1 2 Contents • • • • • • • • • • Introduction History Diversity of intraoral surfaces for bacterial adhesion Dental plaque Changing views of plaque Plaque formation at the ultrastructural level Growth dynamics of dental plaque Individual variables influencing plaque formation Physiologic properties of dental plaque Microbial complexes 3 • • Factors that affect the composition of subgingival biofilms Intraoral equilibrium between cariogenic species and Periodontopathogens • • Microbial specificity of periodontal diseases Association of plaque microorganism with periodontal diseases • • • • • Microbes associated with specific diseases Periodontal Pathogens Microbial diagnostic testing Future advances in periodontal microbiology Conclusion 4 Most unusual infection. Bacteria may attach to the tooth, epithelial surfaces, connective tissues, & to other bacteria. The outer layers of the tooth do not shed & thus microbial colonization is facilitated. Thus, a situation is setup in which microorganisms colonize a relatively stable surface. (google images) 5 Robert koch (1843 – 1910) W D Miller (1853 – 1907) Adolph Witzel(1847 – 1906) 6 G V BLACK (1836 – 1915) J L WILIIAMS (1852 – 1932) 7 Human fetus is usually sterile. Colonization starts at birth. Within hours – facultative & aerobic bacteria. 2nd day – anaerobic bacteria. Within 2 weeks – mature microbiota of gut. After weaning - 10¹⁴ microorganisms with 400 different type of microorganisms. There are 10 times more bacteria than human cells. 8 • There is a positive correlation between the adhesion rate of pathogenic bacteria to different epithelia and the susceptibility of that patient to certain infections. • High turnover rate of epithelial cells prevents accumulation of large masses of microorganisms. • Teeth are natural habitat of for periodontopathogens. 9 • Dental plaque is defined yellow-grayish substance intraoral hard surfaces, restorations.(Carranza 10th clinically as a structured, resilient, that adheres tenaciously to the including removable and fixed edition) • Materia alba refers to soft accumulations of bacteria and tissue cells that lack the organized structure of dental plaque, and it is easily displaced with a water spray. (Carranza 10th edition) • Calculus is a hard deposit that forms by mineralization of dental plaque, and it is generally covered by a layer of unmineralized plaque. (Carranza 10th edition) 10 Composed primarily of microorganisms. 1 gram = 10¹¹ microorganisms 10³ in healthy crevice to 10⁸ in deep pocket. 11 12 Characteristic Supragingival Gram reaction Morphotypes +/Cocci, branching rods, filaments, spirochetes Subgingival Dominated by Dominated by rods and spirochetes Dominated by anaerobes Energy metabolism Facultative with some anaerobes Energy sources Generally ferment carbohydrates Many proteolytic forms Motility Firmly adherent to plaque Adherence less pronounced with matrix many motile forms Can cause caries and gingivitis Can cause gingivitis and periodontitis 13 Response by host Availability of blood products and a low oxidation reduction (redox) potential and GCF substances help and act as nutrients and characterize the anaerobic environment. Microorganisms facing the soft tissue lack a definite intermicrobial matrix and contain primarily gram negative rods and cocci, as well as large numbers of filaments, flagellated rods, and spirochetes. The apical part is dominated by spirochetes, cocci, and rods, whereas in the coronal part, more filaments are observed. 14 • A biofilm is a well organized, cooperating community of microorganisms (Overman 2000). • Biofilms consist of one or more communities of microorganisms, embedded in a glycocalyx, that are attached to a solid surface. • Biofilms have been defined as matrix embedded microbial populations, adherent to each other and/or to surfaces or Interfaces (Costerton et al. 1995). 15 BASIC BIOFILM PROPERTIES (Overman 2000): • Cooperating community of various types of microorganisms. • Microorganisms are arranged in microcolonies. • Microcolonies are surrounded by protective matrix. • Within the microcolonies are differing environments. • Microorganisms have primitive communication system. • Microorganisms in biofilms are resistant to antibiotics, antimicrobials, and host response. 16 LAYERS: Lower plaque layers: Loose layer: Fluid layer: 17 INTERCELLULAR MATRIX 18 EXOPOLYSACCHARIDES 50 – 95% of the dry weight. Maintain the integrity of the biofilms as well as preventing desiccation and attack by harmful agents. It acts as a buffer and assists in the retention of extracellular enzymes. 19 PHYSIOLOGICAL HETEROGENEITY WITHIN BIOFILMS: Cells of the same microbial species can exhibit extremely different physiologic states in a biofilms. pH and the number of metal ions can vary quite remarkably over short distances within a biofilms. Certain microcolonies are completely anaerobic even though composed of a single species and grown in ambient air. 20 • QUORUM SENSING: • Regulation of expression of specific genes through the accumulation of signaling compounds that mediate inter cellular communication. (Prosser 1999). It is dependent on cell density. Quorum sensing signaling molecules produced by putative periodontal pathogens such as P.gingivalis, P.intermedia, and F.nucleatum. (Frias et al 2001). 21 Quorum sensing give biofilms distinct properties: • Expression of genes for antibiotic resistance • Influence community structure by encouraging the growth of beneficial species. • Discouraging the growth of competitors. • Physiological properties of bacteria in the community may be altered. 22 MECHANISM OF INCREASED ANTIBIOTIC RESISTANCE • It differs from species to species, from antibiotic to antibiotic. Few mechanisms are:• The slower rate of growth. • Variation in parameters like nutritional status, growth rate, temperature, pH and prior exposure to sub effective concentrations. • As an ion exchange resin removing antibiotics. • Extracellular enzymes in extracellular matrix, inactivate the susceptible, typically positive charged, hydrophilic antibiotics. • Alteration of genotype and phenotype of the cells growing within a biofilms matrix. 23 SIGNIFICANCE OF MICROBIAL COMMUNITIES • • • A broader habitat range for growth. An increased metabolic diversity and efficiency. An enhanced resistance to environmental stress, antimicrobial agents and the host defenses. Shapiro (1998), Marsh & Bowden (2000). 24 • The formation of the pellicle on the tooth surface. • Initial adhesion and attachment of bacteria. • Colonization and plaque maturation. 25 FORMATION OF THE PELLICLE: The pellicle consists of; Glycoproteins (mucins) Proline-rich proteins Phosphoproteins (statherin) Histidine-rich proteins Enzymes (alpha amylase) Other molecules which acts as adhesion sites. 26 Mechanism involved in pellicle formation include: Electrostatic forces Van der Walls forces Hydrophobic forces 27 INITIAL ADHESION AND ATTACHMENT OF BACTERIA: Phase 1: Transport to the surface; Random contacts may occur. (through Brownian motion, liquid flow or active bacterial movement). Phase 2: Initial adhesion; Initiated by the interaction between the bacterium and the surface, from a certain distance (50 nm), through long range and short range forces. Phase 3: Attachment; A firm anchorage between bacterium and surface will be established by specific interactions (covalent, ionic or hydrogen bonding). Eg: A. viscosus possesses fimbriae that contain adhesions that specifically bind to proline rich proteins of the dental pellicle. Phase 4: Colonization of the surface and biofilm formation. 28 COLONIZATION & PLAQUE MATURATION • Firmly attached microorganisms start growing and newly formed bacterial clusters remain attached, microcolonies or a biofilm can develop. • At least 18 genera from the oral cavity have shown some form of co aggregation. • Highly specific stereochemical interaction of protein and carbohydrate molecules located on the bacterial cell surfaces. • Mediated by lectinlike adhesions and can be inhibited by lactose and other galactosides. 29 Early and Secondary Colonizers: • Interaction of secondary colonizers with early colonizers include the coaggregation of; * Fusobacterium nucleatum with Streptococcus sanguis, * Prevotella loescheii with Actinomyces viscosus, and * Capnocytophaga ochraceus with A.viscosus. • Special examples of coaggregations are the i) “corncob” formation. ii) “Test tube brush” 30 Ultrastructural Aspects: Important changes within first 24 hours. • First 2 to 8 hours – Pioneering streptococci saturate the salivary pellicular binding sites and thus covering 3% to 30% of the enamel surface. • Next 20 hours – a short period of rapid growth is observed. • After 1 day – the term ‘Biofilm’ is fully deserved because organization takes place within it. • 31 • The further growth of the plaque mass occurs preferably by the multiplication of already adhering microorganisms rather than by new colonizers. • The thickness of the plaque increases slowly with time, increasing to 20 to 30 µm after 3 days. (google images) 32 • Clinically it follows an exponential growth curve. • First 24 hours – negligible plaque, covering <3% of the vestibular tooth surface. • During next 3 days – plaque growth increases at a rapid rate, then slows down. • After 4 days – average of 30% of the total tooth crown area will be covered with plaque and no more increase substantially with time. (google images) 33 • Critical role in the normal development of the physiology of the host (McFarland, 2000). • Reduces the risk of infection by acting as a barrier to colonization by exogenous species (termed ‘colonization resistance’) (van der Waaij et al, 1971). • Mechanisms I. More effective competition for nutrients and attachment sites. II. The production of inhibitory factors. III.creation of unfavorable growth conditions for invading species by the normal microflora. 34 • Early plaque formation on teeth follows a typical topographic pattern with initial growth along the gingival margin and from the interdental space. (protected from shear forces). • Plaque formation can also start from surface irregularities like grooves, cracks, perikymata, or pits. • By multiplication, the bacteria subsequently spread out from these initial areas as a relatively even monolayer. 35 • Heavy (fast) plaque formers. • Light (slow) plaque formers. • Simonsson et al (1889) considered no single variable as the great differences in rate of plaque formation. • Zee et al (1997) found no discernible differences except for a more prominent intermicrobial matrix and higher proportions of gram-negative rods in the group of fast growers. • Variation with dentition. • Impact of gingival inflamation. • Impact of patient age. • Spontaneous tooth cleaning. 36 • It is technically impossible to record the dynamics of subgingival plaque formation in an established dentition for the reason that one cannot sterilize a periodontal pocket at present. • Some early studies, using culturing techniques, examined the changes within the subgingival microbiota during the first week after mechanical debridement and reported only partial reduction, followed by a fast regrowth to almost pretreatment levels within 7 days. 37 38 • Periodontal disease status • The local environment • Principle of bacterial transmissioan, translocation, or cross infection • Microbial composition of supra and subgingival biofilms • Translocation and guided tissue regeneration • Translocation and mechanical debridement 39 Periodontal Disease Status: • Perhaps the most influential factor on the composition of the subgingival microbiota is the periodontal disease status of the host. • Clearly, the major difference between health and disease was the increased prevalence and counts of the red complex organisms. 40 The local environment: • One host factor that influences the subgingival environment is pocket depth. • Red complex species, increased strikingly in prevalence and numbers with increasing pocket depth. • The species of the red and orange complexes are also elevated at sites exhibiting gingival inflammation. 41 Principle of bacterial transmission, translocation, or cross infection: • Bacterial fingerprinting clearly illustrates that periodontal pathogens are transmissible within members of a families. • The existence of an intraoral transmission of bacteria (from one niche to another, also called translocation or cross-infection), could jeopardize the outocome of periodontal therapy. • Christersson et al (1985) demonstrated translocation of A.actinomycetumcomitans by periodontal probes in patients with localized aggressive periodontitis. 42 Translocation and Guided Tissue Regeneration: • Nowzari et al (1996) evaluated the amount of guided tissue regeneration and membrane contamination and concluded that healthy group showed significantly less membrane contamination both immediately after insertion as well as at removal after 6 weeks. • Mombelli et all (1997) concluded that pathogens most likely were transferred through saliva from infected untreated periodontal lesions or other niches to the treated sites. 43 Translocation and Mechanical debridement: The one stage, full mouth disinfection concept consists of a combination of the following therapeutic efforts: • Full mouth sacling and root planning within 24 hours to reduce the number of subgingival pathogenic organisms. • Subgingival irrigation of all pockets with a 1% chlorhexidine gel to kill remaining bacteria. • Tongue brushing with an antiseptic to suppress the bacteria in the niche. • Mouth rinsing with an antiseptic to reduce the bacteria in the saliva and on the tonsils. 44 Microbial composition of SUPRA and Subgingival biofilms • The bacteria associated with periodontal diseases reside within biofilms both above and below the gingival margin. • The supragingival biofilms is attached to the tooth surface and is predominated by Actinomyces species. • The nature of subgingival biofilms is more complex with both tooth associated and tissue associated biofilms • As one moves from the supragingival to the subgingival environment and from health to disease, there is a significant decrease in the Actinomyces species and an increase in the proportion of members of the red complex. 45 • Subgingival outgrowth by S.mutans occupying spots that became available after periodontal therapy (e.g increased number of free adhesion/receptor sites). • Creation of a new ecosystem in the subgingival area which is characterized by being more anaerobic, and having a lower redox potential, lower pH and a protein concentrated nutritional environment. • Down growth of S.mutans from the subgingival area, where the species could survive in the saliva. 46 NONSPECIFIC PLAQUE HYPOTHESIS: This hypothesis maintains that periodontal disease results from the “elaboration of noxious products by the entire plaque flora” SPECIFIC PLAQUE HYPOTHESIS: The specific plaque hypothesis states that only certain plaque is pathogenic, and its pathogenicity depends on the presence of or increase in specific microorganisms. 47 Ecological Plaque Hypothesis A change in the nutrient status of a pocket or chemical and physical changes to the habitat are thus considered the primary cause for the overgrowth by pathogens. Axellson vol 3, pg 45 48 Association of Plaque microorganisms with periodontal diseases • Susceptible Host. • Pathogenic species. • The absence, or a small proportion , of beneficial bacteria. 49 (Google images) 50 HIV INFECTION Periodontal disease more prevalent & severe in patients infected with HIV (Greenspan et al 1989, 1993) Pathogenic species same.(Murray et al 1989) Can be treated with conventional periodontal therapies. (Williams et al 1990) (Google images) 51 DIABETES Periodontal disease is more comman in diabetic patients than in non diabetic patients.(Seppala et al 1993). P. intemedia, P. gingivalis, C. rectus were found to be more in diseased sites.(Zambon et al 1988) (Google images) 52 SMOKING More bone loss, attachment loss, deeper periodontal pockets & less gingival bleeding(Hafajee & Socransky 2000). Heal less satisfactorily(Kinane 1997) Red & orange complex species more in smokers than in non – smokers. (Google images) 53 GENETICS Genetic variations or mutations modulate the individuals response. Significant link between genetic marker & periodontal disease(Genco et al 1993). (Google images) 54 PATHOGENS Pathogen with susceptible clonal type & in sufficient numbers. Periodontal pathogens present in healthy sites of periodontal diseased raises question? A actinomycetemcomitans, T. forsythia, P. gingivalis. (Google images) 55 BENEFICIAL BACTERIA Affect disease progression by: Passively occupying niche. Limiting pathogen to adhere to tissue surface. Adversely affecting the growth. Ability to produce virulence factor affected. Degrading virulence factor. 56 Criteria for Identification Koch’s postulates(1870): • The organism must be found in lesions of the disease, • It must be isolated and grown in pure culture on artificial media, • Inoculation of this culture into experimental animals must produce lesions similar to those observed in cases of disease in humans, and • The organism must be recovered from the lesions in the experimental animals. 57 Socransky’s criteria(1978): • • • • • Association with Disease Elimination of the Organism Host Response Animal Pathogenicity Mechanisms of Pathogenicity 58 Microorganisms associated with Specific Periodontal disease 59 GINGIVITIS 104 to 106 bacteria. Gram-negative bacteria. Compared with healthy sites, noticeable increase also occur in the numbers (spirochetes). Pregnancy associated gingivitis is accompanied by dramatic increases in levels of P. intermedia, which uses the steroid as growth factors(Carranza,10th edition). Axellson,pg no 51 60 CHRONIC PERIODONTITIS C. rectus, P. gingivalis, P. intermedia, F. nucleatum and T. forsythia were found to be elevated in the active sites(Carranza,10th ). Sites with chronic periodontitis will be populated with greater proportions of gram-negative organisms and motile bacteria. Certain gram-negative bacteria with pronounced virulence properties have been strongly implicated as etiologic agents e.g. P. gingivalis and Tannerella forsythus. Axellson,pg no 53 61 LOCALIZED AGGRESSIVE PERIODONTITIS • Gram -ve, capnophilic, and anaerobic rods. • The most numerous isolates are several species from the genera Eubacterium, A. Veillonella parvula. • In some populations, a strong case can be made for Aa playing a causative role in localized aggressive periodontitis, especially in cases in which patients harbor highly leukotoxic strains of the organism. • However, some populations of patients with LAP do not harbor Aa, and in still others P. gingivalis may be etiologically more important. 62 naeslundii, F. nucleatum, C. rectus, and GENERALIZED AGGRESSIVE PERIODONTITIS The sub-gingival flora in patients with generalized aggressive periodontitis resembles that in other forms of periodontitis. The predominant subgingival bacteria in patients with generalized aggressive periodontitis are P. gingivalis, T. forsythis A. actinomycetemcomitans, and Campylobacter species. Axellson,pg no 52 63 REFRACTORY CHRONIC PERIODONTITIS • Unusually diverse and may contain enteric rods, staphylococci, and Candida. • Persistently high levels are found of one or more of P. gingivalis, T. forsythis, S. inter-medius, P. intermedia, Peptostreptococcus micros, and Eikenella corrodens. • Persistence of Streptococcus constellatus has also been reported. 64 NECROTIZING ULCERATIVE GINGIVITIS/PERIODONTITIS More than 50% of the isolated species were strict anaerobes with P. gingivalis and F. nucleatum accounting for 7-8% and 3.4%, respectively. Axellson,pg no 53 65 PERIODONTAL ABSCESSES The bacteria isolated from abscesses are similar to those associated with chronic and aggressive forms of periodontitis. An average of approximately 70% of the cultivable flora in exudates from periodontal abscesses are gram-negative and about 50% are anaerobic rods. Periodontal abscesses revealed a high prevalence of the following putative pathogens: F. nucleatum (70.8%), P. micros (70.6%), P. intermedia (62.5%), P. gingivalis (50.0%), and T. forsythis (47.1%). Enteric bacteria, coagulase-negative staphylococci, and Candida albicans have also been detected. 66 Periodontal Pathogens ACTINOBACILLUS ACTINOMYCETEMCOMITANS: ACTINOMYCETEMCOMITANS It was originally named Bacterium actinomycetum comitans (Klinger 1912) which was changed to Bacterium comitans (Lieske 1921) and finally to Actinobacilius actinomycetemcomitans (Topley & Wilson 1929) (Norskov-Lauritsen N(2006) 67 Small, non motile, Gm –ve, saccharolytic, round ended rod that forms small convex colonies with a “star shaped” center when grown on blood agar. Recognised by Newman et al, 1976 (Google images) 68 SEROTYPES: a, b, c, d, e, f. Serotype b with LAP(Zambon et al 1983). Serotype a with chronic periodontitis(Zambon et al 1983). LEUKOTOXIN: Affects PMNS, macrophages, monocytes, lymphocytes. Lowconcentration – apoptosis High concentration - necrosis 69 70 PORPHYROMONAS GINGIVALIS • It is a Gram negative, anaerobic, non motile, asaccharolytic rods that usually exhibit coccal to short rod morphologies. • Group of “black pigmented Bacteroides” Asaccharolytic - P. gingivalis Intermediate level of carbohydrate fermentation - P. intermedia Highly saccharolytic - Prevotella melaninogenica. • P. gingivalis has been shown to induce elevated systemic and local immune responses in subjects with various forms of periodontitis (Haffajee & Socransky 1994). 71 PROTEOLYTIC ACTIVITY • Degrade proteins into short peptides and used metabolically in the generation of energy and as sources of carbon and nitrogen. • Gingipains capable of degrading collagen fragments. • Proteases occur in multiple forms that are found extracellularly or on the bacterial cell surface. • The gingipains specifically cleave proteins at the peptide bond following arginine residues or lysine residues. 72 73 TANNERELLA FORSYTHIA The organism is a Gram negative, anaerobic, spindle shaped, highly pleomorphic rod. Difficult to grow. Need growth factors from other species. B. forsythus was detected more frequently and in higher numbers in active periodontal lesions than inactive lesions (Dzink et al 1988). 74 This species has been shown to produce trypsin like proteolytic activity (Loesche et al 1992) and induce apoptotic cell death (Arakawa et al 2000). Double labeling experiments demonstrated that B. forsythus was both on and in periodontal pocket epithelial cells indicating the species ability to invade. Shares antigen with P. gingivalis. Listgarten et al (1993) found that the species most frequently detected in “refractory” subjects was B. forsythus. 75 SPIROCHETES • These are Gram negative, anaerobic, helical shaped, highly motile microorganisms that are common in many periodontal pockets. • Etiologic agent of acute necrotizing ulcerative gingivitis (Listgarten & Socransky 1964). • At least 15 species of subgingival spirochetes are described. • Pathogen related oral spirochetes” (PROS) were the most frequently detected spirochetes in supra and subgingival plaques of periodontitis patients.(Riviere et al 1991) • Mechanism:o Travel through viscous environment. o Degrade collagen & dentin. o Destroy IgA, IgM, IgG. 76 PREVOTELLA INTERMEDIA Gram negative, short, round-ended anaerobic rod shown to be particularly elevated in acute necrotizing ulcerative gingivitis (Loesche et al 1982), and also in certain forms of periodontitis (Herrera et al 2000). This species appears to have a number of virulence properties exhibited by P. gingivalis and was shown to induce mixed infections.(Hafstrom & Dahlen 1997). It has also been shown to invade oral epithelial cells in vitro (Dorn et al 1998). Strains of P. intermedia that show identical phenotypic traits have been separated into two species, P. intermedia and P. nigrescens (Shah & Gharbia 1992). 77 FUSOBACTERIUM NUCLEATUM • Gram negative, anaerobic, spindle shaped (cigar shaped) rod that has been recognized as part of the subgingival microbiota for over 100 years. (Plaut 1894, Vincent 1899). • Most common isolate found in cultural studies of subgingival plaque samples comprising app. 7-10% of total isolates. (Moore et al 1985). • Mechanism:o Increased secretion of IL-8 (Han et al 2000). o Induce apoptotic cell death in mononuclear and polymorphonuclear cells (Jewett et al 2001). o Cytokine, elastase and oxygen radical release from leukocytes (Sheikhi et al 2000). o Bridging organism. 78 CAMPYLOBACTER RECTUS • Gram negative, anaerobic, short, motile vibrio which utilizes H2 or formate as its energy source. • First described as a member of the “vibrio corroders”, a group of short nondescript rods that formed small convex, “dry spreading” or “corroding” colonies on blood agar plates. • Higher numbers in disease sites as compared with healthy sites and more in sites exhibiting active periodontal destruction.(Rams et al 1993). • Mechanism:o Produce leukotoxin (Gillespie et al 1992). o Stimulate human gingival fibroblasts to produce IL-6 and IL-8 (Ebersole 1996). 79 EIKENELLA CORRODENS • Gram negative, capnophilic, asacharolytic, regular, small rod with blunt ends. • Found more frequently in sites of periodontal destruction as compared with healthy sites and higher levels in active sites (Tanner et al 1987). • Mechanism:- stimulate production of o Matrix metalloproteinase (Dahan et al 2001) o IL-6 and IL-8 (Yumoto et al 1999). 80 PEPTOSTREPTOCOCCUS MICROS P. micros is a Gram positive, anaerobic, small, asaccharolytic coccus. Two genotypes can be distinguished with the smooth genotype being more frequently associated with periodontitis lesions than the rough genotype (Kremer et al. 2000). P. micros was found to be in higher numbers at sites of periodontal destruction as compared with healthy sites (Papapanou et al 2000, Riggio et al 2001). It was shown that P. micros in combination with either P. intermedia or P. nigrescens could produce transmissible abscesses (Van Dalen et al 1998). Produce protease(Grenier 2006) 81 SELENOMONAS SPECIES The selenomonas spp. are Gram negative, curved, saccharolytic rods and may be recognized by their curved shape, tumbling motility and, in good preparations, by the presence of a tuft of flagella inserted in the concave side. Moore et al (1987) described six genetically and phenotypically distinct groups isolated from oral cavity and found S. noxia at a higher proportion of shallow sites (PD>4mm) in chronic periodontitis. 82 EUBACTERIUM SPECIES Suggested as possible periodontal pathogens due to their increased levels in disease sites. (Moore et al 1985). E. nodatum, Eubacterium brachy and Eubacterium timidum are Gram positive, strictly anaerobic, small somewhat pleomorphic rods. Some of these species elicited elevated antibody responses in subjects with destructive periodontitis. (Martin et al 1988) 83 MILLERI STREPTOCOCCI Some of the streptococcal species are associated with and may contribute to disease progression. Milleri streptococci, Streptococcus anginosus, S. constellatus and S. intermidius might contribute to disease progression in subsets of periodontal patients. These species was found to be elevated at sites which demonstrated recent disease progression (Dzink et al 1988). 84 OTHER SPECIES Interest has grown in groups of species not commonly found in the subgingival plaque as initiators or possibly contributors to the pathogenesis of periodontal disease, particularly in individuals who have responded poorly to periodontal therapy. Emphasis have been placed on enteric organisms, staphylococcal species as well as other unusual mouth inhabitants. (Slots et al 1990) 85 VIRUSES Contreras & Slots 2000,Kamma et al 2001 86 Herpesviruses are capable of infecting various types of cells, including polymorphonuclear leukocytes, macrophages, and lymphocytes. The diffuse invasion of Candida fungi and other opportunistic organisms into the gingival tissue of AIDS patients has been demonstrated to be a typical virus-mediated alteration of host defense mechanisms. 87 88 FUNGI Hannula J, Dogan B, Slots (2001) showed geographical differences in the subgingival distribution of C. albicans serotypes and genotypes and suggested geographic clustering of C. albicans clones in Subgingival samples of Chronic Periodontitis patients. Reynaud AH (2001) found a weak correlation between yeasts in periodontal pockets. 89 MIXED INFECTIONS • At the pathogenic end of the spectrum, it is conceivable that different relationships exist between pathogens. • The presence of two pathogens at a site could have no effect or diminish the potential pathogenicity of one or other of the species. • Alternatively, pathogenicity could be enhanced either in an additive or synergistic fashion. • It is not clear whether the combinations suggested in the experimental abscess studies are pertinent to human periodontal diseases 90 PERIIMPLANTITIS High proportion of anaerobic gram negative rods, motile organisms, and spirochetes). Species such as Aa, Pg, Tf, P. micros, C. rectus, Fusobacterium, and Capnocytophaga are often isolated from failing sites. Other species such as Pseudomonas aeruginosa, enterobacteriaceae, Candida albicans and staphylococci, are also frequently detected around implants. 91 Microbial Diagnostic testing 92 MICROBIAL CULTURING • • • • • Gold standard. Positive identification of periodontopathogens. Relative & absolute count. Permits assessment of antibiotic sensitivity. Inability to detect low level of microorganisms, high cost, labour intensiveness, prolonged time, inability to detect certain species. 93 ENZYMATIC ASSAYS • To detect bacteria that produce trypsin like enzyme (BANA) like T. forsythus, T. denticola and P. gingivalis. • Unable to detect the proportion of three bacteria. • Cannot detect presence of other organisms. 94 IMMUNOASSAYS Like Immunofluorescence microscopy, ELISA, Membrane assays, Latex agglutination assays. Higher sensitivity & specificity than culturing. Has low detection thresholds, low cost, rapid, somewhat quantitative. Cannot find antibiotic sensitivity. 95 NUCLEIC ACID PROBES Like Oligonucleotide probe, Whole genomic probe, random cloned probes. Has greater sensitivity than culture methods. Viability of organisms is not required. 96 POLYMERASE CHAIN ASSAYS Like Real time PCR, Multiplex PCR, Hot Start PCR. Most sensitive of any of the above methods. Can also find candidal and enteric microorganisms. 97 FUTURE ADVANCES IN PERIODONTAL MICROBIOLOGY 98 • Specific progress in the field of molecular biology, has led to advances in periodontal microbiology. • Perhaps even more relevant is the present ability to detect microorganisms that cannot be cultivated thus far, which has underscored the limitations of our knowledge of this complex ecologic niche. • Becoming aware that the host response is also of major significance will further improve our understanding of the severity and therapy of periodontal infections. • Finally the recognition of the beneficial activity of several groups of commensal species, such as probiotics, might open new strategies for periodontal therapy. 99 Conclusion • The results of current genome study projects of several periodontopathogens will provide detailed information about the etiology of periodontal diseases, and will likely show new possibilities for the treatment and prevention of periodontal diseases. • In the near future, it is expected that the correlation between biofilm maturation and activation of specific genes of the inner microorganisms will be clarified at the molecular level. 100 References 1. 2. 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