Periodontal Microbiology
Human fetus is sterile but after passing through
birth canal it acquires vaginal and fecal
microorganisms
By 2nd day Anaerobic flora can be detected in
infant edentulous mouth
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Within 2nd week a nearly mature microbiota
is established in the gut of newborn
After weaning(>2yrs), entire human microbial
flora formed by complex collection of more
than 400 types of bacteria.
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Oral cavity is an “Open growth system”
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Oral cavity divided into 5 major niches
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Most species colonize on all above described
niches with the exception of spirochetes
Adherence of bacteria to oral epithelial cells
is directly related to its virulence
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Teeth and Implants are unique from a microbiological point of view for two reason– Provide hard, non-shedding surface that allows for
the development of extensive structured baterial deposit
– Form a unique ectodermal interruptionA special seal of epithelium and connective
tissue exists between external enviroment and internal part of body
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Teeth are primary habitat for periopathogens
Thus teeth can be considered as “Port of entry for Periopathogens
Cariogenic species like S mutans remain restricted to solid surfaces and are called “obligate periphyte”
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Dental Plaque
Dental plaque is a specific, amorphous, granular
deposit which accumulates on the surface of
teeth, dental restoration and dental calculus. - -Glickmann
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WHO defnition
“Dental plaque is a specific but highly variable
entity resulting from growth and colonization of
micro-organisms on surfaces of teeth,restoration
and soft- tissue consisting of various species
microorganisms entangled in extracellular matrix.”
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Dental plaque• Defined clinically as structured, reselient,
yellowish-grayish substance that adhere tenaciously to the intraoral hard surface,including removable and fixed restoration.
• Plaque is primarily composed of bacteria in matrix of salivary glycoproteins and extracellular polysaccharides
• 1gm of dental plaque(wet weight) contains 1011
bacteria
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Thin supragingival dental plaque of a 32-year-old man who had not brushed his teeth for 7 days. A, Unstained plaque is not readily apparent. B, Extent of the plaque becomes apparent when stained with a disclosing solution (i.e.,erythrosine dye)
No of bacteria inSupragingival plaque on single tooth – 109
Gingival Crevice- 103
Deep periodontal pocket- 108
More than 500 distinct microbial species are found in dental plaque
Non bacterial microorganism found in plaque include Mycoplasma species, yeasts, Protozoa and Viruses
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Dental plaque is broadly Classified into
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Supragingival plaque visualized by Disclosing solution
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Plaque-bacteria interaction with tooth surface and Periodontal tissuesDr Saif Khan 1704/07/23
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The enviromental paramaters of subgingival region is different from supragingival region
– Gingival crevice is bathed by GCF which may contains many substances that bacteria may use as nutrients
– Low oxygen tension in the subgingival area– Host inflammatory cells and mediators have
considerable influence on establishment and growth of bacteria
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The apical border of the plaque mass is
separated from junctional epitheliun by a layer
of host leukocytes and the bacteria of this
apical-tooth associated region show an
increased concentration of Gm-ve rods
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Dental Plaque as a Biofilm
• Biofilm is a highly organized structure• Consists of microcolonies of bacterial cells
randomly distributed in a shaped matrix or glycocalyx.
• Lower plaque layer are dense in which microbes are bound together in polysaccharide matrix with other organic and imorganic materials
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• On top of lower layer, loose layer can be seen which can extend into surrounding medium (for teeth and saliva)
• The fluid layer bordering the biofilm has a stationary sublayer and a fluid layer in motion
• Nutrient components penetrate this fluid medium by molecular diffusion
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• The dental plaque biofilm has open fluid filled channels running across the plaque mass
• Act as primitive “circulating system”
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Intercellular Matrix
• Organic Constituent- Consists of– Polysacchrides, Proteins, Glycoprotein & Lipid– Albumin derived from GCF– Lipid material consists of debris from the membranes
of disrupted bacterial & Host cells and possibly Food debris
– Glycoprotein from saliva important component of pellicle
– Polysacchrides produced by bacteria of which Dextran is most predominant form & play major role in maintaining integrity of Biofilm
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Inorganic component• Predominantly Ca and P• Trace minerals Na, K and F• Source of inorganic constituent of supragingival
plaque is primarily saliva• As mineral content increases plaque calcifies to
Calculus• Inorganic portion of subgingival fluid are derived
from crevicular fluid• F content of plaque is basically derived from
external sources such as floridinated toothpaste, rinses, water etc
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Plaque formation at Ultrastructural level1. Formation of Pellicle
– Thin, saliva derived layer– Consists of Glycoprotein (mucins), Proline rich protein,
Phosphoproteins (Statherin), Histidine rich proteins,enzymes
– Forms within seconds of prophylaxis– Forms by selective adsorption of enviromental
macromolecules– Mechanism involved in pellicle formation include
Vanderwall forces & Hydrophobic forces
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2. Initial Adhesion and attachment of bacteriaPhase I : Transport to the surface•Initial contact of bacterium to tooth surface•Random contacts through Brownian movement (40μm/hr)•Through Active bacterial movement
Phase II: Initial Adhesion•Reversible adhesion•Interaction b/w bacteria and surface at certain distance (50nm) through long range and short range forces•Including Vanderwall attractive and electrostatic repulsive forces•Gibbs total energy GTOT = GA + GE
•GTOT is a function of separation distance between negatively charged particle and negatively charged surface in a medium ionic suspension (saliva)•For most bacteria reversible binding takes place 5-20 nm from surface
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Schematic representation of interactions involved in
bacterial adhesion to solid substrata
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Phase III: Attachment•After intial adhesion firm adhesion is established by specific interactions (covalent,ionic or hydrogen•Rough surfaces are more conducive for attachment as bacteria are better protected against sheer force leading change from reversible to irreversible bonding•The bonding between bacteria and pellicle is mediated by by specific extracellular protein components
eg; Streptococci (S sangius) early colonizer binds to acidic proline-rich proteins, also α-amylase and sialic acid
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3. Colonization and plaque maturation
Primary Colonizers: Streptococci and Actinomycetes
Secondary Colonizers: P intermedia, P loescheii, Capnocytophaga, F nucleatum, P gingivalis
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All oral bacteria possess surface molecule or receptor which foster cell to cell interaction
Highly specific stereo chemical interaction of proteins and carbohydrate molecules located on bacterial cell surfaces leading to Coaggregation
Most Co aggregation are among strains of different genera are mediated by Lectin like adhesins and inhibited by Lactose and other galactosides
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Examples of Co-aggregation
Fusobacterium nucleatum with steptocooci sangius,Prevotella loescheii with A viscosus Capnocytophaga ochraceus with A viscosus
Intrageneric co-aggregation in streptocooci
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Fusobacterium nucleatum
S sangius
P loeschiiA viscosus
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Coaggregation
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Growth dynamics of Dental Plaque
• First 2-8 hrs, adherent pioneering streptococci saturate the salivary pellicular binding sites and cover 3-30% of enamel surface
• After 1 day the term Biofilm is fully deserved because organization takes place in it
• After 3 days plaque growth increases at rapid rate and then slows down
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• There is shift towards more anaerobic and gram-negative flora, including an influx of Fusobacteria, filaments, Spiral forms and spirochetes
• In ecological shift within of the biofilm, there is a transition from the early aerobic environment characterized by Gm+ve facultative species to a highly oxygen- deprived environment in which Gm-ve anaerobic microorganism predominate
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Topography of Supragingival Plaque
• Early plaque formation follow typical topograhic pattern with initial growth along gingival margin and interdental space
• Later further there is extension in coronal direction
• This pattern changes when tooth contains surface irregularities such as grooves,cracks, perikymata, or pits
• Surface irregularities can give rise to “individualized plaque growth pattern
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Surface Microroughness
• Rough intraoral surface (crown,implant abutments, denture bases)accumulate and retain more plaque and calculus in terms of thickness area and colony forming unit
• [Ra=0.2µ] is threshold for surface roughness above which bacterial adhesion is facilitated
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Individual variables influencing plaque formation
Heavy(fast) plaque formers Rapid plaque formers demonstrate higher proportion
of Gm-ve rods (35% vs 17%) in 14-day old plaque
Light (slow) plaque formers
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Intersubject variation in plaque formation can be explained by factors such as
• Diet• Food• Smoking• Presence of copper amalgam• Tongue& palate brushing• Colloid stability of bacteria in the saliva• Antimicrobial factors present in saliva• Chemical composition of the Pellicle• Retention depth of dentogingival area
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Variation within the Dentition
Early plaque formation occurs faster in •lower jaw compared to upper•Molar areas•Buccal tooth surfaces as compared to oral sites (esp in upper jaw)•Interdental region compared to buccal or oral surfaces
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Impact of Gingival inflammation
• Early invivo plaque formation is more rapid on tooth surfaces facing inflamed gingival margins than those adjacent to healthy gingival margins
• Increase in crevicular fluid production enhances plaque formation
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Impact of Age
Recent studies show that subject’s age does not
influence de novo plaque formation
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Spontaneous tooth cleaning
• Firm attachment between bacteria and surface this is unlikeky
• Even occlusal part of molars, plaque remains after chewing fibrous food
• Only negligible differences in plaque extension could be observed
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De novo subgingival plaque formation
Recent studies suggest that complex subgingival microbiota, including most periopathogens, is established within 1 week after abutment insertion
Smooth abutments[Ra<0.2µ] were found to harbour less bacteria than less ones, with a slightly higher density of coccoids (i.e nonpathogenic) cell
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Physiological properties of Dental Plaque
The transition from Gm+ve to Gm-ve microorganism observed in structural development of plaque is paralled by physiologic transition in the developing plaque
Early colonizers use oxygen and lower redox potential of the environment which then favors growth of anaerobic species
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Early colonizers use sugar as energy source and saliva as carbon source
Bacteria which predominate in mature plaque or late colonizers are Asaccharolytic and use amino acids and small peptides as energy source
Lactate and formate are by products of metabolism of streptococci and actinomycetes may be used by other microorganism
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Hemin a breakdown product from host hemoglobin is important in metabolism of P gingivalis
Increase in steroid hormone is associated with increase in proportions of Prevotella intermedia in subgingival plaque
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Metabolic interaction among different bacteria species found in plaque and also between host and plaque bacteria
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Ecological plaque Hypothesis Given by Marsh & Co- Workers in 1990
Total amount of dental Plaque and the specific microbial composition of plaque contribute to transition from health to disease
Health –associated dental plaque microflora is considered to be relatively stable overtime and in state of dynamic equilibrium or “ microbial
homeostasis ” Dr Saif Khan 5404/07/23
Ecological plaque Hypothesis
Change in nutrient status of a periodontal pocket or
Chemical and physical changes to habitat can lead over growth of pathogens
Eg: Increase in GCF flow can lead to enrichment of proteolytic species(periopathogens) by providing essential nutrients such as heme containing molecules
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Schematic representation of the ecologicalplaque hypothesis in relation to periodontal disease
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Relationship between the microbial composition of dental plaque in health and disease.
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Non- specific plaque hypothesis Periodontal disease results from elaboration of
noxious products from entire plaque flora
Large amount of plaque produces large amount of noxious product that would overwhelm host’s defense
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Control of periodontal disease depend on control of amount of plaque deposit
The current standard treatment of periodontitis still focuses on the removal of plaque and its product founded on non-specific plaque hypothesis
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Non-specific plaque hypothesis has been discarded because
Some individuals with considerable amount of plaque and calculus as well as gingivitis never developed destructive periodontitis
Individuals with periodontitis demonstrated considerable site specificity in pattern of disease
Individuals with very less plaque developed destructive periodontal disease as in Aggressive periodontitis
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Specific plaque Hypothesis
States that only certain plaque is pathogenic
And its pathogenecity depends on presence of or
increase in specific microorganism
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Plaque harboring specific bacterial
pathogen results in periodontal disease
because these organism produce
substance that mediate the destruction
of host tissue
Eg: A actinomycetemcomitans as pathogen in
localized aggressive periodontitis
Specific Bacterial behaviour in Biofilm:Antibiotic resistance
Microorganisms in biofilm are 1000 to 1500 times more resistant to antibiotics than in their planktonic stage
The mechanism of this increased resistance differs from species to species, from antibiotic to antibiotic, and for biofilm growing in different habitats
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Resistance of bacteria to antibiotics is affected by their
• Nutritional status• Growth rate• Temparature• pH• Prior exposure to subeffective concentration
of anti microbial agents
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Also slower growth of bacterial species in biofilm is
another important mechanism of antibiotic resistance
Biofilm matrix although not significant barrier in itself to
diffusion of antibiotics but have certain properties to
resist diffusion
Biofilm act as ion-exchange resin removing antibiotics
from solution
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Also extracellular enzymes such as β lactamases,
formaldehyde lyase and formaldehyde
dehydrogenase may become trapped and
concentrated in the extracellular matrix thus
inactivating some antibiotics(especially positive
charged hydrophilic antibiotics)
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Some antibiotics such as Macrolide which
are positive charged but hydrophobic
are unaffected by this process
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“Super-resistant” bacteria have been identified
within a biofilm and these cells have multidrug-
resistant pump that can extrude antimicrobials
from the cell
Quorum SensingBacteria in biofilm communicate with each
other
This involves the regulation of expression of specific genes through accumulation of signalling compounds that mediate intercellular communication
When these signalling compounds reach a threshold level(quorum cell density) gene expression is activated
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• Quorum sensing seems to play a role in
expressing genes for antibiotic
resistance and encouraging growth of
beneficial species to the biofilm and
discouraging the growth of competitors
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Schematic representation of the types of interaction that occur in a microbial community, such as dental
plaque
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High density of bacterial cells in biofilm facilitates the exchange of genetic information among cell of the same
species and genera through;
1. Conjugation (sex pilus)2. Transformation (movement of small pieces of DNA
from enviroment into bacterial chromosome)3. Plasmid tranfer4. Transposon transfer (DNA sequence which can
change sequence within the genome)
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Translocation and Mechanical Debridement
• To reduce chance of intraoral transmission, one stage, Full mouth disinfection has been introduced by Leuven group in the 1990s
• This strategy attempts to eradicate, or atleast suppress, periopathogens in short time not only from the periodontal pockets, but also from all their intraoral habitats(mucous membrane, tongue, and saliva)
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One stage, Full mouth disinfection
Full mouth scaling and root planning within 24 hrs to reduce number of subgingival pathogenic organisms
Subgingival irrigation of all pockets with 1% chlorhexidine gel to kill remaining bacteria
Tongue brushing with an antiseptic to suppress the bacteria in the niche
Mouth rinsing with antiseptic to reduce the bacteria in the saliva and on the tonsil
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Benefits of the One stage, Full mouth disinfection
• Pocket depth reduction
• Gain in clinical attachment level
• Microbiologic shift
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The current concept on etiology of Periodontitis considers three factors that determine whether active periodontitis will occur
1. Susceptible Host2. Presence of a Pathogenic species3. Absence or small numbers of beneficial
species
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Role of Beneficial species
Passively occupying
niches otherwise
occupied by pathogenic
bacteria
Actively limiting
pathogens ability to adhere to
appropriate tooth
surface
Adversely affecting
the vitality or growth
of pathogens
Affecting the ability
of pathogenic species to produce virulence
factor
Degrading Virulence
factor produced
by pathogen
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Beneficial species such as S sangius,
Veillonella parvula and C ochreus are
typically found in higher number at
periodontal sites with no attachment loss
where as lower in number where there is
active periodontal destruction
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Criteria for identification of Periodontal pathogen
Kochs PostulatesGiven by Robert Koch as classic criteria by which microorganims are judged causativeMust be routinely isolated from diseased individualMust be grown in pure culture in laboratoryMust produce similar disease when inoculated in susceptible laboratory animalMust be recovered from lesions in diseased laboratory animalStreptococcus mutans has been shown to follow Koch’s postulate as an etiologic agent of dental caries
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koch’s criteria are difficult to apply in periodontal disease because of 3 reasons
1. Inability to culture all the microorganism that have been associated with disease (eg: spirochetes)
2. The difficulties inherent in defining and culturing sites of active disease
3. Lack of good animal model for study of Periodontitis
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Socransky criteria • Proposed criteria by which periodontal microorganism
may be judged to be potential pathogens1. Must be associated with disease, as evident by increase in
the number of organisms at diseased sites2. Must be eliminated or decreased in sites that demonstrate
clinical resolution of disease with treatment3. Must demonstrate a host response, in the form of an
alteration in the host cellular or humoral immune response4. Must be capable of causing disease in experimental animal
model.5. Must demonstrate virulence factors responsible for
enabling the microorganism to cause destruction of periodontal tissue
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Microorganism associated with specific periodontal disease
Fewer coccal cells and more motile rods and spirochetes are found in diseased state than healthy sites by means of phase-contrast or dark-field microscopy
All most all periodontal pathogens except Campylobacter rectus are immobile
Bacteria from healthy periodontal sites consists of gram +ve facultative rods and cocci
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Periodontal Health
Gm+ve facultative species of genera Streptococcus and Actinomycetes(S sangius, S mitis, A viscosus, A naeslundi)
Small proportions of Gm-ve speciesa are also found (P intermedia, F nucleatum,
Capnocytophaga, C ochareus)
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GingivitisMicro biota of dental –plaque induced gingivitis(chronic gingivitis) consists of equal proportions of Gm+ve(56%) & Gm-ve(44%) species
Facultative(59%) & anaerobic(51%) microorganisms
Predominant Gm+ve microorganisms are S sangius, S mitis, S intermedius, S oralis, A viscosus, A naeslundii and P micros
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The predominant Gm-ve microorganisms are F
nucleatum, P intermedia, V parvula as well as
Haemophilus, Campylobacter and Capnocytophaga
Pregnancy associated gingivitis is acute inflammation of
gingivae associatedin pregnancy. There is increase in steroid
hormones in crevicular fluid and dramatic increase in
Prevotella intermedia which uses steroid as growth factor
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Chronic periodontitis
Microscopic examination of plaque from sites with chronic gingivitis consistently revealed elevated proportions of spirochetes
Cultivation of plaque microorganisms from sites chronic periodontitis have reveal high percentages of anaerobic(90%) and gram negative (75%) bacterial species
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Bacteria most often cultivated include P. gingivalis
, T. forsythus,,C rectus, E corrodens, F.
nucleatum, A actinomycetemcomitans, P
micros, Treponema and Eubacterium
C rectus, P gingivalis, P intermedia, F nucleatum & T
forsythia are elevated in active sites
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Detectable levels of P gingivalis, P intermedia,
T forsythia, C rectus and A
actinomycetemcomitans are associated with
disease progression and their elimination by
therapy is associated with improve clinical
outcome
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Also recent studies have documented
association between chronic periodontitis
and viral microrganisms of Herpes
group, most notably Epstein barr virus-1
(EBV-1) and Human cytomegalovirus
(HCMV) are associated with putative
pathogens P gingivalis, T forsythia, P
intermedia and T denticola04/07/23
Microbial Shift during disease
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Localized Aggressive Periodontitis
A actinomycetemcomitans compose of 90% of total cultivable microbiota
P gingivalis, E corrodens, C rectus, F nucleatum, B
capillus, Eubacterium brachy, Capnocytophaga sp
and Spirochetes are also found in significant levels
Herpes viruses, including EBV-1 and HCMV have been associated with LAP
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Necrotizing Periodontal disease
Microbiologic studies indicate high levels of Prevotella intermedia and especially Spirochetes in NUG lesions
Spirochetes penetrate deep into necrotic tissue and unaffected connective tissue
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Microbial specificity in Periodontitis• There is no “black-or-white” situation; most
pathogens might be present, but do not necessarily have to be present for specific form of periodontitis
• Microbial composition can not be used to differentiate different forms of periodontal disease
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Most pathogens can also be detected
in healthy subjects with frequencies
ranging from 10% to 85%. This
automatically reduces the specificity
of microbiologic testing in
periodontology
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Periimplantitis
Inflammatory process affecting the tissue around an
already osseointegrated implant resulting in loss of
supporting bone Healthy periimplant pockets are characterised by
high proportions of coccoid cells, low anaerobic/aerobic ratio, low number of gram anaerobic species and low detection frequency for
periodontal pathogens
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Key characteristics of specific Periopathogens
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