Dental PLAQUE

By Dr. Nitika JainPost Graduate Student

Physiological approaches to the control of oral biofilms

Physiological strategies for the control of oral biofilms

Prevention of low pH

Bacterial homeostasis in plaque biofilms would be less likely to be disrupted if the frequency and depth of acidic conditions following sugar intake could be reduced. This could be achieved physiologically by (a) inhibitors of acid production, (b) consumption of food or drinks containing

non-fermentable sweeteners, and (c) the local generation of base (alkali) in

plaque.

Virulence Factors of Periodontopathogens

Virulence factors of periodontal micro-organisms can be subdivided into (1) factors that promote colonization

(adhesins), (2) toxins and enzymes that degrade host tissues, (3) mechanisms that protect pathogenic bacteria from the host.

Adhesive Surface Proteins and Fibrils

Fimbriae, or pili, are polymeric fibrils composed of repeating subunits that can extend several microns from the cell membrane.

Pili were once thought to be unique to gram-negative bacteria but have now been identified in several gram-positive organisms, including streptococci and actinomyces.

Strains of P. gingivalis produce two types of fimbriae, known as the major and minor fimbriae.

Factors that Promote Tissue Destruction

The gingipains belong to the cysteine protease family, which utilize an active site cysteine residue for catalysis.

Gingipains are classed as “Arg-gingipains” (RgpA and RgpB) or “Lys-gingipain” (Kgp), based on their ability to cleave Arg-Xaa or Lys-Xaa peptide bonds (Xaa represents any amino acid).

Gingipains are multifunctional proteins that play important roles in adhesion, tissue degradation, and evasion of host responses.

Hyperleukotoxic strains include the JP2 clone of A. actinomycetemcomitans, which is uniquely associated with localized aggressive periodontitis.

Strategies for Evading Host Immunity

Pathogenic bacteria have many and varied strategies for evading or subverting the host immune system, including (1) the production of an extracellular capsule, (2) proteolytic degradation of host innate

and/or acquired immunity components, (3) modulation of host responses by binding

serum components on the bacterial cell surface,

(4) invasion of gingival epithelial cells.

Microorganisms Associated With Periodontal Health

The bacteria associated with periodontal health are primarily gram positive facultative species and members of the genera Streptococcus and Actinomyces (e.g., S. sanguis, S. mitis, A. viscosus, and A. naelslundii).

Small proportions of gram negative species are also found, most frequently P. intermedia, F. nucleatum, and Capnocytophaga, Neisseria, and Veillonella spp.

Microscopic studies indicate that a few spirochetes and motile rods also may be found

Certain bacterial species have been proposed to be protective or beneficial to the host, including S. sanguis, Veillonella parvula, and C. ochracea. They are typically found in high numbers at periodontal sites that do not demonstrate attachment loss (inactive sites) but in low numbers at sites where active periodontal destruction occurs. Socransky SS, Haffajee AD; JP 63: 322, 1992.

These species probably function in preventing the colonization or proliferation of pathogenic microorganisms.

One example of a mechanism by which this may occur is the production of H2O2 by S. sanguis; H2O2 is known to be lethal to cells of A. actinomycetecomitans. (Hillman JD, Socransky SS “the relationships between streptococcal

species and periodontopathogenic bacteria in human dental plaque” Arch Oral Biology 30: 791; 1985)

Microorganisms Associated with Specific Periodontal Diseases

Microbiologic procedures clearly demonstrated that the number and proportions of different subgingival bacterial groups varied in periodontal health when compared with the disease state . The total number of bacteria, determined by microscopic counts per gram of plaque, are twice as high in Periodontally diseased sites than in healthy sites.

Microorganisms Associated with Specific Periodontal Diseases

On culturing, bacteria from healthy periodontal sites it consists mainly of Gram positive facultative rods and cocci

( 75%) In gingivitis, (44%) and periodontitis ( 10 – 13%)

Increase in proportions of gram negative rods,13% to 40% in gingivitis and 74% in advanced periodontitis.

Gingivitis:

Model system for experimental gingivitis – described by Loe et al (Loe H; JP 36: 177, 1965) and Theilade et al.

Gingivitis:

The bacteria found in naturally occurring dental plaque-induced gingivitis (chronic gingivitis) consist of roughly equal proportions of gram-positive (56%) and gram-negative

(44%) species, facultative (59%) and anaerobic (41%) microorganisms.

Predominant gram-positive species include S. sanguis, S. mitis, S. intermedius, S. oralis, A. viscosus, A. naelslundii, and Peptostreptococcus micros.

The gram-negative microorganisms are F. nucleatum,P. intermedia, V. parvula, as well as Hemophilus, Capnocytophaga and Campylobacter spp

Pregnancy associated gingivitis : This condition is accompanied by

increases in steroid hormones in crevicular fluid and increases in the P.intermedia, which uses the steroid as growth factors.( Kornman KS , Loesche WJ ; Infect Inhuman 35: 256 , 1982)

Chronic Periodontitis:

Microscopic examination of plaque from sites with chronic periodontitis have consistently revealed elevated proportions of spirochetes .

Cultivation of plaque microorganisms from sites of chronic periodontitis reveals high percentages of anaerobic (90%) gram-negative (75%) bacterial species

In chronic periodontitis, the bacteria most often cultivated at high levels include P. gingivalis, B. forsythus, P. intermedia, C. rectus, Eikenella corrodens, F. nucleatum, A. actinomycetemcomitans, P. micros, and Treponema and Eubacterium.

When Periodontally active sites (i.e., with recent attachment loss) were examined in comparison with inactive sites (i.e., with no recent attachment loss), C. rectus, P. gingivalis, P. intermedia, E. nucleatum, and B. forsythus were found to be elevated in the active sites.

Detectable levels of P. gingivalis, P. intermedia, B. forsythus, C. rectus, and A. actinomycetemcomitans are associated with disease progression and elimination of specific bacterial pathogens with therapy is associated with an improved clinical response.

Recent studies have documented an association between chronic periodontitis and viral microorganisms of the herpes viruses group, most notably Epstein-Barr Virus-1 (EBV-1) and human cytomegalovirus (HCMV). ( Contreras A, Slots J: JPR; 35: 3, 2000)

Presence of subgingival EBV-1 and HCMV are associated with high levels of putative bacterial pathogens, including P. gingivalis, B. forsythus, P. intermedia, and T. denticola

Localized Aggressive Periodontitis:

Localized aggressive periodontitis develops around the time of puberty, is observed in females more often than in males, and typically affects the permanent molars and incisors.

The microbiota associated with localized aggressive periodontitis is predominantly composed of gram-negative, capnophilic, and anaerobic rods.

Microbiologic studies indicate that almost all LJP sites harbor A. actinomycetemcomitans ( Moore WE ; JPR: 22; 235, 1987)

Other organisms found in significant levels include P. gingivalis, E. corrodens, C. rectus, E nucleatum, B. capillus, Eubacterium brachy, and Capnocytophaga spp. and spirochetes.

Herpesviruses, including EBV-1 and HCMV, also have been associated with localized aggressive periodontitis. (Michalowicz BS, Ronderos M; “ human herpes virus and P.

gingivalis are associated with juvenile periodontitis” JP 71: 981, 2000)

After initial colonization of the first permanent teeth to erupt ( molars and incisors), immune defenses are stimulated to produce opsonic antibodies.

Bacteria antagonist to A.a may colonize the periodontal sites and inhibit A.a from further colonization.

A.a may loses its toxin producing capacity for unknown reasons.

A defect in cementum formation may be responsible for the localization of the lesions.

Necrotizing Periodontal Diseases:

Acute inflammation of the gingival and periodontal tissues characterized by necrosis of the marginal gingival tissue and interdental papillae.

Microbiologic studies indicate that high levels of P. intermedia and spirochetes are found in necrotizing

ulcerative gingivitis lesions. Spirochetes are found to penetrate necrotic tissue

and apparently unaffected connective tissue.▪ (Lisgarten MA, Socransky SS; “ relative distribution of bacteria

at clinically healthy and periodontally diseased sites in humans” JCP 5:115; 1978)

Periodontal abscess

Periodontal abscess are acute lesions that result in very rapid destruction of the periodontal tissues.

Often occur in patients with untreated periodontitis but can be seen in maintenance phase or in the absence of periodontitis also ( for example associated with impaction of a foreign objects or with endodontic problems)

Clinical symptoms :

Periodontal pathogens are commonly found in significant numbers in periodontal abscesses include F. nucleatum, P. intermedia, P. gingivalis, P. micros, and B. forsythus. (Newman MG, Socransky SS ; JPR 12: 120, 1997)

Periimplantitis Periimplantitis refers to inflammatory process affecting

tissues around already osseointegrated implant & results in loss of supporting bone.

Healthy periimplant pockets are characterized by high proportions of coccoid cells, low ratio of anaerobic & aerobic species, low numbers of peiodontal pathogens.

Implants with periimplantitis reveals species such as A.a, P. gingivalis, T. forsythia, P. micros, C. rectus, Fusobacterium, & Capnocytophaga are isolated from failing implants.

Other species such as p. aeruginosa, enterobacteriaceae C. albicans, & staphylococci are also detected around implants.

Lee et al. used the cluster method developed by Socransky et al.to analyze the microbiota associated with healthy implants and study the implant and host-related factors able to influence the microbial film.

Rams et al. detected “corn cob” morphology around healthy implants and “brush forms” around failing fixtures.▪ (Rams TE, Link CC Jr (1983) Microbiology of failing

dental implants in humans: electron microscopic observations. J Oral Implantol 11:93–100)

Key characteristics of specific periopathogens

AGGREGATEBACTER ACTINOMYCETECOMITANS:

Forms: multiple biotypes 5 serotypes ( a to e) on the basis of difference in polysaccharides.

Strains from patients in Africa have an increased leucotoxin production.

Culture condition & identification

It grows as white, translucent, smooth, non hemolytic colony on blood agar.

Preferably identified on a specific growth medium ( with vancomycin and bacitracin as antibiotics to suppress other species)under 5 to 10% CO2.

Small, non-motile, Gram-negative saccharolytic, capnophilic, round-ended rod that forms small, convex colonies with a "star-shaped" center

Special pathogenic characterstics

Lipo polysaccharide

Endotoxin

Leukotoxin it forms the pore

in neutrophil granulocytes,

monocytes, and lymphocytes

which consequently die of osmotic

pressure(Baehni et al.

1979), a cytolethal distending

toxin .

Collegenase

destruction of

connective tissue

Protease

able to cleave Ig

G

A. actinomycetemcomitans has been shown, in vitro, to have the ability to invade cultured human gingival epithelial cells (Blix et al. 1992, Sreenivasan et al. 1993), human vascular endothelial cells (Schenkein et al. 2000) and buccal epithelial cells in vivo.

Tannerella forsythia:

It is a non motile, spindle shaped, highly pleomorphic rod & gram negative obligate anaeobe.

Culture condition & identification: It takes 14 days to form minute colonies, only under anaerobic conditions & needs several growth factors (N-acetylmuramic acid) from other species (e.g; F. nucleatum).

Pathogenicity: It produces several Proteolytic enzymes that are able to destroy immunoglobulin & factors of complement system. It also induces apoptotic cell death.

This organism was found in higher numbers in sites of destructive periodontal disease or periodontal abscesses than in gingivitis or healthy sites (Lai et al. 1987, Herrera et al. 2000, Papapanou et al. 2000).

Porphyromonas gingivalis:

Is non motile, pleomorphic (coccal to short) rod & gram negative obligate anaerobe.

P. gingivalis is a member of "black-pigmented Bacteroides" group

The black-pigmented colony is an isolate of Porphyromonas gingivalis

Culture conditions & identification

It grows anaerobically , with dark pigmentation ( brown, dark green, or black) on blood agar because of a metabolic end product from blood( hemin)

Virulence factors

proteases• Destruction of

Ig, complement factors, heme- sequestering proteins, degradation of host cell collagemnase inhibition

hemolysin collagenases

P. gingivalis can inhibit migration of PMNs across an epithelial barrier (Madianos et al. 1997) and has been shown to affect the production or degradation of cytokines by mammalian cells (Darveau et al. 1998, Fletcher et al. 1998, Sandros et al. 2000).

Prevotella intermedia & Prevotella nigrescens:

Prevotella group are short , round ended non motile gram negative rods

They grow anaerobically , with dark pigmentation ( brown black colonies) on blood agar.

The dark-pigmentedcolonies are isolates of Prevotella intermedia

The levels have been shown to be particularly elevated in acute necrotizing ulcerative gingivitis ( Loesche et al. 1982)

in certain forms of periodontitis (Tanner et al. 1979, Dzink et al. 1983, Moore et al. 1985, Maeda et al. 1998, Herrera et al. 2000, Papapanou et al. 2000),

Fusobacterium nucleatum:

F. nucleatum is a Gram-negative, anaerobic, cigar shaped bacillus with pointed ends that has been recognized as part of the subgingival microbiota.

It grows anaerobically on blood agar.

Several serotypes are F. nucleatum ss nucleatum F. nucleatum ss

polymorphum F. nucleatum ss vincentii F. periodonticum

F. Nucleatum on specific medium

F. nucleatum is prevalent in subjects with periodontitis (Papapanou et al. 2000, Socransky et al. 2002) and periodontal abscesses (Her rera et al. 2000, Newman MG, Socransky SS ; JPR 12: 120, 1997).

Invasion of this species into human gingival epithelial cells in vitro was accompanied by an increased secretion of IL-8 from the epithelial cells (Han et al. 2000).

The species can induce apoptotic cell death in mononuclear and polymorphonuclear cells (Jewett et al. 2001) and cytokine, elastase and oxygen radical release from leukocytes (Sheikhi et al. 2000).

Campylobacter rectus:

C. rectus is a Gram-negative, short rod, curved ( vibrio) or helical.The motility is due to polar flagellum.

The organism is unusual in that it utilizes H2 or formate as its energy source.

It was first described as a member of the "vibrio corroders", a group of short nondescript rods that formed small convex, "dry spreading" or "corroding" (pitting) colonies on blood agar plates.

C. rectus has been shown to produce a leukotoxin, and is less virulent and less proteolytic than P. gingivalis

Eikenella corrodens:

E. corrodens is a Gram-negative, capnophilic, asaccharolytic, regular, small rod with blunt ends.

E. corrodens has also been found in association with A. actinomycetemcomitans in some lesions of LJP (Mandell 1984, Mandell et al. 1987).

E. corrodens has been shown to stimulate the production of matrix metalloproteinases (Dahan et al. 2001) and IL-6 and IL-8 (Yumoto et al. 1999).

Peptostreptococcus micros:

P. micros is a Gram-positive, anaerobic, small, asaccharolyticcoccus.

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 in combination with either P. intermedia or P. nigrescens could produce transmissible abscesses (van Dalen et al. 1998).

Selenomonas species:

The organisms 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.

The Selenomonas spp. are Gram-negative, curved, saccharolytic rods.

Moore et al. (1987) described six genetically and phenotypically distinct groups isolated from the human oral cavity.

Selenomonas noxia was found at a higher proportion of shallow sites (PD < 4 mm) in chronic periodontitis subjects compared with similar sites in periodontally healthy subjects (Haffajee et al. 1998). Further, S. noxia was found to be associated with sites that converted from periodontal health to disease (Tanner et al. 1998).

Eubacterium species:

E. nodatum, Eubacterium brachy and Eubacterium timidum are Gram positive, strictly anaerobic, small, pleomorphic rods.

They are often difficult to cultivate, particularly on primary isolation, and appear to grow better in roll tubes than on blood agar plates.

Spirochetes:

Spirochetes is a group of spiral, motile organisms.

They are helical rods 5 to 15µm long with a diameter of o.5µm.

They have 3 to 8 spirals.

Spirochete has been implicated as the likely etiologic agent of acute necrotizing ulcerative gingivitis by its presence in large numbers in tissue biopsies from affected sites (Listgarten & Socransky 1964, Listgarten 1965).

The sample isdominated by large spirochetes with the typical cork-screw appearance.

At least 15 species of subgingival spirochetes have been described, spirochetes are combined in a single group or groups based on cell size; i.e. small, medium or large.

Spirochetes includes T. denticola, T. vincentii, T. socranskii, often associated with periodontitis.

Some spirochetes have capacity to migrate within GCF & penetrate epithelium & connective tissue.

Some have capacity to degrade even dentin T. denticola produces Proteolytic enzymes that

can destroy IgA, IgM, IgG, or complement factors.

DENTAL PAQUE

VIRUSES - Contreras & Slots 2000, Kamma et al 2001

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Four major viral families are associated with the main viral oral diseases of adults, as follows:

1. The group of herpesviruses contains eight different members that all are enveloped double-stranded DNA viruses. In the oral cavity, they are related to different ulcers, tumors, and other oral pathoses.

2.Human papillomaviruses are grouped within five genera and are nonenveloped double-stranded DNA viruses. In the oral cavity, they are related to ulcers, tumors, and oral pathoses.

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DENTAL PAQUE 59

3. Picornaviruses are all nonenveloped, single-stranded RNA viruses. In the oral cavity, they are related to ulcers and different oral pathoses

4. Retroviruses. All retroviruses are enveloped single-stranded RNA viruses. In the oral cavity, they are related to different tumors and oral pathoses.12/27/2011

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Yeast

The majority of isolates are Candida, and the most prevalent species is C. albicans. Together with C. albicans, some of the most common opportunistic fungal pathogens in humans are C. tropicalis, C. glabrata, C. krusei, C parapsilosis, C. guilliermondii, and C. dubliniensis.

Etiologic agents for different types of oral yeast infection

Protozoa

Only a few parasites affect the oral cavity, but an increasing body of literature claims that oral protozoa are more common than previously appreciated.

Depending on the type of infection, the parasitic infectious agents can be divided into two categories: 1.those that induce local infections and those that induce systemic infections with indirect effects on the oral cavity. The former group comprises saprophytes (such as Entamoeba gingivalis and Trichomonas tenax) 2.that have the potential to turn into opportunistic pathogens, or free-living amoebae that occasionally become invasive but seldom present clinically.

Archaea

Archaea are single-celled organisms that are distinct from the bacteria as they are from eukaryotes. The role of archaea in oral diseases is only beginning to be explored. Methanogenic archaea produce methane from hydrogen gas (H2)/carbon dioxide (CO2) and sometimes from formate, acetate, methanol, or methylamine. These organisms have been isolated from patients with periodontal disease by enriching cultures with H2 and CO2.

Subsequently, a clear correlation between the presence of archaeal DNA and periodontal disease was established. (Lepp PW, Brinig MM, Ouverney CC, et al: Methanogenic Archaea and human periodontal disease. Proc Natl Acad Sci U S A 2004;) 101:61-76).Despite these findings, virtually nothing is known about the role of archaea in periodontitis.

Archaea have also been detected in endodontic infections, and again their contribution to disease is unknown. (Vianna ME, Conrads G, Gomes BP, et al: Identification and quantification of archaea involved in primary endodontic infections. J Clin Microbiol 2006; 44:12-74.)

Conclusion

The periodontal microbiota is a very complex ecologic system with many structural and physiologic interactions among the resident bacteria and between the bacteria and the host. It is clearly possible that levels of a particular species may be elevated as a result of environmental changes produces by the disease process and may not be a causative agent. It is also difficult to ascertain the specific etiologic pathogens in periodontitis.

References:

Newman, Takei, Klokkevold, Carranza. Carranza’s Clinical Periodontology, 10th Edition and 11th Edition

Lindhe, Lang, Karring. Clinical Periodontology & Implant Dentistry, 5th Edition.

Philip D Marsh, Michael V Martin, Oral Microbiology, 5th Edition.

Perio 2000 - 49:60, 2009 Perio 2000 - Volume 52 Issue 1, Pages 7 - 121

(Feb 2010) “Advnces in dental research” ADR 1997 11: 176

Philip D. Marsh