This document provides an overview of periodontal microbiology. It discusses the historical background of periodontal microbiology research from the 17th century to modern theories. It describes the structure and composition of dental plaque, including its microbial components, extracellular matrix, and classification. The document outlines the process of plaque formation, including initial bacterial adhesion and attachment, colonization, and maturation into a biofilm. It compares the microbiota and characteristics of supragingival and subgingival plaque. Finally, it briefly discusses peri-implant plaque and biofilm formation.

1. Periodontal Microbiology

2. Contents
 Introduction
 Historical background
 Definitions
 Structure and Composition
 Plaque formation
 Growth dynamics
 Physiological properties
 Peri Implant plaque
 Plaque as a Biofilm
 Microbial specificity of periodontal diseases

3. Introduction
 Oral cavity - >500 bacterial species, mostly commensal & beneficial
(Moore 1994)
 Open growth system
 periodontal diseases unusual human infection. major reason anatomic
feature of a mineralized structure, the tooth, partly exposed to the
external environment and partly in the connective tissues
 Teeth & implants – hard non shedding surfaces, continually held in
immediate proximity to the soft tissues of the periodontium
 Provide port of entry

4. Historical background
 Anton leeuwenhoek (1632-1723) – first described
oral bacteria, related lack of oral hygiene to an
increase in the quantity of these organisms. He also
recommended oral hygiene procedures to keep the
gums healthy (use of salt, toothpicks, quill and
cloth).

5.  Adolph Witzel (1882) – Identified bacteria as cause of
periodontal disease
 WD miller (1890) – first true oral microbiologist,
periodontal disease was a mixed infection of non-specific
normal oral flora (non-specific plaque theory) persisted
largely unchallenged for almost 6 decades
 J leon Williams (1897) – described dental plaque
 GV black (1899) – coined term “gelatinous dental
plaque”

6.  Younger 1905 - first prominent clinicians to recognize
periodontal disease as a bacterial infection
 Rosebury et al. (1950), Bibby (1953) - proposed the
concept of infection of the normal flora, based on
overgrowth in certain patients predisposed by systemic
infection or local trauma or were permitted to increase
because of changes in host or local environmental factors
 1950s - Scandinavian school, led by Waerhaug
importance of bacterial plaque in the etiology of
periodontal diseas

7.  Cross sectional studies: Arno 1958, Ash 1964
Epidemiological studies- Russel 1967, schei 1959
positive correlation between the amount of bacterial
plaque and the severity of gingivitis
 longitudinal studies of Loe et al – 1965, 1966 –
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

8.  Newman et al. 1976, 1977 ; Slots 1976 - 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
 Modern theories of specificity (Loesche 1976) evolved
 Biofilm – Costerton 1999
 Ecological plaque hypotheses PD Marsh 2001

9. Definitions
 In 1800`s plaque described by Williams & Black as “Felt
like mass of micro-organisms observed over the surface
of carious human enamel”
 Lindhe – bacterial aggregations on the teeth or other
solid oral structures
 Bowen (1976) – structured, resilient, yellowish-grayish
substance that adheres teneciously to the intraoral hard
surfaces, including removable and fixed restorations.
 WHO - Variable but specific structural entity resulting
from the colonization and growth of microorganisms of
various species and strains embedded in an extra cellular
matrix.

10. Classification

12. Clinical appearance & distribution
 Adheres firmly to underlying surface, not dislodged
by rinsing or use of sprays
 Color – Whitish to yellowish . Pigmentation from
food chromogens
 Newly formed :translucent, clear, non detectable
 As plaque develops and accumalates – visible as
nodular mass, that varies in colour, primarily seen
along gingival margins
 Detection: Two tone dye, Probe/ Explorer

13. Distribution
 Mandible > Maxilla
 Posteriors > Anteriors
 Buccal > Lingual (Maxilla)
 Interdental regions > buccal or oral surfaces
Can be diffrentiated from:
 Materia alba:
 Calculus

14. Microscopic structure:
 Supragingival plaque: Stratified organization,
multilayered accumulation of bacteria. Gm +ve cocci
& short rods- tooth surface. Gm –ve rods, filaments
& spirochetes predominate in outer surface.
Supragingival plaque
-Calculus formation
-Caries
Marginal plaque
-Gingivitis

15.  Subgingival plaque:
 Tooth associated: characterized by gram-positive
rods and cocci, including Streptococcus mitis, S.
sanguis, A. viscosus, Actinomyces naeslundii, and
Eubacterium spp
- In deeper parts of pocket filamentous organisms
become fewer
-The apical border is separated from the junctional
epithelium by a layer of host leukocytes, and the
bacteria show an increased concentration of gram-
negative rods

17.  Tissue associated : more loosely organized, lack definite
intermicrobial matrix
 Contains primarily gram-negative rods and cocci, as well
as large numbers of filaments, flagellated rods, and
spirochetes
 predominance of species such as S. oralis, S.
intermedius, P. micros, P. gingivalis, P. intermedia,
Bacteroides forsythus, and F. nucleatum
 Host tissue cells may also be found
 Composition depends on pocket depth – coronally-
filaments
apically – spirochetes, cocci, rods
 Tooth associated: Calculus formn & root caries
 Tissue associated: Tissue destruction

19. Composition
1. Water: 80-85% plaque mass ;
50% intracellular, 35% matrix
2. Cells: primarily bacteria, 1 gm (wet weight)= 1011
bacteria
Non bacterial: Mycoplasma spp, yeasts, protozoa,
viruses
(conteras 2000)
Host cells : epithelial cells, macrophages & leucocytes
3. Matrix: Organic
Inorganic

20.  Matrix : 20-30% plaque mass
1. Organic:
Carbohydrates: Dextrans, levans, polysaccharides,
galactose
Lipids
Proteins: Albumin, glycoproteins
Misc: cxtracellular bacterial products, cell remnants, food
2. Inorganic:
Calcium
Phosphorus/ phosphate
Na, Cl, F

21. Plaque formation

22.  Formation of the Dental Pellicle
 Forms within nanoseconds
 Contains – glycoproteins (mucins), PRP`s,
Phosphoproteins, Histidine rich proteins, enzymes (α-
amylase)
 Forms by selective adsorption of the environmental
macromolecules (Scannapieco 1990)
 mechanisms involved in enamel pellicle formation
include electrostatic, van der Waals, and hydrophobic
forces

23. Theories of plaque matrix formation
 Spontaneous precipitation
 Iso electric precipitation of salivary protein
 Chemical changes in salivary proteins
 Effect of calcium ions

24.  Initial adhesion & attachment
 No uniform theory
 Van Loosdrecht et al. 1990, Van Loosdrecht &
Zehnder 1990, Busscher et al. 1990. Rutter &
vincent 1984, Busscher 1987, Schei 1994
 4 stages:
1. Phase 1, Transport to the surface
2. Phase 2. Initial adhesion
3. Phase 3. Attachment
4. Phase 4. Colonization

25.  Phase 1, Transport to the surface
 diffusion by Brownian motion (average displacement
of 40 μm/h)
 Convective transport due to liquid flow (several
orders of magnitude faster than diffusion)
 active bacterial movement (chemotactic activity).

26.  Phase 2. Initial adhesion
 bacterium and a surface interact with each other
from a certain distance (50 nm) through long and
short-range forces.
Long range forces:
Van der waals forces
Electrostatic forces

27.  Short-range interactions
 Primary minimum (<2 nm from the surface)
 short range forces (e.g., hydrogen bonding, ion pair
formation, steric interaction, bridging interaction)
 Bacteria initially adhering in the secondary
minimum, may reach the primary minimum by
passing the energy barrier (B), if it is not too high,
but also by bridging this distance by protruding their
fibrils, fimbriae
 The water film between the interacting surfaces has
to be removed. This dehydrating capacity of bacteria

28.  Phase 3. Attachment
 firm anchorage between bacterium and surface
 Bonding is mediated by specific extracellular
proteinaceous components of the organism
(adhesins) and complementary receptors on the
surface. species-specific (Gibbons & Van Houte 1971,
Van Houte1983)
 Adhesins are often lectins which bind to saccharide
receptors, but some adhesins are thought to bind
proteinaceous receptors (Ellen 1985, Gibbons 1989)

29.  S. sanguis binds to PRP`s & other receptors α
amylase, sialic acid (Hsu 1994, Scannapieco 1995)
 A. viscosus fimbriae bind to PRP`s (Gibbons 1988,
Mergenhagen 1987)
 PRP`s undergo conformational change when
adsorbed –cryptitopes – A. viscosus can recognize
(Gibbons 1988)
 Proteases associated with gingivitis may generate
cryptitopes for Gram-negative organisms and destroy
receptors for benign species (Loesche et al. 1987).

30.  Phase 4. Colonization and plaque maturation
 Interactions among different bacterial cell types
 principal feature – Coaggregation - defined as the
specific cell-to-cell recognition that occurs between
genetically distinct cell types
 Occurs by interaction of protein & carbohydrate
molecules located on cell surface & less specific
interactions – hydrophobic, electrostatic, van der
waals forces

31.  F. nucleatum – S. sanguis
 P. loescheii – A. viscosus
 C. ochraceus – A. viscosus
 Streptococci – intrageneric coaggregation

33.  Coaggregation bridges
 formed when the common partner bears two or more
types of coaggregation mediators

35.  Streptococci, actinomycetes – primary colonizers
Prepare favorable environment for secondary
colonizers
 2ndry colonizers – P. intermedia, F. nucleatum, P.
gingivalis do not colonize clean tooth surface, but
bind to bacteria already bound to tooth
(Kolenbrander 1993)
 Later stages – coaggregation btwn different Gm –ve
species seen – F. nucleatum & P. gingivalis or T.
denticola

36.  Corn cob formn – streptococci adhere to filaments of
B. matruchotti or actinomyces. gingivitis (Cisar 1982)
 bristle-brush formn - central axis of a filamentous
bacterium with perpendicularly associated short
filaments, commonly seen in the subgingival plaque
of teeth associated with periodontitis

39.  Schauder et al 2001: autoinducer-2 was a universal
signal mediating messages among the species in
mixedspecies communities
 collection of molecules formed from the spontaneous
rearrangement of 4,5-dihydroxy-2,3- pentanedione
(DPD)
 autoinducer-2 has been detected in the cell-free
culture supernatants of several oral bacteria
suggesting that, indeed, autoinducer-2 might be a
signal exchanged in mixed-species communities.
 commensal oral bacteria respond to low levels of

41. Growth dynamics of dental plaque
 Ultrastructural aspects:
1st 2-8 hours – streptococci saturate pellicle binding
sites, cover 3-30% of enamel surface (Liljemark
1996)
Next 20 hrs – short period of rapid growth
24 hrs – organized ; biofilm
As bacterial densities approach 2-6 million/mm2 ;
marked ↑ in growth rate – 32 million/mm2
Occurs by multiplication of already adhering mos
(Brecx 1983)

42. Supragingival plaque formn- clinical aspects
 Exponential growth curve (Quirynen 1989)
 1st 24 hrs – clinically negligible (<3% tooth surface)
 Around 3 days – rapid growth rate
 After 4 days – avg 30% tooth surface covered
 Increasing time ecologic shift: Gm +ve facultative to
Gm –ve anaerobic spp.
 Night time – plaque growth rate reduced by 50%

43.  Topography of supragingival plaque:
 Initial growth – along gingival margin & interdental
areas
 Surface irregularities
 Surface roughness – retain more plaque; ↑ motile
organisms, spirochetes, denser packing (Quirynen &
Bollen 1995)
Threshold level for surface roughness Ra 0.2 μm
Surface roughness predominates over surface
energy

45.  Individual variables influencing plaque formation:
 Fast & slow plaque formers
 Zee et al 1997 - enamel blocks bonded, 14-day period
with no oral hygiene, day-1 specimens of the 'rapid'
group showed a more complex supragingival plaque
days 3 to 14, during the maturation period of
supragingival plaque, there were no discernible
differences between the two groups
 Zee et al 1996 – rapid plaque formers higher
proportions of Gm –ve rods
 Simonsson 1989 – saliva of light plaque formers

46.  Gingival inflammation:
Plaque formn more rapid on tooth surfaces facing
inflamed gingival margins (Quirynen 1991,
Ramberg 1995)
 Patients age:
Age does not influence plaque formn (Fransson et al
1996, Holm-pederson 1975)

47.  Subgingival plaque formn:
 Examined using microbiota culture, only partial
reduction (108-105), followed by rapid reegrowth to
pretreatment levels (Goodson 1991, Harper 1987)
remaining bacteria considered source (Petersilka
2002)

48. Characteristic Supragingival Subgingival
Gram reaction +/- Dominated by -
Morph types Cocci, branching rods,
filaments, spirochetes
Dominated by rods and
spirochetes
Energy metabolism Facultative with some
anaerobes
Dominated by anaerobes
Energy sources Generally ferment
carbohydrates
Many proteolytic forms
Motility Firmly adherent to plaque
matrix
Adherence less pronounced with
many motile forms
Causes Can cause caries and
gingivitis
Can cause gingivitis and
periodontitis

49.  Metabolic interactions among plaque bacteria

50.  Peri Implant plaque
 Pristine surface
Quirynen et al 2006: a complex subgingival microbiota
established in a ‘pristine’ peri-implant pocket within
1 week
number was lower than that detected for teeth
week 2, this difference disappeared
From week 2 onwards, only minor increases, red and
orange complexes, further increase up to week 13
could be observed

51.  Quirynen 1996 –
 titanium abutments with different surface roughness
 only the two roughest abutments harbored
spirochetes after 1 month
 After 3 months, subgingivally, the composition of the
flora showed little variation
 results indicate that a reduction in surface roughness
(less than a roughness of 0.2 micron) had no major
effect on the microbiologic composition

52.  Peri-implantitis: microbiota comparable to that of
periodontitis
 high proportion of anaerobic Gram-negative rods,
motile organisms and spirochetes
 Complex microbiota that includes conventional
periodontal pathogens.

53. Biofilm
 Bacteria growing in a microbial community adherent
to a surface do not behave the same as bacteria
growing suspended in a liquid environment.
 Potera (1999) -65% of infections that affect the
human are caused by organisms growing in biofilms
 Defn: Matrix enclosed bacterial populations adherent
to each other and/or to surfaces and inter-surfaces
(Costerton 2000)

54. •Initial colonization followed by lateral spread, vertical direction growth
•Shared resources and activities only possible through biofilm
•Protection from other species, host, and harsh environment
•Need communication – quorum sensing, exchange of genetic information

56. Nature Of Biofilms
 Cooperating community of various types of
microorganisms
 Microorganisms are arranged in microcolonies
 Microcolonies are surrounded by protective matrix
 Within the micro colonies are differing environments
 Microorganisms have primitive communication
system

57. Properties of biofilm
 Structure
 microcolonies of bacterial cells (15–20% by volume),
non-randomly distributed in a shaped matrix or
glycocalyx (75–80% volume)
 presence of voids or water channels
 Nutrients diffuse from the water channel to the
microcolony rather than from the matrix.
 At low shear force, the colonies are shaped liked
towers or mushrooms

58. Exopolysaccharides –the backbone of
the biofilm
 bulk of the matrix
 major role in maintaining the integrity of the biofilm
 Some exopolysaccharides are neutral, such as the
mutan from Streptococcus mutans, whereas others
are highly charged polyanionic macromolecules
 exopolysaccharides can be degraded and utilized by
bacteria within the biofilm
 preventing desiccation and attack by harmful agents.

59.  bind essential nutrients such as cations to create a
local nutritionally rich environment
 The quantity of exopolysaccharides in a biofilm does
not necessarily reflect the proportion of the bacterial
species that produce it

60. Physiological heterogeneity within
biofilms
 same microbial species can exhibit extremely
different physiological states in a biofilm
 pH can vary quite remarkably over short distances
 Bacterial cells within biofilms can produce enzymes
such as b-lactamase against antibiotics or catalases,
superoxide dismutases against oxidizing ions
released by phagocytes
 elastases and cellulases, which become concentrated
in the local matrix and produce tissue damage

61. Quorum sensing
 First suggested by Cooper et al. (1995)
 The regulation of expression of specific genes through the accumulation
of signaling compounds that mediate intercellular communication
(Prosser 1999)
 Dependent on cell density
 few cells low levels of signaling compounds. Once the signaling
compounds reach a threshold level (quorum cell density), gene
expression is activated
 Other mechanisms : Conjugation, transformation, plasmid transfer,
transposon transfer
 conferring tetracycline resistance from, Bacillus subtilis, to
Streptococcus species present in dental plaque grown as a biofilm
(Roberts 1999)

62. Mechanisms of increased antibiotic
resistance of organisms in biofilms
 Bacteria growing in dental plaque display increased
resistance to antimicrobial agents ([Marsh and
Bradshaw,1993; Kinniment et al., 1996)
 Biofilm inhibitory concentration (Anwar et al., 1990;
Nichols, 1994)
 biofilm inhibitory concentration for chlorhexidine
300 times greater when S.sobrinus was grown as a
biofilm compared with the minimum bactericidal
concentration of planktonic cells (Shani et al., 2000)

63.  The structure of a biofilm may restrict the
penetration of the antimicrobial agent
 Some charged inhibitors can bind to oppositely
charged polymers that make up the biofilm matrix
(diffusion-reaction theory). Gilbert 1999
 Agent may also bind to and inhibit the organisms at
the surface of the biofilm, leaving cells in the depths
of the biofilm relatively unaffected.
 Transfer of resistance genes can occur more readily
in biofilm communities

64.  Growth on a surface may also result in the drug
target being modified or not expressed in a biofilm
 Bacteria grow only slowly under nutrient depleted
conditions in an established biofilm and, as a
consequence, are much less susceptible than faster-
dividing cells.
 environment in the depths of a biofilm may be
unfavourable for the optimal action of some drugs
(Gilbert et al.2002)
 matrix in biofilms can also bind and retain
neutralizing enzymes ß-lactamase, IgA protease

65.  Benefits of a community lifestyle
 a broader habitat range for growth
 A more efficient metabolism, e.g. complex host
macromolecules can only be degraded by consortia of
oral bacteria
 increased resistance to stress and antimicrobial
agents
 enhanced virulence ‘pathogenic synergism’ Caldwell
et al., 1997; Shapiro, 1998

66.  Association Of Plaque Microorganisms With
Periodontal Disease

68.  Microbial specificity of periodontal diseases
 Nonspecific Plaque Hypothesis
 Specific Plaque Hypothesis
 Ecological plaque hypothesis

69.  Nonspecific hypotheses
 Non specific and specific hypotheses delineated in
1976 by Walter Loesche
 Supported by epidemiologic studies that correlated
pts age & amnt of plaque with evidence of
periodontitis (Russel 1967, lovadal 1958)
 maintains that periodontal disease results from the
"elaboration of noxious products by the entire plaque
flora”
 Control of periodontal disease depends upon plaque

70.  Specific Plaque Hypothesis
 States that only certain plaque is pathogenic, and its
pathogenicity depends on the presence of or increase
in specific microorganisms
 Plaque harboring specific bacterial pathogens results
in periodontal disease
 Recognition of A.a as pathogen in LAgP (Newman
1977, Slots 1979)

71.  Ecological plaque hypothesis
 P D Marsh 2003
 The interactions between the host and the bacteria is
bi directional
 leads establishment of the climax community
 The climax community does not change unless
changes occur in the environment and the host

73.  Key features:
 selection of ‘pathogenic’ bacteria is directly coupled
to changes in the environment
 diseases need not have a specific aetiology; any
species with relevant traits can contribute to the
disease process
 The role in disease of any subsequently discovered
novel bacterium could be gauged by an assessment of
physiological characteristics
 target the putative pathogens directly, e.g. by

74.  ecological perspective
 alter the local environment by reducing the flow of
GCF by the use of antiinflammatory agents
 site could be made less anaerobic by the use of
oxygenating or redox agents (Ower et al., 1995)

75. References
 Carranza’s Clinical Periodontology – 10th edn
 Clinical Periodontology & Implant Dentistry - 4th edi
 Periodontics. The past Microbiology Part (III)- JCP
1985
 Periodontal microbial ecology - Perio 2000,Vol. 38,
2005, 135–187
 Dental biofilms: difficult therapeutic targets -
Periodontology 2000, Vol. 28, 2002, 12–55
 Are dental diseases examples of ecological
catastrophes? - P. D. Marsh - Microbiology (2003),
149, 279–294