bacterial etiology in periodontium

Evidence of bacterial etiology:
A historical perspective
PRESENTED BY:
SONAL GOYAL
2ND YR POSTGRADUATE
DEPARTMENT OF PERIODONTOLOGY

Contents
 Introduction
 Evidence for a primary role of bacteria in the etiology of
periodontal disease
 Historical perspective
 The decline of interest in microorganisms
 Changing concepts of the microbial etiology of periodontal disease
 Conclusion
 References

Introduction
• Bacteria inhabit the oral cavity from birth to death.
• It is estimated that between 300 and 400 different species are
capable of colonizing the mouth, and any individual may
typically harbor 150 to 200 different species.
• Counts in subgingival sites range from about 10³ in healthy,
shallow sulci to > 10 in deep periodontal pockets.

• In general, these organisms live in harmony with the host, but
in certain circumstances a select group of organisms have the
potential to cause overt disease, including dental caries and
periodontal diseases.
• The number of species that, for example, can initiate destructive
periodontal diseases is limited to perhaps as few as 10 or as
many as 30 species.
Sigmund S, Haffajee AY. Evidence of bacterial etiology a historical perspective. Periodontol
2000 1994;5:7-25.

The tooth provides a surface for the colonization of a diverse
array of bacterial species.
Bacteria may attach to the tooth itself, to the epithelial surfaces
of the gingiva or periodontal pocket, to underlying connective
tissues, if exposed, and to other bacteria attached to these
surfaces.
In contrast to the outer surface of most parts of the body, the
outer layers of the tooth do not shed, and thus microbial
colonization (accumulation) is facilitated.
Thus, a situation is set up in which microorganisms colonize a
relatively stable surface, the tooth, and are continually held in
immediate proximity to the soft tissues of the periodontium.
This poses a potential threat to these tissues and indeed to the
host itself.

Factors such as pH,
Eh and proteolytic
enzymes can affect
the performance of
host defense
mechanisms.
In addition, the
tooth provides
sanctuaries in which
microorganisms can
hide, persist at low
levels during
treatment and then
re-emerge to cause
further problems.
Bacteria in dentinal
tubules, flaws in the
tooth or areas
demineralized by
bacteria are not
easily approached
by the much larger
host cells.
In a similar fashion,
noncellular host
factors must face
diffusion barriers,
lytic enzymes and
absorption by the
mineral structure of
the tooth.
Slots J. Periodontology: past, present, perspectives. Periodontol 2000 2013; 62:7-19.

Mechanical debridement other than
vigorous removal of tooth material
cannot reach organisms in the tooth.
Chemotherapeutic agents also have
difficulty in reaching the species.
In particular, antimicrobial agents that
require the bacteria to multiply would
be adversely affected, since the rate of
growth is suspected to be low.

Evidence for a primary role of bacteria in
the etiology of periodontal disease
I. Acute periodontal infections
• The acute phase of certain human forms of periodontal disease,
such as acute necrotizing ulcerative gingivitis (or Vincent’s
infection or trench mouth)
• It can be alleviated by any of a number of antibiotics, clearly
indicating that microorganisms are the etiological agents of this
periodontal disease.

• In acquired immunodeficiency syndrome:-
• This form of periodontal disease has been termed necrotic
periodontitis and has been shown to be controlled by local
irrigation with antiseptic agents, antiseptic mouthwashes and
systemically administered antibiotics

II. Relation of plaque levels to gingivitis and periodontitis
There is a positive correlation between the amount of bacterial
plaque and the severity of gingivitis and amount of bone loss
• Loe et al. demonstrated that intensive plaque control procedures
essentially eradicated clinical gingivitis. The withdrawal of such
procedures resulted in an increase in bacterial plaque, which was
soon followed by the development of gingivitis. Clinical
gingivitis could also be controlled by means of antibiotics or
antiseptic agents such as chlorhexidine.
2000 1994;5:7-25.

Lindhe & Nyman and
Rosling et al.
demonstrated that the
progress of destructive
periodontitis could be
halted and partially
reversed by surgical
procedures accompanied
by twice-monthly
professional tooth
cleaning.
The Aarhus and
Gothenburg studies
indicated that the
suppression of the total
microbiota could be
effective in controlling
both gingivitis and
destructive periodontitis
in humans.

III. Efficacy of antibiotics in the treatment of periodontitis
Antimicrobial agents have been successfully used for the
treatment of localized juvenile periodontitis adult periodontitis,
refractory periodontitis, recurrent periodontitis and, necrotic
periodontitis.
Although antibiotic treatment was successful in many of the
patients in the different disease groups, specific treatments were
not successful in all subjects, since there are differences in
etiological agents even within the groups outlined above.

• The success of antibiotic therapy in controlling various forms
of periodontitis, the acute phase of acute necrotizing
ulcerative gingivitis and necrotizing periodontitis underlines
the etiological role of microorganisms in these forms of
human periodontal disease.
2000 1994;5:7-25.

IV. Host immune response
If periodontal diseases are caused by microorganisms, it might be
expected that the host would mount an immune response to the
offending species.
Numerous studies indicate that subjects with destructive periodontal
diseases show an elevated serum antibody response to specific
subgingival organisms.
In addition, there appears to be a relationship between responses to
specific organisms and the nature of the periodontal syndromes.

V. The contribution of mechanical irritants
• mechanical irritants such as calculus, overhanging fillings and ill-fitting
dentures played a major role in the pathogenesis of destructive
• When certain microorganisms were introduced into germ-free animals
with calculus formation, accelerated periodontal destruction occurred
• studies of ligature induced disease in rats, beagles and monkeys
demonstrated that inflammatory changes and loss of periodontal
attachment occurred in direct proximity to massive accumulations of
bacteria.

VI. Pathogenic potential of plaque bacteria
• Human bacterial plaque has demonstrated pathogenic potential
when implanted into extraoral sites such as subcutaneous
destructive abscesses in humans
• A number of toxic products can be detected in dental plaque,
including endotoxins , cell wall mucopeptides, fatty and organic
acids, H,S, NH, indole, amines and leukotoxin.
2000 1994;5:7-25.

• In addition, enzymes have been shown to be produced by whole
plaque or individual microorganisms from plaque that can be
demonstrated to hydrolyze a wide variety of tissue constituents.
• Finally, it must be pointed out that the bacterial masses that
accumulate at or in the gingival sulcus possess an array of
antigens and possibly polyclonal activators capable of
triggering sequences of host-mediated events that have been
postulated as mechanisms of tissue destruction.

VII. Studies in experimental animals: rodents
• Studies in experimental animals have added considerable support to
the hypothesis of the significant role of microorganisms in the
etiology of periodontal disease.
• The use of antibiotics or antiseptics in a variety of animal model
systems controls the soft tissue pathology and most of the hard
tissue destruction in these animals

Periodontal disease can be transmitted from an animal harboring
the disease to one initially free of it by caging diseased and
disease-free animals together or by the implantation of plaque or
specific microorganisms derived from the plaque of diseased
animals.
In addition, certain microorganisms isolated from human
periodontal pockets can initiate periodontal destruction in several
animal model systems

VIII. Studies in experimental animals: dogs and primates
Studies in the beagle and monkey have indicated that daily
removal of bacterial plaque can prevent the onset of gingivitis
and destructive periodontal diseases
Antibiotic or antiseptic administration without mechanical
debridement also controlled gingivitis and retarded attachment
loss

Placement of a ligature in subgingival sites of beagles and
monkeys resulted in massive plaque accumulation,
inflammation, pocketing and loss of the attachment apparatus,
including the alveolar bone
These events occur rapidly, usually within 60 days, and the
attachment loss can be prevented by the administration of
systemic tetracycline
2000 1994;5:7-25.

IX. Studies in experimental animals: immunization
• If bacteria are etiological agents of periodontal diseases, then
immunization against the whole organism or components of the
cell might be expected to alter the level of periodontal
destruction in humans and experimental animals.
• Several studies indicated that immunization by organisms or
their constituents diminished the severity of periodontal disease,
particularly in rats monoinfected with P gingiualis or in ligature-
induced disease in monkeys

bacteria are important in the etiology of disease but that the
outcome, protection or tissue damage, is affected by the nature
and level of the immune response
The question is no longer whether microorganisms cause
periodontal diseases but rather which specific organisms are
responsible for which specific disease forms.
2000 1994;5:7-25.

Historical perspective
Although the majority of
evidence cited was developed
in the last 30 years, the search
for the etiological agents of
periodontal diseases has been
in progress for over a century.
The search started in the
golden age of microbiology
(about 1880-1920), when the
etiological agents of many
medically important infections
were determined.

The early search:
• Investigators in the period from 1880 to 1930 suggested 4
distinct groups of microorganisms as possible etiological agents;
amebae, spirochetes, fusiforms and streptococci
• Although a greater variety of improved techniques are available,
different techniques can emphasize the importance of different
organisms.
Heaton B, Dietrich T. Causal theory and etiology of periodontal diseases. Periodontol 2000
2012;58:26-36.

The identification of a
suspected pathogen was
heavily influenced by
the nature of the
techniques available.
The major
techniques at that
time were wet
mount or stained
smear microscopy
and limited cultural
techniques.
The different
techniques
suggested different
etiological agents

Amebae
• Certain groups of investigators used stained smears to seek amebae
in bacterial plaque.
• They found higher proportions of amebae in lesions of destructive
periodontal diseases than in samples taken from sites from healthy
mouths or mouths with gingivitis
2012;58:26-36.

Other approaches used agents such as emitin administered
systemically or locally
Local therapies included the use of dyes or other antiseptic
agents to decrease the numbers of amebae in the oral cavity.
On the basis of this association with destructive lesions, they
suggested therapies directed at controlling amebae.

The role of amebae in periodontal disease was questioned by some
authors, because amebae were found in sites with minimal or no
disease and could not be detected in many sites with destructive
disease and because of the failure of emitin to ameliorate the signs
of the disease
2000 1994;5:7-25.

Spirochetes
• spirochetes may be etiological agents of destructive periodontal
disease.
• Therapies were proposed that sought to control disease by the
elimination or suppression of these microorganisms.
• In a classic example of this type of approach, Kritchevsky & Seguin
proposed the systemic administration of neosalvarsan (compound
606), an arsenic-containing antimicrobial preparation, coupled with the
use of subgingival scaling to control destructive periodontal disease.

• The investigators cited and others claimed success in
controlling advanced destructive periodontal disease by
combining local and systemic therapy. Others questioned the
relationship of spirochetes to periodontal diseases
2000 1994;5:7-25.

Fusiforms
• The third group of organisms which were frequently suggested
to be etiological agents of destructive periodontal diseases
including Vincent’s infection were the fusiforms.
• These organisms were originally recognized on the basis of
their frequent appearance in microscopic examination of
subgingival plaque samples.

Streptococci
• The fourth group of microorganisms proposed as etiological
agents of periodontal diseases in this era were the streptococci.
• These microorganisms were proposed on the basis of cultural
examination of samples of plaque from subgingival sites of
2012;58:26-36.

• The selection of the streptococci may have been predicated on the fact
that cultural techniques at that time were much less effective than the
cultural techniques available today.
• Currently, more than 70% of the microorganisms enumerated
microscopically can be cultivated from supra- and subgingival plaque
samples.
• Thus, when samples were taken from destructive periodontal lesions,
only the organisms that were most easily cultivated would consistently
be detected.

Vaccines
• For the first 3 decades of the twentieth century, physicians and
dentists commonly used vaccines in attempts to control bacterial
infections.
• Three types of vaccines were used for the control of periodontal
diseases.
• These included vaccines prepared from pure cultures of streptococci
and other oral organisms, autogenous vaccines and stock vaccines
such as Van Cott’s vaccine, Goldenberg’s vaccine or Inava
Endocorps vaccine
2012;58:26-36.

Inava Endocorps was made up of a
mixture of 7 organisms including
Staphylococcus aureus, Escherichia coli
and Klebsiella pneumoniae.
Goldenberg’s vaccine was composed of
streptococci, staphylococci, enterococci,
Micrococcus tetragenus, Friedlander’s
bacillus and other species and was
designed to be injected at the lesion site.
Typically, 1-3 drops were injected
slowly, each containing about 109
bacteria.

Autogenous vaccines were prepared
from the dental plaque of patients with
destructive periodontal diseases.
Plaque samples were removed from the
diseased site (sometimes extracted root
tips were used), “sterilized” by heat
and/or by immersion in iodine or
formalin solutions and then re-injected
into the same patient, either in the local
periodontal lesion or systemically

• Proponents of all 3 techniques claimed great efficacy for the
vaccination methods used, whereas others using the same
techniques were more skeptical
2000 1994;5:7-25.

Other forms of therapy directed against oral microorganisms
• The difficulty in controlling microorganisms in the absence of
specific antimicrobial agents gave rise to a series of rather
remarkable treatment procedures.
• For example, ultraviolet light was widely used to attempt to
control the oral microbiota and to improve the well-being of the
local tissue

Dental practitioners used electrochemical techniques
caustic agents such as phenol, sulfuric, trichloracetic or
chromic acids, nascent copper and castor oil soap
(sodium ricolineate) and even radium was used to
combat root canal infections
The recent interest in controlling the epithelium to
maximize reattachment had antecedents in this era.
One technique that appears to have been common was
the use of sodium sulfide to “dissolve” the epithelial
lining of the pocket and permit reattachment

Invasion - the early years
• One of the more interesting phenomena of research is the fact that
research workers keep rediscovering the same phenomena in a
cyclical fashion.
2000 1994;5:7-25.

Treatment of periodontal diseases
using systemic or local
antimicrobial agents as well as
mechanical debridement was
common in the late 1800s and
early 19OOs, rare in the middle
of the twentieth century and then
rediscovered in the 1970s.
In a similar fashion, invasion of
the periodontal tissues by
bacteria was thought to be
important in the pathogenesis of
periodontal diseases in the early
19OOs, forgotten and then
rediscovered

Invasion of spirochetes deep into the lesions of Vincent’s angina
were clearly documented
It was thought that the spirochetes moved into the connective
tissues first and were followed by fusiform-shaped species.

The decline of interest in microorganisms
• The initial enthusiasm for the hunt for the etiological agents of
destructive periodontal diseases subsided slowly, and by the
mid-1930s virtually no workers were involved in this quest.
• This state was eloquently described by Belding & Belding in
the aptly titled “Bacteria-dental orphans”
2000 1994;5:7-25.

During the period from the mid-1920s to the early 1960s, the
attitude toward the etiology of periodontal disease changed.
In the first 2 decades of this period, it was thought that
periodontal disease was due to some constitutional defect on
the part of the patient, to trauma from occlusion, to disuse
atrophy or to some combination of these factors
Bacteria were thought to be merely secondary invader in this
process or, at most, contributors to th inflammation observed
in periodontal destruction

Nonspecific plaque hypothesis
• Treatment of patients based on the notion of constitutional defects
or trauma from occlusion was not effective in controlling
• Clinicians recognized that plaque control was essential in the
satisfactory treatment of periodontal patients.

• During the late 1950s, a group of clinicians, sometimes
referred to as plaque evangelists, heavily emphasized the need
for plaque control in the prevention and treatment of
• Thus, once again bacteria were thought to play a role in the
etiology of destructive periodontal disease, but as nonspecific
causative agents.
2012;58:26-36.

According to this nonspecific plaque
hypothesis, any accumulation of
microorganisms at or below the gingival
margin would produce irritants, leading to
inflammation.
The inflammation, in turn, was responsible for
the periodontal tissue destruction.
The specific species of microorganisms that
accumulated on the teeth was not considered
to be particularly significant, providing that
their numbers were sufficiently large to trigger
a destructive process.

Mixed anaerobic infections
• Beginning in the late 1920s, a series of oral and medical
microbiologists began to work on a variant of the hypothesis of
nonspecific infections of periodontal disease.
• These investigators believed that periodontal disease was the
result of mixed infections.

• This hypothesis had been considered since the late 1800s, when
microscopic observations by Vincent in France suggested that
certain forms of periodontal disease, particularly acute
necrotizing ulcerative gingivitis were due to a complex of
microorganisms dominated by fusiforms and spirochetes.
• This group of infections were known as fusospirochetal
infections

• Macdonald et al. were able to produce transmissible mixed infections
in the guinea pig groin using combinations of pure cultures.
• The critical mixture of 4 organisms included a Bacteroides
rnelaninogenicus strain, a gram-positive anaerobic rod and 2 other
gram-negative anaerobic rods.
• This combination of organisms was completely different from those
used by Smith and Proske & Sayers to cause transmissible infections.
• These results led to the concept that mixed infections might be
bacteriologically nonspecific but biochemically specific.

• Any combination of microorganisms capable of producing an
array of destructive metabolites could lead to transmissible
infections in animals and, by extension, to destructive
periodontal infections in humans.
• Later, experiments suggested that members of the B.
Meluninogenicus group were the key species in these infections.
• Other organisms could be substituted for the gram-positive and
gram-negative rods as long as the B. rnelaninogenicus strains
were present

Transmissibility of hamster periodontal disease
• In the 1960s, interest in the specific microbial etiology of
periodontal disease was rekindled by 2 groups of experiments.
• Keyes & Jordan demonstrated that periodontal disease could be
transmitted in the hamster from animals with periodontal
disease to animals without periodontal disease by caging them
together.
2000 1994;5:7-25.

• They then demonstrated that swabs of plaque or feces from
diseased animals were effective in transmitting the disease to
animals free of disease.
• Finally, it was demonstrated that a pure culture of an organism
that later became known as Actinomyces viscosus was capable
of causing destructive periodontal disease in animals free of
disease
• Other species isolated from the plaques of hamsters with
periodontal disease did not have this capability.

Invasion in acute necrotizing ulcerative gingivitis
• At about the same time, Listgarten demonstrated that
spirochetes could be detected in practically pure cultures in the
lesions of acute necrotizing ulcerative gingivitis both in the
connective tissue and within the adjacent epithelium.
2000 1994;5:7-25.

• The spirochetes that invaded these lesions were relatively
homogeneous with respect to cell size, the ultrastructural
characteristics of their outer cell envelope and the number of
axial fibrils (endoflagella) observed in thin sections.
• Such findings suggested that there might be more specificity
to the microbial etiology of periodontal disease than had been
accepted for the previous 4 decades.

The emphasis in the 1960s was on the
mechanical control of plaque
accumulation.
This approach was consistent with the
prevailing concept that periodontal disease
was due to a nonspecific accumulation of
bacteria on tooth surfaces.
This concept is very much in evidence
today and still serves as the basis of
preventive techniques in most dental
practices.
It is also clear that nonspecific plaque
control is not able to effectively prevent
all forms of periodontal disease.

Copious plaque-forming microorganisms as etiological agents
of periodontal disease
• The findings of Keyes and Jordan stimulated a new concept of
• The organisms responsible for the destruction observed in the
hamster clearly differed from other organisms by their ability to
form large amounts of bacterial plaque both in the hamster and in
in vitro test systems

Parallel studies of Streptococcus mutans, a prime
etiological agent of dental caries, demonstrated that it
too was capable of forming large amounts of plaque
in the hamster model system as well as large amounts
of plaque, dental caries and, most importantly,
periodontal destruction in gnotobiotic rat systems
Thus, a concept emerged that microorganisms that
were capable of forming large amounts of plaque in
vivo and in vitro should be considered as prime
suspects in the etiology of periodontal diseases.
In vitro test systems were devised to seek plaque-
forming microorganisms.

Actinomyces, particularly A. viscosus and
Actinomyces naeslundii, also demonstrated the
property of in vitro plaque formation, but
would do so in the presence of sucrose as well
as many other carbohydrates.
These findings reinforced the concept that
organisms that formed abundant plaque were
responsible for destructive periodontal disease.
Unfortunately, later research findings revealed
major discrepancies in this hypothesis.

Changing concepts of the microbial etiology
of periodontal disease
• By the end of the 1960s, it was generally accepted that dental
plaque was in some way associated with human periodontal
disease.
• It was believed that the presence of bacterial plaque initiated a
series of as yet undefined events that led to the destruction of
the periodontium.
2012;58:26-36.

The composition of plaque
was thought to be
relatively similar from
patient to patient and from
site to site within patients.
Variability was
recognized, but the
true extent of
differences in
bacterial composition
was not appreciated.
It was thought that
the major event
triggering destructive
periodontal disease
was an increase in
mass of bacterial
plaque, possibly
accompanied by a
diminution of host
resistance.

• Indeed, in the mid-1960% the classic studies of Loe et al.
convincingly demonstrated that plaque accumulation directly
preceded and initiated gingivitis.
• Many investigators believed that gingivitis was harmful and
led to the eventual destruction of the periodontal tissues,
probably by host-mediated events.
2000 1994;5:7-25.

•Earlier considerations of plaque suggested that it was a
heterogeneous bacterial mass that led to disease when allowed to
overgrow.
•Recent studies have revealed that a relatively simple microbiota
consisting of coccal and short rod forms is associated with
healthy periodontal sites

Recognition of differences between plaques
• The once prevalent view that dental plaque composition was
reasonably consistent from individual to individual and site to
site is clearly not valid.
• The impression that plaque is reasonably similar in
composition was derived in part from studies of the infectious
potential of plaque when injected subcutaneously into
experimental animals and partly because investigators
examined the microbial composition of pooled dental plaque.

• Difficulties that delayed recognition of the differences in plaque
composition between health and disease were more technical in
nature and included problems in the dispersion of plaque
samples (while maintaining cell viability) and the recovery of
microorganisms that were often oxygen-sensitive or fastidious
in their growth requirements.
2000 1994;5:7-25.

Technical and conceptual advances have permitted a
more realistic view of dental plaque.
Such advances include the routine use of continuous
anaerobiosis techniques, better techniques of sample
taking and dispersion and improvements in culture media
and methods of identifying organisms.
Microbiological sampling from discrete sites and
microscopy of sections of in situ human plaque have
proven to be synergistic approaches that have permitted
a clearer understanding of the localization and patterns of
colonization of possible subgingival pathogens.

Re-introduction of the hypothesis of bacterial specificity in the
etiology of periodontal diseases
• The recognition of differences in the composition of bacterial
plaque from subject to subject and site to site within subjects
led to a series of investigations.

Newman et al. and Slots demonstrated that
the microbial composition of subgingival
plaque taken from diseased sites differed
substantially from the samples taken from
healthy sites in subjects with localized
juvenile periodontitis.
Tanner et al. and Slots demonstrated that the
microbiota recovered from lesion sites from
subjects with adult periodontitis differed
from the microbiota from healthy sites in the
same subjects and also from lesion sites in
localized juvenile periodontitis subjects.

Conclusion
“Those who cannot remember the past are condemned to
repeat it” (Santayana). This warning is particularly relevant
in periodontal research.
The cyclical interest in specific subgingival
microorganisms as the etiological agents of periodontal
diseases was responsible for a long delay in identifymg
these agents and defining methods for their control.
Today, there is a real concern that, once again, researchers
could be swayed away from studies of cause and control
into fashionable but blind alleys.
It is important that researchers in this field not be
sidetracked by lack of immediate gratification and the
influence and comments of groups whose knowledge of the
biology of periodontal diseases is limited.

• Ultimately, it must be recognized that the control of infectious
diseases has inevitably been brought about by the control of
the organisms that caused them.
• The unique anatomical features of periodontal diseases may
make this control somewhat difficult, but the investigator
should not be deterred from the ultimate microbiological
control.

References
1. Sigmund S, Haffajee AY. Evidence of bacterial etiology a
historical perspective. Periodontol 2000 1994;5:7-25.
2. Moore WEC, Moore LVH. The bacteria of periodontal
diseases. Periodontol 2000 1994;5:66-77.
3. Vargas Segura AI, Ilyina A, Segura Ceniceros EP, et al.
Etiology and microbiology of periodontal diseases: A review.
African J microbio res 2015;9:2300-2306

4. Heaton B, Dietrich T. Causal theory and etiology of periodontal
diseases. Periodontol 2000 2012;58:26-36.
5. Oppermann RV, Haas AN, Kuchenbecker C, et al.
Epidemiology of periodontal diseases in adults from Latin
America. Periodontol 2000 2015;67:13-33.
6. Hujoel P, Zena LG, Cunha-Cruz J, et al. Historical perspectives on
theories of periodontal disease etiology. Periodontol 2000
2012;58:153-160.
7. Slots J. Periodontology: past, present, perspectives. Periodontol
2000 2013; 62:7-19.

bacterial etiology in periodontium

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