Transcript DENTAL PLAQUE IS A MICROBIAL BIOFILM

DENTAL PLAQUE IS A MICROBIAL BIOFILM
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Microbial biofilm: a structured community of bacterial cells enclosed in a self-produced
polymeric matrix and adherent to an inert or living surface.
Dr. Bill Costerton
There is a strong clinical correlation between the bacterial plaque
composition and the innate host defense status
Healthy plaque
(mostly gram positive
bacteria)
Innate host response results in
tissue protection
Periopathogenic
plaque (mostly gram
negative anaerobes)
Innate host response results in
tissue and bone destruction
Commensal and periopathogenic bacteria result in a significantly different
innate host response
The oral microbial community is the best characterized microbial
consortium associated with the human host.
highly organized biofilm
consortium (Kolenbrander,
NIDCR)
microbial shift disease
Socransky (Forsyth)
Two major forms of periodontal disease
gingivitis: inflammation that leads to
redness and swelling of the periodontal
tissue surrounding the tooth root surface
reversible without professional treatment
periodontitis: inflammation that leads
to periodontal tissue destruction and
alveolar bone loss, requires periopathogens
and host susceptibility factors
Gingivitis
Gingivitis is a milder and reversible
form of periodontal disease that only
affects the gums. It develops as toxins
in plaque that irritate gums, making
them red, tender, swollen, and likely
to bleed easily. It can usually be
eliminated by daily brushing, cleaning
between your teeth, and regular
dental cleanings.
Classification of gingival diseases
A. Dental plaque-induced gingival diseases
1. associated with dental plaque only
2. hormonal changes associated gingivitis
puberty
pregnancy
3. medication induced gingivitis (gingival overgrowth)
Phenytoin (taken to control seizures),
cyclosporine (taken by people who have had organ transplants)
nifedipine (taken to control blood pressure and heart rhythm abnormalities)
4. malnutrition induced gingivitis
Vitamin C deficiency (scurvy);
Niacin deficiency (pellagra)
Classification of gingival diseases
B. Non dental plaque-induced gingival lesions
1. specific bacterial origin (gonorrhoeae)
2. viral origin (herpes, HIV)
3. fungal origin (Thrush, candidiasis)
4. genetic origin (hereditary gingival fibromatosis)
5. gingival manifestations of systemic conditions (allergic reactions)
6. traumatic lesions
Periodontitis
Gingivitis may lead to more serious, destructive forms of periodontal disease, called Periodontitis. There are
several forms of periodontitis, with the most common being chronic adult periodontitis. The gum line
recedes, which can expose the tooth's root. Exposed roots can become susceptible to decay and sensitive
to cold and touch. The sulcus deepens into a pocket in the early stage of periodontal disease. Plaque that
collects in these pockets can be difficult to remove during regular brushing and interdental cleaning. In some
cases, so much ligament and bone are destroyed that the tooth becomes loose. Usually, your dentist can still
treat the disease at this point.
Classification of Periodontal Diseases
A. Chronic periodontitis localized and generalized
B. Aggressive periodontitis localized and generalized
C. Periodontitis as a manifestation of systemic disease
(ex. neutropenia, Down’s syndrome)
D. Necrotizing periodontal diseases
necrotizing ulcerative gingivitis (NUG), necrotizing ulcerative periodontitis (NUP)
(trench mouth fusiform-spirochete variety) 1-week course of Metronidazole
E. Abscesses of the periodontium
gingival, periodontal
Site specific consortium patterns will be defined by microbial biogeography
Questions:
Site specific recolonization patterns
Frequency of specific bacteria
at different sites (F. nucleatum on
molars)
Adult type chronic
periodontitis is a highly
localized disease
Horizontal and vertical transmission
Reservoirs
Correlations to health and disease
Nature Reviews Microbiology 4, 102-112 (February 2006) |
Microbial biogeography: putting microorganisms on the map
Bacterial factors associated with periodontitis
sufficient numbers of bacteria are present to exceed
disease threshold (ie 108 bacteria/pocket)
periopathogens are of a virulent clonal type
Invasion: P. gingivalis can invade epithelial cells and gain access to
the connective tissue of the periodontium
Toxins: Aa produces a leukotoxin that kills neutrophils strong correlation
To disease
Enzymes: P. gingivalis produces a protease that degrades host tissues
including antibodies and other host defense proteins
Toxic products: gram negative bacteria release LPS that can induce bone
resorption
Localized juvenile aggressive periodontitis (LJP)
There is good evidence for a bacterial etiological agent:
Aggregatibacter actinomycetemcomitans strain JP-2
A.a. strainJP-2 has a deletion in the leukotoxin promoter region which results in high
levels of leukotoxin secretion. Leukotoxin lyses neutrophils
Longitudinal 2 year study
examining Jp-2 colonization and
susceptibility to disease
JP-2 is endemic in Morocco
and African Americans
Risk Factors for Periodontitis
People who smoke or chew tobacco are more likely to have periodontal disease. And it's
more likely to be more severe than in those who do not use any tobacco products.
Some systemic diseases, such as diabetes, can lower your body's resistance to infection,
making periodontal diseases more severe
Many medications, such as steroids, some types of anti-epilepsy drugs, cancer therapy drugs,
some calcium channel blockers, and oral contraceptives can affect the gums. In addition,
medications that reduce your salivary flow can result in a chronically dry mouth, which can irritate
your oral soft tissues. Let your dentist know about your medications and update your
medical history files at the dental office when any changes occur
Bridges that no longer fit properly, crooked teeth or fillings that have become defective
can contribute to plaque retention and increase the risk of developing periodontal disease.
Pregnancy or use of oral contraceptives increases hormone levels that can cause gum tissue
to be more sensitive to the toxins and enzymes produced by plaque and can accelerate growth
of some bacteria. The gums are more likely to become red, tender and swollen, and bleed easily
Heredity. Sometimes you may do everything right and still develop periodontal disease.
In that case, you — along with close to one-third of the population — may have inherited
a predisposition to gum problems.
There is a characteristic shift in the microbial
population from mostly gram positive bacteria
in supra-gingival plaque to mostly gram
negative bacteria in sub-gingival plaque
supra-gingival plaque
Streptococcus sanguis
Streptococcus mitis
sub-gingival plaque
Fusobacterium nucleatum
Eikenella corrodens
Actinomyces naeslundii
Porphyromonas gingivalis
Actinomyces viscosus
Prevotella intermedia
Bacterial species associated with different periodontal
clinical states
Health:
Gingivitis
Streptococcus
sanguis
Actinomyces
species
Porphyromonas gingivalis
Streptococcu
s mitis
Streptococcus
species
Bacteroides forsythus
Veillonella
parvula
Veillonella
species
Actinomyeces
naeslundii
Actinomyces
viscosus
Rothia
dentocarios
a
Fusobacterium
species
Prevotella
intermedia
Periodontitis
Treponema dentacola
Periodontal tissue is highly vascularized giving the tissue
access to serum soluble and cellular components
of the innate host defense system
Paradigm for bacterial interactions with the periodontium
Bacteria live on the tooth and tooth root surface and release
components that interact with host tissue (PRR ligands)
Composition of bacterial polymicrobial community contributes to
the type of host tissue responses (PRR)
Innate Immunity Adaptive Immunity
• Non-specific*
• Minutes to hours
• Receptors are germline
origin
• Complement
• Inflammation
“buys time” for and
Initiates adaptive immunity
• Specific
• 2-3 days
• Receptors are generated
randomly with clonal
expansion
• T, B, and dendritic cells
• Antibody and cellular
immunity
Lipopolysaccharide is a potent stimulator
of the host inflammatory response
• Over 200 years ago spontaneous remission of “neoplasms”
was found to be associated with acute infection
• The discovery that infections were caused by
bacteriainitiated therapeutic studies with bacterial fractions
and led to the discovery of endotoxin and “hemorrhagic
necrosis” of tumors
• Lipopolysaccharide from Gram-negative bacteria was
found to be the active anti-tumor component and led to the
discovery of the potent cytokine TNF-a
• The complete chemical synthesis of E. coli lipid A in 1985
confirmed LPS as a peotent activator of host defenses
The pattern recognition
hypothesis provides a conceptual
framework to understand host
responses to LPS
Characteristics of Pattern
Recognition Receptor Ligands
• Shared by large group of pathogens
• Conserved and essential structures, attempts to
modify result in death of microorganism or loss of
virulence
• Distinct from self antigens
The immune system evolved to discriminate
infectious nonself from noninfectious self.
Janeway, Jr., Immunology Today 13:11-16, 1992.
Charles A.
The host does not recognize
bacteria; it records bacterial
colonization and movement by
their structures.
Bacterial Recognition by the
Innate Host Response System
• Lipopolysaccharide (LPS)
• Lipoteichoic Acid (LTA)
• Peptidoglycan
GRAM POSITIVE CELL
ENVELOPE
secreted exoenzymes to perform extracellular digestion
Lysozyme
Lipoteichoic acid
Teichoic acid
Cytoplasmic membrane
Cytoplasm
GRAM NEGATIVE
CELL ENVELOPE
Lysozyme
Porin
Lipopolysaccharide
Outer
Membrane
(Braun) Lipoprotein
peptidoglycan
degradative enzyme
Inner
(cytoplasmic)
membrane
Cytoplasm
Structure of Lipopolysaccharide
The ENDOTOXIN
vary
conserved
different bacteria
have different lipid A structures
Pattern Recognition Receptors
Functions:
opsonization
activation of complement and coagulation cascades
phagocytosis
activation of inflammation
induction of apoptosis
Examples of pattern recognition receptors
serum soluble
C-reactive protein (CRP)
serum amyloid protein (SAP)
mannan-binding lectin (MBL)
lipopolysaccharide
binding protein (LBP)
sCD14
CRP and SAP bind phosphorylcholine
on bacteria and act as opsonins, also bind C1q
to activate complement
MBL is member of the collectin family, has a C type
lectin domain, binds mannose residues and
activates complement by the alternate pathway
LBP and sCD14 shuttle LPS and other microbial
and host components to activation
and neutralization pathways
Examples of pattern recognition receptors
cell associated
Macrophage mannose receptor (MMR)
Macrophage scavenger
receptor (MSR)
mCD14
Toll like receptors (TLR)
MMR has a C type lectin domain,
binds mannose on microorganisms
and mediates phagocytosis
MSR binds polyanionic ligands
(RNA, LPS, LTA), mediates phagocytosis
mCD14 and TLR’s function in a complex
to deliver activating signal to cells in response to
binding microbial components
LPS interactions with TLR4
Host cells respond to LPS
Examples of Bacterial Components “recognized”
by the Toll Like Receptor component of the
innate host defense system
TLR-2
peptidoglycan
bacterial
lipoproteins
lipoteichoic acid
TLR-4
E. coli type LPS
P. gingivalis LPS
TLR-5
flagellin
TLR-9
bacterial DNA
different combinations of TLRs may augment or inhibit responses to other
bacterial components
TLRs recognize molecular pattern associated with bacterial pathogens. TIR
domain-containing adaptors define the specificity of TLR signaling.
LPS
peptidoglycan
lipoproteins
LBP
sCD14
TLR6
or TLR4
TLR1
TLR2
TLR5
MD-2
TLR9
cell
membrane
MyD88
Cytoplasm
IRAK
TRAF6
The type of bacterial component
will influence the type of response.
MKK
p38
NF-kB
AP-1
NF-kB
Nucleus
Inflammatory mediators
inflammatory cytokines
Toll Signaling Pathways
cell adhesion molecules
chemokines
Activate:
antimicrobial peptides
costimulatory molecules
MHC molecules
How may commensal bacteria influence the periodontal
innate host response ?
Two approaches are being employed:
Immunohistochemical analysis of healthy tissue
Studies in germ free animals
Immunohistochemical analysis of clinically healthy periodontal tissue
mCD14
LBP
Co-expression of LBP and mCD14 peptides in gingival tissues.
LBP expression is confined to the cytoplasm in gingival epithelium
mCD14 is mainly confined to the cells in the epithelium-connective tissue interface.
Dr. Jin University of Hong Kong
Expression of TLR 2 and TLR 4 in healthy and diseased periodontal tissue
Negative control
chronic periodontitis
TLR 2 is found in
gingival epithelium
chronic periodontitis
TLR 2 expresses in
both gingival epithelium
and connective tissues
chronic periodontitis
TLR 2 is found in
connective tissue
clinically healthy tissue
TLR 2 is found in the
gingival epithelium
clinically healthy tissue
TLR 2 is found in the
gingival epithelium
chronic periodontitis
TLR 4 is found in
connective tissue
clinically healthy tissue
No TLR 4 is found
One contribution of commensal oral flora to innate immunity
may be to induce innate defense detection mechanisms
Healthy Tissue
Diseased Tissue
sCD14
++++
++
mCD14
++++
++
LBP*
++++
++
IL-1b
++
++++
++
++++
++
++++
Detection
Amplification
TNFa
TLR 2 & 4
Immunohistochemical and in situ analysis of diseased and healthy human
periodontal tissue. Laboratory of Dr. Jin Li Jian, Hong Kong University
The innate host response can discriminate between
commensals and pathogens by recognition of different
microbial patterns
Pattern recognition
specificity due to a host and
microbial regulated dialogue
LBP, CD14, and TLR microbial ligand binding affinities
TLR 1-9, different activation pathways
with different microbial components
TLR expression patterns
epithelial, endothelial, monocyte
Ex. epithelial cells do not have TLR 4
Germ free mice contain No bacteria
Keeping germ-free mice in an isolator.
germ free mice are born by sterile Caesarean section
breed in a sterile isolator with sterile food, water and bedding
.
It is important to note that germ-free animals have no bacteria in the intestine or on other
body surfaces, whereas specific-pathogen-free (SPF) mice are only devoid of known
mouse pathogens and do contain intestinal bacteria.
Germ free mice have been employed to determine
effects of commensal colonization of the intestine
Early studies in germ-free mice demonstrated that:
commensal bacteria have a direct impact on the morphology of the intestine
(the villi of the small intestine are longer and the crypts are shorter and contain fewer
cells in germ-free animals)
commensal bacteria have a direct impact on the development and function of
the intestinal immune system
(commensal bacteria are required for the complete development of Peyer’s patches,
the lamina propria, and the intraepithelial spaces, all three of the main immune elements
found in the intestine)
commensal bacteria are responsible for degradation of mucus glycoproteins
commensal bacteria have effects on intestinal motility
Rapid microbial induction of angiogenesis in small intestinal villi of adult
ex-germ-free mice
A, B, and C are confocal scans of capillary network present in the upper third of
small intestinal villi; D is the quantitation of villus capillary network density
Thaddeus S. Stappenbeck, Lora V. Hooper, and Jeffrey I. Gordon*
2002 Proceedings National Academy of Science: 15451–15455
Do commensal bacteria actively interact with the innate host defense system?
A study examining innate host defense mediator expression in germ
free and conventionally reared mice was performed
mouse gingival tissue was removed for RT-PCR analysis
Dixon, D., Reife, R., Cebra, J., and Darveau, R. 2004 Commensal bacteria influence
innate status within gingival tissues: A pilot study J. of Periodontology 75(11):14861492
Germ free mice display significantly lower IL-1b protein, consistent with
the innate host response system responding to commensal
bacteria.
Immunohistochemical analysis confirms IL-1b expression in clinically
healthy mouse periodontal tissue
Expression of IL-1β in periodontal ligament cells. a): 10×; b) and c): 40×.
IL-1b was expressed in periodontal fibroblasts
We suspect IL-1b may represent an global “indirect regulator of
innate defense mediator expression
Summary of Microbial Colonization and Innate Defense Status
Dental plaque is a highly organized site specific consortium of
microbial species that evolved to live with each other and the host
in the oral cavity
There is a strong correlation between the type of microbial consortiums
found in sub gingival plaque and the innate defense mediator expression
In both clinically healthy and diseased periodontal tissue
Germ free mice can be employed to identify innate host defense mediators
that are expressed in response to commensal colonization compared to
those that are developmentally expressed by the host.
Pathogens disrupt commensal bacterial interactions
subverting normal innate host responses
invade and gain access to protected compartments
alter existing commensal / host innate defense dialogue
P. gingivalis: invades gingival epithelial and microvascular endothelial cells, produces
proteases including hemolysins, induces bone loss in animal models of infection