Chapter 1: Introduction

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2 1.1 Biofilms John Donne ( ) famously wrote No man is an island, entire of itself ; words that summarise an absolute truth of humanity- it is impossible to thrive and flourish in isolation. An idea that we, as humans, still do not completely understand has been accepted and incorporated since times immemorial into the lives of the most seemingly primitive creatures - Microorganisms. For most of the history of microbiology, microorganisms have primarily been characterized as planktonic, freely suspended cells and described on the basis of their growth characteristics in nutritionally rich culture media (Donlan, 2002). The extent to which microbial growth and development occurred on surfaces, as complex communities had not been clearly fathomed (Hall-Stoodley et al., 2004). It was in the 1970s that scientists began to appreciate that in most natural environments, majority of bacterial biomass exist in the form of surface-associated microbial communities (Costerton et al., 1999). Such a population of surface-associated well organised, cooperating communities of microorganisms then came to be referred to as a Biofilm, a term coined by Bill Costerton in 1978 (Kolter, 2010; Kokare et al., 2009; Chandki et al., 2011). The widespread recognition that biofilms possessed the ability to impact a plethora of varied environments from water pipes to catheters and stents of patients led to a curiosity about molecular mechanisms underlying the formation and maintenance of these communities (Costerton et al., 1999). This interest triggered the development of various imaging techniques and experimental models that have now elucidated that biofilms are not simply passive assemblages of cells that are stuck to surfaces, but are structurally and dynamically complex biological systems (Hall- Stoodley et al., 2004). In 2002, Donlan and Costerton offered the most salient description of a biofilm by describing it as a microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other, are embedded in a matrix of extracellular polymeric substances that they have produced, and exhibit an altered phenotype with respect to growth rate and gene transcription (Donlan and Costerton, 2002). The biofilm mode of existence has developed as a survival strategy where in the cooperative communal nature of a microbial community provides advantages to the participating microorganisms. These advantages include broader habitat range for growth, an enhanced resistance to School of Science, SVKM s NMIMS (deemed-to-be) University Page 1

3 antimicrobial agents and host defence and an enhanced virulence (Donlan and Costerton, 2002). 1.2 Mouth as a microbial habitat It has been estimated that the human body is made up of cells of which only 10% are mammalian while the rest are the microorganisms that make up the resident microflora of the host (Marsh, 2000a; Avila et al., 2009). The composition of this microflora varies at distinct habitats like urogentital tract, human gut, skin, etc but is relatively consistent over time at each individual site among individuals (Marsh, 2000a). The oral cavity is one such site of the body whose distinct resident microflora has been of great interest due to its vast diversity (Bowden and Hamilton, 1998). The following unique features of the mouth make it a unique microbial habitat: 1. Within the oral cavity, mucosal surfaces like lips, cheek and palate are subject to continuous shedding of cells or desquamation which impedes accumulation of biofilms. In contrast, teeth provide hard non-shedding surfaces that allow accumulation of higher quantities of biomass. Such differences in the type of subtratum and the environmental factors they are exposed to, leads to local variations in microbial composition of biofilms at different locations within the oral cavity (Marsh, 2000a; Liljemark and Bloomquist, 1996). 2. The constant flushing of saliva within the mouth has a substantial effect on the oral microflora. Saliva has a ph range ( ) that encourages the growth of many microorganisms. Components of saliva influence the development of biofilms by the following mechanisms: (Marsh, 2000a; Scannapieco, 1994) a. Aggregating microbes to facilitate their clearance from the mouth b. Adsorbing to teeth surface to form an acquired pellicle to which microorganisms can attach c. Serving as a primary source of nutrients d. Mediating microbial inhibition or killing 3. In addition to saliva, the gingival crevicular fluid (GCF), a plasma derived fluid that flows through the junctional epithelium, provides microbes in the gingival crevice School of Science, SVKM s NMIMS (deemed-to-be) University Page 2

4 with nutrients and carries host immune components that play an important role in regulating the microflora therein (Marsh, 2000a). The oral cavity is not a homogenous environment. It is characterised by differences among sites in key ecological factors like adhesion ligands, ph, nutrients, redox potential, oxygen and temperature. To persist in the oral cavity, bacteria must be able to tolerate rapid and substantial environmental fluctuations (Lemos et al., 2005). Different sites of the mouth like lips, palate, cheek, tongue and the different teeth surfaces provide distinct habitats, thereby resulting in the development of distinct sitespecific microbial communities. The properties of the habitat influence the type and number of species colonising it. Factors affecting the growth of microorganisms in the healthy oral cavity are enlisted in Table 1.1. Table 1.1: Key environmental factors affecting the growth of microorganisms in the healthy oral cavity (Marsh, 2000b) Factor Range Comment Temperature C Oxygen is abundant at mucosal surfaces Oxygen 0 21% Redox potential (Eh) to < mv ph Gradients exist in dental plaque enabling obligate anaerobes to grow. Gradients exist within plaque; lowest value recorded in gingival crevice. Plaque ph falls during dietary sugar metabolism Sub-gingival plaque ph rises during inflammation. Nutrients Endogenous Exogenous Peptides, proteins and glycoproteins in saliva and gingival crevicular fluid. Dietary sugars facilitate selection of acidogenic and acid-tolerating species in plaque causing a reduction in plaque ph and demineralisation of enamel. School of Science, SVKM s NMIMS (deemed-to-be) University Page 3

5 Once the early colonisers form their niche, they may modify the surrounding environment, making it suitable for other species to colonize. Thus, a bidirectional relation exists between the habitat and the microbial community (Takashi, 2005). 1.3 Oral Microflora The oral microbial community is diverse. Oral microbes are predominantly bacteria but fungi, viruses, mycoplasmas and even protozoa (Marsh, 2000a) and archaea (Kulik et al., 2001) can also be found. Previous studies utilizing culture-dependent and culture independent molecular techniques have estimated the diversity within the oral cavity to consist of over 700 species or phylotypes, however, not all of these have been identified (Kulik et al., 2001; Aas et al., 2005; Kreth et al., 2009). Numerous factors impede the identification of this vast number of species. The most challenging hurdles are as follows: Many of the species are non-culturable with available laboratory technologies. Genomic similarities do not allow for organismal determination based on short read lengths. In spite of these limitations, efforts are under way to identify and characterize the microorganisms with the largest representation within the communities of healthy mouths (Jenkinson and Lamont, 2005;Avila et al; 2009). The different bacterial genera commonly found in the oral cavity have been enlisted in Table 1.2. School of Science, SVKM s NMIMS (deemed-to-be) University Page 4

6 Table 1.2: Bacterial genera found in the oral cavity (Marsh and Martin, 1999) Cocci Rods Gram positive Abiotrophia Enterococcus Peptostreptococcus Streptococcus Staphylococcus Stomatococcus Actinomyces Bifidobacterium Corynebacterium Eubacterium Lactobacillus Propionibacterium Pseudoramibacter Rothia Gram negative Moraxella Neisseria Veillonella Actinobacillus Haemophilus Bacteroids Campylobacter Leptotrichia Cantonella Prophyromonas Capnocytophaga Prevotella Cantipedia Selenomonas Desulphovibro Simonsiella Desulphobacter Eikenella Fusobacterium Treponema Wolinella Some of these bacteria tend to predominate in certain distinct microbial habitats within the oral cavity as summarized in Table 1.3 School of Science, SVKM s NMIMS (deemed-to-be) University Page 5

7 Table 1.3: Distinct microbial habitats within a healthy mouth (Marsh, 2000a) Habitat Lips, palate, cheek Tongue Teeth Predominant microbial groups Streptococcus spp. Neisseria, Veillonella Streptococcus spp. Actinomyces Veillonella Obligate anaerobes Simonsiella Streptococcus spp. Actinomyces Veillonella, Eubacterium Obligate anaerobes Spirochaetes Haemophili Comments Desquamation restricts biomass Surfaces have distinct cell types Candida act as opportunistic pathogens Staphylococcus may be present Highly papillated surface-reservoir for anaerobes Non-shedding surfaces that promote biofilm formation Distinct surfaces for colonisation (fissures, approximal, gingival crevice) which support a characteristic flora due to their intrinsic biological properties. Teeth harbour the most diverse oral microbial communities In spite of being well equipped with an array of host defences provided by both the innate and adaptive arms of the immune system, all mucosal and dental surfaces of the mouth are naturally colonised by a diverse collection of micro-organisms. There is evidence to suggest that the resident bacteria have developed mechanisms to evade host defences to ensure longer survival in the oral cavity (Macotte and Lavoie, 1998). Studies show that there exists an active communication between some of the resident bacteria and mucosal cells that down regulates potentially damaging proinflammatory host responses to the normal oral microflora, while the host retains the ability to respond to genuine microbial insults (Cosseau et al., 2008; Marsh, 2012). School of Science, SVKM s NMIMS (deemed-to-be) University Page 6

8 The oral resident flora has developed mechanisms that permit a beneficial relationship with the host which include: Colonisation resistance : Resident oral bacteria prevent the establishment of exogenous microorganisms within the mouth. The natural oral microflora is better adapted at attachment to oral surfaces and more efficient at metabolising the available nutrients for growth. They can also produce inhibitory factors and create hostile environments that restrict colonisation by potential microbial invaders (Marsh, 1992; Lemos et al., 2005; Marsh and Percival, 2006). Contributions to gastrointestinal and cardiovascular health: Recent findings suggest that the resident oral bacteria contribute to the maintenance of healthy gastrointestinal and cardiovascular systems via the metabolism of dietary nitrate. Approximately 25% of ingested nitrate is secreted in saliva where some oral resident bacteria reduce nitrate to nitrite. Nitrite can affect a number of key physiological processes including the regulation of blood flow, blood pressure, gastric integrity and tissue protection against ischemic injury (Lundberg, 2009; Marsh 2012). Therefore, it is imperative, antimicrobial agents used to treat oral health issues are used judiciously with respect to doses and duration of treatments, to ensure that beneficial resident microflora of the mouth are not indiscriminately killed. 1.4 Dental plaque The diverse oral microflora were initially thought to be free living but were later understood to be thriving in the form of one of the most complex surface associated microbial communities of the human body - Dental plaque. In 1683, The Dutch naturalist Anton van Leeuwenhoek ( ) was the first to open the world s eyes to the world of microorganisms by examining samples of his own dental plaque using his homemade microscope (Kuramitsu et al., 2007; Huang et al., 2011). Clinically, dental plaque is the soft, tenacious deposit that forms on tooth surfaces and which is not readily removed by rinsing with water (Bowen, 1972). Microbiologically, Marsh School of Science, SVKM s NMIMS (deemed-to-be) University Page 7

9 (2004) has saliently defined plaque as the diverse community of microorganisms found on the tooth surface as a biofilm, embedded in an extracellular matrix of polymers of host and microbial origin. From a microbial physiology aspect, oral microbial communities are classical examples of biofilms. Just as initially proposed by Costerton et al., (1995) the behaviours displayed by oral microbial organisms grown in liquid culture are very different from those of the same organisms grown on a solid surface or within a community such as dental plaque (Davey and O Toole, 2000; Islam et al., 2007). Dental plaque is characterised by unique properties enlisted in Table 1.4 Table 1.4: Properties of dental biofilms (Marsh, 2004; Garcia-Godoy and Hicks, 2008) Property Open architecture Protection from host defences Enhanced resistance to antimicrobial agents Neutralization of inhibitors Gene transfer Novel gene expression Coordinated gene responses Spatial and Environmental Heterogeneity Broad Habitat Range Pathogenic Synergism Efficient Metabolism Mechanism of Action Presence of channels and voids Extracellular polymers form a functional matrix Increased resistance to antibiotic agents and chlorhexidine Beta-lactamase production by bacteria to protect sensitive neighbouring bacteria Drug-resistant genes and increased ability to take up DNA Synthesis of novel proteins Production of cell-cell signalling molecules (competence-stimulating peptide, autoinducer-2) ph and oxygen concentration gradients and coadhesion Obligate anaerobes in an aerobic environment Enhanced virulence and resistance to stress Catabolism of complex macromolecules by bacterial community working in concert School of Science, SVKM s NMIMS (deemed-to-be) University Page 8

10 As a result of these properties, dental plaque possesses a highly resilient nature. In the presence of good dental hygiene practices, it is destroyed on a daily basis yet quickly and repeatedly re-establishes itself (Palmer et al., 2006). Of all oral microbial ecosystems, dental plaque has been the major focus of oral microbiological research probably because of its characteristic features as a complex polymicrobial biofilm and its association with dental caries and periodontal diseases Microbial composition of dental plaque Based on its location and its relationship with the gingival margin, plaque can be broadly classified into two categories: Supragingival plaque: Plaque situated superior to the gingival margin Subgingival plaque: Plaque situated inferior to the gingival margin. It may or may not be attached to the epithelium or tooth surface (Reddy, 2008). School of Science, SVKM s NMIMS (deemed-to-be) University Page 9

11 Figure 1.1: Diagrammatic representation depicting the classification of plaque on the basis of their location and relationship with gingival margin (Marsh and Martin, 1999) School of Science, SVKM s NMIMS (deemed-to-be) University Page 10

12 Microbial constituents vary among the two types of plaque due to differences in their biological properties. The comparisons between supragingival plaque and subginigival plaque have been summarized in Table 1.5 Table 1.5: Comparison between supragingival plaque and subgingival plaque (Takahashi, 2005; Aas et al., 2005; Kuramitsu et al., 2007; Reddy, 2008) Supragingival Plaque Subgingival Plaque Surface for microbial GCF-coated tooth Saliva-coated tooth adhesion GCF-coated epithelium Matrix 50% Matrix Little or no matrix Flora Mostly Gram positive Mostly Gram negative Motile bacteria Few Common Anaerobic/ Aerobic Aerobic unless thick Highly anaerobic Nutrition Saliva GCF Carbohydrates Desquamated epithelium ph Neutral/ Acidic Neutral Oxygen concentration High/ Low Low Metabolic property of microbial ecosystem Saccharolytic Asaccharolytic/ Proteolytic Aggregatibacter actinomycetemcomitans Tannerella forsythia Streptococcus sanguinis Campylobacter spp. Streptococcus mutans Dominant bacterial Capnocytophoga spp. Streptococcus mitis species Eikenella corrodens Streptococcus salivarius Fusobacterium nucleatum Lactobacillus Porphyromonas gingivalis Prevotella intermedia Treponema denticola School of Science, SVKM s NMIMS (deemed-to-be) University Page 11

13 In the cases of both, supragingival and subgingival plaque, the microbial communities on teeth and gingival tissues can accumulate high concentrations of bacterial metabolites. These include fatty acid end products, ammonia, hydrogen peroxide, oxidants and carbon dioxide within their local environments, which further influence the bacterial species within the microbial community, as well as the host (Kuramitsu et al., 2007) Formation of dental plaque Formation of dental plaque is a dynamic process involving continuous attachment, growth, detachment and reattachment of oral microorganisms, but can be divided into several stages. As delineated by Marsh (2000a), these stages are: a) Acquired enamel (or dental) pellicle formation: Within minutes of tooth eruption or professional cleaning of tooth, molecules of salivary and microbial origin selectively adsorb to the tooth surface. Li et al. (2004a) have identified different molecules of the acquired pellicle which include albumin, amylase, carbonic anhydrase II, secretory Immunoglobulin A (siga), Immunoglobulin G (IgG), Immunoglobulin M (IgM), lactoferrin, lysozyme, proline-rich proteins (PRP), statherin, histatin 1 and mucous glycoprotein. Some of these like PRP, amylase, mucins and statherin function as receptors for bacterial adhesins (Scannapeico et al., 1994). Glucosyltransferases can also be found in the active form in the enamel pellicle where they catalyze the synthesis of glucan that serves as a ligand for glucan binding proteins on Streptococci. b) Passive transport of microorganisms to the pellicle: The primary colonizers of the biofilm attach to the receptors of the pellicle. They are mostly Gram positive cocci and rods and filaments and a small number of gram-negative cocci (Li et al., 2004b; Sbordone and Bortolaia 2003). c) Reversible bacterial adhesion: This results from long-range (10-20 nm) physico-chemical interactions between the bacterial surface and the pelliclecoated tooth. The interplay of repulsive electrostatic forces (both surfaces are negatively charged) and Van der Waals attraction result in a weak net attraction. This can be augmented by cation bridging and hydrophobic School of Science, SVKM s NMIMS (deemed-to-be) University Page 12

14 interactions or further weakened by hydration forces (Jenkinson and Lamont, 1997). As soon as the pioneer bacteria attach to the pellicle, they begin to excrete extracellular polysaccharides (EPS), which helps the bacteria stay bound together and attach to the pellicle (Huang et al., 2011). d) Irreversible bacterial adhesion: The reversible adhesion, discussed above, is followed by a much stronger, irreversible attachment. Short-range (<1 nm) stronger, specific stereochemical interactions involving bacterial surface components (adhesins) and cognate receptors on the pellicle begin to occur. Lectin-like adhesion, which involves binding of carbohydrate (glycosidic) receptors by bacterial polypeptide adhesins are a commonly observed interaction (Jenkinson and Lamont, 1997). e) Late colonization: This stage is characterised by two crucial physical interactions - co-aggregation and co-adhesion. Co-aggregation is the cell-cell recognition between genetically distinct bacteria in a planktonic suspension, whereas co-adhesion refers to the recognition between a planktonic cell and a surface-attached cell (Foster and Kolenbrander, 2004). These interactions involve adhesin-receptor interactions between approaching bacteria and already attached early colonizers, increasing the diversity of the biofilm. The cohesion process results in characteristic morphological structures such as corncobs and test-tube brushes (Seniviratne et al., 2011) and may facilitate metabolic interactions. f) Multiplication of the attached microorganisms and confluent growth: The bacterial cells continue to divide until a three-dimensional mixed-culture biofilm forms that is spatially and functionally organized. Metabolism of microorganisms modifies the local environment and creates gradients in key parameters (oxygen, redox potential, ph, nutrients, and metabolic end products) creating micro-environments that enable coexistence and growth of diverse bacteria with conflicting needs (Marsh and Bradshaw, 1997). Synthesis of extracellular polysaccharides also takes place, resulting in the formation of intercellular matrix. The spatial arrangement of the cells and School of Science, SVKM s NMIMS (deemed-to-be) University Page 13

15 intercellular matrix will determine the architecture of the biofilm (Marsh, 2004). g) Active detachment of bacteria: The detachment of bacteria from biofilms is essential to allow colonization of new habitats. Detachment may occur in multiple ways: detachment as single cells in a continuous predictable fashion (erosion), sporadic detachment of large groups of cells (sloughing) or as an intermediate process whereby large pieces of biofilm consisting of about 10 4 cells are shed from the biofilm in a predictable manner (Thomas and Naikishi, 2006). Bacteria within the biofilm can produce enzymes that break specific adhesins, enabling cells to detach into saliva and probably colonize elsewhere (Marsh, 2004). School of Science, SVKM s NMIMS (deemed-to-be) University Page 14

16 Figure 1.2: Diagrammatic representation depicting pattern of biofilm development in dental plaque (Rickard et al., 2003) School of Science, SVKM s NMIMS (deemed-to-be) University Page 15

17 1.4.3 Structure of dental plaque In order to gain a better understanding, conventional techniques like light and electron microscopy have been used to visualise biofilms. However, biofilm specimen preparation (dehydration, fixation, and staining) may result in artifacts, shrinkage and distortion. With the advent of confocal laser scanning microscopy (CLSM), it has become possible to observe living biofilms while they grow and metabolize and is aided with information from modern staining methods. In addition to fluorescent staining methods, it is possible to use dyes that bind selectively to either dead or live bacteria allowing the investigator to understand the distribution of viable and non viable cells within the biofilm (ten Cate, 2006). The current concept of biofilm structure is based on the pioneering studies done by the Bozeman Montana Center for Biofilm Engineering (Costerton et al., 1995). They showed the biofilm as a thin basal layer on the substratum, in contact and occasionally penetrating the acquired pellicle, and with columnar, mushroom-shaped multi-bacterial extensions into the lumen of the solution, separated by regions ( channels ) seemingly empty or filled with extracellular polysaccharide (EPS) Interactions between resident flora within dental plaque Microorganisms within the dental biofilm are spatially arranged in close proximity to each other, which facilitates interactions among them. These interactions are crucial to maintenance of stability of the biofilm. The interactions include: (Kuramitsu et al., 2007) Competition between bacteria for nutrients Synergistic interactions which may stimulate the growth or survival of one or more residents Production of an antagonist by one resident which inhibits the growth of another Neutralization of a virulence factor produced by one organism by another resident Interference in the growth-dependent signalling mechanisms of one organism by another School of Science, SVKM s NMIMS (deemed-to-be) University Page 16

18 In addition to these interactions, bacteria additionally communicate with each other through a phenomenon called Quorum sensing. Quorum sensing is a process of chemical communication among bacteria, which is defined as a gene regulation in response to cell density, which influences various functions, like virulence, acid tolerance and biofilm formation (Hojo et al., 2009). Quorum sensing signalling serves intra-species communication and is often highly specific. The quorum-sensing systems found in oral bacteria include signal molecules called autoinducers like AI-2 and streptococcal competence stimulating peptide (CSP) (Olsen, 2006). The luxs gene expressing AI-2 is conserved among many species of bacteria, including Streptococcus mutans, Streptococcus gordonii, Streptococcus oralis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and other oral microorganisms (Huang et al., 2011). AI-2 produced is detected by a large number of diverse bacteria, hence it is called a universal language used for cross-species communication (Yung-Hua and Xiaolin, 2012). The competence stimulating peptide (CSP) is known to induce alarmones- intracellular signal molecules that are produced due to harsh environmental factors. These alarmones can convey sophisticated messages in a population including the induction of altruistic cellular suicide under stressful conditions (Huang et al., 2011). These quorum sensing systems are indispensible in mature biofilms characterised by high cell density and the presence of a varied array of species. Efforts are under way to develop therapies that interference with these quorum sensing mechanisms to deal with oral biofilm related ailments and diseases (Olsen, 2006) Microbial homeostasis Once resident plaque microflora develop at a healthy site within the mouth, its species composition is characterized by a degree of stability or balance among the component species, in spite of regular minor environmental stresses caused due to dietary components, oral hygiene, host defences, diurnal changes in saliva flow, etc. (Marsh, 2006). This stability, termed as Microbial homeostasis, develops not due to indifference among the component species but rather results from a dynamic balance of microbial interactions, including synergism and antagonism as well as subtle cellcell signalling (Marsh, 1992). Mature dental plaque biofilm acts as a community or a unit rather than as a sum of the properties of individual bacterial members (Seneviratne et al., 2011). In a state of Microbial homeostasis, dental plaque does not School of Science, SVKM s NMIMS (deemed-to-be) University Page 17

19 have the ability to cause harm but in fact promotes good oral health, since they share a beneficial relationship with the host as previously described Perturbations to dental plaque Plaque preferentially accumulates at stagnant or retentive sites unless it is removed by diligent oral hygiene. As the plaque mass increases, the buffering and antimicrobial properties of saliva are less able to penetrate plaque and protect the enamel and gingival tissues. Insufficient oral hygiene, aging processes, genetic factors, changes in dietary intake as well as changes in immunity of host can encourage the plaque microbiota into a diseases associated state. There is a shift in the balance of the predominant bacteria in plaque away from those associated with health and microbial homeostasis breaks down (Garcia-Godoy and Hicks, 2008). Current hypotheses to explain the role of plaque bacteria in the etiology of diseases The fact that periodontitis and dental caries, the most prevalent diseases in humans, are dental plaque-mediated diseases is very well established (Sbordone and Bortolaia, 2003). However, despite 120 years of active research, there has been on-going controversy as to which bacteria within the biofilm are involved in causation of these diseases. Two hypotheses have been proposed in this respect: Specific plaque hypothesis The "Specific Plaque Hypothesis" proposed by Loesche (1976) stated that, out of the diverse collection of organisms comprising the resident plaque microflora, only a few species are actively involved in disease. It therefore entails that treatment involving an antimicrobial component should be aimed at diagnosis and then elimination of causative organisms (Marsh, 2006). Non-Specific Plaque Hypothesis This hypothesis proposed by Theilade (1986) purports that caries are an outcome of the overall activity of the multiple species present within the heterogeneous plaque microflora and not a specific organism. In keeping with this hypothesis, the flora would need to be suppressed either continuously or periodically using agents that School of Science, SVKM s NMIMS (deemed-to-be) University Page 18

20 ensure maximum lethality of flora. This could lead to over usage of broad-spectrum agents or the combination of agents (Loesche, 1999; Marsh, 2006). As an alternative to the two main schools of thought mentioned above, a hypothesis has been proposed that reconciles the key elements of the two earlier hypotheses. Ecological Plaque Hypothesis A direct relationship exists between the environment and the balance and behaviour of resident plaque microflora. A shift in the homeostatic balance of the resident microflora due to a change in local environmental conditions can lead to the selection or enrichment of previously minor components of the oral biofilm thereby causing diseases. This concept led to the proposal of a modified hypothesis by Marsh (1991, 1994) called the "Ecological Plaque Hypothesis". Common factors that disrupt homeostasis include frequent exposure to nutrients like fermentable carbohydrates, consistently low ph within the oral cavity and low availability of oxygen. Manipulation of physiological factors that drive changes in the oral environment could lead to some degree of control over the composition of the plaque community, and lead to the identification of new physiological strategies to maintain the beneficial properties of the biofilm (Marsh and Bradshaw, 1997). 1.5 Plaque mediated diseases Much attention has been focused on the identification of bacteria which cause oral diseases. It is of equal importance that bacteria associated with health also be identified so that a microbiological goal for therapy can be established (Aas et al, 2005; Filoche et al., 2010). However, it is difficult to define a normal flora given the prevalence and complexity of these diseases, although recent research has indicated that there is a distinctive bacterial flora in a healthy oral cavity which is different from that of diseased oral cavities (Filoche et al., 2010). Figure 1.3 depicts transitions in the composition of predominant plaque microflora in health and disease. School of Science, SVKM s NMIMS (deemed-to-be) University Page 19

21 Figure 1.3: Transitions in the composition of predominant plaque microflora in health and disease (Marsh, 1992) Figure 1.4: Accumulation of dental plaque beyond levels compatible with oral health (Walsh, 2006) School of Science, SVKM s NMIMS (deemed-to-be) University Page 20

22 1.5.1 Plaque mediated dental caries Dental caries is a transmissible bacterial disease process caused by acids from bacterial metabolism diffusing into enamel and dentine and dissolving the mineral (Featherstone, 2008). Dental caries was once thought to be a simple disease with the mutans streptococci group being attributed as the sole etiological agent. Caries research was dominated by treatments targeted at these species (Loeche et al., 1975; Minahi and Loesche, 1977; Nishikawara et al., 2006; Filoche et al., 2010). The group Mutans streptococci includes Streptococcus mutans and Streptococcus sobrinus. Their key virulence factors include synthesis of water insoluble glucans from sucrose, adherence, acidogenicity and aciduricity (Nishikawara et al., 2006). However, it has now been established that the presence of high numbers of mutans streptococci alone is insufficient for the development of caries (Seneviratne et al., 2011). The direct cause of dental caries is cariogenic plaque which develops when normally low populations of acidogenic and aciduric bacterial species, previously in balance with the oral environment, increase following high frequency carbohydrate exposure (Loesche, 1986; Filoche et al., 2010). Cariogenic plaques (CP) harbour high levels of S. mutans, S. mitis, Bifidobacterium spp, low levels of Streptococcus sanguinis and moderate to high levels of Actinomyces species. Levels of lactobacilli steadily increase as the lesion progresses (Minahi and Loesche, 1977; Seneviratne et al., 2011; Filoche et al., 2010). The metabolism of fermentable carbohydrates by these microbiota result in the acidification of plaque (ph<5). The acid induced demineralization of the enamel and dentin cause cavitation in the teeth. School of Science, SVKM s NMIMS (deemed-to-be) University Page 21

23 Figure 1.5: Use of ecological plaque hypothesis to explain the incidence and prevention of caries. (Marsh and Bradshaw, 1997) Figure 1.6: Severely damaged teeth due to carious lesions and cavitations. (Szczawinska-Poplonyk et al., 2011) School of Science, SVKM s NMIMS (deemed-to-be) University Page 22

24 1.5.2 Plaque mediated periodontal disease Periodontal disease reflects a cellular inflammatory response of the gingival and surrounding connective tissue to the bacterial accumulations on teeth (Filoche et al., 2010; Kornman, 2008). These inflammatory responses are clinically classified as gingivitis and periodontitis. Apart from the non-exfoliating surface of teeth being an ideal substratum for plaque formation, they are also a link to the deep periodontal space, offering microorganisms an easy route of entry into dentinal tubules and enamel enamel fissures. These regions are easily colonized by microbes, but difficult to reach for the host defence mechanisms (Sbordone and Bortolaia, 2003). The accumulation of plaque around the gingival margin leads to inflammation or infection of the gums called gingivitis. This inflammatory response leads to an increased flow of GCF which, in addition to introducing components of the host response, also provides a novel source of potential nutrients for the microflora (Marsh and Bradshaw, 1997). Additionally release of GCF leads to a reduction in the redox potential (Eh) which is preferred by periodontopathic bacteria like Porphyromonas gingivalis allowing them to overgrow other organisms in the dental plaque (Marsh and Bradshaw, 1997; Seneviratne et al., 2011). When left untreated, the infection and inflammation spread from the gingival to the ligaments and bone that support the teeth leading to periodontitis. Loss of support causes the teeth to become loose and eventually fall out. Periodontitis is the primary cause of tooth loss in adults (Savage, 2009). Periodontal disease is characterised by a significant increase in the prevalence of obligate anaerobic Gram negative bacilli, especially proteolytic species (Marsh and Bradshaw, 1997) like Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia, Aggregatibacter actinomycetecomintans and Fusobacterium nucleatum (Marsh 1992, Filoche et al., 2010). School of Science, SVKM s NMIMS (deemed-to-be) University Page 23

25 Figure 1.7: Use of ecological plaque hypothesis to explain the incidence and prevention of periodontal diseases (Marsh and Bradshaw, 1997) Figure 1.8: Accumulation of plaque around the gingival margin leading to gingivitis and periodontitis (Preshaw et al., 2012) School of Science, SVKM s NMIMS (deemed-to-be) University Page 24

26 1.5.3 Plaque and systemic health The periodontium responds to dental plaque by the process of inflammation. Plaque releases a variety of biologically active products, such as bacterial lipopolysaccharides, chemotactic peptides, protein toxins, and organic acids. These molecules stimulate the host to produce a variety of responses, among them the production and release of potent agents known as cytokines (Panagakos and Scannapieco, 2011). Considering the chronic nature of these diseases and the exuberant local and systematic host response to the microbial assault, it is reasonable to hypothesize that these infections may influence systemic health and disease (Scannapieco, 1998; Panagakos and Scannapieco, 2011). A number of epidemiological studies have suggested that an infected oral cavity can act as the site of origin for dissemination of pathogenic organisms to distant body sites, especially in immuno-compromised hosts or hosts undergoing immunosuppressive treatments (Li et al, 2000). In 1891, Miller proposed the theory of focal infection which stated that foci of sepsis were responsible for the initiation and progression of a variety of inflammatory diseases. In spite of this theory being largely unsubstantiated, there has been a renewed interest in investigating relationships between systemic and oral health (Barnet, 2006). With normal oral health and dental care, only small numbers of mostly facultative bacterial species gain access to the bloodstream. In the absence of oral hygiene, the numbers of bacteria colonizing the teeth, especially supragingivally, could increase 2- to 10-fold (Li et al., 2000). Under normal circumstances, the host possesses barrier systems that work together to inhibit and eliminate penetrating dental plaque bacteria. However, as a result of advanced periodontitis, a thin, highly permeable, and frequently ulcerated pocket epithelium is the only barrier between the bacterial biofilms and the underlying connective tissues. The strands of the pocket epithelium are easily broached, allowing large doses of bacterial toxins and other products access to the tooth supporting connective tissues and blood vessels thereby introducing bacteria into the bloodstream, leading to an increase in the prevalence and magnitude of bacteremia (Li et al., 2000; Jin et al., 2003). Once the bacteria and toxins gain access to the bloodstream, they may further lead to cardiovascular diseases, infective endocarditis, bacterial pneumonia and diabetes School of Science, SVKM s NMIMS (deemed-to-be) University Page 25

27 mellitus. Periodontal disease is also linked to adverse pregnancy outcomes (Li et al., 2000; Jin et al., 2003; Panagakos and Scannapieco, 2011). 1.6 Approaches to control of dental plaque Considering the impact of dental plaque on oral and systemic health, there is a need to devise therapies dedicated to its control. The various approaches towards the control of dental plaque have been discussed below: Mechanical control of dental plaque Mechanical plaque control still remains the basic element for the prevention and control of plaque build up (Vacaru et al., 2003). Toothbrushing is the most commonly used measure for plaque control in daily oral self care (Creeth et al., 2009, Imai et al., 2012). However, in spite of regular brushing, it is possible that plaque may still remain as tooth brushes are unable to penetrate intact interdental areas. Power toothbrushes can serve as an alternative to manual toothbrush by delivering more enhanced plaque removal due to the mode of action, better compliance and/or, by correcting poor brushing technique (Sharma et al., 2012). Use of tongue scrapers, dental floss, interdental tooth brushes and tooth picks are recommended as adjuncts to toothbrushing (manual or power) to achieve better results (Imai et al., 2012). A professional tooth cleaning every 6-8 weeks aids in periodic removal of plaque from all tooth surfaces using mechanically driven instruments. Regular dental visits are recommended to keep oral health in check. These visits generally include plaque evaluation, oral hygiene instructions, probing depth measurements, registration of bleeding on probing, scaling (plaque removal) if required and tooth polishing (Westfelt, 1996). Limitations in mechanical control: (Asadoorian, 2006; Bansal et al., 2012; Creeth et al., 2009; Teles and Teles, 2009; Imai et al., 2012) Results obtained from brushing are subject to a variety of factors like type of brush, duration and technique of brushing, manual dexterity of the user, etc. Only 2-10% of the population perform interdental cleaning on daily basis using floss or tooth picks. School of Science, SVKM s NMIMS (deemed-to-be) University Page 26

28 Epidemiological data have suggested that mechanical oral self care does not achieve its theoretical potential for controlling bacterial plaque accumulation and gingival disease Chemical control of dental plaque Studies indicate that self performed mechanical plaque removal is inefficient and leaves room for improvement. Efforts directed towards development of chemical agents of plaque control have provided a plethora of agents that maybe used as an adjunct to mechanical plaque control to reduce or prevent oral disease (Teles and Teles, 2009; Asadoorian, 2006). Five categories of agents for approaches have been considered: (Asadoorian, 2006; Mhaske et al., 2012) Antibiotics aimed at inhibition or killing of specific bacteria Broad spectrum antiseptics aimed at killing or preventing proliferation of all plaque organisms Single or combinations of enzymes that could modify plaque structure or activity Modifying agents that are non enzymatic which act as dispersing or denaturing agents that can alter structure or metabolic activity of plaque Agents that could affect bacterial attachment to pellicle surface Table 1.6: Commonly used antibiotics in oral care and their mode of action (Soares et al., 2012) Antibiotic Penicillins Tetracylines Macrolides Metronidazole Mode of action Inhibition of peptidoglycan synthesis Inhibition of amino acid and protein synthesis Alterations in cytoplasmic membrane leading to leakage of cell contents Inhibition of protein synthesis Formation of redox intermediate intracellular metabolites that target RNA, DNA and cellular proteins School of Science, SVKM s NMIMS (deemed-to-be) University Page 27

29 Table 1.7: Commonly used antiseptics in oral care and their mode of action (Marsh, 1992; Asadoorian, 2006; Ishiyama et al., 2012; Mhaske et al., 2012) Active agents Examples Mode of action Phenolic compounds Triclosan Cell wall disruption Induce leakage of cell contents Bacterial enzyme inhibition Bis-biguanides Chlorhexidine Cell wall disruption Precipitation of cytoplasm contents Cetylpyridinium chloride (CPC) Cell wall disruption Quaternary Domiphen bromide Alteration of cytoplasm ammonium compounds (DB) contents Benzethonium chloride (BC) Bacterial enzyme inhibition Halogens Fluoride, Iodine Interferes with acid production of acidogenic bacteria Oxygenating agents Peroxides Cytotoxicity due to generation of reactive oxygen species Limitations in chemical control Chlorhexidine, often referred to as the gold standard for oral care is known to cause side effects like staining of the tongue, teeth and restorations, perturbation of taste and also supragingival calculus (Marsh, 1992; Bansal et al, 2012). The dosage of a chemical agent is determined as per its laboratory evaluated Minimum Inhibitory Concentration (MIC) or Minimum Bactericidal Concentration (MBC). However within the oral cavity, a chemical agent may School of Science, SVKM s NMIMS (deemed-to-be) University Page 28

30 remain at MIC levels for a very short duration due to loss of agent via expectoration and constant swallowing (Marsh, 1992; Marsh 2012). Long term use of broad spectrum antimicrobials may lead to disruption of the natural balance of the oral microflora, colonization by exogenous organisms and development of microbial resistance (Marsh, 1992; Bansal et al., 2012) Biological control of dental plaque a) Probiotic therapy Instead of using chemical agents that may disrupt the homeostasis of the resident plaque flora, probiotic therapy is being explored as an alternative. Three main modes of action have been proposed to contribute to the effects of probiotics: (Sugano et al., 2012) Production of antimicrobial substances against pathogens Competitive exclusion mechanisms Modulation of host defence systems Studies suggest that Lactobacillus salivarius T12711 and Streptococcus salivarius K12 possess the potential to be used as non-cariogenic probiotics for maintaining a healthy ecosystem for the oral microflora, thereby preventing the colonization of periodontopathic bacteria (Burton et al., 2006; Islam et al., 2007; Sugano et al., 2012). Additionally, a new class of pathogen-selective molecules, called specifically (or selectively) targeted antimicrobial peptides (STAMPs) were developed to selectively kill Streptococcus mutans within multi-species dental plaque. Competence stimulating peptide (CSP) produced by S. mutans was selected as the STAMP targeting domain to ensure the targeted delivery of the antimicrobial peptide. This ensured the elimination of S. mutans without affecting closely related non cariogenic oral streptococci, indicating the potential of these molecules to be developed into probiotic antibiotics (Eckert et al., 2006). School of Science, SVKM s NMIMS (deemed-to-be) University Page 29

31 b) Vaccines Secretory IgA is the principal immune component of major and minor gland salivary secretions and thus would be considered to be the primary mediator of adaptive immunity in the salivary milieu apart from other immunoglobulins like IgG and IgM which are derived from the gingival crevicular fluid. In addition to this, gingival sulcus also contains various cellular components of the immune system like lymphocytes, macrophages and neutrophils (Gambhir et al., 2012). The mode of action of these antibodies is inhibition of the adherence and possibly metabolic activities of pathogenic bacteria of the oral cavity (Islam et al., 2007). With a view of developing vaccines, organisms crucial in the etiology of caries and periodontal diseases are designated as targets. The virulence factors contributing to the pathogenecity of these organisms are recognised as the key antigens for development of vaccines. The research focus is mainly on the incorporation of these antigens into mucosal immune systems and delivery with or without adjuvants to mucosal IgAinductive sites. P. gingivalis and S. mutans are being explored as targets for vaccine development due to their roles in periodontal diseases and caries respectively (Islam et al., 2007; Sugano et al., 2012). c) Replacement therapy Recombinant DNA technology had aided in the development of one of the most promising new approaches to maintaining homeostasis within the oral cavity- Replacement therapy. Genetic engineering is used to modify the wild type strain of a pathogen into an effector strain such that it is deficient of its virulence factors but posses excellent colonization potential. An effector strain should posses the following properties: (Hillman et al., 2000) It must not cause disease itself or otherwise predispose the host to other disease states by disrupting the ecosystem in which it resides. It must persistently colonize the host tissue at risk and thereby prevent colonization or outgrowth of the pathogen to levels necessary for it to exert its pathogenic potential. In situations where the pathogen is itself a member of the indigenous flora, an effector strain should aggressively displace the resident pathogen It should possess a high degree of genetic stability. School of Science, SVKM s NMIMS (deemed-to-be) University Page 30

32 When introduced into the oral cavity, the effector strain will colonize the niche, thereby preventing colonization and outgrowth of wild-type strain (Islam et al., 2007). Using this approach, a harmless strain is permanently implanted in the host s oral flora. Studies on S. mutans effector strains have shown promising results (Hillman et al., 2007) Herbal control of dental plaque Considering the limitations of synthetic agents on plaque control, efforts are underway to identify novel nature-based anti-plaque strategies. Medicinal plants have, since time immemorial, provided mankind with an array of uses in amelioration of diseases. Most developing countries still majorly rely on traditional medicine (Palombo, 2009; Bansal et al., 2012). Multiple studies have reported the use of plants in the form of crude extracts or as isolated phytochemicals in the treatment of oral diseases. Presence of phytochemicals like flavonoids, tannins, alkaloids and essential oils have been reported to be responsible for the potential of plant extracts in improving oral health (Ferrazzano et al., 2009; Palombo, 2009). Sanguinarine and essential oils like thymol, menthol and eucalyptol are already making their presence felt as potent oral care agents comparable to synthetic agents like Chlorhexidine (Marsh, 1992; Bansal et al., 2012; Asadoorian, 2006). Advantages of Herbal control: Herbal products have a larger public acceptance due its nature based approach and minimal side effects. There is no dearth in availability of medicinal plants in India thereby ensuring a sustainable supply of economic medicines. 1.7 Biofilm Models in Oral Care Research Anti-plaque agents, synthetic or natural, are evaluated primarily in the laboratory using conventional microbiological methods that allow the determination of MIC or MBC values which are usually quoted as the primary indicator of their potential efficacy (Marsh, 1992; Kinniment et al., 1996; Haraszthy et al., 2006). However, School of Science, SVKM s NMIMS (deemed-to-be) University Page 31

33 these agents may markedly vary in their efficacy when used in vivo. Reasons for this discrepancy include: (Wilson, 1996; Sbordone and Bortolaia, 2003) Biofilms within the mouth are polymicrobial and more resistant. The effectiveness of antimicrobial agents decreases with increasing age of the biofilm. Presence of extracellular polysaccharide matrix hinders penetration of the agents. Growth of microbiota within the biofilm is very slow, leading to slow uptake of the agent. Due to these factors, many oral care agents fail clinically in spite of promising results during laboratory evaluation. Therefore, it is now apparent that standardised tests such as determination of MIC are no longer appropriate on their own to fully characterise susceptibility of plaque to new therapeutics (Pratten and Ready, 2010). Additionally, in vivo experimental studies on natural dental plaque is inconvenient due to difficulties experienced during volunteer studies (Sissons, 1997). These difficulties have given incentive to the development of range of biofilm models (Sissons, 1997; Wimpenny, 1997; Pratten and Ready, 2010). These models may be designed and set up to mimic various characteristics of the oral cavity thereby allowing better understanding of the underlying mechanisms of biofilm formation and the measure that need to be taken for its control (Pratten and Ready, 2010). Prediction of in vivo plaque behaviour towards a therapeutic agent is made possible when evaluated within an experimental biofilm model (Sissons, 1997). Efforts have continually been directed towards the development of novel anti-plaque agents. However, at present, the need for side effect free anti-plaque agents as well as the resolution of issues regarding inadequacies in efficacy testing protocols are areas that have become the prime focus of oral care research. School of Science, SVKM s NMIMS (deemed-to-be) University Page 32