Chandra D. Sykes DDSGraduate Endodontics

Biofilm and its Relevance In Persistant Endodontic Infection

Definitions: Biofi lm: A collection of microorganisms, extracellular

polysaccharide substance, and organic mater located at the interface in solid-liquid, gas-liquid, or liquid-liquid biphasic systems.

    

Stages of Biofilm FormationThere are f ive stages of b iof i lm development:

m in it ia l attachment

m i r rever s ible attachment

m Growth

m EPS Production

m disper s ion

Bacterial interactionsSingle-celled organisms generally exhibit two distinct modes of

behavior:Planktonic-single cells float or swim independently in some liquid

medium. Attached state-cells are closely packed and firmly attached to each

other and usually form a solid surface.

Supragingival biofilm formation Acquired pell icle- makes the surface receptive to colonization by specific

bacteria. • sal ivary mucins , such as [MUC.sub.5]B and [MUC.sub.7], contribute to the

formation of acquired pellicle

• statherin- a salivary acidic phosphoprotein

• prol ine-rich proteins (PRP) -promote bacterial adhesion to tooth surfaces.

formation begins within minutes of a professional prophylaxis; within 1 hour, microorganisms attach to the pellicle.

Extracellular Polysaccharide Substance This matrix protects the cells within it and facilitates communication

among them through biochemical signals

This is an SEM micrograph of an extracellular matrix forming around a cocci type bacterial infection. The bacteria in the left portion of the micrograph are almost completely encapsulated with the matrix. In other regions, the matrix is holding the bacteria together, but has not yet completely encapsulated them. Bacteria in such a matrix behave very differently from free floating bacteria. It is believed that the matrix allows communication between the individual bacterium. 

This SEM micrograph shows an Extracellular Polysaccharide Substance (EPS). A bacterial biofilm is held together by a material excreted by the bacteria known as Extracellular Polysaccahride Substance (EPS). This photograph shows a nice view of an EPS matrix holding a colony of Cocci bacteria together. The EPS has developed to the degree that it is completely encapsulating many of the individual bacterium. The colony has developed to the point that it is many levels deep.

QUORUM SENSING

A system by which bacteria communicate. Signaling molecules — chemicals similar to pheromones that are produced by an individual bacterium — can affect the behavior of surrounding bacteria.

Sessile cells in a biofilm “talk” to each other via quorum sensing to build microcolonies and to keep water channels open.

The final stage of biofilm formation is known as development, and is the stage in which the biofilm is established and may only change in shape and size. This development of biofilm allows for the cells to become more ant ib iotic res istant

Failure of an antimicrobial agent to rapidly or completely penetrate a biofilm is perhaps the most intuitively appealing explanation for biofilm resistance.

Four mechanisms that confer antimicrobial tolerance to cells living in a biofilm:

1. EPS2. Physiological state of biofilm

3. Metabolic heterogeneity4. Persisters ????

Resistance to antimicrobial agents 1,000- fold greater than planktonic cells

Failure of an agent to penetrate the full depth of the biofilm

Cells in a biofilm experience nutrient limitation and therefore exist in a slow-growing or starved state

Oral biofilms are more resistant to: chlorhexidine, amine fluoride, amoxycillin, doxycycline,and

metronidazole than planktonic cells

Concerning periodontal diseaseSocransky and colleagues (1998,2000)- recognized that early plaque

consists predominantly of gram-positive organisms and that if the plaque is left undisturbed it undergoes a process of maturation resulting in a more complex and predominantly gram-negative flora. These investigators assigned the organisms of the subgingival

microbiota into groups, or complexes, based on their association with health and various disease severities

Color designations were used to denote the association of particular bacterial complexes with periodontal infections: o Blue, yellow, green, and purple complexes-early colonizers of the

subgingival florao Orange and red complexes-late colonizers associated with

mature subgingival plaque (Socransky SS et al., J Clin Periodontol. 1998, Socransky SS, Haffajee AD., Periodontol 2000)

Red complex organisms Porphyromonas gingivalis Tannerella forsythensis Treponema denticola organisms are found in greater numbers in diseased sites and in

more advanced periodontal disease

Concerning Endodontics Bacterial biofilms have been found to develop on root

surfaces outside the apical foramen and be associated with refractory periapical periodontitis.

Porphyromonas gingivalis, Tannerella forsythensis, and Fusobacterium nucleatum were associated with extraradicular biofilm formation and refractory periapical periodontitis

Mature Biofilm

Significance of biofilm Contributing to host tissue damage:As the biofilm matures and proliferates, soluble compounds produced by

pathogenic bacteria penetrate the sulcular epithelium. These compounds stimulate host cells to produce chemical mediators associated with the inflammatory process: Interleukin-1 beta (IL-1[beta]), prostaglandins, tumor necrosis factor alpha (TNF-[alpha]), and matrix

metalloproteinases

As the inflammatory process continues, additional mediators are produced, and more inflammatory cell types such as neutrophils, T cells, and monocytes are recruited to the area.

Proinflammatory cytokines are produced in the tissues as a response to the chronic inflammatory process, and these proteins may further escalate the local inflammatory response and affect the initiation and progression of systemic inflammation and disease.

The result of this chronic inf lammation is a breakdown of gingival col lagen and accumulat ion of an inf lammatory inf i ltrate, leading to the cl inical s igns of gingivit is. In some individuals, the inf lammatory process wil l a lso lead to the breakdown of collagen in the periodontal l igament and resorption of the supporting alveolar bone.

Significance of biofilm Potential to spread

Seeding dispersal: Programmed detachment of planktonic bacterial cells caused by local hydrolysis of the extracellular polysaccharide matrix, and conversion of a subpopulation of cells into motile planktonic cells

Clumping dispersal: A physical detachment pathway in which a fragment of a

microcolony, simply detaches from the biofilm and is carried by the bulk until it lodges in a new location and initiates a new sessile population.

Biofilm in root canal surfaces oral microorganisms are able to colonize root canals by adhering to the dentin

walls

Biofilm formation in root canals is probably initiated at some time after the first invasion of the pulp chamber by planktonic oral organisms after some tissue breakdown. At this point, the inflammatory lesion frontage that moves successively toward the apex will provide the fluid vehicle for the invading planktonic organisms so these can multiply and continue attaching to the root canal walls . Hypothesis: Svensäter and Bergenholtz

Aggregations of microorganisms can be seen adhering to the inner walls of an accessory canal, thus demonstrating the retention of these biofilm communities

Molven et al, reported the microbial colonization of the external root apex of teeth with pulp necrosis and periapical lesion by cocci, bacilli, cocci-bacilli, filament, spirochetes, and also the presence of bacterial biofilm on the apical 2 mm of the external root surface in 83.3% of the cases.

Sjogren et al, reported a success rate of 86% in case of a necrotic pulp with a periapical radiolucency.

 The necrotic pulp tissue becomes a       favorable environment for microbial       proliferation due to the presence of     organic residue or nutrients, which      act as substrate or culture medium.

Gram-negative bacteria are more      frequent than Gram- positive       bacteria. 

Facultative or strict anaerobic      microorganisms are more frequent      than aerobic microorganisms, and     the presence of bacilli and filaments    is equivalent to that of cocci

Periradicular Biofilms

extraradicular biofilms average thickness of 30 to 40µm thickness

Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry

Treatment of persistent periapical infectionsTo reduce the load of bacteria residing in the lateral

canals or deltas within the apical third

Resection of the apical root t ip :• To physically eradicate the biofilm layer residing on the root surface

Conclusion

Dental biofilm is a complex, organized microbial community that is the primary etiologic factor for the most frequently occurring oral diseases such as, dental caries, periodontal diseases, and apical endodontic pathosis. It is imperative to understand and to realize the complexity and nature of the biofilm, especially the role it plays in harboring and protecting the microorganisms, thus, contributing to persistent infections.