describes the known periodontal pathogens, novel pathogens and note on role of virus and fungi in pathogenicity in periodontitis

1. Periodontal
pathogens
DR LAKKIREDDY VASAVI REDDY
II MDS

2. Contents:
 Introduction
 History
 Uncertainty in defining the periodontal pathogens
 Criteria for determination
 Virulence factors
 Microbial complexes
 Microbes in detail
 Novel pathogens
 Role of viruses
 Role of fungi
 Conclusion

3. Introduction:
 Periodontitis is a polymicrobial disease involving a complex interaction
between the oral microorganisms organized in a biofilm structure and the
host immune response.
 As with any other infection, identification of the microbial pathogens
associated with the etiology of periodontitis is the first step toward the
development of effective therapeutic approaches.
 The establishment of a microorganism as a true pathogen should be based
on 2 main levels of evidence: (1) the organism should be present in higher
prevalence and/or levels in disease than in health (“association” studies),
and (2) its suppression or elimination should reduce or stop disease
progression

4.  It is important to underline that periodontal diseases appear to be initiated
by a relatively limited number of periodontal pathogens in the complex
dental biofilm.
 Clinical and experimental evidences confirm that certain bacterial strains in
the periodontal environment can induce gingival tissue inflammation and
bone destruction. These bacterial strains are defined as periodontal
pathogens
 These microbes possess numerous potent virulence factors aimed at
neutralizing local host defenses and destroying periodontal tissues

7. Uncertainty in defining
the pathogens:
 The complexity of the subgingival microbiota.
 Sample taking
 Difficulties in cultivation, characterization and identification of micro-
organisms of plaque.
 Mixed infections
 Opportunistic microbial species
 Disease activity.
 Multiple periodontal diseases in different subjects
 The possibility of multiple diseases in a subject.
 The carrier state
 Virulent factors.
 Genetic virulence elements

8. Criteria for defining pathogens
 This in destructive periodontal diseases were initially based on Koch’s
postulates 1980.
 These postulates were:
(1) The agent must be isolated from every case of the disease
(2) It must not be recovered from cases of other forms of disease or non
pathogenically, and
(3) After isolation and repeated growth in pure culture, the pathogen must induce
disease in experimental animals.
(4) Re‐isolated from the inoculated, diseased experimental host and identified as
being identical to the original specific causative agent.

9. Socransky’s criteria, (1999)
 Association - must be associated with disease as evident by increase in
number of organisms at diseased site
 Elimination - be eliminated /decreased in sites which shows disease
resolution with treatment
 Host response – in the form of alteration in host cellular or humoral
immune response.
 Virulence – demonstrate virulence factors capable of causing destruction
of tissue.
 Animal studies - be capable of causing disease in experimental animal
models.
 Risk assessment – risk conferred by the presence of an organism may be
assessed.

10.  According to published reports, Aggregatibacter actinomycetemcomitans
and Porphyromonas gingivalis are the two species classified as periodontal
pathogens that fulfill all of the criteria listed above.

11. Virulence factors:
 Virulence from the Latin, “virulentus” or “full of poison.”
 Defined as the relative ability of an organism to cause disease or to interfere with a
metabolic or physiological function of its host.
 Poulin and Combs (1999) defined the concept of virulence in terms of the type of
molecules being produced by the microbe. As such, they defined virulence in terms of
“virulence factors,” that is, components of a microbe, which when present harm the host,
but when absent (i.e., mutation) impair this ability.

12. Virulent V/s Avirulent microbe (Holt and Ebersole 2005)
 The characteristic endproducts of bacterial metabolism; the chemical composition of bacterial components; the
ability of the intact bacterium or its parts to overwhelm host defense mechanisms, its invasiveness, and its ability
to kill
Virulence factors can have a multitude of functions:
 The ability to induce microbe–host interactions
 The ability to invade the host
 The ability to grow in the confines of a host cell
 The ability to evade / interfere with host defenses

13.  The virulence capacities of bacteria depend on the production of certain
factors for adhesion such as adhesins, lectins, fimbriae and vesicles.
 Agents that directly damage the periodontal tissues are proteases, alkali and
acid phosphatases produced by microorganisms, fatty and organic acids, IgG-
and IgA-proteases, chondroitinsulfatase and toxic products (endotoxins,
leukotoxin, mucopeptides of the bacterial wall, end-products of metabolism
such as H2 S, NH4 , indole).
 Mobility is an essential virulent factor of certain pathogenic bacteria and allows
them to invade the epithelium and connective tissue (spirochetes).

14. Microbial complexes:

15. Classification of periodontal
pathogens:

16. Based on pathogenicity:

17. According to
Lindhe:

18. Distribution:

20. “The Green
Complex”
AAC serotype a
Eikenella corrodens
Capnocytophaga gingivalis
Capnocytophaga sputigene
Capnocytophaga ochracea
Camphylobacter concisus

21. Aggregatibacter actinomycetem comitans
 ATCC number: 33384
 Small, non-motile, Gram-negative, saccharolytic, capnophilic,
coccobacillus.
 Prominent member of the HACEK group of pathogens.
 It is derived from the Greek word. actes- meaning Ray, because of the
star on the top of agar colony. Mycetes- meaning fungus;
 Comitans- latin word, meaning in common with, or accompanying
Actinomycetes spp., reflects the association of Actinobacillus with
Actinomycetes.

22. Historical aspects:
Norskov-Lauritsen & Kilian 2006- Aggregatibacter

23.  Culture characteristics: chocolate and blood agar with visible
colonies appearing after 48-72 hours.
 It forms small (0.4-1.0 mm in diameter), white, smooth, circular,
convex, non hemolytic and translucent colonies on solid media
with a slightly irregular edge.
 Specific growth media with bacitracin & vancomycin under 5%-
10% carbon dioxide.
 On prolonged incubation of 5-7 days colonies may develop a
central density with a four-six pointed star-shaped or as crossed
cigars appearence.
 On gram staining , the organism appear as pale staining, gram-
ve coccobaccilli

24.  Structural characteristics: reveal features typical gram-negative organisms
including an outer cytoplasmic membrane, a periplasmic space, and an inner
cytoplasmic membrane.
 The outer membrane is covered by a ruthenium staining material possibly a
carbohydrate microcapsule as well as membrane vesicles, "blebs",
morphologically identical to lipopolysaccharide vesicles.

25.  Serological characteristics:
• 6 serotypes- a,b,c,d,e,f, recently g.
• Serotype a, b are most common in oral cavity. Serotype c constitutes 10% human
isolates. Whereas serotype d, e, f are less common.
• Most subjects are infected with one serotype only and AAC infections seen to be
relatively serotype stable.
• Researchers have observed that serotype b was related to LAP, serotype a was more
commonly detected in samples from adult subjects while serotype c was related
periodontal health in adults (Zambon et al 1983).

26.  Virulence factors:

30. Lindhe and Slots listed the following destructive mechanisms that may
be used by AAC in AgP.
 AAC can produce substances that can kill PMNs and monocytes,
thereby compromising the patient’s ability to fight invading
bacteria of their products. This leukotoxin may be counteracted by
the development of serum antibodies.
 Endotoxins from AAC can induce shwartzman’s reaction,
macrophage toxicity, platelet aggregation, complement activation
and bone resorption.
 produce potent proteolytic enzymes that can destroy collagen,
activate the complement system or degrade immunoglobulins.
 produce a fibroblast – inhibiting factor that impairs the defence
mechanisms (i.e. fibroblast cytotoxicity).

31.  Transmission:
• pathogens cluster in families.
• However, recent evidence suggested the possibility that
people with periodontitis may cause periodontal
breakdown in their spouses.
• Route: salivary and mucosal contact or an inanimate
object.
• The role of saliva as a transport vehicle - AAC can be
cultured from salivary samples, which indicate that
these bacteria are able to survive in saliva during
transportation to a new host.

32. Antibiotic resistance:
 Studies my Roe et al have demonstrated the resistance of several strains of
A. a to tetracyclines.
 It demonstrated the conjugal transfer of TetB determinant.

33. Eikenella corrodens
ATCC number: 23834
Etymology: Eiken: Named after M. Eiken, who first named
the type species of the genus as bacteroides.

34. Historical aspect:
 Microbes exhibiting characteristics similar to E.corrodens were first
described by Henriksen and Holm.
 Eiken was the first to characterize this sp. in detail in 1958. He
proposed the name Bacteroides corrodens.
 Jackson and Goodman proposed to assign the facultatively
anaerobic B.corrodens to a new sp. E.corrodens

35.  Gram negative, capnophilic, asacchrolytic, non-spore forming,
non-motile, small rod with blunt ends.
 It was first described in 1948 as a slow growing anaerobic,
gram negative rod.
 E.corrodens is a commonly found commensal in the human
oral cavity.
 The microorganism has a distinguishing feature ie the ability
to pit or corrode the agar in plated culture. The colonies grow
in the little grooves.

36. Colony characteristics:
• E.corrodens is a pleomorphic bacillus that sometimes
appears coccobacillary.
• It grows in aerobic( require hemin) and anaerobic
conditions, but requires an atmosphere containing atleast
10% carbon dioxide.
• E.corrodens must be incubated for atleast 2-3 days before
the colonies grow to size sufficient for counting.
• The colonies are small and greyish and produce a greenish
discoloration of the agar and smells faintly of bleach.
• Pitting of agar- characteristic feature. A typical corroding
colonies are seen distinct from other species.

37.  Structure:
 E.corrodens possesses a typical gram negative bacteria cell envelope
consisting of an inner membrane, a peptidoglycan layer, and an outer
membrane which may be covered by an additional polysaccharide slime
layer.
 The presence of fimbriae(pili) is unclear. Henrichsen and Blom showed that
E.corrodens possesses fimbriae with a diameter of appx. 5nm.
 The presence of a polar fimbriae correlated with the corroding colony
morphology as well as with the twitching type of motility seen.
 It has been stated possible that the “pili” may be an artefact of a dehydrated
slime layer during preparation for microscopy.

38. Virulence factors:
 Lipopolysaccharide
• The chemical composition of LPS has been determined by a study by
Progulske and Holt using gel electrophoresis and concluded that this
organism expressed both high and low molecular weight LPS.
• Another study by Chen and Wilson on LPS has shown that E.corrodens
consists primarily of low molecular weight forms. According to them
there are 16 different LPS phenotypes and structural heterogeneity can
be appreciated.

39.  Exopolysaccharide layer
• Exopolysaccharides that adhere strongly to the bacterial surface
are referred to as capsules, and more loosely bound ones are
called slime layers.
• They function mainly as antiphagocytic substances which impede
engulfment by PMN’s and mononuclear phagocytes.
• E.corrodens has a slime layer whose extract has been shown to
suppress the immune response.
 Outer membrane proteins (OMP)
• E.corrodens is known to have a principal outer membrane protein
(POMP). The POMP is known to exhibit dose-dependent
stimulation or depression of phagocytosis and induce platelet
aggregation.

40.  Adhesins
• First step to initiating infection is the entry into and attachment
to the host cell surfaces.
• E.corrodens LPS exhibits hemagglutinating activity and may
play a role in adhesion of the organism to epithelial cells.
• Slots and Gibbons first demonstrated that E.corrodens attaches
to human gingival crevicular epithelial cells in vitro.
• The adhesion is mediated through a lectin-like bacterial protein
& a galactose-like receptor on the surface of cells.

41. Capnocytophaga species
Etymology: kapnos: smoke
cytophaga: bacterial genus name
“bacteria requiring carbon dioxide”

42.  They are a group of gram-negative, facultative
anaerobic fusiform rods.
 Culture characteristics: They require carbon
dioxide for growth and culture and hence the name.
Anaerobic gas mixtures have been used for isolation.
They are viable if atleast 5% carbon dioxide is
present.
 Trypticase soy agar with 5% sheep blood has been
reported to give maximal growth.
 On the basis of biochemical, morphological and DNA
based homology studies, Leadbetter and his
colleagues proposed that the genus contains three
species: C. ochracea, C. gingivalis and C. sputigena

43. Occurrence and pathogenicity:
• The principal natural habitat of Capnocytophaga sp in man is the oral cavity.
C.ochracea occurs in the subgingival sulcus of healthy adults.
• Elevated levels of Capnocytophaga sp are isolated from cases of periodontitis.
• C.ochracea has been correlated with the presence of disease in LJP patients.
• LJP patients are usually known to harbor 5 human oral species namely:
C.gingivalis, ochraceae, sputigena, granulosa and haemolytica.

44. Virulence factors:
• The interaction between Capnocytophaga sp and host defences is complex.
• It produces a dialyzable substance which impairs neutrophil chemotaxis.
• C.ochracea is able to degrade IgA1 and IgG which would enable the organism
to induce local paralysis of the host defenses.
• C.sputigena produces a fibroblast proliferation inhibitory factor and an
endotoxin.

45. Orange
complex
organisms
Prevotella intermedia
Peptostreptococcus micros
Fusobacterium nucleatum
Campylobacter rectus
Eubacterium nodatum

46. Peptostreptococcus micros
 Family: Clostridiaceae
Etymology: Pepto: digest/cook
streptococcus: bacterial genus name
micros: small/little
“The digesting bacteria”

47. • Gram positive, obligate anaerobic, small, non-spore forming, non-motile,
asacchrolytic coccus.
• Occurrence: short chains in pairs or individually
• Commensal organisms in humans, living predominantly in the mouth, GIT
and urinary tracts.
• Under immunosuppressed or traumatic conditions these organisms become
pathogenic.
• Peptostreptococcus as known to cause brain, liver and lung abscesses as
well as generalized necrotizing soft tissue infections.

48.  Colony characteristics: Medium sized, greyish colonies with a glistening
surface. Entire margin is seen. Colonies are elevated and convex in shape.
 Two genotypes: smooth and rough
(Rough genotype: Kremer et al 2000)
 They are slow growing bacteria with increasing resistance to
antimicrobial drugs (Haffajee et al, 1998).
 Frequently detected in sites with inc. tissue destruction as compared to
healthy/ gingivitis sites ( van Wilkenhoff et al 2002, Lee et al 2003, etc).
Their levels were seen to be decreased at successfully treated sites
(Haffajee et al 1988a).

49. Virulence :
 P.micros is able to adhere to epithelial cells and to other periodontopathogens,
including Porphyromonas gingivalis and Fusobacte rium nucleatum (Kremer et
al. 1999; Kremer and van Steenbergen 2000).
 P.micros cells have also the ability to bind A. actinomycetemcomitans
lipopolysaccharide on their surface, thus signifi cantly increasing their capacity
to induce TNF-a production by human macrophages (Yoshioka et al. 2005).
 It was also showed that the P.micros cell wall preparation induced intracellular
signaling pathways, leading to an increased production of proinflammatory
cytokines, chemokines and MMP-9 by macrophages
 Other traits like proteolytic and plasmin acquired activity, gelatinase and
hyaluronidase activity is being studied.

50. Prevotella intermedia
 Family: Prevotellaceae
 Etymology: Prevotella, named after the French microbiologist, A.
R. Prevot, a pioneer in anaerobic microbiology

51.  Gram negative, short, anaerobic, non-motile, rod shaped singular
bacteria originally grouped under the bacteroides genus.
 This bacterium is a common commensal in the gingival crevice and is
often isolated from cases of gingivitis and purulent lesions of the mouth.
 Culture characteristics: Strict anaerobes, requires haemin for growth.
Black pigmented bacteria.

52.  Prevotella bacteria colonize by adhering to other bacteria in
addition to epithelial cells , creating a larger infection in an already
infected area.
 Prevotella cells have a natural antibiotic resistant genes, which
prevent extermination.
 It has been detected commonly in cases of ANUG (Loesche et al
1982), progressing sites in CP (Lopez et al 2000) and by
immunohistological methods in the intercellular spaces of
periodontal pockets of rapidly progressive periodontitis subjects
(Hillman et al 1998).
 Elevated serum antibodies to this species have been observed in
some subsets of refractory periodontitis (Haffajee et al 1988b).

53. P intermedia and pregnancy
 Kornman and Loesche showed that during pregnancy increased levels of
progesterone and estrogen paralleled gingival conditions and proportions
of P.intermedia.
 Jansen et al demonstrated a 55 fold increase in the proportion of
P.intermedia in pregnant women when compared to non-pregnant
controls.
 Whereas Johnsson et al demonstrated no difference in the levels of
P.intermedia in pregnant women and controls.

54.  Strains of P.intermedia that have identical phenotype have been
separated into 2 species: P.intermedia and P.nigrescens (Shah &
Gharbia 1992).
 Newly described species: Prevotella pallens (Kononen E et al 2000)
 Newer studies which discriminate between the two sp. might
strengthen the relationship of one or both the sp. to periodontal
disease pathogenesis.

55. Virulence:
 In vitro invasion of Prevotella intermedia to human gingival epithelial cells has been
observed (Dorn et al. 1998), and intracellular division of Prevotella intermedia in cultured
human gingival fibroblasts has been observed by Dogan et al. (2000).
 Prevotella intermedia induced proinflammatory cytokine expression in human gingival
epithelial cells (Sugiyama et al. 2002) and human periodontal ligament (hPDL) cells
(Yamamoto et al. 2006; Guan et al. 2006).
 In P. intermedia, several proteases have been described, among them being trypsin-like
serine proteases, a dipeptidyl peptidase IV, CPs (Shibata et al. 2003; Guan et al. 2006;
Deschner et al. 2003) and a new cysteine protease from the cysteine-histidine-dyad
class, interpain A (Mallorquí-Fernández et al. 2008).
 Prevotella intermedia also possess various types of fi mbriae (surface appendages).
Some of these surface structures mediate the adherence of the organism to several
mammalian erythrocytes, resulting in the agglutination of the erythrocytes (Leung et al.
1999).

56. Fusobacterium nucleatum
 Family: Fusobacteriaceae
 Etymology: fusus: spindle
bacterios: bacteria

57.  Filamentous, facultative anaerobic, gram-negative, non-spore forming
oral bacterium found in the normal flora of the mouth.
 Its is an oral bacterium indigenous to the human oral cavity and plays a
role in periodontal disease.
 Classic cigar-shaped cells with pointed ends. Splindle-shaped or
fusiform rods with variable lengths.
 Key component of plaque due to its abundance and ability to
coaggregate with other species in the oral cavity.

58.  Culture characteristics: Grows best on trypticase containing
medium, peptone and yeast extract. They obtain energy from
fermentation of sugars or amino acids and produce butyric acid
as a by-product.
 F nucleatum is the most common isolate in subgingival plaque
samples comprising appx 7-10% of total isolates from different
clinical conditions. (Dzink et al,1985 and Moore et al,1985)
 F.nucleatum is prevalent in subjects with periodontitis (Boutaga,
2006) and periodontal abscesses (Herrera, 2000) and decreased
after successful periodontal therapy ( Van der Velden, 2003).

59.  High number and frequency in periodontal lesions.
 Fusobacteria have a potent LPS.
 Production of tissue irritants and its synergism with other bacteria in
mixed infection.
 As fusobacteria can remove sulphur from cystiene and methionine to
produce odoriferous hydrogen sulfide and methyl mercaptan.
 OMP are of great interest with respect to coaggregation, cell mutation
and antibiotic susceptibility.
 Ability to form a bridge between early and late colonizers.

60. Virulence:
 The ability to form a biofi lm and coadhere could be an important
virulence mechanism, and may explain the fi nding in a study on alkali-
resistant bacteria that F. nucleatum is capable of surviving at pH 9.0 (Zilm
and Rogers 2007).
 It was recently also suggested that F. nucleatum facilitates invasion of host
cells by P. gingivalis (Saito et al. 2008).
 Apart from its metabolic versatility, its cell-surface properties enable it to
attach to epithelial cells, collagen, gingival epithelial cells.

61. Campylobacter rectus
 Family: Campylobateraceae
 Etymology: Kampulos: bent
Bacter: bacterium

62.  The campylobacter genus contains medically important species that are
important human pathogens and once classified as Vibrios.
 Gram negative, anaerobic, capnophilic, short, motile vibrio with a single
polar flagellum.
 It was first described as a member of vibrio corroders, a group of short rods
that formed small convex, “corroding” or “pitting” colonies on blood agar
plates.
 The organism is unusual in that it utilizes hydrogen or formate as its energy
source.

63.  C. rectus is strongly associated with periodontal disease.
 It has an un usual and distinctive 150kDa protein para-crystalline
cell surface which forms a S-layer, postulated as an important
virulence factor.
 C. rectus helps to initiate periodontitis by increasing the expression
of pro-inflammatory cytokines.
 C. rectus is widely distributed in the subgingival sites of primary,
mixed and permanent dentition (Umeda 2004).

64.  C.rectus has been shown to be present in higher numbers in samples
from diseased sites as compared to healthy sites (Moore et al,
Papapanau et al, Ihara et al, Suda et al) or in site that were converting
from periodontal health to disease (Tanner et al).
 Tanner, Haffajee, Levy, Colombo - found to be in lower numbers after
successful periodontal therapy.
 Gajardo et al (2005) - higher numbers in cases with GAP than in
other forms of periodontitis.
 Virulence: Like AAC, C.rectus has shown to produce a leukotoxin
(Gillespie 1992). It is capable of stimulating human gingival
fibroblasts to produce IL-6 and IL-8.

65.  Systemic connection
• Elevated IgM antibody to C.rectus in fetal chord blood has
been associated with increased rate of prematurity (Madianos
et al).
• Increased levels of C.rectus along with Peptostreptococcus
micros in subgingival plaque of pregnant women was
associated with an increased risk of pre-term low birth
weight (Buduneli et al,2005).
• C.rectus along with other species has been detected in
atherosclerotic vessels (Fiehn et al, 2005).

66. Eubacterium nodatum
 Family: Eubacteriaceae
 ATCC no: 33099
 Etymology: eu: good/beneficial
bacterium: bacteria
nodum: entangled

67.  Gram positive, strictly anaerobic, regular or irregular rods.
 Difficult to cultivate and grow better on roll tubes than on blood agar plates.
 Difficult to culture and isolate, due to which paucity of information relating to
their association with periodontitis.
 E nodatum, E brachy, E timidum are the most common species associated with
destructive periodontal disease.

68.  Han et al found that Eubacterium species were the only organism
that were significantly increased in Chinese localized early-onset
periodontitis patients.
 Haffajee et al in 2006 in their study confirmed the strong
association of Pg and T forsythis with chronic periodontitis and
emphasized a strong association of E nodatum and T denticola with
these organisms.
 Moore at al found increased levels of E nodatum at diseased sites.
They were detected at 39% of diseased sites and comprised 1.3% to
5.7% of the flora, which was an increase of 31 to 68 times over
health.

69. “Red complex”:
Porphyromonas gingivalis
Tannerella forsythia
Treponema denticola

70. Porphyromonas gingivalis
 Family: Porphyromonadaceae
 ATCC number: 33277
Historical perspective:
 Darveau- “Bonafide periopathogen”.
 Initially only one genera ie. Bacteroides was known, Later Bacteroides,
Fusobacterium, Leptotrichia
 Pg was delineated from B.melaninogenicus (Burdon 1928)
 Basonym of Pg: Bacteroides gingivalis (Coykendall et al in 1980)
 Termed by Shah and Collins in 1988.

71.  Gram negative, non motile, short
non-spore forming, obligate
anaerobic, pleomorphic rods.
 Site specificity: Normal habitat of
Pg in oral cavity is gingival sulcus
and is not usually found in the
supragingival plaque. Von
Winkelhoff showed the absence of
Pg in supragingival plaque of
patients with bone loss despite high
levels in subgingival plaque.

72.  Culture characteristics: Grown anaerobically with dark pigmentation on
media containing lysed blood (blood agar).
 The colonies are initially white to cream coloured but become pigmented due
to concentration of protoheme into a deep red to black colour.
 When grown on carbohydrates and proteins they produce acidic end
products such as butyric acid, propionic acid and acetic acid.

77.  Transmission: vertical and horizontal
 Due to difficulties in detecting Pg from children by culture
methods, there is less published data on vertical transmission.
 Tiute-McDonnell et al (1997) used PCR assay to detect oral Pg
from members of 104 randomly selected multi-generation
families. Results showed that finding an infected family member
substantially, increased the relative risk of detecting Pg from other
family members.

78. Horizontal transmission: Can occur between siblings or spouses.
 Two studies, Petit et al (1993) & Saarrela (1996) both reported that siblings
harbored identical genotypes of Pg.
 The reasons for the strain identity in siblings may be that the siblings have
transmitted the strain to each other or that each sibling has acquired the strain
directly from bacterium-positive parent.
 However, the study designs do not permit distinction between the horizontal and
vertical modes of transmission

79.  Biochemical properties:
• Colonizes where oxygen tension is low but nitrogenous substances are present in
abundance. Subgingival environment is ideal as endogenous nutrients are rich in
peptides and amino acids.
• Production of large amounts of protoheme is the characteristic of the genus Pg.
More the concentration of hemin the more the virulence potential.
• Arginine is the primary substrate of this species and can synthesize vit B12.
• This species has been shown to produce trypsin-like proteolytic activity (BANA
positive) (Loesche et al 1992).

81. Tannerella forsythia
 Family: Porphyromonadaceae
 ATCC No: 43037
 Gram negative, spindle-shaped, highly pleomorphic rod.

82. Historical aspect:
First isolated in 1979 by Tanner
et al at the Forsyth Institute from
subjects having adv periodontitis
and was described as “Fusiform
Bacteroides”
The species name was given to
honour the institute where the
bacteria was first cultured and
was initially called as B.forsythus
The genus name was given after
Anne Tanner who described the
sp and was thereafter renamed
as Tannerella forsythensis

83.  Colony characteristics: highly difficult to culture and it takes appx
7-14 days for minute colonies to develop. The growth of Tf was
shown to be enhanced by co-cultivation with Fn (Socransky et al
1988).
 The growth of Tf is stimulated by the addition of N-
acetylmuramic acid in the medium.
 The cells are regularly shaped short gram-negative rods rather
than pleomorphic cells with a slightly convex border and a
depressed center (Tanner and Izard 2006).

84.  Structural characteristics:
• Tf has a distinct ultrastructure. As other gram-negative species it
shows the presence of a serrated S-layer that is easily visible by EM.
This S layer contributes to the pathogenecity of Tf.
• It has shown to produce trypsin like proteolytic activity.
• The S-layer has been shown to mediate hemeagglutination,
adhesion/invasion of epithelial cells, murine subcutaneous abscess
formation.
• Tf in co-cultures of macrophage and epithelial cells leads to the
expression of pro-inflammatory cytokines, chemokines, PGE2 and
MMP-9 (Bodet et al 2006).

85.  T.forsythia biofilm formation and maintenance: It forms
biofilms with Fn. The structure and thickness of Tf biofilms is
influenced by Fn.
 Both sps co-aggregate when in planktonic forms and this
interspecies binding appears to be critical in the formation of
Tf-Fn biofilms
 Host response to Tf: Tf produces a cysteine protease encoded
by the prtH gene, which plays a role in the transition from
commensal organisms to pathogen.
 Higher levels of these genes were associated with significant
future attachment loss.
 It also expresses a cell surface associated protein designated as
BspA, which is an important virulence factor. This interacts
with monocytes through TLR signaling.

87. Treponema denticola
 Family: Spirochaetaceae
Etymology: Trepos: to turn
Nemo: thread-like
dentos: tooth
cola: to inhabit

88.  Spirochetes are spiral shaped bacteria with periplasmic
flagella that originate at opposite poles of the bacterium and
overlap near the middle of the cell.
 Spirochetes are gram negative, anaerobic, helical shaped,
highly motile microorganisms that are common in
periodontal pockets.
 Spirochetes have been considered as possible periodontal
pathogens since the late 1800’s and in 1980’s a resurgence of
interest for using spirochetes as diagnostic indicators of
disease activity and /or therapeutic efficacy was developed.

89.  Culture characteristics: There are presently 10 cultivable
species of spirochetes (Ellen and Galimanas 2005). These
species require very complex media. Infusions of animal
organs, trypsin digests of casein, various fatty acids and growth
factors and serum.
 Spirochetes are difficult to culture and are hence Treponema
are seen under dark field microscopy.

90.  Healthy sites showed no or very few spirochetes, sites with
gingivitis showed low to moderate levels and deep pockets
harbored large number of these organisms.
 Spirochetes are known to be localized in areas adjacent to the
epithelial lining of the periodontal pocket. This facilitates in
attachment and invasion into the adjacent tissues.
 Spirochetes such as Treponema have been shown to be at the
forefront of periodontal lesions as demonstrated in sections of
undisturbed plaque samples (Noiri et al 2001).

91.  T.denticola was found to be more common in periodontitis
sites as compared to healthy sites, more common in
subgingival than supragingival plaque (Albandar et al1997,
Haffaajee et al 1998, Yuan et al 2001).
 They were also seen elevated in healthy sites which
progressed to gingivitis (Riviere et al 1988).
 It was shown to decrease in number in periodontally treated
sites but not change or increase in non-responsive sites
(Simonson et al 1992).

92.  The “pathogen-related oral spirochetes” (PROS) were the most
frequently detected spirochetes in supra- and subgingival plaque
of periodontitis patients.
 The PROS were also detected in plaque samples from ANUG
and tissue biopsies from ANUG lesions using
immunohistochemical techniques (Riviere et al 1991a).
 PROS were also shown to have the ability to penetrate a tissue
barrier in in vitro systems.

93.  Virulence factors:
• Tissue invasion is the hallmark of spirochetal infections, whether the
portal of entry is the skin, genital mucosa or periodontium.
• The virulence factors that determine the invasiveness are the various
proteins involved in the synthesis and energetics of flagellar motility,
chemotaxis proteins and the chymotrypsin-like protease, dentilisin.
• The serine protease, dentilisin, has a wide range of protein substrates
including fibronectin, laminin and fibrinogen (Ishihara et al 1996).
• Dentilisin has the capacity to activate host MMP’s that may degrade
ECM. (Encoded by the prtP gene)

94. • The inhibitory mechanism has been traced to a T.denticola immunosuppressive
protein Sip which induces irreversible arrest of t-cells in the G1 phase of the cell
cycle and the subsequent apoptosis.
• Major OMP’s include cytoskeletal reorganization, cell shrinkage, membrane
blebbing and loss of volume.
• Porins are typical bacterial surface proteins which form channels through which
ions and small nutrient solutes may be transported across the lipid rich outer
membrane.
• The major outer sheath protein antigen (Msp) of T.denticola is one of the better
characterized virulence determinants of oral treponemes.
• T.denticola has a very well-characterized enzyme cystalysin which catabolizes L-
cysteine to produce pyruvate, ammonia and hydrogen sulfide.

95.  Successful treatment of periodontal infections is accompanied
by a decrease in the numbers and proportions of oral
spirochetes as a group and individual species.
 This reduction is known to be so consistent that it has been used
in some studies as a measure of compliance in determining
whether subjects used the prescribed antibiotics or not (Loesche
et al 1993).

96. Novel pathogens:
 Dialister pneumosintes
 Filifactor alocis
 T. lecithinolyticum
 Solobacterium moorei
 Cryptobacterium curtum
 Mitsuokella dentalis
 Salmonella sputigena
 Synergistes
 Porphyromonas endodontalis
 Slacia exugia

97. Cryptobacterium curtum
 Cells are short Gram-positive rods. Occasionally
Gram-variable are in stationary phase.
 Obligatory anaerobic, non-motile and non-
sporing. Catalase negative and asaccharolytic.
 Individual cells occur singly or in masses.
 C. curtum is characterized as opportunistic
pathogen with a typical occurrence in the oral
cavity, involved in dental and oral infections such
as periodontitis, inflammation and abscess.
 Nakazawa et al. in their study proposed novel
Eubacterium-like isolates, from the periodontal
pocket of an adult patient with periodontal
disease and necrotic dental pulp and suggested
that it should be classified in a new genus and
species C. curtum.

98. Filifactor alocis (A thread like organism
inhabiting in a furrow)
 Filum-thread; factor- a maker; alox - a
furrow; referring to its isolation from a
crevice of the gums.
 Originally isolated in 1985 from human
gingival crevice as Fusobacterium alocis, it
was reclassified into genus Filifactor based
on 16S rRNA and named as F. alocis.
 It is a fastidious, Gram-positive, obligatory
anaerobic rod possessing trypsin-like
enzymatic activity similar to P. gingivalis
and T. denticola. F. alocis has the ability to
survive in periodontal pocket and share
common virulence properties with
Fusobacterium

99.  This organism has been found in elevated numbers in AgP (77.8%)
and CP (76.7%) compared with periodontally healthy individuals due
to its potential to withstand oxidative stress and inflammatory
microenvironment provided by periodontal pocket
 Filifactor is attributed as the second most prevalent in CP and third
most prevalent in generalized aggressive periodontitis and
proposed to be an excellent marker organism for periodontal
disease.
 A study by Dahlén and Leonhardt in 2006 concluded that F. alocis
should be added to the 12 species used for routine diagnostics of
periodontitis-associated bacterial flora

100. T. lecithinolyticum (Lekithos-egg yolk-
effecting the breakdown of egg yolk)
 It is a small saccharolytic spirochaete possessing
phospholipase A and C activities.
 T. lecithinolyticum activates matrix metalloproteinase-2 in
human gingival fibroblasts and periodontal ligament cells
and induce activation of osteoclast by a prostaglandin E2-
dependent mechanism.
 Major surface protein and prcA-prtP gene are considered as
virulence factors of this organism, which exhibit
chymotrypsin like protease activity stimulating various
inflammatory cytokines namely interleukin-1, 6, 8 and
intercellular adhesion molecule-1.
 The prevalence of this organism is more pronounced in
rapidly progressive periodontitis and CP compared to
healthy individuals.

101. Mitsuokella dentalis
 M. dentalis is a nonmotile, nonspore forming, Gram-negative rods
approximately 0.7 μm wide by 1-2 μm long.
 Flynn et al. reported that M. dentalis is a constituent of the pathogenic
microbiota in human periodontitis.
 M. dentalis has a low virulence potential as a periodontal pathogen. It does
not have the ability to activate latent human fibroblast type, neutrophil
interstitial procollagenases that lead to degradation of Type I collagen that
is an essential step for periodontal tissue invasion and disease progression.

102. Dialister pneumosintes
 As small, Gram-negative rod that grows with
punctiform, circular, convex, clear, transparent,
shiny, smooth colonies on blood agar.
 Activate immune-mediated cells to release
proinflammatory cytokines, prostaglandins, matrix
metalloproteinases (MMP'S) that eventually lead to
periodontal connective tissue destruction, and
resorption of alveolar bone.
 D. pneumosintesis reported to be significantly
higher in prevalence among patients with refractory
periodontitis, rapidly progressing periodontitis
suggesting its role in disease pathogenesis

103. Selenomonas sputigena
 S. sputigena has evolved as a chief periodontal pathogen due to its
virulence factors and its key role in coaggregation and maturation of
plaque.
 Lipopolysaccharides of S. sputigena could be one of the multitude of
pathogenic factors involved in periodontal disease.
 It induces release of interleukin 6 (IL-6), IL-1α in macrophages thereby
provoking inflammation.
 Its association with chronic periodontitis was confirmed by its high
prevalence among periodontal pocket microbiota

104. Solobacterium moorie
 S. moorei can be a major source of malodorous compounds in halitosis by
producing VSCs through a process involving the β-galactosidase activity of
the bacterium and an exogenous source of proteases
 It adheres to oral epithelial cells through adhesins. It can also induce the
secretion of IL-8 in gingival epithelial cells, promote osteoclast
differentiation, and inhibit proliferation of osteoblasts

105. Viruses :
 Periodontal diseases have a multifactorial etiology.
 They encompass a variety of infectious entities with various clinical
manifestations, natural histories and response to treatment.
 Bacteria have long been proposed as inciting agents of gingival inflammation
and tissue injury, both of which underlie the pathogenesis of periodontitis.
 In the past decade the viral etiology of periodontal disease is gaining interest.
 Herpes virus seems to be the most important DNA viruses in periodontal
pathology.

106.  Present in gingival tissue, gingival cervicular fluid and subgingival plaque, in the presence of
periodontal disease (Cappuyns et al. 2005).
 Coinfection of two viruses like EBV and CMV is also responsible for progressive periodontitis.
 HHV also cooperate with specific bacteria in periodontal tissue breakdown. A co-infection of
active Herpes viruses and periodontopathic bacteria may constitute a major cause of
periodontitis and explain a number of the clinical characteristics of the disease.
 The ability of an active virus infection to alter the periodontal immune responses may
constitute a crucial pathogenetic feature of periodontitis. An active viral infection can exert
direct cytopathogenic effects on key cells of the periodontium; induce the release of
proinflammatory cytokines

109. Fungi :
 Although the main reservoir of candida sp is believed to be the buccal mucosa
, the micro organism can co-aggregate with bacterial in subgingival and
adhere epithelial cells.
 Such interactions are associated with with capacity of candida spp to invade
gingival conjuctive tissue, and may be important in micrbial colonization that
contribute to progression of oral alteration caused by diabetes mellitus, certain
drugs and immunosuppressive disease like AIDS.
 In addition immune defecience can result in proliferation of candida spp and
germination of forms that are more virulent and have higher capacity to
adhere to and penetrate cells in host tissue
 Baros et al investigated the genetic diversity and exoenzymes C. albicans and
C. dubilinesis isolated from systemically healthy patients with periodontitis
 Mucormycosis is seen as most destructing bone disease.

111. Organisms associated with other
periodontal diseases:
Necrotizing periodontal diseases:
 Microbial samples from NPD lesions have demonstrated a constant and a variable
part of the flora.
 The"constant flora" primarily contained Treponema sp., Selenomonas sp.,
Fusobacterium sp. and P. intermedia, and the "variable flora" consisted of a
heterogeneous array of bacterial types (Loesche et al. 1982).
 The role of HCMV has also been demonstrated in the pathogenesis.

112. Acute necrotizing ulcerative gingivitis (ANUG):
 Treponema species 32%; B intermedius 24%; Selenomonas species
6%; Fusobacterium species 3% (Loesche et al.1982).
Abscesses of the periodontium:
 Periodontal abscesses are acyte lesions that result in rapid
destruction.
 Significant numbers of F nucleatum, P intermedia, P gingivalis, P
micros and T forsythia are found.

113. Linear gingival erythema:
 Both fungi like C. albicans, and a number of periopathogenic
bacteria consistent with those seen in conventional periodontitis, i.e.
Pg, Pi, AAC, F nucleatum and C rectus (Murray et al. 1988, 1989,
1991).
Peri-implantitis:
 Opportunistic periodontal pathogens such as AAC, Pg, Tf, PI, P
micros and F nucleatum have been identified in association with peri-
implantitis (van Winkelhoff & Wolf 2000, van Winkelhoff et al. 2000).
 Partially edentulous peri-implant pockets – rapid appearance of
spirochetes.

114. Beneficial species:
Can affect the pathogenic bacteria by
 Passively occupying a niche
 Actively limiting a pathogen’s ability to bind to host surfaces
 By adversely affecting the vitality and growth of pathogen
 By affecting the ability of pathogen to produce virulence factors
 By degrading the virulence factors produced by pathogen
In periodontal microbiology, a bacterial strain is considered beneficil when its
prevalence is high than in disease.
s. sanguis, s. mitis, veilonella, capnocytophaga ochracea, actinomyces are
recovered from periodntally healthy sites.

115. Conclusion:
 The key to successful periodontal therapy and maintenance is elimination or reduction
of pathogenic bacteria from periodontal pockets and establishment of microbiota
compatible with periodontal health.
 On the other hand, the presence of certain microorganisms in the periodontal pockets
(P. gingivalis, T. forsythia, P. intermedia, Treponema denticola, A.
actinomycetemcomitans, Candida spp.) allows the evolutional potential of the
periodontal disease to be estimated.
 Therefore, the identification of subgingival pathogenic strains in gingivitis and
periodontitis could aid in the better differentiation of the different periodontal diseases.
 That is why, it is important to perform both qualitative and quantitative determination of
well-known periodontopathogens in the periodontal pockets. In addition, prediction of
the disease progression would allow targeted preventive therapy.
 Further emphasis on role of virus , fungi and archea has to be established in
pathogenesis of periodontal disease.
 Recognition of beneficial species may open up new strategies like probiotics and
microbial replacement therapies.

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