1

2
 Contents
 Introduction

 History

 Microbial Biofilm

 Structure of Mature Dental Plaque Biofilm

 Accumulation of Dental Plaque Biofilm

 Factors Affecting Supra-gingival Dental Plaque Formation

 De Novo Subgingival Plaque Formation

 Characterstics of Biofilm Bacteria

 Metabolism of Dental Plaque Bacteria

 Communication between Biofilm Bacteria

 Interactions and Antimicrobial Resistance

3
• Microbiologic Specificity of Periodontal Diseases
• Specific Plaque Hypothesis(1976)

• Non Specific Plaque Hypothesis(1986)

• Ecologic Plaque Hypothesis(1994)

• Keystone Pathogen Hypothesis(2012)

• Criteria for identification of Perio-Pathogens

• Transition From Health to Disease

• Host susceptibility

• Pathogenic Bacteria

• Beneficial Species

• Periodontal Health

• Gingivitis, Chronic Periodontitis, Localized Aggressive Periodontitis,

Aggressive periodontitis, Necrotizing Periodontal Diseases

• Peri-implantitis

4
 Virulence Factors of PerioPathogens

◦ Adhesive surface proteins and fibrils

◦ Factors that promote tissue destruction

◦ Evasion of Host Immunity

 Future Advances in Periodontal Micriobiology

◦ Oral microbiology and Genomics

 Conclusion

 References

5
 INTRODUCTION
 Periodontal diseases are multifactorial infections elicited by a complex of bacterial species that
interact with host tissues and cells causing the release of a broad array of inflammatory
cytokines, chemokines , and mediators, some of which lead to destruction of the periodontal
structures, including the tooth supporting tissues, alveolar bone, and periodontal ligament.

(Stanley C. Holt and Jeffrey L. Ebersole)

 Wide variety in the composition of the sub-gingival microflora.

(van Winkelhoff and de Graaff 1991).

 The environmental influences on plaque composition has led to concepts on disease
prevention that have embraced ecological principles.

(Marsh, 2003)

6
INTRODUCTION(contd)
Establishment of Microflora
 Human fetus is usually sterile.

 Colonization starts at birth.

 Within hours – facultative & aerobic bacteria.

 2nd day – anaerobic bacteria.

 Within 2 weeks – mature microbiota of gut.

(Text book of Oral Microbiology: PD Marsh & MV Martin)

 After weaning - 10¹⁴ microorganisms with 400 different type of microorganisms.

 There are 10 times more bacteria than human cells.

(Carranza’s Clinical Periodontology) – 10th edi.

7
 Streptococcus salivarius and Streptococcus mitis first and most dominant oral
microbes to colonise oral cavity

 Nesisseria , Actinomyces and Staphylococcus are among first colonizers of oral

cavity.

 Species colonizing after teeth eruption S. sanguis, Lactobacillus , S. oralis.

 It is estimated that more than 500 different species are capable of colonizing adult
mouth and one individual are capable of harbouring 150 or more different species.

8
 It has been estimated that nearly 700 bacterial taxa and species, which show some

structural organization in the biofilms ,can colonize the oral cavity of humans,

although still in depth researches , about the multitude of bacteria compete,

coexist, and ⁄ or synergize to initiate this chronic disease process.

9
 Several different types of surfaces are available for bacterial adhesion: soft tissues,
such as mucosae, skin, and cornea, and hard tissues, such as teeth and nails.

 On the basis of physical and morphologic criteria, the oral cavity can be divided into
six major ecosystems (also called niches), each with the following distinct ecologic
determinants:

1. The intraoral, supragingival, hard surfaces (teeth, implants, restorations, and

prostheses)

2. Subgingival regions adjacent to a hard surface, including the periodontal/peri-

implant pocket (with its crevicular fluid, the root cementum or implant surface, and
the pocket epithelium)

3. The buccal, palatal epithelium, and the epithelium of the floor of the mouth

4. The dorsum of the tongue

5. The tonsils

6. The saliva 10
Clinical Periodontology Carranza -11th
edi.
11
HISTORY
 1890 W.D miller published the book “The microorganisms of human mouth”

 J. Leon Williams who stated “dental plaque is a gelatinous accumulation of bacteria and
the obvious association of plaque build up and periodontal disease.

 1880 - 1930 - Golden age of microbiology .

J L WILIIAMS (1852 – 1932)

W D Miller (1853 – 1907)

12
Using different techniques available (wet mounts or stained smear microscopy),
scientists identified 4 potential pathogens –

Amoebae

Spirochetes

Fusiforms

Streptococci

Basis for Specific Plaque Hypothesis

13
 Haffajee and Socransky J
Periodontol 1992; 63:322-331

14
 USES OF VACCINES

 These included vaccines prepared from pure cultures of streptococci, and other oral
organisms, autogenous vaccines, and stock vaccines.

 Administered systemically or locally in the periodontal tissues.

 1920 to 1940 it was thought that periodontal disease was due to some constitutional
defect on the part of the patient, trauma from occlusion, disuse atrophy or some
combination of these factors
(Periodontics, 6th ed. St. Louis: C.V. Mosby: 1987:147-197)

15
 1930-1970 – failed to identify a specific organisms as etiologic agent of periodontal
disease ( Tanner 1978)

 Next 100 years, additional etiologic agents of lung infections were discovered; some
as recently as 1977, when the causative agent of Legionnaire's disease was
discovered. .(Thelaide 1986)

 Studied and specific microbial complexes suggested.

(Slots 1986, Loesche 1976).

 In the mid-1960s, it was suggested that a specific spirochete might be the cause of
acute necrotizing ulcerative gingivitis.
(Listgarten 1965)
Non specific plaque hypothesis was proposed according to gross accumulation of
dental plaque to cause periodontal destruction.

16
 Early 1970s- Drs. Jeffcoat and Goodson; however, taking samples from sites (or
subjects) which are in remission and interpreting them as if they were obtained
from sites undergoing active destruction could lead to erroneous conclusions.
 (J Periodontol 1992; 63:322-331)

Periodontal diseases have been discussed at length. In brief, investigators have

been handicapped by the dual problems of technical difficulty and inadequate
understanding of the biology of destructive periodontal diseases.
( Brenner DJ, Hillman JD 1973)

 It was be faced with discriminating the pathogen(s) from among the 300 to
400 candidate species encountered in subgingival plaque.

17
The classic approach to this problem has been to use the criteria known as
Koch's postulates (1870)
1)Be routinely isolated from diseased individual

2)Be grown in pure culture in laboratory

3)Produce a similar disease when inoculated into susceptible laboratory animal

4) Be recovered from lesions in a diseased laboratory animal.

18
 Socranky Criteria(1978)

1. Be associated with disease, as evident by increases in the number of organisms at

diseased sites

2. Be eliminated or decreased in sites that demonstrate clinical resolution of disease

with treatment

3. Induce a host response , in form of an alteration in host cellular or humoral immune

response

4. Be capable of causing disease in experimental animal models

5. Produce demonstrable virulence factors responsible for enabling the microorgansim

to cause destruction of periodontal tissues.

19
EVOLVING CONCEPT

20
 Current concept of Periodontal Microbiology

◦ a susceptible host,

◦ the presence of pathogenic species, and the absence of so-called "beneficial

bacteria"

◦ Bacterial Interactions

◦ It is generally accepted that the oral biofilm in association with anaerobic bacteria is
the main etiological factor in periodontal disease

◦ Teeth are primary habitat of Perio Pathogens.

◦ Cariogenic species like S. mutans remain restricted to solid surface and is called
Obligate Periphyte.

21
 DEFINITIONS

Materia alba is a White cheese like ,soft accumulations of salivary proteins, some

bacteria ,many desquamated cells and occasionally disintegrating food debris that

lack the organized structure of dental plaque, and it is easily displaced with a water

spray. (Carranza 10th edition)

Calculus is a hard deposit that forms by mineralization of dental plaque, and it is

generally covered by a layer of unmineralized plaque. (Carranza 10th edition)

22
 Dental plaque is defined clinically as a structured, resilient, yellow-grayish substance

that adheres tenaciously to the intraoral hard surfaces, including removable and

fixed restorations (Carranza 10th edition)

 Dental plaque is a specific, amorphous, granular deposit which accumulates on the

surface of teeth ,dental restoration and dental calculus

(Glickman 1964)

 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 entangles in extracellular matrix

(WHO 1978 )

23
 It is defined as structured, resilient yellowish –gray substance that adhere
tenaciously to the intra-oral hard surface , including removable and fixed
restoration.

(W H Bowen, 1976)

24
 A biofilm is a well organized, cooperating community of microorganisms.

(Overman 2000).

 Biofilms consist of one or more communities of microorganisms, embedded in a

glycocalyx, that are attached to a solid surface.

 Biofilms is a microbial derived sessile community characterized by cells that are

irreversibly attached to substratum or interface or each other , embedded in a matrix of

extracellular polymeric substances that they have produced and exhibit altered

phenotype and respond to growth rate and transcription.

 Biofilm is a matrix enclosed bacterial population which are adherend to each other or

to interfaces. (Costerton et al. 1978).

25
 Biofilm are composed of microbial cells encased within a matrix of extracellular

polymeric substances such as polysaccharides, proteins and nucleic acids.

(Carranza 11th edi)

 Biofilm is relatively undefinable microbial community, associated with tooth surface

or any other hard, non shedding surface. (Widerer and Charkelis 1989)

26
CLASSIFICATION

27
Characteristic Supragingival Subgingival
Gram reaction +/- Dominated by -
Morphotypes Cocci, branching rods, Dominated by rods and
filaments, spirochetes spirochetes
Energy metabolism Facultative with some Dominated by anaerobes
anaerobes
Energy sources Generally ferment Many proteolytic forms
carbohydrates
Motility Firmly adherent to plaque Adherence less pronounced
matrix with many motile forms
Response by host Can cause caries and gingivitis Can cause gingivitis and
periodontitis

28
 Supra gingival plaque shows stratified organisation of multi-layered accumulation of

bacterial morphotypes .Gram positive cocci and short rods at tooth surface.

(P D Marsh 2000)

 Gram negative rods and filaments and spirochetes in outer surface of mature plaque

 Availability of blood products and a low oxidation reduction (redox) potential and

GCF substances help and act as nutrients and characterize the anaerobic

environment.

29
 Tooth associated cervical plaque - Filaments dominate

 Outer layer S. mitis , S. sanguis Actinomyces , A. naeslundi

 Deeper –Filaments predominate

30
 STRUCTURE AND COMPOSITION OF PLAQUE

 Composed primarily of microorganisms.

 1 gram = 10¹¹ microorganisms

 10³ in healthy crevice to 10⁸ in deep pocket.

 More than 500 microbial species are found in dental plaque

 It is primarily composed of bacteria in matrix of salivary glycoproteins and extracellular

polysaccharides.

 1gm of dental plaque contains 1011

 Non bacterial microorganisms -> mycoplasma, yeasts, protozoa, viruses

 Heterogenous structure microcolonies of bacterial cells , having primitive

circulatory system having open fluid-filled channels running through plaque mass.

31
INTERCELLULAR MATRIX

32
Exopolysaccharides
 50 – 95% of the dry weight.

 Maintain the integrity of the biofilms as well as preventing desiccation and attack by
harmful agents.

 It acts as a buffer and assists in the retention of extracellular enzymes.

 Composition of dental plaque also varies on distinct anatomical surfaces (e.g.

fissures, approximal and smooth surfaces, gingival crevice, dentures) due to the
prevailing physical and biological properties of each site

[Bowden et al., 1975; Slots, 1977; Theilade et al., 1982)

33
 Uneven patterns of penetration of radiolabelled fluoride, sucrose and phosphate were
found in plaque generated naturally on an in situ biofilm model in volunteers

[Robinson et al., 1997]

 Diffusion of glucans of increasing molecular size was retarded in laboratory mixed

culture biofilms

[Thurnheer et al., 2003]

 use of two-photon excitation microscopy coupled with fluorescent life-time imaging

demonstrated considerable heterogeneity in pH over relatively short distances
[Vroom et al., 1999].

34
 Cells of the same microbial species can exhibit extremely different physiologic states
in a biofilms.

 pH and the number of metal ions can vary quite remarkably over short distances
within a biofilms.

 Certain microcolonies are completely anaerobic even though composed of a single

species and grown in ambient air.

35
PLAQUE FORMATION AT
ULTRASTRUCTURAL LEVEL
 THE FORMATION OF THE PELLICLE ON THE TOOTH SURFACE.-Acquired
pellicle formation

 INITIAL ADHESION AND ATTACHMENT OF BACTERIA. Reversible adhesion

involving weak long-range physicochemical interactions between the cell surface
and the pellicle, which can lead to stronger adhesin-receptor mediated attachment

 Co-adhesion resulting in attachment of secondary colonizers to already attached cells

 COLONIZATION AND PLAQUE MATURATION- Multiplication and biofilm

formation (including the synthesis of exopolysaccharides) and, on occasion,
Detachment.

36
FORMATION OF PELLICLE
The pellicle consists of;

Glycoproteins (mucins)

Proline-rich proteins

Phosphoproteins (statherin)

Histidine-rich proteins

Enzymes (alpha amylase)

Other molecules which acts as adhesion sites

37
 Mechanism involved in pellicle formation include:-

Electrostatic forces

Van der Walls forces

Hydrophobic forces

Adsorption of host and bacterial molecules to the tooth surface. This conditioning film (the
acquired pellicle) forms immediately following eruption or cleaning [AlHashimi and
Levine, 1989] and directly influences the pattern of initial microbial colonization. Modern
techniques offer the opportunity to more fully explore the distribution and composition of
pellicle components [Li et al., 2003].

38
 Within 1 minute after pellicle starts forming and within 2 hrs pellicle is in

equilibrium with adsorption and detachment.

 The conformational changes that may occur following adsorption of molecules, and

the impact of this on their properties, are now amenable for study: for example, the

molecular structure of glucans changes when glucosyltransferases are adsorbed to a

surface

[Vacca-Smith et al., 1996; Kopec et al., 2001]

39
INITIAL ADHESION AND
ATTACHMENT OF BACTERIA
Phase 1: Transport to the surface;
 Random contacts may occur. (through Brownian motion, liquid flow or active bacterial
movement).initial interactions are non specific
Phase 2: Initial adhesion;
 Initiated by the interaction between the bacterium and the surface, from a certain
distance (50 nm), through long range and short range forces.
 Passive transport of oral bacteria to the tooth surface. Weak, long-range
physicochemical interactions between the microbial cell surface and the pellicle-coated
tooth create a weak area of net attraction that facilitates reversible adhesion
[Busscher and van der Mei, 1997].
 After 4-8 hrs ,60% to 80% bacteria are genus streptococci

40
 Subsequently, strong, short-range interactions between specific molecules on the
bacterial cell surface (adhesins) and complementary receptors in the pellicle can result
in irreversible attachment

[Jenkinson and Lamont, 1997; Lamont and Jenkinson, 2000]

BACETRIA WHICH CAN SURVIVE WITHOUT OXYGEN ARE PRESENT

HAEMOPHILUS

NEISSERIA

ACTINOMYCES

VEILLONELLA

Oral bacteria generally possess more than one type of adhesin on their cell surface and
can participate in multiple interactions both with host molecules and similar receptors
on other bacteria by coadhesion.
41
 Primary 1. Streptococcus gordonii
2. Streptococcus intermedius
Colonizers 3. Streptococcus mitis
4. Streptococcus oralis
5. Streptococcus sanguinis
6. Actinomyces gerencseria
7. Actinomyces israelii
8. Actinomyces naeslundii
9. Actinomyces oris
10. Aggregatibacter actinomycetemcomitans serotype a
11. Capnocytophaga gingivalis
12. Capnocytophaga ochracea
13. Capnocytophaga sputigena
14. Eik enella corrodens
15. Actinomyces odontolyticus
16. Veillonella parvula

42
Phase 3: Attachment;
A firm anchorage between bacterium and surface will be established by specific
interactions (covalent, ionic or hydrogen bonding).
(Bradshaw 2000)

 Eg: A. viscosus possesses fimbriae that contain adhesions that specifically bind to proline
rich proteins of the dental pellicle.

 Co-adhesion of later colonizers to already attached early colonizers. This stage also
involves specific interbacterial adhesin-receptor interactions (often involving lectins)

 increase in the diversity of the bioilm and to the formation of unusual morphological
structures, such as corn-cobs and rosettes

[Kolenbrander et al., 2000].

43
 Co-adhesion may also facilitate the functional organization of dental plaque.
Bacteria engage in a range of antagonistic and synergistic biochemical interactions

[Marsh and Bradshaw, 1999].

 The efficiency of metabolic interactions among bacteria in food chains may be

enhanced if they are brought into close physical contact. Likewise, the co-adhesion
of obligately anaerobic bacteria to oxygen-consuming species can ensure their
survival in overtly aerobic oral environments

[Bradshaw et al., 1998].

44
Secondary 1. Campylobacter gracilis
2. Campylobacter rectus
Colonizers 3. Campylobacter showae
4. Eubacterium nodatum
5. Aggregatibacter actinomycetemcomitans serotype
b
6. Fusobacterium nucleatum ssp nucleatum
7. Fusobacterium nucleatum ssp vincentii
8. Fusobacterium nucleatum ssp polymorphum
9. Fusobacterium periodonticum
10. Parvimonas micra
11. Prevotella intermedia
12. Prevotella loescheii
13. Prevotella nigrescens
14. Streptococcus constellatus
15. Tannerella forsythia
16. Porphyromonas gingivalis
17. Treponema denticola

45
 Phase 4: Colonization of the surface and biofilm formation.
 Multiplication of the attached micro-organisms. Cell division leads to confluent
growth and, eventually, a three-dimensional spatially and functionally organized,
mixed-culture biofilm. Polymer production results in the formation of a complex
extracellular matrix made up of soluble and insoluble glucans, fructans and
heteropolymers.

 Such a matrix is a common feature of biofilms and makes a significant contribution

to the known structural integrity and general resistance of biofilms; the matrix can be
biologically active and retain nutrients, water and key enzymes within the biofilm

[Allison, 2003].

 Further studies are required to fully understand the influence of the matrix on the
architecture and properties of dental plaque.

46
 When viewed by conventional light or electron microscopy, mature dental plaque
appears as a densely packed structure

[Listgarten, 1999; Marsh and Nyvad, 2003];

 Recent application of novel microscopic techniques has demonstrated a more open

architecture . Endogenous substrates (derived from saliva or gingival crevicular fluid)
are the main source of nutrients for oral bacteria

[Beighton et al., 1986]

 catabolism requires the concerted and sequential action of groups of microbes with
complementary enzyme profiles

[Bradshaw et al., 1994; Marsh and Bowden, 2000]

 plaque functions as a true microbial community. Active detachment. Bacteria can

respond to environmental cues and detach from surfaces, enabling cells to colonize
elsewhere

47
 Enzymes produced by sessile bacteria can hydrolyse the specific adhesins that

anchor cells to the surface.

[Cavedon and London, 1993; Lee et al., 1996].

 Once established, the resident plaque microflora remains relatively stable over time

and is of benefit to the host [Marsh, 2000].

 The resident microflora of all sites plays a critical role in the normal development of

the physiology of the host and also reduces the chance of infection by acting as a

barrier to colonization by exogenous (and often pathogenic) species.

48
EARLY AND SECONDARY
COLONIZERS
 Interaction of secondary colonizers with early colonizers include the coaggregation
of;
 * Fusobacterium nucleatum with Streptococcus sanguis,
 * Prevotella loescheii with Actinomyces viscosus, and
 * Capnocytophaga ochraceus with A.viscosus.

TERTIARY COLONIZERS - P.gingivalis, C. rectus, E. corrodens, A.

actinomycetemcomitans

 Special examples of coaggregations are the

 i) “corncob” formation.
 ii) “Test tube brush”

49
CORN- COB (SUPRA GINGIVAL PLAQUE)

50
Coaggregation
 Coaggregation is a direct interaction and is distinct from agglutination, which
occurs when cells are stuck together by molecules in solution.
 At least 18 genera from the oral cavity have shown some form of Coaggregation.
(P E Kolenbrander, 1993)
 All oral bacteria possess surface molecules that foster some sort of cell-cell
interaction
( J Periodontal 1994 )
 Strong cell-cell binding is then determined by the presence of adhesin proteins or
carbohydrates on one partner and complementary receptor proteins or
carbohydrates on the other.

 Many coaggregations between strains of different genera are mediated by

lectin-like adhesins (proteins that recognize carbohydrates) and can be
inhibited by lactose and other galactosides.

51
 Fusobacteria coaggregate with all other human oral bacteria.

 Veillonella spp., Capnocytophaga spp. and Prevotella spp. bind to

streptococci and/or Actinomyces.

 F. nucleatum with S. sanguinis

 Prevotella loescheii with A. oris

 Capnocytophaga ochracea with A. oris

52
53
55
GROWTH DYNAMICS OF DENTAL
PLAQUE
Ultrastructural Aspects: Important changes within first 24 hours.
 
 First 2 to 8 hours – Pioneering streptococci saturate the salivary pellicular binding
sites and thus covering 3% to 30% of the enamel surface.

 Next 20 hours – a short period of rapid growth is observed.

 After 1 day – the term ‘Biofilm’ is fully deserved because organization takes
place within it.

56
COMMUNICATION BETWEEN CELLS

 SMALL DIFFUSIBLE PEPTIDES

 HORIZONTAL GENE TRANSFER

 AUTO INDUCER

(P D Marsh 1993)

57
• QUORUM SENSING:

• It was first mentioned by FUQUA ET AL

• Regulation of expression of specific genes through the accumulation of signaling
compounds that mediate inter cellular communication. (Prosser 1999).

 It is dependent on cell density.

 Quorum sensing signaling molecules produced by putative periodontal pathogens

such as P.gingivalis, P.intermedia, and F.nucleatum.
(Frias et al 2001).

58
 Biofilm Regulation of Gene Expression Bacteria.

 Minimal threshold level of individual cell mass is required to initiate a concerted

population response.

 The signal molecule used for communication is called “auto-inducer”

 Process is auto induction.

 The binding of bacteria to specific receptors can trigger significant changes in both
bacterial and host cell patterns of gene expression, e.g. following the initial attachment of
Escherichia coli to uro-epithelial cells [Abraham et al., 1998].

 Surface-associated responses are now being identified in plaque bacteria, although the
magnitude of this shift in gene expression may be less than that observed in free-living
species because of the absolute dependence of oral bacteria on a biofilm lifestyle [Burne,
1998].

59
 The exposure of Streptococcus gordonii to saliva resulted in the induction of genes
(sspA/B) encoding adhesins that can bind to salivary glycoproteins and engage in co-
aggregation with Actinomyces spp.
(Du and Kolenbrander, 2000)
 Changes in protein profile following attachment have been identified in
Streptococcus mutans using a whole-cell proteomic approach
[Svensater et al., 2001].
 Proteins involved in a range of biochemical functions including protein folding and
secretion, amino acid and fatty acid biosynthesis, and cell division were up-
regulated.
 Similarly, genes associated with glucan (gtfBC) and fructan synthesis (ftf) in S.
mutans were differentially regulated in biofilms [Li and Burne, 2001].

60
 There was little influence of surface growth in early biofilm formation (48 h), but gtf
expression was markedly up-regulated in older (7-day) biofilms, whereas ftf activity
was repressed.

 This was interpreted as an indirect effect of biofilm growth on gene expression, i.e.
the altered phenotype was probably due to changes in local environmental conditions
within the biofilm (e.g. sugar concentration, pH) rather than due to attachment per se
[Li and Burne, 2001].

 Thus, biofilm growth can have both direct and indirect influences on gene expression
by oral bacteria.

61
 In S. mutans, quorum sensing is mediated by a competence stimulating peptide (CSP)
[Li et al., 2002].

 This peptide also induced genetic competence in S. mutans so that the transformation
frequency of biofilm-grown S. mutans was 10- to 600-fold greater than for planktonic
cells

[Li et al., 2001]

 Lysed cells in biofilms could act as donors of chromosomal DNA, thereby increasing
the opportunity for horizontal gene transfer in dental plaque.

 This quorum sensing system also functions to regulate acid tolerance in S. mutans
biofilms [Li et al., 2002]

 It has been proposed that S. mutans, upon exposure to low pH, could release CSP and
initiate a co-ordinated ‘protective’ response among neighbouring cells to such a
potentially lethal stress.

 CSPs are specific for cells of the same species, but other communication systems may
function between different taxa (Kolenbrander et al)

62
 Genes encoding autoinducer 2 have been detected in several genera of gram-positive
and gram-negative bacteria so that autoinducer 2 may have a broader species range,
although its role in plaque remains to be determined.

 However, mutants of the luxS gene that encodes for the autoinducer 2 synthase in S.
mutans and S. gordonii had an impaired ability to produce monospecies biofilms in
vitro

[Blehert et al., 2003; Merritt et al., 2003].

 Future research will identify more of these sophisticated communication networks,

and it has been suggested that analogues of the signalling molecules could be used as
novel therapeutic agents to manipulate the properties of biofilms.

 Gene Transfer Cells also communicate with one another in biofilms via horizontal
gene transfer. As discussed above, signalling molecules such as CSP markedly
increase the ability of recipient cells in biofilms to take up DNA [Li et al., 2002].

63
Quorum sensing give biofilms distinct properties:

 Expression of genes for antibiotic resistance

 Influence community structure by encouraging the growth of beneficial species.

 Discouraging the growth of competitors.

 Physiological properties of bacteria in the community may be altered.

64
MECHANISM OF ANTIBIOTIC
RESISTANCE
• It differs from species to species, from antibiotic to antibiotic.

 Antimicrobial Resistance- A major finding of clinical relevance with respect to

micro-organisms growing on a surface is their increased resistance to antimicrobial
agents [Gilbert et al., 1997, 2002; Ceri et al., 1999].

 Conventionally, the sensitivity of bacteria to antimicrobial agents is determined on

cells grown in liquid culture by the measurement of the minimum inhibitory
concentration or minimum bactericidal concentration.

65
 The further growth of the plaque mass occurs preferably by the multiplication of
already adhering microorganisms rather than by new colonizers.

 The thickness of the plaque increases slowly with time, increasing to 20 to 30 µm

after 3 days.

66
 As yet, however, these proposals have not been widely accepted, and there are no
generally agreed standardized methods by which these concentrations could be
determined.

 Bacteria growing in dental plaque also display increased resistance to antimicrobial

agents, including those used in dentifrices and mouth rinses

[Marsh and Bradshaw, 1993; Kinniment et al., 1996)

 For example, the biofilm inhibitory concentration for chlorhexidine and amine
fluoride was 300 and 75 times greater, respectively, when S. sobrinus was grown as a
biofilm compared with the minimum bactericidal concentration of planktonic cells
[Shani et al., 2000].

67
 Similarly, it was necessary to administer 10– 50 times the minimum inhibitory
concentration of chlorhexidine to eliminate S. sanguinis (previously S. sanguis)
biofilms within 24 h [Larsen and Fiehn, 1996].

 The age of the biofilm can also be a significant factor; older biofilms (72 h) of S.
sanguinis were more resistant to chlorhexidine than younger (24 h) biofilms

 Biofilms of oral bacteria are also more resistant to antibiotics (e.g. amoxycillin,
doxycycline, metronidazole) [Larsen, 2002; Larsen and Fiehn, 1996].

 The mechanisms behind the increased resistance of biofilms to antimicrobial agents

are the subject of much research .[Gilbert et al., 2002].

68
Few mechanisms are:-
 The slower rate of growth.
 Variation in parameters like nutritional status, growth rate, temperature, pH and prior
exposure to sub effective concentrations.
 As an ion exchange resin removing antibiotics.
 Extracellular enzymes in extracellular matrix, inactivate the susceptible, typically
positive charged, hydrophilic antibiotics.
 Alteration of genotype and phenotype of the cells growing within a biofilms matrix .

69
 Cells can become resistant due to mutations affecting the drug target, the presence of
efflux pumps or to the production of modifying enzymes etc., but even innately
sensitive bacteria become resistant when growing on a surface.

 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.

70
 The 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.

 As stated earlier, bacteria growing on a surface display a novel phenotype, and this

can result in a reduced sensitivity to inhibitors, while the transfer of resistance genes

can occur more readily in biofilm communities such as dental plaque. Growth on a

surface may also result in the drug target being modified or not expressed in a

biofilm.

71
72
 Bacteria grow only slowly under nutrient depleted conditions in an established biofilm

and, as a consequence, are much less susceptible than faster-dividing cells. In addition,

it has also been proposed that the environment in the depths of a biofilm may be

unfavourable for the optimal action of some drugs

[Gilbert et al., 2002].

 The matrix in biofilms can also bind and retain neutralizing

[Allison, 2003].

At present, it is not clear whether some or all of these effects account for the observed

resistance of cells in plaque biofilms

73
DE NOVO SUPRAGINGIVAL
PLAQUE FORMATION
 Clinically it follows an exponential growth curve.

 First 24 hours – negligible plaque, covering <3% of the vestibular tooth surface.

 During next 3 days – plaque growth increases at a rapid rate, then slows down.

 After 4 days – average of 30% of the total tooth crown area will be covered with
plaque and no more increase substantially with time.

74
 Critical role in the normal development of the physiology of the host (McFarland,
2000).

 Reduces the risk of infection by acting as a barrier to colonization by exogenous

species (termed ‘colonization resistance’) (van der Waaij et al, 1971).

 Mechanisms
I. More effective competition for nutrients and attachment sites.
II. The production of inhibitory factors.
III. creation of unfavorable growth conditions for invading species by the normal
microflora.

75
TOPOGRAPHY OF SUPRAGINGIVAL
PLAQUE
 Early plaque formation on teeth follows a typical topographic pattern with initial
growth along the gingival margin and from the interdental space. (protected from
shear forces).

 Plaque formation can also start from surface irregularities like grooves, cracks,
perikymata, or pits.

 By multiplication, the bacteria subsequently spread out from these initial areas as a
relatively even monolayer.

76
VARIABLES AFFECTING
PLAQUE FORMATION

 Considered no single variable as the great differences in rate of plaque formation.

Simonsson et al (1889)
 Found no discernible differences except for a more prominent intermicrobial matrix
and higher proportions of gram-negative rods in the group of fast growers. Zee et al
(1997)
 Variation with dentition.
 Impact of gingival inflamation.
 Impact of patient age.
 Spontaneous tooth cleaning.

77
DE NOVO SUBGINGIVAL PLAQUE
FORMATION
 Some early studies, using culturing techniques, examined the changes within the
subgingival microbiota during the first week after mechanical debridement and
reported only partial reduction, followed by a fast regrowth to almost pretreatment
levels within 7 days.

78
PLAQUE HYPOTHESIS
NON- SPECIFIC PLAQUE HYPOTHESIS
 NSPH are part of a controversy that took place for over a century

(Miller, 1890 Loesche, 1976 Theilade, 1986 ).

 At the end of the nineteenth century the most common idea about dental infections
was that they were caused by the non-specific overgrowth of all bacteria in dental
plaque.

(Black, 1884 , 1899; Miller,1890; Loesche, 1986).

 This idea is referred to as the “Non-specific plaque hypothesis” (NSPH)

(Loesche,1976) and was based on work of researchers such as Black (1884) and
Miller (1890).

79
 Applying the NSPH it was postulated that it was the quantity of plaque that determined

the pathogenicity without discriminating between the levels of virulence of bacteria.

 Believing this, the host would have a threshold capacity to detoxify bacterial products

(e.g., saliva neutralizing acid) and disease would only develop if this threshold was

surpassed and the virulence factors could no longer be neutralized

(Theilade, 1986).

 The conclusion was that if any plaque has an equal potential to cause disease, the best

way of disease prevention would be non-specific mechanical removal of as much

plaque as possible by e.g., tooth brushing or tooth picking.

80
SPECIFIC PLAQUE HYPOTHESIS
 In the 1970s, culture-based techniques and microscopy allowed discrimination of
specific bacterial species and opened the hunt for disease-related micro-organisms.

 In 1976, Walter J. Loesche announced the “Specific Plaque Hypothesis” (SPH),

postulating that dental caries was an infection with specific bacteria in the dental
plaque of which the most relevant were “mutans streptococci” (main
species: Streptococcus mutans and Streptococcus sobrinus) and lactobacilli
(Loesche, 1976).

81
 This hypothesis proposed that use of antibiotics against specific bacterial species could

cure and prevent caries

(Loesche and Nafe, 1973; Loesche, 1976, 1986; Loesche et al., 1977).

 Furthermore antibiotics reduced the abundance of cariogenic bacteria but failed to

eliminate them thus as soon as the treatment was stopped, abundance increased (Loesche

and Nafe, 1973; Loesche et al., 1977), while a long period of treatment leads to

antibiotic resistance (Kornman and Karl, 1982).

82
 These suggested “specific-pathogens” are part of the indigenous microflora and

unlike foreign pathogens cannot be eliminated from the oral cavity (van Palenstein

Helderman, 1984).

 This extended the SPH to periodontal diseases which were proposed to be

inflammations caused by specific periopathogens and antibiotic treatment would be

effective (Loesche, 1986).

83
 In the decade after the SPH was introduced, potential periopathogens included:

protozoa, spirochetes, streptococci, and actinomyces. In addition, Gram-negative,

anaerobic rods including black-pigmented Bacteriodes such as Bacteriodes

melaninogenicus (renamed to Prevotela melaninogenica) and others from the

genus Wolinella (re-classified as Campylobacter) and facultative anaerobic,

 Gram-negative rods of the genera Capnocytophaga, Eikenella and Actinobacillus 

(van Palenstein Helderman, 1981; Socransky et al.,1982; Slots and Genco, 1984 and

reviewed by Theilade, 1986) were identified as periopathogens.

84
 However, these findings were limited due to the large number of uncultivable species
(~50%) (Siqueira and Rôças,2013) and the bias toward easily cultivable species
(Handelsman, 2004).

 The finding of different species related to periodontal disease led to the idea that oral
disease could be initiated by a number of specific pathogens (Socransky, 1977;
Theilade, 1986).

 This idea was further investigated over the next decades and led to the famous
Socransky-complexes which include bacterial clusters based on their association with
periodontal disease (Socransky et al., 1998).

85
ECOLOGICAL PLAQUE HYPOTHESIS
 “CHANGE IN THE ENVIRONMENT CONDITIONS CAN LEAD TO
ECOLOGICAL SHIFT.”

 In 1994 Philip D. Marsh proposed a hypothesis that combined key concepts of the
earlier hypotheses.

 In his “Ecological Plaque Hypothesis” (EPH), disease is the result of an imbalance in

the total microflora due to ecological stress, resulting in an enrichment of some “oral
pathogens” or disease-related micro-organisms (Marsh, 1994).

 This idea was not entirely new since Theilade, in the review concluded that
“increased virulence of plaque (leading to disease) is due to a plaque ecology
unfavorable to the host and favorable for overgrowth by some of the indigenous
bacteria having a pathogenic potential” (Theilade, 1986)..

86
 Marsh expanded this theory and related the changes in microbial composition to

changes in ecological factors such as the presence of nutrients and essential cofactors,

pH and redox potential (Marsh, 1994, 2003).

 Marsh also considered the reverse: the bacteria in dental plaque affect the environment.

For instance, early colonizers of supragingival dental surfaces, are usually facultative

anaerobic bacteria that use the oxygen, producing carbon dioxide and hydrogen

(Alexander, 1971; Marsh,2003).

87
 This lowers the redox potential giving strict anaerobes a chance to settle and multiply
in the biofilm. Bacterial growth is dictated by the environment, which in turn is
influenced by bacterial metabolism, leading to mutual dependencies in health but
also a chain of events that lead to diseases.

 However, like the other hypotheses, the traditional EPH does not address the role of

genetic factors of the host that significantly contribute to the composition of dental

plaque and to susceptibility to disease (Mason et al., 2013).

88
KEYSTONE PATHOGEN
HYPOTHESIS
 The concept of keystone species is derived from basic ecological studies. Certain
species have an effect on their environment that is disproportional relative to their
overall abundance (Paine, 1969 Power et al., 1996; Darveau et al., 2012).

 George Hajishengallis and colleagues applied this concept to (oral) microbiology by

proposing “The Keystone-Pathogen Hypothesis” (KPH) (Hajishengallis et al., 2012).
The KPH indicates that certain low-abundance microbial pathogens can cause
inflammatory disease by increasing the quantity of the normal microbiota and by
changing its composition (Hajishengallis et al., 2012).

89
 For instance ,Porphyromonas gingivalis is shown to be able to manipulate the native

immune system of the host

 By doing so it was hypothesized that it does not only facilitate its own survival and

multiplication, but of the entire microbial community.

 In contrast to dominant species that can influence inflammation by their abundant

presence, keystone pathogens can trigger inflammation when they are present in low

numbers (Hajishengallis et al., 2012).

90
 When disease develops and advanced stages are reached, the keystone pathogen are

detected in higher numbers (Socransky et al., 1998). Importantly, even though their

absolute number increases, keystone pathogens can decrease in levels compared to

the total bacterial load which increases as plaque accumulates in periodontitis

(Abusleme et al., 2013).

91
 The KPH was developed by observing the properties of the “red complex” (Socransky
et al., 1998) bacterium P. gingivalis.

  P. gingivalis was able to colonize by itself, but was not able to trigger disease
without the presence of other bacterial species. This indicates that (some of) the
commensal microbiota is essential in the disease process.

 Evidence of P. gingivalis acting as a keystone pathogen was also obtained in rabbit

models (Hasturk et al., 2007) and non-human primates (Page et al., 2007).

92
HOST SUSCEPTIBILITY
 Genetic factors

 Increased prevalence of P.gingivalis, A. actinomycetemcomitans. T.forsythia,

E.coli,C.albicans in smokers

 Teughels and co-workers 100% increase in A.a. colonization when epithelial cells

are infected with h CMV.

93
BENEFICIAL SPECIES
 GRAM POSITIVE FACULTATIVE BACTERIA-STREPTOCOCCUS (S. mitis S.

sanguis )& ACTINOMYCES (A. viscosus & A. naeslundii)

 Small proportions gram negative species P. intermedia, F. nucleatum ,

Capnocytophagia, Nesisseria and Veillonella sp.

 Few spirochetes and motile rods

94
BENEFICIAL BACTERIA

Affect disease progression by:-

 Passively occupying niche.

 Limiting pathogen to adhere to tissue surface.

 Adversely affecting the growth.

 Ability to produce virulence factor affected.

 Degrading virulence factor.

95
 PERIODONTAL PATHOGENS

AGGREGATIBACTER ACTINOMYCETEMCOMITANS:
ACTINOMYCETEMCOMITANS
 Bacterium actinomycetem comitans was described by KLINGER as coccobacillary
bacteria isolated together with Actinomyces from actinomycotic lesions of man .

 It was reclassified as Actinobacillus actinomycetemcomitans by TOPLEY &

WILSON and as Haemophilus actinomycetemcomitans by Potts et al.

 Recent studies in 2006 involving multilocus sequence analysis by Nørskov Lauritsen

N and Kilian M have shown a phylogenetic similarity of Actinobacillus
actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis

96
 ROBERT KOCH’S POSTULATES(1884) states that for an organism to produce a
disease, it must

 (i) occur only in that disease,

 (ii) be able to grow in pure culture,

 (iii) be recovered from the host.

 Aggregatibacter actinomycetemcomitans did not meet these needs as it did not exist in
isolation in the disease.

 However, it met the criteria of a potential pathogen proposed by

 Sigmund Socransky which states that the pathogen must,

 (i) be associated with a disease by increase in number of organisms at sites,

 (ii) be eliminated in sites after treatment and

 (iii) demonstrate virulence factors to cause periodontal tissue destruction

97
.

This resulted in the addition of the genus aggregatibacter (aggregate, to come together;

bacter, bacterial rod; aggregatibacter, rod shaped bacterium that aggregates with others), to

the family pasteurellaceae in 2006 to cover gram negative, non motile, facultatively anaerobic

rods or coccobacilli that were previously known as haemophilus (actinobacillus or bacterium)

actinomycetemcomitans (now aggregatibacter actinomycetemcomitans),

98
TAXONOMY
 The genus aggregatibacter is taxonomically in the

FAMILY – PASTEURELLACEAE

ORDER PASTEURELLALES

CLASS GAMMAPROTEOBACTERIA

PHYLUM PROTEOBACTERIA.

Five sero groups of Aggregatibacter actinomycetemcomitans were classified by

Taichman based on surface polysaccharide located on the O-side chain of

lipopolysaccharide using tube agglutination studies

99
 SEROTYPES a,b,c,d,e,f

 Serotype b with LAP(Zambon et al 1983).

 Serotype a with chronic periodontitis(Zambon et al 1983).

 Serotypes a, b and c are most prevalent in the oral cavity. A particular clone of

serotype b with enhanced leukotoxic activity is predominantly associated with cases

of localized aggressive perioodontitis . Serotype c is found in healthy subjects

100
101
Structure
 0.4 ± 0.1X 0.1 ± 0.4 micrometers in size

 SURFACE ULTRASTRUCTURE Aggregatibacter actinomycetemcomitans possess

fimbriae, vesicles and extracellular amorphous materials.

102
 VESICLES
A.actinomycetemcomitans has numerous vesicles or blebs which

are lipopolysaccharide in nature.

 Highly leukotoxic strains tend to have more vesicles. These vesicles contain endotoxin which

has a bone resorption activitiy and a bacteriocin termed actinobacillin.

 These vesicles also exhibit adhesive properties and function as delivery vehicles for toxic

materials

103
 Virulence is defined as the ability of a microbe to cause infection. Generically, the virulence

attributes of microbial pathogens include the ability to enter a

 Extra oral infections are caused byA.actinomycetemcomitans due to its ability to reduce oxygen in the

tissues. Brain meninges, septicemia, UTI, osteomyelitis, endocarditis and abscesses are common.

 A. actinomycetemcomitans mediated endocarditis is a chronic

syndrome with fever, chills, anorexia, weight loss, heart murmur and

night sweats. Aggregatibacter actinomycetemcomitans expresses

a Human Shock Protein like molecule, HSP 60 that cross reacts

with antibodies to human HSP 60

104
CULTURE MEDIUM
 MGB (trypticase soy broth) with malachite green and bacitracin was the earliest media used

to culture (A.a).

 It was then followed by medium with trypticase soy agar, serum with bacitracin and

vancomycin (TSBV).

 Exclusive growth of A.a was found in a particular culture medium which contained TSBV,

spiramycin, fucidic acid and carbencillin. RPMI – 1640 and Dulbecco’s modified Eagle

medium are used now with a generation time of 246 and 346 min

105
106
VIRULENCE FACTORS

 Leukotoxins (LtxA) - The Aa leukotoxin is a member of a family of pore-

forming toxins, characterized by a series of glycine- rich repeats in the C-

terminal portion of the protein that are involved in cation binding and

appear to be essential to toxin activity. This family exoprotein family is

found among numerous genera of gram-negative bacteria and may play a

significant role in periodontal disease pathogenesis.

107
 The leukotoxin appears to be mainly present in the outer cell membrane,in the

extracellular outer membrane vesicles.

 Aa strains vary in their ability to produce the leukotoxin and the strains can

be classified into leukotoxin producing – strains and nonleukotoxin-producing

strains.

 The mechanism of leukotoxicity includes:

◦  Membranolytic activity producing pores in the target cell.

◦  Phospholipids act as the receptor for the toxin whose activity result in a rapid

influx of Ca2+ into the cell.

◦  Necrosis and apoptosis.

108
 Exposure of neutrophils and monocytes/ macrophages, to strains that produce large

amount of LtxA, result from the ability of LtxA to form pores in the membrane of the

target cells, leading to osmolysis caused by water influx into the cells

109
CYTOLETHAL DISTENDING TOXIN (CDT) –

Cytolethal distending toxin is encoded by a locus of three genes, cdtABC.

The toxin itself is encoded by cdtB, while cdtA and cdtC appear to encode

proteins that mediate interaction between the cytolethal distending toxin complex

and the host cell surface.

Several reports indicate that the Cdt complex of Aa is composed of all three

proteins CdtA, -B and -C, in equal proportions.

 CdtB exhibits toxicity when artificially injected into cells all three proteins are

required for maximal toxin activity

110
 Mechanism of action –

 The active subunit, CdtB, exhibits DNase I activity.

 CdtA and CdtC is less clear, both proteins possess putative mucin-like

carbohydratebinding domains that predict interaction with the host cell surface.

 cdt action predicts that CdtA and CdtC interact with the host membrane and facilitate

CdtB entry into the cell.

 After CdtB enters the cell, it is transported into the nucleus by an active process that

requires amino acid residues in its N terminus.

111
 In the nucleus, CdtB causes DNA damage through its DNase activity

 Induction of apoptosis. Induction of apoptosis in lymphocytes occurs through capase

activation.

 In human gingival fibroblasts, Aa Cdt is able to stimulate the production of receptor

activator of nuclear factor-KB ligand, and this activation is independent of

interleukin-1, interleukin-6, tumor necrosis factor-α, or prostaglandin E2 expression .

112
 Lipopolysaccharide (LPS) –

 It is a major integral component of the outer membrane of gram negative bacteria.

 The LPS of Aa has a broad spectrum of immunological, endotoxic activities.

 These activities include

◦  Stimulation of in vitro bone resorption.

◦  The production of IL-1, and prostaglandin (PGE2) from macrophages.

◦  Polyclonal activation of B-lymphocytes.

113
 The bone resorptive activities of this LPS are the result of stimulation of PGE2 ,IL-I

release from osteoblasts and other cells.

 Aa is known to activate the complement cascade by the alternative pathway which

in turn generates prostaglandins and this is the probable mechanism of bone

resorption in case of periodontitis.

114
 Surface–associated material (SAM) –

 It has been shown that proteins associated with the outer surfaces of some but not all

putative periodontal pathogens are potent inducers of bone resorption and tissue

pathology

 The SAM of Aa is composed - Bacterial capsule and - Other molecules loosely bound to

the outer surface of the external membrane.

 Several proteins and peptides. These proteins are active in very low concentrations and

it is presumed that their actions are important in the pathogenesis of periodontitis by

stimulating alveolar bone resorption.

115
 These inhibit:

 Periodontal ligament regeneration and repair.

 Promote B-lymphocytes and plasma cell proliferation.

 Stimulate T suppressor cells.

 Suppress immunoglobulin production.

 Resistance to complement - mediated killing.

 Escape the anti-bacterial actions of the immune system by surviving within epithelial

cells and other periodontal mammalian cells.

116
 Chemotactic inhibition factors

 A.actinomycetemcomitans produces factors which inhibit the chemotaxis of PMNs.

 These are known as chemotactic inhibition factors.

 These factors could reduce the number of PMNs in the local lesion available to
phagocytose and kill these bacteria.

 Extracellular outer membrane vesicles - Aa produces numerous extracellular outer

membrane vesicles which are shed from the surface of the bacteria. These vesicles
contain

  leucotoxin

  LPS

 Their small size permits them to cross epithelial barriers such as the pocket epithelium

117
 Proteases that degrade immunoglobulins- A.actinomycetemcomitans produces

proteolytic enzymes which degrade immunoglobulins. This could reduce the local

effectiveness of antibodies produced against these bacteria.

 Collagenase Aa produces a collagenolytic proteinase which can attack collagen.

 Thus these proteases may inhibit the proliferation

118
ADHESINS
 Adhesins are proteinaceous in nature and mediate the attachment of bacteria to

specific receptors on epithelial cells. These are

 Associated with the outer membrane of the bacterium

 Are released into the medium in the form of vesicles.

 The identities of these molecules are unknown and the relationship between the

bound and secreted adhesion molecules is yet to be determined.

119
FIMBRIAE
 FIMBRIAE They are small filamentous cell surface appendages. They occur in a

peritrichous array, measuring more than 2 micrometers diameter and in bundles .

 It forms two types of colonies;

 colonies with star shaped interior (star positive)

 nonfimbriated strains (star negative).

 The most abundant protein in the fimbriae is 304-a with molecular mass 6.5k Da.

120
 Fimbriae gives the bacteria a rough morphology, the morphological type associated

with fresh isolates adhesion of Aa to epithelial cells involves multiple determinants.

Aa exhibits more fimbriae when grown anaerobically than when cultured in CO2.

Like many other gram-negative bacteria,

 Aa may exhibit fimbriae, small filamentous cell surface appendages associated with

bacterial colonization of host tissues.

 Aa fimbriae occur in peritrichous arrays, may be more than 2 pm in length and 5 nm

in diameter and often occur in bundle

121
VESICLES

 A prominent feature of the surface of Aa is vesicles (blebs).

 These structures, which are lipopolysaccharide in nature, originate from and are

continuous with the outer membrane.

 Vesicles are also released into the external environment in large numbers.

122
 The surface of HIGHLY LEUKOTOXIC

 A. actinomycetemcomitans strains has an abundance of extracellular membranous

vesicles, in contrast to minimally or non-leukotoxic strains, which have few or no

vesicles.

 Furthermore, vesicles per se exhibit leukotoxic activity.

 Other biologically active components of A. actinomycetemcomitans vesicles are

endotoxin,bone resorption activity and a bacteriocin, termed actinobacillin.

123
PORPHYROMONAS GINGIVALIS

 It is a Gram negative, anaerobic, non motile, asaccharolytic rods that

usually exhibit coccal to short rod morphologies.

 Group of “black pigmented Bacteroides”

 Asaccharolytic - P. gingivalis

 Intermediate level of carbohydrate fermentation - P. intermedia

 Highly saccharolytic - Prevotella melaninogenica.

 P. gingivalis has been shown to induce elevated systemic and local

immune responses in subjects with various forms of periodontitis

(Haffajee & Socransky 1994).

124
PROTEOLYTIC ACTIVITY
 Degrade proteins into short peptides and used metabolically in the generation of
energy and as sources of carbon and nitrogen.
 Gingipains capable of degrading collagen fragments.
 Proteases occur in multiple forms that are found extracellularly or on the bacterial
cell surface.
 The gingipains specifically cleave proteins at the peptide bond following arginine
residues or lysine residues.

125
GINGIPAIN
 Gingipains are trypsin-like cysteine proteinases produced by Porphyromonas

gingivalis, a major causative bacterium of adult periodontitis.

 HRgpA and RgpB products of 2 distinct but related genes, rgpA and rgpB,

respectively, are specific for Arg-Xaa peptide bonds.

 HRgpA and RgpB induce vascular permeability enhancement through activation of

the kallikrein/kinin pathway and activate the blood coagulation system, which,

respectively, are potentially associated with gingival crevicular fluid production and

progression of inflammation leading to alveolar bone loss in the periodontitis site

126
 Kgp is the most potent fibrinogen/fibrin degrading enzyme of the 3 gingipains in human

plasma and is involved in the bleeding tendency at the diseased gingiva.

 HRgpA activates coagulation factors and degrades fibrinogen/fibrin more efficiently

than RgpB due to the adhesion/hemagglutinin domains, which have affinity for

phospholipids and fibrinogen.

 Gingipains degrade macrophage CD14, thus inhibiting activation of the leukocytes

through the lipopolysaccharide (LPS) receptor, and thereby facilitating sustained

colonization of P. gingivalis.

 Gingipains play a role in bacterial housekeeping and infection, including amino acid

uptake from host proteins and fimbriae maturation.

127
 Based on the important activities of gingipains in the bacterial infection and the pathogenesis

of periodontitis, the bacterial proteinases can be targets for periodontal disease therapy.

 Immunization with RgpB, HRgpA, or a portion of HRgpA catalytic domain attenuated P.

gingivalis induced disorders in mice.

 In addition, a trypsin-like proteinase inhibitor retarded P. gingivalis growth specifically.

Gingipains are potent virulence factors of P. gingivalis, and are likely to be associated with

the development of periodontitis.

 It is, therefore, suggested that gingipain inhibition by vaccination and gingipain-specific

inhibitors is a useful therapy for adult periodontitis caused by P. gingivalis infection

128
129
 Gram-negative oral anaerobe that is involved in the pathogenesis of periodontitis, an

inflammatory disease that destroys the tissues supporting the tooth which eventually

may lead to tooth loss.

 Among the over 500 bacterial species living in the oral cavity, a bacterial complex

named “red complex” and composed of Porphyromonas gingivalis, Treponema

denticola, and Tannerella forsythia has been strongly associated with advanced

periodontal lesions

130
 Porphyromonas gingivalis can locally invade periodontal tissues and evade the host

defense mechanisms. In doing so, it utilizes a panel of virulence factors that cause

deregulation of the innate immune and inflammatory responses.

 Porphyromonas gingivalis rapidly adheres to the host cell surface followed by

internalization via lipid rafts and incorporation of the bacterium into early

phagosomes. Porphyromonas gingivalis activates cellular autophagy to provide a

replicative niche while suppressing apoptosis

131
 The ability of Porphyromonas gingivalis to cause adult periodontitis is determined by

its arsenal of virulence factors. Biofilm formation and bacterial dipeptidyl peptidase

IV (DPPIV) activity contribute to the pathogenic potential of Porphyromonas

gingivalis.

 Furthermore, biofilm formation may enhance Porphyromonas gingivalisvirulence

through increased DPPIV activity. Because of their importance for bacterial

colonization and growth, biofilm formation and DPPIV activity could present

interesting therapeutic targets to tackle periodontitis

132
 Porphyromonas gingivalis serotypes K1 and K2 .

 This important information suggests that these serotypes could elicit a greater bone

resorption in vivo and have a significant role in the periodontitis pathogenesis.

  Porphyromonas gingivalis K1 and K2 serotypes induce a strong Th1-Th17-response.

 These Porphyromonas gingivalis serotypes induce higher osteoclasts activation,

133
Virulence and Growth of Porphyromonas
gingivalis: Role of Iron

  Iron utilized by this pathogen in the form of heme has been shown to play an

essential role in its growth and virulence.

  Porphyromonas gingivalis does not produce siderophores. Instead, it employs

specific outer membrane receptors, proteases (particularly gingipains), and
lipoproteins to acquire iron/heme.

 Specific proteins involved in iron and heme capture have been described

134
 Additionally, the proteolytic activities of gingipain R and gingipain K contribute to
processing/maturation of various cell-surface proteins of Porphyromonas gingivalis,
such as fimA fimbrilin (a subunit of major fimbriae), 75-kDa protein (a subunit of
minor fimbriae),

 hemagglutinins, and the hemoglobin receptor protein, which are important for the
bacterium to colonize and proliferate in the gingival crevice and to invade the
periodontium.

 Levels of lysosomal proteinases such as cathepsins B, H, L, and G and medullasin

were determined in gingival crevicular fluid from periodontitis patients and
experimental gingivitis subjects by activity measurement and sensitive immunoassay

135
Virulence and Growth of Porphyromonas
gingivalis: Role of Adhesins

 Retention and growth of Porphyromonas gingivalison diverse surfaces are facilitated by

a repertoire of adhesins including fimbriae, hemagglutinins, and proteinases.

 Histatins are human salivary gland peptides with antimicrobial and anti-inflammatory

activities.

 histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and attenuates

HagB-induced chemokine responses in human myeloid dendritic cells. Thus histatin 5 is

capable of attenuating chemokine responses which may help control oral inflammation

 Porphyromonas gingivalis produces large amounts of arginine- and lysine-specific

cysteine proteinases in cell-associated and secretory forms.

136
 Porphyromonas gingivalis contains exceedingly high concentrations of cysteine

proteinases with trypsin-like activity which has been implicated as a virulence factor

in adult-onset periodontitis

 Several of these enzymes are apparently expressed as active proteolytic products

following processing of larger precursor proteins.

 In addition, more recent data have suggested a close relationship between some of

these enzymes and two other potential virulence factors of Porphyromonas

gingivalis: hemagglutinins and collagenases.

137
 Surface components of Porphyromonas gingivalis have contact with host tissues and
cells because of the outermost cell elements.

 As a result, such components of Porphyromonas gingivalis are potentially important

in the occurrence of periodontal diseases.

  Porphyromonas gingivalis fimbriae are a critical factor for mediation of interaction

of this bacterial organism with host tissues, as Porphyromonas gingivalis promotes
both bacterial adhesion to and invasion of targeted sites. 

 Porphyromonas gingivalis fimbriae are likely to interrupt the cellular signaling via

extracellular matrix proteins/integrins in periodontal regions. Fimbriae are also
thought to be critically important in invasive events of this bacterial organism to host
cell

138
Porphyromonas gingivalis fimbriae
 are capable of binding to salivary enzymes, extracellular matrix proteins, and
commensal bacteria as well as also strongly adhering to cellular alpha5beta1-integrin

 Following adhesion to alpha5beta1-integrin, Porphyromonas gingivalis is captured

by cellular pseudopodia which enable invagination through an actin-mediated
pathway.

  Porphyromonas gingivalis fimbriae are classified into 6 genotypes (types I to V and

Ib) based on the diversity of the fimA genes encoding each fimbria subunit.
Intracellular Porphyromonas gingivalis with type II fimbriae has been found to
clearly degrade integrin-related signaling molecules.

139
 A majority of periodontitis patients were found to carry type II fimA fibriae

of Porphyromonas gingivalis, followed by type IV; type II fimA fimbriae

of Porphyromonas gingivalis were found significantly more often in patients with

more severe forms of periodontitis

140
 Lipopolysaccharide of Porphyromonas
gingivalis
 The lipopolysaccharide of Porphyromonas gingivalis is a key factor in the
development of periodontitis.

 Gingival fibroblasts, which are the major constituents of gingival connective tissue,
may directly interact with Porphyromonas gingivalis and its bacterial products,
including lipopolysaccharide, in periodontitis lesions.

 Due to its ability to potently activate host inflammatory and innate defense
responses, it has been proposed to function as an important molecule that alerts the
host of potential bacterial infection.

 However, although highly conserved, lipopolysaccharide contains important

structural differences among different bacterial species that can significantly alter
host responses .

141
 Plasminogen activator inhibitor type 1 (PAI-1) mRNA binding protein expression
was increased in gingiva from periodontitis patients.

 Porphyromonas gingivalis lipopolysaccharide increases the expression of EphB4

while inhibiting the expression of EphrinB2.

  Porphyromonas gingivalis LPS induces proinflammatory cytokines, such as IL-1β,

IL-6, and IL-8, which induce periodontal tissue destruction. Periodontal ligament
stem cells (PDLSCs) play an important role in periodontal tissue regeneration and
are expected to have future applications in cellular therapies for periodontitis. 

142
Cysteine Proteases of Porphyromonas
gingivalis
 The cysteine proteases of Porphyromonas gingivalis are extracellular products of an
important etiological agent in periodontal diseases.

 They are significant targets of the immune responses of affected individuals and are
viewed by some as potential molecular targets for therapeutic approaches to
periodontal diseases .

 These enzymes are involved in both the destruction of periodontal tissues and
interrupting host-defense mechanisms through the degradation of immunoglobulins
and complement factors

143
Recent Research on Porphyromonas
gingivalis

 specifically between Porphyromonas gingivalis and Aggregatibacter

actinomycetemcomitans, as they are the major causes of gingivitis.

focusing on the fimbriae, specifically finding ways to inhibit the minor fimbriae

production as that would prevent the formation of a biofilm on the tooth .

Another major area of research is finding out how current methods to destroy these

pathogens work such as ammonium nitrate.

144
TANNERELLA FORSYTHIA

 The organism is a Gram negative, anaerobic, spindle shaped, highly

pleomorphic rod.

 Difficult to grow.
 Need growth factors from other species.

 B. forsythus was detected more frequently and in higher numbers in active

periodontal lesions than inactive lesions (Dzink et al 1988).

145
 This species has been shown to produce trypsin like proteolytic activity (Loesche et al 1992)

and induce apoptotic cell death (Arakawa et al 2000).

 Double labeling experiments demonstrated that B. forsythus was both on and in periodontal

pocket epithelial cells indicating the species ability to invade.

 Shares antigen with P. gingivalis.

 Listgarten et al (1993) found that the species most frequently detected in “refractory” subjects

was B. forsythus.

146
 SPIROCHETES

 These are Gram negative, anaerobic, helical shaped, highly motile

microorganisms that are common in many periodontal pockets.
 Etiologic agent of acute necrotizing ulcerative gingivitis (Listgarten &
Socransky 1964).
 At least 15 species of subgingival spirochetes are described.
 Pathogen related oral spirochetes” (PROS) were the most frequently
detected spirochetes in supra and subgingival plaques of periodontitis
patients.(Riviere et al 1991)
 Mechanism:-

o Travel through viscous environment.

o Degrade collagen & dentin.
o Destroy IgA, IgM, IgG.

147
PREVOTELLA INTERMEDIA

 Gram negative, short, round-ended anaerobic rod shown to be

particularly elevated in acute necrotizing ulcerative gingivitis
(Loesche et al 1982), and also in certain forms of
periodontitis (Herrera et al 2000).

 This species appears to have a number of virulence properties

exhibited by P. gingivalis and was shown to induce mixed
infections.(Hafstrom & Dahlen 1997).

 It has also been shown to invade oral epithelial cells in vitro

(Dorn et al 1998).

 Strains of P. intermedia that show identical phenotypic traits

have been separated into two species, P. intermedia and P.
nigrescens (Shah & Gharbia 1992).

148
 FUSOBACTERIUM NUCLEATUM
 Gram negative, anaerobic, spindle shaped (cigar shaped) rod that has been
recognized as part of the subgingival microbiota for over 100 years. (Plaut 1894,
Vincent 1899).

 Most common isolate found in cultural studies of subgingival plaque samples

comprising app. 7-10% of total isolates. (Moore et al 1985).

 Mechanism:-
o Increased secretion of IL-8 (Han et al 2000).
o Induce apoptotic cell death in mononuclear

and polymorphonuclear cells (Jewett et al 2001).

o Cytokine, elastase and oxygen radical release

from leukocytes (Sheikhi et al 2000).

o Bridging organism.

149
CAMPYLOBACTER RECTUS

 Gram negative, anaerobic, short, motile vibrio which utilizes

H2 or formate as its energy source.

 First described as a member of the “vibrio corroders”, a group

of short nondescript rods that formed small convex, “dry
spreading” or “corroding” colonies on blood agar plates.

 Higher numbers in disease sites as compared with healthy sites

and more in sites exhibiting active periodontal destruction.
(Rams et al 1993).

 Mechanism:-
o Produce leukotoxin (Gillespie et al 1992).
o Stimulate human gingival fibroblasts to produce

IL-6 and IL-8 (Ebersole 1996).

150
EIKENELLA CORRODENS
 Gram negative, capnophilic, asacharolytic, regular, small rod with blunt
ends.

 Found more frequently in sites of periodontal destruction as compared

with healthy sites and higher levels in active sites (Tanner et al 1987).

 Mechanism:- stimulate production of

o Matrix metalloproteinase

(Dahan et al 2001)
o IL-6 and IL-8 (Yumoto et al 1999).

151
PEPTOSTREPTOCOCCUS MICROS

 P. micros is a Gram positive, anaerobic, small, asaccharolytic coccus.

 Two genotypes can be distinguished with the smooth genotype being more
frequently associated with periodontitis lesions than the rough genotype
(Kremer et al. 2000).
 P. micros was found to be in higher numbers at sites of periodontal
destruction as compared with healthy sites (Papapanou et al 2000, Riggio
et al 2001).
 It was shown that P. micros in combination with either P. intermedia or P.
nigrescens could produce transmissible abscesses (Van Dalen et al 1998).
 Produce protease(Grenier 2006)

152
SELENOMONAS SPECIES

 The selenomonas spp. are Gram negative, curved, saccharolytic rods and
may be recognized by their curved shape, tumbling motility and, in good
preparations, by the presence of a tuft of flagella inserted in the concave
side.

 Moore et al (1987) described six genetically and phenotypically distinct

groups isolated from oral cavity and found S. noxia at a higher proportion
of shallow sites (PD>4mm) in chronic periodontitis.

153
EUBACTERIUM SPECIES

 Suggested as possible periodontal pathogens due to their increased levels in

disease sites. (Moore et al 1985).

 E. nodatum, Eubacterium brachy and Eubacterium timidum are Gram positive,

strictly anaerobic, small somewhat pleomorphic rods.

 Some of these species elicited elevated antibody responses in subjects with

destructive periodontitis. (Martin et al 1988)

154
  MILLERI STREPTOCOCCI

 Some of the streptococcal species are associated with

and may contribute to disease progression.

 Milleri streptococci, Streptococcus anginosus, S.

constellatus and S. intermidius might contribute to
disease progression in subsets of periodontal patients.

 These species was found to be elevated at sites which

demonstrated recent disease progression (Dzink et al
1988).

155
 OTHER SPECIES

 Interest has grown in groups of species not commonly found in the subgingival plaque as

initiators or possibly contributors to the pathogenesis of periodontal disease, particularly in

individuals who have responded poorly to periodontal therapy.

 Emphasis have been placed on enteric organisms, staphylococcal species as well as other

unusual mouth inhabitants. (Slots et al 1990)

156
Color Complexes

157
 VIRUSES
Contreras & Slots 2000,Kamma et al 2001

158
159
160
 Herpesviruses are capable of infecting various types of cells, including

polymorphonuclear leukocytes, macrophages, and lymphocytes.

 The diffuse invasion of Candida fungi and other opportunistic organisms into

the gingival tissue of AIDS patients has been demonstrated to be a typical

virus-mediated alteration of host defense mechanisms.

161
162
 FUNGI

 Hannula J, Dogan B, Slots (2001) showed geographical differences in the

subgingival distribution of C. albicans serotypes and genotypes and suggested

geographic clustering of C. albicans clones in Subgingival samples of Chronic

Periodontitis patients.

 Reynaud AH (2001) found a weak correlation between yeasts in periodontal

pockets.

163
Microorganisms associated with
Specific Periodontal disease

164
 Listgarten et al in 1965 studied the structure of microbiota from extracted

natural teeth to study assiciated pathogen.

 First finding was association of medium sized spirochetes with NUG and

LJP

165
GINGIVITIS

 104 to 106 bacteria.

 Gram-negative bacteria.

 Compared with healthy sites, noticeable increase also occur in the numbers
(spirochetes).

 Pregnancy associated gingivitis is accompanied by dramatic increases in levels of

P. intermedia, which uses the steroid as growth factors(Carranza,10 th edition).

166
CHRONIC PERIODONTITIS

 C. rectus, P. gingivalis, P. intermedia, F. nucleatum and T. forsythia were found to be elevated in the active

sites(Carranza,10th ).

 Sites with chronic periodontitis will be populated with greater proportions of gram-negative organisms and

motile bacteria.

 C. rectus, Eikennella corrodens , F. nucleatum, A. actinomycetemcomitans, P.micra, E.nodatum

 Certain gram-negative bacteria with pronounced virulence properties have been strongly implicated as

etiologic agents e.g. P. gingivalis and Tannerella forsythus.

167
LOCALIZED AGGRESSIVE PERIODONTITIS

 In 1976 , Slots studies predominant cultivable organisms in juvenile periodontitis

 Gram -ve, capnophilic, and anaerobic rods.

 The most numerous isolates are several species from the genera Eubacterium, A.
naeslundii, F. nucleatum, C. rectus, and Veillonella parvula.

 Aa playing a causative role in localized aggressive periodontitis, especially in cases

in which patients harbor highly leukotoxic strains of the organism.

 However, some populations of patients with LAP do not harbor Aa, and in still
others P. gingivalis may be etiologically more important.

168
 Elevated levels of local and systemic antibodies to

A. actinomycetemcomitans

 They were more in active sites then inactive sites.

169
GENERALIZED AGGRESSIVE
PERIODONTITIS

 The sub-gingival flora in patients with generalized aggressive peri­odontitis resembles

that in other forms of periodontitis.

 The predominant subgingival bacteria in patients with generalized aggressive

periodontitis are P. gingivalis, T. forsythis A. actinomycetemcomitans, and
Campylobacter species.

170
 REFRACTORY CHRONIC
PERIODONTITIS
 Unusually diverse and may contain enteric rods, staphylococci, and Candida.

 Persistently high levels are found of one or more of P. gingivalis, T. forsythis, S.

inter-medius, P. intermedia, Peptostreptococcus micros, and Eikenella corrodens.

 Persistence of Streptococcus constellatus has also been reported.

171
 NECROTIZING ULCERATIVE
GINGIVITIS/PERIODONTITIS

 More than 50% of the isolated species were strict anaerobes with P. gingivalis and
F. nucleatum accounting for 7-8% and 3.4%, respectively.

172
PERIODONTAL ABSCESSES

 The bacteria isolated from abscesses are similar to those associated with chronic and
aggressive forms of periodontitis.

 An average of approximately 70% of the cultivable flora in exudates from

periodontal abscesses are gram-negative and about 50% are anaerobic rods.

 Periodontal abscesses revealed a high prevalence of the following putative

pathogens: F. nucleatum (70.8%), P. micros (70.6%), P. intermedia (62.5%), P.
gingivalis (50.0%), and T. forsythis (47.1%).

 Enteric bacteria, coagulase-negative staphylococci, and Candida albicans have also

been detected.

173
 MIXED INFECTIONS

 At the pathogenic end of the spectrum, it is conceivable that different relationships exist between

pathogens.

 The presence of two pathogens at a site could have no effect or diminish the potential

pathogenicity of one or other of the species.

 Alternatively, pathogenicity could be enhanced either in an additive or synergistic fashion.

 It is not clear whether the combinations suggested in the experimental abscess studies are

pertinent to human periodontal diseases

174
 PERIIMPLANTITIS

 High proportion of anaerobic gram negative rods, motile organisms, and spirochetes).

 Species such as Aa, Pg, Tf, P. micros, C. rectus, Fusobacterium, and Capnocytophaga are

often isolated from failing sites.

 Other species such as Pseudomonas aeruginosa, enterobacteriaceae, Candida albicans and

staphylococci, are also frequently detected around implants.

175
Microbial Diagnostic testing

176
MICROBIAL CULTURING

 Gold standard.

 Positive identification of periodontopathogens.

 Relative & absolute count.

 Permits assessment of antibiotic sensitivity.

 Inability to detect low level of microorganisms, high cost, labour intensiveness, prolonged
time, inability to detect certain species.

177
ENZYMATIC ASSAYS

 To detect bacteria that produce trypsin like enzyme (BANA) like T. forsythus, T.

denticola and P. gingivalis.

 Unable to detect the proportion of three bacteria.

 Cannot detect presence of other organisms.

178
 IMMUNOASSAYS

 Like Immunofluorescence microscopy, ELISA, Membrane assays, Latex

agglutination assays.

 Higher sensitivity & specificity than culturing.

 Has low detection thresholds, low cost, rapid, somewhat quantitative.

 Cannot find antibiotic sensitivity.

179
NUCLEIC ACID PROBES

 Like Oligonucleotide probe, Whole genomic probe, random cloned probes.

 Has greater sensitivity than culture methods.

 Viability of organisms is not required.

180
 POLYMERASE CHAIN
ASSAYS
 Like Real time PCR, Multiplex PCR, Hot Start PCR.

 Most sensitive of any of the above methods.

 Can also find candidal and enteric microorganisms.

181
FUTURE ADVANCES IN
PERIODONTAL MICROBIOLOGY

182
 Specific progress in the field of molecular biology, has led to advances in periodontal

microbiology.

 Perhaps even more relevant is the present ability to detect microorganisms that cannot be

cultivated thus far, which has underscored the limitations of our knowledge of this

complex ecologic niche.

 Becoming aware that the host response is also of major significance will further improve

our understanding of the severity and therapy of periodontal infections.

 Finally the recognition of the beneficial activity of several groups of commensal species,

such as probiotics, might open new strategies for periodontal therapy.

183
Conclusion

 The results of current genome study projects of several periodontopathogens will

provide detailed information about the etiology of periodontal diseases, and will likely

show new possibilities for the treatment and prevention of periodontal diseases.

 In the near future, it is expected that the correlation between biofilm maturation and

activation of specific genes of the inner microorganisms will be clarified at the

molecular level.

184
 References
 Carranza’s Clinical Periodontology; 10 th edition. Elsevier publication.

 Textbook of Clinical Periodontology, Glickman; 6 th edition. Periodontal therapy; Henry M.

Goldman, Cohen; C. V. Mosby company.

 Clinical Periodontology and Implant dentistry, Jan Lindhe, 4 th edition. Blackwell munksgaard.

 Periodontal therapy; Henry M. Goldman, Cohen; C. V. Mosby company.

 Periodontics. Medicine, Surgery, and Implants. Rose, Mealey, Genco, Elsevier publication.

 PD Marsh: Plaque as a biofilm: pharmacological principles of drug delivery and action in the
sub- and supragingival environment. Oral Diseases (2003) 9 (Suppl. 1), 16–22

185
 Pamela R. Overman . Biofilm: A New View of Plaque. JCDP 2000;1, 3;1-8.

 Marsh PD : Dental plaque: biological significance of a biofilm and community life-style. J

Clin Periodontol 2005; 32 (Suppl. 6): 7–15.

 Casey Hein. Perio Pathways Etiology Fast-forwarded: The Host-bacterial

Interaction; Theory and the Risk Continuum. Contemporary Oral Hygiene;
2004. Dec;16-20.

 Peter M. Loomer; Microbiological diagnostic testing in the treatment of

periodontal diseases. Periodontology 2000;2004,34;49-56.

186
 Anne. D. Haffajee & sigmund.S. Socransky. Microbial etiological agents of

Destructive periodontal diseases. Periodontology 2000, Vol. 5, 1994, 78-11.

 Tatsuj Nishihara & Takeyoshi koseki. Microbial etiology of Periodontitis.

Periodontology 2000, Vol. 36, 2004, 14–26.

 Ohguchi, Ishihara, Masahiro. J Periodont Res 2003; 38: 191-197.

 William M, Hendersson B. FEMS Microbiol Lett 1995; 17: 365-379.

 Fives-Taylor P, Meyer D, Mintz K. J Dent Res 1995; 9: 55-62.

187
 Dzink, J. L., Socransky, S. S. & Haffajee, A. D .(1988) The predominant cultivable

microbiota of active and inactive lesions of destructive periodontal diseases. J. Clin.

Periodontol. vol. 15, pp. 316-323, 0303-6979

 Asikainen, S. & Chen, C. (1999) Oral ecology and person-to-person transmission of

Actinobacillus actinomycetemcomitans and Porphyromona gingivalis.

Periodontology 2000 vol. 20, pp. 65-81, 0906-6713

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