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Periodontal Microbiology Susan Kinder Haake, Michael G. Newman, Russell]. Nisengard, and Mariano Sanz CHAPTER OUTLINE DENTAL PLAQUE: A HOST ASSOCIATED BIOFILM Macroscopic Structure and Composition of Dental Plaque Formation of Dental Plaque Microscopic Structure and Physiologic Properties of Dental Plaque Significance of the Biofilm Environment ASSOCIATION OF PLAQUE MICROORGANISMS WITH PERIODONTAL DISEASES Microbial Specificity of Periodontal Diseases Microorganisms Associated with Specific Periodontal Diseases Conclusions from Studies of the Association of Microorganisms with Periodontal Diseases CRITERIA FOR IDENTIFICATION OF PERIODONTAL PATHOGENS FUTURE ADVANCES IN PERIODONTAL MICROBIOLOGY

DENTAL PLAQUE: A HOST_ ASSOCIATED BIOFILM

Macroscopic Structure and Composition of Dental Plaque

Fig. 6-1 A, One-day-old plaque. Microcolonies of plaque bacteria extend perpendicularly away from tooth surfaces. B, Developed supragingival plaque showing overall filamentous nature and microcolonies (arrows) extending perpendicularly away from tooth surface. Saliva-plaque interface shown (S). (A, From Listgarten M: Development of dental plaque on epoxy resin crowns in man. A light and electron microscopic study. J Periodontol 1975; 46:10.B, Courtesy Dr. Max Listgarten, Philadelphia, Penn.)

Fig. 6-2 Histologic section of plaque showing nonbacterial components such as white blood cells (arrow) and epithelial cells (asterisk), interspersed among bacteria (13). (Courtesy Dr. Max Listgarten, Philadelphia, Penn.)

Fig. 6-3 Vertical section through a 4-day human plaque sample Fig. 6-3 Vertical section through a 4-day human plaque sample. An intraoral device designed for in vivo generation of plaque biofilms on enamel was used. Confocal microscopy enabled visualization of the section of plaque without the dehydration steps used in conventional histologic preparations. Notice the channels (white arrows) that traverse from the plaque surface through the bacterial mass (M; grey-white areas) to the enamel surface. An area in which the bacterial mass appears to attach to the enamel surface (A) is indicated. Scale bar = 25 um. (From Wood et al: Architecture of intact natural human plaque biofilms studied by confocal laser scanning microscopy. J Dent Res 2000; 79:21. Courtesy Dr. Simon Wood, Leeds, England.)

Formation of Dental Plaque Dental plaque may be readily visualized on teeth after 1 to 2 days with no oral hygiene measures. Plaque is white, grayish, or yellow and has a globular appearance. Formation of the dental pellicle on the tooth surface is the initial phase of plaque development. All surfaces of the oral cavity, including all tissue surfaces as well as surfaces of teeth and fixed and removable restorations, are coated with a glycoprotein pellicle. This pellicle is derived from components of saliva and crevicular fluid as well as bacterial and host tissue cell products and debris. The mechanisms involved in enamel pellicle formation include electrostatic, van der Waals, and hydrophobic forces. The hydroxyapatite surface has a predominance of negatively charged phosphate groups that interact directly or indirectly with positively charged components of salivary and crevicular fluid macromolecules."'

Initial Colonization of the Tooth Surface. Within a few hours, bacteria are found on the dental pellicle. The initial bacteria colonizing the pellicle-coated tooth surface are predominantly gram-positive facultative microorganisms such as Actinomyces viscosus and Streptococcus sanguis. These initial colonizers adhere to the pellicle 21,24,63 through specific molecules, termed adhesins, on the bacterial surface that interact with receptors in the dental pellicle. For example, cells of A. viscosus possess fibrous protein structures called fimbriae that extend from the bacterial cell surface. In this ecologic succession of the biofilm, there is a transition from the early aerobic environment characterized by gram positive facultative species to a highly oxygen-deprived environment in which gram-negative anaerobic microorganisms predominate.

Secondary Colonization and Plaque Maturation Secondary colonizers are the microorganisms that do not initially colonize clean tooth surfaces, including Prevotella intermedia, Prevotella loescheii, Capnocytophaga spp., Fusobacterium nucleatum, and Porphyromonas gingivalis. Extensive laboratory studies have documented the ability of different species and genera of plaque microorganisms to adhere to one another, a process known as coaggregation. Well-characterized interactions of secondary colonizers with early colonizers include the coaggregation of F. nucleatum with S. sanguis, 3 5 P. loescheii with A.viscosus, 103.104 and Capnocytophaga ochracea with A. viscosus.

Microscopic Structure and Physiologic Properties of Dental Plaque

Fig. 6-7 Left Diagrammatic representation of the histologic structure of subgingival plaque. Right, Histologic section of subgingival plaque. Arrow with box, Sulcular epithelium. White arrow, Predominantly gram-negative unattached zone. Black arrow, Tooth surface. Asterisk, redominantly gram-positive attached zone. (From Listgarten M: Development of dental plaque on epoxy resin crowns in man. A light and electron microscopic study. J Periodontol 1975; 46:10.)

Fig. 6-8 Minute lesion on surface of root (resorption cavity) previously covered by attached plaque. Note microorganisms (single arrows) within resorption cavity. Cemental mounds can easily be identified (double arrows). (Courtesy Dr. J. Sottosanti, La Jolla, Calif.)

Fig. 6-9 Scanning electron photomicrograph of cross section of cementum (C) with attached subgingival plaque (AP). Area shown is within a periodontal pocket. (Courtesy Dr. J. Sottosanti, La Jolla, Calif.)

Fig. 6-10 Scanning electron micrograph of cocci and filaments associated with surface of pocket epithelium in a case of marginal gingivitis. (Magnification x3000.)

Fig. 6-11 Scanning electron micrograph of frontal view of pocket wall showing short rods on epithelial surface. (Magnification x 10,000.)

Fig. 6-12 Schematic illustration of metabolic interactions among different bacterial species found in plaque, as well as between the host and plaque bacteria. These interactions are likely to be important to the survival of bacteria in the periodontal environment.

Significance of the Biofilm Environment

ASSOCIATION OF PLAQUE MICROORGANISMS WITH PERIODONTAL DISEASES Microbial Specificity of Periodontal Diseases Nonspecific Plaque Hypothesis

Specific Plaque Hypothesis Microorganisms Associated with Specific Periodontal Diseases

Fig. 6-15 Cultivable subgingival microbiota associated with periodontal health and disease. A, Distribution of gram-positive and gram-negative rods and cocci. B, Distribution of anaerobic, facultative, gram-positive and gram-negative species. (Adapted from Slots J, Rams TE: Microbiology of periodontal disease. I n: Slots J, Taubman MA (eds): Contemporary Oral Microbiology and Immunology. St Louis, Mosby, 1992.)

Periodontal Health The bacteria associated with periodontal health are primarily grampositive facultative species and members of the genera Streptococcus and ctinomyces (e.g., S. sanguis, S. mitis, A. viscosus, and A. naeslundii). Small proportions of gramnegative species are also found, most frequently P. intermedia, F. nucleatum, and Capnocytophaga, Neisseria, and Veillonella spp. Microscopic studies indicate that a few spirochetes and motile rods also may be found. Certain bacterial species have been proposed to be protective or beneficial to the host, including S. sanguis, Veillonella parvula, and C. ochracea. One example of a mechanism by which this may occur is the production of H202 by S. sanguis; H202 is known to be lethal to cells of A. actinomycetemcomitans.

Gingivitis After 8 hours without oral hygiene, bacteria may be found at oncentrations of 10 3 to 104 per square millimeter of tooth surface and will increase in number by a factor of 100 to 1000 in the next 24-hour period. The initial microbiota of experimental gingivitis consists of gram-positive rods, gram-positive cocci, and gram-negative cocci.

Chronic Periodontitis Studies in which untreated populations were examined over long time intervals indicate disease progression at mean rates ranging from 0.05 to 0.3 mm of attachment loss per year Localized Aggressive Periodontitis. Periodontitis as a Manifestation of Systemic Disease Necrotizing Periodontal Diseases. Abscesses of the Periodontium

Conclusions from Studies of the Association of Microorganisms with Periodontal Diseases

CRITERIA FOR IDENTIFICATION OF PERIODONTAL PATHOGENS In the 1870s, Robert Koch developed the classic criteria by which a microorganism can be judged to be a causative agent in human infections. These criteria, known as Koch's postulates, stipulate that the causative agent must: 1. Be routinely isolated from diseased individuals 2. Be grown in pure culture in the laboratory 3. Produce a similar disease when inoculated into susceptible laboratory animals 4. Be recovered from lesions in a diseased laboratory animal

1. Be associated with disease, as evident by increases in Sigmund Socransky, a researcher at the Forsyth Dental Center in Boston, proposed criteria by which periodontal microorganisms may be judged to be potential pathogens. 94 According to these criteria, a potential pathogen must: 1. Be associated with disease, as evident by increases in the number of organisms at diseased sites 2. Be eliminated or decreased in sites that demonstrate clinical resolution of disease with treatment 3. Demonstrate a host response, in the form of an alteration in the host cellular or humoral immune response 4. Be capable of causing disease in experimental animal models 5. Demonstrate virulence factors responsible for enabling the microorganism to cause destruction of the periodontal tissues

FUTURE ADVANCES IN PERIODONTAL MICROBIOLOGY