Microbiology of periodontal diseases

The research on the microbiology of periodontal health and diseases has been the focus of intense investigation for several decades. Microbial biofilm in the gingival sulcus around teeth is the most important cause of periodontal diseases. It is a structured community of bacterial cells enclosed in a self-produced polymeric matrix 1. Although more than 700 different species are present in subgingival microbiota 2-4, only a few of these species are actually involved in the initiation and progression of the periodontal disease process.  Moreover, research on the etiopathogenesis of periodontal diseases has suggested the role of environmental 5, behavioral 6, 7, and genetic 8 risk factors in periodontal disease progression, but most, if not all forms of periodontitis, should be viewed primarily as infectious diseases. Many technological advances have occurred in molecular techniques in the last few decades which have provided us the capability of performing high-throughput analysis of a large number of samples, circumventing some of the limitations of cultural techniques. These techniques are especially useful in studying periodontal microbiology because there are many periodontal pathogens which are not cultivable and require molecular techniques to identify their presence in the bacterial sample. In the following discussion, we shall study the role of microorganisms in the etiopathogenesis of periodontal diseases and details of those microorganisms which have been most commonly associated with periodontal disease progression.

Historical aspect

As already described in the chapter 3 Dental plaque and its development as biofilm, the period from 1880 to 1930 is known as the ‘golden age of microbiology’ 9. Scientists identified four different groups of potential etiologic agents (amoebae, spirochetes, fusiforms, and streptococci) for various periodontal diseases using the techniques available at that time (wet mounts or stained smear microscopy). Researchers suggested the specific plaque hypothesis based on these findings. However, with advancements in bacterial identification techniques, many other bacterial species were identified in dental plaque derived from periodontitis patients. Studies conducted between 1930 and 1970 failed to identify a specific organism as the etiologic agent of periodontal diseases which led to the proposal of non-specific plaque hypothesis, according to which gross accumulation of dental plaque would be necessary and sufficient to cause periodontitis.

Later on, with the advancements in the field of microbiology, immunology, and molecular biology, numerous studies concluded a putative pathogenic role of many bacteria, mainly Gram-negative species in the etiopathogenesis of periodontal diseases. These species include A. actinomycetemcomitans, tannerella forsythia, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus, Fusobacterium nucleatum, and Treponema denticola. Virulence factors produced by these microorganisms have been identified and their role in periodontal destruction is well established. These findings led to the return to the theory of specificity in the microbial etiology of periodontal diseases. Presently, many bacterial species have been identified which contribute to the microbial etiology of periodontal tissue destruction. In the following sections, we shall read about these microorganisms.

 

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Basic structural components of bacterial cell:

Although, bacterial cells are smaller and simpler in structure as compared to eukaryotic cells, the bacteria are an exceedingly diverse group of organisms that differ in size, shape, habitat and metabolism. All bacterial cells are surrounded by at least one membrane, the cytoplasmic membrane enclosing the cytoplasm, but most cells are surrounded in addition by a thick cell wall (the Gram-positive) and another group by a thin cell wall followed by a second membrane, the outer membrane (the Gram-negative), where both membranes are separated by the periplasm. So, Gram-positive bacteria consist of three compartments, the cytoplasm, the cytoplasmic membrane and the extracytoplasm, while Gram-negative bacteria contain two additional compartments, the periplasm, and the outer membrane. Following are the basic structural components of bacterial cells:

  1. Capsule: Found in some bacterial cells, this additional outer covering protects the cell when it is engulfed by other organisms and helps the cell adhere to surfaces and nutrients. It is visible with negative (background) staining. It protects against chemicals and desiccation, store waste products and protects the bacterium from attack by phagocytic cells. It also helps bacteria to form colonies.
  2. Cell wall: In both Gram-negative and Gram-positive bacterial cells, the cell wall is located on the outside of the inner membrane, but is further surrounded by the outer membrane in Gram-negative bacteria. The major components of the bacterial cell wall are long glycan strands that are cross-linked by short peptides containing amino acids in both the D- and L-isoform and the whole ensemble is called peptidoglycan or murein, forming the murein sacculus.
  3. Cytoplasm: A gel-like substance composed mainly of water that also contains enzymes, salts, cell components, and various organic molecules.
  4. Cell membrane or Plasma membrane – Surrounds the cell’s cytoplasm and regulates the flow of substances in and out of the cell. It has a typical unit membrane structure composed of proteins and lipids, basically similar to the membrane that surrounds all eukaryotic cells. In electron micrographs, it appears as a triple-layered structure of lipids and proteins that completely surround the cytoplasm.
  5. Pili: Pili (singular: pilus), also called fimbriae, are hair-like appendages built by protein subunits called pilin or fimbrin and usually extend from the surface of Gram-negative bacteria, with a diameter ranging from 2 nm to 8 nm. They function in bacterial cell-to-cell interactions, adhesion to specific receptors on host cells, uptake or transfer of genetic material and twitching motility (a form of locomotion that is powered by extension and retraction of the pilus filament). They also provide receptors for bacteriophages.
  6. Flagella: Used for the locomotion by many motile bacteria. It is a rigid, hollow cylinder of protein, the base of which rotates propelling the cell along.
  7. Ribosomes: Cell structures responsible for translation during protein synthesis.
  8. Plasmids: Plasmids are small circular pieces of DNA which are present in some bacteria, containing genes, additional to those in the chromosome; some bacteria contain more than one plasmid. Plasmids are known to carry genes which may help the bacterium to survive in adverse conditions and carry genes for antibiotic resistance. Plasmids can be transferred to another bacterium by conjugation, transformation or transduction.
  9. Nucleoid region: Area of the cytoplasm that contains the single bacterial DNA molecule.
  10. Endospore: It is a hard and resistant outer covering formed by some bacteria, which ensures their survival in severe conditions of drought, toxic chemicals and extremes of temperature, e.g. Bacillus anthracis spores, which cause the disease anthrax, are known to be viable even after 50 years in the soil.
  11. Mesosomes: Tight infoldings of the plasma membrane that may be the site of respiration and involved in cell division and the uptake of DNA; they might be an artifact of preparation for electron microscopy.

Bacterial taxonomy

Systematic classification and categorizations of organisms into ordered groups is called taxonomy. A working knowledge of bacterial taxonomy is essential for understanding various bacterial species involved in the periodontal disease progression. Although higher organisms are classified according to their evolutionary pathways (i.e. phylogenetically), bacteria cannot be similarly categorized because of the insufficiency in their morphological features. Bacterial classification is somewhat artificial in that they are categorized according to phenotypic features, which facilitate their laboratory identification. These comprise

  • Morphology (Cocci, Bacilli, spirochetes etc.)
  • Staining properties (Gram-positive, Gram-negative etc.)
  • Spore formers or non-spore
  • Cultural requirements (aerobic, facultative anaerobic, anaerobic etc.)
  • Biochemical reactions (saccharolytic and asaccharolytic according to sugar fermentation reaction)
  • Antigenic structure (serotypes).

The Gram positive bacteria

 

The Gram negative bacteria

Establishing an organism as periodontal pathogen

Periodontal diseases are caused by a variety of microorganisms that reside at or below the gingival margin in the form of plaque biofilm. Because there is a complex microbiota involved in the formation of plaque biofilm, first we need to distinguish the pathogenic bacterial species from the host-compatible species. Secondly, we need to devise treatment methods that can decrease the pathogens while maintaining host-compatible species. But, how to establish an association of an organism with the pathogenesis of a particular periodontal disease?

To establish an association of an organism with disease pathogenesis, three rules for experimental proof for the pathogenicity of an organism were presented in 1883 by the German bacteriologist, Robert Koch, and a fourth rule was appended by Smith (1905)10. These rules of proof are often referred to as Koch’s Postulates. These are,

  1. The suspected causal organism must be constantly associated with the disease.
  2. The suspected causal organism must be isolated from an infected host and grown in pure culture.
  3. When a healthy susceptible host is inoculated with the pathogen from pure culture, symptoms of the original disease must develop.
  4. The same pathogen must be re-isolated from the host infected under experimental conditions.

However, periodontal diseases are not caused by a single organism which is the cornerstone of Koch’s postulates. Indeed, these disease entities are the result of mixed infections. To better identify periodontopathogenic bacterial candidates, Koch’s postulates were replaced with Socransky’s postulates 11. These include the following:

  1. The organism must be found in relatively high numbers in proximity to the periodontal lesion.
  2. The organism must be absent, or present in much smaller numbers in periodontally healthy subjects or in subjects with other forms of periodontal disease.
  3. The organism must have high levels of serum, salivary and gingival crevicular fluid antibody developed against it in periodontally diseased subjects.
  4. The organism must be found to produce virulence factors in vitro, which can be correlated with clinical histopathology.
  5. The organism must mimic similar pathogenic properties in an appropriate animal model.
  6. Clinical improvement following treatment must eliminate the putative pathogen from the periodontal lesion.

Various micro-organisms are involved in the pathogenesis of periodontal diseases. But along with them, many micro-organisms are beneficial in nature as they promote periodontal health and are known as commensals. So, the presence of pathogens and absence of commensal bacterial species in the periodontal arena initiates the periodontal disease process. One of the most important characteristics of commensal bacteria is their ability to prevent the host immune system from being activated. One proposed mechanism is the development of tolerance 12, which includes the generation of suppressor T-lymphocytes and the presence of inhibitory cytokines, mainly transforming growth factor-β and interleukin (IL)-10.

Subgingival microbial complexes

In a landmark study, Socransky et al. (1998) 13 examined over 13,000 subgingival plaque samples from 185 adult subjects and used DNA hybridization methodology and community ordination techniques to demonstrate the presence of specific microbial groups within dental plaque. The presence and levels of 40 subgingival taxa were determined in plaque samples using whole genomic DNA probes and checkerboard DNA-DNA hybridization. According to the presence of an organism and the related periodontal status, these organisms were grouped into five complexes (Figure 5.3),

1st complex (Red complex) - tannerella forsythia, P. gingivalis, and T. denticola.

2nd complex (Orange complex) - Eubacterium nodatum, Campylobacter rectus, Campylobacter showae, Streptococcus constellatus and Campylobacter gracilis.

3rd complex (Yellow complex) - Streptococcus sanguis. S. oralis, S. mitis, S. gordonii and S. intermedius.

4th complex (Green complex) - Campylobacter concisus, Eikenella corrodens and Actinobacillus actinomycetemcomitans serotype a.

5th complex (purple complex) - Veillonella parvula and Actinomyces odontolyticus.

  1. actinotnycetemcomitansserotype b, Selenomonas noxiaand Actinomyces naeslundii genospecies 2 (A. viscosus) were outliers with little relation to each other and the 5 major complexes.

Figure 5.3 The sub-gingival microbial complexes

Subgingival microbial complexes

Out of these groups of species the Actinomyces species, yellow, green and purple complexes are early colonizers of the tooth surface whose growth usually precedes the multiplication of the predominantly Gram-negative orange and red complexes. The orange and red complexes are comprised of the species thought to be the major etiologic agents of periodontal diseases.

Microorganisms associated with periodontal health

As discussed in the chapter 2 “Dental plaque and its development as biofilm”, the early dental plaque has been shown to be dominated (60%-90%) by Streptococcus species. Other bacterial species found in early plaque include Eikenella species, Haemophilus species, some Prevotella species, Capnocytophaga species, Propionibacterium species, and Veillonella species 14. It has been shown that Actinomyces species is the predominant species after 2 hours of biofilm formation in healthy subjects 15. As the maturation of plaque takes place, organisms also called ‘tertiary colonizers’ can be isolated from the plaque samples. These include A. actinomycetemcomitans, P. intermedia, Eubacterium species, Treponema species, and Porphyromonas gingivalis. F. nucleatum serves to bridge between the early and late colonizers.

Typically, healthy gingival sites have a bacterial pattern which is similar to, as described for the relatively immature supragingival plaque (Table 5.1). The majority of the bacterial counts from plaque samples from healthy sites consistently have bacterial species that belong to streptococci, Actinomyces species (especially, A. viscosus and A. naeslundii) and Veillonella species 16-18. It has been demonstrated that if a plaque sample is taken from healthy periodontal sites and is examined in a wet mount using phase-contrast or dark field microscopy, the bacteria seen are primarily non-motile with a ratio of motile to non-motile forms of about 1:40 19. This study also provides evidence that in diseased periodontal sites the number of motile forms of bacteria increases.  There are many other bacterial species that can be identified in plaque samples from healthy periodontal sites, but these are only minor and transient components of maturing dental plaque 17. It must be noted that some bacterial species which are considered as periodontal pathogens may be found associated with gingival health. Furthermore, in areas where recently scaling and root planing has been done, the microflora is almost similar to that of healthy periodontal sites 19. Most of the bacterial species isolated from dental plaque in healthy sites belong to purple and yellow complexes.

Microorganisms associated with periodontal diseases

Specific microorganisms have been implicated in various periodontal conditions by various studies. However, it must be remembered that because periodontal diseases are primarily the result of complex microbial infection, the majority of these organisms are common in various periodontal conditions.  Attempts have been made to make some general comparisons of the subgingival microbial profiles associated with chronic and aggressive forms of periodontitis (Table 5.1). Mombelli et al. (2002) 20 in a systemic review, tried to make a comparison between chronic and aggressive periodontitis on the basis of presence or absence of most commonly identified putative periodontal pathogens P. gingivalis, A. actinomycetemcomitans, P. intermedia, T. forsythia and C. rectus in patients with chronic and aggressive periodontitis. The results of the study could not conclude any relation of any of these organisms specifically to chronic or aggressive periodontitis. However, commonly identified organisms in gingivitis, chronic periodontitis, and aggressive periodontitis can be listed based on extensive research work done on the microbiology of various periodontal conditions.

Gingivitis:

The early studies on gingivitis demonstrated that with the development of gingivitis, there is a gradual microbial shift from a Gram-positive dominated microflora to more complex flora that contains significantly higher numbers of Gram-negative and spiral forms of bacterial species 21. Later studies, on the development of gingivitis, elaborated that the bacterial culture of plaque gradually shifts from predominantly streptococcus to that dominated by Aggregatibacter species 17, 22. Furthermore, it was also observed that although the number of Aggregatibacter species increases with the increase in plaque mass, but bleeding on probing was associated with increased numbers of A. viscosus and pigmented Bacteroides. More extensive studies on gingivitis have demonstrated that sites with gingivitis have increased numbers of F. nucleatum, Eubacterium timidum, Treponema species and Bacteroides species 23. Most of the bacterial species isolated from gingivitis associated plaque primarily belong to Aggregatibacter, purple and yellow complexes with partial representation from the orange complex.

Chronic periodontitis:

Extensive research has been done to identify the microbiota associated with chronic periodontitis. The untreated chronic periodontitis patients have a predictable pattern of subgingival plaque maturation. Although, the composition of microbiota may vary a little depending upon how the ecosystem during plaque maturation has been disrupted by oral hygiene methods, most of the patients with chronic periodontitis demonstrate almost consistent bacterial species in subgingival plaque samples.  The most consistent microorganisms isolated from chronic periodontitis cases include P. gingivalis and T. forsythia 24. Other organisms that have been shown to be associated with chronic periodontitis include F. nucleatum, P. intermedia, Winonalla parvula, Campylobacter species, Haemophilus species, Selenomonas species and Treponema species.

The recent non-culture based identification techniques have identified organisms from Archaea domain in association with periodontal diseases. These organisms are prokaryotes that physically resemble bacteria, but have different nucleotide sequences, hence cannot be considered in bacteria domain. Instead, they are genetically closer to microbes in the Eukarya domain. In chronic periodontitis cases, approximately 19-73 % of subgingival sites have been shown to harbor Archaea 25-28. It must be noted that Archaea have no direct pathogenic effects, but these can contribute to the overall pathogenicity of subgingival biofilms by various syntrophic interactions with other microorganisms present in the plaque 29.

Another organism that has been identified with non-culture techniques is Filifactor alocis 30.  Filifactor alocis is a fastidious, Gram-positive, obligately anaerobic rod which was repeatedly identified in periodontal lesions, using DNA-based methods. It has been suggested to be a marker for periodontal deterioration. Plaque samples derived from healthy, chronic periodontitis and aggressive periodontitis patients were subjected to PCR analysis. Results showed that patients suffering from generalized aggressive periodontitis or chronic periodontitis harbored F. alocis whereas it was rarely detected in the control group. The authors concluded that F. alocis is a contributor to the pathogenetic structure of biofilms accounting for periodontal inflammation and can be considered as an excellent marker organism for periodontal disease.

Aggressive periodontitis:

The localized and generalized forms of aggressive periodontitis result in rapid periodontal destruction. The most commonly implicated organism in aggressive periodontitis is Aggregatibacter actinomycetemcomitans 31, 32. This organism is often found in high numbers in sites with aggressive periodontal destruction and is rarely seen in healthy subjec8ts, those with plaque-induced gingivitis, or edentulous patients 33-35. Other organisms that are routinely isolated from aggressive periodontitis cases include Capnocytophaga species and Prevotella species. Studies have shown that infection with A. actinomycetemcomitans is not substantially reduced following the non-surgical periodontal therapy 36, 37, but may be reduced by systemic antibiotic and surgical therapy 35, 37, 38. These findings indicate that A. actinomycetemcomitans invades the tissue and cannot be removed simply by non-surgical periodontal therapy. In juvenile diabetes with aggressive periodontitis, Capnocytophaga species appears to play an important role 39. Along with these organisms, Wolinella recta, E. corrodens, and T. forsythia have also been identified in areas with aggressive periodontal destruction 40-43.

Refractory periodontitis:

According to the American Academy of Periodontology (AAP) 44, refractory periodontitis is not a single disease entity. The term refers to destructive periodontal diseases in patients who, when longitudinally monitored, demonstrate additional attachment loss at one or more sites, despite well-executed therapeutic and patient efforts to stop the progression of the disease. It has been reported that microbiota of the refractory periodontitis, on an average, is generally similar to that observed in chronic periodontitis 45. However, some differences have been pointed out. Magnusson et al. (1991) 46 and Colombo et al. (1998b) 45 have pointed out high levels of Streptococcus constellatus/intermedius in refractory periodontitis lesions while Gordon et al. (1985) 47 have observed high levels of motile organisms and black pigmented species in these cases.  High levels of Aggregatibacter actinomycetemcomitans were observed by van Winkelhoff et al. (1992) 48 in refractory periodontitis cases. Winkel et al. (1997)49 found high levels of T. forsythia in these cases. Haffajee et al. (1988) 50 in a study on 13 refractory periodontitis subjects found that the microbial profile of these patients, primarily contained members of the red and/or orange complexes. Thus, it can be summarized that the microbial profile of refractory periodontitis patients is not very different from that of chronic and aggressive periodontitis. However, according to the specifically identified organisms, individual treatments may have to be designed for every patient.

Necrotizing ulcerative gingivitis and periodontitis:

Necrotizing periodontal diseases are the result of microbial infection in association with attenuated immune response commonly due to malnutrition, stress or HIV infection. There are specific organisms that have been isolated from necrotizing periodontal lesions. These include Prevotella intermedius and Treponema species (intermediate-sized oral treponemes), Selenomonas species and Fusobacterium species 51, 52. The microorganisms isolated from HIV-associated periodontitis have been found to be similar to that observed in conventional periodontitis patients 53-55. Furthermore, Candida albicans infection has also been associated with necrotizing ulcerative gingivitis in HIV seropositive patients 56.

Table 5.1 Microbiota commonly associated with various periodontal conditions

Periodontal Health
Gingivitis
Chronic periodontitis
Aggressive periodontitis
Gram positive organismsGram-positive organismsGram-positive organismsActinobacillus actinomycetemcomitans
Porphyromonas gingivalis
Streptococcus oralis
Streptococcus sanguis
Streptococcus mitis
Actinomyces gerencseriae
Actmomyces naeslundi
Lactobacillus species
Actinomyces naeslundi
Peprostreptococcus micros
Streptococcus onginosus
Eubacterium brachy
Eubacterium nodatum
Mogibacterium timidium
Parvimonas micra
Peptostreptococcus stomatis
Parvimonas micra
Tannerella forsythia
Prevotella intermedia
Campylobacter rectus
Peptostreptococcus micros
Campylobacter concisus
Prevotella nigrescens
Gram-negative organismsGram-negative organismsGram-negative organismsEikenella corrodens
Selenomonas sputigena
Fusobacterium nucleatum
Fusobacterium species
Prevotella nigrescens
Veillonella species
Fusobacterium nucleatum
Prevotella intermedia
Winonalla parvula
Campylobacter species
Haemophilus species
Selenomonas species
Treponema species
Tannerella forsythia
Fusobacterium nucleatum
Porphyromonas gingivalis
Prevotella intermedia
Prevotella loescheii
Dialister pneumosintes
Campylobacter rectus
Treponema species

Factors that determine initiation and progression of the periodontal disease

There are many factors that determine the establishment and proliferation of microorganisms in a host. These can be broadly divided into pathogen and host-related factors. The pathogen should have properties which enable it to overcome the host immune response and to adhere and proliferate on or in the host. The host related factors include the defense mechanism which prevents the pathogen to establish itself in the host and proliferate. These factors are affected by the local and environmental factors which may or may not favor the establishment of infection.  Let us now discuss pathogen virulence, host susceptibility and other factors which affect the occurrence of a disease.

Virulence of microorganism:

Virulence factors refer to the properties that enable a microorganism to establish itself on or within a host of a particular species and enhance its potential to cause disease. These factors, primarily perform three functions,

  1. Invade the host
  2. Evade host defenses
  3. Cause disease

These factors include cell surface proteins that mediate the bacterial attachment, cell surface carbohydrates, and proteins that protect an organism, bacterial toxins and hydrolytic enzymes that may contribute to the pathogenicity of the bacterium. Bacterial adherence to the host is the first step in the development of the disease. Many pathogenic bacteria colonize mucosal sites by using pili (fimbriae) to adhere to the cells. In the context of periodontal disease progression, initial adherence, colonization and growth of microorganisms in dental biofilm is the first step in disease progression. The attachment of bacteria to dental pellicle exhibits a great deal of specificity and appears to involve specific receptors on the bacterial and pellicle surfaces. Plaque accumulation may be mediated by bacterial extracellular polysaccharides, salivary components as well as direct cell-to-cell binding. Salivary components play a dual role in plaque formation. They can mediate bacterial attachment to the pellicle or plaque periphery, but by binding to unattached bacteria they can also diminish the attachment of such bacteria. As the plaque matures, bacterial coaggregation can be seen. Coaggregation prevalent among bacteria isolated from the human oral cavity was first reported by Gibbons and Nygaard in 1970 57. Once established in a biofilm, these factors start producing their respective virulence factors which trigger the host immune response. It must be noted that different strains of a species vary in their virulence. Certain clonal types of a particular bacterial species are more commonly found in subjects with periodontitis as compared to periodontally healthy patients. It has been demonstrated in animal experiments that the virulence of different strains varies for P. gingivalis 58-61.

To cause disease, a pathogen should have all the necessary genetic information regarding virulence factors which may enable the establishment of that pathogen in the host. If some of the elements of this genetic information are lacking in a pathogen, these could be received from other strains of that species (or possibly other species) via phage, plasmids or transposons 62Finally, the organism should be located at a right location in sufficient numbers (which varies from species to species) to initiate the disease process.

Many bacterial species are equipped with various virulence factors to overcome the host defense mechanisms. These virulence factors vary amongst bacterial species and within a species from strain to strain. Certain toxins such as leukotoxins cause damage to polymorphonuclear leukocytes (PMN’s), which make the first line of defense against bacterial invasion in the tissue. For example, leukotoxin by A. actinomycetemcomitans destroys leukocytes, thus facilitating bacterial invasion 63. These leukotoxins have also been found to be effective against monocytes 63 and mature T and B lymphocyte cell lines 64. Furthermore, specific mechanisms that interfere with killing mechanisms of PMN’s have been developed by various bacterial species 65-67. Anti-phagocytosis factors such as bacterial capsule prevent the bacterial phagocytosis by macrophages. Specific antibody response (IgA and IgG) has been detected against certain bacterial species such as P. gingivalis, P. intermedia, P. melaninogenica, and Capnocytophaga species. Many putative periodontal pathogens have been shown to possess proteases which can destroy these specific antibodies, thereby facilitating bacterial invasion 68-70.

To effectively evade the host defense system, an organism should have sophisticated mechanisms to prevent its inactivation or killing. Complex mechanisms have been explained for various organisms which modulate or suppress the immune response, thus ensuring bacterial survival 71, 72. Once, a periodontal pathogen overcomes the host immune response; it may cause tissue damage by producing various toxins and enzymes that degrade the components of the connective tissue.

Host susceptibility:

A susceptible host is the one who responds to the infection in a different way as compared to normal individuals. Recent research has enumerated various “host susceptibility factors” based on various new methods for comparing the immunological responses in subjects with periodontitis and healthy individuals. These factors include poorly regulated immune response and defective PMN function. Systemic conditions like diabetes mellitus, HIV infection, etc. have been shown to have a direct relation to periodontal disease progression.

Local factors:

The local environment in the subgingival area is a major factor that determines the establishment and survival of various bacterial species. Although, mostly the subgingival environment is host-compatible and favors the growth of commensals, sometimes due to microorganism related or host related factors the subgingival environment becomes conducive for the growth of putative periodontal pathogens.

Transmission of bacterial pathogens

It is a well-known fact that development of the disease can be prevented if the organism causing that disease can be prevented from entering the body. Thus, if we can prevent the entry of periodontal pathogens in the oral cavity, the progression of the periodontal disease process can be prevented. But from where these organisms reach the oral cavity? The most commonly identified periodontal pathogens are not usually found in our surrounding environments such as soil, water, and air. Hence, most probably, these microorganisms are transmitted from one person to another. The transmission of the organisms can be horizontal or vertical. The vertical transmission is from parents to offspring. Any mode of transmission other than the parent-offspring transmission is referred to as horizontal transmission.

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Other than bacterial transmission, there are various other modes by which the bacterial genetic material coding for various virulent factors can be transferred from one bacterial clonal type to another. These gene transfer mechanisms include bacteriophage, transformation, plasmids and conjugative transposons 62.

Major periodontal pathogens

Although, periodontal diseases are caused by various bacterial species in a complex microbiota in the form of a biofilm, following is the description of most commonly identified putative periodontopathogens (Table 5.2),

Aggregatibacter actinomycetemcomitans

This bacterium was first isolated by Klinger 81 from a cervicofacial actinomycotic lesion in 1912 and was named Bacterium actinomycetemcomitans. Topley and Wilson (1929) 82 reclassified it as Actinobacillus actinomycetemcomitans and Potts et al. (1985) 83 as Haemophilus actinomycetemcomitans. In 2006 it was again reclassified based on multilocus sequence analysis by Nørskov-Lauritsen N and Kilian M (2006) 84. Because of the phylogenetic similarity of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis, genus Aggregatibacter (aggregate, to come together; bacter, bacterial rod; aggregatibacter, rod-shaped bacterium that aggregates with others) was added to the family pasteurellaceae. This genus includes Gram-negative, non-motile, facultatively anaerobic rods or coccobacilli that were previously known as Haemophilus.

Morphological Structure:

Structurally, A. actinomycetemcomitans are Gram-negative, small non-motile coccobacilli that grow singly, in pairs, or in small clumps and are variously described as fastidious and CO2-requiring bacteria. These are small, short (0.4×1 µm) straight or curved rods with rounded ends 85. Electron microscopic studies have demonstrated membrane vesicles that appear to release from the cells. On blood agar, colonies are small, gray to white, translucent, smooth and non-hemolytic. Growth is stimulated by the addition of CO2 and species grows well in 5-10 % of CO2 86These ferment a range of sugars, including glucose and fructose, but not sucrose and lactose. Acid end products include lactate, succinate, acetate and propionate. A. actinomycetemcomitans is grown in anaerobic cultures and from plaque, it can be rapidly identified by fluorescent labelled antisera. It can also be identified by the use of DNA probes.

When grown in culture, these form small colonies, approximately 0.5-1.0 mm in diameter. Colonies are translucent (or transparent) with irregular edges appearing smooth, circular and convex. The colonial morphology of fresh isolates is distinctive with the internal star-shaped or crossed cigar morphology which forms embedding in the agar that gives A. actinomycetemcomitans its name 85.

Taxonomy of A. actinomycetemcomitans:

King and Tatum (1962) 87 provided the first detailed biochemical and serological description of A. actinomycetemcomitans. As already stated, these bacteria are closely related to Haemophilus aphrophilus. Differentiation is done by its ability to grow in the absence of haemin and factor V (Nicotinamide adenine dinucleotide). Serological studies have been attempted to determine the antigenic relationship between A. actinomycetemcomitans and Haemophilus aphrophilus. Using agglutination studies, 24 different serogroups and 6 major agglutination antigens of A. actinomycetemcomitans were identified. Currently, A. actinomycetemcomitans strains are classified into six serotypes, a, b, c, d, e, and f, the specificity of which is defined by surface polysaccharides 88-92. A new serotype i.e. serotype g of A. actinomycetemcomitans has recently been identified 93. The predominant A. actinomycetemcomitans serotypes seem to be a, b, and c 34, 94-99.

Most leukotoxic strains are of serotype b. It is important to note that serotype b of A. actinomycetemcomitans has been found more frequently and detected in higher numbers in active periodontitis lesions, whereas serotype a and c have a stronger association with periodontal health 100. Taxonomic classification of bacterial serotypes is based on the following biochemical characteristic features:

  1. Fermentation of different carbohydrates.
  2. Multivariate analysis of chemotaxonomic data.
  3. Cellular fatty acid composition.
  4. The composition of lipopolysaccharides.
  5. Analysis of total proteins.
  6. Multilocus enzyme electrophoresis.
  7. Respiratory quinones.

 Surface Ultrastructure of A. actinomycetemcomitans:

The surface ultrastructure of A. actinomycetemcomitans demonstrates following components,

  • Fimbriae: They may exhibit fimbriae which are around 2 µm in length and 5nm in diameter. Fimbriated strains produce colonies with star-shaped interior structure. These are required for attachment of the bacterium to host.
  • Vesicles: These are a prominent feature of actinomycetemcomitans. These structures are lipopolysaccharides in nature and originate from and are continuous with the outer membrane. A. actinomycetemcomitans species with high leukotoxic production have abundant extracellular membrane vesicles. Vesicles per se exhibit leukotoxic activity. Other components of vesicles are endotoxin, bone resorption activity, bacteriocin, and actinobacillin.
  • Extracellular amorphous material: Certain actinomycetemcomitans have an amorphous material that frequently embeds adjacent cell in the matrix.

Virulence Factors associated with A. actinomycetemcomitans 101:

A. actinomycetemcomitans has many virulent factors which facilitate attachment and proliferation of these bacteria in the host. These include,

  • Adhesion factors
  • Bacteriocin
  • Bone resorption factors
  • Collagenases
  • Cytotoxins
  • Extracellular membrane vesicles
  • Fc binding proteins
  • Leukotoxin
  • Lipopolysaccharides (LPS)
  • Immunosuppressive factors
  • Products that Inhibit PMN function.
  • Factors facilitating penetration of epithelial cells
  • Cytolethal distending toxin

These are discussed as follows,

Adhesion of A. actinomycetemcomitans:

It is executed by adhesins which are bacterial cell surface components. They interact and bind to very specific receptors in saliva, on the surface of the tooth, on extracellular matrix proteins and on epithelial cells. Distinct adhesins may be expressed by bacterium under different environmental conditions 102. Most of the adhesins are proteinaceous in nature. A. actinomycetemcomitans strains with fimbriae adhere three to four folds better than those without fimbriae.

Bacterial aggregation during plaque formation:

The bacterial aggregation during plaque formation is very specific. Bacteria assert in the plaque biofilm by means of aggregation, inter-generic and intra-generic coaggregation and interactions with distinct and bacteria specific salivary binding proteins. Following initial colonization, bacteria including A. actinomycetemcomitans attach to the initial colonizers.

Adhesion to epithelial cells:

Most of the A. actinomycetemcomitans strains bind strongly to the epithelial cells 102. Binding is rapid, which reaches saturation within 1 hour 103. Cell surface entities that mediate adhesion are fimbriae, extracellular amorphous material and extracellular vesicles 102.

Binding to extracellular matrix proteins:

In order to initiate disease, A. actinomycetemcomitans must bind to the extracellular matrix. A major component of the extracellular matrix is collagen 104. The major fibers forming the extracellular matrix are collagen Type I, II, III, V, XI. Less abundant, but equally important are noncollagenous glycosylated proteins, fibronectin, and laminin. Outer membrane proteins are important for binding of A. actinomycetemcomitans to collagen and other proteins but binding is highly specific.

Table 5.2 Periodontal pathogens, their virulence factors, and their action

Micro-organism
Adhesion/
Colonization
Virulent factors
Actions
Aggregatibactor actinomyce-
temcomitans
Pill

Vesicle

Capsular antigen
Leukotoxin

Cytolethal distending toxin (CDT)

Fc-binding protein

Collagenases

LPS

Proteases
Kills neutrophils, lymphocytes and monocytes

Host cell cytotoxicity



Deactivation of Ig’s


Disintegration of collegen

Potent stimulator of IL-1, PGE-2 and TNF-α

Cleavage of Ig’s.
Host cell cytotoxicity
P. GingivalisPill

Vesicle

Capsular antigen
Proteases (gingipains)

LPS

Hemolysins

Volatile sulfur
compounds

Ammonia

Indole
Degradation of Ig, complement factors.


Potent stimulator of IL-1, PGE-2 and TNF-α

Causes agglutination and lysis of erytherocytes.

Host cell cytotoxicity


Host cell cytotoxicity

Host cell cytotoxicity
P. IntermediaBacterial surface adhesinsProteases

Hemolysins

LPS
Degrade matrix components, host cell receptors and Ig’s

Lysis of RBC’s

Potent stimulator of IL-1, IL-6 and IL-8 release. Causes periodontal tissue destruction and alveolar bone resorption.
T. DenticolaMajor surface protein (MSP)

Fibronectin-binding adhesins
LPS

Proteases

Dentilisin

Major surface protein (MSP)

Phospholipase C (PLC)

H2S and methyl mercaptan
Potent stimulator of IL-1, PGE-2 and TNF-α

Degradation of extracellular matrix

Pleiotropic effects. Crucial for the virulence of T.denticola

Cytotoxic for a wide variety of cells


Directly or indirectly damages tissue by hydrolysis of membrane phospholipids

Cytotoxic effects that is primarily due to inhibition of cytochrome oxidases.
T. ForsythiaS-layer (TfsA and TfsB)

Leucine-rich-repeat protein (BspA)
Lipoproteins (BfLP)

Leucine-rich-repeat protein (BspA)
Activates gingival fibroblasts to produce elevated levels of interleukin-6 and TNF-α.

Causes release of pro-inflammatory cytokines from monocytes and chemokines from osteoblasts

Bacteriocins:

These are proteins produced by bacteria that are lethal for other strains and species of bacteria. A. actinomycetemcomitans bacteriocins are active against S. sanguis, S. uberis and A. viscosus 105, 106. Bacteriocins increase the permeability of the cell membrane of the target bacteria leading to the leakage of DNA, RNA and macromolecules essential for growth.

Bone resorption factors:

A. actinomycetemcomitans stimulate bone resorption in periodontal tissues by various mechanisms;

  • Lipo-polysaccharides.
  • Proteolysis sensitive factor in micro-vesicles.
  • Surface-associated

The surface-associated material has recently been identified as the molecular chaperone, GroEL. The chaperone appears to act in a direct way with the major bone resorbing cell population, the osteoclast 107-109.

Collagenase:

As collagen is the most abundant component of extracellular matrix, its destruction leads to the degradation of extracellular matrix. A. actinomycetemcomitans produces a collagenolytic protein, which can attack collagen. Collagenase is primarily produced by two important periodontal pathogens, A. actinomycetemcomitans and P. gingivalis 110, 111.

Cytotoxins:

Fibroblasts are the most important source of collagen in the extracellular matrix. Cytotoxins act on fibroblasts and inhibit their proliferation. The cytotoxin produced by A. actinomycetemcomitans has been identified as a 50 kDa protein that inhibits DNA synthesis of fibroblasts 112.

Extracellular membrane vesicles:

Almost all strains of A. actinomyceterncomitans have these vesicles. Growth conditions alter the formation and morphology of vesicles. These vesicles contain leukotoxins, endotoxins, factors involved in bone resorption activity and bacteriocins 105, 106.

Fc binding proteins:

These bind to the Fc portion of an antibody and hence, inhibit phagocytosis. Fc portion also activates the complement system, so all these pathways are blocked.

Leukotoxin:

The ability of A. actinomycetemcomitans extracts to cause the death of leukocytes was first shown more than 36 years ago 63, 113. Leukotoxin is a member of RTX (repeat in toxin) family of toxins which are responsible for the pore forming leukotoxic and haemolytic activity 114. The leukotoxin gene (Ltx A) resides on operon consisting of 4 genes C, A, B and D 115. Ltx B and Ltx D are involved in transporting the toxin to the surface of the cell while Ltx C post –transitionally activates the toxin. The expression appears to be regulated by the presence of oxygen and is induced under anaerobic conditions 116. The mechanism of leukotoxicity 117 includes:

  • Membranolytic activity, producing pores in the target cell.
  • Phospholipids act as the receptor for the toxin whose activity results in a rapid influx of Ca2+ into the cell.
  • Necrosis and apoptosis.

The function of systemic antibodies……………….

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Lipopolysaccharides (LPS):

These are endotoxins having the potential to modulate the host responses and contribute to the tissue destruction. LPS causes tissue destruction by following mechanisms 121,

  • Stimulation of in vitro bone resorption.
  • The production of IL-1, TNF and prostaglandin (PGE2) from macrophages.
  • Polyclonal activation of B-lymphocytes.

The bone resorptive activities of this LPS 122, 123 are the result of stimulation of PGE2 and IL-1 release from osteoblasts and other cells. Along with this A. actinomycetemcomitans is also 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 124.

 

 

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Lipopolysaccharides (LPS) and cytokine induction:

Lipopolysaccharides are potent inducers of cytokine production 121. Let’s try and understand the structure of LPS. LPS is built up of three……

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Lipid A.

  • Core region.
  • O-specific side chain.

These separate building blocks have widely different compositions and structures, and this is reflected in their biological activities and functions.

Lipid A. It is an important part of LPS and the ‘‘endotoxic’’ activities of LPS are largely due to the lipid A region, although this activity can be modulated by other regions of the molecule. Lipid A is a general term for a family of β(1-6)-linked disaccharides to which are attached medium to long-chain fatty acids (10- to 28-carbon chain length) linked to the sugar residues by ester or amide linkages and containing glycosidic and nonglycosidic phosphoryl groups.

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Immunosuppressive factors:

A. actinomycetemcomitans produces proteins that inhibit DNA, RNA and protein synthesis by human lymphocytes. It is believed that it affects immunoglobulin production by activating B-cells that downregulate the ability of B- and T-cells to respond to mitogens. In addition, leukotoxin impairs the ability of lymphocytes to respond to mitogens by inhibiting DNA, RNA, protein, IgG and IgM synthesis.

Products that Inhibit PMN function:

A. actinomycetemcomitans produces low molecular weight compounds that inhibit PMN leukocyte chemotaxis .The inhibitory activity is aborted by treatment with proteinase K, which means that the compounds are proteinaceous in nature. Along with this, A. actinomycetemcomitans has been shown to be capable of inhibiting PMNs from producing some potent antibacterial agents that are gained when the PMNs fuse with lysosomes 101.

Penetration of epithelial cells:

Research work has shown that A. actinomycetemcomitans can penetrate the gingival epithelial cells 125, 126. Entry of the bacteria into the cell permits them to either transit the epithelial cell barrier or persist and grow in a protected cellular environment. Studies done by Meyer et al. (1997) 127, 128 suggest that primary receptor for A. actinomycetemcomitans invasion is transferrin receptor. Invasion through integrins is the secondary pathway of entry. A. actinomycetemcomitans invasion of epithelial cells is a highly dynamic and complex process. It involves the attachment of the organism to the host cell with the initiation of some form of signalling, binding to a receptor, entry in a vacuole, escape from the vacuole, rapid multiplication, intracellular spread, exit from the cell and cell to cell spread 128.

Cytolethal distending toxin (CDT):

It has been shown that A. actinomycetemcomitans also produces a 60-kDa protein, which downregulates both T and B-cell responsiveness through the activation of a subpopulation of B-lymphocytes 129. The cytolethal distending toxin is encoded by a locus of three genes, cdt A,B,C 130, 131. It impairs the lymphocyte function by arresting its cell cycle. The active subunit, CdtB, exhibits DNase I activity. While the role of CdtA and CdtC is less clear, both proteins possess putative mucin-like carbohydrate-binding domains that predict interaction with the host cell surface.

 

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Clonal types in pathogenesis of periodontal diseases:

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The first step in the pathogenesis of periodontal disease is the adherence of bacteria to periodontal tissue. Multiple clonal types may exist for a particular bacterial species, all of which may not be similarly virulent. Amano and co-workers (2000) 132 have reported that both disease-associated and non-disease associated genotypes exist in P. gingivalis, suggesting that there is a significant predominance of P. gingivalis with type II fimA in periodontitis patients. Highly leukotoxic clones of A. actinomycetemcomitans have been identified 133. Other studies have demonstrated the production of leukotoxin from A. actinomycetemcomitans is regulated in part by environmental factors, such as atmosphere, nutrition supply, and pH 134-136. The optimal pH for the growth of A. actinomycetemcomitans is reported to be in the range of 7.0–8.0 135.

 Porphyromonas gingivalis

P. gingivalis is a Gram-negative, anaerobic, non-motile, asaccharolytic and black pigmented rod that form greenish-black colonies on blood agar plates 137. It is one of the major pathogens of chronic periodontitis138, 139. This organism has been included in the red complex, which is strongly associated with periodontal destruction. In both periodontitis and healthy subjects, P. gingivalis can be recovered in low frequency from the subgingival flora, tongue, buccal mucosa, tonsils and saliva 140-142.

Structural components:

Capsule:

It is an anti-phagocytosis virulence factor. Six distinct capsular serotypes have been described (K1-K6) 143, 144 Capsules from three P. gingivalis strains have been purified. They are mainly composed of sugars like galactose, glucose, glucosamine, rhamnose and mannose. A strong relationship exists between the extent P. gingivalis encapsulation and its ability to act as a pathogen. Increased encapsulation is related to decreased auto-agglutination, increased resistance to phagocytosis, serum resistance and decreased induction of PMN leukocyte chemiluminescence. Studies in a mouse infection model have revealed that encapsulated P. gingivalis strains are more virulent than non-encapsulated strains 58, 145-147. The components of the bacterial capsules have been used to make the vaccine.

Outer membrane:

The outer membrane of P. gingivalis contains at least 20 major proteins, ranging from 20-90 kDa. These proteins have a significant effect on fibroblasts. They stimulate their proliferation. Because of this, a 24 kDa protein has been given name “fibroblast activating factor”. P. gingivalis also produces a 75 kDa major outer membrane protein that exists as a high molecular weight polymer. It has been found that these proteins can stimulate polyclonal B-cell invasion and can elicit IL-1 production. These outer membrane proteins are also important in plaque formation and coaggregation. P. gingivalis plays an important role in the formation and maintenance of plaque biofilm. Gibbson and Nygaard (1970) 57 demonstrated that there was a specific interaction of P. gingivalis with various Gram-positive and Gram-negative bacteria. Coaggregation between P. gingivalis and A. actinomycetemcomitans was found to be important for the initial subgingival biofilm formation. P. gingivalis has an absolute growth requirement of haemin (iron). It produces hemolysin associated with its outer membrane which lysis the blood cells.

Lipopolysaccharide (LPS):

The analysis of LPS from 6 different strains of P. gingivalis indicate that it contains sugars like rhamnose, mannose, and galactose. LPS has been studied for various immunological properties. Chemically, it is composed of 3 parts O-antigen, core and lipid A. Endotoxin activity is confined to lipid A, while significant immunological activities are due to O antigen. LPS also interacts with CD14 receptors on cells and initiate the immune reaction.

Bacterial fimbriae:

Fimbriae or pili are proteinaceous, filamentous appendages that protrude outwards from the bacterial cell surface and play a crucial role in virulence by stimulating bacterial attachment to host cells or tissues 148. Fimbriae of P. gingivalis were first recognized on the outer surface by electron microscopic observation 149, 150. They are arranged in a peritrichous pattern on P. gingivalis. Fimbrae is composed of 1000 units of fimbrillin. P. gingivalis fimbriae possess a strong ability to interact with host proteins (such as salivary proteins, extracellular matrix proteins), epithelial cells, and fibroblast, which promote the colonization of P. gingivalis in the oral cavity 151, 152. Animal experiments conducted strongly implicate fimbriae as an important virulence factor. Environmental factors like temperature, pH, haemin limitation, serum, saliva, osmotic effects and effects of Ca++ limitation have a significant effect on fimbrae formation. P. gingivalis fimbriae are classified into six genotypes based on the diversity of the fimA genes encoding each fimbrillin (types I to V, and type Ib), and that P. gingivalis with type II fimA is most closely associated with the progression of chronic periodontitis 153-155.

Proteinases:

P. gingivalis produces a large number of hydrolytic, proteolytic and lipolytic enzymes that are produced essentially by all of the known strains. Many of these enzymes are exposed on the surface of the bacterium where they come in contact with the host cells and tissues. These enzymes play a significant role in periodontal disease progression, including dissemination of P. gingivalis into deeper tissues. Classification of proteinases is relied upon their catalytic functions. To date, four proteinases have been recognized: serine, aspartate, thiol, and metalloproteinases. The arg- and lys- proteinases have been given a common name Gingipains. There are atleast 3 different genes which encode for the proteolytic activity of P. gingivalis. These genes encode for 2 cystein proteinases, arginine- gingipain (Arg-gingipain A & B; Rgp-A and Rgp-B) and lysine-gingipain (lys-gingipain, Kgp). Arg-gingipain is encoded by two genes and lys- gingipain by one gene 156. Movement of cysteine proteinases from the bacterial cytoplasm to the membrane involves the secretory pathway. Arg- and lys- proteinases or the gingipains belong to trypsin-like proteinases.

Colonization of P. gingivalis:

 Despite the host defense mechanisms in saliva and GCF, P. gingivalis can adhere and then colonize in gingival crevices to a variety of surface components lining the gingival crevicular cells and the tooth surface. The adhesion is mainly mediated by the fimbriae, although other bacterial components such as vesicles, hemagglutinin, and proteases may play an adjunctive role 157. P. gingivalis is capable of co-aggregating with Actinomyces naeslundii genospecies 2 (Actinomyces viscosus), Streptococcus gordonii, and Streptococcus mitis 158, 159.

Virulence factors associated with P. gingivalis:

There are various virulence factors that have been demonstrated to be the key elements in the ability of P. gingivalis to evade the host defense and cause periodontal disease progression. These virulence factors include the following,

  • Fimbriae
  • Proteinases
    • Gingipains
    • Collagenases
    • Gelatinases
    • Dipeptidyl aminopeptidase IV
  • Hemagglutinin
  • Capsule lipopolysaccharide (LPS)
  • Outer membrane vesicles (OMV’s)

Pathogenic process of P. gingivalis:

The initial event in the pathogenicity of P. gingivalis is its interaction (adherence) in the oral cavity 160, 161. This process is facilitated by fimbriae, proteases, hemagglutinins and lipopolysaccharide 158. P. gingivalis fimbriae help in the bacterial interactions with the host cells and also with other bacteria. Bacterial adherence to mucosal and tooth surfaces, as well as bacterial co-aggregation,  are the essential steps for colonization of various oral bacterial species. Fimbriae are involved in each of these processes 163, as well as cell adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and P- and E-selectins. P. gingivalis major fimbriae have been shown to be necessary for bacterial invasion in the host cells 163.

Proteinases:

P. gingivalis proteinases have been reported to exhibit enzymatic activity against a broad range of host proteins, including host proteinase inhibitors, immunoglobulins, iron transporting/sequestering proteins, extracellular matrix proteins, bactericidal proteins and peptides, and proteins involved in the coagulation, complement, and kallikrein/kinin cascades 164. The majority of this activity is due to cysteine proteinases, referred to as gingipains.

Gingipains:

Gingipains were originally considered as “trypsin-like proteases”, but actually, they comprise a group of cysteine endopeptidases 156 that have been reported to account for at least 85% of the general proteolytic activity displayed by P. gingivalis and 100% of the expressed “trypsin-like activity.” They are either secreted or membrane bound and are arginine or lysine-specific. Gingipains are present in large quantities .

Structural description of gingipains:

The mature form of RgpA possesses both, catalytic and hemagglutinin domain, while RgpB possesses only a catalytic domain. Hemagglutinin domain is responsible for the adherence of microorganism to erythrocytes. There is a high degree of homology between the catalytic domains of RgpA and RgpB at both the DNA and protein levels, while the hemagglutinin domain of RgpA is similar to the P. gingivalis hemagglutinin domain of Kgp.

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Mechanism of action of Gingipains:

  • Vascular permeability enhancement: R-gingipains are very potent factors of vascular permeability enhancement. This activity is induced through plasma prekallikrein activation and subsequent bradykinin release 166, 167. Gingipain K by itself is not able to induce vascular permeability in human plasma, but working in association with R gingipains, it can induce vascular permeability by cleaving bradykinin directly from high molecular weight kininogen 168. So, gingipains are responsible for the increase in gingival crevicular fluid production at periodontitis sites infected with gingivalis.
  • Cleavage of complement components: RgpA is a very efficient enzyme in the generation of C5a through direct cleavage of C5. C5a is a potent chemotactic factor that likely contributes to the significant leukocyte infiltration at P. gingivalis induced periodontitis lesions. It also degrades C3 and in this way eliminates the creation of C3-derived opsonins, thus rendering P. gingivalis resistant to phagocytosis 168, 169 this ultimately results in massive accumulation of neutrophils in the inflamed periodontal tissue which contributes to very high levels of active granular proteinases (elastase, cathepsin G, gelatinase, and collagenases) in gingival crevicular fluid 170, 171 that may be responsible for connective tissue destruction.
  • Change in the subgingival environment: The massive accumulation of neutrophils leads to generation of the high level of active granular proteinases which enables subgingival plaque bacteria to thrive due to the presence of high concentrations of peptides and amino acids, thus further aggravating tissue destruction 172.
  • Degradation of clotting factors: Recent studies indicate that RgpA is capable of activating factor X and suggest that this gingipain could be responsible for the production of thrombin 173. Fibrinogen is a major target for Kgp. In vitro, this enzyme degrades the fibrinogen A alpha-chain within a minute 174, thus rendering it nonclottable. It has been postulated that the nonrestricted activity of gingipain K in periodontal pockets contributes to a bleeding tendency, especially because it also very efficiently destroys the pro-coagulant portion of high-molecular-weight kininogen 174.
  • Stimulation of different cell types to produce inflammatory mediators: More recently, it has been reported that P. gingivalis gingipains can activate different cell types leading to the secretion of inflammatory mediators 175-177. In one recent study, it has been found that P. gingivalis gingipains induced an inflammatory response in macrophages through activation of intracellular kinases. Along with that, it was shown that both the Arg- and Lys-gingipain preparations induced the production of TNF-α and IL-8 by macrophages 178.

Collagenase:

The collagenases produced by P. gingivalis are capable of degrading collagen Type I. Proteolytic enzymes like gingipains, collagenases and hyaluronidases destroy periodontal tissue directly or indirectly, leading to the progression of the disease 158, 179. Numerous studies have demonstrated the collagenase activity of P. gingivalis 180-183. It has been shown that inactivation of gingipain R (both RgpA and RgpB) completely eliminates the capacity of P. gingivalis to cleave native collagen Type I, suggesting that these two enzymes are responsible for the collagenase activity of P. gingivalis. Furthermore, gingipain K was shown to have little or no involvement in the degradation of collagen Type I by P. gingivalis 184. P. gingivalis produced collagenase is a therapeutic target of low dose doxycycline in host modulation therapy.

Gelatinase:

There is a scanty literature regarding gelatinases produced by P. gingivalis. However, it is considered as an important component of total proteolytic activity demonstrated by this organism.

Dipeptidyl aminopeptidase IV:

It is a serine protease that cleaves X-Pro or X-Ala dipeptide at the N-terminal end of the polypeptide chain 185. It does not have gelatinase activity itself, but has exopeptidase activity 186. These proteases along with other proteases produced by P. gingivalis degrade connective tissue, facilitating the invasion by P. gingivalis.

Hemagglutinin:

P. gingivalis RgpA and Kgp have hemagglutinin domains. Their ability to hemagglutinate erythrocytes has been well documented 187, 188. Proteinase-hemagglutinin complexes may thus be important in the uptake of essential growth factors by the bacteria, via hemagglutination 189.

As gingipains are responsible for evasion of the host response, thus, it indicates that inhibition of gingipains should be a useful tool, both to assess the contribution of their proteolytic activities to the virulence of the bacterium and to facilitate the development of new therapeutic approaches to periodontal diseases. Research has been done to find out therapeutic agents that inhibit gingipain activity. Among this series of compounds used for gingipain inhibition, it has been found that KYT-1 and KYT-36 had the most potent and selective inhibitory activities of Rgp and Kgp, respectively 190.

Capsule lipopolysaccharide (LPS):

P. gingivalis like other Gram-negative bacteria are sheathed by a capsular LPS. This LPS capsule is outer membrane component recognized by the host cell, thus initiating intracellular signalling events. The affinity of LPS to its pattern recognition receptors, such as the TLRs and CD14, facilitates its recognition and discrimination from other bacterial species by the host immune system. P. gingivalis LPS has been shown to stimulate pro-inflammatory cytokine production by monocytes in vitro 191-194. However, it is a weaker cytokine stimulator as compared to the LPS of other Gram-negative bacteria 195. Structurally, LPS of P. gingivalis is different from other Gram-negative bacteria in terms of O-antigen, acylation patterns and receptor-activating capacities of the lipid A component 196.

Outer membrane vesicles (OMV’s):

P. gingivalis produces OMVs that carry virulence factors to the cell surface. These vesicles are released in an extracellular manner, retaining the full components of outer membrane constituents, including lipopolysaccharide, muramic acid, capsule, fimbriae, and gingipains 197, 198. These measure between 50 and 250 nm in diameter, but are usually around 50 nm. These vesicles are primarily involved in communication with the host cells and other members of microbial biofilms, resulting in the transmission of virulence factors into these host cells and the formation of bacterial communities in plaque biofilm.

 

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Vaccination against P. gingivalis:

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Many researchers have investigated a group of cell surface carbohydrates designated as K-antigens 199, lipopolysaccharides 200, 201 and various proteins, including fimbriae 202 the 53-KDa and 67K-Da cell surface proteins 203 hemagglutinin 204 and cysteine proteases referred to as gingipains 205. Gingipains are present in large quantities on the cell surface of P. gingivalis and have the highest potential to be used as a vaccine antigen.

Gingipains as candidate for periodontal vaccine:

As gingipains are expressed on the outer membrane of P. gingivalis, they are potential candidates for the periodontal vaccine. Gingipain vaccines are mainly DNA vaccines. Arg-gingipain-encoding genes have been cloned from various P. gingivalis strains 206, 207The sequence analysis shows that two separate genes located on the chromosome of P. gingivalis encode Rgp (rgpA and rgpB). As different gingipains contain a catalytic domain and a haemagglutinin domain or any one of them, studies have been done to find out the protective host response against these after immunization 208-211. The antibody response has been documented against these antigens. Further studies are required in this direction to analyze the effectiveness of the vaccine and its practical implications.

Tannerella forsythia

This organism is an anaerobic, fusiform bacterium which was first isolated in the mid-1970s at The Forsyth Institute from subjects with progressing advanced periodontitis and was described as ‘fusiform Bacteroides’ by Tanner et al. (1979) 212. It belongs to the Cytophaga-Bacteroides family and was initially named as Bacteroides forsythus. Later on, it was reclassified as Tannerella forsythia by Sakamoto et al. (2002) 213, based on 16S rRNA phylogenetic analysis. The major problem with the isolation and cultivation of this organism is that it has anaerobic growth requirements for cultural detection. Most of the studies done on this organism are based on non-cultural approaches (immunoassays and DNA-based assays). There are many studies which have implicated Tannerella forsythia as a causative agent for progressive clinical attachment loss associated with periodontitis 6, 7, 40, 214-215. So far, the virulence factors identified for T. forsythia include,

  • Trypsin-like proteases 216
  • PrtH proteases 217 ,
  • Sialidases SiaH 218, 219 and NanH 220,
  • A leucine-rich repeat cell-surface-associated and secreted protein BspA 221,
  • An apoptosis-inducing activity 222,
  • Alpha-D-glucosidase and N-acetyl-beta-glucosaminidase 223,
  • A hemagglutinin 224,
  • Components of the bacterial S-layer 225, 226, and
  • Methylglyoxal production 227.

Following is a brief description of a few of these virulence factors:

Proteases:

T. forsythia is an asaccharolytic organism and requires the presence of peptides and free amino acids for its growth. It procures these essential substrates from the host by degradation of host proteins. Two proteases produced by T. forsythia have been identified which perform this function. These are a trypsin-like protease and PrtH proteases. Along with degradation of host proteins for their growth, these also act as important virulence factors for host invasion in multiple ways which include 228,

  • By causing periodontal connective tissue destruction
  • By activating host degradative enzymes
  • By cleaving components involved in innate (cytokines/chemokines, complement factors) immunity
  • By modifying host cell proteins to expose cryptotopes for bacterial colonization

Surface-layer associated glycoproteins:

The surface components of T. forsythia play a vital role in its virulence. These include,

S-layer:

The organism possesses a surface layer which is made up of serrated structural subunits (about 10 nm wide and 10 nm high) in either oblique or tetragonal lattices 229. This layer is usually described as S-layer. It consists of two high molecular weight glycoproteins (220 and 210 kDa). These glycoproteins are encoded by tfsA and tfsB genes, respectively 230-231. Along with providing protective shielding, this layer also helps in bacterial adhesion as the bacteria lack surface appendages such as fimbriae. It has been shown that S-layer promotes epithelial cell adherence and invasion 225, 226. The S-layer associated glycans facilitate co-aggregation of the organism with F. nucleatum during biofilm formation 232.

Leucine-rich repeat BspA protein:

This is a surface as well as secreted protein that act as an important virulence factor for T. forsythia. Short term used for Bacteroides surface protein A is BspA. This protein has the ability to interact with host factors and/or components. It has been shown to bind to the extracellular matrix component, fibronectin and the clotting factor fibrinogen 221. BspA also facilitates bacterial adherence and invasion into epithelial cells 233. Furthermore, this protein has also been shown to mediate surface interaction with F. nucleatum during biofilm formation 234. By activating the TLR-2-dependent pathway, BspA has been shown to trigger the release of bone-resorbing pro-inflammatory cytokines from monocytes 235 and IL-8 from gingival epithelial cells 236. Hence, it can be concluded that BspA due to its interaction with host immune response plays an important role in the pathogenesis of periodontal tissue breakdown.

Surface lipoproteins:

The surface lipoproteins are involved in the release of pro-inflammatory cytokines (IL-6 and TNF-α) from human gingival fibroblasts and monocytic cells 237. Furthermore, these also have been shown to induce cellular apoptosis 237. The cell apoptosis induced by surface lipoproteins is triggered by activation of caspase-8 which is an initiator of the caspase cascade in apoptosis.

Glycosidic activity:

T. forsythia expresses a variety of glycosidases including neuraminidases SiaHI 219, neuraminidases NanH 220, α-D-glucosidase and N-acetyl β-D glucosaminidase (hexA) 223. These enzymes are responsible for the degradation of various host oligosaccharides and proteoglycans, the products of which provides nutrition to various bacterial species in the plaque bacterial community. Furthermore, the exposure of glycosidases also results in exposure of epitopes for adherence of bacteria.

Treponema denticola

Oral Treponemes are the members of the Spirochaetes phylum having three families (Spirochaetaceae, Brachyspiraceae, and Leptospiraceae) and nine genera. Out of these, genus Treponema includes the causative agents of syphilis and periodontal pathogens. Spirochetes have a distinct shape and structure. Their length varies from 5 to 20 µm and diameter ranges from 0.1 to 0.5 µm. Structurally, they are similar to Gram-positive bacteria consisting of inner and outer membranes. However, most of the species lack lipopolysaccharide (LPS) and instead have lipoproteins and glycolipids 238, 239.  The ultrastructure of spirochetes is characterized by the presence of internal flagella known as endoflagella. Two distinct bundles are formed by endoflagella within the periplasmic space which insert into the inner membrane at opposite ends of the cell. These bundles extend up to two third the length of the cell, wrapping around the inner membrane in a right-handed manner. Among all the oral spirochetes, one of the most studied is T. denticola which possesses the features needed for adherence, invasion, and damage of the periodontal tissues. This periodontal pathogen mostly inhabits the deeper periodontal pockets and is not an early colonizer of subgingival plaque 240. It is typically found in dense subgingival bacterial biofilms at the interface of the biofilms and the gingival epithelium 241, 242. It specifically binds to both P. gingivalis and T. forsythia with a similar avidity which explains its usual presence with these two periodontal pathogens. It binds weakly to Fusobacteria and the interaction is mediated by a carbohydrate moiety, the major sheath protein (Msp) 243, 244. It also binds to early-colonizers like Streptococcus crista 245. Various virulent factors have been identified in T. denticola which makes it an important periodontal pathogen. These include,

  • Major sheath protein (Msp)
  • Leucine-rich repeat proteins
  • Transposases
  • Lipooligosaccharides (LOS)
  • Periplasmic flagella
  • Dentilisin
  • Cystalysin
  • Trypsin-like proteases
  • Lipoproteins
  • Outer sheath vesicles
  • Factor H binding protein
  • Immunomodulation

These virulence factors are discussed as follows,

Major sheath protein (Msp):

The major outer sheath protein (Msp) is a primary virulence determinant in T. denticola. It is the principal immunogenic surface antigen that forms oligomeric protein complexes in the outer membrane of this bacterium 246. Msp has been shown to have matrix protein binding properties in vitro 247 and can cause transient transmembrane ion fluxes and depolarization of host cells 248, 249. Hence, it has been implicated as a mediator of the interaction between the spirochete and the gingival epithelium in periodontal diseases.

Leucine-rich repeat proteins:

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Transposases:

Transposases are a family of enzymes that “cut and paste” mobile genetic elements from one position to another within the genome. It has been observed that 25 out of 35 putative transposases genes were upregulated in a biofilm as compared to the planktonic cell, indicating that these participate in novel gene-regulatory mechanisms in a biofilm 251.

Lipooligosaccharides (LOS):

The outer sheath of T. denticola does not have a typical lipopolysaccharide framework. Instead, it contains lipooligosaccharides (LOS) which consist of two regions Lipid A and R Polysaccharide (core glycolipid) and lacks the O antigen. LOS has been shown to stimulate the activation of NO (nitric oxide) and TNF-α in LPS -responsive and -nonresponsive mouse macrophages. The macrophage activation by LOS is similar to lipopolysaccharide activation 252.

Periplasmic flagella:

T. denticola has usually two periplasmic flagellae attached at each end. The flagellar filament of T. denticola consists of 3 core proteins: FlaB1, FlaB2 and FlaB3. These flagella facilitate translocation of the bacteria in highly viscous environments. Furthermore, it has been shown that periplasmic flagella do not get immobilized by flagella-specific antibodies produced by the host response to infection 253.

Dentilisin:

It is a protease located on the cell surface of T. denticola that cleaves at phenylalanyl/alanyl and prolyl/alanyl bonds 254. The active site of these serine proteases contains a His-Asp-Ser motif in chymotrypsin that is believed to be responsible for catalyzing the hydrolysis of peptide bonds. It contributes to disease progression by disrupting or modulating intercellular host signalling pathways and degrading host cell matrix proteins. It is also involved in the activation of complement pathway by cleaving C3.

Cystalysin:

It is a hemolytic protein capable of haemo oxidizing hemoglobin and causing lysis of human erythrocytes 255. The production of H2S is considered to be the major function of cystalysin, which can thus be regarded as an important virulence factor 256, 257.

Trypsin-like proteases:

The enzyme responsible for trypsin-like activity in T. denticola is oligopeptidase B which is encoded by opdB (TDE2140). This enzyme has been shown to cleave C-terminal to Arg-residues 258. The exact role of this enzyme in the virulence of T. denticola still remains to be clarified.

Lipoproteins:

These are the most abundant membrane-associated proteins found in spirochetes. 20 lipoproteins have been detected in T. denticola in a study by Veith et al. (2009)259 including 2 hemin-binding proteins (HbpA, HbpB), 2 putative extracellular solute-binding lipoproteins, 4 putative oligopeptide/dipeptide  ABC transporter peptide-binding proteins, the dentilisin complex-associated polypeptide (PrcA) and 11 uncharacterized putative lipoproteins. The exact role of these lipoproteins in the virulence of T. denticola still needs to be clarified.

Outer sheath vesicles:

The outer sheath vesicles are produced by bacteria in response to changes in the external environment 260, 261. These vesicles are potent virulence factors because they possess adhesins,  toxins,  and  proteolytic  enzymes,  which facilitate bacterial  invasion and  aggregationare  cytotoxic,  and  can modulate the host immune response 262. Furthermore, these vesicles help in bacterial aggregation by delivering proteases and toxins which are active against other bacterial species, thus facilitating to secure a niche in the competitive environment of subgingival plaque 263, 264. These vesicles also facilitate remote delivery of labile signaling molecules and prevent their degradation by other microorganisms 265. The outer sheath vesicles of T. denticola have also been implicated in tissue invasion 262, 266. These have been shown to disrupt the tight junctions in epithelial cell monolayers, hence facilitating connective tissue invasion 267.

Factor H binding protein:

  1. denticola possesses a small surface-exposed lipoprotein, FhbB. This lipoprotein binds complement regulatory proteins of the factor H (FH) family. It cleaves factor H into subfragment of ~50 kDa. This lipoprotein, also down-regulates the production of C3b. Thus, this factor is involved in the immunomodulation.

Immunomodulation:

  1. denticola can evade various aspects of innate immunity by preventing efficient binding of antimicrobial peptides, such as β-defensins. It has been demonstrated that young patients with generalized severe periodontitis exhibited lower serum levels of antibodies against T. denticola as compared to those in healthy subjects 268. The diminished antibody response against T. denticola in patients with aggressive periodontitis is suggestive of its immunomodulatory properties. Furthermore, T. denticola has also been reported to affect the migration of neutrophils 270.
 

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Invasiveness of bacteria:

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Prevotella intermedia

P. intermedia are Gram-negative, black-pigmented, obligate anaerobic rods which have been implicated as putative periodontal pathogens 51, 52, 281-284. These are called as “black pigmented bacteria” because they form shiny and smooth colonies with gray, light brown or black color on blood agar plate 285. P. intermedia were formerly known as Bacteroides intermedia or Bacteroides melaninogenicus. They co-aggregate with other Gram-positive oral bacteria such as P. gingivalis and Actinomyces viscosus during plaque formation. This bacterial species appears to have multiple virulence factors exhibited by P. gingivalis and has been shown to induce mixed infections on injection in laboratory animals 286. Furthermore, it has also been shown to invade oral epithelial cells in vitro 279. Along with periodontal infections, P. intermedia has also been reported to colonize in the respiratory tract and has been found to be associated with cystic fibrosis and chronic bronchitis 287, 288. It utilizes estradiol and progesterone as a growth factor, instead of vitamin K 289. There are various virulence factors associated with P. intermedia. Some of these are,

  • Lipopolysaccharides
  • Proteinases
  • Immunomodulators

Lipopolysaccharides:

LPS is a major constituent of the outer membrane of P. intermedia. It is an important virulence factor which can trigger a number of host cells, especially mononuclear phagocytes, to produce and release a wide variety of pharmacologically active mediators, including IL-1β, IL-6, IL-8, and, most importantly, tumor necrosis factor a (TNF-α) 290, IL-10 291, IL-6 and IL-8 292, 293.

Proteinases:

Proteinases produced by P. intermedia contribute in tissue destruction and increased availability of nutrients. The proteinases produced by P. intermedia display trypsin-like and dipeptidyl peptidase activities 294. These proteinases have also been shown to possess properties of cysteine proteinases 295-297.

Immunomodulators:

Interpain A is a secreted protein composed of 868 amino acid residues 298. It has been shown to degrade C3 complement of complement system 299. Hence, the organism has been shown to modulate the innate immune response which facilitates its survival in the host tissues.

Renaming and re-classification of periodontal pathogens

 

Previous Classification New Classification Reference
Bacteroides gingivalis Porphyromonas gingivalis Shah and Collins, 1988 300
Bacteroides endodontalis Porphyromonas
endodontalis
Shah and Collins, 1988 300
Bacteroides intermedius  P. intermedia Shah and Collins, 1990 301
Bacteroides melaninogenicus Prevotella melaninogenica Shah and Collins, 1990 301
Bacteroides denticola Prevotella denticola Shah and Collins, 1990 301
Bacteroides loescheii Prevotella loescheii Shah and Collins, 1990 301
Wolinella recta Campylobacter rectus Vandamme, et al., 1991 302
Wolinella curva Campylobacter curvus Vandamme, et al., 1991 302
Actinobacillus actinomycetemcomitans Aggregatibactor actinomycetemcomitansNørskov-

 

Role of viruses in pathogenesis of periodontal diseases

The research during the last few decades has pointed out that viruses may also be involved in the pathogenesis of periodontal diseases. These findings have been possible because of the introduction of molecular identification techniques, primarily the Polymerase chain reaction (PCR)-based techniques. Various viral pathogens have been identified which may play an active role in periodontal tissue destruction. Most of the well-investigated viruses as causative agents for periodontitis belong to herpesvirus family. The viruses within the herpes virus family have been divided into three subfamilies according to pathogenicity, the type of cell wherewith they were infected and the properties of their growth: alpha, beta and gamma subfamilies. The alpha subfamily consists of Herpes simplex virus type 1 (HSV-1), type 2 (HSV-2) and Varicella zoster virus (VZV). These viruses have a rapid growth pattern and they remain latent in sensory nerve ganglia. The beta herpesvirus subfamily consists of human cytomegalovirus (CMV), human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7). These have a slow replication rate, usually produce large multinucleated cells and remain latent in lymphoreticular tissue, salivary glands,  kidneys and other tissues. The gamma herpesviruses include Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8). They are located in lymphoid tissues latently. A high proportion of inflammatory cells derived from periodontally diseased sites have been shown to be infected by viruses, including HCMV, EBV-1, EBV-2, HHV-6 and HSV 303.

Other viruses that have been shown to be present in periodontitis associated sites include papillomaviruses 304-306, human immunodeficiency virus (HIV) 307, 308, human T-lymphotropic virus type 1 309, hepatitis B virus 310, hepatitis C virus 311 and torque teno virus 312. Table 5.3 describes various viruses and their clinical manifestations.

Viral infections and their oral manifestations

Viral diagnostic methods:

Various diagnostic methods have been used to identify viruses in periodontal diseases. The earlier virus identification techniques included cell culture to detect characteristic cytopathic effects and morphological determination of intra-cytoplasmic changes in clinical specimens. The presence of herpesvirus in periodontal specimens has also been confirmed by using flow cytometry and immunofluorescence staining. Presently, molecular methods such as Polymerase chain reaction (PCR) techniques are used to identify the presence of specific virus in a specimen. There are several types PCR techniques used to identify specific DNA sequences. These techniques have been discussed in detail in “Advanced diagnostic techniques”. The main problem associated with PCR techniques is that they only detect the viruses they are designed to detect. There are many viruses which have been identified or are yet to be identified in the oral environment. The newer techniques that have been introduced to overcome these limitations aremicroarrays’ andpyro-sequencing’. Microarrays use probes that detect different viruses (or other agents) and can be applied to a slide and the sample DNA or RNA hybridized onto the slide, thus offering the possible detection of all known viruses. Pyro-sequencing involves sequencing of complete nucleic acids present in the sample so that the recognizable viral sequences can be identified by searching relevant databases. The limitations associated with these methods are their reduced sensitive than PCR techniques and higher cost as compared to conventional PCR techniques. However, efforts are being made to reduce the cost associated with pyro-sequencing technique.

Viruses in periodontal diseases

Evidence for viral involvement in periodontitis:

The evidence for the involvement of viruses in the etiopathogenesis of periodontal diseases has been derived from various findings. The following evidence suggests that viruses may be actively involved in the development of periodontitis,

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The dynamics of viral communities in periodontal health and disease:

As already stated earlier in this article, oral cavity provides diverse niches that harbor a wide range of microbiota 322. These microorganisms create a complex biofilm where a complex interaction between different bacterial species can be observed. The pathogenesis of periodontal disease depend more on these bacterial interactions and changes in the subgingival environment due to these interactions rather than individual bacterial species identified. Similarly, the viral population in the subgingival environment should be analyzed as a whole in their virulence potential in causing periodontal disease. Although, herpesviruses are the most commonly identified viruses in the oral cavity, but there is a much larger population of viruses present, the majority of which are bacteriophages 323, 324. Most of these bacteriophages primarily belong to Caudovirus families: Siphoviridae (generally lysogenic with intermediate host ranges), Podoviridae (typically lytic with relatively narrow host ranges) and Myoviridae (typically lytic with relatively broad host ranges) 325, 326. Presently, there is insufficient clinical data describing the combined pathogenic potential of various viruses and bacteriophages present in the subgingival arena and this field is wide open for research. However, specific viruses have been investigated in periodontal health and disease. Let us now discuss in detail various periodontal conditions and viruses associated with them.

Periodontal health:

The most commonly identified viruses in healthy or slightly inflamed periodontal sites belong to herpesvirus family. In the presence of previous attachment loss, herpesviruses can be detected even in areas with slight inflammation. Furthermore, healthy periodontal sites in a periodontitis patient may harbor more number of herpesviruses as compared to periodontal sites in an individual with a healthy periodontium without previous periodontitis 327. The healthy periodontal sites usually do not harbor CMV 328. Periodontal sites with gingivitis have been shown to harbor EBV and CMV 319, 329.

It must be noted here that various studies have demonstrated a wide variation in the occurrence of various viruses in different periodontal conditions. This variation is primarily attributed to diagnostic difficulties and a natural fluctuation of periodontal herpesviruses 330.

Aggressive periodontitis:

Many investigations have been done to identify the presence of viruses in aggressive periodontitis cases.  Localized aggressive periodontitis Afro-Caribbean adolescent population was studied to find out the presence of subgingival herpesviruses. It was observed that CMV and P. gingivalis had a synergistic relationship that influenced the risk of both the occurrence and the extent of disease 331. The authors concluded that in Afro-Caribbean adolescent population, there was a strong relationship between the presence of aggressive periodontitis and presence of CMV and P. gingivalis. These findings were further supported by other studies 319, 332, 333.

Another study by Kamma et al. (2001) 320 was done on 16 subjects with early onset periodontitis. The results of the study demonstrated that EBV, CMV and EBV-CMV co-habitation was significantly associated with sites with an aggressive periodontal breakdown. It has been demonstrated that genome copy-counts as high as 8.3 x 108 and 4.6 x 105 for EBV and CMV, respectively, can be present at a site with advanced periodontal destruction 334, 335. Viruses other than EBV and CMV that have been detected at the site of advanced periodontal destruction include Herpesviruses 6, 7 and 8 336. Along with herpesviruses, non-herpesviruses also have been identified at sites with advanced periodontal destruction 306.

Chronic periodontitis:

As for aggressive periodontitis, the most commonly isolated viruses in chronic periodontitis belong to herpesvirus family, including herpes simplex virus-1, EBV type 1 and CMV 337. The antibody response against these viruses has also been identified. In GCF samples from periodontitis sites, an antibody response against EBV was detected in 32% of samples and against CMV in 71% samples 338.

Viruses other than those belonging to herpesvirus family isolated from periodontitis sites include human T-lymphotropic virus type 1 309, hepatitis B virus 310, hepatitis C virus 311, papillomaviruses 304- 306, human immunodeficiency virus (HIV) 307, 308, and torque teno virus 312.

HIV-associated periodontitis:

The HIV-positive immuno-compromised patients usually demonstrated various bacterial and viral diseases which are opportunistic and are not usually observed in immunocompetent patients. HIV-induced immunosuppression causes re-activation of herpesvirus 339. EBV-1 has been found more frequently in subgingival sites of HIV-positive patients than in subgingival sites of HIV-negative patients 340. It has been shown that the most commonly identified herpesvirus in HIV-associated periodontitis cases is CMV, which was identified in 81% of HIV-associated periodontitis lesions 341. Furthermore, apart from its presence in HIV-positive immunocompromised patients, CMV has also been implicated in acute periodontitis 342, in periodontal abscess formation 343, in mandibular osteomyelitis 343 and in refractory chronic sinusitis 344. EBV-2 was detected in 57% biopsies of HIV-positive patients and was not detected in biopsies from non-HIV associated periodontitis patients 341. Other viruses that have been detected in HIV-positive periodontitis patients include Human herpesvirus-8, herpes simplex virus, EBV, CMV 336, 340, 345, 346.

Necrotizing ulcerative gingivitis and periodontitis:

The necrotizing periodontal diseases are primarily observed in immunocompromised patients due to malnutrition, psychosocial stress or HIV infection. In severe cases the necrosis of a large portion of the oro-facial structure can take place, resulting in a condition known as ‘noma’ or ‘cancrum oris’. Many viruses have been identified from periodontal lesions from these patients. In a study done on malnourished children herpes simplex virus, EBV and CMV were detected in 23%, 27% and 59% of cases, respectively 347. The herpesvirus is usually acquired by children in early childhood, which, along with various putative bacterial species can cause necrotizing ulcerative gingivitis in malnourished children.

 

 

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Difficulties in establishing the etiology of periodontal disease:

As discussed above, many bacterial species have been identified as potential periodontal pathogens. However, there are many problems in determining which organisms are important in the pathogenesis of periodontal disease. These include 348,

  • More than 700 species of microorganisms have been identified from the periodontal pockets of different individuals with a single periodontal site harboring 30-100 species.
  • The proliferation of bacterial species is determined by the habitat. The environmental factors play a major role in the establishment of periodontopathogenic microflora.
  • There are many bacterial species which are difficult to grow in culture or cannot be grown in culture media. They are identified with the help of molecular identification methods recognizing their DNA sequences. But, without viable microbes, antibiotic sensitivities cannot be determined.

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Conclusion

It is clear from the above discussion that various micro-organisms act as major etiological factors in the initiation and propagation of periodontal diseases. Although periodontal diseases are multifactorial diseases, microbial etiology should be viewed as their most important etiology. Recent molecular technologies have enabled us to understand the pathogenic mechanisms associated with various micro-organisms. However, much still has to be learned. We need to investigate the behavior of the microorganisms in biofilms and the expression of their virulence factors. In future, a better understanding of molecular mechanisms involved in the microbial etiopathogenesis of periodontal diseases is expected.

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