MicroBioLogy of Periodontal Diseases

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Contents •

Introduction

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History

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Diversity of intraoral surfaces for bacterial adhesion

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Dental plaque

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Changing views of plaque

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Plaque formation at the ultrastructural level

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Growth dynamics of dental plaque

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Individual variables influencing plaque formation

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Physiologic properties of dental plaque

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Microbial complexes 3

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Factors that affect the composition of subgingival biofilms

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Intraoral equilibrium between cariogenic species and Periodontopathogens

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Microbial specificity of periodontal diseases

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Association of plaque microorganism with periodontal diseases

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Microbes associated with specific diseases

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Periodontal Pathogens

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Microbial diagnostic testing

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Future advances in periodontal microbiology

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Conclusion

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 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)

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Robert koch (1843 – 1910)

Adolph Witzel(1847 – 1906) W D Miller (1853 – 1907)

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G V BLACK (1836 – 1915)

J L WILIIAMS (1852 – 1932)

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     

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.

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• 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.

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• 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.

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Characteristic Supragingival

Subgingival

Gram reaction

+/-

Dominated by -

Morphotypes

Cocci, branching rods, filaments, spirochetes

Dominated by rods and spirochetes

Energy metabolism Facultative with some anaerobes

Dominated by anaerobes

Energy sources

Generally ferment carbohydrates

Many proteolytic forms

Motility

Firmly adherent to plaque Adherence less pronounced with matrix many motile forms

Response by host

Can cause caries and gingivitis

Can cause gingivitis and periodontitis 13

 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:

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INTERCELLULAR MATRIX

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  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.

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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.

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• 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).

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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.

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An increased metabolic diversity and efficiency.

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An enhanced resistance to environmental stress, antimicrobial agents and the host defenses.

Shapiro (1998), Marsh & Bowden (2000).

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• The formation of the pellicle on the tooth surface. • Initial adhesion and attachment of bacteria. • Colonization and plaque maturation.

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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.

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Mechanism involved in pellicle formation include: Electrostatic forces  Van der Walls forces  Hydrophobic forces

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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”

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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.

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• Clinically it follows an exponential growth curve. • First 24 hours – negligible plaque, covering 4mm) in chronic periodontitis.

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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)

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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).

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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)

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VIRUSES  Contreras & Slots 2000,Kamma et al 2001

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 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.

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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.

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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

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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.

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Microbial Diagnostic testing

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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.

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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.

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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.

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NUCLEIC ACID PROBES  Like Oligonucleotide probe, Whole genomic probe, random cloned probes.  Has greater sensitivity than culture methods.  Viability of organisms is not required.

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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.

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FUTURE ADVANCES IN PERIODONTAL MICROBIOLOGY

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• 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.

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References 1.

Carranza’s Clinical Periodontology; 10th edition. Elsevier publication.

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Textbook of Clinical Periodontology, Glickman; 6th edition. Periodontal therapy; Henry M. Goldman, Cohen; C. V. Mosby company.

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Clinical Periodontology and Implant dentistry, Jan Lindhe, 4th edition. Blackwell munksgaard.

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Periodontal therapy; Henry M. Goldman, Cohen; C. V. Mosby company.

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Periodontics. Medicine, Surgery, and Implants. Rose, Mealey, Genco, Elsevier publication.

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PD Marsh: Plaque as a biofilm: pharmacological principles of drug delivery and action in the sub- and supragingival environment.101 Oral

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Pamela R. Overman . Biofilm: A New View of Plaque. JCDP 2000;1, 3;1-8.

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Marsh PD : Dental plaque: biological significance of a biofilm and community life-style. J Clin Periodontol 2005; 32 (Suppl. 6): 7–15.

10. Casey Hein. Perio Pathways Etiology Fast-forwarded: The Hostbacterial Interaction; Theory and the Risk Continuum. Contemporary Oral Hygiene; 2004. Dec;16-20. 11. Peter M. Loomer; Microbiological diagnostic testing in the treatment of periodontal diseases. Periodontology 2000;2004,34;4956. 102

12. Anne. D. Haffajee & sigmund.S. Socransky. Microbial etiological agents of Destructive periodontal diseases. Periodontology 2000, Vol. 5, 1994, 78-11. 13. Tatsuj Nishihara & Takeyoshi koseki. Microbial etiology of Periodontitis. Periodontology 2000, Vol. 36, 2004, 14–26. 14. Google images

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