**5. Recent advances in anti-plaque agents: Chemoprophylactic agents, antimicrobial peptides, anti-quorum sensing approach and probiotics/replacement therapy**

Control of oral biofilms is essential for maintaining oral health and preventing dental caries, gingivitis and periodontitis. However, oral biofilms are not easily controlled by mechanical means and represent difficult targets for chemical control (Socransky, 2002). With the exception of chlorhexidine and fluoride, few of the existing oral prophylactic agents have significant effects (Petersen & Scheie, 1998; Wu & Savitt, 2002; Scheie, 2003). A likely explanation for this low efficiency is due to the fact that microorganisms organized in biofilms possess characteristics that differentiate them from planktonic cells, such as higher

which is positively charged, binds tightly to negatively charged bacteria membrane, causing

Fig. 2. Scanning electron micrograph of planktonic *S. mutans* UA159 cells exposed to chlorhexidine at 4.5 µg/ml. After 6h of CHX incubation, several wilted cells with spilled

*Inhibition of nucleic acids synthesis.* Some antibiotics interfere with the processes of DNA transcription and replication of microorganisms. Some drugs with this mode of action have limited use due to interference with DNA and RNA of mammals. Others, such as rifampin and quinolones, are more widely used in chemotherapy by having a higher degree of

*Inhibition of Synthesis of Essential Metabolites*. The enzymatic activity of a specific microorganism can be competitively inhibited by a substance (antimetabolites) that closely

Control of oral biofilms is essential for maintaining oral health and preventing dental caries, gingivitis and periodontitis. However, oral biofilms are not easily controlled by mechanical means and represent difficult targets for chemical control (Socransky, 2002). With the exception of chlorhexidine and fluoride, few of the existing oral prophylactic agents have significant effects (Petersen & Scheie, 1998; Wu & Savitt, 2002; Scheie, 2003). A likely explanation for this low efficiency is due to the fact that microorganisms organized in biofilms possess characteristics that differentiate them from planktonic cells, such as higher

**5. Recent advances in anti-plaque agents: Chemoprophylactic agents,** 

intracellular material could be observed (15 kV and 13.000x of magnification).

resembles enzyme's normal substrate (Russell and Hugo, 1994).

**antimicrobial peptides, anti-quorum sensing approach and** 

its disruption (Gilbert & Moore, 2005).

selective toxicity (Silver, 1967).

**probiotics/replacement therapy** 

resistance to several antimicrobial agents; most studies so far use study models with planktonic cells, not reproducing the reality of the oral cavity. In addition, antimicrobials for oral use must have adequate diffusion in biofilms to be effective (Marsh, 2005).

Thus, many of these studies need to be revalidated, taking into account the oral environment. Recent approaches to the study of microbial gene expression and regulation in non-oral microorganisms have elucidated systems for transduction of stimuli in biofilms, such as two-component systems and quorum sensing (two-component and quorumsensing systems) that allow the coordinated gene expression in these structures. These studies based on understanding the regulation and expression in microbial biofilms can potentially benefit the development of new strategies for prevention and treatment of diseases caused by oral biofilms. Thus, the intervention should be directed at targets such as surface adhesion, colonization, co-adhesion, metabolism, growth, adaptation, maturation, climax community and detachment, and strategies must be based on surface modification, immunization, replacement therapy , interference with two-component systems and quorum sensing (Scheie, 2004).

These new drugs must be highly specific, have little ability to induce resistance in microorganisms and produce minimal effects on vital functions of human cells. In therapeutic approaches, the main target should be the mature and established biofilm. In this case, genes and proteins essential for viability of microorganisms represent the traditional targets for designing these antimicrobial drugs. Among these potential agents are included bacteriophages, inhibitors of the biosynthesis of fatty acids and antimicrobial peptides (Hancock, 1999, Payne et al. 2001; Sulakvelidze & Morris, 2001). In prophylactic approaches, the main targets are the pathogenic microorganisms directly involved in the formation of mono or multi-species biofilms. Promising targets for this purpose would be the two-component systems and quorum sensing, whose inference could be used to ensure the ecological balance in the biofilm, allowing the maintenance of health-related microbiota (Marsh, 2010). This approach would have a selective toxicity, since these systems are present in most microorganisms, but not in mammalian cells, which use other mechanisms of signal transduction.

Another important strategy is the modification of tooth surface or, more precisely, the film acquired from the enamel to prevent bacterial colonization and thus biofilm formation. The film acquired from enamel has binding sites for oral bacteria through specific and nonspecific binding mechanisms. An in vitro study showed that the combination of alkylphosphate and a nonionic surfactant changes the characteristics of tooth surface, making it less attractive for microorganisms. However, the clinical efficacy of these agents has been low, probably due to difficulties in obtaining the active components of these agents (Olsson, 1998).

Some properties of topical antimicrobial agents for oral use are essential to their success as high substantivity in the oral sites of biological action, low acute and chronic toxicity, and low permeability, being overall associated with their mechanism of action. Clinical activity of the antimicrobial agent depends on the drug formulation that must have a quick and efficient release vehicle. The supragingival plaque, film acquired from enamel and saliva may be primary sites of action for these agents, but the detailed understanding of these interactions is limited. These antimicrobials are retained by electrostatic bonds to carboxylic

Microbial Dynamics and Caries: The Role of Antimicrobials 213

functions or virulence. Efforts are being made for manufacturing active and passive vaccines, especially for tooth decay. In active immunization, an attenuated antigen induces a protective immune response when administered. In passive immunization, the ready

Studies on animals and humans using approaches with active and passive immunization have been successful, especially in passive immunization where there is impediment to recolonization of microorganisms related to dental caries (Koga et al. 2002; Smith, 2002) and also in periodontal disease (Booth et al., 1996). The vehicles for passive immunization, such as milk from immunized cows (Shimazaki et al., 2001) and transgenic plants (Ma et al., 1998), have been tested with promising results. Similarly, it was shown that recombinant chimeric microbial vectors that are non-virulent, but express antigens of *S. mutans* (Huang et al., 2001, Taubman et al., 2001) or *P. gingivalis* (Sharma et al., 2001) promoted protection

One of the issues that still need to be solved is about which immune system should be stimulated, if the systemic immune system or that associated with mucosal. In the case of an anti-caries vaccine, it would be more interesting the oral administration and based on induction of the immune system associated with mucosa. A vaccine against periodontal disease should probably involve the systemic immune system and that associated with mucosa. A major problem is that approaches to immunization are usually directed against epitopes of isolate bacteria; however, both tooth decay and periodontal disease are diseases whose etiologic agent consists of a multispecies microbiota (Marsh, 1994). Moreover, since microorganisms have the ability to form biofilms and adapt to this environment, this can lead to changes in antigenicity which could affect the durability of protection induced by

An alternative approach are a new class of antibiotics called of antimicrobial peptides (AMP) that can be used against these microorganisms (White et al., 1995, Yount & Yeaman, 2004; Hancock & Sahl, 2006; Gardy et al., 2009); however, a poor understanding of the fundamental principles of the action mechanisms and structure-activity relationship of these drugs (Shai, 2002; Bechinger, 2009) has reduced the development of MPAs that can be used clinically.

The potential advantages of using antimicrobial peptides as antimicrobial drugs are significant (Hamill et al., 2008, Easton et al., 2009). They have a broad spectrum of activity against many strains of Gram positive and negative bacteria, including strains resistant to other drugs, and are also active against some fungi. Moreover, their interactions with bacterial components do not involve binding sites to specific proteins and thus do not induce resistance. The AMP bioavailability is reduced because they cannot be taken orally; however, topical applications and injections are available. Recently, AMP has been tested in clinical trials for various applications in oral candidiasis (Demegen Pharmaceuticals, 2010), infections associated with catheters (Melo et al., 2006) and infections in implant surfaces (Kazemzadeh-Narbat et al., 2010). These clinically tested AMPs are derived from natural peptides, this fact being responsible for its biggest drawback, which is the high production cost compared to other chemical antibiotics. For this reason, there is a critical need for development of new AMPs, powerful, small and with simple composition. The potential for the rational design of these drugs is often limited due to little knowledge about the details of

against tooth decay and loss of alveolar bone in experimental animals.

antibody is administered (Sheie, 2004).

immunization.

its mechanism of action.

acids and phosphate and sulfate residues of proteins and glycoproteins in the oral mucosa, film acquired from enamel and plaque. The non-ionic antibacterials are retained by adsorption to lipophilic regions in these receptor sites. The ability of these antiplaque agents have to keep an optimal concentration in saliva over a long period, in addition to remaining in the bioactive form at the action sites, such as the teeth surfaces is extremely important and influence in the clinical effectiveness of these agents (Cummins & Creeth , 1992).

The analysis of retention characteristics and antimicrobial properties of clinically proven antiplaque agents suggest that they act multifunctionally and at multiple sites. Thus, they reduce growth and metabolism of bacteria in plaque, saliva and tooth surface, but also reduce the adhesion of potential settlers. Two generic routes to increase the antiplaque activity of these agents have received attention. Firstly the use of a combination of antimicrobial agents with similar but complementary activities uses only one route and mode of action. A second potential route is the use of a polymer that serves as auxiliary retention of only one antimicrobial used (Cummins, 1991b).

The total oral retention, salivary profile and agent concentrations on the plaque, film acquired from enamel and oral mucosa are not only indicators of biological activity in vivo, but they serve as potential indicators for this activity. This means that the increased release of a specific agent in vivo is not predictive of its increased clinical efficacy (Cummins, 1991b). The increased activity on the site or sites of biological action combined with agent's residence time in the oral cavity are the best predictors of agent's clinical activity (Creeth & Cummins, 1992).

Replacement therapy has been suggested as a strategy for replacement of pathogenic microorganisms modified to become less virulent. Some requirements for this type of approach are important, such as: the replaced organism must not cause disease by itself; it must persistently colonize and must possess a high degree of genetic stability. DNA technology has enabled to produce potential candidates for replacement therapy in the prevention of dental caries. Among these, there is the super-colonizing strain of *S. mutans*. This strain produces mutacin, which allows it to replace the wild-type strain efficiently. It lacks the enzyme lactate dehydrogenase and therefore is unable to produce lactate (Hillman et al., 2000). Other ureolitic recombinant strains have been constructed and are capable of hydrolyzing urea to ammonia, thereby offsetting the environment acidification (Clancy et al., 2000).

A possible future approach would be to use genetically modified microorganisms for releasing molecules that could interfere with pathways such as signal transduction of twocomponent and quorum sensing. However, it is important to emphasize that there is the possibility of a genetically modified strain subsequently undergoes transformation in oral biofilms and then becomes a pathogenic opportunistic strain (Scheie, 2004).

Immunization against oral diseases as dental caries and periodontal disease has been extensively studied in recent decades (Koga et al. 2002; Smith, 2002). The goal would be inhibiting or reducing the virulence of some microbial etiological agents. Several molecules involved in various stages of the pathogenesis of caries and periodontal disease could be susceptible to immune intervention and serve as targets for production of vaccines. Thus, it would be possible to eliminate microorganisms of the oral cavity with antibodies able to block adhesins or receptors involved in adhesion, or metabolically modify important

acids and phosphate and sulfate residues of proteins and glycoproteins in the oral mucosa, film acquired from enamel and plaque. The non-ionic antibacterials are retained by adsorption to lipophilic regions in these receptor sites. The ability of these antiplaque agents have to keep an optimal concentration in saliva over a long period, in addition to remaining in the bioactive form at the action sites, such as the teeth surfaces is extremely important and

The analysis of retention characteristics and antimicrobial properties of clinically proven antiplaque agents suggest that they act multifunctionally and at multiple sites. Thus, they reduce growth and metabolism of bacteria in plaque, saliva and tooth surface, but also reduce the adhesion of potential settlers. Two generic routes to increase the antiplaque activity of these agents have received attention. Firstly the use of a combination of antimicrobial agents with similar but complementary activities uses only one route and mode of action. A second potential route is the use of a polymer that serves as auxiliary

The total oral retention, salivary profile and agent concentrations on the plaque, film acquired from enamel and oral mucosa are not only indicators of biological activity in vivo, but they serve as potential indicators for this activity. This means that the increased release of a specific agent in vivo is not predictive of its increased clinical efficacy (Cummins, 1991b). The increased activity on the site or sites of biological action combined with agent's residence time in the oral cavity are the best predictors of agent's clinical activity (Creeth &

Replacement therapy has been suggested as a strategy for replacement of pathogenic microorganisms modified to become less virulent. Some requirements for this type of approach are important, such as: the replaced organism must not cause disease by itself; it must persistently colonize and must possess a high degree of genetic stability. DNA technology has enabled to produce potential candidates for replacement therapy in the prevention of dental caries. Among these, there is the super-colonizing strain of *S. mutans*. This strain produces mutacin, which allows it to replace the wild-type strain efficiently. It lacks the enzyme lactate dehydrogenase and therefore is unable to produce lactate (Hillman et al., 2000). Other ureolitic recombinant strains have been constructed and are capable of hydrolyzing urea to ammonia, thereby offsetting the environment acidification (Clancy et

A possible future approach would be to use genetically modified microorganisms for releasing molecules that could interfere with pathways such as signal transduction of twocomponent and quorum sensing. However, it is important to emphasize that there is the possibility of a genetically modified strain subsequently undergoes transformation in oral

Immunization against oral diseases as dental caries and periodontal disease has been extensively studied in recent decades (Koga et al. 2002; Smith, 2002). The goal would be inhibiting or reducing the virulence of some microbial etiological agents. Several molecules involved in various stages of the pathogenesis of caries and periodontal disease could be susceptible to immune intervention and serve as targets for production of vaccines. Thus, it would be possible to eliminate microorganisms of the oral cavity with antibodies able to block adhesins or receptors involved in adhesion, or metabolically modify important

biofilms and then becomes a pathogenic opportunistic strain (Scheie, 2004).

influence in the clinical effectiveness of these agents (Cummins & Creeth , 1992).

retention of only one antimicrobial used (Cummins, 1991b).

Cummins, 1992).

al., 2000).

functions or virulence. Efforts are being made for manufacturing active and passive vaccines, especially for tooth decay. In active immunization, an attenuated antigen induces a protective immune response when administered. In passive immunization, the ready antibody is administered (Sheie, 2004).

Studies on animals and humans using approaches with active and passive immunization have been successful, especially in passive immunization where there is impediment to recolonization of microorganisms related to dental caries (Koga et al. 2002; Smith, 2002) and also in periodontal disease (Booth et al., 1996). The vehicles for passive immunization, such as milk from immunized cows (Shimazaki et al., 2001) and transgenic plants (Ma et al., 1998), have been tested with promising results. Similarly, it was shown that recombinant chimeric microbial vectors that are non-virulent, but express antigens of *S. mutans* (Huang et al., 2001, Taubman et al., 2001) or *P. gingivalis* (Sharma et al., 2001) promoted protection against tooth decay and loss of alveolar bone in experimental animals.

One of the issues that still need to be solved is about which immune system should be stimulated, if the systemic immune system or that associated with mucosal. In the case of an anti-caries vaccine, it would be more interesting the oral administration and based on induction of the immune system associated with mucosa. A vaccine against periodontal disease should probably involve the systemic immune system and that associated with mucosa. A major problem is that approaches to immunization are usually directed against epitopes of isolate bacteria; however, both tooth decay and periodontal disease are diseases whose etiologic agent consists of a multispecies microbiota (Marsh, 1994). Moreover, since microorganisms have the ability to form biofilms and adapt to this environment, this can lead to changes in antigenicity which could affect the durability of protection induced by immunization.

An alternative approach are a new class of antibiotics called of antimicrobial peptides (AMP) that can be used against these microorganisms (White et al., 1995, Yount & Yeaman, 2004; Hancock & Sahl, 2006; Gardy et al., 2009); however, a poor understanding of the fundamental principles of the action mechanisms and structure-activity relationship of these drugs (Shai, 2002; Bechinger, 2009) has reduced the development of MPAs that can be used clinically.

The potential advantages of using antimicrobial peptides as antimicrobial drugs are significant (Hamill et al., 2008, Easton et al., 2009). They have a broad spectrum of activity against many strains of Gram positive and negative bacteria, including strains resistant to other drugs, and are also active against some fungi. Moreover, their interactions with bacterial components do not involve binding sites to specific proteins and thus do not induce resistance. The AMP bioavailability is reduced because they cannot be taken orally; however, topical applications and injections are available. Recently, AMP has been tested in clinical trials for various applications in oral candidiasis (Demegen Pharmaceuticals, 2010), infections associated with catheters (Melo et al., 2006) and infections in implant surfaces (Kazemzadeh-Narbat et al., 2010). These clinically tested AMPs are derived from natural peptides, this fact being responsible for its biggest drawback, which is the high production cost compared to other chemical antibiotics. For this reason, there is a critical need for development of new AMPs, powerful, small and with simple composition. The potential for the rational design of these drugs is often limited due to little knowledge about the details of its mechanism of action.

Microbial Dynamics and Caries: The Role of Antimicrobials 215

number of *S. mutans* in the mouth (Caglar et al., 2008) in order to decrease the incidence of tooth decay. Other bacterium normally found in the mouth, and important in this sense, is *Streptococcus salivarius*, which produces a bacteriocin that inhibits anaerobic Gram-negative bacteria and that in vivo was shown also to reduce the level of halitosis (Burton et al., 2006). In conclusion, microbiota analysis methods independent of culture have allowed to understanding the diversity of the oral microbiota. So far, most studies have focused on the microbiota composition in the disease, but a better understanding of this microflora in health is required and also as probiotic organisms are capable of restoring and maintaining health in such environment. Thus, future studies are still needed, especially for analyzing the interactions between species and how to use this knowledge to develop new products

Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. (2005). Defining the normal bacterial

Ahmer BMM. (2004). Cell-to cell signaling on Escherichia coli and Salmonella enterica. *Mol* 

Al-Hashimi I, Levine MJ. (1989). Characterization of in vivo salivary-derived enamel

Bassler BL. (2002). Small talk. Cell-to-cell communication in bacteria. *Cell*, Vol. 109, p. 421-

Bechinger B. (2009). Rationalizing the membrane interactions of cationic amphipathic

Bjarnsholt T, Jensen PO, Burmolle M, Hentzer M, Haagensen JA, Hougen HP, et al. (2005).

Booth V, Ashley FP, Lehner T (1996). Passive immunization with monoclonal antibodies

Bowden GH, Hardie JM, Slack GL. (1975). Microbial variations in approximal dental plaque.

Bowden GH. (1999). Controlled environment model for accumulation of biofilms of oral

Bradshaw DJ, Marsh PD, Schilling KM, Cummins D. (1996). A modified chemostat system to study the ecology of oral biofilms. *J Appl Bacteriol*, Vol. 80, No. 2, pp. 124-30. Bradshaw DJ, Marsh PD, Watson GK, Allison C. (1998). Role of Fusobacterium nucleatum

communities during aeration. *Infect Immun*, Vol. 66, No. 10, pp. 4729-32. Broadley SJ, Jenkins PA, Furr JR, Russell AD. (1995). Potentiation of the effects of

ethambutol. *Journal of Medical Microbiology*, Vol. 43, pp. 458–460.

antimicrobial peptides by their molecular shape. *Current Opinion in Colloid &* 

*Pseudomonas aeruginosa* tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent. *Microbiology*; Vol.

against *Porphyromonas gingivalis* in patients with periodontitis. *Infect Immun,* Vol.

and coaggregation in anaerobe survival in planktonic and biofilm oral microbial

chlorhexidine diacetate and cetylpyridinium chloride on mycobacteria by

flora of the oral cavity. *J Clin Microbiol*, Vol. 43, n. 11, pp. 5721-32.

for prevention and treatment of oral diseases.

*Microbiol*, Vol. 52, p.933-945.

151:(Pt 2), pp. 373−83.

64, pp. 422-427.

pellicle. *Arch Oral Biol*, Vol. 34, n. 4, pp. 289-95.

*Interface Science*, Vol. 14, pp. 349–355.

*Caries Res*, Vol. 9, n. 4, pp. 253-77.

bacteria. *Methods Enzymol*, Vol. 310, pp. 216-24.

**7. References** 

424.

Since tooth decay is an infection, it would be logical to treat the disease with antibiotics or antimicrobials, such as antimicrobial peptides. However, most of these agents are not selective, have broad spectrum of action on the microorganisms such as chlorhexidine, iodopovidine, fluoride, penicillin or other antimicrobial/antibiotics. Importantly, the agents described above does not sterilize the oral cavity, since it is exposed to the external environment where there are many microbes, it is not a sterile space. Thus, the use of broadspectrum agents for treating dental caries can suppress the infection, but will never eliminate it entirely (Luoma et al., 1978). In this context, due to the limitations of traditional strategies in the management of dental caries, a "probiotic" approach of the disease is necessary. The term "probiotic" used here means that mechanisms are used to selectively remove only the pathogen responsible for disease in an attempt to keep the oral ecosystem intact. Most efforts in this sense are derived from studies that have attempted to genetically modify strains of *Streptococcus mutans*, turning them into strains that in addition to not producing acids, still competing for the same ecological niche that wild strain of *S. mutans*  (Hillman, 2002).

In theory and experimentally in laboratory animals, when this substitute organism is introduced, it completely shifts the wild *S. mutans* causing the disease. This action stops the decay process and also prevents the re-emergence of disease-causing organisms, eliminating the possibility of re-infection, since the "normal microbiota is complete." Another way to remove pathogens is developing specific antimicrobials for certain targets (Eckert et al., 2006). The basic principle is developing a cheap molecule that targets only the organism of interest, in this case *S. mutans, S sobrinus*, or other pathogens.

In the case of the oral cavity and tooth decay, this system is attractive from the perspective of eliminating all pathogens, thus preventing the re-growth of the original infection. There are also laboratory and clinical evidence demonstrating that when the biofilm's bacterial ecosystem is free of *S. mutans*, this bacterium finds it difficult to be reintroduced due to competitive inhibition with other microorganisms (Keene & Shklair, 1974; Shi, 2005). A criticism to probiotic approaches is that they target only one of the pathogens involved with the disease, being not directed at other pathogens that may be involved with the beginning of the process, as the case of dental caries.
