Periodontitis

Periodontitis as a Biofilm Infection

Casey Chen, DDS, PhD

Copyright 2001 Journal of the California Dental Association.

Microbial biofilms are a major concern of infections associated with implantable medical devices as well as with many non-implant related chronic infectious diseases in humans. Dental plaque is also a biofilm. Dental plaque is probably one of the best characterized biofilms occurring on the surface of human tissues. This article will examine the impact of biofilm research on concepts of microbial etiology and the treatment implications for periodontitis.

Biofilm formation has become a focus of intense research in recent years. Biofilms are the predominant mode of existence of most bacteria in nature.¹ A biofilm can be defined as a community of bacteria embedded in exopolysaccharide that adheres onto an inert or living surface. The ubiquitous biofilms are found on the surfaces of rocks, oil and water pipelines, sewage treatment systems, plants, animals and humans. Microbial biofilms are a major concern of infections associated with implantable medical devices as well as with many non-implant related chronic infectious diseases in humans.²

Dental plaque is also a biofilm. Dental plaque is probably one of the best characterized biofilms occurring on the surface of human tissues. From the perspective of conducting scientific research, dental plaque has the advantages of being universally present on tooth surfaces, being easily accessible, and containing sufficient complexity for researchers to examine the basic principle of biofilm formation. Historically, the studies of dental plaque and the studies of biofilms have had different emphases. The time seems ripe for examining the impact of biofilm research on concepts of microbial etiology and the treatment modalities of periodontitis.

Teleology of Biofilms

The terms sessile and planktonic are often used to distinguish the two modes of existence of bacteria. Sessile bacteria refer to the attached bacteria in biofilms. Planktonic bacteria are free-floating single-cell bacteria. Many microbial species exist predominantly as sessile bacteria but release small numbers of planktonic cells for dispersion. The question of why bacteria form biofilms may have many answers (speculations). Several possible reasons are described below.

Homeostasis. Immobilized biofilms offer some semblance of stability for bacteria with regard to nutrient availability, temperature, mineral concentration, oxygen concentration, and pH. In a relatively stable environment, sessile bacteria need not waste energy in reacting to the constantly changing conditions that would be encountered by free-floating planktonic cells.

Growth and metabolic dependence. Synergistic relationships have been observed among different bacterial species forming biofilms.³ A bacterial species may create a favorable environment, e.g., an anoxic condition, to support the growth of other organisms, e.g., obligate anaerobic bacteria. The presence of one bacterial species may provide additional adherence sites for other bacteria through coaggregation.^4,5 Different bacteria in biofilms may be metabolically dependent on each other. For example, vitamin K produced by certain Prevotella species is used by subgingival Porphyromonas gingivalis for growth.⁶

Protection. Biofilms can protect sessile bacteria from harmful substances. Exopolysaccharide appears to be an essential component of biofilms. An important function of exopolysaccharide is the protection of the sessile bacteria from antibiotics and bactericides.^7-9

Diversification as a survival strategy. Bacteria of the same species localized in different parts of the biofilm are in different physiologic states and express different phenotypes. The phenotype diversification may help the survival of the bacteria if the environment changes suddenly. Bacteria may also acquire new genetic traits to increase diversities. Genetic exchange has been shown to occur in biofilms.¹⁰ Close proximity of different bacterial species in biofilms may facilitate genetic exchange and provide bacteria opportunities for acquisition of new genetic traits.

Selected Characteristics of Microbial Biofilms

A thorough discussion of biofilm characteristics can be found in several excellent reviews of this topic.^1-3 The following review of selected features of biofilms is intended to provide a framework for the subsequent discussion of dental plaque as a biofilm.

Microscopic structural characteristics. The study of biofilm structure used to be performed predominantly by electron microscopy. A major drawback of electron microscopy studies is the need to dehydrate samples, which may distort the spatial relationship among bacteria in biofilms. Also, the structure of the loose, highly hydrated exopolysaccharide in biofilms is not usually revealed by electron microscopy. As a consequence, such studies may show biofilms to be a collection of random aggregates of bacteria without distinct features.

With the advent of the digital imaging devices such as confocal scanning laser microscopy and the use of various nontoxic fluorescent probes, fully hydrated bacterial biofilms can be observed in situ.¹¹ Biofilms display distinct architectural structures that consist of a variable distribution of cells and cell aggregates, the associated exopolysaccharide, void spaces, and water channels.^1,3 The existence of void spaces and water channels allows the diffusion of nutrients and waste products. The detailed structures of biofilms vary among different bacterial species and also are dependent on growth conditions.

The microscopic study of biofilms has entered an exciting era in which different technologies converge to give a more realistic picture of biofilms. For example, fluorescent in situ hybridization utilizes specific fluorescence-tagged DNA probes to localize individual bacterial species in biofilms.¹² The combination of fluorescent in situ hybridization and confocal scanning laser microscopy may be used to map the location of target bacterial species within a mixed-species biofilm in reconstructed three-dimensional images.

Cell-to-cell communication. Bacteria can coordinate with each other and act as a group when a critical density of bacterial cells is reached. Research on quorum sensing systems showed that the vast majority of gram-negative bacteria utilize N-acyl homoserine lactones as signaling molecules.^13,14 The signaling molecules are produced at a low level by each bacterium and are freely diffusable through bacterial envelopes. When the N-acyl homoserine lactones are accumulated and reach a critical threshold value (due to higher cell densities), the molecule triggers and activates a transcriptional activator that in turn induces the expression of target genes. Quorum sensing systems have been shown to play a role in biofilm formation.¹⁵ P. aeruginosa has two quorum sensing systems, the lasR-lasI and the RhlR-RhlI systems. Specific inactivation of the lasR-lasI system allowed the mutant to adhere to a glass surface, but rendered the mutant unable to form a mature, structured, thick, and biocide-resistant biofilm.¹⁵ There is evidence that the N-acyl homoserine lactones are produced by P. aeruginosa biofilms in vivo.¹⁶ The involvement of quorum sensing systems in biofilm formation seems logical. Genes involved in biofilm formation, which requires increase of cell density and cell-to-cell contact, may be useless if only few cells in planktonic phase exist.

Resistance to antimicrobial agents. Sessile bacteria exhibit several hundred- to thousand-fold higher resistance to antimicrobial agents in comparison to planktonic cells of the same species.^2,7 Several hypotheses have been proposed to explain the mechanisms of the increased resistance.^2,7-9 Exopolysaccharide may retard the diffusion of certain antibiotics and biocides. Some of the sessile bacteria are slow-growing or in a dormant state due to nutrient limitations. The starved cells are metabolically inactive and may be more resistant to antibiotics. It is also speculated that some sessile bacteria display a distinct biofilm phenotype that is inherently resistant to antimicrobial agents. The expression of this resistance phenotype is a result of a programmed response to growth in a biofilm and is not related to the starvation or the metabolic activity of the bacteria. The resistance phenotype persists even after the sessile cells are removed from biofilms and grown in enriched media.

Microbial Etiology of Periodontitis

Periodontitis is a chronic bacterial infection that affects supporting structures of the teeth, including gingivae, periodontal ligament, and alveolar bone. The disease is one of the most common bacterial infections among humans.¹⁷ A tremendous effort has been directed toward identifying the bacterial causative agents of periodontitis and understanding their pathogenic mechanisms.¹⁸ It should be remembered that in scientific research, answers are often influenced by the way in which the questions are framed. In the following discussion, the conventional viewpoint of microbial periodontal etiology is presented.

Conceptual framework of infectious diseases. To understand infectious diseases, it is easier to use as a model an acute infectious disease in which a single pathogen is the sole cause. A good example may be primary syphilis, in which the causative agent, Treponema pallidum, invades the genitalia of the infected individual and causes a localized lesion called chancre. The disease occurs within days (i.e., acute) of infection by T. pallidum. No other bacterial species are involved in the pathogenesis of syphilis. Nor does biofilm formation play a role in the infections. Once the role of T. pallidum in syphilis is defined, subsequent studies may be devoted to examining the detailed pathogenic mechanisms. Attention may focus on identifying the bacterial virulence factors, the host immune response, and how each parameter modulates the disease progression. In general, medical science has a relatively good grasp on the pathogenesis of single-pathogen infectious diseases and is largely successful in treating this type of acute bacterial infection with antimicrobial therapy. However, there are other bacterial infectious diseases operating under different principles.

Consensus list of periodontal pathogens. The causal relationship between oral bacteria and periodontitis is difficult to determine for a number of reasons.¹⁹ Briefly, more than 500 taxa of oral bacteria may be cultivable from gingival crevices.²⁰ There may be several hundred more noncultivable bacteria in gingival crevices as well. These noncultivable bacteria may be detected and characterized by polymerase chain reaction cloning and sequencing of their 16S rRNA genes.^21,22 The sheer numbers of different gingival bacteria needed to be evaluated may overwhelm anyone trying to decide whether a bacterium, or a consortium of bacteria, is the cause of periodontitis. Furthermore, periodontitis is typically, but not always, a slowly progressing disease. There is a time gap between the initial infection by the periodontal pathogens and the clinical manifestation of the disease. The causal relationship between the bacteria and the disease becomes obscure. Also, many oral bacteria are considered commensal organisms due to their low pathogenicity and frequent occurrence in healthy individuals. Yet, the commensal bacteria may cause periodontitis under the "right" condition such as poor oral hygiene, poor host immune response, or deepened periodontal pockets. It is difficult to determine when commensal organisms play the role of nonpathogens and when they play the role of causative agents in the disease. Finally, it would have been relatively easy to substantiate the etiologic role of a putative periodontal pathogen if the bacteria could be selectively eradicated by periodontal therapy. However, periodontal therapy is usually accompanied by a major change in subgingival microbiota. The etiologic role of bacteria cannot be inferred from the clinical outcomes of periodontal treatment. In spite of these difficulties, one of the most remarkable achievements in periodontal research is the delineation of the role of a number of specific bacterial species in periodontitis. This is due in part due to advancements in anaerobic culture techniques, bacterial taxonomy, and molecular identification and characterization of subgingival bacteria. It is worth noting that the following consensus list of important periodontal pathogens was derived using a set of modified Koch’s postulates as proposed by Socransky.²³ A periodontal pathogen should possess the following characteristics:

* Association with disease. The bacterium should be present at high levels in diseased individuals and either absent or present in lower levels in healthy controls.

* Elimination of the organism. Elimination or suppression of the organism should result in the arrest of the disease.

* Host response. Increased or decreased host immune response to a specific bacterium is suggestive of a significant role of the bacterium in disease.

* Animal pathogenicity. The pathogenicity of the bacterium could be inferred from the ability of the bacterium to cause disease in experimental animals.

* Mechanisms of pathogenicity. The bacterium should possess characteristics that could contribute to the pathogenesis of periodontal disease.

The resultant list of periodontal pathogens²⁴ is shown in Table 1. The bacteria are categorized based on the strength of the evidence compiled from numerous clinical and microbiological studies.

Periodontitis as a Biofilm Infection

The modified Koch’s postulates, the resultant list of periodontal pathogens, and the implied concept of microbial etiology of periodontitis are influenced by the conceptual framework of single-pathogen acute infections caused by planktonic bacterial cells. However, periodontitis is more similar to bacterial biofilm infections than to acute infections. A list of selected biofilm infections in humans is listed in Table 2.

Biofilm infections share some common features.² A critical early step of the disease involves the forming of biofilms on inert surfaces or living tissues. Commensal bacteria are frequently involved in biofilm infections. Clinically, biofilm infections are slow to progress and difficult to treat. The bacteria in biofilm infections are frequently resistant to antimicrobial agents that are effective against planktonic bacteria. The bacteria and the infections relapse after the cessation of the drug therapy. The host immune response is ineffective against biofilm infections and may even be harmful to the host. All these characteristics of biofilm infections apply to periodontitis.

The view of periodontitis as a biofilm infection does not change the importance of the consensus periodontal pathogens. It does modify the view of microbial etiology and may have some influence over the treatment modality of periodontitis. Dental plaque may be viewed as being a primitive multicellular organism. Some part (readers: periodontal pathogens) of this multicellular organism causes great damage to periodontal tissue. Other parts of the primitive multicellular organism may be essential for the survival of the entire organism. The goal of periodontal therapy will remain the elimination of periodontal pathogens. Nevertheless, one may choose to target the weaknesses of the primitive multicellular dental plaque (which may not be the pathogens) to achieve the goal.

Dental plaque is a ubiquitous structure formed on the surface of oral tissues by oral bacteria.^25,26 Dental plaque is made up predominantly of bacterial cells but also includes other minor components such as bacterial enzymes, bacterial metabolic products, host salivary proteins, immunoglobulins IgG and IgA, and complement components. This paper accepts a priori that dental plaque is a biofilm. Nevertheless, it will be helpful to review the supporting evidence for dental plaque as a biofilm. The following discussion begins with a description of the orderly process of plaque formation, followed by a brief review of the biofilm features of dental plaque.

Orderly process of plaque formation. More details have been learned about the formation of dental plaque than any other naturally formed biofilm. Plaque formation is an extremely complex process. Several excellent reviews are available for interested readers.^6,26,27 Only a brief description of the process is provided here. Immediately after removal of bacteria on the tooth surface by prophylaxis, a ubiquitous layer of dental pellicles is formed on the tooth surface. The early bacterial colonizers, mostly facultative gram-positive streptococci and Actinomyces species, adhere to the dental pellicles on the tooth surface. Following the adherence of early colonizers, the plaque increases its cell numbers mainly by bacterial growth. The early colonizers provide a variety of niches for the adherence and growth of late bacterial colonizers. The plaque continues to increase in thickness by both adherence and bacterial growth. The microbial composition of plaque gradually becomes more diversified and includes an increasing number of gram-negative bacteria and obligate anaerobic organisms. The inter-species and inter-generic bacterial coaggregations play a significant role in the plaque maturation process.^4,5,28 After several days to two to three weeks, the plaque reaches its full potential and becomes a mature bacterial community.

The orderly process of plaque formation suggests specific interactions. Many bacteria express pili (fimbriae), which are proteinacious hair-like structures projecting from the bacterial surface. Bacteria may also express nonfimbrial adhesins on the cell envelope. Both types of adhesins mediate the attachment of the bacteria to receptors in dental pellicles or on the surface of other bacteria. For example, Actinomyces naeslundii expresses two types of pili, type 1 pili mediate the attachment to dental pellicles and type 2 pili bind streptococci.²⁹ The specific interactions are also found between salivary components of dental pellicles and the early colonizers. α-amylase binds Streptococcus gordonii.²⁶ The salivary protein statherin binds Actinomyces viscosus.²⁶ Proline-rich proteins also mediate the adherence of a number of actinomyces and streptococci.²⁶ It is important to recognize that the specific interactions in the plaque formation may provide some opportunities to disrupt the formation process.

Microscopic structural characteristics of dental plaque. Electron microscopy studies have some drawbacks in identifying the structural characteristic of biofilms. Nevertheless, it is worth revisiting the seminal work of these type of studies of dental plaque by Listgarten.^30,31

With electron microscopy, the mature supragingival plaque appeared as a layer of dense and predominantly filamentous organisms adhering to the enamel surface (Figures 1 and 2). The filamentous organisms were long and oriented with their longitudinal axis perpendicular to the tooth surface. The bacterial cells were held together by extracellular matrix. The surface of the plaque contained distinctive corncob formations indicative of interbacterial species coaggregation (Figure 3). The subgingival plaque is a natural extension of supragingival plaque. There was a gradual change from the dense, thicker, predominantly filamentous supragingival plaque, to the thinner, less densely packed, and less organized subgingival plaque (Figures 4 and 5). The adhering layer of the subgingival plaque contained short filamentous bacteria. The surface of the subgingival adherent layer was covered with distinctive bacteria comprising many flagellated bacteria and spirochetes. The surface of the subgingival adherent layer also contained bristle brush and test-tube brush formations (Figure 6). The presence of exopolysaccharide in subgingival plaque was also evident.

The supra- and subgingival plaque shown in electron micrographs appear to be more compact than the single-species biofilms examined by confocal scanning laser microscopy in vitro. The lack of void spaces and water channels may be an artifact due to the collapse of bacterial aggregate from dehydration during the sample preparation or a result of a higher nutrient contents in gingival crevices supporting the growth of a higher density of bacteria. Also, the presence of numerous different bacterial species, each favoring a different ecological niche, may reduce the voids in the biofilms. Nevertheless, it is clear that dental plaque exhibits an orderly structure. The distribution of different bacterial morphotypes in dental plaque shows discernible patterns.

More recently, confocal scanning laser microscopy has been employed to examine the structure of natural dental plaque. Wood and colleagues³² examined the architecture of 4-day-old dental plaque formed in volunteers wearing a device attached to the buccal surface of molars. The device contained an enamel substrate to allow for plaque formation. The confocal scanning laser microscopy results showed a highly heterogeneous distribution of bacterial mass. There were void spaces throughout the plaque, and some appeared to open to the surface of the plaque, allowing exchange of fluids. Large bacterial masses resembling mushroom structures surrounded by open spaces and channels were also observed.

These microscopic structural studies presented above support the concept that supra- and subgingival plaque are biofilms. The naturally formed dental plaque shows more complexity than in vitro biofilms of single or limited species, and it may have different structural characteristics. The studies also suggest that specific interactions were involved in plaque formation, as would be expected from biofilms.

Cell-to-cell communication in dental plaque. It is reasonable to assume that bacteria in dental plaque may utilize certain cell-to-cell communication systems in order to coordinate their behaviors. Although direct evidence for bacterial communication in plaque is lacking, cell-density-dependent behaviors of oral bacteria have been demonstrated in naturally formed subgingival plaque on the tooth surface. Bloomquist and colleagues³³ examined bacterial growth patterns of the plaque formed from two to 24 hours on enamel pieces placed on the tooth surfaces of healthy volunteers. Following rapid adherence of oral bacteria onto the enamel surface to a density of 2.5 to 6.3x10⁵ cells/mm, there was a period of relatively slow cell growth. A rapid burst of cell growth occurred when the cell density reached the level of 2.5 to 4 x 10⁶/mm, and the growth rate declined at densities beyond 6.3 x 10⁶ cells per mm². It was postulated that the cell-density-dependent burst of cell proliferation was a demonstration of cell-to-cell communication. The mechanism of this postulated cell-to-cell communication was not known. There may be a tremendous difference in behavior between young plaque and mature plaque. Therefore the issue of biofilm behavior of dental plaque is not settled by the previous studies.

Antimicrobial resistance of bacteria in dental plaque. A limited number of in vitro studies showed that oral streptococci in biofilms were more resistant to chlorhexidine in biofilms than planktonic cells.^34,35 The resistance phenotype of sessile subgingival bacteria may also be inferred from clinical studies of adjunct systemic antibiotics therapy for periodontitis.^36-38 Although antibiotic therapy resulted in a proportional increase of subgingival bacteria resistant to the corresponding drugs, the antibiotics did not eliminate all susceptible subgingival bacteria. While there are many plausible explanations, these findings may be explained by the higher resistance of sessile bacteria than planktonic cells to antibiotics.

Treatment Implications

The concept of microbial etiology of periodontitis has undergone a tremendous change from nonspecific plaque hypothesis in which the quantity of the plaque is considered critical, to the specific plaque hypothesis in which a limited number of periodontal bacterial species (i.e., the quality) are recognized as the cause of the disease. Still, the available treatment modalities remain essentially nonspecific. Plaque removal, commonly achieved by scaling and root planing, is an important step in periodontal therapy. Periodontal osseous surgery eliminates deep pockets to allow effective plaque removal regiments (i.e., brushing and flossing). Various chemical agents with broad spectrum of antimicrobial activities are used for oral rinsing and subgingival irrigation. Even systemic or local delivery of antibiotics, which markedly modify the composition of the subgingival microbiota, can be considered a nonspecific therapy. Eradication of selective, specific periodontal pathogens has always been the "unspoken" or "unachievable" goal of periodontal therapy.

Biofilm research is at an infancy stage and has not resulted in a noticeable change in periodontal treatment modalities but will have an impact to the future periodontal treatment. Table 3 provides an outline of clinical implications based on the biological properties of biofilms. It should be noted that the goal of eradicating periodontal pathogens and the importance of microbial diagnosis will remain unchanged. What will likely change is how these periodontal pathogens are controlled.

The first four biofilm properties listed in Table 3 suggest the idea that it may be possible to interfere with the plaque formation process and/or weaken the plaque structure. It could be done by killing key bacterial members of the plaque, by blocking the specific interactions between bacterial and host molecules, by disrupting cell-to-cell communication, and by attacking non-cellular structural components of the biofilms with chemical agents. In particular, the idea that the disruption of bacterial cell-to-cell communications may interfere with biofilm formation is the most promising and exciting area of research. It may one day be possible to use subgingival irrigation solutions containing inhibitors of the bacterial communication pathways to interfere with plaque formation. The fifth biofilm property suggests that antimicrobial agents alone are often ineffective in treating periodontitis without concomitant plaque removal by mechanical means. Biofilm research has further identified additional means to help remove biofilms. For example, electric current applied to biofilm has been shown to reduce the resistance of biofilm bacteria to antimicrobial agents.³⁹ The last biofilm property suggests that vaccination may not be a good strategy of periodontal therapy. It may even suggest that elevated immune responses to bacterial biofilms may be potentially harmful to the host. Modulating host immune response to biofilms may reduce tissue damages in disease.

Conclusion

Biofilms are the preferred mode of growth for many bacteria in nature, including periodontal pathogens. Although early periodontal microbiologists did not use the term "biofilms" to describe dental plaque, they clearly noted the distinct structure of the plaque, the orderly process, and the specific interactions involved in plaque formation. These are now considered salient features of biofilms.

The previous focus of the periodontal microbiology research has been in identifying and classifying subgingival bacteria and delineating the causal relationship of the bacteria and periodontitis. The consensus list of periodontal pathogens represents a remarkable achievement of dental research. The next breakthrough will likely result from the convergence of different disciplines of biofilms research. Ultimately, it may be learned how individual periodontal pathogens cause periodontitis, how the dental plaque behaves as a community, what is the most effective means to disrupt the plaque formation, and what methods could be used to convert pathogenic plaque to one compatible with oral health.

Author

Casey Chen, DDS, PhD, is an associate professor in the Department of Periodontology at the University of the Southern California School of Dentistry.

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To requested a printed copy of this article, please contact/Casey Chen, DDS, PhD, USC School of Dentistry, Periodontology, 925 W. 34th St., Los Angeles, CA 90089.

Table 1. Consensus Periodontal Pathogens²⁴

Table 2. Partial List of Human Infections Involving Biofilms²

Table 3. Clinical Implications of Periodontal Therapy From the Perspective of Periodontitis as a Biofilm Infection

Legends

Figures 1 and 2

Figure 1. Supragingival plaque of a periodontitis patient. The enamel surface (E) is on the right side of the micrograph. A dense layer of bacteria, mostly filamentous, adheres to the enamel. Corncob formations are visible on the surface of the plaque. 850x magnification. From Listgarten,30 used with permission. Figure 2. Magnified view (1,500x) of the adherent bacterial layer in Figure 1. From Listgarten,30 used with permission.

Figures 3 and 4

Figure 3. Magnified view (1,500x) of corncob formations on the surface of the adherent bacterial layer in Figure 1. From Listgarten,30 used with permission. Figure 4. Subgingival plaque of a periodontitis patient. The cementum surface (C) is on the right side of the micrograph. Bacteria adhere to the cementum surface appear to be less dense and less filamentous. The test-tube brush formations are found on the surface of the adherent bacterial layer. 600x magnification. From Listgarten,30 used with permission.

Figures 5 and 6

Figure 5. Magnified view (2,000x) of the surface of the adherent bacterial layer in Figure 4. MC: Mammalian cells, mostly macrophages and neutrophils, adhere to the bacterial layer. From Listgarten,30 used with permission. Figure 6. Magnified view (1,500x) of the test-tube brush formations. From Listgarten,30 used with permission.