Lakhasly

Application of modern research techniques, has allowed fuller understanding of many important phe- nomena occurring on the tooth surface near the gingi- val margin and in the gingival sulcus, the site of bacte- rial dental calculus deposits. This techniques has also contributed to the knowledge of the highly complex structure of dental plaque. Dental plaque was one of

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the first structures formed by bacteria that has been described as a biofilm (Marsh, 2005; ten Cate, 2006; Hoiby et al., 2011). Over 700 bacterial species have been isolated from the human oral cavity and the majority of them are associated with dental biofilm. Many years of research have shown that this biofilm is a highly specialized, co-ordinated, multi-species form of microorganism life, permanently located on the tooth surface in a matrix, surrounded by a layer of extracel- lular polysaccharides (EPS). It is created by the layered growth of microorganisms existing as separated micro- colonies, mainly bacteria capable of adhering to each other, which can form a community where spatially dis- tributed populations can interact. When organized in biofilms, the oral micro-organisms are less susceptible to antimicrobials and more resistant to immunologi- cal defense systems (Davies, 2003; Dufour et al., 2013; Stoodley et al., 2002).
Microorganisms in the oral cavity form two types of biofilm on the surface of the tooth: the supra-gin- gival plaque and the sub-gingival plaque, which differ significantly in the composition of the bacterial flora. Supragingival plaque is dominated by Gram positive bacteria, including Streptococcus mutans, Streptococcus salivarius Streptococcus mitis and Lactobacillus, while subgingival plaque is dominated by Gram-negative anaerobic bacteria, such as Actinobacillus, Campylo- bacter spp., Fusubacterium nucleatum, Porphyromonas gingivalis (Hojo et al., 2009; He and Shi, 2009; Marsh, 2012). The cause of dental caries is usually the supra- gingival microbiome. The sub-gingival microbiome is associated with gingivitis and periodontal disease (Abusleme et al., 2013).
The formation and development of biofilm takes place in three main steps: attachment of the initial pio- neer species, which leads to an increase in the biofilm mass due to colonization, co-adhesion, co-aggregation of other species of microorganisms, production of extracellular polysaccharides and separation of bacte- ria from the surface of the biofilm and their spread in the environment of the oral cavity. (Hoiby et al., 2011; Scheie and Petersen, 2004; ten Cate, 2006).
Since microorganisms are not able to colonize the cleaned tooth surfaces, which are deprived of any exter- nal components, the presence of the acquired pellicle is necessary for adhesion. The acquired pellicle is formed on the tooth surface immediately upon their brushing or professional cleaning treatments. This process is esti- mated to take minutes. It consists of saliva and proteins from gingival fluid adsorbed on to the cleaned surface. Gingival fluid is composed of different host-derived molecules rich in proline, tyrosine, histidine, includ- ing proteins and agglutinins which act as a source of receptors that are recognized by various oral bacteria, mucins and other glycoproteins. The initial stage of bac-
terial adhesion to the tooth surface includes interaction of the superficial substances of microorganism with the components of saliva contained in the acquired pellicle. As the attached bacteria cells grow and divide, many will start to express a biofilm phenotype, which will include the secretion of extracellular polymeric sub- stances (EPS) with polypeptides, carbohydrates and nucleic acids (Hojo, 2009; Kolebrander et al., 2010).
In the initial non-specific phase of biofilm forma- tion, when bacteria that form it are located at a consid- erable distance apart, the electrostatic, hydrophobic and van der Waals forces allow for reversible adhesion of microorganisms (Hics et al., 2003; Scheie and Petersen, 2004). This initial phase of adhesion becomes irrevers- ible later due to a specific reaction between bacteria adhesins and PRPs (proline-rich glycoproteins) on the surface of the acquired pellicle. Accumulation of biofilm microorganisms is very fast as a result of the interspecies aggregation of streptococci with actino- mycetes, as well as agglutination of microorganisms within one species, which leads to aggregation of new bacterial species with the already settled organisms. Among the early colonizers of the biofilm, different species of Streptococcus spp., Eikenella spp., Actinomyces spp., Haemophilus spp., Prevotella spp., Capnocytophaga spp., Priopionibacterium spp., and Veillonella spp. have been identified in 60–90% of cases. The later colonizers include Fusobacterium nucleatum, Actinobacillus spp., Prevotella spp., Eubacterium spp., Treponema spp. and Porphyromonas spp. (Kolebrander et al., 2010; Marsh, 2012; Nasidze et al., 2009).
Several salivary components have been shown to have a role in microbial adhesion to the pellicle, for instance, salivary oligosaccharide-containing glyco- proteins may serve as receptors for oral streptococci and the salivary proline-rich protein 1 and statherin have been implicated as receptors for type 1 fimbrie of Actinomyces viscosus (Gibbons et al., 1988; Marsh, 2005; Scheie and Petersen, 2004). The wide range of streptococcal species produce the most well-studied oral adhesins, including antigen I/II, PaG, SspA, amyl- ase-binding proteins, and type 1 fimbrie-associated protein. Adhesins produced by other oral bacteria have also been identified. Bacterial forms colonize the pel- licle acquired during the day, which is transformed into dental biofilm.
A major role in the formation of biofilm is attrib- uted to extracellular polysaccharides (EPS: Exopolysac- charides) containing, among others, mannose and gly- cosidic residues, which form a bacterial capsule or are released into the environment, where they become part of mucus. The composition of EPS varies depending on the bacterial strain and environmental conditions. Consist of exopolisaccharides, proteins and extracel- lular DNA. The biofilm matrix is increased by further

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precipitation of salivary glycoproteins, mainly through the creation of extracellular disaccharides. A particu- larly important role in the creation of plaque mass and adhesion of bacteria is attributed to polysaccharide polymers-soluble ones such as glucan and fructan, and insoluble ones, such as mutan (Hojo et al., 2009; Marsh, 2011). The cariogenic streptococci form extra- cellular polymers in the enzymatic reactions involving α-glucosidases. For example, S. mutans produces two glucosidases. One leads to the formation of insoluble glucan with α-1,3-glucan-binding called mutan. The other it creates a bond α-1,6 characteristic of the solu- ble glucan known as dextran. Dextran acts as a sub- strate for further metabolism in the event of shortage of saccharides in food. Sticky mutan is involved in increasing of the plaque mass and enhances adhesion of the cariogenic microorganisms. From fructose, with involvement of β-fructosidase, a polymer called fructan or levan is derived. Fructan, like dextran, constitutes an extracellular metabolic substrate. S. mutans produces a rare soluble fructan called inulin (Biswas et al., 2005; Takashi and Nyvad, 2008).
Extracellular polysaccharides are created also by other bacteria in the oral cavity, such as S.sanguis and A. viscosus, but a major role in their production is attributed to S. mutans. One of the important func- tions of EPS, in addition to the significant role in the processes described above, is the protection of these microorganisms against the host defense system and this is especially meaningful for the pathogenic nature of cariogenic microorganisms. The coating made of pol- ysaccharides, due to its hydrophilic nature, effectively protects bacteria from phagocytosis (Allison, 2003).
After the adhesion phase, the process of building of the biofilm structure begins with microbial multipli- cation and differentiation (Belda et al., 2012, Filoche et al., 2010). Immediately after adhesion of bacteria to the surface or to other bacteria, activation or inhibi- tion of specific gene expression occurs, with biofilm maturation being associated with changes in activity of particular genes in relation to the environmental con- ditions. Changes in gene expression involve the occur- rence of relevant phenotypic traits. For example, due to changes in the environment such as limited access to oxygen, the metabolism of cells changes: the activity of anaerobic metabolic pathways of glycolysis increases, and the synthesis of certain enzymes is inhibited (Ling et al., 2010; Roberts and Mullany, 2010). Production of flagellin, the protein included in the flagella of Gram- negative bacteria, is inhibited. As a result, certain struc- tures disappear, e.g. flagella, which are no longer needed in this “stationary” form of existence within the biofilm (Marsh, 2005).
Microorganisms in biofilm are arranged in orderly, complex structures that are highly differentiated, con-
sisting of bacterial microcolonies. The mature biofilm, coated with a layer of exopolysaccharides, on the sur- face of the tooth is sometimes composed of a countless number of microcolonies, separated from one another by numerous fluid filled channels, which creates a unique communication system of biofilm cells. The fluid circulating in the channels flows past microcolo- nies, delivering nutrients, oxygen, enzymes, metabo- lites, signal molecules and removing waste products (Wood et al., 2000; Stoodley et al., 2002). In addition, it acts as a protection for its inhabitants against exter- nal factors including antibiotics, antibodies, bacte- riophages, leukocytes. Thus it plays a triple role: sur- rounds, fastens and protects. The mutual proximity of the cells encourages exchange of genetic information by transfer of plasmids, including those encoding for resistance to antimicrobial substances and antibiotics (Davies, 2003; Roberts and Mullany, 2010).
Biological interactions have a role in structuring communities. They can occur within a community, across communities between microbiota and host, and between microbiota and their preators (Biswas et al., 2005; Kolebrander et al., 2012; Kuboniqa et al., 2012).
A very important property of bacterial biofilm, much better developed than in the case of planktonic cells, is the ability to communicate with each other and regulate metabolic processes. Quorum sensing (QS) signaling represents a signaling pathway that is activated as a response to cell density. The stimuli of QS systems are signal molecules called autoinducers, their concentration is a function of microbial density. It depends on the cell population density in a particular site. Bacteria have complex gene expression programs which are up-regulated when they co-habit in dense populations. For example, their activity is changed and slow down their metabolism which is essential to pre- vent an accumulation of metabolic waste or the deple- tion of nutrients (Irie and Parsek, 2008; Kolebrander et al., 2002; Scheie and Petersen, 2004). Bacteria are able to accurately identify the chemical nature of the signals and their threshold concentration in the environment, which allows for their specific growth and control of physiological and metabolic processes of the entire population. Streptococci have evolved various means of coping with the deleterious effects of environmen- tal stressors and avoiding the host immune system. Recently several studies have shown that streptococci colonizing the mouth and upper respiratory tract are able to mount complex stress responses in order to per- sist and successfully survive competition in their eco- logical niche. Using a small quorum sensing peptide pheromone acting as a stress-inducible ‘alarmone’, oral streptococci synchronize the gene expression of a spe- cific group of cells to coordinate important biological activities. (Dufour et al., 2013). Bacteria, thanks to the

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chemical signals they send, are able to control impor- tant life processes such as bioluminescence, production of virulence factors, symbiosis, conjugation, mobility, spore formation, colony growth and biofilm formation (Kolebrander et al., 2002; Marsh, 2005; Spoering and Gilmore, 2006). Gram-positive and Gram-negative bac- teria have developed completely different QS transmis- sion systems for signaling molecules. Functions of auto- inducers in Gram-negative bacteria are fulfilled by acyl homoserine lactones (AHLs), and in Gram-positive by specific oligopeptides and auto inducers-2 (AI-2). AHL diffuses into cytoplasm and up-regulates the transcrip- tion of specific genes thus influencing the production of vital proteins. In S. mutans, QS is mediated by a com- petence-stimulating peptide (CSP). It was shown that when CSP reached a threshold level, a subpopulation of the bacteria lysed. By doing so they provided DNA to the environment which was then taken up by other competent bacteria, which is essential for exchange of genetic material and evolutionary process. These spe- cific signaling molecules are used for communication between cells of the same or different bacterial popula- tions or strains. Bacteriocins might also function as QS signals and have a direct effect on biofilm composition (Irie and Parsek, 2008; Li et al., 2002; Miller and Bassler, 2001; Spoering et al., 2006).
The formation of the layer of microorganisms is a dynamic process. Adhesion, growth, removal and re- attachment constitute a continuous process and thus dental plaque microbiom undergoes a constant reor- ganization. This naturally-constructed biofilm consor- tia of bacteria may reach a thickness of 300–500 cells on the surface of the teeth. The surface of mature bio- film may release individual cells, which can then form a biofilm on other sites, teeth, tooth surfaces, gingival sulcus or transform into the planktonic form. In this manner biofilm constitutes a subsisting source, con- tinuously sowing bacteria into the body fluids and to the environment.
Bacteria living in the biofilm form usually do not give up to the defense mechanisms activated by the immune system, and antibiotic action is also limited (Hojo et al., 2009; Roberts and Mullany, 2010). Only occasionally do antibiotics or other antimicrobial meas- ures manage to penetrate the sticky polysaccharide sub- stance surrounding the biofilm. The variety of environ- mental conditions and the diversity of bacteria species in biofilms provide a sufficient protection against antimicrobial agents (Marsh, 2012). Concentrations of antimicrobial agents used to destroy the biofilm com- ponents must be very high, even up to several hundred times greater than necessary for destruction of the free- living bacteria (Roberts and Mullany, 2010; Scheie and Petersen, 2004). The best method to fight dental biofilm is to prevent colonization of the tooth surface, which
constitutes a potential base for biofilm formation, and thereby inhibit its development. However, it is essen- tial to comply with the generally accepted principles of dental caries prophylaxis.