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Oral Biofilms: The "Balancing Act"

Margaret I. Scarlett, DMD

President
Scarlett Consulting International
Atlanta, Georgia

Overview of Biofilms
It has been said that the mouth is a petri dish. There is a coating of bacteria, viruses, and fungi covering all of the soft and hard tissues in the mouth. This coating may be thick, as can be seen with dental plaque, or invisible, such as in the normal oral mucosa. Rather than acting like bacteria or other organisms which are planktonic, or free-floating and non-adherent, these organisms clump together to form a very sticky biofilm.

New techniques for characterizing the organisms of the mouth have been used to gain more information about how the bacteria in oral biofilms work for good oral health. These include tests such as in situ fluorescent hybridization, laser technology, digital imaging, scanning electron microscopy, ribonucleic acid and deoxyribonucleic acid microprobe technologies, confocal microscopy of the mouth, and polymerase chain reaction. Alternatively, when the balance of bacteria within a biofilm with the host immune and inflammatory responses is upset, this can change the potential for oral health or disease in the mouth.^1-3

Biofilm may contain more than 700 species of bacteria, many of which have not been cultured outside of the mouth.¹ The composition of bacteria within the biofilm seems to be unique for to each particular individual. Bacterial colonization by different bacteria may even vary by site within the oral cavity.^1,4 The biofilm is formed when organisms attach to either hard or soft surfaces in the mouth. While initially colonized by one species of organism, the biofilm matures into a mixed species community of organisms.

Life Cycle of Biofilms
The cycle of biofilm formation is simple. After each bacterial cell attaches, it creates a new surface on which other bacteria can adhere. A change occurs after more cells are attached to the surface. More cells come to the same area and become part of the biofilm. Not unlike people, bacteria behave differently as a group than they do individually. The biofilms produce waste products in large amounts, which must be processed to maintain a healthy community. The cells in the biofilm communicate efficiently through certain identified signaling mechanisms from specific bacteria.^2,3

The first bacteria to adhere to a surface and colonize the biofilm are unique and may vary from one person to another. After initial colonization, the composition of subsequent organisms in the biofilm will be more diverse, but there will be common organisms, characteristically balanced between aerobes (oxygenated) and anaerobes (non-oxygenated).⁴ As the biofilm becomes thicker, the secretion of chemical agents provides communication by certain bacteria among cells in the biofilm. Scientists have discovered that specific bacteria secrete a type of chemical signal, such as autoinducer-2, to exchange information about the biofilm.

After bacteria have formed a cohesive biofilm, communication among them may continue. These are modulated by other chemical agents, such as adhesins and receptors, which impact the growth of the biofilm and the type of bacteria that grow within a particular biofilm. Scientists have identified chemical communication both among bacteria and between the biofilm and the host.⁵

The biofilm exhibits some resilency to outside threats. When exposed to antimicrobials or antibiotics, biofilm may also provide a substrate for gene transfer among bacteria to confer resistance. Bacteria can transfer the genes for resistance to neighboring susceptible bacteria. In this way, gene transfer converts a previously avirulent organism into a highly virulent pathogen.⁵

The progression of the biofilm becomes more complex over time; the migration of single cells and their attachment creates an early structure for the biofilm, which then forms a mature biofilm. The composition of the cells in the biofilm becomes more heterogeneous, with some variation. In maturity, the biofilm has some detachment or even streamers of clusters of cells. This causes seeding of the cells in other sites within the oral cavity. This creates new biofilms in other areas as the cycle continues.⁶

The maturation of bacteria may be problematic in balancing the biofilm on the healthy side of the equation. Bacteria within the biofilm may express new, more virulent phenotypes as it develops. The new testing techniques identified earlier have detected the presence of new organisms not previously identified. Growth conditions are dependent on the depth of biofilms—where nutrients and oxygen are usually limited—and waste products from neighbors. The lack of oxygen favors the anaerobic bacteria, which are implicated in periodontal disease. Gram-negative anaerobes, such as Porphyromonas gingivalis or Actinomyces Actinomycetemcomitans, in biofilm have been found in high numbers in diseased gingival tissue. As the biofilm becomes thicker, antimicrobials or antibiotics cannot reach the cells, favoring the growth of more anaerobes. Therefore, the bacteria found at the bottom of the biofilm look and act different than the types of organisms located at the surface or when the biofilm first forms.⁶

Significance of Biofilms
The percentage of oral disease conditions attributable to biofilms is not precisely known; however, it is estimated that bacterial biofilms may cause up to 65% of all infections in the body.⁷ There is no reason to believe that the percentage of infections of the oral cavity is any different than the rest of the body.

Bacteria embedded within biofilms are protected from many of the natural host defenses and are notoriously difficult for the host to remove because of the sticky substance that holds the biofilm together as a group. Contact with a surface triggers the release of key bacterial enzymes that catalyze the formation of sticky polysaccharides that promote colonization and protection.⁸ From 50% to 90% of a biofilm is composed of this polysaccharide-rich “glue,” called extracellular polymeric substance (EPS). EPS contains organisms and non-organic material, and with its hydrated form provides more protection from any host immune response. Fibrous strands of these polysaccharides stream out from certain bacteria in the biofilm. These strands are composed largely of exopolysaccharide intercellular adhesions (PIAs).⁹ The strands effectively block host immune response by transfer of the genetic material previously mentioned, as well as phagocytosis and death of a key host defense cell, the polymorphonuclear cell (PMN).

Immunity and Biofilms
Host immune factors provide balance and protective or defensive factors against the biofilm. This occurs in three ways:

• by phagocytosis or engulfment of the organism by macrophages;
• from lysing reactions by protective proteins that lyse cells at the surface;
• synthesis of antimicrobial peptides and key cytokines.¹⁰

A key cell in host immunity is the PMN. However, PMN functions are impaired by the biofilm, with reduced oxygen and hydrogen peroxide production.¹¹ PMNs produce cytokines and phagocytose individual organisms on the surface of a biofilm. However, PMNs cannot penetrate the biofilm very well if it is thick. In contrast, macrophages are a secondary defense that can engulf cells in the biofilm, while antibodies and immunoglobulins are also produced to kill cells. Other peripheral blood cells, such as basophils and eosinophils, produce chemicals, such as histamines, to destroy bacteria.¹²

The biofilm is a multifaceted three-dimensional film that adheres to oral surfaces, recolonizes easily, and is difficult to remove. The physical structure of biofilms is so complex that the host immune responses may only be effective at the outer surface of the biofilm. This may occur because host-protective antibodies and other serum or salivary proteins are unable to penetrate the thickness of the biofilm. Scientists have found that host antibodies are ineffective in killing organisms within a biofilm, when they are highly effective in killing planktonic or individual forms of the organism.^12-15 In addition, host macrophages cannot absorb bacterium growing within a complex polysaccharide matrix attached to a solid surface. This causes the host macrophage to release large amounts of pro-inflammatory enzymes and cytokines, leading to inflammation and destruction of nearby tissues.¹³ Synthetic peptides in attacking biofilms may be important to supplement the cellular defense systems of naturally produced peptides.¹⁴

Fortunately, the host immune system has other defensive factors—such as T-helper cells which produce a key cytokine, interleukin-2 (IL-2)—that respond to produce immunoglobulins, antibodies, and other cytokines to kill biofilm organisms or regulate an immune response. Inflammatory complement proteins produced in this process also play a role in the phagocytosis of biofilm organisms. These may also activate natural killer cells that deliver chemical barriers to inactivate organisms within the biofilm. PMNs produce neutralizing antibodies, and have the capacity to increase oxygen capacity and hydrogen peroxide. Because oral biofilm is impacted by low pH, lack of essential nutrients, and oxygen, these protective factors are important in down-regulating the response to the biofilm in the tissue.¹⁵

The range of both defensive and protective factors by the host immune response and the mediating inflammatory response to the biofilm and its byproducts may determine health or disease in the oral cavity. The complex production of immunoglobulins and migration of key inflammatory cytokines is important in this regard. Inflammatory responses of increased vascularization and increased endotoxin release may be important, particularly in areas such as the gingival sulcus.¹³ Scientists have shown that not only do bacteria increase after eating, but endotoxins from oral organisms increase four-fold after eating.¹⁶ This means that there is at least a transitory inflammatory response that is created after each meal or snack. The host immune system is protective by keeping the by-products of the biofilm in check and attracting both PMNs and macrophages to engulf pathogens within a biofilm. However, the host phagocytosis by PMNs and macrophages is impeded by increases in endotoxins and increases in vascularity as a result of local tissue inflammation from the biofilm. In this regard, cytokines such as IL-2, IL-10, and others are important mediators for regulating inflammatory responses to the biofilm. When the inflammatory response increases to a certain level (that is individually host-dependent), then the inflammatory chemicals can produce tissue lysing with concomitant tissue damage. This is a common feature of localized periodontal infection with deep pocketing. Moreover, local inflammatory responses can impact systemic inflammatory factors, as with diabetic patients. New information is emerging about the role of various cytokines and inflammatory factors in patients with immunocompromised or systemic health issues.^1,4,13,15

The cycle of biofilm, from initial attachment to maturation and then to detachment, is a complex process and the response to this is host-dependent. Meanwhile, the initial immune response usually occurs first by PMNs, then macrophages and other T and B immune cells occur concurrent with biofilm. As a result of chemicals produced by the host immune response to the biofilm and by-products of the biofilm, an inflammatory response is generated that impacts local inflammation and perhaps systemic inflammatory factors.

Conclusion
Recognizing the role of biofilm in oral health and reducing its impact on the host immune response is important in maintaining good patient health and is an emerging science in dentistry. The rapid increase of knowledge in biofilms means that oral health teams will soon have a better understanding of how to modulate oral health and disease processes in clinical practice.

References
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