R eview Article

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DENTAL PLAQUE..........UNVEILING THE BIOFILM INSIDE

Shantipriya Reddy Professor and Head, Sanjay Kaul Professor, Prasad MGS Reader, Hrishikesh Asutkar Postgraduate student, Nirjhar Bhowmik Postgraduate student, Jaya Senior Lecturer, Department of Periodontics,
Dr. Syamala Reddy Dental College, Hospital and Research Center, Bengaluru, Karnataka, India. Correspondence: Nirjhar Bhowmik, Postgraduate student, Department of Periodontics, Dr. Syamala Reddy Dental College, Hospital
and Research Center, Bengaluru, Karnataka, India. E-mail: nirbhowmik@yahoo.co.in Received Feb 08, 2012; Revised Mar 13, 2012; Accepted Mar 24, 2012

ABSTRACT Dental plaque is the diverse microbial community, embedded in a matrix of host and bacterial polymers, growing on teeth as a biofilm. Dental plaque develops naturally, and contributes to the host defences by preventing colonization by exogenous species. The composition of dental plaque varies at distinct surfaces as a result of the inherent biological and physical properties at these sites; the balance of the predominant bacterial populations shifts in disease. Bacteria growing on a surface display a novel phenotype; one consequence of which is an increased resistance to antimicrobial agents. Such biofilm-associated traits, affect the mode of action and efficacy of antimicrobials. Agents with a broad spectrum of activity in laboratory studies may display a far narrower inhibitory profile in the mouth. This may result in a selective inhibition of species implicated in disease, or reduced production of virulence factors, while preserving the benefits associated with a resident oral microflora. Key Words: Dental plaque, open architecture, diffusion reaction theory, selective inhibition.

Changing Views of Dental Plaque Over the past 50 years, the understanding and characterization of dental plaque have undergone significant evolution. Loesche 6 proposed both a nonspecific and a specific plaque hypothesis for periodontal disease initiation and progression. The nonspecific plaque hypothesis proposed that the entire microbial community of plaque that accumulated on tooth surfaces and in the gingival crevice contributed to the development of periodontal disease. Plaque bacteria produced virulence factors and noxious products that initiated inflammation, challenged the host defence system, and resulted in the destruction of periodontal tissues. Under this hypothesis, the quantity of plaque was considered to be the critical factor in the development of periodontal disease. Thus, increases in the amount of plaque (quantity), as opposed to specific pathogenic microorganisms (quality) found in the plaque, were viewed as being primarily responsible for inducing disease and disease progression 7,8. Studies on the microbial aetiology of various forms of periodontitis support the specific plaque hypothesis, which proposes that only certain microorganisms within the plaque complex are pathogenic. Despite the presence of hundreds of species of microorganisms in periodontal pockets, fewer than 20 are routinely found in increased proportions at periodontally diseased sites. These specific virulent

bacterial species activate the hosts immune and inflammatory responses that then cause bone and soft tissue destruction 6,8,9. Socransky and colleagues 4,10 recognized that early plaque consists predominantly of gram-positive organisms and that if the plaque is left undisturbed it undergoes a process of maturation resulting in a more complex and predominantly gram-negative flora. These investigators assigned the organisms of the subgingival microbiota into groups, or complexes, based on their association with health and various disease severities4,10. Colour designations were used to denote the association of particular bacterial complexes with periodontal infections. The blue, yellow, green, and purple complexes designate early colonizers of the subgingival flora. Orange and red complexes reflect late colonizers associated with mature subgingival plaque. Certain bacterial complexes are associated with health or disease10,11. For example, the bacteria in the red complex are more likely to be associated with clinical indicators of periodontal disease such as periodontal pocketing and clinical attachment loss. Plaque Recognized as a Biofilm (Table: 1) Research over the past decade has led to the recognition of dental plaque as a biofilm - a highly organized accumulation of microbial communities attached to an environmental surface. Biofilms are organized to maximize

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energy, spatial arrangements, communication, and continuity of the community of microorganisms. Biofilms protect bacteria living within their structures and thereby provide an advantage over free-floating (planktonic) bacteria. The slimy extracellular matrix produced by biofilm bacteria encloses the microbial community and protects it from the surrounding environment, including attacks from chemotherapeutic agents. Chemotherapeutic agents have difficulty penetrating the polysaccharide matrix to reach and affect the microorganisms1, 11-13. Thus, the matrix helps to protect bacteria deep within the biofilm from antibiotics and antiseptics, increasing the likelihood of the colonies survival. Furthermore, the extracellular matrix keeps the bacteria banded together, so they are not flushed away by the action of saliva and gingival crevicular fluid. Mechanical methods, including toothbrushing, interdental cleaning, and professional scaling procedures, are required to regularly and effectively disrupt and remove the plaque biofilm. Antiseptics, such as mouthrinses, can help to control the biofilm but must be formulated so as to be able to penetrate the plaque matrix and gain access to the pathogenic bacteria. Biofilms have a definite architectural structure. The bacteria are not uniformly distributed throughout the biofilm; rather, there are aggregates of microcolonies that vary in shape and size. Channels between the colonies allow for circulation of nutrients and by-products and provide a system to eliminate wastes14, 15. Microorganisms on the outer surface of biofilms are not as strongly attached within the matrix and tend to grow faster than those bacteria deeper within the biofilm. Surface microorganisms are more susceptible to detachment, a characteristic that facilitates travel to form new biofilm colonies on nearby oral structures and tissues. Bacteria in biofilm communicate with each other by a process called quorum sensing. This dynamic, sophisticated communication system enables bacteria to monitor each others presence and to modulate their gene expression in response to the number of bacteria in a given area of the biofilm8. In addition, as a result of quorum sensing, portions of the biofilm can become detached in order to maintain a cell density compatible with continued survival. Stages of Biofilm Formation The growth and development of biofilm are characterized by 4 stages: initial adherence, lag phase, rapid growth, and steady state. Biofilm formation begins with the adherence of bacteria to a tooth surface, followed by a lag phase in which changes in genetic expression (phenotypic shifts) occur. A period of rapid growth then occurs, and an exopolysaccharide matrix is produced. During the steady state, the biofilm reaches growth equilibrium. Surface detachment and sloughing occur, and new bacteria are acquired.

Initial Adherence and Lag Phase The first phase of supragingival biofilm formation is the deposition of salivary components, known as acquired pellicle, on tooth surfaces. This pellicle makes the surface receptive to colonization by specific bacteria. Salivary glands produce a variety of proteins and peptides that further contribute to biofilm formation. For example, salivary mucins, such as MUC5B and MUC7, contribute to the formation of acquired pellicle16, 17, and statherin, a salivary acidic phosphoprotein, and proline-rich proteins promote bacterial adhesion to tooth surfaces18. Acquired pellicle formation begins within minutes of a professional prophylaxis; within 1 hour, microorganisms attach to the pellicle. Usually, gram-positive cocci are the first microorganisms to colonize the teeth. As bacteria shift from planktonic to sessile life, a phenotypic change in the bacteria occurs requiring significant genetic up-regulation (gene signaling that promotes this shift). As genetic expression shifts, there is a lag in bacterial growth. Rapid Growth During the rapid growth stage, adherent bacteria secrete large amounts of water-insoluble extracellular polysaccharides to form the biofilm matrix. The growth of microcolonies within the matrix occurs. With time, additional varieties of bacteria adhere to the early colonizers - a process known as coaggregation and the bacterial complexity of the biofilm increases. These processes involve unique, selective molecular interactions leading to structural stratification within the biofilm. Coaggregation and subsequent cell division also increase the thickness of biofilm19-21. Steady State/Detachment During the steady state phase, bacteria in the interior of biofilms slow their growth or become static. Bacteria deep within the biofilm show signs of death with disrupted bacterial cells and other cells devoid of cytoplasm; bacteria near the surface remain intact. During this phase, crystals can be observed in the interbacterial matrix that may represent initial calculus mineralization 22. As noted above, during the steady state stage, surface detachment and sloughing also occur, with some bacteria travelling to form new biofilm colonies. Biofilm Structure Extracellular Polymeric Substances Biofilms are composed primarily of microbial cells and EPS. EPS may account for 50% to 90% of the total organic carbon

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of biofilms23 and can be considered the primary matrix material of the biofilm. EPS may vary in chemical and physical properties, but it is primarily composed of polysaccharides. Some of these polysaccharides are neutral or polyanionic, as is the case for the EPS of gram-negative bacteria. The presence of uronic acids (such as Dglucuronic, D-galacturonic, and mannuronic acids) or ketallinked pryruvates confers the anionic property24. This property is important because it allows association of divalent cations such as calcium and magnesium, which have been shown to cross-link with the polymer strands and provide greater binding force in a developed biofilm23. In the case of some gram-positive bacteria, such as the staphylococci, the chemical composition of EPS may be quite different and may be primarily cationic. Hussain et al25, found that the slime of coagulase-negative bacteria consists of a teichoic acid mixed with small quantities of proteins. EPS is also highly hydrated because it can incorporate large amounts of water into its structure by hydrogen bonding. EPS may be hydrophobic, although most types of EPS are both hydrophilic and hydrophobic24. EPS may also vary in its solubility. Sutherland24 noted two important properties of EPS that may have a marked effect on the biofilm. First, the composition and structure of the polysaccharides determine their primary conformation. For example, many bacterial EPS possess backbone structures that contain 1,3- or 1,4--linked hexose residues and tend to be more rigid, less deformable, and in certain cases poorly soluble or insoluble. Other EPS molecules may be readily soluble in water. Second, the EPS of biofilms is not generally uniform but may vary spatially and temporally. Leriche26 et al. used the binding specificity of lectins to simple sugars to evaluate bacterial biofilm development by different organisms. These researchers results showed that different organisms produce differing amounts of EPS and that the amount of EPS increases with age of the biofilm.EPS may associate with metal ions, divalent cations, other macromolecules (such as proteins, DNA, lipids, and even humic substances) 23. EPS production is known to be affected by nutrient status of the growth medium; excess available carbon and limitation of nitrogen, potassium, or phosphate promote EPS synthesis24. Slow bacterial growth will also enhance EPS production24 because EPS is highly hydrated, it prevents desiccation in some natural biofilms. EPS may also contribute to the antimicrobial resistance properties of biofilms by impeding the mass transport of antibiotics through the biofilm, probably by binding directly to these agents27. Biofilm Architecture Tolker-Nielsen and Molin noted that every microbial biofilm community is unique28 although some structural attributes

can generally be considered universal. The term biofilm is in some ways a misnomer, since biofilms are not a continuous monolayer surface deposit. Rather, biofilms are very heterogeneous, containing microcolonies of bacterial cells encased in an EPS matrix and separated from other microcolonies by interstitial voids (water channels). Liquid flow occurs in these water channels, allowing diffusion of nutrients, oxygen, and even antimicrobial agents. This concept of heterogeneity is descriptive not only for mixed culture biofilms (such as might be found in environmental biofilms) but also for pure culture biofilms common on medical devices and those associated with infectious diseases. Stoodleyet al. defined certain criteria or characteristics that could be considered descriptive of biofilms in general, including a thin base film, ranging from a patchy monolayer of cells to a film several layers thick containing water channels29. The organisms composing the biofilm may also have a marked effect on the biofilm structure. For example, James et al showed that biofilm thickness could be affected by the number of component organisms. Pure cultures of either K. pneumoniae or P. aeruginosa biofilms in a laboratory reactor were thinner (15 and 30 respectively), whereas a biofilm containing both species was thicker (40 ). Jones et al. noted that this could be because one species enhanced the stability of the other. Biofilm architecture is heterogeneous both in space and time, constantly changing because of external and internal processes. Tolker-Nielsen et al.32 investigated the role of cell motility in biofilm architecture in flow cells by examining the interactions of P. aeruginosa and P. putida by confocal laser scanning microscopy. When these two organisms were added to the flow cell system, each organism initially formed small microcolonies. With time, the colonies intermixed, showing the migration of cells from one microcolony to the other. The microcolony structure changed from a compact structure to a looser structure over time, and when this occurred the cells inside the microcolonies were observed to be motile. Motile cells ultimately dispersed from the biofilm, resulting in dissolution of the microcolony. Biofilm and Oral Disease Biofilms can cover surfaces throughout the oral cavity. Microcolonies exist on oral mucosa, the tongue, biomaterials used for restorations and dental appliances, and tooth surfaces above and below the gingival margin. It is important for oral health professionals to communicate to their patients that both dental caries and periodontal disease are infectious diseases resulting from dental plaque biofilm accumulation. Each of these diseases requires specific strategies for prevention and treatment. With respect to periodontal disease, dental plaque biofilm demonstrates a succession of microbial colonization with

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changes in bacterial flora observed from health to disease. Bacterial species contained in the yellow, green, and purple complexes appear to colonize the subgingival sulcus first and predominate in gingival health. In contrast, orange complex bacteria are associated with gingivitis and gingival bleeding. Interestingly, bacteria of the orange complex may also be associated with red complex microorganisms including Porphyromonas gingivalis, Tannerella forsythensis, and Treponema denticola, organisms found in greater numbers in diseased sites and in more advanced periodontal disease. As the biofilm matures and proliferates, soluble compounds produced by pathogenic bacteria penetrate the sulcular epithelium. These compounds stimulate host cells to produce chemical mediators associated with the inflammatory process; Interleukin-1 beta (IL-1 ), prostaglandins, tumor necrosis factor alpha (TNF), and matrix metalloproteinases are mediators that recruit neutrophils to the area via chemotaxis and cause increased permeability of gingival blood vessels, permitting plasma proteins to migrate from within the blood vessels into the tissue. As the gingival inflammatory process continues, additional mediators are produced, and more inflammatory cell types such as neutrophils, T cells, and monocytes are recruited to the area. Proinflammatory cytokines are produced in the tissues as a response to the chronic inflammatory process, and these proteins may further escalate the local inflammatory response and affect the initiation and progression of systemic inflammation and disease. The result of this chronic inflammation is a breakdown of gingival collagen and accumulation of an inflammatory infiltrate, leading to the clinical signs of gingivitis. In some individuals, the inflammatory process will also lead to the breakdown of collagen in the periodontal ligament and resorption of the supporting alveolar bone. It is at this point that the lesion progresses from gingivitis to periodontitis, continuing the same challenge from proinflammatory mediators as with chronic gingivitis. Thus, controlling dental plaque biofilm is essential to preventing and reversing gingivitis as well as preventing and managing periodontitis. The Established Community: Biofilm Ecology The basic structural unit of the biofilm is the microcolony. Proximity of cells within the microcolony (or between microcolonies) provides an ideal environment for creation of nutrient gradients, exchange of genes, and quorum sensing. Since microcolonies may be composed of multiple species, the cycling of various nutrients (e.g., nitrogen, sulfur, and carbon) through redox reactions can readily occur in aquatic and soil biofilms. The primary colonizers which are gram positive organisms are facultative anaerobes in nature and they make the environment

conducive for the late colonizers. It has been proposed that as a microbial biofilm develops the community will ultimately form a more stable climax community. As the community is able to adapt appropriately to outside perturbations the term microbial homeostasis has been suggested to reflect stability within a climax community. Gene Transfer Biofilms also provide an ideal niche for the exchange of extra-chromosomal DNA (plasmids). Conjugation (the mechanism of plasmid transfer) occurs at a greater rate between cells in biofilms than between planktonic cells3537 . Ghigo38 has suggested that medically relevant strains of bacteria that contain conjugative plasmids more readily develop biofilms. He showed that the F conjugative pilus (encoded by the tra operon of the F plasmid) acts as an adhesion factor for both cell-surface and cell-cell interactions, resulting in a three dimensional biofilm of Escherichia coli. Plasmid-carrying strains have also been shown to transfer plasmids to recipient organisms, resulting in biofilm formation; without plasmids these same organisms produce only microcolonies without any further development. The probable reason for enhanced conjugation is that the biofilm environment provides minimal shear and closer cell-to-cell contact. Since plasmids may encode for resistance to multiple antimicrobial agents, biofilm association also provides a mechanism for selecting for, and promoting the spread of, bacterial resistance to antimicrobial agents. Quorum Sensing Cell-to-cell signalling has recently been demonstrated to play a role in cell attachment and detachment from biofilms. Xie et al.39showed that certain dental plaque bacteria can modulate expression of the genes encoding fimbrial expression (fimA) in Porphyromonas gingivalis. P. gingivalis would not attach to Streptococcus cristatis biofilms grown on glass slides. P. gingivalis, on the other hand, readily attached to S. gordonii. S. cristatus cell-free extract substantially affected expression of fimA in P. gingivalis, as determined by using a reporter system. S. cristatus is able to modulate P. Gingivalis fimA expression and prevent its attachment to the biofilm. Davies et al.40 showed that two different cell-to-cell signalling systems in P. aeruginosa, lasR-lasI and rhlR-rhlI, were involved in biofilm formation. At sufficient population densities, these signals reach concentrations required for activation of genes involved in biofilm differentiation. Mutants unable to produce both signals (double mutant) were able to produce a biofilm, but unlike the wild type, their biofilms were much thinner, cells were more densely packed, and the typical biofilm

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architecture was lacking. In addition, these mutant biofilms were much more easily removed from surfaces by a surfactant treatment. Addition of homoserine lactone to the medium containing the mutant biofilms resulted in biofilms similar to the wild type with respect to structure and thickness. Stickler et al.41 also detected acylated homoserine lactone signals homoserine lactone signals in biofilms of gramnegative bacteria on urethral catheters. Yung-Hua et al.42 showed that induction of genetic competence (enabling the uptake and incorporation of exogenous DNA by transformation) is also mediated by quorum sensing in S. mutans. Transformational frequencies were 10600 times higher in biofilms than planktonic cells. Predation and Competition Bacteria within biofilms may be subject to predation by free-living protozoa, Bdellovibrio spp., bacteriophage, and polymorphonuclear leukocytes (PMNs) as a result of localized cell concentration. Murga et al.43 demonstrated the colonization and subsequent predation of heterotrophic biofilms by Hartmannella vermiformis, a free-living protozoon. Predation has also been demonstrated with Acanthamoeba spp. in contact lens storage case biofilms44. James et al.31 noted that competition also occurs within biofilms and demonstrated that invasion of a Hyphomicrobium sp. biofilm by P. putida resulted in dominance by the P. putida, even though the biofilmassociated Hyphomicrobium numbers remained relatively constant. Stewart et al.45 investigated biofilms containing K. pneumoniae and P. aeruginosa and found that both species are able to coexist in a stable community even though P. aeruginosa growth rates are much slower in the mixed culture biofilm than when grown as a pure culture biofilm. P. aeruginosa grow primarily as a base biofilm, whereas K. pneumoniae form localized microcolonies (covering only about 10% of the area) that may have greater access to nutrients and oxygen. Apparently P. aeruginosa can compete because it colonizes the surface rapidly and establishes a long-term competitive advantage. K. pneumoniae apparently survives because of its ability to attach to the P. Aeruginosa biofilm, grow more rapidly, and out-compete the P. Aeruginosa in the surface layers of the biofilm. Dispersal Biofilm cells may be dispersed either by shedding of daughter cells from actively growing cells, detachment as a result of nutrient levels or quorum sensing, or shearing of biofilm aggregates (continuous removal of small portions

of the biofilm) because of flow effects. The mechanisms underlying the process of shedding by actively growing cells in a biofilm are not well understood. Gilbert et al.53 showed that surface hydrophobicity characteristics of newly divided daughter cells spontaneously dispersed from either E. coli or P. aeruginosa biofilms differ substantially from those of either chemostat-intact biofilms or resuspended biofilm cells. These researchers suggested that these differences might explain newly divided daughter cells detachment. Hydrophobicity was lowest for the newly dispersed cells and steadily increases upon continued incubation and growth. Alginate is the major component of the EPS of P. aeruginosa. Inducing alginate lyase expression substantially decreased the amount of alginate produced, which corresponded with a significant increase in the number of detached cells. The authors54 suggested that the role of algL (the gene cassette for alginate lyase production) in wild type P. aeruginosa may be to cause a release of cells from solid surfaces or biofilms, aiding in the dispersal of these organisms. Polysaccharidase enzymes specific for the EPS of different organisms may possibly be produced during different phases of biofilm growth of these organisms. Detachment caused by physical forces has been studied in greater detail. Brading et al.55 have emphasized the importance of physical forces in detachment, stating that the three main processes for detachment are erosion or shearing (continuous removal of small portions of the biofilm), sloughing (rapid and massive removal), and abrasion (detachment due to collision of particles from the bulk fluid with the biofilm). Characklis 56 noted that the rate of erosion from the biofilm increases with increase in biofilm thickness and fluid shear at the biofilm-bulk liquid interface. Sloughing is more random than erosion and is thought to result from nutrient or oxygen depletion within the biofilm structure 55. Sloughing is more commonly observed with thicker biofilms that have developed in nutrient-rich environments 56. Possible Strategies to Control Oral Biofilm. Disruption of biofilm matrix The mechanical disruption of the biofilm matrix has been one of the oldest and most successful means to control oral biofilms, the main advantage of the technique is it is simple to perform, gives consistent results, helps to reduce the bacterial load, no development of resistance so may be performed regularly as home care procedure or as professional oral prophylaxis. Control of nutrients Addition of base-generating nutrients (arginine) Reduction of GCF flow through anti-inflammatory agents Inhibition of key microbial enzymes

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Control of biofilm pH Sugar substitutes Antimicrobial agents Fluoride Stimulate base production Control of redox potential Redox agents Oxygenating agents Conclusion The key to a more complete understanding of the role of microorganisms in dental diseases such as periodontal diseases and caries may depend on a paradigm shift away from concepts that have evolved from studies of classical medical infections with a simple and specific (e.g. single species) aetiology to an appreciation of ecological principles. The development of plaque-mediated disease at a site may be viewed as a breakdown of the homeostatic mechanisms that normally maintain a beneficial relationship between the resident oral microflora and the host. When assessing treatment options, an appreciation of the ecology of the oral cavity will enable the enlightened clinician to take a more holistic approach and consider the nutrition, physiology, host defences, and general well-being of the patient, as these will affect the balance and activity of the resident oral microflora. Future episodes of disease will occur unless the cause of any breakdown in homeostasis is recognized and remedied. Table 1: Basic Biofilm Properties. 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 biofilm are resistant to antibiotics, antimicrobials, and host response.
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