Chandra D' Sykes DDS

1
Biofilm and its Relevance In Persistant
Endodontic Infection

• Chandra D. Sykes DDS
• Graduate Endodontics

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.5B 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

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.
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 ????

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-1beta), 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.

18
(No Transcript)
19
(No Transcript)
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
  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

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.

22

• 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

23
Periradicular Biofilms

• extraradicular biofilms average thickness of 30
  to 40µm thickness
• Department of Restorative Dentistry and
  Endodontology, Osaka University Graduate School
  of Dentistry

24
(No Transcript)
25
(No Transcript)
26
(No Transcript)
27
(No Transcript)
28
(No Transcript)
29
(No Transcript)
30
(No Transcript)
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.