Microbiology and Immunology Exam 2 Class 3 [The Microbiology of Periodontal Disease]

What is Periodontium

Tissue that surround and support teeth

- Gingiva

- Alveolar bone

- Cementum

- Periodontal ligament

Collar of fibrous soft tissue that invests the cervical region on teeth and is contiguous with its periodontal ligament and lining alveolar mucosa`

Gingiva

Collar-like band of epithelium, which located apical to the base of gingival sulcus. It usually terminates at the CEJ in healthy conditions or close to CEJ

Junctional epithelium

The junctional epithelium function

- attaches to the tooth and provides a secure seal around the tooth surface

- Barrier against plaque bacteria

The junctional epithelium ______ migrates in periodontitis

apically

Form a dense fibrous network to provide the shape of gingiva and attachment to teeth

gingival fibers

Vascular and cellular connective tissue that surround the root and alveolar bone

Rich in: Cellular components and fibers

Periodontal ligament

Functions of the periodontal ligaments

- anchor teeth to the bone

- support and maintenance of gingival tissue

- Transmission of occlusion (chewing) force

- Absorption of occlusion force

Provides osseous support

Alveolar bone

The shallow V-shaped space that is coronal to the attachment of the junctional epithelium and bounded by the tooth on one side and the sulcular epithelium on the other.

Gingival sulcus

Exudate similar to serum, nutrients for bacteria and inflammatory molecules

Found in the gingival sulcus

Gingival crevicular fluid

Most common types of periodontal disease

- Gingivitis

- Periodontitis

Gingivitis

Inflammation that resides in gingiva and has not caused damage to the tooth supporting structures- cementum, PDL, and alveolar bone. No attachment loss so it is reversible

Periodontitis

Inflammation that results in the destruction of tooth supporting structures with bone and attachment loss and thus irreversible

When you put in your probe for a patient with gingivitis what is reason for having a bigger number

This is due to swelling of the tissue not due to a bigger pocket

Gingival Health without clinical attachment loss

When you probe the bottom of the sulcus is coronally positioned to the CEJ and so the junctional epithelium attaches to the enamel as well as a little bit to the root surface

- The root structure is not exposed

- No attachment loss

Periodontitis with clinical attachment loss

When you probe the sulcus is apical to the CEJ. Damage of the junctional epithelium and some bone loss. Junctional epithelium will be detached from the tooth and migrate apically.

- The root structure is now exposed

- Attachment loss

A biofilm-induced inflammatory disease which is the major form of tooth loss

Periodontal disease

Alveolar bone loss occurs when

bacteria are present

No bacteria =

Bacteria =

Minimal bone loss

More bone loss

NHANES studies

National Health and Nutrition Examination Survey

Based on NHANES study Periodontal disease is tightly associated with

flossing (plaque bacteria control practice)

- Non-flossers have more disease activity

We can conclude from the NHANES study that

more bacteria control = less disease activities

Other than bacteria the _______ response is also critical in periodontal disease

host immune

The plaque bacteria can activate a range of

different cells including host immune cells which can produce a lot of inflammatory mediators

If host response is disregulated (for whatever reason)

Inflammation will be more exaggerated and can lead to periodontitis

In oral cavity approximately _____ species most of oral bacteria are ________ under normal circumstances, living in harmoney with other bacteria and a host.

700, commensal bacteria

When this harmony is disrupted

dysbiosis disease can occur

Ratio of bacteria to human cell

10:1

TOP HAT: Which of the following statements is correct about facultative vs obligate anaerobes

Both can survive when oxygen is present

Neither can survive when oxygen is present

Both can survive when oxygen is absent

Neither can survive when oxygen is absent

Both can survive when oxygen is absent

TOP HAT: Which of the following statements is correct about Gram negative bacteria

Have a thicker cell wall than Gram positive bacteria

Have an outer lipid membrane

Appear to be purple in Gram staining

Do not produce endotoxins

Have an outer lipid membrane

TOP HAT: Pellicle for biofilm development is produced by

Host

Bacteria

Virus

Host (mainly from saliva)

TOP HAT: Compared to planktonic bacteria, the same bacteria in biofilm or plaque generally grow

Slower

Faster

About the same

Slower

Biofilm grow slower because they are competing for nutrients (his words later in the lecture)

Tooth Deposits: Materia Alba

- White-cheese like accumulation

- Salivary proteins, some bacteria, desquamated epithelial cells, food debris

- Lack organized structure

- Easily displaced with a water spray

Tooth Deposits: Plaque (biofilms)

- Primarily composed of bacteria in a matrix of glycoproteins and extracellular polysaccharides

- Biofilm

- Impossible to remove by rinsing or with the use of sprays

Tooth Deposites: Calculus

- Hard deposit that forms via mineralization of dental plaque

- Generally covered by a layer of unmineralized dental plaque

A structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface

Biofilm

Primary etiology of caries, gingivitis, periodontitis and peri-implantitis

Bacterial plaque

The biofilm is highly structured

This is true

Biofilms: Structured communities

Colonized by a diverse range of bacteria

- Heterogeneous microcolonies

- Efficient metabolic network

- Temporal pattern of colonization

- Communication (quorum sensing)

Adhesion of biofilms

- adhesion to matrix

- adhesion to other bacteria

Components of biofilm

Water: 97%

Microbial cells: 2-5%

Polysaccharides: 1-2%

Proteins: <1-2%

DNA and RNA <1-2%

Process of plaque formation

1. Formation of the acquired pellicle on the tooth surface

2. Initial adhesion/attachment of bacteria

3. Colonization/plaque maturation

The formation of the pellicle on the tooth or soft surface

- Thin layer of organic materia

- From Saliva (proline-rich and histidine-rich proteins, glycoprotein mucin-receptors for bacteria to adhere)

- Forms on tooth surface within minutes

- Bacteria-free, acellular

- Provides a scaffold

The initial adhesion/attachment of bacteria

- Occurs within 2 hours via bacterial movement, sedimentation, nonspecific hydrophobic, electrostatic, and van der waals forces-- reversible

- Strong attachment specific binding between adhesions from bacteria and (mucins) receptors in pellicle-- irreversible

Colonization/ Plaque formation

- Coadhesion: bacteria adhering to other bacteria that are already in the plaque mass (also through specific receptors-adhesins among bacteria)

- Coaggregation: Process by which genetically distinct bacteria become attached to one another via specific molecules in suspension (corncob and bristle brush)

Structural features of biofilm

- Has water channels as circulatory system in the deep layers of the film

- Has heterogeneity in the distribution of different species

- Enhanced tolerance of environmental stress, antimicrobial agents, and the host defenses (protection)

Communication between biofilm bacteria is through

Quorum sensing

Quorum sensing

bacteria secrete a signaling molecule that accumulates in the local environment and triggers a response such as a change in the expression of specific genes once they reach a critical threshold concentration

Quorum sensing is a method to control bacteria growth due to

Sense of pressure on the population of bacteria

Quorum sensing can modulate the

drug resistance genes

Colonization is not a random process

This is true

Early colonizers are

Streptococcus species

or other facultative anaerobes

- Gram positive

Secondary colonizers common features

All gram negative strict anaerobic bacteria

Subgingival Microbial Complexes study

Found:

- Associations among species analyzed via cluster analysis

- Yellow (strepptococci species)

- Orange complex (late colonizers): bridges the early colonizers and red complex

- Red complex: is closely associated with periodontal disease

Bacteria closely related to periodontal disease

P. gingivalis

T. forsythia

T. denticola

Where are biofilms found

Supragingival

Subgingival

Supragingival

At or coronal to the gingival margin

Subgingivial

Apical to the gingival margin

Subgingivial plaque

1. Tooth attached

2. Unattached, loosely adherent, planktonic

3. Epithelial-associated

4. Connective tissue-associated (tissue invasion)

5. Alveolar-associated

Subgingival Biofilms compared to Supragingival Biofilms

- Limited access to oral cavity (anerobiosis)

- Increased gingival crevicular fluid

- Minimal mechanical removal by GCF flow due to presence of pocket wall

- Bacteria without adhesion may colonize

- Saliva is a limited source of new organisms

The bacteria within biofilms are _____-______ times more resistant to antimicrobial agents than their planktonic counterparts

1000-1500

Lacking transport system for an antibiotic; lacks the target of the antibiotic molecule; or outer membrane that covers cell wall of Gram negative bacteria

Inherent (natural) resistance

Acquired by means of mutation in its own DNA or acquisition of resistance-conferring DNA from another source; vertical gene transfer and horizontal gene transfer

Acquired resistance

Acquired resistance to antibiotic via:

- Vertical gene transfer

--spontaneous mutation in Bacterial DNA that is directly transferred to all progeny

- Horizontal gene transfer

-- Conjugation

-- Transformation

-- Transduction

Biofilm: why are the bacteria so resistant to antibiotic

- Slow growth in biofilm (nutrient limitation, stress)

- Failure of the antimicrobial to penetrate the biofilm (glycocalyx and exopolysacharide matrix)

- heterogeneity of biofilm structure

- Quorum sensing

- Exchange of genetic materials

Antibiotics used in periodontics

Amoxicillin: inhibition of bacterial cell wall formation

Metronidazole: Interference with bacterial DNA

Ciprofloxacin: Interference with DNA replication

Clindamycin: Inhibition of protein synthesis (50s rRNA)

Doxycycline: Inhibition of protein synthesis (30s rRNA)

Azithromycin: Inhibition of protein synthesis (23 rRNA)

For antibiotics to work they require the bacteria to be in the ____ phase

Log (exponential)

- Bacteria in biofilms are not in the Log phase (they are growing slowly)

Etiology of Periodontal Diseases 4 main hypothesis

1. Non-specific plaque hypothesis

2. Specific plaque hypothesis

3. Ecologic plaque hypothesis

4. Key stone pathogen hypothesis

Non specific plaque hypothesis (1950-1970)

- This theory correlated both age and the amount of plaque with evidence of periodontitis

- Large amounts of plaque would cause a higher production of noxious products, which would overwhelm the host's defense

- Control of the disease depends on the reduction of the total amount of plaque

Specific plaque hypothesis (1960-)

- The pathogenicity of dental plaque depends on the presence of or an increase in specific microorganisms

- Improvements in sampling and detection of bacteria

-- recognition of A. actinomycetemcomitans in aggressive periodontitis

-- Association of "Socransky's red-complex" in periodontitis

Ecologic plaque hypothesis ( 1994)

- Both the total amount of dental plaque and the specific microbial composition of plaque may contribute to the transition from health to disease

- The health-associated dental plaque = microbial homeostasis

- The host response may be brought by an excessive accumulation of nonspecific dental plaque, by plaque independent host factors or by environmental factors

- Microenvironment changes (host response) lead to dysbiosis

Keystone Pathogen Hypothesis (2011-)

- Certain low-abundance microbial pathogens can orchestrate inflammatory disease by remodeling a normally benign microbiota into a dysbiotic one

- P. gingivalis in specific pathogen-free mice can cause periodontal bone loss even when the proportion of P.g is very small in the total microbiota

- Interspecies communication between keystone pathogens and other members of the community is one of the key factors that leads to overgrowth of the more pathogenic microbiota

For disease to happen we need

1. Host susceptibility

2. Pathogenic bacteria

Pathogenic Bacteria

- T. forsythis

- T. denticola

- P. gingivalis

- A. actinomycetemcomitans

Socransky's Criteria for Microorganisms to be judged to be potential pathogens

- Be associated with disease, as evidenced by increases in the number of organisms at diseased sites

- Be eliminated or decreased in sites that demonstrate the clinical resolution of disease with treatment

- Induce a host response in the form of an alteration in the host cellular or humoral immune response

- Be capable of causing disease in experimental animal models

- Produce demonstrable virulence factors that are responsible for enabling the microorganism to cause the destruction of the periodontal tissues

The transition from health to Disease Bacterial profiles:

Health:

- G+ facultative species

- Streptococci, Actinomyces species

Gingivitis:

- Similar portions of G+/G- and facultative/anaerobic

- Increased Actinomyces, decreased Streptococci

Periodontitis:

- High % of G- anaerobes

- Rods, spirohcetes

Microbial shifting from health to periodontitis

From G+ to G-

From cocci to rods

From nonmotile to motile organisms

From facultative anaerobes to obligate anaerobes

From carbohydrate fermenting to proteolytic species

Certain bacterial species are protective or beneficial to the host

S. sanguinis

Veillonella parvula

C. ochraces

Bacteria associated with Gingivitis

Equal proportion of G+ and G- species

Equal proportion of facultative and anaerobic

Periodontal pathogens begin to be present (P. gingivalis, T forsythia, P. intermedia, C. rectus, Treponema spp) but in smaller amounts then periodontitis

Bacteria associated with Periodontitis

Plaque from sites with periodontitis showed elevated proportions of spirochetes

High percentage of anaerobic G- bacterial species

P. gingivalis and A. actinomycetemcomitants can invade host tissue cells

Qualifying bacteria based on Socransky's Periodontopathogen Criteria: P. gingivalis, A. actinomycetemcomitans, T. forsythia

Virulence factors

1. Factors that promote colonization (adhesins)

2. Toxins and enzymes that degrade host tissue

3. Mechanisms that protect pathogenic bacteria from the host

Virulence factors: Adhesion

Fimbriae or pili: polymeric fibrils that provide the points of contact on the host cells and tissues

Highly immunogenic

Virulence factors: Tissue destruction-promoting factors

Bacterial proteases activate the host enzymes

Trypsin-like protease activity

Example: Gingipain (cysteine protease) from P. gingivalis multifunctional proteins that play important roles in adhesion, tissue degradation, and the evasion of host responses

Virulence factors: Strategies for evading host immunity

Production of an extracellular capsule

Proteolytic degradation of host innate or acquire immunity components

Modulation of host responses by avoiding binding serum components on the bacterial cell surface

Invasion of gingival epithelial cells

P. gingivalis

G- obligate anaerobic, rod

nonmotile

Black pigment on blood agar (requires heme to grow)

Tissue invader/intragingival

Increased in active periodontal disease sites

Produces virulence factors

P. gingivalis main Virulence factors

- LPS: affects inflammatory response

- Capsule: evasion of phagocytosis

- Fimbriae: adhesion and biofilm formation

- Gingipains: degradation of protein immune molecules

- Hemolysis and hemagglutinins: adhesion, and heme acquisition and supply

A. actinomycetemcomitans

G- facultative anaerobic coccobacillus

nonmotile

Tissue invader/ intragingival

Increased in active periodontal sites

Major pathogen for molar-incisor patter periodontitis

A. actinomycetemcomitans main virulence factors

Leukotoxin: (kills all the below)

- PMNs (neutrophils) cell death, degranulation with massive lysosome enzymes

- Monocytes/macrophages: activation of inflammasome complex to secrete IL-1beta

- Lymphocytes: apoptosis

Cytolethal distending toxin (CDT):

- Causes DNA damage, cell cycle arrest, and apoptosis of affected cells (gingival epithelial cells, gingival fibroblasts and periodontal ligament fibroblasts)

- Stimulates pro-inflammatory and osteolytic cytokine production

A. actinomycetemcomitans cause localized aggressive periodontitis

This is true

Virulence factors that promote colonization: P. gingivalis

Fimbriae

Pili

Exoploysaccharide

Outer membrane proteins

Virulence factors that are toxins and enzymes that degrade host tissue: P. gingivalis

Collagenase

Trypsin-like protease

Gelatinase

Gingipain

Hemolysin

Hemagglutanins

Virulence factors mechanisms that protect pathogenic bacteria from host: P. gingivalis

Enzymes

Gingipain

Capsules

LPS

Virulence factors that promote colonization: A. actinomycetemcominats

Adhesins

Invasins

Bacterocins

Virulence factors that are toxins and enzymes that degrade host tissue: A. actinomycetemcominats

Cytotoxins

Heat shock proteins (HSPs)

Collagenase

Bone resporption agents

Virulence factors mechanisms that protect pathogenic bacteria from the host: A. actinomycetemcominats

Leukotoxin

LPS

Chemotactic inhibitors

Cytolethal distending toxin (CDT)

Immunosuppressive proteins

Fc-binding proteins

Oral/periodontal microbiome can be translocated extraoraly by

1. Enteral (GI) route

2. Circulation (bacteremia)

- Tooth extraction >80% incidence

- Toothbrushing 28% incidence

- Professional cleaning ~70% incidence

- Transient (40-60 min) in healthy individuals

-- Association not causation--

Necrotizing periodontal disease

- Painful acute lesions

- Severe inflammation

- Necrosis of the gingival tissue

- Increased bleeding tendency

Bacteria associated with with Necrotizing periodontal disease (not specific from periodontitis)

- Fusobacterium nucleatum

- Spirochetes (treponema)

- Prevotella intermedia

In necrotizing periodontal disease we have not

identified a specific bacteria causing the disease