**3. Communication microbial biofilms:** *Quorum sensing* **mechanisms**

In the dental biofilm bacteria do not exist as independent entities, but as a coordinated and metabolically integrated microbial community (Marsh & Bowden, 2000). This interaction provides enormous benefits to the participating organizations compared to the same bacteria grown in planctonic form, including: a wider range of habitat for growth, increased diversity, increased metabolic efficiency and greater resistance to environment, antimicrobial agents and host defenses (Shapiro, 1998; Marsh & Bowden, 2000).

The heterogeneity and high bacteria density within biofilms promote genotypic and phenotypic changes through releasing of self-inducers in the environment, leading to modification of gene expression (virulence genes of exoenzymes, exopolysaccharides) and at the same time, the acquisition of a important competitive advantage for survival and perpetuation in natural environments, highly competitive (eg, oral cavity, intestine), where hundreds of species coexist (Shei & Petersen, 2004). These virulence factors mediated by self-inductors were called "quorum sensing" (Fuqua et al., 1994).

The *quorum sensing* (QS) is a communication process among microorganisms mediated by population density. The main QS' regulation mechanism is given by production of autoinducers released into the external environment, where they accumulate, and the interaction with its receptor, which may be intracellular or present in cell surface (Nealson et al, 1970; Hens et al., 2007). When its concentration reaches a certain threshold, it promotes the activation or repression of several genes causing cells to exhibit new phenotypes (Redfield, 2002).

Microbial Dynamics and Caries: The Role of Antimicrobials 209

The discovery that *S. mutans* performs quorum-sensing of the system ideal in growing biofilms led us to investigate other features of this system in biofilm formation and biofilm physiology. The strategy for control of microorganisms by interfering in QS systems

Antimicrobials can be bactericidal (kill the microorganism directly) or bacteriostatic (prevent the microbe growth). In the case of bacteriostatic drugs, host defenses such as phagocytosis and antibody production usually destroy the microorganism. With the suspension of the second type of drug, bacteria can grow back. For bacteriostatic and bactericidal actions are apparent it is necessary to determine the MIC (Minimum Inhibitory Concentration) and MBC (minimum bactericidal concentration). As the therapeutic activity of antibiotics depends, among other factors, on their concentrations in body fluids, MICs and CBMs are essential determinations, since the establishment of the antibiotic regimen depends on them. The MIC and MBC are estimated in vitro, but used to determine bacteriostatic and

In Biofilms, MIC and MBC of antimicrobial agents usually must be greater than those required for plancttonic cells, due to its greater resistance to these drugs. In addition, optimal antimicrobials indicated for diseases that have bacteria organized in biofilms as etiological agent, must have good distribution in these structures. The main mechanisms of action of antimicrobials include: inhibition of cell wall synthesis, inhibition of protein synthesis, plasma membrane damage, inhibition of the synthesis of nucleic acids and

*Cell Wall Inhibition*. The bacterium's cell wall consists of a network of macromolecules called peptidoglycan, which is found exclusively in bacteria's cell wall. Penicillin and other antibiotics prevent complete synthesis of peptidoglycan, consequently, the cell wall becomes fragile and cell undergoes lysis. As penicillin targets the synthesis process, only cells in active growth will be affected by this antibiotic. And as human cells do not have

*Inhibition of Protein Synthesis*. Protein synthesis is a characteristic common to all cells, both prokaryotes and eukaryotes, not presenting therefore a suitable target for selective toxicity. Eukaryotic cells have 80S ribosomes and prokaryotic cells have 70S ribosomes. The difference in the ribosome structure is responsible for selective toxicity to antibiotics that affect protein synthesis. However, the mitochondria (important cytoplasmic organelles) also has the 70S ribosomal unit similar to bacteria units. Antibiotics that act on the 70S ribosome may therefore have adverse effects on host cells. Among the antibiotics that interfere are the clorofenicol, erythromycin, streptomycin, and tetracycline (Nakamura & Tamaoki, 1968).

*Damage to the plasma membrane.* Certain antibiotics, especially polypeptide antibiotics, promote changes in the permeability of plasma membrane. These changes result in the loss of major metabolites of the microbial cell. For example, polymyxin B disrupts the plasma membrane by binding to membrane phospholipids (Lambert & Hammond, 1973). Likewise, planktonic cells, when exposed to higher concentrations of the chlorhexidine (CHX), suffer membrane rupture (Figure 2). This observation can be explained by the fact that CHX,

peptidoglycan, penicillin has low cytotoxicity to the host cell (Broadley et al. 1995).

presents an important alternative for control of oral biofilms.

bactericidal concentrations of antibiotics in body fluids (Maillard, 2002).

inhibition of the synthesis of essential metabolites (Maillard, 2002).

**4. Antimicrobials: Mechanisms of action** 

The first indication that bacteria communicate through chemical signals came from studies of Nealson et al. (1970). They studied the bioluminescence regulation in the marine bacterium *Vibrio fischeri*, which has a symbiotic relationship with marine animals such as squid. In this regard, the host uses the light produced by the bacterium, to attract preys and partners or ward off predators, while the *V. fischeri* obtains necessary nutrients from its host (Nealson et al., 1970).

The luminescence is observed only when bacteria colonize the host's organs and by increasing the number of bacteria in the medium they are able to perceive cell density by detecting the auto-inducer concentration. Upon reaching a threshold concentration of selfinduction, it is enough to trigger the process of gene transcription (Swem et al., 2009).

The self-inducers involved in this process may be of different chemical nature, in gramnegative organisms the signaling molecules are derived from N-acyl homoserine lactone (AHL) and its regulation occurs through homologous proteins LuxR and LuxI. The first protein acts as an enzyme (AHLsintetase) and second, when connecting to the AHL, forms the AHL-LuxR the complex, which is responsible for the activation and expression of numerous genes. In gram-positive, self-inducers usually correspond to small peptides (hepta and octapeptides). These peptides are usually secreted by carriers bound to ATP (ABC). Some interact with membrane-bound kinases sensors carrying a flag through the membrane; others are transported into the cell by oligopeptide permeases, which then interact with intracellular receptors (Swem et al., 2009; Rock Road, et al., 2010).

In QS systems via AHLs, the variation in the acyl chain (chain length, degree of oxidation and saturation) may confer some specificity to these communication systems. Thus, there seems to be some cross-talk among bacteria belonging to different genera. Part of this crosstalk may represent a way by which bacteria acquire information about the total population, allowing a response to competitors or prospective members (Williams, 2007). *E. coli* does not synthesize AHLs but express a homologous biosensor LUXR (SDIA). It is speculated that this system allows *E. coli* detecting communication signals of other gram-negative and exploiting such information for its own benefit (Ahmer, 2004).

The gram-negative bacterium, *Streptococcus mutans*, a major pathogen of dental caries, performs the quorum-sensing by releasing mediator peptides of gene expression. The signaling system involves at least six gene products encoded *comCDE*, *comAB and comX* (Cvitkovitch et al., 2003). The OMCC gene encodes a precursor peptide, which when cleaved and exported release a signal peptide, 21 amino acid or stimulating competence peptide (CSP). Through the quorum-sensing, it was found that the competence-stimulating peptide (CSP) was necessary for proper formation of *S. mutans* biofilm in addition to its virulence characteristics (Li et al., 2001).

The quorum sensing systems control a variety of microbial processes such as sporulation, virulence, biofilm formation, conjugation and production of extracellular enzymes (Miller & Bassler, 2001). Bacteria use QS to coordinate gene expression within species. Moreover, the same detection signals are used to inhibit or activate transcription programs between competing bacteria strains and other existing species in the same microenvironment (Bassler, 2002). Communication can still cross the borders of the kingdom, as QS effector molecules that can alter the eukaryotic transcription programs, found in epithelial cells and immune effector cells (Williams, 2007; Shin et al., 2005).

The first indication that bacteria communicate through chemical signals came from studies of Nealson et al. (1970). They studied the bioluminescence regulation in the marine bacterium *Vibrio fischeri*, which has a symbiotic relationship with marine animals such as squid. In this regard, the host uses the light produced by the bacterium, to attract preys and partners or ward off predators, while the *V. fischeri* obtains necessary nutrients from its host

The luminescence is observed only when bacteria colonize the host's organs and by increasing the number of bacteria in the medium they are able to perceive cell density by detecting the auto-inducer concentration. Upon reaching a threshold concentration of selfinduction, it is enough to trigger the process of gene transcription (Swem et al., 2009).

The self-inducers involved in this process may be of different chemical nature, in gramnegative organisms the signaling molecules are derived from N-acyl homoserine lactone (AHL) and its regulation occurs through homologous proteins LuxR and LuxI. The first protein acts as an enzyme (AHLsintetase) and second, when connecting to the AHL, forms the AHL-LuxR the complex, which is responsible for the activation and expression of numerous genes. In gram-positive, self-inducers usually correspond to small peptides (hepta and octapeptides). These peptides are usually secreted by carriers bound to ATP (ABC). Some interact with membrane-bound kinases sensors carrying a flag through the membrane; others are transported into the cell by oligopeptide permeases, which then

In QS systems via AHLs, the variation in the acyl chain (chain length, degree of oxidation and saturation) may confer some specificity to these communication systems. Thus, there seems to be some cross-talk among bacteria belonging to different genera. Part of this crosstalk may represent a way by which bacteria acquire information about the total population, allowing a response to competitors or prospective members (Williams, 2007). *E. coli* does not synthesize AHLs but express a homologous biosensor LUXR (SDIA). It is speculated that this system allows *E. coli* detecting communication signals of other gram-negative and

The gram-negative bacterium, *Streptococcus mutans*, a major pathogen of dental caries, performs the quorum-sensing by releasing mediator peptides of gene expression. The signaling system involves at least six gene products encoded *comCDE*, *comAB and comX* (Cvitkovitch et al., 2003). The OMCC gene encodes a precursor peptide, which when cleaved and exported release a signal peptide, 21 amino acid or stimulating competence peptide (CSP). Through the quorum-sensing, it was found that the competence-stimulating peptide (CSP) was necessary for proper formation of *S. mutans* biofilm in addition to its virulence

The quorum sensing systems control a variety of microbial processes such as sporulation, virulence, biofilm formation, conjugation and production of extracellular enzymes (Miller & Bassler, 2001). Bacteria use QS to coordinate gene expression within species. Moreover, the same detection signals are used to inhibit or activate transcription programs between competing bacteria strains and other existing species in the same microenvironment (Bassler, 2002). Communication can still cross the borders of the kingdom, as QS effector molecules that can alter the eukaryotic transcription programs, found in epithelial cells and

interact with intracellular receptors (Swem et al., 2009; Rock Road, et al., 2010).

exploiting such information for its own benefit (Ahmer, 2004).

immune effector cells (Williams, 2007; Shin et al., 2005).

characteristics (Li et al., 2001).

(Nealson et al., 1970).

The discovery that *S. mutans* performs quorum-sensing of the system ideal in growing biofilms led us to investigate other features of this system in biofilm formation and biofilm physiology. The strategy for control of microorganisms by interfering in QS systems presents an important alternative for control of oral biofilms.
