PLAQUE AS A BIOFILMAND

MICROBIOLOGY OF PERIODONTAL DISEASES

Navneet Randhawa

MDS Final year

INTRODUCTION• It is estimated that about 700 different spp. are capable of colonizing the

mouth.

• An individual may harbor 150 or more different spp.

• The ecological relationship between periodontal microbiota & host –benign .

• A subset of organisms either introduced, overgrow or exhibit new properties –leading to periodontal diseases.

EVIDENCE OF INFECTIOUS NATURE OF PERIODONTAL DISEASE

1. Acute periodontal infections: - alleviated by anti microbial therapy.

2. Positive co-relation of plaque levels to gingivitis and amount of bone loss. (Loe et al 1965)

3. Efficacy of antibiotics in treatment of periodontitis:

4 HOST IMMUNOLOGIC RESPONSE: ↑serum antibody response (Taubman et al 1994 )

5. Toxic products (endotoxins, H2S , NH3, indole, amines, leucotoxins ) - demonstrate pathogenic potential.

6. STUDIES IN EXPERIMENTAL ANIMALS: Induce periodontal disease

UNIQUE FEATURES OF PERIODONTAL INFECTIONS

• Mineralized tooth structure passes through the integument so that part of it is exposed to the external environment while part is within C.T.

• Non shedding tooth surface.- provides ‘sancturies’ in which micro-organisms can hide.

• Causative agent reside in biofilms outside the body.

• Formidable problem for host and therapist.

HISTORICAL PERSPECTIVE

• 1683 Antonie van Leeuwenhoek

• “Poor oral hygiene – large no. of bacteria”

“I didn’t clean my teeth for three days and then took the material that had lodged in small amounts on the gums above my front teeth…. I found a few living animalcules…”

The search for the etiological agents of periodontal diseases started

in the ‘golden age of microbiology’ [app. 1880-1920]

• 1890 W.D. Miller “pyorrhoea alveolaris is not caused by a specific bacterium”

• Invesigators from the period of 1880-1930 suggested four distinct groups of micro-organisms. Viz; amoeba,spirochetes,fusiforms,sreptococci.

• 1925 -1950

Decline of interest in microorganisms

Bacteria : secondary invaders/contributors to inflammation

Emphasized on occlusal factors,calculus & poor host defense

1950 Waerhaugh : Dental plaque in the initiation & progression of pdldiseases

• 1950-1975 : Non specific hypothesis

• 1970s : Improvement in anaerobic culture technique.Distinct microfloras in pdl health &gingivitis-Specific plaque hypothesis

• 1980s : A.a , P.g, P.intermedia , T.forsythus periodontitis

These classic studies provided

the initial impetus to perform

large scale studies attempting to

relate specific microorganisms to

the etiology of periodontal

disease

PLAQUE AS A BIOFILM

BIOFILM

Biofilm

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

‘ Matrix enclosed bacterial populations adherent to each other

and to surfaces or interfaces’

Costerton et al,1994

• Biofilms are ubiquitous and form on virtually all surfaces immersed in natural aqueousenvironment

• Biofilms are the preferred method of growth for most bacteria.

• Protection from :

- competing microorganisms

- environmental factors

-toxic substances

• Facilitate :

-processing and uptake of nutrients

-cross feeding

-removal of potentially harmful metabolites

-development of appropriate physicochemical

environment

PLAQUE

• Dental plaque as a naturally occuring microbial deposit represents a ‘true biofilm’

“Highly specific variable structural entity formed by the sequential colonization of microorganism on tooth surface, epithelium & restorations”. (WHO)

“Clinically as a structured resilient, yellow greyish substance that adheres tenaceously to the intraoral hard surfaces, including removable & fixed restorations”.

COMPOSITION OF DENTAL PLAQUE:

• Composed primarily of microorganisms within an intercellular matrix

• 1g 2 * 10 bacteria

Polysaccharides

Proteins

Organic Glycoproteins

Lipid

Intercellular matrix

Calcium

Inorganic Phosphorus

Na,K,F

FORMATION OF DENTAL PLAQUE

• Formation of pellicle on tooth surface

• Initial adhesion & attachment of bacteria

• Colonization & plaque maturation

FORMATION OF PELLICLE

Initial phase of plaque formation

Forms with in nanosecond - vigorously polished teeth

Derived from components of saliva + crevicular fluid +bacterial & host tissue cell products Components-

Glycoprotein (mucin)

Proline rich protein

Enzymes (α-amylase)

Other molecules

• Studies of early (2-hour) enamel pellicle reveal that its amino acid composition differs from that of saliva, indicating that the pellicle forms by selective adsorption of the environmental macromolecules(Scannapieco1990).

• The mechanisms include

• Electrostatic,

• Van der Waals, and

• Hydrophobic forces.

hydroxyapatite surface - negatively charged phosphate groups

Interact with positively charged components of salivary and crevicular fluid macromolecules.

Pellicles function as-

a protective barrier,

providing lubrication

preventing tissue desiccation.

provide a substrate to which bacteria in the environment attach.

The specific component of pellicle depend on underlying surface.

Thus the characteristic of the underlying hard surface are transferred through the

pellicle layers and influence initial bacterial adhesion.(Pratt et al 1991)

Absolom et al (1987) even observed a clear relationship between the type of proteins adsorbed in the pellicle & the free energy of the substratum surface.

2]Initial adhesion and attachment of bacteria:

Phase 1: Transport to the surface

-Brownian motion

-Sedimentation of bacteria

-Liquid flow

-Active bacterial movement

Phase 2: Initial adhesion

• Initial adhesion- reversible.

• Initiated by the interaction between the bacterium and the surface.

• Long-range (>50nm):non-specific interaction : Van der Waals attractive forces.

• Shorter-range (10-20nm) interactions: Van der Waals attraction forces + electrostatic repulsion →weak area of attraction →reversible adhesion

PHASE 3: ATTACHMENT

• After initial, a firm anchorage -specific interactions (covalent, ionic or hydrogen bonding)

• This follows direct contact or bridging- true extracellular filamentous appendages (10nm)

• On a rough surface bacteria are better protected against shear forces so that a change from reversible to irreversible bonding occurs more easily & more frequently.

SECONDARY COLONIZATION & PLAQUE MATURATION

Secondary colonizers

Prevotella intermedia,

Prevotella loescheii,

Capnocytophaga spp.,

Fusobacterium nucleatum, and

Porphyromonas gingivalis.

adhere to cells of bacteria already in the plaque

COAGGREGATION

Highly specific stereochemical interaction -protein and carbohydrate molecules located on the bacterial cell surfaces

Eg

Fusobacteria coaggregate with all other human bacteria.

Veillonellae, capnocytophagae & prevotella bind to streptococci & actinomycetes. (Kolenbrader et al1995)

Most coaggregation

Mediated by lectinlike adhesins

Inhibited by lactose & other galactosides.

Well characterized interaction include the coaggregation of–

Fusobacterium nucleatum with S.sanguis,

Prevotella loescheii with A. viscosus

Capnocytophaga ochraceus with A. viscosus

Streptococci show intrageneric coaggregationbind to the nascent monolayer of already bound streptococci.

CORN COB TEST TUBE BRUSH

• Sequential colonization :‘bridging species’.

• Thus as plaque matures an ecologic shift occurs in the biofilm from theearly aerobic environment characterized by gram positive facultativespecies to a highly oxygen deprived environment where gram negativeanaerobic organisms predominate

CLASSIFICATION OF PLAQUE

SUPRAGINGIVAL SUBGINGIVAL

Supragingival

Direct contact with the gingival margin is referred to as marginal plaque.

Demonstrates stratified organisation of bacteria.

Gram +ve cocci & short rods predominate at the tooth surface.

Gram -ve rods & filaments as well as spirochetes - outer surface of mature plaque mass.

Subgingival:

• Formed below the gingival margin,

• Present between the tooth & sulcular tissue

• Subgingival microbiota differ in composition: Local availablity of blood product

Low redox potential anaerobic environment.

• Subgingival plaque can be divided in to

• Attached/tooth associated

• Unattached plaque

• Epithelial associated

• Within CT

• On bone surface

PLAQUE HYPOTHESIS

NON SPECIFIC PLAQUE HYPOTHESIS:

• In 1900s Any accumulation of microorganisms at or below the gingival margin would produce irritants leading to inflammation

• Periodontal disease results from the ‘elaboration of noxious products by the entire plaque flora’.

When only small amount of plaque the noxious product neutralized by

host.

Similarly large amounts of plaque noxious product overwhelm the host

defense.

Limitations

• Some individuals with considerable amount of plaque & gingivitis never developed destructive periodontitis.

• Site specificity in the pattern of disease

SPECIFIC PLAQUE HYPOTHESIS[WALTER LOESCHE 1979]

• Only certain plaque is pathogenic & its pathogenicity depends on the presence of or increase in specific microorganism.

• This concept predicts that plaque harboring specific bacterial pathogens results in a periodontal disease because these organism produce substances that mediate the destruction of host tissue.

• Acceptance of this hypothesis was spurred by recognition of A.a as a pathogen in localized aggressive periodontitis.

ECOLOGICAL PLAQUE HYPOTHESIS [MARSH 1991]

• A change in a key environmental factor will trigger a shift in the balance of the resident microflora & this might predispose a site to disease.

Increased Increased High GCF flow, bleeding

plaque inflammation temp

Overgrowth of gram –ve

anaerobes

• Microbial specificity in disease would be due to the fact that only certain species are competitive under the new environmental condition

ECOLOGICAL

SHIFT

Special Bacterial Behaviour in a biofilm:

1)Antibiotic resistance:

a] Slower rate of growth within the biofilm

b] Matrix resists diffusion of antibiotics

acts as an ion exchange resin

c] Beta lactamase,formaldehyde lyase & formaldehyde dehydrogenase may

become conc.

d] Super resistant bacteria with ‘multi drug efflux pumps’

3)High density of cells facilitate exchange of genetic information:

-conjugation

-transformation

-plasmid transfer

-transposon transfer

• When within a biofilm bacteria exhibit both favourable & antagonistic relations with the other species

Favourable interactions:

- provides of growth factors

-facilitates attachment

Antagonistic interactions:

-enzymes which inhibit binding

-factors that kill other species

QUORUM SENSING

• Quorum sensing in bacteria "involves the regulation of expression of specific genes through the accumulation of signaling compounds that mediate inter cellular communication” (Prosser 1999).

• Quorum sensing - dependent on cell density

• Few cells: signaling compounds at low levels

• Auto induction ↑concentration

• Once the signaling compounds reach threshold level (quorum cell density), gene expression is activated.

FACTORS AFFECTING THE COMPOSITION OF BIOFILM

• Disease status- ↑red and orange complex

• Pocket depth: red and orange complex↑ with ↑ pocket depth

• Transmission:

Two types of transmission are recognized:

• "vertical“- that is transmission from parent to offspring, "horizontal”- passage of an organism between individuals outside the parent—offspring relationship.

• Acquisition of new strains of pathogenic species can occur at both young and older ages. If the newly acquired strain is more virulent than the pre-existing strain of that species, then a change in disease pattern could occur

• Host susceptibility

HIV infection and diabetes

• Studies such as these suggest that altered host susceptibility → change the rate of disease progression(periodontal pathogens –same)

Smoking

• ↑red and orange complex

• Tobacco- diminish the local and systemic immune response

PERIODONTAL MICROBES

ACQUISITION OF MICROFLORA

• Mouth of fetus- sterile.

• No. of organisms rapidly increase following 6- 10 hrs after birth.

• Anaerobic bacteria -2nd day

Birth

• >2 yrs complex microflora is formed

• More than 400 different types of bacteria

Infancy and early childhood • Greatest increase with

the eruption of permanent teeth.

• Bacteria occupy several niches

Adolescence

CLASSIFICATION OF ORAL MICROBES.

bn

GRAM + VE GRAM - VE

PROKARYOTES FACULTATIVE ANAEROBES OBLIGATE ANAEROBES FACULTATIVE ANAEROBES OBLIGATE ANAEROBES

COCCI

STREPTOCOCCUS

S.MUTANS

S.SANGUIS

S.ORALIS

S.MITIS

S.INTERMEDIUS

PEPTOSTERPTOCOCCUS NISSERRIA VEILLONELLA

V.PARVULA

RODS

ACTINOMYCES

A.NAESLUNDI

A.VISCOSUS

A.ISRAELLI

A.ODONTOLUTICUS

LACTOBACILLUS

L.ORIS

L.ACIDOPHILLUS

L.SALIVARIUS

L.BUCCALIS

EUBACTERIUM

ACTINOBACILLUS :A.a

EIKENELLA:E.corrodens

CAPNOCYTOPHAGA

CAMPHYLOBACTER

PORPHYROMONAS: P.g

PREVOTELLA :P.i

BACTEROIDES : T.f

FUSOBATERIUM : F.n

SELENOMONAS

SPIROCHETES &

MYCOPLASM

MYCOPLASM

SPIROCHETES OF ANUG

T.Denticola

T.Socranskii

EUKARYOTES CANDIDA

CANDIDA ALBICANS

ETNAMOEBA TRICHOMONAS

CURRENT CONCEPT OF THE ETIOLOGY OFPERIODONTITIS:

SUSCEPTIBLE PRESENCE OF

HOST PATHOGEN

ABSENCE OF

BENEFICIAL Spp.

ROLE OF BENEFICIAL SPECIES:

• Occupies the niche

• Limits pathogens’ ability to adhere

• Affects vitality & growth of pathogen

• Affects ability of pathogen to produce virulence factors

• Degrades virulence factors produced by pathogens

CURRENT SUSPECTED PATHOGENS OF DESTRUCTIVE PERIODONTAL DISEASE.

DEFINING PERIODONTAL PATHOGENS…

• Periodontal disease occurs in area normally inhabited by many bacteria, hence – difficult to identify specific microbes.

• KOCH’S POSTULATES 1870

1. The causative agent must be routinely isolated from diseased individuals

2. Be grown in pure cultures in lab.

3. Must produce a similar disease when inoculated in susceptible lab animal.

4. Be recovered from lesions in a diseased lab animal.

PROBLEMS IN APPLYING THESE POSTULATES IN PERIODONTAL

DISEASES…i. In ability to culture all the micro-organisms that have been associated with the

disease.(eg: oral spirochetes).

ii. Difficulty in defining and culturing the sites of active disease.

iii. Lack of animal model system to study periodontitis.

SOCRANSKY (1977) PROPOSED AN ALTERNATIVE CRITERIA TO IDENTIFY KEY ORGANISMS IN PERIODONTAL

INFECTIONS…

i. Organism must be associated with disease as evident by increase in no. of organism at disease sites.

ii. Must be eliminated or decreased in sites that demonstrate clinical resolution of disease with treatment.

iii. Must demonstrate a host response in form of an alteration in the host cellular and humoral immune response.

iv. Must be capable of causing disease in experimental animal model

v. Must demonstrate virulence factors responsible for enabling the micro organism to cause destructive periodontal disease.

SOCRANSKY ET AL 1987 REVIEWED REASONS FOR UNCERTAINITY ON DEFINING

PERIODONTAL PATHOGENS…1. Over 500 spp may be cultured from periodontal pockets of different individuals

and one may recover 30-100 spp from a single site.

2. Many spp- difficult to grow and identify.

3. Physical contraints of pocket makes it diffcult to collect a representative sample.

4. Site within a subject do not appear to be progressing equally at all times- time of

sampling may play a critical role.

5. there appears to be multiple destructive periodontal disease that mostly cannot be differentiated on a clinical basis.

6. opportunistic spp my grow as a result of disease rather than as the cause.

7.some of the infections are mixed- difficult to evaluate the role of single spp.

8.strains of putative pathogens may differ from virulence.

9.more virulent strains of a spp may harbor plasmids that might confer virulence properties.

HILL’S POSTULATES

• Given current obstacles etiology of periodontitis – pathogenic microbial cummunity concept +hills criteria of causality

MICROBIAL COMPLEXES• Socransky et al 1998 examined over 13000 sub gingival plaque samples from 185 adults and

recognized 6 closely associated groups of bacterial spp using DNA hybridization technique.

• Kolenbrander et al 2006 cell to cell recognition in oral bacteria is not random and each strain has a defined set of partners.

• Functionally similar adhesins found on bacteria of different genera may recognisethe same receptors on other bacterial cells helping in coaggregation.

DESCRIPTION OF MICRO ORGANISMS

AGGREGATIBACTER ACTINOMYCETEMCOMITANS

Actinobacillus actinomycetemcomitans.

• Klinger (1912) first isolated from cervicofacial actinomycosis lesions.

• Henrich and Pulverer(1959) were the first to demonstrate that A. a was part of the normal oral flora and indicated that it could colonize teeth, mucosa and the oropharyx.

• With respect to periodontal disease, A. a was first implicated as the cause of juvenile periodontitis in 1976 by Newman et al. and by Slots .

Isolated together with A.israelii, hence the name which means ‘together with actinomyces’.

Gram –ve, small, non-motile, saccharolytic, capnophilic, round-ended rod .

Culture condition & identification: grows as white, translucent, smooth, non hemolytic colony on blood agar with star shaped internal structure(Actino=star)

• The primary oral ecological niche for Aa is dental plaque.

• High levels- in pockets

• Low levels- in other oral surfaces, tongue, saliva

• It has not been cultured from edentulous mouths

• It does not belong to indigenous microbiota of any other body site.

• Can cause non oral infections such as endocarditis, abscess in lungs, head & neck and abdominal areas.

• Distribution pattern- generally isolated areas, as IgG response to Aa is protective and limit infection.

• Conventional srp is not effective in eliminating Aa due to ability of Aa to invade the gingival c.t. (christerson et al)

• Hence effective therapy involves antibiotics alone or in combination with surgery (zambonet al 1986)

Adhesin- Aaebinds to CHO

receptor on buccalepithelial cell

Moves to supragingival

plaque by Flp 1

Fimbrae along with CHO polymer PGA mediate attachment

to hard surface

Moves to sub gingival plaque

From here may attach to & invade epithelial lining of pocket & enter c.t

Finally may leave oral cavity &

contribute to or cause endocarditis

SEROTYPES• 6 serotypes- a,b,c,d,e and f.

• Most subjects infected with only one serotype

• Serotype ‘a’ and ‘b’ are most common in oral cavity while serotype ‘c’ is important in extra oral infections.

• While serotype ‘b’ was more common in LAP individuals serotype ‘a’ was common in chronic periodontitis.

• Intrafamilial transmission- family members seem to be infected by the same serotype

TRANSMISSION OF A.A

• Vertical or horizontal

• Route of transmission: Salivary and mucosal contact or on inanimate object.

VIRULENCE FACTORS PRODUCED BY A.A…

• Can be classified as_

i. Factors that assist in colonizing the dental plaque and gingival sulcus-bacterial capsule and fimbrae

ii. Factors helping it to evade host defence mechanism-leukotoxin

iii. Factors causing tissue destruction-LPS endotoxin(bone resorption), Collagenase( connective tissue breakdown) Reduction in collagen density

VESICLES

• Blebs-LPS in nature

• Originate & continuous with outer membrane.

• Vesicles function as delivery vehicles for A.a toxic materials

• vesicles

ADHESINS

Among adherence factors there are bacterial capsules and fimbriae .

FIMBRIAE

• Associated with adhesion

Non fimbriated also exhibit adhesive properties

Anaerobically grown Aa produce more fimbriae than the Aa grown in aerobic environment. (scannoapieco et al)

SUSTENINS

BACTERIOCINS

• Proteins produced by bacteria…..lethal to other bacteria

• Active against S.sanguis/A.viscosus

• MOA : permeability of cell membrane of target bacteria

• Leakage of DNA/RNA/macromolecules essential for growth

EVASINS

CYTOTOXINS

• (-) fibroblast proliferation

• Leukotoxin : heat labile factor, that can destroy PMNs

• Aa strains from LAP pts exhibited higher leukotoxin production than from healthy subjects

• More common in young patients

LEUKOTOXIN

Species specific / cell specific

Binds to neutrophils/monocytes/lymphocytes

Pores in the membrane of target cells

Hampers the ability of cell to sustain osmotic homeostasis

IMMUNOSUPPRESSIVE FACTORS

• A.a produces protein

• Inhibits DNA/RNA synthesis in T cells

• Inhibits IgG & IgM synthesis by lymphocytes

RED COMPLEX BACTERIA

T . Denticola

T. Forsythia

P.Gingivalis

PORPHYROMONAS GINGIVALIS

• Gram-negative, anaerobic, non-motile, asaccharolytic rods - member of the much investigated "black-pigmented Bacteroides" group.

• Initially grouped into a single species, B. melaninogenicus.

• At present only known porphyromonas spp isolated from humans that produce phenylacetic acid as metabolic end product.

• Non-oral infections- endocarditis

• Habitat :• Mouth with poor oral hygiene preferred over clean tooth surface, need G+ve bacteria.• Not seen in edentulous mouth

• Levels increase with age so infrequent in children, acquired in later life

Association: • Commonly encountered in sites with destructive periodontal disease• Refractory sites (haffajee et al, 1988) • Advanced periodontal disease (Slots 1977)• Highly inflamed gingivitis lesions (mayrand 1981)• Acute periodontal abscess (newman and sims 1979)

• Able to invade human gingival epithelial cells (duncan et al 1993) -found in higher number in epithelial cells recovered from periodontal pocket (dzink et al, 1989)

VIRULENCE FACTORS1. Capsule: polysaccharide capsule helps in protection against phagocytosis.

2. Fimbriae: helps in binding to host cells and saliva coated tooth

3. Proteinases: a large no. of hemolytic, proteolytic and lipolytic substances are produced.• 4 proteolytic substances are recognized.

• Serine• Aspartate• Thiol• Metalloprotienases

• Out of these collegenase, aminopeptidase, trypsin like protease are critical.

Cysteine proteinases (gingipains)

• Cleave polypeptides after arginine and lysine residue and classified as

• Arginine (Arg-) specific proteinases

• Lysine (Lys-) specific proteinases

Effects:

• ↑ vascular permeability ---↑ GCF

• Chemotactic for PMNL so ↑ conc.of PMNL at sites of tissue destruction

• Arg- gingipain distrupts oxidative burst mechanism of PMNLs.

• Only known prokaryote to inhibit this PMN function.

PATHOGENICITY OF P.GINGIVALISADHESION & COAGGREGATION

• 1st step

• Fimbriae(major adhesive determinant)

• Proteases

• Hemagglutinins

• LPS

• Arg – gingipain also helps in binding of fimbriae to host cells

• Capable of coaggregating- strept. Spp & actinomyces spp.

• Vesicles- bridges non aggregating spp like S.aureus, certain types of candida

SUSTENINS• Sustenance of spp is crucial for its survival

• Biomolecules released to physiologic requirements of these bacteria

• Critical virulent factors

• Proteases- nutrition for bacterial growth in confines of their ecological environ.

• Important for survival of bacterias and collateral damage to host.

• Gingipains-originally considered as trypsin like protease

• Comprises of a group of cysteine endopeptidases that account for 85% of proteolytic activity of P. gingivalis.

• Soluble and cell associated and product of 3 genes rpg A, rpg B, kpg encoding these proteinases.

END PRODUCTS OF METABOLISM

• P.g utilizes peptides

• Produce ammonia & organic acids

• Used by other members in subgingival biofilm

P.g peptidylarginine deiminase

NH4

• –ve impact on neutrophil function

• Pathogen growth

• P.g- H2S, CH3SH

• Halitosis

• Toxic to host cells

• Degrade disulfide bonds

EVASINS

• P.g alters the secretion & accumulation of selected chemokines

• Interefers with MCP-1

• IL-8 expression

• chemokine level : Ability of host to recognise bacterial species

• Ability of PMNs to remove bacterial cell

• Destruction of neutrophils before it can act-----this provides the bacteria with proteolytic nutrients for metabolism & growth

• Attack & degrade C3a & C3b

• Directly activates C5 C5a

• P.g forms small outer membrane vesicles that contains bacterial proteases

• Degrade host C5a receptors that are present on the surface of attacking neutrophils

• Neutrophil loses its phagocytic function

• PMNs death & degranulation

• Release PMNs associated hydrolytic enzymes

• Further affect host cell & tissue destruction

• Gingipain R from P.g

• Cleaves CD14

• LPS induced IL-8 secretion from gingival fibroblast

• Immune evasion by

• P.g secretes heat stable molecule ( Geatch et al )

• Inhibit host cell growth / proliferation

• Promote apoptosis (lymphocytes)

• P.g metabolism

• Butyric acid

• Inhibit Immune cell function &Lymphocyte proliferation

• LPS of P.g causes increase in IL1,TNFα, PGE2 capable of activating osteoclasts.

• Putrify salivary glycoproteins

• Undermining their function in maintaining homeostasis

Effects on blood factors , coagulation and clotting

• Gingipain –activates blood clotting pathways

• PMNs accumulation in actively resorbing periodontitis site

• LPS thrombin & IL-I in GCF tissue destruction

• Gingipain R activates factor IX, X, prothrombin

• Gingipain degrade fibrinogen very rapidly (Pike et al)

• Results non clotting host condition

• Active against fibrin gel

OTHER VIRULENCE FACTORS….

• Various other factors are collagenases, endotoxins, trypsin like enzyme,

fibrinolysin, proteases that destroys Ig, fatty acids, NH3, hydrogen

sulphide.

• Induces elevated systemic and local immune response in periodontitis

subjects.not seen in normal individual

TANERELLA FORSYTHUS• Previously known as B. forsythus

• First described in 1979 (Tanner et al. 1979) as a "fusiform" Bacteroides at The Forsyth Institute.

• Gram-negative, anaerobic, spindle-shaped, highly pleomorphic rod.

• Difficult to grow, often requiring 7–14 days for minute colonies to develop.

• The growth of the organism was shown to be enhanced by co-cultivation with F. nucleatum and commonly occurs with this species in subgingival sites (Socransky et al. 1988).

• The species was shown to have an unusual requirement for N-acetylmuramic acid (Wyss 1989).

ASSOCIATION:

• Found in higher numbers • in sites of destructive periodontal disease or

• periodontal abscesses than in gingivitis or healthy sites (Lai et al. 1987).

• in active periodontal lesions than inactive lesions (Dzink et al. 1988)

• Refractory periodontitis cases. (Listgarten et al,1993).

• Subjects who harbored T. forsythus were at greater risk for alveolar bone loss, attachment loss and tooth loss (Machtei et al. 1999

• Studies using checkerboard DNA–DNA hybridization techniques to examine subgingivalplaque samples demonstrated that T. forsythus was the most common species detected from periodontal pockets (Dibart et al. 1998).

• Listgarten et al 1993 found that T. forsythia was the spp most frequently detected in refractory periodontitis.

• Serum Ab to this organism was often extremely elevated in subset of refractory periodontitissubjects.

S LAYER- MEDIATES HEME AGGLUTINATION, ADHESION/INVASION OF EPITHELIAL CELLS, MURINE SUBCUTANEOUS ABSCESS FORMATION.

ADHESINS

• Coaggregates with F.nucleatum

• Increase colonisation in subgingival biofilms

SUSTENINS

• Produces enzymatic peptidase

• Degrades BANA

• Described as trypsin like proteases : ( - ) by serine protease

• Sialidase from T.f

• Cleaves α-ketosidic linkages between sialic acid & glycosyl residues of host glycoproteins/glycolipids

• Modified / degraded proteoglycans : nutrition for members of oral microbiota

EVASINS

• T.f produce lipoproteins

• Activates gingival fibroblasts\

• increase IL-6 & TNF-alpha

• Also kappa B production by fb

TREPONEMA DENTICOLA

ADHESINS

• Coaggregation b/w P.g & T.d is mediated by fimbriae binding protein-DENTILYSIN

• Helps transport of Pg to deeper regions

• Binds to ECM proteins like laminin fibronetin /heparin

• And host cells like gingival fibroblast

• Collagen binding proteins of T.d binds Type I,IV,V----------adherence /colonozation (Li et al)

• Produces major outer sheath proteins

host cells

• lectin like proteins

• Binding activity (-) mannose/galactose residues

• This protein might have porin activity –enables transportation of molecules into /out of host cells

SUSTENINS

• Possess peptidases associated with its outer sheath

• Chymotripsin like protease ( Uilto et al )

• T.denticola + erythrocyte---resulting in cell lysis & competing with host for available hemin derived iron

• Synthesize 2 low iron induced outer membrane proteins : HbpA & HbpB—binds hemin

EVASINS

• Chymotrypsin like protease + proteins on outer sheath

• Chymotrypsin like protease complex

• Adhesion

• Degradation of humoral proteins ( basement membrane components)

• Activates MMPs

• Forms vesicles with hyaluronidase

Effects on host innate & immune mechanism

• T.denticola on fibroblast

• Cell rounding

• Formation of surface blebs

• Detachment from cell surface (Weinberg & Holt)

• Major sheath protein complex of T.d interferes with collagen attachment via β-integrin

• Direct effect on host response

• T.d produces immunosuppressive proteins (Sip)

• Lymphocyte proliferation

• By inducing an arrest at G1 phase in human T-cells

• Irreversible

• Activates apoptotic pathway in these cells

• May be resistant to β-defensins -1 & -2

PREVOTELLA INTERMEDIA• Second black-pigmented Bacteroides to receive considerable interest

• Gram-negative, short, round-ended anaerobic rod

• Association:• acute necrotizing ulcerative gingivitis ( Loesche et al. 1982),

• certain forms of periodontitis (Tanner et al. 1979, Herrera et al)

• progressing sites in chronic periodontitis (Tanner et al)

• rapidly progressive periodontitis subjects (Hillmann)

• Elevated serum antibodies to this species have been observed in some but not all subjects with refractory periodontitis (Haffajee et al. 1988b).

Elimination:

Berglundh et al. (1998) demonstrated that improved clinical parameters after the use of mechanical therapy and systemically administered amoxicillin and metronidazole were associated with a decrease of periodontal pathogens including P. intermedia.

Successful treatment of peri-implantitis with local delivery of tetracycline also significantly decreased the frequency of detection of P . intermedia (Mombelli et al. 2001)

• This species appears to have a number of the virulence properties exhibited by P . Gingivalis

• shown to induce mixed infections on injection in laboratory animals (Hafstrom& Dahlen 1997).

Pregnancy gingivitis

Physiological level of steroid hormone increases

vascular permeability increases

E/P substitute menadione as a growth factor in P.i

• P. intermedia can utilize albumin, mucin and glucose as sources of energy

• Modify its pathogenic factors (proteolytic activity, depending on the nutrients in the environment)

• This nutritional flexibility may explain why P.intermedia resides in both subgingival/supragingival areas

SPIROCHETES

• Gram-negative, anaerobic, helical shaped, highly motile microorganisms

• implicated as the likely etiologic agent of acute necrotizing ulcerative gingivitis by its presence in large numbers in tissue biopsies from affected sites (Listgarten & Socransky 1964, Listgarten 1965)

Cultural studies suggested that T. denticola and a "large treponeme" were found more frequently in patients with severe periodontitis than in healthy or gingivitis sites (Moore et al. 1982).

FUSOBACTERIUM NUCLEATUM• Gram-negative, anaerobic, spindle shaped rod

• Association:• periodontitis (Papapanou,Socransky)• periodontal abscesses (Herrera et al. 2000).

• Elimination: Successful treatment of peri-implantitis with local delivery of tetracycline was associated with a significant reduction in frequency of detection in several species including F. nucleatum(Mombelli et al. 2001).

• Invasion of this species into human gingival epithelial cells in vitro was accompanied by an increased secretion of IL-8 from the epithelial cells (Han et al. 2000).

CAMPYLOBACTER RECTUS

• Earlier called Wolinella rectus

• Gram-negative, anaerobic, short, motile vibrio.

• Utilizes H2 or formate as its energy source.

• It was first described as a member of the "vibrio corroders", a group of short nondescript rods that formed small convex, "dry spreading" or "corroding" (pitting) colonies on blood agar plates.

• Association:

• Higher numbers in disease sites as compared with healthy sites (Moore et al. 1983)

• In sites exhibiting active periodontal destruction (Dzink et al. 1985)

• In periodontitis patients with NIDDM.

• Elimination: found in lower numbers

• After successful periodontal therapy (Tanner et al. 1987)

• Treatment of peri-implantitis with local delivery of tetracycline (Mombelli et al.)

• Shown to produce a leukotoxin (Gillespie et al. 1992)

• Capable of stimulating human gingival fibroblasts to produce IL- 6 and IL-8

EIKENELLA CORRODENS• Gram-negative, capnophilic, asaccharolytic, regular, small rod with blunt ends

• Also found in osteomyelitis, RC infections, CNS infections

• Association:• periodontal destruction as compared with healthy sites (Savitt & Socransky 1984)• active sites (Dzink et al. 1985, Tanner et al. 1987) • in sites of subjects who responded poorly to periodonal therapy (Haffajee et al. 1988)• with Aa in some lesions of LJP (Mandell 1984)

• Stimulate the production of matrix metalloproteinases (Dahan et al. 2001) and IL-6 and IL-8 (Yumoto et al. 1999).

PEPTOSTREPTOCOCCUS MICROS

• Gram-positive, anaerobic, small, asaccharolytic coccus.

• associated with mixed anaerobic infections in the oral cavity and other parts of the body (Finegold1977).

• Two genotypes • smooth genotype (associated with periodontitis lesions)• rough genotype

• Association:• periodontal destruction as compared with gingivitis or healthy sites (Moore et al. 1983)• actively breaking down sites (Dzink et al. 1988).

EUBACTERIUM SPECIES• G+ve, strictly anaerobic, small, somewhat pleomorphic rods

• Possible periodontal pathogens due to their increased levels in disease sites, particularly those of severe periodontitis (Moore et al. 1982)

• Difficult to cultivate, particularly on primary isolation, and appear to grow better in roll tubes than on blood agar plates

• Elicited elevated antibody responses in subjects with different forms of destructive periodontitis (Tew et al. 1985a,b, Vincent et al. 1986, Martin et al. 1988)

OTHER SPECIES

• All periodontal pathogens have not yet been identified.

• Particularly in individuals who have responded poorly to periodontal therapy, there are chances of infection with unusual species.

• Emphasis has been placed on enteric organisms, staphylococcal species

MICRO ORGANISM ASSOCIATED WITH

SPECIFIC PERIODONTAL DISEASE

MICROBIAL SHIFT DURING DISEASE:

HEALTH GINGIVITIS PERIODONTITIS

Gram +ve Gram –ve

Cocci Rods

Non motile Motile

Facultative anaerobes Obligate anaerobes

Fermenting Proteolytic

Gingivitis

Actinomyces species

Streptococcus

species

Veillonella species

Fusobacterium

species

Prevotella

intermedia

Health

Streptococcus

sanguis

Streptococcus mitis

Veillonella

parvula

Actinomyeces

naeslundii

Actinomyces

viscosus

Periodontitis

Porphyromonas gingivalis

Tanerella forsythus

Treponema denticola

BACTERIAL SPECIES ASSOCIATED WITH DIFFERENT PERIODONTAL CLINICAL

STATES

PREGNANCY GINGIVITIS• Dramatic increase in levels of P.intermedia.

• Other microbes reported : P.melaninogenica, F.nucleatum, T. forsythia.

• Periodontal abscess• F. nucleatum, P. intermedia, P. gingivalis, P. micros, and B. forsythus

CHRONIC PERIODONTITIS• High percentages of anaerobic (90%) gram-negative (75%) bacterial species

• Bacteria most often cultivated at high levels include P. gingivalis, B. forsythus, P. intermedia, C. rectus, Eikenellacorrodens, F. nucleatum, A. actinomycetemcomitans, P. micros, and Treponema and Eubacterium

• C. rectus, P. gingivalis, P. intermedia, f. nucleatum, and B. forsythus were found to be elevated in the active sites

• Recent studies have documented an association between chronic periodontitis and viral microorganisms of the herpes viruses group, most notably Epstein-Barr Virus-1 (EBV-1) and human cytomegalovirus (HCMV).

AGGRESSIVE PERIODONTITIS

• predominantly gram-negative, capnophilic, and anaerobic rods.

• almost all LJP sites harbor A. actinomycetemcomitans, which may comprise as much as 90% of the total cultivable microbiota.

• Other organisms found in significant levels include P. gingivalis, E. corrodens, C. rectus, E nucleatum, B. capillus, Eubacterium ,and Capnocytophaga spp. and spirochetes

• Herpes viruses, including EBV-1 and HCMV, also have been associated

NECROTIZING PERIODONTAL DISEASE:

• Microbiologic studies indicate that high levels of P. intertmedia and spirochetes in necrotizing ulcerative gingivitis lesions.

• Spirochetes are found to penetrate necrotic tissue and apparently unaffected connective tissue.

• In NUG F. nucleatum is also found in association

PERIODONTITIS IN MEDICALLY COMPROMISED PATIENTS

• HIV PERIODONTITIS

• HIV periodontitis lesions reveal spirochetes, Fusobacterium species, A.a, W.recta, P.micros and P.intermedia each averaging

5-20% of the subgingival microflora.

• Some lesions also yield B.fragilis, F.necrophorum, E.aerofaciens, Clostridium species, enterococci and P.aeuroginosa and

C.albicans.

• DIABETES MELLITUS

• Capnocytophaga species and P. intermedia have been related to the initial breakdown in periodontium.

• W. recta, A.actinomycetemcomitans and P. gingivalis may also play a role

PERIODONTAL ENDODONTIC LESION

• Reflects microbiota of the separate endodontic and periodontal lesions

(Legoff 1997)

• Accordingly it might be impossible to obtain a sample that is

representative of either of the lesion.

PERIIMPLANTITIS

• Healthy perimplant pockets : high proportions of coccoid cells, a low ratio of anaerobic/aerobic species, low detection frequencies for pathogens.

• Around failing implants : A.a., P.gingivalis, T. forsythia, P.micros, C.rectus, Fusobacterium and Capnocytophaga

• Implant associated with refractory periodontitis : P.aeruginosa, C.albicans, staphylococci aureus

PARASITESEntamoeba gingivalis

• Protozoan & normal commensal

• ↑elderly individuals and in diseased mouths.

• Earlier- critical role

Now- opportunistic pathogen

• Found in soft calculus, periodontal pockets and with diseased tonsils.

Trichomonas tenax

• Only parasitic flagellate found in the oral cavity.

• Numbers are seen to increase in periodontal pockets.

VACCINE ???

• Conventional therapy : painful & frequently unsuccessful

• Recurrence of destructive process

• Prevention & control of periodontitis

• The three types of vaccine that were employed for the control of periodontal disease-

1. Vaccines prepared from pure cultures of streptococcus & other oral micro organism

2. Autogenous vaccines prepared from plaque of patients with destrucive periodontal disease

3. Stock vaccines such as Van Cott’s vaccine, Goldenberg’s vaccine, Inava endocorp vaccine-adminstered systemically/locally.

• A recent report suggested that the hemagglutinin domain was implicated in the nuclear targeting of this protease

• Vaccines are mainly peptide & DNA vaccines (Scragg 2002)

• DNA vaccine induces cellular immunity & humoral immunity

• Peptide vaccine induces humoral immunity

• Further analysis is required to evaluate at which stage these immune responses prevent P. gingivalis infection

• Recent data indicate that the gingipains (RgpA and Kgp) of P. gingivalis are potential candidates for a vaccine

CLINICAL IMPLICATIONS• Aid in designing the measure to prevent these diseases

• Determination of risk factors, • e.g. high levels of p.gingivalis in a smoker

• To monitor and evaluation of treatment outcome• Declining level of suspected pd pathogen

• To target therapy• Development of vaccine

• Use of antimicrobials

ADVANCES IN MICROBIOLOGY

• DNA based methodology for identification and detection of specific bacteria and virus offers remarkable advantage

• No. of samples examined and no of micro organisms identified have increased dramatically

• Also recognition of beneficial activity of several groups such as probiotics might open new strategies in Pdl therapy.

CONCLUSION

• A full understanding of the microbial factors, their

pathogenicity as well as host factors are of the essential importance for

pathogenesis of periodontal disease

• Microbial diagnosis may play a vital role in treating cases of refractory

periodontitis / periodontitis related to specific microorganisms

REFERENCES1. Contemporary Oral Microbiology and Immunology – Slots & Taubman.

2. Clinical periodontology and implant dentistry: jan lindhe 4th edition

3. Clinical periodontology: carranza 10th edition

4. Periodontology 2000, vol 38

5. Periodontology 2000, vol 55

6. Text book of Microbiology : Ananth Narayanan, C.K. Jayram Paniker.

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