Periodontal pathogens

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

1. Periodontal pathogens Dr Lakkireddy Vasavi reddy II MDS

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

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

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

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

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

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

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

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

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

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

12. Microbial complexes:

13. Classification of periodontal pathogens:

14. Based on pathogenicity:

15. According to Lindhe :

16. Distribution:

17. “The Green Complex”

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

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

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

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

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

23. Virulence factors:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

39. Orange complex organisms

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

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

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

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

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

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

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

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

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

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

50. Fusobacterium nucleatum Family: Fusobacteriaceae Etymology: fusus : spindle bacterios : bacteria

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

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

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

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

55. Campylobacter rectus Family: Campylobateraceae Etymology: Kampulos : bent Bacter : bacterium

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

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

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

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

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

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

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

63. “Red complex”:

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

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

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

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

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

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

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

71. Historical aspect:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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