Biofilm and its Relevance In Persistant Endodontic Infection

1. Chandra D. Sykes DDS Graduate Endodontics Biofilm and its Relevance In Persistant Endodontic Infection

2. Definitions: Biofilm: A collection of microorganisms, extracellular polysaccharide substance, and organic mater located at the interface in solid-liquid, gas-liquid, or liquid-liquid biphasic systems.

3. Stages of Biofilm Formation There are five stages of biofilm development: initial attachment irreversible attachment Growth EPS Production dispersion

4. Bacterial interactions Single-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.

5. Supragingival biofilm formation Acquired pellicle- makes the surface receptive to colonization by specific bacteria. salivary mucins, such as [MUC.sub.5]B and [MUC.sub.7], contribute to the formation of acquired pellicle statherin- a salivary acidic phosphoprotein proline-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.

6. Extracellular Polysaccharide Substance This matrix protects the cells within it and facilitates communication among them through biochemical signals

7. 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.

8. 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.

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

10.  Four mechanisms that confer antimicrobial tolerance to cells living in a biofilm: 1. EPS 2. Physiological state of biofilm 3. Metabolic heterogeneity 4. Persisters ???? The "persister" is a hypothetical cell state in which microorganisms are protected from all types of antimicrobial insults. Persisters may constitute one percent or less of a biofilm population. Though small in relative numbers, this protected subpopulation is sufficient to reseed the biofilm in the event of catastrophic chemical or physical challenge.�No one has yet identified a persister cell, but persisters have alternately been imagined as spore-like cells that are dormant and incapable of growth and as phenotypic variants that grow as rapidly as the parent strain (Spoering and Lewis, 2001) The "persister" is a hypothetical cell state in which microorganisms are protected from all types of antimicrobial insults. Persisters may constitute one percent or less of a biofilm population. Though small in relative numbers, this protected subpopulation is sufficient to reseed the biofilm in the event of catastrophic chemical or physical challenge.�No one has yet identified a persister cell, but persisters have alternately been imagined as spore-like cells that are dormant and incapable of growth and as phenotypic variants that grow as rapidly as the parent strain (Spoering and Lewis, 2001)

11. 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

12. Concerning periodontal disease Socransky 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: Blue, yellow, green, and purple complexes-early colonizers of the subgingival flora 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)

13. Red complex organisms Porphyromonas gingivalis Tannerella forsythensis Treponema denticola organisms are found in greater numbers in diseased sites and in more advanced periodontal disease

14. 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

15. Mature Biofilm

16. 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 inflammation is a breakdown of gingival collagen and accumulation of an inflammatory infiltrate, leading to the clinical signs of gingivitis. In some individuals, the inflammatory process will also lead to the breakdown of collagen in the periodontal ligament and resorption of the supporting alveolar bone.

17. 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.

20. 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: Svens�ter 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

21. 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 mediumThe 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

23. Periradicular Biofilms extraradicular biofilms average thickness of 30 to 40�m thickness Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry

31. Treatment of persistent periapical infections To reduce the load of bacteria residing in the lateral canals or deltas within the apical third

32. Resection of the apical root tip: To physically eradicate the biofilm layer residing on the root surface

33. 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.