinted from (Kolenbrander, et al., 2006). See also Plate 10.1. caister.com/biofilmsbooks Human Oral Multi-species Biofilms | 177 salivary components, such as lactoferrin, histatins and lysozyme, have antimicrobial properties thought to hinder invasion of the oral environment by bacteria not specifically adapted to this niche (Scannapieco, 1994). Salivary components such as proline-rich proteins are adsorbed onto tooth surfaces to form the acquired enamel pellicle, a protein film containing molecules specifically recognized by bacterial adhesins during the initial colonization of teeth (Scannapieco, 1994). In fact, because pellicle formation is so rapid (within minutes), oral biofilm bacteria are not in direct contact with the tooth surface. Tooth surfaces in and near the gingival sulci are contacted by a second non-salivary secretion called gingival crevicular fluid (GCF). Like saliva, this secretion is a complex mixture, but GCF is closer in composition to serum than to saliva. GCF is roughly 30-fold higher in protein concentration than is saliva, and GCF is the major source of immunoglobulin G in the oral cavity (Cimasoni, 1983). Flow rates of GCF are very low (0.5–2.4 ml per day across the entire oral cavity) compared to saliva (0.5–2.0 ml per minute), and GCF is the primary fluid within the sulcus. Thus, the hard tissue environments of subgingival (bathed in GCF) and supragingival (bathed in saliva) sites are different from each other and their topology, in turn, is different from the soft tissue surface structure. The constant flow of saliva and GCF make it imperative for oral bacteria to adhere to a surface to prevent being swallowed. The differences in molecular composition of the acquired pellicle on the supragingival surface, GCF-bathed subgingival surface, and soft tissue surface mediate specific bacterial adherence preferences called tissue tropism.