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
Dr Saif Khan 209/10/13

 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.
09/10/13 Dr Saif Khan 3

Oral cavity is an “Open growth
system”

Oral cavity divided into 5 major niches

 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

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 interruption
A special seal of epithelium and connective
tissue exists between external enviroment
and internal part of body

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”

Dental Plaque
Dental plaque is a specific, amorphous, granular
deposit which accumulates on the surface of
teeth, dental restoration and dental calculus.
- -Glickmann

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.”

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

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 in
Supragingival plaque on single tooth – 109
GingivalCrevice-
103
Deepperiodontalpocket-
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

Dental plaque is broadly Classified into

Supragingival plaque visualized by
Disclosing solution

aque-bacteria interaction with tooth surface and Periodontal tissDr Saif Khan 1709/10/13

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

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

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

• 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

• The dental plaque biofilm has open fluid filled
channels running across the plaque mass
• Act as primitive “circulating system”

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

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

Plaque formation at Ultrastructural level
1. 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

2. Initial Adhesion and attachment of bacteria
Phase 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

Schematic representation of interactions involved in
bacterial adhesion to solid substrata

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

3. Colonization and plaque maturation
Primary Colonizers: Streptococci and Actinomycetes
Secondary Colonizers: P intermedia, P loescheii,
Capnocytophaga, F nucleatum, P gingivalis

 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

Examples of Co-aggregation
 Fusobacterium nucleatum with steptocooci
sangius,Prevotella loescheii with A viscosus
 Capnocytophaga ochraceus with A viscosus
 Intrageneric co-aggregation in streptocooci

Dr Saif Khan 36
Fusobacterium
nucleatum
S sangius
P loeschii
A viscosus
09/10/13

Coaggregation

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

• 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

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

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

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

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

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

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

Impact of Age
Recent studies show that subject’s age does not
influence de novo plaque formation

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

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

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

 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

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

Metabolic interaction among different bacteria species found in plaque and
also between host and plaque bacteria

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 5409/10/13

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

Dr Saif Khan 56
Schematic representation of the ecological
plaque hypothesis in relation to periodontal disease
09/10/13

Dr Saif Khan 57
Relationship between the microbial composition of dental plaque in health and
disease.
09/10/13

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

Dr Saif Khan 59
 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
09/10/13

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

Specific plaque Hypothesis
 States that only certain plaque is pathogenic
 And its pathogenecity depends on presence of or
increase in specific microorganism

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

Resistance of bacteria to antibiotics is affected
by their
• Nutritional status
• Growth rate
• Temparature
• pH
• Prior exposure to subeffective concentration
of anti microbial agents

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

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)

 Some antibiotics such as Macrolide which
are positive charged but hydrophobic
are unaffected by this process

“Super-resistant” bacteria have been identified
within a biofilm and these cells have multidrug-
resistant pump that can extrude antimicrobials
from the cell

Quorum Sensing
Bacteria 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

• 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

Schematic representation of the types of interaction
that occur in a microbial community, such as dental
plaque

Dr Saif Khan 72
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 tranfer
4. Transposon transfer (DNA sequence which can
change sequence within the genome)
09/10/13

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)

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

Benefits of the One stage, Full mouth
disinfection
• Pocket depth reduction
• Gain in clinical attachment level
• Microbiologic shift

The current concept on etiology of
Periodontitis considers three factors that
determine whether active periodontitis
will occur
1. Susceptible Host
2. Presence of a Pathogenic species
3. Absence or small numbers of beneficial
species

Role of Beneficial
species
Role of Beneficial
species
Passively
occupying
niches
otherwise
occupied by
pathogenic
bacteria
Passively
occupying
niches
otherwise
occupied by
pathogenic
bacteria
Actively
limiting
pathogens
ability to
adhere to
appropriate
tooth
surface
Actively
limiting
pathogens
ability to
adhere to
appropriate
tooth
surface
Adversely
affecting
the vitality
or growth
of
pathogens
Adversely
affecting
the vitality
or growth
of
pathogens
Affecting
the ability
of
pathogenic
species to
produce
virulence
factor
Affecting
the ability
of
pathogenic
species to
produce
virulence
factor
Degrading
Virulence
factor
produced
by
pathogen
Degrading
Virulence
factor
produced
by
pathogen

Dr Saif Khan 80
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
09/10/13

Criteria for identification of Periodontal pathogen
Kochs Postulates
Given by Robert Koch as classic criteria by which
microorganims are judged causative
Must be routinely isolated from diseased individual
Must be grown in pure culture in laboratory
Must produce similar disease when inoculated in
susceptible laboratory animal
Must be recovered from lesions in diseased laboratory
animal
Streptococcus mutans has been shown to follow Koch’s
postulate as an etiologic agent of dental caries

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

Socransky criteria
• Proposed criteria by which periodontal microorganism
may be judged to be potential pathogens
1. Must be associated with disease, as evident by increase
in the number of organisms at diseased sites
2. Must be eliminated or decreased in sites that
demonstrate clinical resolution of disease with
treatment
3. Must demonstrate a host response, in the form of an
alteration in the host cellular or humoral immune
response
4. 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

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

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)

Gingivitis
Micro 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

Dr Saif Khan 87
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
09/10/13

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

Dr Saif Khan 89
 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 sites09/10/13

Dr Saif Khan 90
 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
09/10/13

Dr Saif Khan 91
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 denticola
09/10/13

Microbial Shift during disease

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

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

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

Dr Saif Khan 96
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
09/10/13

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

Key characteristics of specific
Periopathogens

Periodontal microbiology

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