Dental biofilm (plaque) – development,

structure, composition and properties

Assoc. Prof. Dr. N. Gateva, PHD

The resident MOs gain a secure, warm, nutritious habitat from the host

Benefits to the host:- contribute to food digestion, nutrition, - regulation of human metabolism, - differentiation of the host mucosa, - immune development and function, and prevention of colonization by exogenous (often pathogenic) microbes - produce vitamins (vitamin B12, thiamine and riboflavin, vitamin K required for blood coagulation)

The resident microflora:

• Make an active contribution to the maintenance of health.

• This relationship between the resident microbiome and the host is dynamic.

• In health - the composition of resident populations is remarkably stable.

• This can be perturbed by changes in lifestyle, immune status or by broad spectrum antibiotic therapy. • Such perturbations have been associated with a number of

clinical disorders such as obesity, allergy and a variety of inflammatory disease.

Bacterial lifestyles• In order to persist, oral microorganisms have to attach to a surface and

grow, otherwise they will be lost from the habitat.

• Most bacteria live in complex communities called biofilms.

• A biofilm is a well-organized 3D structured community of bacteria that adheres to surfaces and is embedded in an extracellular slime layer.

• Once a bacterium attaches to a surface, it activates a whole different set of genes that gives the bacterium different characteristics from those that it had as a free-floating organism.

• More than 99% of all bacteria on earth live as attached bacteria (in biofilm).

• The dental plaque is an example of such biofilm.

Definition of dental biofilm

• It is a microbial ecosystem or biofilm composed of densely packed microbial structure of insoluble salivary glycoprotein, microbial intracellular products and to some extend epithelial cells and debris arranged in an organized complex inter cellular matrix. (WHO)

•Dental plaque can be defined as the soft deposits that form the biofilm adhering to the tooth surface or other hard surfaces in the oral cavity, including removable and fixed restorations. (Carranza 9th edition)

Dental biofilm

• is a complex microbial community with specific bacterial growth on a polysaccharide matrix attached to the enamel through the acquired pellicle.

The structure of biofilm communities

A biofilm community comprises of:

•bacterial microcolonies;

• an extracellular slime layer;

• fluid channels;

• a primitive communication system.

Bacterial microcolonies

• Oral bacteria attach to a surface and to each other

• They cluster together to form sessile, mushroom-shaped microcolonies that are attached to the surface at a narrow base

• Each microcolony is a tiny, independent community containing thousands of compatible bacteria.

Bacterial microcolonies• Different microcolonies may contain different

combinations of bacterial species.

• 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.

• The biofilm structure provides a range of customized living environments (with differing pH, nutrient availability, and oxygen concentration) within which bacteria with different physiological needs can survive.

Extracellular slime layer. • Is protective barrier thatcovers mushroom bacterialmicrocolonies.

• It protects microorganisms inthe microcolony fromantibiotics, antimicrobialagents and host defensemechanisms.

• The matrix that integrates and supports microbial colonies is made up of polysaccharides, proteins and Deoxyribonucleic acid (DNA) secreted bybacteria.

Fluid channels: • A series of fluid channels penetrates the

extracellular slime layer.

• They provide nutrients and oxygen for the bacterial microcolonies and facilitate movement of bacterial metabolites, waste products, and enzymes within the biofilm structure.

• Special microorganisms are responsible for maintaining the 3-D architecture of the biofilm.

• They release an abundance of molecules that protect the fluid channels from sticking or filling with other microorganisms.

Primitive communication system:

• To communicate with other bacterial microcolonies each bacterial microcolony uses chemical signals.

• This forms a primitive communication system of biofilm.

Composition of Dental biofilm:

• Primarily of microorganisms

• Organic matter • Polysaccharide

• Glycoprotein

• Lipid

• Albumin

• Inorganic matter • Sodium

• Potassium

• Fluoride

Formation and development of dental biofilms

Microbial colonization in the mouth

• The moment a baby passes through the birth canal and takes its first breath, microbes begin to reside in its mouth.

• Later, as the teeth erupt, additional bacteria establish colonies on the tooth surfaces.

Dental biofilm formation:

Formation of pellicle

Attachment of single bacterial cells (0–24 h).

Growth of attached bacteria leading to the formation of distinct microcolonies(4–24 h).

Microbial succession (and coadhesion) leading to increased species diversity concomitant with continued growth of microcolonies (1–14 days).

Climax community/mature biofilm (2 week or older).

Dental biofilm formation:

Formation of pellicle

The Pellicle • Is an acellular, proteinaceous film, derived from saliva, which

forms on a ‘naked’ tooth surface.

• It is a thin coating of salivary proteins that attach to the tooth surface within minutes after a professional cleaning.

• The major constituents of the pellicle are salivary glycoproteins, phosphoproteins, lipids and, • to a lesser extent, components from the gingival

crevicular fluid.

• Salivary proteins that form the pellicle have affinity for oral streptococci and serve as receptors for bacterial adhesins.

1. Pellicle

• It acts like double-sided adhesive tape, adhering to the tooth surface on one side and on the other side, providing a sticky surface that facilitates bacterial attachment to the tooth surface.

1. Pellicle formation

• Following pellicle formation, bacteria begin to attach to the outer surface of the pellicle.

• Bacteria connect to the pellicle and each other with hundreds of hairlike structures called fimbriae or with bacterial-binding proteins -adhesins.

• The act of attaching to a solid surface stimulates the bacteria to excrete an extracellular slime layer that helps to anchor them to the surface and provides protection for the attached bacteria.

Function of pellicle:

• The pellicle plays an important modifying role in caries and erosion because of its permeable-selective nature restricting transport of ions in and out of the dental hard tissues.

• The pellicle can influence plaque`s formation.

• Glycoproteins in the pellicle serve as a food for microorganisms and for bacterial cell receptors. • They can bind immunoglobulins, complement system proteins, and lysozyme

from the saliva or gingival crevicular fluid.

• Thus, the pellicle becomes part of the local immune defense of the body.

Function of pellicle:

• The pellicle serves for the adhesion of the bacterial cells. • It is accomplished with the help of electrostatic forces as

well as through specific interactions of the bacterial cells with the proteins of the pellicle.

• Salivary proteins that form the pellicle have affinity for oral streptococci and serve as receptors for bacterial adhesins.

• They support the colonization of bacterial cells.

Dental biofilm formation:

Formation of pellicle

Attachment of single bacterial cells (0–4 h).

2.2.

• Bacteria connect to the pellicle and each other

• with hundreds of hair like structures called fimbriae or pills

• with bacterial-binding proteins -adhesins.

• Once they stick, the bacteria begin producing substances that stimulate other free-floating bacteria to join the community.

Initial adhesion and bacteria attachment

Attachment of bacteria

• Attaching bacteria to the tooth surface is the first step in the formation of the dental plaque.

• Bacterial species have surface structures:

• Fibers/filaments that help to attach them to different surfaces.

• Adhesins.

Microbial colonization

• Specific way of a bacterial cell`s attachment on the tooth surface: • the bacteria have a surface

recognition system -components of the bacterial surface - adhesins that allows:

• to bind to additional molecules of the pellicle - receptors.

Attachment of bacteriaInitial microbial

colonization

Enamel

Pellicle

Dental biofilm formation:

Formation of pellicle

Attachment of single bacterial cells (0–24 h).

Growth of attached bacteria leading to the formation of distinct microcolonies (4–24 h).

Microbial colonization

• of teeth requires that bacteria adhere to the surface.

• begins with formation of 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.

• The biofilm grows primarily through cell division of the adherent bacteria, rather than through the attachment of new bacteria

Primary colonizers

• The first microorganisms, which can adhere

• They are able to stick directly to the acquired pellicle.

• are not pathogenic.

• The earliest colonizers are overwhelmingly cocci (spherical cells), especially streptococci.

Tooth surface

Primary colonizers

• Are aerobic cocci and rods.

• Cocci are probably the first to adhere because they: • are small and round

• therefore, have a smaller energy barrier to overcome than other bacterial forms.

• Of the early colonizers, Streptococcus sanguis often appears first, followed by S. mutans.

Primary colonizers

Primary colonizers provide new

binding sites for the adhesion of

new oral bacteria.

Their metabolic activity modifies

the local microenvironment.

Early colonizers (Streptococci and

species of Actinomyces) use

oxygen.

This way they reduce the oxidative-

reduction potential of the

environment, which then favors

the growth of anaerobic species.

Primary colonizers:

• Gr (+) facultative bacteria predominate:

• Ss: Str. sanguinis - most represented;

• Av: Actinomyces - after 24h

• Provide new binding sites for adhesion.

• Gr (+) facultative cocci and rods co-aggregate and multiply.

Primary colonizers :• The surface receptors of Gr

(+) facultative cocci and rods allow the subsequent adhesion of Gr (-) organisms that have limited ability to bind directly to the pellicle.

• Fn: Fusobacterium nucleatum

• BI: Prevotellaintermedia

Biofilm

Pellicle

Primary colonization:

As the colonies grow, larger areas of comfortable enamel surfaces are covered

Part of the surface microorganisms, which are not very tightly attached, become readily removable by flushing forces

They are replaced by new bacterial cells from bacterial growth

The formation of the first microbial colonies takes up to 24 hours.

Microbial colonies:

• In the beginning - there is accumulation of cells in separate areas above the enamel.

• In order to be able to structure in colonies, new mechanisms of adhesion are put into operation - between individual cells coaggregation and coadhesion.

Secondary colonization• In just two days the dental biofilm doubles in size.

• The increased plaque and continue accumulation of microorganisms in colonies begins to gradually deplete the oxygen in the deeper layers.

• This becomes a prerequisite for the settlement of other microorganisms for which anaerobic conditions are appropriate.

• After the first day, other microbial species start to settle in addition to cocci and this process is called secondary colonization.

Secondary colonization

Primary colonization

In 2 days – the dental plaque doubles in size

Its thickening and accumulation of MO -depletes oxygen

These is prerequisite for settlement of anaerobic MOs

Diversification of the microbial composition

• Gr(+) и Gr(-) microbial species

Until day 7, Streptococci are the predominant species

Secondary colonizers• A second wave of bacterial colonizers adheres to bacteria that are already

attached to the pellicle. • They may be able to colonize only an existing bacterial layer and are unable

to act as primary colonizers. • Surface receptors of primary colonizers probably provide a means of

attachment for secondary colonizers onto the initial bacterial layer. • Bacteria attach by strong lectin-like, cell-to-cell interactions with similar or

dissimilar bacteria that are already attached (the primary colonizers). • The secondary colonizers also attach to the established pioneer species via

adhesin–receptor interactions (termed coaggregation or coadhesion).

Secondary colonization:

• Diversity of MO increases

• Plaque`s aging and maturation leads to:

• Changes in environmental conditions

• Secondary settlements predominantly of Gr(-) obligates anaerobes.

• As result - the pathogenicity of plaque biofilm increase.

Microbial colonization:

• Starts with microbial adhesion – primary colonizer.

• Some bacteria are unable to bind directly to the conditioning film, but are able to interact with molecules on bacteria that are already attached (co-adhesion), also by adhesin-receptor interactions

• The basis of secondary colonization is the mechanism of co-adhesion or coaggregation.

Coaggregation

• is the ability of new bacterial colonizers to adhere to the previouslyattached cells.

• The bacteria cluster together to form sessile, mushroom-shapedmicro colonies that are attached to the tooth surface at a narrowbase.

• The result of coaggregation is the formation of a complex array ofdifferent bacteria linked to one another.

Coaggregation:

• One bacterium, Fusobacterium nucleatum, can co-adhere with almost all other bacteria found in dental plaque

• It is considered to be a key bridging organism between early and later coloniser

• If the plaque is allowed to remain undisturbed, it eventually becomespopulated with secondary colonizers that are the likely etiologicagents of caries, gingivitis, and periodontitis, the destructive form ofinflammatory periodontal disease.

• With it, the diversity of microorganisms grows by predominantly Gr(-) obligate anaerobes - rods and filamentous microorganisms• Tannerella, Actinomyces, Treponema, Fusobacterium).

• With secondary colonizers, the pathogenicity of plaque biofilmincreases.

Dental biofilm formation:

Formation of pellicle

Attachment of single bacterial cells (0–24 h).

Growth of attached bacteria leading to the formation of distinct microcolonies (4–24 h).

Microbial succession (and coadhesion) leading to increased species diversity concomitant with continued growth of microcolonies (1–14 days).

Dental biofilm formation:

Formation of pellicle

Attachment of single bacterial cells (0–24 h).

Growth of attached bacteria leading to the formation of distinct microcolonies (4–24 h).

Microbial succession (and coaggregation) leading to increased species diversity concomitant with continued growth of microcolonies (1–14 days).

Climax community/mature biofilm (2 week or older).

Mature biofilm• After the 2 week the dynamic changes in the composition of the

plaque decrease and stop.

• After the 21 day, the plaque acquires a degree of stability and no longer increases.

• Flushing forces continuously remove the most superficial layers of bacteria.

• Different microorganisms and microcolonies establish their relationships and form the complex community called the climax community.

• It creates a favorable environment for the mutual life of all microorganisms.

Mature biofilm• A self-sustaining and independent from macroorganism nutrient

medium is created, with nutritional synergy between different microbial species.

• The complex microbial community gains strong resistance to all oral antimicrobial mechanisms, including the immune defense mechanisms of the macroorganism.

• From this point onwards, we are talking about a mature plaque biofilm.

Adaptation of microorganisms to the life inplaque biofilm• MO have complex communication systems that regulate their

division, growth and function.

• These systems are called signaling and are triggered by various stimuli• from the environment,

• can be also unknown molecules.

• They can be emitted from a group of microorganisms and directed toother so-called target cells.

• Signal systems regulate the gene expression of microorganisms bystimulating or suppressing it depending on the situation.

Regulatory system

• A two-component regulatory system composed of histidinekinase and a regulatory response.

• Signal system at critical microbial concentration.

• Such systems have both Gr(+) and Gr(-) microorganisms • In most oral streptococci and some periodontal

pathogens.

Two-component regulatory system

• Only performs interspecies communication and plays animportant role in transmitting signals during microbialadaptation to the biofilm.

• It acts in certain places on the cell membrane.

• When an external stimulus reaches this sensitive membraneregion, its reception takes place via the histidine-kinasereceptor.

• It induces a response that stimulates or suppresses a specific gene that leads to a particular cellular expression.

Signal system at critical microbial concentration (susceptibility to microbial quorum).

• This system is a way of signaling that is activated as a response to the critical accumulation of cellular elements.

• It acts inside the microbial species and among different species. The stimuli of this system are signal molecules called autoinductors. • are produced in a constant quantity and their concentration depends on microbial

density.

• The perception of signals only occurs when they reach a critical threshold of saturation.

• The term quorum is used to indicate that the presence of a certain number of MOs is required to trigger signals that will change the microbial population.

Subgingival biofilm formation:

• Following a few days of undisturbedplaque formation, the gingival marginbecomes inflamed and swollen.

• These inflammatory changes result in the creation of a deepened gingival sulcus.

Subgingival biofilm:

• The biofilm extends into this subgingivalregion and flourishes in this protectedenvironment, resulting in the formation ofa mature subgingival plaque biofilm.

• Gingival inflammation does not appearuntil the biofilm changes from onecomposed largely of Gr(+) bacteria to onecontaining Gr(-) anaerobes.

Subgingival biofilm:

• A subgingival bacterial microcolony arepredominantly composed of Gr (-) anaerobic bacteria.

• They become established in the gingival sulcusbetween 3 and 12 weeks after the beginning ofsupragingival plaque formation.

• Most bacterial species currently suspected ofbeing periodontal pathogens are anaerobic, Gr (-) bacteria.

Subgingival biofilm:

• Gr (-) anaerobic (e.g., oxygen-intolerant) speciespredominate in the subgingival plaque during the laterphases of plaque development.

• They may also be present in early plaque, • Treponema, Porphyromonas, Prevotella, Fusobacterium species.

• There is evidence that oxygen does not penetrate morethan 0.1 mm into the dental plaque, a fact that mayexplain the presence of anaerobic bacteria in earlyplaque.

Microbial differences in mature plaque biofilm• Plaque bacteria vary in number and proportions from time to time and

from site to site within the mouth of any one individual.

• The diversity is even greater between individuals, between races, and between supra- and subgingival plaque.

• Different tooth surfaces create different conditions for microorganisms.

• There are differences in the degree of protection from the flushing forces, and in the biological and chemical factors affecting microbial growth.

• These differences determine the composition of microbial communities byforming different oral niches: • on the approximal surfaces of the teeth.

• on fissures, groves and pits.

• in the area of gingival sulcus.

Approximal plaque biofilm

• Varies considerably from one side to another.

• At all approximal surfaces, anaerobic species are significantly morethan aerobes.

• Predominant microorganisms are of the genus Actinomyces, and mainly of the group of A. naeslundii.

• The number of streptococci is considerably lower than in earlybacterial deposits.

• A relatively high amount of Veillonella is detected.

Approximal plaque biofilm

• Quantitative and qualitative differences in the microbial species of the approximal plaque can be so great that they convert any approximal surface into a unique one.

• These local variations in the composition of the microflora may explain why some surfaces have high carious activity with possible cavity formation, whereas a neighboring approximal surface remains free from clinically detectable caries.

Plaque biofilm on occlusal surface

• The colonization of the fissure with S.mutans and S.sanguis depends entirely on their salivary concentration.

• Once established in the fissures, their presence does not depend on it.

• There are no significant differences with the mature plaque on the smooth surfaces. • Gr(+) cocci and rods predominate.

• There are significant differences in the microbial composition between the fissures of the various teeth as well as in the various branches of a one and the same fissure.

• Because this each fissure is accepted as a unique separate eco-niche.

Plaque biofilm on occlusal surface

• In deep fissures are found differences in the microbial composition at the entrance and at the bottom.

• At the fissure entrance the percentage of streptococcus is significantly higher than that within the fissure. • This explains the morphological findings of the initial carious lesion of the fissure,

which begins at the entrance not at the bottom.

• At the entrance, cocci and rods are arranged palisaded and perpendicular, • very similar to their arrangement on the smooth surfaces. • They are mixed with filamentous microorganisms.

• Within of fissure, the filaments are very few • mainly cocci and rods are present.

• Cocci are grouped in colonies, and the plaque matrix differs significantly in quantity and density.

Subgingival plaque biofilm:

• Bacteria in the subgingival plaque are several motile and non-motile species that do not form distinctive microcolonies.

• They are located on the surface of primary layer of adherent bacterial cells from which they are separated by an abundant intercellular matrix.

• Microorganisms in the subgingival plaque are influenced by the composition of gingival fluid, desquamated epithelial cells and bacterial products.

Subgingival plaque biofilm:

• The predominant MOs in the subgingival plaque are S. mitis, S. sanguis, A. naeslundii, A. odontolyticus, A. meyeri, A. georgiae, Rothiadentocariosus, Eubacterium spp., Fusobacterium spp., Treponema spp.

• Periodontal pathogens are also present in the subgingival plaque.

• In these oral niches Gr(-) anaerobes predominate, which colonize these niches during the late phase of plaque formation. They can also be found in the early plaque.• These are Treponema, Porphyromonas, Prevotella, Fusobacteruim spp.

Classification

By location on tooth

Supra gingival plaque

Coronal Marginal

Sub gingival plaque

Attached Unattached

By pathogenic effects

Cariogenic plaque

Periodontal diseases

Calculogenicplaque

Composition

Pathogenic potential of plaque biofilm• The role of plaque biofilm in the etiology of the two major oral

diseases - dental caries and periodontal disease - has been proven.

• The bacteria that are associated with caries differ from those that areassociated with periodontal disease.

• The pathogens involved in periodontal disease are geographicallylocated deep in the sulcus and are different species with differentmetabolisms.

• The plaque that is responsible for caries is generally locatedsupragingivally and is acidogenic.

Supragingival plaque biofilm - cariogenic potential.

• In the supragingival plaque predominate processes related tocarbohydrate metabolism. Frequent intake of fermentablecarbohydrates results in maintaining a low pH level in the plaque.

• By sugars intake in the supragingival plaque occurs glycolysis andaccumulation of acids. These acids quickly and only for a short period of time cause a drop in pH below the critical level, causinginitiation of dental caries.

Subgingival plaque biofilm - pathogenicpotential in periodontal disease

• Bacteria suspected of causing periodontal disease have proteolytic properties and are represented by:• Treponema, Porphyromonas, Prevotella, Fusobacterium spp.,

Spirochetes.

• They are defined as periodontal pathogens.

The pathogenic mechanisms that cause periodontal disease are:

• Direct cytotoxicity by production of proteases, cytotoxins and other virulent microbial factors:• Hydrolytic enzymes (collagenase, fibrinolysin, protease, hyaluronidase, chondroitin

sulfatase) cause alteration of the periodontal tissue;

• Endotoxins, mucopeptidases, lipoteichoic acid stimulate an inflammatory response and have osteolytic activity;

• Capsule - suppresses phagocytosis increasing bacterial survival within host cells, and facilitates adhesion;

• Microbial products:• leukotoxin - causes cytolysis of neutrophils and monocytes;

• anti-neutrophil factor - suppresses neutrophil chemotaxis;

• microbial metabolic products - organic acids, hydrogen sulphate, indole, ammonia, toxins that have toxic effects on gingival sulcus epithelial cells.

The pathogenic mechanisms that cause periodontal disease are:• Induction of tissue damage indirectly by acute non-specific

inflammatory response.

• Induction of tissue damage through specific immunopathological mechanisms of chronic inflammatory process in the gingiva and the possibility of progressive destruction of the entire periodontal tissue.

Factors, which favor dental biofilm accumulation

Tooth`s irregularities

Restorative materials

Removable prosthodontic constructions

Calculus Anormal tooth

positionTooth in eruption

Cavitation Orthodontic

appliance

Control and Removal of Dental Plaque Biofilms

• Bacterial microcolonies are protected by one another and by the extracellular slime layer and are unusually resistant to antibiotics (administered systemically), antimicrobials (administered locally), and the body's defense system.

• The slime layer may prevent the drugs from penetrating fully into the depth of the biofilm.

• Bacteria can develop resistance to antimicrobial drugs by producing a thicker protective slime layer.

• The slime layer may protect the bacteria against leukocytes (defensive cells of the body's immune system).

Control and Removal of Dental Plaque Biofilms

• Antibiotic or antimicrobial therapy usually will not kill the biofilm.

• The biofilm can be destroyed by simply wiping it off (disrupting their attachment to a surface).

• Physical removal of bacterial plaque biofilms is the most effective means of control.

• Subgingival plaque within pockets cannot be reached by brushes, floss, or oral rinses.

• Therefore, frequent periodontal debridement of subgingival root surfaces by a dental hygienist or dentist is an essential component in the treatment of periodontitis.

Conclusion:

Dental biofilm Time Gingivitis

Gingivitis Time Periodontitis

Periodontitis Time Tooth lost