Chapter 1: Introduction - shodhganga.inflibnet.ac.inshodhganga.inflibnet.ac.in/bitstream/10603/72540/8/06.chapter 1.pdf · Chapter 1: Introduction ... his homemade microscope (Kuramitsu

Chapter 1: Introduction


School of Science, SVKM’s NMIMS (deemed-to-be) University Page 1

1.1 Biofilms

John Donne (1572–1631) 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, co-

operating 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



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 1014 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 (6.75-7.25) 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



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 site-

specific 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 35–36°C

Oxygen 0–21%

Oxygen is abundant at mucosal surfaces Gradients exist in dental plaque enabling obligate anaerobes to grow.

Redox potential

(Eh)

+ 200 to < - 200 mV

Gradients exist within plaque; lowest value recorded in gingival crevice.

pH 6.75-7.25 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.



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.



Table 1.2: Bacterial genera found in the oral cavity (Marsh and Martin, 1999)

Gram positive Gram negative

Cocci

Abiotrophia

Enterococcus

Peptostreptococcus

Streptococcus

Staphylococcus

Stomatococcus

Moraxella

Neisseria

Veillonella

Rods

Actinomyces

Bifidobacterium

Corynebacterium

Eubacterium

Lactobacillus

Propionibacterium

Pseudoramibacter

Rothia

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



Table 1.3: Distinct microbial habitats within a healthy mouth (Marsh, 2000a)

Habitat Predominant

microbial groups Comments

Lips, palate,

cheek

Streptococcus spp.

Neisseria,

Veillonella

Desquamation restricts biomass

Surfaces have distinct cell types

Candida act as opportunistic pathogens

Staphylococcus may be present

Tongue

Streptococcus spp.

Actinomyces

Veillonella

Obligate anaerobes

Simonsiella

Highly papillated surface-reservoir for

anaerobes

Teeth

Streptococcus spp.

Actinomyces

Veillonella,

Eubacterium

Obligate anaerobes

Spirochaetes

Haemophili

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 pro-

inflammatory 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).



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 (1632-1723) 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



(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 Mechanism of Action

Open architecture Presence of channels and voids

Protection from host

defences Extracellular polymers form a functional matrix

Enhanced resistance to

antimicrobial agents

Increased resistance to antibiotic agents and

chlorhexidine

Neutralization of inhibitors Beta-lactamase production by bacteria to protect

sensitive neighbouring bacteria

Gene transfer Drug-resistant genes and increased ability to take

up DNA

Novel gene expression Synthesis of novel proteins

Coordinated gene responses Production of cell-cell signalling molecules

(competence-stimulating peptide, autoinducer-2)

Spatial and Environmental

Heterogeneity

pH and oxygen concentration gradients and co-

adhesion

Broad Habitat Range Obligate anaerobes in an aerobic environment

Pathogenic Synergism Enhanced virulence and resistance to stress

Efficient Metabolism Catabolism of complex macromolecules by

bacterial community working in concert



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.

1.4.1 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).



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)



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

adhesion Saliva-coated tooth

GCF-coated tooth

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

Carbohydrates

GCF

Desquamated epithelium

pH Neutral/ Acidic Neutral

Oxygen concentration High/ Low Low

Metabolic property

of

microbial ecosystem

Saccharolytic Asaccharolytic/ Proteolytic

Dominant bacterial

species

Streptococcus sanguinis

Streptococcus mutans

Streptococcus mitis

Streptococcus salivarius

Lactobacillus

Aggregatibacter

actinomycetemcomitans

Tannerella forsythia

Campylobacter spp.

Capnocytophoga spp.

Eikenella corrodens

Fusobacterium nucleatum

Porphyromonas gingivalis

Prevotella intermedia

Treponema denticola



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).

1.4.2 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 pellicle-

coated 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



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



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 104

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).



Figure 1.2: Diagrammatic representation depicting pattern of biofilm

development in dental plaque (Rickard et al., 2003)



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).

1.4.4 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



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). 1.4.5 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 cell-

cell 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



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.

1.4.6 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



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.



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)



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.



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)



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).



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)



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



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:

1.6.1 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.



• Epidemiological data have suggested that mechanical oral self care does not

achieve its theoretical potential for controlling bacterial plaque accumulation

and gingival disease.

1.6.2 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 Mode of action

Penicillins Inhibition of peptidoglycan synthesis

Tetracylines Inhibition of amino acid and protein synthesis

Alterations in cytoplasmic membrane leading to leakage of cell

contents

Macrolides Inhibition of protein synthesis

Metronidazole Formation of redox intermediate intracellular metabolites that

target RNA, DNA and cellular proteins



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


Precipitation of cytoplasm

contents

Quaternary

ammonium compounds

Cetylpyridinium chloride

(CPC)

Domiphen bromide

(DB)

Benzethonium chloride

(BC)


Alteration of cytoplasm

contents

Halogens Fluoride, Iodine

Bacterial enzyme inhibition

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



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).

1.6.3 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).



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 IgA-

inductive 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.



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).

1.6.4 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,



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.

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