Dental Biofilms

Benjamin Lim

What is a Biofilm?

A biofilm is made up of a community of microogrganism stuck to each other and/or a surface. As these cells adhere to each other and the surface, they produce an extracellular matrix of polymeric substances. The biofilm extracellular polymeric substance is sometimes referred to as "slime," and contains a polymeric jumble of DNA, proteins and polysaccharides. (Costerton et al, 1995)

Dental Biofilm

As soon as a tooth erupts or when one cleans his teeth, an enamel pellicle is formed. An enamel pellicle is a protein film on the surface of enamel when negatively-charged glycoproteins from saliva adheres to the tooth surface. The enamel pellicle also contains several components derived from the saliva, such as cystatins, histatins, lysozyme, proline-rich and mucinous proteins. It is a bacteria-free layer and serves two purpose: 1. provide lubrication during mastication, 2. Protect the tooth from demineralization due to its selective-permeability, restricting transport of ions in and out of the dental hard tissues.

A dental pellicle is usually 0.1 to 1 micrometers thick. It slowly matures and develops into a dental biofilm as bacteria begin to colonize it. (Garcia & Hicks, 2008) The development of a dental biofilm is discussed in the following section.

Please refer to the section on "Function on Saliva" for a greater understanding of the dental pellicle.

Development Of Dental Biofilm

Biofilms are formed in 3 basic stages (See Fig. 1):

Biofilm formation

Fig. 1 Development of a Dental Biofilm.
Stage 1. Pellicle Formation; Stage 2. Bacteria Colonization; Stage 3 Biofilm Maturation; Stage 4. Maturation II; Stage 5. Dispersion. ( Davies, 2007)

Stage 1. Pellicle Formation

As discussed earlier, a pellicle is formed as soon as the teeth are cleaned. It is an acellular protein film that forms on the surface enamel by selective binding of negatively- charged glycoproteins from saliva that prevents continuous deposition of salivary calcium phosphate. Bacteria and other microorganisms then begins to adhere to this layer (Garcia & Hicks, 2008).

Stage 2. Bacteria Colonization

The pellicle-coated teeth is subsequently colonized by a number of gram-positive bacteria, including several strains of Streptococci, attaching to the negatively charged glycoproteins in the enamel pellicle.(Fig. 2) These "primary colonizers" then produce proteins which allow for attachment and adherence to the pellicle-coated tooth surface. Protein rich glycoprotein have anchoring receptors that allow microorganisms to attach firmly to their surfaces. The attachment is mainly by electrostatic, hydrophobic ionic and van der Waals forces.(Fejerskov & Kidd, 2003) The bacterial surface appendages structures (fibrils, Fimbriae), lectin-like interactions, surface protein, antigen, electrostatic interactions, hydrophobic interactions may have a profound effect on the adhesion and colonization to tooth surface.

Dental Biofilms - Cariology
Fig. 2 Simplified explanation of the the principle of selective adherence of bacteria to the enamel. Successful attachment occurs
when surface characteristics of the bacteria fit with the component in the pellicle (Fejerskov & Kidd, 2003)

The types of attachment of bacteria onto the biofilm layer can be classified into specific and non-specific types. Specific types refer to microscopic interactions as the interactions between stereochemically complementary surface components occurring over extremely short distances allowing specific ionic, hydrogen and possibly chemical bonds. Non-specific interactions refers to overall macroscopic surface properties as charge or surface free energy. These interactions can extend over relatively longer distances. It is impossible at the present stage to give a more precise description of what is meant by 'extremely short' and 'considerably large' separation distances, mainly because the bacterial cell surface cannot be considered as the flat surface of a sphere. As soon as any kind of surface appendage can bridge the separation distance (or a considerable part of it) any definition of separation distance looses its meaning. (Busscher & Weerkamp, 1987) Fig. 3 summarises the types of attachment interactions.

specific and non-specific interaction
Fig 3. Schematic representation of interactions involved in bacterial adhesion to solid substrata (Busscher & Weerkamp, 1987)

At separation distances > 50 nm: only attractive Van der Waals forces operate. It is too far for the opposing surfaces to recognize specific surface components.

At separation distances between 10-20 nm: interactions occur due to electrostatic repulsion. Adhesion is reversible in this state, but due to a rearrangement on the bacterial cell surface leading to specific short-range interactions, adhesion changes gradually to less reversible or essentially irreversible. Water films between the interacting surfaces must be removed by the role of hydrophobicity and hydrophobic surface component at the end, which enables specific short-range interactions to occur.

At separation distances < 1.5 nm: specific interactions occur only at this extremely small distances, where the energy barrier has been overcome. Special interactions upon the direct contact lead to an irreversible bonding. The capability to do so will be very much strain-dependent.
(Busscher & Weerkamp, 1987)

Plaque on the surface then increases as the primary colonizers divide, and as secondary colonizers attach to the primary colonizers, and multiply in number as well. Secondary colonizers include Actinomyces spp. and gram-negatives such as Prevotella intermedia and Capnocytophagaspecies (as well as many others).

Tertiary colonizers may include species of Porphyromonas, Campylobacter, and Treponema. By this time, metabolic reactions in the biofilm may lead to predictable structures in some members of the plaque: for example, a "corn cob" like array of cocci bound to filaments within the biofilm.

This process allows for bacterial diversity in dental plaque with aerobic and anaerobic organisms coexisting. Between 300 and 500 bacteria colonize dental biofilms, with many organisms still undergoing identification and characterization (Garcia & Hicks, 2008).

Stages 3 and 4. Biofilm Maturation

As the biofilm matures (1 to 2 weeks or older), the inner layer of the tooth surface becomes densely packed with gram-positive bacteria next to the tooth surface while the outer layer of the biofilm is usually loosely structured and varies in composition. The most striking change in the composition of biofilm is, however, the shift of a Streptococcus-dominated biofilm to one dominated by Actinomyces. This shift in microbial population is known as microbial succession.

The principle of such a phenomenon is that the pioneer bacteria creates an environment that is more attractive to secondary invaders but increasingly less favourable for their own species. Some factors contributing to this may be due to the lack of nutrients, the accumulation of inhibitory products or the lowering of oxygen concentration due to the thickening of bacteria deposits.

As such, there is a gradual replacement of the pioneer microbial community to species more suitable for the modified environment. Figure 4 shows the mircobial succession of some bacteria found in the dental biofilm. (Fejerskov & Kidd, 2003)

Dental Biofilms - Cariology

Fig. 4 Relative proportions of selected microorganisms developing in plaque in relation to their atmospheric requirements (Fejerskov & Kidd, 2003)

and Composition

A biofilm commnuity is contains 4 basic components (Fig. 5): 1. Bacterial micro-colonies, 2. An extracellular slime layer, 3. Fluid Nutrient channel, 4. Primitive communicative system. (Nield-Gehrig, 2003)

Structure of Biofilm
Fig. 5 Structure of a Polymicrobial Biofilm (Nizet & Esko, 2009)

Bacteria Micro-colonies

As the bacteria attach to a surface and to each other, they cluster together to form sessile, mushroom-shaped microcolonies. Different microcolonies may contain different combinations of bacterial species. Each microcolony is a tiny, independent community containing thousands of compatible bacteria. Bacteria found in the center of a microcolony may live in a strict anaerobic environment, while other bacteria at the edges of the fluid channels may live in an aerobic environment (Fig 6). The biofilm structure provides a range of customized living environments (with differing pHs, nutrient availability, and oxygen concentration) within which bacteria with different physiological needs can survive (Nield-Gehrig, 2003).

In biofilms, bacteria live under nutrient limitation and in a dormant state in which defense molecules (e.g. antimicrobial peptides) produced by the immune system and pharmacologic antibiotics are less effective. Moreover, the extracellular matrix can bind and inactivate these same agents, contributing to the persistence of the biofilm and difficulty in medical treatment of biofilm infections, such as those that arise on catheters and other medical devices (Nizet & Esko, 2009).

Dental Biofilms - Cariology
Fig. 6 Illustration showing the aerobic regions of a biofilm near
the fluid nutrient channel and anaerobic regions near the centre of the biofilm.(Nield-Gehrig, 2003)

Extracellular Slime Layer

Surrounding every bacteria microcolonies is the extracellular slime layer, also known as the Exopolysaccharide Structured(EPS) matrix. It acts as a protective barrier, protecting the bacterial microcolonies from antibiotics, antimicrobials, and host defense mechanisms. EPS may account for 50% to 90% of the total organic carbon of biofilms.(Nield-Gehrig, 2003)

The EPS is a complex and extremely imperative component of all biofilms as it provides architectural structure and mechanical stability to the attached population. The matrix is composed of cells, water and secreted/released extracellular macromolecules. In addition, a range of enzymic and regulatory activities can be found within the matrix. Together, these different components and activities are likely to interact and in so doing create a series of local environments within the matrix which co-exist as a functional consortium. The matrix architecture is also subject to a number of extrinsic factors, including fluctuations in nutrient and gaseous levels and fluid shear. Together, these intrinsic and extrinsic factors combine to produce a dynamic, heterogeneous microenvironment for the attached and enveloped cells. (Allison, 2003)

The EPS is synthesized by biofilm bacteria. It varies greatly in their composition and in their chemical and physical properties. The EPS contains uronic acids (D-glucuronic, D-galacturonic, or D-mannuronic acids) or ketal-linked pyruvate which are negatively-charged. Inorganic residues, such as phosphate or sulfate, also contribute to the negative charge. It is thought that the carbon in the EPS itself can serve as the primary carbon reserve for biofilm microorganisms during substrate deprivation(Nizet et at, 2009). This property is important because it allows association of divalent cations such as calcium and magnesium, which have been shown to cross-link with the polymer strands and provide greater binding force in a developed biofilm.EPS production is known to be affected by nutrient status of the growth medium; excess available carbon and limitation of nitrogen, potassium, or phosphate promote EPS synthesis. (Allison, 2003)

In addition, the EPS is also highly hydrated because it can incorporate large amounts of water into its structure by hydrogen bonding. EPS may be hydrophobic, although most types of EPS are both hydrophilic and hydrophobic.EPS may also vary in its solubility. The composition and structure of the polysaccharides determine their primary conformation. For example, many bacterial EPS possess backbone structures that contain 1,3- or 1,4-β-linked hexose residues and tend to be more rigid, less deformable, and in certain cases poorly soluble or insoluble. Other EPS molecules may be readily soluble in water. The EPS of biofilms is not generally uniform but may vary spatially and temporally. (Donlan, 2002)

Fluid Nutrient Channel

Biofilms are very heterogeneous, containing micro-colonies of bacterial cells enveloped in an EPS matrix. Within the EPS, there are voids which refers to the absence of bacterial clusters. A series of fluid channels penetrates the EPS through these instititial voids(Fig 7). These channels allow liquid flow, providing nutrients and oxygen for the bacterial micro-colonies and facilitate movement of bacterial metabolites, waste products, and enzymes within the biofilm structure(Nield-Gehrig, 2003).

Dental Biofilms - Cariology
Fig. 7 Illustration showing the fluid channels passing through the EPS,
facilitating the movement of nutrients in and out of the biofilm (Nield-Gehrig, 2003)

Primitive Communication System

Each bacterial microcolony uses chemical signals to create a primitive communication system used to communicate with other bacterial microcolonies (Fig. 8).

Dental Biofilms - Cariology

Fig. 8 Bacteria uses chemical signals to communicate with each other(Nield-Gehrig, 2003)

Some more common forms of communication within bacterial colonies include gene expression, cell-cell signaling and antibiotic resistance. Specific bacteria within the biofilm are able to act with other species to help or impair the host (Fig. 9)

i. Biofilm regulation of gene expression. Most studies have been performed on bacteria that predominate in supragingival plaque (e.g.streptococci). During the initial stages of biofilm formation by S. mutans (first 2 h following attachment), 33 proteins were differentially expressed (25 proteins were up-regulated; 8 proteins down-regulated). There was an increase in the relative synthesis of enzymes involved in carbohydrate catabolism; these might be needed for energy generation, although these molecules are multi-functional and can also act as adhesins when located on the cell surface. In contrast, some glycolytic enzymes involved in acid production were down-regulated in older (3 day) biofilms, while proteins involved with a range of biochemical functions including protein folding and secretion, amino acid and fatty acid biosynthesis, and cell division were upregulated. This findings demonstrate that the biofilm environment can have an effect on gene expression by plaque bacteria. (Marsh, 2005)

ii. Cell-cell signaling and gene transfer. In addition to the many conventional metabolic interactions (synergistic and anatagonistic) that have been well catalogued to occur among oral bacteria, organisms from plaque have also been shown to communicate with one another in a cell density-dependent manner via small diffusible molecules. In the case of S. mutans, it was discovered that the competence-stimulating peptide (CSP) was necessary for proper biofilm formation of S. mutans. It was also shown that this CSP-mediated cell-cell signaling causes the transformation frequency of S. mutans be 10- to 600- fold greater in biofilms than in planktonic cells. This shows that the quorum-sensing pathway in S. mutans needed for genetic competence is key in the proper formation of dental plaque biofilms. (Marsh, 2005)

iii. Antibiotic resistance. Bacteria growing in dental plaque also display an increased tolerance to antimicrobial agents The age of the biofilm can also be a significant factor; older biofilms (72 h) of S. sanguinis were more resistant to chlorhexidine than younger (24 h) biofilms. Confocal microscopy of in situ established natural biofilms showed that chlorhexidine only affected the outer layers of cells in 24 and 48 h plaque biofilms, suggesting either quenching of the agent at the biofilm surface or a lack of penetration. Biofilms of oral bacteria are also more tolerant of antibiotics (e.g. amoxycillin, doxycycline, minocycline, metronidazole) than planktonic cells, although the degree of resistance can vary with the organism, the model system and the inhibitor used. (Marsh, 2005)

Dental Biofilms - Cariology
Fig. 9 Schematic representation of the types of interaction that occur in a microbial community, such as dental plaque, growing as a biofilm.Bacteria adhere by adhesin–receptor interactions to the acquired pellicle or to already attached cells (co-adhesion). Bacteria interact synergistically to metabolize complex host molecules, and food webs can develop, enabling the efficient cycling of nutrients. Bacteria communicate via diffusible signalling molecules and by gene transfer; bacteria can also engage in cross-talk if in contact with host cells. Cells in biofilms are less susceptible to antimicrobial agents and the host defences; this may be because of physical properties of the biofilm or to protection from neighbouring cells, e.g. because of the secretion of neutralizing enzymes to making sensitive cells appear resistant (‘‘R’’), or following horizontal gene transfer. The environmental heterogeneity generated within biofilms encourages genotypic and phenotypic diversity, which enhances their ability to persist in the face of assault from the innate and adaptive immune responses, from antimicrobial attack, and from environmental stress (Marsh, 2005)

Cariogenic features of Dental Biofilm

Demineralization of Enamel Surface

Caries formation begins when fermentable dietary carbohydrates is made available to acid-producing (acidogenic) bacteria. Dental biofilms contain these cariogenic bacteria and some examples of such bacteria include S. mutans, Streptococcus sobrinus, Lactobacillus species and Actinomyces species, as well as to a lesser extent Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii and Streptococcus anginosus. These bacteria are able to survive low pH environments (aciduric). They ferment dietary carbohydrates into lactate. When dietary carbohydrates is in abundance, steptococci produces mainly lactate, a plaque acid that drives the demineralization process.

As more acid is produced, the concentration of hydrogen ions increase significantly, lowering the pH within the biofilm fluid and along the interface between the biofilm and enamel surface from 7.0 to 5.0. The critical pH(around pH 5.0) at which enamel undergoes dissolution is reached. This increase in hydrogen ions creates a driving force to diffuse hydrogen ions into the fluid in the pores surrounding the hydroxyapatite(HAP) in the subsurface enamel. This results in the demineralization of the subsurface enamel whereby calcium and phosphate moves towards the enamel surface, resulting in the egress of calcium and phosphate from the enamel subsurface to the overlying biofilm (Fig 10). Dental plaque is now rich in calcium and phosphate deposits (Garcia & Hicks, 2008).

Dental Biofilms - Cariology
Fig. 10 Demineralization of tooth (South Beach Dental, 2009)

The process of demineralization and remineralization occurs continuously, with periods of demineralization interspersed with remineralization. The effects of demineralization can be reversed if there is adequate time between acidogenic challenges to allow for remineralization to occur (Fig 11).

Dental Biofilms - Cariology
Fig. 11 Diagram showing the balance between the pathological and protective processes of caries formation (Featherstone, 1999).

Remineralization of Enamel Surface

Remineralization of the enamel surface begins after the resting pH of the dental plaque is restored. The hydrogen ion concentration equilibrates between the dental biofilm and the fluid in the pores surrounding the HAP crystals of the demineralized subsurface enamel. There is no longer a driving force for demineralization.
Calcium and phosphate ions are transported passively into the subsurface enamel from the saliva and biofilm. The primary driving force for the remineralization of enamel surface is by the supersatured calcium and phosphate concentrations in saliva and plaque as compared with the less saturated fluid in the subsurface enamel pores (Garcia & Hicks, 2008).

Please refer to section "Function of Saliva" for videos on the demineralization and remineralization of the enamel.



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