DR. SWAPNIL BORKAR
I-YEAR POST GRADUATE STUDENT
DEPARTMENT OF PERIODONTICS & ORAL IMPLANTOLOGY
S.D.K.S DENTAL COLLEGE
Need to understand microbial etiology
Postulate of Robert Koch and Limitation
Virulence : History
Virulence factor : Type
Outer Membrane Proteins
Outer membrane vesicles
Lipopolysaccharide And Associated
Peptidoglycan as aVF
PhysiologicalVirulence of commensal
VF and Host response
VF of P. gingivalis
Microbial etiology of periodontal diseases is
needed to be well understood because of two
Knowledge of the etiological agents of periodontal
diseases would help in the selection of appropriate
It would provide a useful therapeutic approach to
control and prevent periodontal diseases.
(manufacturing of vaccines against various pathogens prior
to the onset of diseases.)
Microbiology of periodontal diseases is difficult to
understand and study due to difficulty in sample collection,
cultivation and identification of isolates.
Periodontal infections are mixed infections and
microbiota is very complex, making hard to distinguish
between secondary invaders and true pathogens.
Periodontal diseases appears to be episodic and thus,
there is difficulty in differentiating between active and
inactive site for sampling. 4
In 1890 Robert Koch postulated
guidelines to establish a standard
for evidence of causation in infectious
disease (based on early work by Henle):
The microorganisms should be constantly
associated with the lesion of the disease.
It should be possible to isolate the bacterium in pure culture from the
Inoculation of such pure culture into suitable animal model should
reproduce the lesion of disease.
It should be possible to reisolate the microbes in pure culture from the
lesions produced in the experimental animals. 5
Limitations of Koch’s postulates :
◦ Inability to culture all organisms (large size spirochetes in
periodontitis can not be grown in pure culture.)
◦ Lack of good model system :host range in restricted to humans or to
animal species in which human diseases cannot be reproduced (p.
gingivalis does not usually coloninize in animals.)
◦ The actual state of periodontal disease progression can be difficult to
◦ The same clinical sign and symptoms my be produce by several
organisms which may give rise to variety of clinical disease
◦ Thus the inherent problem with koch’s postulates in
showing causality is the primarily concern with the infecting
agent and lack of consideration for the environment or the
influence of the host in disease development.
Socransky’s criterion :
Sigmund Socransky, a researcher at the forsyth dental center
in Boston, proposed criteria by which periodontal
microorganisms my be judge to be potential pathogens.
Association with the lesion : association is the 1st
requirement for a microbe to cause a periodontal lesion.
Pathogen should be associated with disease, as evident by inc. in
the no. organisms at diseased sites.
Elimination of suspected organism : organism should be
eliminated or decreased in sites that demonstrate clinical
resolution of disease with treatment.
Host response to organisms: microbe should be able to
demonstrate a host response, in the form of an alteration
in the host cellular or humoral immune response.
Animal studies : pathogen should be capable of causing
disease in experimental animal models.
Virulence factor : potential pathogens should be
capable of producing virulence factor which cause
destruction of the periodontal tissues.
It has been estimated that nearly 700 bacterial taxa,
phylotypes and species, which show some structural
organization in the biofilms (Patridge et al.1985), can
colonize the oral cavity of humans, although it remains
unclear how this multitude
of bacteria compete,
coexist, and or synergize
to initiate chronic and
with periodontal health
The term “virulence” is generally defined as the relative ability of a organism
to cause disease or to interfere with a metabolic or physiologic function of its host.
The word is derived from the Latin,‘virulentus’, or ‘full of poison’.
Vir is a latin word meaning strength.
In this context it means "very destructive". Humans are the most virulent
organism ever produced on this planet.
Thus virulence refers to the ability of a microbe to express pathogenicity
(eg; virulent), which is contrasted with nonpathogenic or avirulent organisms.
The absence of a susceptible host renders a ‘potentially’ virulent microbe,
As early as 1924, Kolmer attempted to define virulence as characteristic
comprising at least two intrinsic microbial factors, toxicity, and aggressiveness,
or the ability to invade a susceptible host.
Poulin & Combs defined the concept of virulence in terms of the type of
molecules being produced by the microbe.
As such, they defined virulence in terms of ‘virulence factors’, that is,
components of a microbe which when present harm the host, but when
absent (i.e. mutation) impair this ability. This mutation does not affect the
ability of the microbe to grow and reproduce.
Thus virulence is not a separate property of the microbe, but is a complex
interaction between the microbe and its host; this interaction being dependent
upon many extrinsic factors of the environment.
Virulence factors can have a multitude of functions:
The ability to induce microbe-host interactions (attachment)
The ability to invade the host
The ability to grow in the confines of a host cell
The ability to evade/interfere with host defenses
Virulence is defined as:
Property of invasive power [H Zinsser. 1914]
Strength of the pathogenic activity [WW Ford 1927]
Relative capacity to enter and multiply in a given host [T. Smith.
Pathogenicity: the capacity of a microbe to produce disease
[Watson-DW Brandly 1949 ]
Synonym for pathogenicity: the capacity of a microbe to cause
disease [Youmans, GP., Paterson, PY Sommers, HM.
Degree of pathogenicity [Wood-WB and Davis-BD. 1980]
Relative capacity to overcome available defenses
[Sparling PF. 1983]
Percent of death per infection [Ewald, PW. 1993]
Disease severity as assessed by reduction in host fitness
postinfection [Read AF. 1994]
Measure of the capacity to infect or damage a host [Lipsitch
M Moxon ER. 1997]
Relative capacity to cause damage in a host [Casadevall A
Virulence factor is defined as:
Microbial products that permit a pathogen to cause
disease [H. Smith. 1977]
A component of a pathogen that when deleted specifically
impairs virulence but not viability [Youmans, GP.,
Paterson, PY, Sommers, HM. 1975]
A component of a pathogen that damages the host; can
include components essential for viability including modulins
[Casadevall A Pirofski LA1999, Henderson
B; Poole S;Wilson M 1996]
Important to the role of these prokaryotes in attacking
the host is the ability of several of them to directly attack
host tissues by proteolytic digestion, as well as their ability
to elaborate large amounts and types of “virulence
outer membrane proteins
Which act both directly and indirectly through the
activation of a variety of macromolecules that themselves are
destructive to the host.
The elaboration of several of these virulence factors
appears to be closely regulated by the expression of host
factors (i.e., hemin) that appear in several in vivo animal
models of pathogenesis to control the virulence of the
specific microbial species.
For the most part the establishment of a bacterial
infection whether in the respiratory, alimentary, urogenital
tract, or in the oral cavity (associated with hard and soft
tissue surfaces) requires at least five integrated events:
(1) an initial colonization of the tissue surface;
(2) penetration of this surface either directly or indirectly;
(3) emergence and multiplication of the invading bacterium in
(4) the eventual damage of the host's tissues; and
(5) the survival of the invading bacterium in the ecological
niche by evasion of the host's defense mechanisms.
The ability to adhere to host cells and resist physical removal
or the establishment of pathogen at the appropriate portal of
Pathogens usually colonize host tissues that are in contact
with the external environment.
Virulence factor that promote
Using pili (fimbriae) to adhere to host cells
Using adhesins to adhere to host cells
Using biofilms to adhere to host cells
The ability of a pathogen to invade tissue
1. Mechanisms for colonization (adherence and initial
2. Production of extracellular substances (“invasins”) that
promote the immediate invasion of tissues
3. Ability to bypass or overcome host defense mechanisms
which facilitate the actual invasive process.
The identification of pathogen (s) of an infectious disease, including
periodontal diseases, leads inevitably to the question ‘how do these
organisms cause the disease?’
By far the greatest number of studies that have sought virulence
factors of known or presumed periodontal pathogens have
examined factors produced by P. gingivalis.
Holt and Ebersol 2000: have provided a global overview of the
microbial factors that are thought to cause virulence in bacterial
infections and have described specific examples of such factors that
are produced by periodontal species, in particular, the “Red
Complex” species P. gingivalis,T. forsythia, andT. denticola
The classic progression of the development of periodontitis with its
associated formation of an inflammatory lesion is characterized by
a highly reproducible microbiological progression of a Gram-
positive microbiota to a highly pathogenic Gram-negative one.
Among these "putative periodontopathic species" are members of
While members of the genera Actinomyces and Streptococcus may not
be directly involved in the microbial progression, these species do
appear to be essential to the construction of the network of
microbial species that comprise the subgingival plaque matrix.
The Gram-negative eubacteria that are now recognized as putative
periodontopathogens have developed a plethora of mechanisms by
which they can invade the host, and evade destruction or
neutralization by the host's defenses and survive in this complex
environment of diverse microbial species that compete and synergize
for survival. 39
These mechanisms include the development of surface slimes and capsules
that function to interfere with host defenses (phagocytosis), as well as
initiate the formation of abscesses.
Essential in many cases to the emergence and survival of a bacterium in a
host environment is its adherence to tissue surfaces.
The periodontal pocket is unique in that it contains four types of
colonizing surfaces: mineralized tissues (cementum and dentin), keratinized
epithelium (oral sulcular epithelium), nonkeratinized epithelium (junctional
epithelium), as well as the surfaces of the eubacteria themselves.
Without a doubt, all of the species that invade or "pass through“ the oral
cavity have access to the periodontal pocket; however, only those
eubacteria that are able to successfully colonize these different surfaces
will survive in this environment.
Adhesion to these surfaces usually (but not always)
occurs by the interaction of thin surface appendages to
specific host receptors, fimbriae and capsules.
The invading bacterium has also developed complex
macromolecules in its surface layer that play an active
role in the infective process.
These macromolecules include lipopolysaccharides, outer
membrane-associated proteins, enzymatic molecules, toxins,
The outer membrane macromolecules are not only
detrimental to host cell integrity and viability, but may
also have a direct effect on the character of the resident
Both the Gram-positive cell wall and Gram negative cell wall
(cell envelope) are complex structures.
The Gram-negative cell envelope consists of a trilaminar outer
membrane and an underlying peptidoglycan layer.
The thin peptidoglycan layer that maintains cell shape is linked
to the outer membrane by a lipoprotein of low molecular
weight through its diaminopimelic acid in the peptide of the
The linkage of the lipoprotein is through an interaction
between the N-terminal lipid of the lipoprotein and the
hydrophobic (i.e., lipophilic) domain of the outer membrane.
Several unique and physiologically essential proteins are
major components of the outer membrane.
These include the porins, integral/matrix proteins, and the
In addition to the possible regulation of bacterial virulence
through the surfaced layer, eubacteria also regulate
virulence by controlling the synthesis of specific outer
membrane proteins (OMPS).
They accomplish this by the action of specific signals from
their immediate growth environment.
Numerous investigators have shown that iron regulates the
expression of unique OMPS that are essential for a variety
of cell functions, including transport and virulence.
McKee et al have shown that the virulence of P. gingivalis
strain W50 as expressed in a mouse abscess model was
regulated by the level of hemin in the growth medium
Excess hemin (i.e., concentrations above 2.5 µg/ml) in the
growth medium resulted in increased P. gingivalis virulence,
while low hemin concentrations (i.e., up to 0.5 µg/ml)
produced cells with decreased virulence.
Bramanti and Holt (unpublished) showed that the
pretreatment of mice with the iron chelating agent Desferal
protected them from lethal doses of bacteria grown in either
limited or excess hemin.
These data provide strong evidence that support the role of
iron and iron availability in the pathogenesis of P. gingivalis.
The majority of the studies have been concentrated
on P. gingivalis.
Unfortunately, many of these studies have involved the
use of cell extracts, and for the most part
interpretation of the results remains unclear since
these extracts contain a variety of bacterial
components in addition to the outer membrane.
In one of the few studies of the biological activity of
the oral Eikenella spp., E. corrodens, Progulske et al1984.
isolated both the LPS and outer membrane and found
both exhibited potent bone resorptive activity
Outer Membrane Proteins
Porphyromonas and Bacteroides
Kennell and Holt and Williams and Holt have made an
exhaustive examination of the distribution of both the omps
and major outer membrane proteins (momps) in a large
number of the oral P. gingivalis strains.
What was evident in their results was that the
distribution of both outer membrane proteins in these P.
gingivalis strains was very complex when compared with, for
example, E. coli. In addition, there was significant strain
Several studies of the cell envelope of F. nucleatum have
revealed that in addition to the usual complement of omps, F.
nucleatum also contained at least two momps in the 40 to 46
One of the few studies of the protein composition of the
cell envelope from A. actinomycetemcomitans was that of
DiRienzo and Spieler.
The cell envelope polypeptides were remarkably
conserved across the strains, with three momps (Env-1, -2,
and -3) being observed.
Progulske and Holt 1984 andWagley et al have examined
the distribution of omps of E. corrodens and observed that the
momps occurred at approximately 33.5 kDa.
Borinski and Holt 1990. have studied the surface of clinical
isolates and ATCC strains of W. recta.
The membrane consisted of three momps with relative
molecular migrations of 43, 47, and 51 kDa calculated by linear
One of the few studies of the chemistry and biology of the
outer sheath of Treponema denticola was that of Masuda and
The outer sheath of the oral treponemes consisted of
tripartate vesicles with a polygonal fine structure in a
photomicrograph of negatively stained Treponema denticola, strain GM-1.The coiled cell is
surrounded by a thin, loose-fitting outer sheath (OS) that encloses a series of endoflagella
(EF) that emerge from each end of the cell.The cytoplasmic region is enclosed in the protoplasmic
cylinder (PS). Note in the inset that the outer sheath is constructed of a
regular hexagonal array of subunits, the components of which probably comprise the
major polypeptide of the sheath, the 64 kDa protein. Bar =100 nm; inset bar = 10 nm.
Structure and Composition
The putative periodontopathic bacteria all release outer
membrane vesicles into their surroundings.
These vesicles are a direct outgrowth or evagination of the
The vesicle has the capacity to entrap the contents of the
periplasmic space (location of the cells numerous bacterial
proteolytic and hydrolytic enzymes), and along with its LPS
and other outer membrane proteins is a formidable
virulence factor. 52
The fact that it is capable of concentrating many
of the hydrolytic, phosphorylytic, and proteolytic enzymes
from the periplasmic space in structures that range in size
between 20 to 500 nm in diameter makes it a significant
potential participant in the progressive events of periodontal
The vesicle could interact with other bacteria or
penetrate into host tissues, resulting in the release of small
peptides that are used by many different oral bacteria for
This, along with its LPS, permits it to find its way
into deeper tissues of the periodontium and other host
tissues where it can elaborate its complement of destructive
enzymes and outer membrane-associated LPS 54
LIPOPOLYSACCHARIDE AND ASSOCIATED
Introduction and structure
The lipopolysaccharide (LPS, endotoxin) has been one
of the most studied of the prokaryotic outer membrane
The cell envelope consists of two basic layers: the outer
membrane and a thin peptidoglycan layer.
The outer membrane contains diffusion pores, matrix
proteins, lipopolysaccharide, and lipoproteins.
The lipoprotein chemically connects the outer
membrane with the peptidoglycan and functions to
stabilize it. 56
Interactions of LPS
Fibroblast and Epithelial Cell Interactions
The LPS appears to be a significant mediator of inflammatory
host responses, especially the stimulation of a variety of
cytokines that are closely linked to the inflammatory
Meikle et al. 1986 have proposed that the destruction of the
periodontium is the result of the interaction of bacterial
products (LPS, outer membrane, etc.) and inflammatory cells.
The outcome of this interaction is the production of IL-1, a
cytokine responsible for the production of a variety of
metalloproteases, lymphocyte differentiation and
proliferation, eicosanoid production, and bone resorption.
Clearly, the outcome of these interactions is tissue damage
and bone destruction
Peptidoglycan as aVirulence
Barnard and Holt1984. have provided the only
observations that the isolated peptidoglycan from
several of the putative periodontal pathogens functions
as a virulence factor.
All of the peptidoglycans produced a dose-dependent
Herpes virus in PDL disease
Since the mid 1990s, herpes viruses have emerged as putative pathogens in
various types of periodontal disease JPR 2000
In particular, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV)
seem to pay important roles in the etiopathogensis of severe types of
progressive periodontitis in adults
localized and generalized aggressive (juvenile) periodontitis
acute necrotitising ulcerative gingivitis
periodontal abscesses and
some rare types of advanced periodontitis associated with medical disorders.
HCMV infects periodontal
monocytes/macrophages and T-lymphocytes, and
EBV infects periodontal B-lymphocytes.
Herpes-virus-associated periodontal sites tend to
harbor elevated levels of periodontopathic
bacterisa, including P. gingivalis,T. forsythia,T.
denticola, Prevotella intermedia,A.A. comitans.
PATHOGENESIS OF HERPESVIRUS ASSOCIATED PERIODONTAL DISEASE
Physiological virulence of commensal opportunistic
P. gingivalis appears to play a significant role in the progression
of chronic periodontitis.
In fact, Darveau et al1997, classified this small, gram-negative,
black-pigmented anaerobe as a bona fide periodontal
Specifically, P. gingivalis has been shown to be capable of
adhering to a variety of host tissues and cells, and to invade
these cells and multiply.
To accomplish this, P. gingivalis employs several bacterial
components: fimbriae, proteases, hemaglutinins, and
P. gingivalis is capable of coaggregating with Actinomyces
naeslundii, Streptococcus gordonii, and S. mitis. Various other
oral streptococci can also coaggregate with P. gingivalis
Namikoshi et al 2003, have shown that a 40 kDa outer
membrane protein from P. ginigvalis is critical for
coaggregation with s. gordinii.
Coaggregation of P. gingivalis with both S. oralis and S.
gordonii is inhibited by chlorhexidine and hydrogen
P. gingivalis effect changes in host cell
structures/functions, and bacterial macromolecular
responses appear to contribute substantially to the
success and breadth of P. gingivalis as periodontal
pathogen across human population.
T. denticola are rapidly motile, obligatory anaerobic Gram-
negative bacteria and have been estimated to account for
approximately 50% of the total bacteria present in a
During periods of oral health the number and distribution
of these types of bacteria are low or nearly undetectable.
However, during gingivitis and the progression to
periodontitis there is large increase in the number,
proportion of the total population, and diversity of species.
Armitage et al was one of the first to establish a positive
relationship between the percentage of spirochetes in
periodontal pockets and clinical measures of inflammation,
increased dental plaque, increasing gingival exudate, bleeding
on probing, periodontal pocket depth, and the loss of
connective tissue attachment.
T. denticola interacts with other oral bacterial species,
notably P. gingivalis and F. nucleatum.
This coaggregation likely plays a role in the progression of
PDL disease (i.e. biofilm formation) Kigure et al 1995
T. denticola possesses several peptidases associated with its
A prolyl-phehnylalanine specific protease appears to be
important in T.denticola virulence
Uitto et al 1986: called this protease a chemotrypsin-like
This spirochete displays numerous properties that enhance
its ability to interact with host tissue and contribute to
changing the inflammatory environment of infected
They are the third member of the ‘Red Complex’ as detailed
by Socransky et al 1998:
Tanner et al described it as a gram negative anaerobic
fusiform isolated from the human oral cavity.
It was frequently isolated along with P. gingivalis from cases of
active chronic periodontitis Grossi et al 2001 and has been
associated with severe periodontal disease.
T. forsythia colonization has been associated with Dialister
Pneumosintes, a new periodontal putative pathogen, JPR 2002
particularly in older individuals with destructive periodontal
A. actinomycetemcomitans armamentarium of virulence factors
ensures its survival in the oral cavity and enables it to promote
Factors that promote A. actinomycetemcomitans colonization
and persistence in the oral cavity include adhesins, bacteriocins,
invasins and antibiotic resistance.
It can interact with and adhere to all components of the oral
cavity (the tooth surface, other oral bacteria, epithelial cells or
the extracellular matrix).
The adherence is mediated by a number of distinct adhesins
that are elements of the cell surface (outer membrane proteins,
vesicles, fimbriae or amorphous material). 70
A. actinomycetemcomitans enhances its chance of colonization
by producing actinobacillin, an antibiotic that is active against
both streptococci and Actinomyces, primary colonizers of the
A. actinomycetemcomitans resistance to tetracyclines, a drug
often used in the treatment of periodontal disease, is on the
Periodontal pathogens or their pathogenic products must be
able to pass through the epithelial cell barrier in order to reach
and cause destruction to underlying tissues (the gingiva,
cementum, periodontal ligament and alveolar bone).
A. actinomycetemcomitans is able to elicit its own uptake into
epithelial cells and its spread to adjacent cells by usurping
normal epithelial cell function.
Once the organisms are firmly established in the gingiva, the
host responds to the bacterial onslaught, especially to the
bacterial lipopolysaccharide, by a marked and continual
inflammatory response, which results in the destruction of
the periodontal tissues.
A. actinomycetemcomitans has at least three individual
factors that cause bone resorption (lipopolysaccharide,
proteolysis-sensitive factor, and GroEL), as well as a number
of activities (collagenase, fibroblast cytotoxin, etc.) that elicit
detrimental effects on connective tissue and the extracellular
It is of considerable interest to know that A.
actinomycetemcomitans possesses so many virulence factors,
but unfortunate that only a few have been extensively
VIRULENCE FACTORS AND HOST RESPONSE
Neutrophil-Mediated Host Response to Porphyromonas gingivalis:
Alpdogan Kantarci andThomas E.Van Dyke
Virulence factors of Porphyromonas gingivalis such as the
gingipains, fimbrillin peptides, capsule polysaccharides,
lipopolysaccharides, haemagglutinating and haemolysing
activities, toxic products of metabolism, outer membrane
vesicles, and other enzymes have important roles in eliciting
host responses in various ways.
These factors significantly affect epithelial/endothelial cells, but
their major effect is observed on the modulation of
Periodontitis represents an important model for neutrophil-
mediated host tissue injury.
In this model, neutrophils, primed or stimulated by the
presence or persistence of infection, express an elevated and
This, in turn, leads to tissue destruction mediated by
It is essential to understand the mechanisms underlying the
interactions between the neutrophils and the microbial
virulence factors to be able to develop rational, novel
Bacterial pathogens involved in periodontal diseases exert a
part of their destructive effect by triggering and inducing
host cells to elevate their secretion of MMPs.
Pathogen-secreted phospholipase (PLC) is one bacterial
product that may trigger this host response. Ding et al1995
Bacterial PLC may induce degranulation of PMN, MMPs and
increase MMP expression in oral epithelial cells.
The released proteases can be converted into active form
by the proteases of plaque bacteria.
Thereby, the pathogenic oral bacteria may indirectly
participate in the destruction of periodontal tissues
Periodontal disease: bacterial virulence factors, host
response and impact on systemic health – Graves et al
Teeth are coated with a biofilm that contains periodontal
Pathogens express virulence factors which enable them
to invade and replicate within epithelial cells and to
invade the underlying connective tissue.
This stimulates production of prostaglandins and
cytokines that induce tissue loss.
In addition, these bacteria have the potential to modulate
the course of systemic diseases such as atherosclerosis
and to contribute to low birthweight and preterm labor.
Detection of important virulence factors by using the host’s
It has recently been recognized, that many pathogens express
virulence genes only when they are in their human or animal
The virulence factors that were expressed only when in the
host were consistently being missed by traditional methods.
Periodontitis is a disease attributable to multiple infectious
agents and interconnected cellular and humoral host immune
However, it has been difficult to unravel the precise role of
various putative pathogens and host responses in the
pathogenesis of periodontitis.
Even though specific infectious agent are of key importance in
the development of periodontitis, it is unlikely that a single
agent or even a small group of pathogens are the sole cause
or modulator of this heterogeneous disease.