The Oral Cavity From a Microbe’s Perspective

With the exception of those microorganisms that are present in feces and in secretory fluids, all bacteria maintain themselves within their host by adhering to a surface. This principle also applies to the oral cavity. From an ecologic viewpoint, the oral cavity, which communicates with the pharynx, should be considered as an “open growth system” with an uninterrupted ingestion and removal of microorganisms and their nutrients. A dynamic equilibrium exists between the adhesion forces of microorganisms and a variety of removal forces that originate from the following sources: (1) swallowing, mastication, or blowing the nose; (2) tongue and oral hygiene implements; (3) the wash-out effect of the salivary, nasal, and crevicular fluid outflow; and (4) the active motion of the cilia of the nasal and sinus walls. Most organisms can only survive in the oropharynx when they adhere to either the soft tissues or the hard surfaces (i.e., teeth, restorative or prosthetic materials, and implants).

The ability of a bacterium to adhere to its host is crucial for the induction of infectious diseases, such as gingivitis or periodontitis.³¹⁰ Oral bacteria and especially pathogenic bacteria, such as Porphyromonas gingivalis (Figure 8-1, H and J) and Aggregatibacter actinomycetemcomitans (Figure 8-1, K), have a large battery of virulence factors, one of which is the ability to adhere to hard intraoral surfaces and/or to the oral mucosae (Figure 8-2).^{56,58,105,358}

On the basis of physical and morphologic criteria, the oral cavity can be divided into six major ecosystems (also called niches), each with the following distinct ecologic determinants:

In health, there is a core set of microorganisms that are almost universally present in these ecosystems. This core microbiome includes members of the phyla Firmicutes (Streptococcus spp., Veillonella spp., and Granulicatella spp.), Proteobacteria (Neisseria spp., Campylobacter spp., and Haemophilus spp.), Actinobacteria (Corynebacterium spp., Rothia spp., and Actinomyces spp.), Bacteroidetes (Prevotella spp., Capnocytophaga spp., and Porphyromonas spp.) and Fusobacteria (Fusobacterium spp.).149,200,433

Table 8-2 summarizes several publications that discuss the detection frequency of periodontopathogens in these different niches. Most species (with the exception of spirochetes) (Figure 8-1, L) are able to colonize all of them. Some periodontopathogens (e.g. Fusobacterium nucleatum [Figure 8-1, G], Prevotella intermedia [Figure 8-1, H]) are involved in the etiology of tonsillitis, and most periodontopathogens are able to colonize the maxillary sinus.^37,404

The soft-tissue surfaces are actively involved in the process of bacterial adhesion and colonization.¹⁴⁶ They employ a variety of mechanisms to prevent the adhesion of pathogenic organisms, with shedding being one of the most important. The high turnover rate of the intraoral epithelial cells, especially of the gingiva, prevents the permanent accumulation of large masses of microorganisms on these surfaces. In essence, this is a natural cleansing mechanism. However, bacteria can also adhere to host cells and form a commensal relationship that is beneficial for both parties.

The host vaginal epithelial cells, for example, supply glucose for the colonized lactobacilli, which in turn produce acid. A lowering of the pH prevents the growth of many other species that have deleterious effects on the vaginal environment.³¹⁷ As such, these endogenous bacteria and their products can be considered necessary and beneficial components of a healthy body.

In periodontal pockets, studies have shown high numbers of bacteria attached to pocket epithelial cells in vivo. Areas of gingival inflammation are characterized by an increased number of adhering bacteria.82,392 These adhering bacteria can also infiltrate the pocket wall in relatively large numbers and reach the underlying stroma (Figure 8-3).^99,232,327 In general, there is a positive correlation between the adhesion rate of pathogenic bacteria to different epithelia and the susceptibility of the affected patient to certain infections.²⁶³

Women prone to urinary tract infections, for example, harbor five times more bacteria per cell in adhesion assays of Escherichia coli to different epithelial cells of their urogenital tract (periurethral, vaginal, or uroepithelial cells). Similar observations have been made regarding the adhesion of Streptococcus pneumoniae to nasopharyngeal epithelial cells of children prone to recurrent otitis media infections as well as regarding the adhesion of Haemophilus influenzae to buccal cells of subjects prone to acute bronchitis.69,381

There are some indications that the same may be true for periodontal infections. Isogai and coworkers reported a significantly lower adherence rate of P. gingivalis and P. intermedia strains to gingival epithelial cells in rats that were resistant to gingivitis compared with susceptible rats.¹⁵⁵ An in vitro study of cultured human pocket epithelial cells (Figure 8-4) showed a similar tendency when patients who were resistant to periodontitis were compared with patients with severe periodontal breakdown.²⁹⁹

Bacteria also adhere to hard tissues. In the human body, teeth and nails are the only naturally occurring nonshedding surfaces. Artificial nonshedding surfaces of medical importance are prosthetic devices such as catheters, artificial joints, dental implants, and heart valves. From a microbiological viewpoint, teeth and implants are unique for two reasons: (1) they provide a hard, nonshedding surface that allows for the development of extensive structured bacterial deposits; and (2) they form a unique ectodermal interruption. There is a special seal of epithelium (junctional epithelium) and connective tissue between the external environment and the internal parts of the body. The accumulation and metabolism of bacteria on these hard surfaces is considered the primary cause of caries, gingivitis, periodontitis, periimplantitis, and, sometimes, bad breath.

In the periodontal pocket, different strategies contribute to bacterial survival, such as adhesion to the pocket epithelium and, when dentine is encountered, the colonization of the dentine tubules.²⁹⁰ The crevicular fluid with its constant outflow does not favor the maintenance of unattached bacteria in the periodontal pocket.

It has been suggested that teeth are the primary habitat for periodontopathogens, because soon after a full-mouth tooth extraction in patients with severe periodontitis, key pathogens such as A. actinomycetemcomitans and P. gingivalis disappeared from the oral cavity as determined by bacterial culturing techniques.⁷¹ Prevotella intermedia and other black-pigmented Prevotella spp. did remain, but at lower detection frequencies and numbers (see Table 8-2).

The same applies to edentulous infants and wearers of full dentures in whom significant proportions of periodontopathogens have been recorded, with the exception of A. actinomycetemcomitans and P. gingivalis.70,179 Therefore, teeth were considered as a “porte d’entrée” for periodontopathogens.

However, studies involving the use of molecular tools to detect and quantify oral bacteria seem to indicate that A. actinomycetemcomitans and P. gingivalis are not entirely eradicated after full-mouth extraction. They may remain colonizers of the oral cavity, but, when teeth are lost, their relative numbers decrease.³⁰⁴

Alternatively, cariogenic species were thought to be relatively restricted to solid surfaces (see Table 8-2). Therefore, S. mutans (Figure 8-1, M) was often considered an obligate periphyte.³⁸⁰ In some studies, this species was only detected from the time that the deciduous teeth erupted in the oral cavity.⁵⁰ In a longitudinal observation of adults with severe dental caries, the cariogenic species fell below detection level after full-mouth extraction but reappeared a few days after denture insertion.⁵¹ On the basis of these reports and on their own observations, Caufield and Gibbons concluded that most of the S. mutans cells in the saliva or on the tongue are derived from the biofilm present on the teeth and that the mucosae could not act as a reservoir for the infection of teeth by those organisms.⁵⁴ They suggested a “window of infectivity” for the acquisition of S. mutans at a mean age of 26 months (range, 9 to 44 months).⁵³ This observation has been supported by a few clinical studies, which showed that the initial colonization of S. mutans varied between 7 to 36 months, which is the time period that coincides with the eruption of the primary teeth.^7,49,101 On the other hand, longitudinal studies by Wan and coworkers^408,409 and Law and Seow¹⁹⁸ showed that S. mutans colonization increased with the age of children, without any discrete window of infectivity. There is now clinical evidence that S. mutans can be detected in the mouths of predentate children, before the eruption of the first tooth.^243,408,410

Bacteria and Their Biofilm Mode of Living

To many people, the word microbiologist conjures images of someone in a laboratory coat swilling a flask of bacterial cells that are growing happily as pure cultures in nutrient-rich broth. In nature, bacteria rarely enjoy such an easy life. The major struggle faced by bacteria lies in obtaining sufficient nutrients to support growth. Competition is rife, and most microbial communities contain many different species, sometimes 100 or more, sharing the same site. Nutrients tend to concentrate at interfaces, and consequently the densest bacterial populations are located at interfaces of various kinds. In aquatic ecosystems, the major interfaces are the solid–liquid boundaries, such as the surfaces of submerged rocks or soil particles, and the air–water interface that is present in open systems, such as oceans or lakes. Bacteria grow well in the laboratory at the solid–gas interface on the surface of an agar plate. However, desiccation rapidly kills most bacterial cells, and microbial growth at solid–gas interfaces is therefore generally restricted to areas where moisture is available. The human body provides several interfaces that support microbial populations, of which the most important in healthy individuals are the skin, gut, mouth, and female urogenital tract. As mentioned previously, the oral cavity is somewhat unique in this regard, because it provides hard, nonshedding surfaces (teeth) that are accessible for microbial colonization.

The importance of surfaces for microbial growth was recognized as early as the 1920s, when a number of workers independently noted that bacteria growing on glass slides submerged in soil were different from those that could be cultured in broth.¹⁹⁷ However, it was not until around 50 years later that sessile microbial populations were considered to be sufficiently different from free-living microorganisms to merit their own name, and the term biofilms was coined (Figure 8-5 and Figure 8-6, B and C). Biofilms are composed of microbial cells encased within a matrix of extracellular polymeric substances, such as polysaccharides, proteins, and nucleic acids.

Biofilm bacteria are often up to 1000 times more resistant to antimicrobial agents than their planktonic counterparts.9,92 Bacteria that grow in multispecies biofilms interact closely with neighboring cells. Sometimes these interactions are mutually beneficial, as is the case when one organism removes another’s waste products and uses them as an energy source. In other instances, bacteria compete with their neighbors by secreting antibacterial molecules such as inhibitory peptides (bacteriocins) or hydrogen peroxide (H₂O₂). In addition, the biofilm mode of growth facilitates cell–cell signaling and deoxyribonucleic acid (DNA) exchange between bacteria. It is clear that microbial ecology within biofilm communities is highly complex and that, in many cases, knowledge is only emerging at this point.

Biofilms are heterogeneous: variations in biofilm structure exist within individual biofilms and between different types of biofilms. However, a number of structural features that are common to many biofilms have been noted. For example, biofilms frequently contain microcolonies of bacterial cells. Water channels are commonly found in biofilms, and these can form a primitive circulatory system that removes waste products and brings fresh nutrients to the deeper layers of the film. Surface structures, such as fronds, can dissipate the energy of fluid flowing over the biofilm and lead to the rapid blockage of vessels.⁸¹ In mixed-species biofilms, there is often heterogeneity in the distribution of different species. Steep chemical gradients exist, such as those of oxygen or pH, and these produce distinct microenvironments within the biofilm.

Microbial populations on the surfaces of teeth (dental plaque) are excellent examples of biofilm communities (Figure 8-7). The architecture of a dental plaque biofilm has many features in common with other biofilms. It is heterogeneous in structure, with clear evidence of open fluid-filled channels running through the plaque mass^64,65,425 (Figure 8-5). Nutrients make contact with the sessile (attached) microcolonies by diffusion from the water channels to the microcolony rather than from the matrix. The bacteria exist and proliferate within the intercellular matrix through which the channels run. The matrix confers a specialized environment that distinguishes the bacteria that exist within the biofilm from those that are free-floating; this is the so-called “planktonic state” in solutions such as saliva or crevicular fluid. The biofilm matrix functions as a barrier. Substances produced by bacteria within the biofilm are retained and concentrated, which fosters metabolic interactions among the different bacteria.

The intercellular matrix consists of organic and inorganic materials derived from saliva, gingival crevicular fluid, and bacterial products.

Organic constituents of the matrix include polysaccharides, proteins, glycoproteins, lipid material, and DNA.²¹⁵ Albumin, which probably originates from crevicular fluid, has been identified as a component of the plaque matrix. The lipid material consists of debris from the membranes of disrupted bacterial and host cells, bacterial vesicles, and possibly food debris. Glycoproteins from the saliva are an important component of the pellicle that initially coats a clean tooth surface, but they also become incorporated into the developing plaque biofilm. Polysaccharides produced by bacteria also contribute to the organic portion of the matrix. They play a major role in maintaining the integrity of the biofilm.

The inorganic components of plaque are predominantly calcium and phosphorus, with trace amounts of other minerals such as sodium, potassium, and fluoride. The source of inorganic constituents of supragingival plaque is primarily saliva. As the mineral content increases, the plaque mass becomes calcified to form calculus (Figure 8-8). Calculus is frequently found in areas of the dentition adjacent to salivary ducts (e.g., the lingual surface of the mandibular incisors and canines, the buccal surface of the maxillary first molars), which reflects the high concentration of minerals available from saliva in those regions. The inorganic components of subgingival plaque are derived from crevicular fluid (a serum transudate). The calcification of subgingival plaque also results in calculus formation (Figure 8-9). Subgingival calculus is typically dark green or dark brown, which probably reflects the presence of blood products that are associated with subgingival hemorrhage.

The importance of these biofilms for oral diseases, such as dental caries and periodontitis—together with the relative ease with which tooth surface biofilms can be accessed—has led to dental plaque becoming one of the most highly studied biofilm systems. It is anticipated that, by understanding the mechanisms involved in the accumulation of dental plaque and the transition from health to disease, it will be possible to improve our control over these processes and to further restrict plaque-associated oral diseases.

• Supragingival plaque is found at or above the gingival margin; when in direct contact with the gingival margin, it is referred to as marginal plaque.

• Subgingival plaque is found below the gingival margin, between the tooth and the gingival pocket epithelium.