Dental plaque (bacteria biofilm) - genesis and structure. Microbiology of dental plaque - differences between immature and mature plaque. Microbial metabolism and plaque biofilm pathogenic potential. Prof. d-r Kabaktchieva - 2014y.

The dental professional comes into contact with two of the most widespread of all human diseases - dental caries and periodontal diseases

a single pathogenic microorganism. gingivitis caries caries Unlike typical infectious diseases, dental caries and periodontal diseases are not caused by a single pathogenic microorganism.

a naturally acquired bacterial biofilm that develops on the teeth Dental caries and periodontal diseases result from the accumulation of many different species of bacteria that form dental plaque a naturally acquired bacterial biofilm that develops on the teeth .

Dental plaque Dental plaque is a multi-species biofilm, Some bacterial species may be of greater relevance in the development of caries and periodontal diseases.

To understand the role of dental plaque in caries must first know how dental plaque forms ? how changes in the proportions of different plaque bacteria can contribute to the development of this oral disease?

Dental Plaque: A Microbial Biofilm Most natural surfaces have their own coating of microorganisms, or biofilm, adapted to their individual habitats.

Bacterial adhesion to surfaces primarily involves two types of reactions: physicochemical and biochemical. These same interactions occur in the formation of plaque and calculus on oral structures.

in the case of bacterial plaque, All living cells in nature, including bacterial plaque cells, have a net-negative surface charge. The cells can, therefore, be attracted to oppositely charged surfaces as skin. in the case of bacterial plaque, have attracting between the surfaces of cells , teeth, and soft tissues of the oral cavity.

slime layers which prolong the existence of The microorganisms within bacterial plaque can produce : extracellular coatings, such as slime layers which prolong the existence of biofilms ; a variety of surface fibrils, or appendages, that extend from their cell walls. These mechanisms mediate attachment of bacteria to a substrate by providing additional attachment structures between the tooth surface and the plaque, thus allowing the formation of adherent matrices.

Bacterial Colonization of the Mouth Microorganisms found in the oral cavity are naturally acquired from the environment. Bacteria are acquired from the atmosphere, food, human contact. Bacteria form colonies between saliva and hard tissues such as erupted teeth, and exposed root cementum and dentin.

- the reduced enamel epithelium and Prior to eruption, the external surface of tooth enamel is lined by remnants of the enamel-forming organ. These tissue remnants are: - the reduced enamel epithelium and - the basal lamina

The basal lamina connects the epithelium to the enamel surface. The basal lamina is also continuous with organic material that fills the microscopic voids in the superficial enamel. This subsurface organic material appears as a fringe-like structure attached to the basal lamina and is composed of residual enamel matrix proteins. This material is referred to as a subsurface pellicle. The pellicle originates from local cells during tooth formation; therefore, it is considered to be of endogenous origin.

The transmission electron micrograph demonstrates : SSP B AP ES The transmission electron micrograph demonstrates : remnants of the subsurface pellicle (SSP) the acquired pellicle (AP) They are between the enamel surface (ES) and the bacterial cells (B) of the dental plaque.

Figure . Junction of reduced enamel epithelium and enamel. RA HD EM ES BL Figure . Junction of reduced enamel epithelium and enamel. The reduced ameloblasts (RA) are attached to the enamel by hemidesmosomes (HD) and a basal lamina (BL). EM, enamel matrix remnants form a subsurface pellicle; ES, enamel space.

- digested by salivary and bacterial enzymes When the tooth emerges into the oral cavity, the remnants of the reduced enamel epithelium are: - worn off - digested by salivary and bacterial enzymes

This coating is also referred to as a pellicle. An erupted tooth immediately becomes covered by a thin, microscopic coating of saliva materials. The salivary components become adsorbed to the surface of the enamel within seconds. This coating is also referred to as a pellicle.

Because the pellicle is acquired after eruption of teeth, it is said to have an exogenous origin the pellicle was formed by a substance from outside the tooth, rather than during the development of teeth.

The oral bacteria can form colonies in the acquired pellicle.

The Acquired Pellicle The coating of salivary origin that forms on exposed tooth surfaces is called the acquired pellicle. It is acellular and consists primarily of glycoproteins derived from saliva B АР ЕS

A glycoprotein is a protein molecule that includes an attached carbohydrate component.

Oral fluids and small molecules can slowly diffuse through the acquired pellicle into the superficial enamel; If the pellicle is displaced, ( by a prophylaxis), the pellicle begins to reform immediately; It takes about a week for the pellicle to develop its condensed, mature structure, which may also incorporate bacterial products.

Colonization of the acquired pellicle can be beneficial for the bacteria, because the pellicle components can serve as nutrients. For example: proline-rich salivary proteins may be degraded by bacterial collagenases. This action releases peptides, free amino acids, + salivary mucins may enhance the growth of dental plaque organisms, such as actinomycetes and spirochetes.

The carbohydrate components of certain pellicle glycoproteins may serve as receptors for proteins that bind bacteria to surfaces - e.g., adhesins, Тhereby the adhesins contributing to bacterial adhesion to the tooth.

The binding sites on the pellicle, are also host proteins, including: immunoglobulins (i.e., antibodies), the enzyme lysozyme, and proteins of the complement system. These host proteins originate from saliva and gingival sulcus fluid.

competing for the binding sites. Аn antagonistic relationship often exists between different types of bacteria competing for the binding sites. For example: it has been shown that some streptococci synthesize and release proteins called bacteriocins, which can inhibit some strains of Actinomyces and Actinobacillus species.

Dental Plaque Formation All bacteria that initiate plaque formation come in contact with the organically coated tooth surface by chance. Forces exist that tend either to allow bacteria to accumulate on teeth or to remove them. Shifts in these forces determine whether more or less plaque accumulates at a given site on a tooth.

Bacteria tend to be removed from the teeth during mastication of foods, by the tongue, and by toothbrushing and other oral hygiene activities. For this reason, bacteria tend to accumulate on teeth in sheltered, undisturbed environments, which basically are sites at risk. These sites include the occlusal fissures, the surfaces apical to the contact between adjacent teeth, and in the gingival sulcus.

Therefore, it is no coincidence that the major plaque-based diseases - caries and inflammatory periodontal diseases arise at these sites where plaque is most abundant and stagnant.

Initial plaque formation may take as long as 2 hours. Colonization begins as a series of isolated colonies, often confined to microscopic tooth surface irregularities.

With the aid of nutrients from saliva and host food, the colonizing bacteria begin to multiply. About 2 days are required for the plaque to double in mass, During which time the bacterial colonies have been growing together.

The most dramatic change in bacterial numbers occurs during the first 4 or 5 days of plaque formation. After approximately 21 days, bacterial replication slows, and plaque accumulation becomes relatively stable.

The increasing thickness of the plaque limits the diffusion of oxygen to the entrapped original, oxygen-tolerant populations of bacteria. As a result, the organisms that survive in the deeper aspects of the developing plaque are either facultative or obligate anaerobes.

The forming bacterial colonies are rapidly covered by saliva. When seen with the scanning electron microscope, growing colonies protrude from the surface of the plaque as domes .

of dome formation in the plaque. . “Domes” have appearance of a cluster of igloos beneath newly fallen snow Scanning electron micrograph of dome formation in the plaque. .

- human cells such as epithelial cells (desquamated cells) In individuals with poor oral hygiene, superficial dental plaque may incorporate : - food debris , - human cells such as epithelial cells (desquamated cells) - leukocytes. This debris is called materia alba, which literally means "white matter.“ Unlike plaque, it is usually removed easily by rinsing with water. cloud

At times, the plaque demonstrates staining, which is caused by chromogenic bacteria, which produce a brown pigment.

Molecular Mechanisms of Bacterial Adhesion The initial bacterial attachment to the acquired pellicle ( A) is thought to involve physicochemical interactions (e.g., electrostatic forces and hydrophobic bonding) between molecules of the amino acids phenylalanine and leucine.

details A. A side chain of a phenylalanine component of a bacterial protein interacts via hydrophobic bonding with a side chain of a leucine component of a salivary glycoprotein in the acquired pellicle.

The hydrophobicity of some streptococci, is caused by cell wall-associated molecules including glucosyltransferase, an enzyme that converts the glucose portion of the sugar, sucrose, into extracellular polysaccharide. Some glucosyltransferases have been designated as hydrophobins. These polysaccharides include "sticky" glucans that, through hydrogen bonding, are thought to contribute to the mediation of bacterial adhesion (Fig. C). Once the bacteria adhere, they are often "entombed" as additional glucan is produced.

C. The host's dietary sucrose is converted by the bacterial enzyme, glucosyltransferase, to the extracellular polysaccharide, glucan, which has many hydrophobic groups and can interact with amino acid side-chain groups, such as serine, tyrosine, and threonine. in the acquired pellicle.

Molecular Mechanisms of Bacterial Adhesion

B. The negatively charged carboxyl group of a bacterial protein is attracted to a positively charged calcium ion (i.e., electrostatic attraction), which in turn is attracted to a negatively charged phosphate group of a salivary phosphoprotein in the acquired pellicle.

Bacteria also have external cell-surface proteins termed adhesins, which have lectin-like activity, because they can bind to carbohydrate components of glycoproteins. Тhe adhesins may be located on bacterial surface appendages, such as fimbriae (Figure D). Fimbria-associated adhesins probably mediate bacterial adhesion via ionic or hydrogen-bonding interactions.

D. The fimbrial surface appendage extends from the bacterial cell to permit the terminal adhesin portion to bind to a sugar component of a salivary glycoprotein

Facultative anaerobes can exist in an environment with or without oxygen; obligate anaerobes cannot exist in an environment with oxygen. Lectins are plant proteins with receptor sites that bind specific sugars.

Another molecular mechanism of bacterial adhesion is calcium bridging Another molecular mechanism of bacterial adhesion is calcium bridging. (Figure B). In this process positively charged, divalent calcium ions in the saliva help to link the negatively charged cell surfaces of bacteria to the negatively charged acquired pellicle

Bacteria in the Dental Plaque The bacteria colonize the teeth in a reasonably predictable sequence. The first to adhere are primary colonizers, sometimes referred to as "pioneer species”. These are microorganisms that are able to stick directly to the acquired pellicle.

Those that arrive later are secondary colonizers. They may be able to colonize an existing bacterial layer, but they are unable to act as primary colonizers. Generally speaking, the primary colonizers are not pathogenic.

If the plaque is allowed to remain undisturbed, it eventually becomes populated with secondary colonizers that are the likely etiologic agents of dental caries and periodontal diseases. The earliest colonizers are cocci (spherical bacteria), especially streptococci, which constitute 47% to 85% of the cells found during the first 4 hours after professional tooth cleaning. These organisms tend to be followed by short rods and filamentous bacteria.

Тhe most abundant colonization is on the proximal surfaces, in the fissures of teeth, and in the gingival sulcus region. Cocci are probably the first to adhere because they are small and round and, therefore, have a smaller energy barrier to overcome than other bacterial forms.

gram-negative cocci such as Veillonella tend to increase. The first colonizers tend to be aerobic (oxygen-tolerant) bacteria including Neisseria and Rothia; The streptococci, the gram-positive facultative rods, and the actinomycetes are the main organisms in plaque found in early fissures and approximal plaque. As plaque oxygen levels fall, the proportions of gram-negative rods (e.g., fusobacteria) and gram-negative cocci such as Veillonella tend to increase. .

Of the early colonizers: Streptococcus sanguis often appears first, followed by Streptococcus mutans Both depend on a sheltered environment for growth and the presence of extracellular carbohydrate (e.g., sucrose). Sucrose is used to synthesize intracellular polysaccharides that serve as an internal source of energy, as well as external polysaccharide coats.

The polysaccharide coating helps protect the cell from the osmotic effects of sucrose. In addition, the coating reduces the inhibitory effect of toxic metabolic end products, such as lactic acid, on bacterial survival.

Тhe non-motile cells, including streptococci and actinomycetes, come into contact with the tooth randomly, motile cells such as the spirochetes are likely to be attracted by chemotactic factors (e.g., nutrients). Surface receptors probably provide a means of attachment for secondary colonizers onto the initial bacterial layer.

Bacteria that cannot adhere easily to the tooth initially via organic coatings can probably attach by strong lectin-like, cell-to-cell interactions with similar or dissimilar bacteria that are already attached (i.e., the primary colonizers).

Gram-negative, anaerobic species such as Treponema, Porphyromonas, Prevotella, and Fusobacterium species predominate in the subgingival plaque during the later phases of plaque development, but they may also be present in early plaque. There is evidence that oxygen does not penetrate more than 0.1 mm into the dental plaque, a fact that may explain the presence of anaerobic bacteria in early plaque.

Dental Plaque Matrix The organisms are positioned perpendicular to the tooth surface as a result of competitive colonization. The bacterial cells in the biofilm are surrounded by an intercellular plaque matrix (Figure – see next).

Figure . An electron micrograph showing palisades (P) of bacteria perpendicular to the enamel surface (ES), bacterial cells that are probably secondary colonizers (SC), the intercellular plaque matrix (IPM), the acquired pellicle (AP).

salivary and serum proteins/ glycoproteins represent minor components. The matrix is composed of both organic and inorganic components that originate primarily from the bacteria. Polysaccharides derived from bacterial metabolism of carbohydrates are a major constituent of the matrix, salivary and serum proteins/ glycoproteins represent minor components.

Some bacteria on the surface of the biofilm aggregate into distinctive structures that include arrangements of cocci ("corn-cob" configurations) and rods ("test-tube brush" configurations) radially arranged around a central filament (Figure see next).

Figure : A. Cross section of "corn cob" from 2-month-old plaque. A coarse fibrillar material attaches the cocci (C) to the central filament (CF).. B. Coarse "test-tube brush" formations consisting of central filament (CF) surrounded by large, filamentous bacteria with flagella uniformly distributed over its body (LF). Background consists of a spirochete-rich microbiota (S).

Dental Plaque Metabolism For metabolism to occur, a source of energy is required. For the caries-related S. mutans and many other acid-forming organisms, this energy source can be sucrose.

Almost immediately following exposure of these microorganisms to sucrose, they produce: acid, intracellular polysaccharides, which provide a reserve source of energy for each bacterium extracellular polysaccharides including glucans (dextran) and fructans (levan).

Glucans can be viscid substances that help anchor the bacteria to the pellicle, as well as stabilize the plaque mass. Fructans can act as an energy source for any bacteria having the enzyme levanase.

The glucans constitute up to approximately 20% of plaque dry weight, Levans - about 10%, and Bacteria the remaining 70% to 80%. The glucans and fructans are major contributors to the intercellular plaque matrix.

Plaque organisms grow under adverse environmental conditions, which include: - varying pH, temperature, ionic strength, oxygen tension, nutrient levels, antagonistic elements, such as competing organisms , the host inflammatory-immune response.

Variations of the aforementioned conditions can affect the primary and extended adherence of microorganisms to the surface, as well the diffusion of essential elements (i.e., oxygen and nutrients), all of which are required for the prolonged existence of bacterial biofilm.

The plaque organisms must find a safe haven in relation to their neighbors and the oral environment. Such a favorable location is termed an ecological niche.

With dietary sugars entering the plaque, anaerobic glycolysis results in acidogenesis (acid production) and accumulation of acid in the plaque. If no acid-consuming organisms (e.g., Veillonella) are available to use the acids, the plaque pH drops rapidly from 7.0 to below 4.5. This drop is important because enamel begins to demineralize between pH 5.0 and 5.5.

One possible outcome of the drop in pH may be the dissolution of the mineralized tooth surface adjacent to the plaque, resulting in carious cavitation of the tooth. This process provides the bacteria access to the inorganic elements (e.g., calcium and phosphate) needed for their nutritional requirements.

Until supragingival plaque mineralizes as dental calculus, it can be removed by mechanical debridement (e.g., toothbrushing, flossing, or use of interdental aids).

coloring

Dental plaque cannot be removed by rinsing alone. As the plaque matures, it becomes more resistant to removal with a toothbrush. almost 3 times as much pressure to remove it on the third day as on the first. Зъб.плака не може да бъде отстранена само с изплакване. С узряването на плаката тя става по-резистентна на отстраняване с помощта на четка. Почти три пъти повече налягане е необходимо да се отстрани плаката на третия ден в сравнение с това необходимо при първия ден .

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