**4. Antimicrobials: Mechanisms of action**

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

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

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

*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,

Microbial Dynamics and Caries: The Role of Antimicrobials 211

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

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

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

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

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

oral use must have adequate diffusion in biofilms to be effective (Marsh, 2005).

(Scheie, 2004).

transduction.

(Olsson, 1998).

which is positively charged, binds tightly to negatively charged bacteria membrane, causing its disruption (Gilbert & Moore, 2005).

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

*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 selective toxicity (Silver, 1967).

*Inhibition of Synthesis of Essential Metabolites*. The enzymatic activity of a specific microorganism can be competitively inhibited by a substance (antimetabolites) that closely resembles enzyme's normal substrate (Russell and Hugo, 1994).
