1.1 Dental Caries - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/13195/10/10_chapter...

Review of Literature

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1.1 Dental Caries

Dental caries is a localized and progressive decay of the teeth. Not only does it

cause many people to experience a great deal of pain, it leads to continuous

discomfort through inconvenient treatment. WHO’s report on the Global Problem of

Oral Diseases, notes that oral diseases such as dental caries (tooth decay),

periodontitis (gum disease) and oral and pharyngeal cancers are global health problem

in both the industrialized and the developing countries, especially among poorer

communities (Petersen, 2003). Dental caries is a major oral affliction in developing

countries, affecting 60-90% of the school children and the vast majority of adults. An

estimated five billion people worldwide have experienced dental caries (Petersen,

2003). Figure 1.1 highlights the dental caries experience among 12-year-old children

in the six WHO regions in the year 2000, based on the DMFT (Decayed, Missing and

Filled Teeth) Index, which measures the lifetime experience of dental caries

in permanent dentition. In developing countries like India, the rate of dental caries is

rising and since more than 80% of the world’s children live in these countries, dental

caries is considered to be a major public health problem (Cirino and Scantlebury,

1998).

1.2 How dental caries are caused?

Dental caries is a multifactoral infectious disease in which the active agent or

agents are members of the indigenous oral flora (Figure 1.2). Oral cavity harbors a

rich and diverse microbial flora because of its ideal humidity and temperature, the

frequent passage through it of most nutrients needed by many microbial species and

presence of several ecological niche. The presence of a myriad of microorganisms is a

natural part of proper oral health. Oral microbes can adhere to surfaces throughout the


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Figure 1.1: Dental caries levels (DMFT) of 12-year-olds worldwide. World Health

Organization. (Petersen PE: The World Oral Health Report. Geneva,

Switzerland:World Health Organization; 2003)


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oral cavity (Loesche, 1986). These include the tongue, epithelial cells lining roof of

the mouth and the cheeks, and enamel of the teeth.

1.3 Microbial and host ecology

Under the conditions in which the human-oral microflora relationship evolved,

the bacteria that inhabit the human mouth appear to have a commensal or even

mutualistic relationship with their human host and with each other. The mouth can be

considered an ideal environment for the growth of microorganisms, since it is warm

and moist and has a constant influx of nutrients through saliva and food intake. In

fact, it has been calculated that there are as many as 4 x 1010 organisms in each gram

of plaque removed from the teeth (Gibbons 2005). These organisms consist of, on an

average, more than 400 species that live together through the exploitation of very

specific ecological niches. For example, Lactobacilli are known to favour the dorsum

of the tongue, while Streptococcus mutans requires a solid, non-shedding surface for

colonization, as demonstrated by the rapid appearance of S. mutans in the mouth of

toothless infants when obturators are inserted to fix cleft palates (Tanzer et al. 2001).

This complex relationship even consists of bacteria whose presence is contingent on

other “pioneer” bacterial species. It has even been demonstrated that certain oral

microorganisms can cooperate in a mutualistic manner for a common benefit, as when

species collaborate by using different species-specific enzymes to break down

complex host molecules that could not be metabolized by a single species (Marsh,

1994).


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Figure 1.2: Dental caries is a multifactoral infectious disease.


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The ecology of the mouth, however, does not just involve interactions among

microorganisms. In fact, the host plays a large role in maintaining a uniform

ecosystem, especially through the saliva. Saliva is a complex mineral- and protein-

rich solution that delivers nutrients to the many bacterial species within the mouth

while also protecting host surfaces. During mastication, increased saliva flow prevents

changes in oral pH, because the buffer bicarbonate is present in saliva and acts as an

acid sink at a time when acidic products are being introduced into the mouth. Urea

and the peptide sialin are both also present in low concentrations in saliva and

produce ammonia when hydrolyzed, a basic product capable of raising pH. (Loesche,

1986). This basicity and buffering counteracts the lactic acid produced by anaerobic

bacteria in the mouth during the fermentation that occurs when nutrients are

introduced, offsetting decay of the teeth caused by this acid. Saliva also contains

glycoproteins that are known to be antibacterial, such as lysozyme and

lactoperoxidase. These compounds act independently of the host's immune system,

and are able to destroy invasive bacteria without harming the ecological balance of

the oral cavity, since indigenous bacteria have evolved resistance (Loesche, 1986).

A remarkable example of the balanced relationship between saliva and oral

microbiota is the fact that saliva is actually supersaturated with calcium and phospate

ions, which precipitate to form hydroxyapatite and remineralize the teeth. This

supersaturated solution should theoretically result in uncontrollable tooth growth as

more and more calcium phosphate precipitates onto the teeth. However, proteins

containing proline and a peptide called statherin, both of which are present in saliva,

have been shown to slow the rate of precipitation of these ions to a rate which

perfectly matches the rate of decay induced by bacteria during normal lactic acid

formation (Loesche, 1986). Under these conditions, teeth should be caries-free while


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the mouth remains colonized. The extensive decay and rampant levels of S. mutans in

patients suffering from xerostomia, or reduced saliva flow, is a striking example of

the role of saliva in caries prevention (Marsh, 1994). In fact, patients undergoing

radiation for head and neck cancers are often used as models for the progression of

decay, since severe xerostomia is an almost universal side effect of such treatment

(Loesche,1986).

1.4 Pathogenic Bacteria

Historically, cavities were attributed to a general overgrowth of oral bacteria,

termed the “non-specific plaque hypothesis.” However, this was disproved by Keyes

(1960), who compared the bacterial make-up of caries-active and caries-inactive

hamsters and found much higher proportions of a group of bacteria termed the

“mutans streptococci” in the cariogenic hamsters (Keyes, 1960). He further showed

that hamsters without caries did not develop them until exposed to caries-active

hamsters or their faeces. The mutans streptococci could be isolated from these newly

caries-active hamsters, and cultures of the bacteria would also cause caries in caries-

free hamsters. Keyes' hamster experiments showed that these bacteria fulfilled Koch's

postulates for infectious disease and led to the adoption of the “specific plaque

hypothesis,” which states that only certain bacterial species are responsible for

cariogenic behavior (Gibbons 2005).

This breakthrough made researchers eager to discover which bacterial species

were responsible for caries formation. Caries are formed when the rate of decay of the

teeth caused by the lactic acid produced by anaerobic bacteria exceeds the rate of

repair initiated by the phosphate and calcium ions in saliva. Lactic acid production

surges when sucrose is introduced into the mouth during meals or snacks, resulting in


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an overall drop in oral pH. Thusly, if acidity is a prerequisite for caries formation,

then only species that thrive in an acidic environment, known as acidophilic species,

can play a role in producing them. In fact, when cultures simulating a community of

oral bacteria were pulsed with glucose to produce a constant pH lower than 5, the

acidophilic species Lactobacillus casei, Veilonella dispar, and especially

Streptococcus mutans were able to dominate niches previously occupied by other,

avirulent species, and became irreversibly over-represented in the population (Marsh,

1994).

1.5 Streptococcus mutans

Kingdom: Bacteria

Phylum: Firmicutes

Class: Cocci

Order: Lactobacillales

Family: Streptococcaceae

Genus: Streptococcus

Species: mutans

Streptococcus mutans has been implicated most of all as the initiator of dental

caries. Streptococcus mutans is a Gram-positive, facultatively anaerobic bacteria

commonly found in the human oral cavity. The natural habitat of S. mutans is the

human mouth. The organism can be isolated frequently from faeces in human

(Finegold et al., 1975). It was first observed by Clarke who found a small, chained

cocco bacillus which was more oval than spherical in shape. He suggested that these

microorganisms were mutant streptococci and called them Streptococcus mutans

(Clarke, 1924). The cells are spherical or ovoid, 0.5-2.0 µm in diameter, occurring in


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pairs or chains when grown in liquid media, and stain Gram-positive (Figure 1.3). The

optimum temperature for growth is 37°C, and growth is usually restricted to 25-45°C.

What makes Streptococcus mutans such a potent initiator of caries? A variety

of virulence factors unique to the bacterium have been isolated that play an important

role in caries formation. First, S. mutans is an anaerobic bacterium known to produce

lactic acid as part of its metabolism. Then there is the ability of S. mutans to bind to

tooth surfaces in the presence of sucrose by the formation of water-insoluble glucans,

a polysaccharide that aids in binding the bacterium to the tooth. Mutant strains

developed to produce water-soluble glucans instead have extremely diminished

cariogenicity, especially on the smooth surfaces of the teeth which require greater

tenacity for binding to occur (Loesche 1986). Water-insoluble glucan has also been

found to lower the calcium and phosphate concentration of saliva, decreasing its

ability to repair the tooth decay caused by bacterial lactic acid (Nogueira et al. 2004).

The most important virulence factor, however, is the acidophilicity of Streptococcus

mutans. Unlike the majority of oral microorganisms, S. mutans thrives under acidic

conditions and becomes the dominant bacterium in cultures with permanently reduced

pH. Additionally, unlike many species present in plaque, whose metabolisms slow

considerably at such a low pH, the metabolism of S. mutans actually improves, as the

proton motive system used to transport nutrients through its cell wall in environments

of low pH or high glucose concentration is modulated by hydrogen ion content, which

increases with acidity (Hamilton and Svesater, 1998). In this way, S. mutans can

actually continue to lower or maintain the oral pH at an unnaturally acidic value,

leading to conditions favorable for its own metabolism and unfavorable for other

species it once coexisted with. It is this lowered pH that results in demineralization

and cavitation of the teeth, both of which increase with increased rates of S. mutans.


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Figure 1.3: A microscopic view of Streptococcus mutans (Source of image: http://microbiologyfall2010.wikispaces.com/file/view/Streptococcus-mutans.jpg/185 8 99615/Streptococcus-mutans.jpg)


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Under acidic conditions, S. mutans succeeds in creating a cycle that is favorable for

itself and unfavourable for others involved in the oral ecology – becoming, in effect, a

pathogen.

1.5.1. Virulence factors

1.5.1.1 Adhesion

The adhesion of S. mutans within dental plaque can be mediated via sucrose-

independent as well as sucrose dependent means. Sucrose-independent adhesion to

salivary components within the acquired enamel pellicle may initiate the attachment

process, but sucrose-dependent adhesion may be primarily responsible for

establishing colonization to the tooth surface (Figure 1.4). Sucrose intake is one of the

factors that correlate with the level of infant colonization (Wan et al., 2003).

Adhesion to pre-formed glucan on the tooth surface may also facilitate colonization

(Schilling and Bowen, 1992). S. mutans is most frequently transmitted to infant

children from their mothers, and S. mutans can generally be recovered from the oral

cavity following a window of infectivity (Caufield, 1997). The ability of S. mutans to

synthesize glucans from sucrose increases the efficiency of adhesion and enhances the

proportion of S. mutans within dental plaque. Thus, sucrose-dependent adhesion plays

a prominent role in initiating the changes in plaque ecology that can lead to dental

caries.

1.5.1.1.1 Sucrose-independent adhesion

Sucrose-independent adhesion of S. mutans is thought to be most profoundly

influenced by antigen I/II, an 185kDa surface protein. Similar proteins are found on

most oral streptococci (Ma et al., 1991) and have been designated by a variety of


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names including P1, SpaP, Sr, PAc, and antigen B. Proteins within the antigen I/II

family share structural similarity based on amino acid domains, but display variable

functionality with respect to binding salivary agglutinins, salivary pellicle

components, and other plaque bacteria (Whittaker et al., 1996; Peterson et al., 2002).

The alanine-rich and proline-rich domains are thought to be primarily responsible for

the interaction between antigen I/II and salivary components (Yu et al., 1997;

Hajishengallis et al., 1994; Crowley et al., 1993; Nakai et al., 1993; Brady et al.,

1992). The evidence for a role of antigen I/II in adhesion is based primarily on

studying the adhesion of S. mutans to saliva-coated hydroxyapatite (Ohta et al., 1989;

Lee et al., 1989; Douglas and Russel, 1982). A notable example is provided by

Bowen et al. (1991) who demonstrated that an isogenic mutant lacking P1 (antigen

I/II) did not bind as well as the wild-type to saliva-coated hydroxyapatite, but bound

equally well as the wild-type to saliva-coated hydroxyapatite that also contained in

situ synthesized glucan. Additionally, the P1 mutant was as cariogenic as the wild-

type in rats fed a high (56%) sucrose diet. A subsequent report by Crowley et al.

(1999) that a P1 mutant was less virulent in rats fed a 5% sucrose diet might have

seemed contradictory. But a reported role for antigen I/II in dentinal tubule invasion

provided a potential explanation for the reduction in virulence in rats fed a low

sucrose diet and supported the designation of P1 as a virulence factor. Additionally,

we have noted that the loss of P1 results in a change in the structural architecture of in

vitro-formed biofilms. If a similar change occurs in vivo it might have an influence on

virulence (Love et al., 1997).

1.5.1.1.2 Sucrose-dependent adhesion

The action of glucosyltransferases (GTFs) in the synthesis of glucans is the

major mechanism behind sucrose-dependent adhesion. The GTFs possess activity


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that results in the splitting of sucrose, the only natural substrate for the GTFs, into

glucose and fructose (Figure 1.4) (Monchois et al., 1999). The glucose moiety is then

added to a growing polymer of glucan called the extracellular polysaccharides (EPS)

(Figure 1.5). The energy rich glycosidic bond between the glucose and fructose

moieties supplies the free energy needed for the synthesis of EPS. S. mutans possesses

three GTFs encoded by gtfB, gtfC, and gtfD. Other members of the MS, for example

S. sobrinus, harbor four genes encoding GTFs. Collectively, the GTFs synthesize both

water soluble and water-insoluble glucans. The water-soluble glucan is a

predominantly linear polymer linked by alpha- 1,6-glycosidic linkages that resembles

dextran. The water insoluble variety, also called mutan, has a higher degree of

branching and predominantly alpha-1,3-linkages. Both types of polymers are thought

to contribute to sucrose dependent colonization and caries but the water-insoluble

glucan may be of primary importance for smooth surface caries (Munro et al., 1995).

Several groups have demonstrated that S. mutans strains inactivated in one or more gtf

genes have diminished virulence when tested in rodent models of caries (Yamashita et

al., 1993; Munro et al., 1991; Tanzer et al., 1974).

The ability of glucan to facilitate adhesion of S. mutans may be due to

hydrogen bonding of the glucan polymers to both the salivary pellicle and the bacteria

(Rolla et al., 1998). In vitro in the presence of sucrose, S. mutans becomes coated

with glucan. Presumably, sucrose-dependent adhesion can involve binding by glucan-

coated bacteria, or attachment of S. mutans to glucan present within the dental plaque

(Schilling and Bowen, 1992; Schilling et al., 1989; Rolla et al., 1983). This glucan

could be synthesized by extracellular GTFs that bound the salivary pellicle, S. mutans

that had previously adhered via sucrose-independent means, or perhaps by other

oral streptococci. It is not known why S. mutans requires multiple GTFs, but there is


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Figure 1.4: Utilization of sucrose by Streptococcus mutans and pathogenesis of dental

caries.


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Figure 1.5: Streptococcus mutans embedded in the extracellular polysaccharides

(EPS), which plays a major role in sucrose dependent adhesion.

Bacteria Glucans (EPS)


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evidence that the different GTFs have differing affinities for the bacterial surface or

salivary pellicle (Vacca- Smith and Bowen, 1998), and that a particular ratio of each

is necessary for optimal sucrose-dependent adhesion (Ooshima et al., 2001) The basis

for glucan-binding by individual bacteria is still subject to speculation. The search for

a cell surface glucan receptor led to the isolation of several nonenzymatic glucan-

binding proteins and confirmed the glucan-binding abilities of the GTFs. However,

most of these proteins are exported and lack cell wall anchoring motifs. The glucan-

binding domain (GBD) of the GTFs (Janecek et al., 2000) consists of a carboxyl

terminal region of amino acid repeats that is shared by at least two non-enzymatic

glucanbinding proteins, GbpA (Banas et al., 1990) and GbpD (Shah and Russel,

2002). It is possible that another non-enzymatic glucan-binding protein, GbpC, acts as

a cell surface glucan receptor, though an aggregation phenotype attributed to GbpC is

only observed under certain stressful growth conditions (Sato et al., 1997). Another

possibility is the wall-associated protein WapA, originally antigen A (Russel, 1979).

Although it has not been described as a glucan-binding protein the inactivation of the

wapA gene resulted in a reduction in aggregation and adhesion to a smooth, glass

surface (Qian and Dao, 1993). Alternatively, the WapA may make a contribution to

sucrose-dependent adhesion indirectly. The disruption of other genetic loci also has

been correlated with diminished sucrose-dependent adhesion (Tao and Tanzer, 2002).

The mechanism of contribution of these loci is uncertain, but may border on the

interruption of general cell housekeeping functions.

1.5.1.2 Non-enzymatic glucan binding proteins

With the increasing interest for the search for a cell surface receptor for glucan

led many groups to screen S. mutans culture supernatants or cell extracts, often via


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affinity chromatography, for proteins capable of binding glucan or mediating dextran-

dependent aggregation. In addition to the GTFs, non-enzymatic glucan-binding

proteins (GBPs) were recovered. The first of these was GbpA (Ruusel RRB, 1979);

subsequently GbpB (Smith et al., 1994), GbpC (Sato et al., 1997), and GbpD (Shah

and Russel, 2002) were described. Several GBPs have been noted in other MS as

well. Interestingly, non-MS oral streptococci may possess one or more GTFs but non-

enzymatic GBPs have not been identified among these species. Since glucan plays

such a prominent role in the caries process, proteins capable of binding glucan were

hypothesized to contribute to sucrose dependent adhesion and possibly to the cohesive

nature of the dental plaque biofilm. There is now evidence in support of these ideas,

though strain differences and differences in model systems may delay the task of

assigning a precise role to an individual GBP.

1.5.1.3 Carbohydrate metabolism

Besides the proteins and enzymes that contribute to sucrose-dependent

adhesion, there exist other proteins involved in the metabolism of sucrose, glucans, or

other carbohydrates that are considered potential virulence factors. These include a

fructosyltransferase (Ftf), a fructanase (FruA), an extracellular dextranase (DexA),

and proteins responsible for intracellular polysaccharide accumulation (Dlt1-4).

Sucrose can enter S. mutans via three different transport mechanisms (Colby and

Russel 1997). One of these, the multiple sugar metabolism system, is encoded by an

operon that includes a sucrose phosphorylase (GtfA), and an intracellular dextranase

(DexB) (Tao et al., 1993). This system is capable of transporting isomaltosaccharides

that can be generated by the action of the extracellular dextranase DexA (Colby and

Russel, 1997). In an analogous manner to the glucans, the Ftf can synthesize fructan


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from sucrose, and FruA can liberate the fructose for transport into the cell.

Nevertheless, there is no firm evidence that S. mutans can propagate solely on a diet

of extracellular glucan or fructan.

1.5.1.4 Acidogenicity

S. mutans contains a complete glycolytic pathway and can produce lactate,

formate, acetate, and ethanol as fermentation products (Ajdic et al., 2002). The

precise distribution of fermentation products will depend on growth conditions with

lactate being the major product when glucose is abundant (Dashper and Reynolds,

1996). Strains deficient in lactate dehydrogenase (LDH) display reduced cariogenicity

(Johnson et al., 1980; Fitzgerald et al., 1989) and the absence of lactate

dehydrogenase (LDH) is lethal (Hillman et al., 1996). The velocity with which S.

mutans produces acid when tested at a pH in the range from 7.0 to 5.0 exceeds that of

other oral streptococci in most instances (de Soet et al., 2000). The relative

acidogenicity of S. mutans can vary from one isolate to another and strict correlations

between acidogencity and caries experience is lacking (Kohler et al., 1995).

Nonetheless it is generally thought that the acidogenicity of S. mutans leads to

ecological changes in the plaque flora that includes an elevation in the proportion of

S. mutans and other acidogenic and acid-tolerant species. This cariogenic flora will

reduce plaque pH to lower levels than will a healthy plaque flora upon the ingestion

of fermentable carbohydrate, and the recovery to a neutral pH will be prolonged

(Loesche, 1986; Graf, 1970; Stephen, 1944). Sustained plaque pH values below 5.4

favor the demineralization of enamel and the development of dental caries.


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1.5.1.5 Acid-tolerance

Accompanying the acidogenicity of S. mutans is its aciduricity or acid-

tolerance. S. mutans retains glycolytic capabilities even at pH levels that are growth

inhibitory (as low as pH 4.4) (Bender et al., 1985). As with its acidogenicity, it is the

extent of its aciduricity, rather than its novelty, that distinguishes S. mutans from the

other oral streptococci. The acid tolerance of S. mutans is largely mediated by an

F1F0-ATPase proton pump but also involves adaptation with an accompanying

change in gene and protein expression. Together they constitute the acidtolerance

response (ATR). In vitro the ATR has been shown to protect the organisms from a

sub-lethal pH challenge (Svesater et al., 1997) and acid shock or growth at acidic pH

has been associated with changes in the expression of over 30 proteins (Wilkins et al.,

2002; Hamilton and Svesater, 1998). The full scope of the changes associated with

acid tolerance is still under investigation. Evidence is also accumulating that acid-

tolerance may be aided by the synthesis of water-insoluble glucan and the formation

of a biofilm. S. mutans cells within a biofilm were better able to survive an acid

challenge than planktonically grown bacteria (McNeill and Hamilton, 2003). This

may be related to quorum sensing systems efficiently inducing the ATR, and physical

characteristics of the biofilm. Hata and Mayanagi (2003) reported that the speed of

diffusion of hydronium ions is proportional to the amount of waterinsoluble glucan

produced by S. mutans. These results highlight the connection between different

virulence properties of S. mutans and indicate that the role of glucan extends beyond

promoting adhesion. These differences may also reflect the basis for why the glucan-

synthesizing capacity of S. mutans evolved differently than those for the other oral

streptococci.


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1.5.1.6 Maintaining intracellular pH

The consequence of excretion of acid by products of metabolism is the

acidification of the external environment of S. mutans. External protons are capable of

permeating streptococcal membranes and acidifying the cytoplasm. Glycolytic

enzymes and other cellular functions are sensitive to inhibition by low intracellular

pH. Therefore, the activity of the membrane-bound proton translocating ATPase, or

F-ATPase, is critical for establishing and maintaining a pH gradient across the

cytoplasmic membrane. Bender et al. have shown that the efficiency of this enzyme at

different pH values correlates well with the acid-tolerance of various oral streptococci

(Bender et al., 1985). As the pH falls, there is increased activity of the S. mutans F-

ATPase (Hamilton and Buckley, 1991; Belli and Marquis, 1991) which helps

maintain a pH gradient of approximately one pH unit (Dashper and Raynolds, 1992;

Hamilton and Buckley, 1991). At the same time the fatty acid profile of the membrane

shifts, decreasing permeability to protons (Quivey et al., 2000) , and excretion of

acidic end products increases (Dashper and Raynolds, 1996). Catabolites other than

glucose may counter the effects of the F-ATPase when introduced via symport along

with a proton (Belli and Marquis, 1994). It is interesting to speculate whether the

presence of an extracellular glucan-digesting dextranase can ensure a supply of

glucose during times when exogenous glucose is not available.

1.5.1.7 Biofilm formation

Dental plaque is the community of microorganisms found on a tooth surface as

a biofilm, embedded in a matrix of polymers of host and bacterial origin (Figure 1.6)

(Socransky and Haffajee, 2002; Marsh, 2004). Of clinical relevance is the fact that

biofilms are less susceptible to antimicrobial agents, while microbial communities can


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display enhanced pathogenicity (pathogenic synergism) (van Steenbergen et al.,

1984). The structure of the plaque biofilm might restrict the penetration of

antimicrobial agents, while bacteria growing on a surface grow slowly and display a

novel phenotype, one consequence of which is a reduced sensitivity to inhibitors

(Gilbert et al., 2002). Plaque is natural and contributes (like the resident microflora of

all other sites in the body) to the normal development of the physiology and defenses

of the host (Marsh, 2000).

Dental plaque is a biofilm and many variables associated with growth in a

biofilm, including adhesion, nutrient flow, and co-aggregation can influence growth

rate, gene expression, and quorum sensing in ways that differ from life in a planktonic

environment. Consequently, recent investigations have begun to examine the

expression of virulence genes within biofilms. For instance, variability of gene

expression in response to the environment has been observed for gtfB and gtfC genes.


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Figure 1.6: Streptococcus mutans participates in the formation of biofilms on tooth

surfaces. a) Initial attachment of S. mutans to tooth surfaces. b)

Accumulation of mutans streptococci on tooth surfaces in the presence of

sucrose. c) Acid production by S. mutans. (Copyright © 2006 Nature

Publishing Group Nature Reviews Immunology)


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Evidence suggests that these genes can be both independently transcribed and

co-transcribed. Using the chloramphenicol acetyltransferase (cat) reporter gene,

Hudson and Curtiss (Hudson and Curtiss, 1990) demonstrated increased expression of

the gtfB/C genes in response to sucrose or when bound to an artificial tooth pellicle.

Burne et al. (1997) saw a similar stimulation with sucrose in biofilms, though the

time-frame of the increase and the CAT specific activity varied between 48 hr and 7-

day biofilms. But a plasmid-based luciferase reporter system found no change in gtfB

and gtfC expression in the presence of sucrose (Goodman and Gao, 2000). Fujiwara

et al. (2002) reported that sucrose reduced the expression of gtfB and gtfC when tested

using batch cultures and reverse transcription (RT)-PCR. These studies highlight the

difficulty inherent in attempting to model the in vivo environment and trying to

understand the precise contributions of even a single virulence factor. Another

approach to investigating S. mutans virulence has been to search for genes required

for biofilm development. This approach casts a wider net than focusing on

carbohydrate metabolism and has yielded some interesting results. Disruption of

genes involved in various two-component and quorum sensing signaling systems have

affected the biofilm-forming capacity of S. mutans (Merritt et al., 2003; Li et al.,

2002a; Li et al., 2002b; Wen and Burne, 2002; Yoshida and Kuramitsu, 2002;

Bhagwat et al., 2001) . Similar observations have been made in other bacterial species

(Davies et al., 1998; Rachid et al., 2000). These data suggest that genes encoding the

GTFs, and genes encoding other exported proteins directly involved in biofilm

development, may be globally regulated.

1.5.1.8 Development of dental plaque biofilm

Dental plaque forms via an ordered sequence of events, resulting in a

structurally- and functionally-organized, species-rich microbial community (Marsh,


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2004). Distinct stages in plaque formation include: acquired pellicle formation;

reversible adhesion involving weak long-range physicochemical interactions between

the cell surface and the pellicle, which can lead to stronger adhesin-receptor mediated

attachment; co-adhesion resulting in attachment of secondary colonizers to already

attached cells (Kolenbrander et al., 2000); multiplication and biofilm formation

(including the synthesis of exopolysaccharides) and, on occasion, detachment. The

increase in knowledge of the mechanisms of bacterial attachment and co-adhesion

could lead to strategies to control or influence the pattern of biofilm formation.

Analogs could be synthesized to block adhesin-receptor attachment or co-adhesion,

and the properties of the colonizing surfaces could be chemically modified to make

them less conducive to microbial colonization. However, cells can express multiple

types of adhesin (Zhang et al., 2005; Hasty et al., 1992), so that even if a major

adhesin is blocked, other mechanisms of attachment may be invoked. Furthermore,

although adhesion is necessary for colonization, the final proportions of a species

within a mixed culture biofilm such as dental plaque will depend ultimately on the

ability of an organism to grow and outcompete neighboring cells.

Once formed, the overall composition of the climax community of plaque is

diverse, with many species being detected at individual sites. Molecular ecology

approaches, in which 16S rRNA genes are amplified from plaque samples, have

identified >600 bacterial and Archae taxa, of which approximately 50% are currently

unculturable (Wade, 1999). Once plaque forms, its species composition at a site is

characterized by a degree of stability or balance among the component species, in

spite of regular minor environmental stresses, e.g., from dietary components, oral

hygiene, host defenses, diurnal changes in saliva flow, etc. This stability (termed

microbial homeostasis) is not due to any biological indifference among the resident


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organisms, but is due to a balance imposed by numerous microbial interactions,

including examples of both synergism and antagonism (Marsh et al., 1989). These

include conventional biochemical interactions such as those necessary to catabolise

complex host glycoproteins and to develop food chains, but in addition, more subtle

cell-cell signalling can occur. This signalling can lead to coordinated gene expression

within the microbial community, and these signalling strategies are currently being

viewed as potential targets for novel therapeutics (Suntharalingam and Cvitkovitch,

2005; Kolenbrander et al., 2002).

1.5.2 Quorum sensing: Regulation and pathogenic potential of Streptococcus

mutans

Streptococcus mutans belongs to the taxonomically outdated group of

‘viridans streptococci’ (Facklam, 2002). Like other members of this group (i.e.

Streptococcus sanguinis), Streptococcus mutans persistently colonizes the surfaces of

the oral cavity once it has been passed predominantly from mother to child.

(Emanuelsson et al., 1998; Grönroos et al., 1998) This feature relies on the capability

of the bacteria to specifically and firmly bind to the tooth surface by protein adhesins

and subsequently, produce a water-insoluble, sticky extracellular matrix of complex

polysaccharides. The mixture of adherent bacteria and matrix molecules form a

typical biofilm (Kolenbrander et al., 2002). Like other streptococci, S. mutans can

metabolize sugar only by fermentation. Unlike other streptococci, S. mutans continues

the fermentation process until high amounts of lactic acid (a pH value close to 4) have

been reached. The lactic acid dissolves calcium ions present in the teeth and results in

formation of caries. This pathologic process is the basis of one of the economically

most important diseases in industrialized countries. In the last two years, it became

evident that biofilm formation and acid tolerance of S. mutans are both crucially


25

associated with QS-regulation. S. mutans was found to possess a QS-regulation

system that closely resembles the com regulon of S. pneumoniae. At one genomic

locus there is a comCDE operon encoding the ComC signaling peptide, the

membrane-associated ComD sensor kinase, and the ComE regulator (Li et al., 2001a).

At a different site, there is a comAB operon that is translated into a transmembrane

processor/transporter unit (Peterson et al., 2002). Finally, at another site the comX

gene encodes a RNA polymerase cofactor (Ajdic et al., 2002).As in S. pneumoniae,

ComC is constantly produced and exported by ComAB, and reaches its critical

threshold concentration at cell densities typical for early- to mid-log phase cells. Then

its sensing and the subsequent regulation by ComD/ComE induces a positive

autoregulatory loop on comCDE and comAB transcription (i.e. a signal amplification

and a regulatory acceleration). In addition, comX expression leads to an increased

formation of ComX-RNA polymerase complexes and, as a consequence, to a more

efficient binding to and transcription of genes with com-boxes in their promoters

(Figure 1.7). Using defined S. mutans com mutants, synthetic ComC signaling

molecules and the appropriate environmental conditions, Li et al. showed in a series

of experiments that the com system exhibits the typical regulatory features of a QS-

regulatory unit and is a prerequisite for the uptake of foreign DNA (natural

competence) (Li et al., 2002). The competence is more pronounced and

simultaneously, the tolerance to pH values of 4 is better when the cells live at a high

cell density in a biofilm. Finally, a normal biofilm will only be formed with a

functional com regulon. Thus, the com regulon appears to be a key player in the

ability of S. mutans to cause caries lesions.


26

Figure 1.7: General pathway of the Com-driven quorum-sensing regulation in

viridans streptococci. The com regulon comprises several genes and

operons located at different loci of the streptococcal genome. The

precursor of the signaling molecules, ComC, is processed and secreted

by the activity of the membrane-associated ComAB machinery. The

competence stimulating peptide (CSP) interacts with membrane-

associated histidine kinase ComD. This interaction results in the

phosphorylation of the regulator ComE (ComE~P). The activated

regulator ComE~P positively (+) stimulates the transcription rate of the

Com-dependent promoters (Pcom). This leads to the increased

production of the alternative sigma factor ComX, which in turn by

interaction with the RNA polymerase core enzyme results in differential

transcription of many secondary Com-dependent genes. The regulon is

not drawn to scale.


27

1.5.3 Dental Prophylaxis: Strategies for managing caries

Strategies for the prevention of disease include the following:

1.5.3.1 Habit and hygiene

The lifestyle of a person and behavioural factors that manipulate the oral

hygiene undoubtedly, influence the susceptibility to dental caries (Selwitz et al.,

2007). Continuous brushing and flossing of teeth removes the bacteria and

fermentable substances as well. The continuous flow of saliva reduces the cariogenic

flora on the tooth surface. Saliva also acts as a buffering agent during the continuous

acid production in the oral cavity (Kleinberg, 2002). Individuals with dry mouth

syndrome are hence more prone to dental caries. This has been observed with people

following radiation treatment of head and neck cancer, using narcotics and patients

with Sjogren’s syndrome (Dreizen and Brown, 1976; Lowenthal, 1967).

Dental caries is correlated with sugar uptake in the diet. Increase in

urbanization has led to the replacement of crude sugar by refined sugar that worsened

the situation (Peterson, 2003). Numerous studies indicate the linear correlation of

sugar consumption with dental caries in population worldwide (Burt, 1993; Marthaler,

1990). However, starchy foods and fresh fruits are reported to be less cariogenic.

Foods that involve extensive mastication stimulate production of saliva and hence

have low cariogenic potential. Intake of fibrillar and firm fruits like apple and carrots

act as natural toothbrush, as they clean the tooth surface during chewing. Substitution

of fermentable carbohydrates by xylilitol, saccharin and aspartame has been

efficiently used for reducing caries (Naylor, 1986). Fluids like juices and milk seems


28

to be less cariogenic as they are lesser retained in oral cavity. Consumption of

carbonated drinks however pose a higher risk to dental caries (Sowers et al., 2006).

1.5.3.2 Anti-microbial therapies

The concept of reducing plaque utilizing antimicrobial agents seems like a

reasonable strategy. Stannous fluoride and amine fluoride as well as numerous

metallic ions have demonstrated antimicrobial effectiveness (Ghaffar et al. 1997;

Scheie et al., 1989). Tin, zinc, copper and others have demonstrated anti-microbial

effects, but none have demonstrated anti-caries effectiveness. Essential oils, a mixture

of thymol, eucalyptol, methyl salicylate and menthol have been demonstrated to be

effective in preventing the build-up of supra-gingival plaque and gingivitis (Charles et

al., 2000; Walker, 1988), but have not demonstrated effectiveness in the reduction of

caries lesions. Triclosan has also demonstrated effectiveness in reducing plaque and

gingivitis, but has also failed to produce reductions in caries (Gjermo and Saxton,

1991). Chlorhexidine, a bisguanide, is a broad spectrum antimicrobial that functions

by disturbing the cell membrane of bacteria.

Studies have demonstrated that chlorhexidine, administered frequently enough

and in high enough doses, can reduce the Streptococcus mutans bioburden in the

mouth (Ribeiro et al., 2007). Unfortunately, clinical studies focusing on actual caries

reductions have been ambiguous.

1.5.3.3 Inhibition of acid production

This can be attained by use of fluoride containing products or other metabolic

inhibitors. Fluoride not only improves enamel chemistry but also inhibits several key

enzymes, especially those involved in glycolysis and in maintaining intracellular pH.


29

Fluoride can reduce the pH fall following sugar metabolism in plaque biofilms, and in

so doing, prevent the establishment of conditions that favor growth of acid-toleratant

cariogenic species (Svante et al., 2003).

1.5.3.4 Antibiotics

Ever since the establishment of dental caries as an infectious disease,

antibiotics are constantly in use for treating it. Chlorhexidine digluconate (CHX) had

been considered as a gold standard in this field. It has been used with the aim of

elimination or suppression of mutans streptococci in the oral cavity. CHX damages

outer cell layers but is insufficient to induce lysis or death (El-Moung et al., 1985).

Moreover, it crosses cell wall or outer membrane by passive diffusion and attacks

bacterial cytoplasm, followed by leakage of cellular constituents. However, high

concentration causes coagulation of intracellular constituents. As a result cytoplasm

becomes congealed with a consequent reduction in leakage. Thus there is a biphasic

effect on membrane permeabilities: In the presence of CHX, high rate of leakage is

observed initially but as CHX concentration soars, leakage decreases because of the

coagulation of cytosol (Mc Donnell and Russell, 1999). The best results in reducing

dental plaque have been observed with chlorhexidine gels and mouthwashes

(Emilson, 1994). As disparities in its efficacy against varying subjects have been

observed, CHX cannot be regarded as single line defense for the cure of caries

(Twetman, 2004).

The pioneering work of McClure and Hewitt indicated the usefulness of

penicillin in preventing experimentally induced caries (1946). Many other antibiotics

with activity against gram-positive bacteria depress the development of dental caries

in experimental animals (Fitzgerald, 1989). S. mutans has been reported to be highly


30

susceptible to penicillin, methicillin, ampicillin, erythromycin, cephalothin and many

other antibiotics (Little et al., 1979). More antibiotics are now screened for their

effects against oral biofilms (Nguyen, 2005).

1.5.3.5 Probiotic therapies

Enormous strides have been made in comprehending the cure for dental caries

by the aid of genetic engineering. The sequencing of complete S. mutans genome

marks the beginning of an era of fashioned therapies (Ajidic et al., 2002). An ideal

drug for caries prevention should aim at maintaining the normal homeostasis of the

oral cavity and reducing the virulence of S. mutans (Figure 1.8).

1.5.3.6 Vaccines

Immune defense in dental caries is mediated mainly by secretory IgA present

in saliva and generated by mucosal immune system. The mode of action of these

antibodies is inhibition of adherence and possibly metabolic activities of S. mutans

(Russell et al., 1999). In view of vaccine development against caries, the virulence

factors, specifically the adherence motivating factors are recognized as key antigens.

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 (Smith, 2003). Novel strategies using Ag I/II, Gtfs and glucan binding proteins

are designed for pursuing vaccine goal as these are main proteins that mediate the

attachment of bacteria. Induction of salivary IgA and circulating IgG antibodies have

been observed by oral or intranasal immunization with these antigens in many animal

models (Jespersgaard, 1999). Similar antigen preparations in human trials have shown

successful induction of salivary S-IgA (Childers et al., 2002; 1997). Tailored vaccines


31

Figure 1.8: An ideal approach for the control of dental caries. S.mutans – , S.

mutans in oral cavity aggregated on the surface of teeth and ferment

sugars to produce acid leading to pathogenicity. Sucrose moiety- ,

Sucrose moieties facilitate aggregation of bacterial cells on the tooth

surface. Drug candidate- , A potent drug combating pathogens to reduce

the cariogenicity. The drug should specifically interact with S. mutans

and inhibit both adhesin and polysaccharide mediated attachment. It

should act as a buffering agent and control the acid production by oral

microbes (Islam et al., 2007).


32

possessing immunogenic sites of the virulence determinant traits have showed

significant caries-preventive results (Katz et al., 1993). However, chimeric proteins

using both AgI/II and Gtfs, has shown most encouraging response (Zhang et al.,

2002). DNA vaccines using these proteins are also developed and assayed for their

anticariogenic spectrum (Fan et al., 2002). Better efficiency of fusion DNA vaccine

coding for both AgI/II and glucosyltransferases in rats has also been reported (Guo et

al., 2004).

Numerous approaches for passive immunization are employed to avoid

complications that might arise from active immunization (Ferretti et al., 1980).

Immunity through antibodies for glucosyltransferases available through cow’s milk

(Loirmaranta et al., 1998), hen’s eggs (Hatta et al., 1997; Hamada et al., 1991) and

plantibodies (Ma et al., 1998; 1995) has been reported. Efficient delivery systems are

being developed for a continuous and controlled release of vaccines. Both animated

(Huang et al., 2001) and non-animated vehicles as liposomes (Michalek et al., 1992)

and microparticles (Smith et al., 2000) have been assessed in animal models.

1.5.3.7 Replacement therapy

In the post-genomic era, recombinant DNA technology is being used for

finding an answer to dental caries. The recent word in vogue is replacement therapy.

Genetic engineering is being used to tailor the effector strain for replacement therapy

of dental caries, which acts as a vaccine and should not be pathogenic. Moreover, it

colonizes the niche, thereby preventing colonization and outgrowth of wild type

strains. Using this approach, a harmless strain is permanently implanted in the host’s

oral flora. Once established, the effector strain competes with the wild-type strain and

prevents its outgrowth (Hillman, 2002).


33

An effector strain with all these properties, BCS3-L1 was constructed from a

clinical isolate JH1140 with high mutacin activity and genetic stability. The

mutagenesis approach has also been used successfully by constructing a lactate

dehydrogenase mutant, which has been reported to reduce cariogenic potential

(Johnson et al., 1980). The resultant strain thus produced, was deficient in lactic acid

production and had elevated mutacin levels. Colonization studies with the mutant

strain BCS3-L1 shows no pathogenic hazards as histopathological examination

showed no detectable carious lesions (Hillman et al., 2000).

1.5.3.8 Medicinal herbs

For thousands of years, natural products have been used in traditional

medicine all over the world and predate the introduction of antibiotics and other

modern drugs. The antimicrobial efficacy attributed to some plants in treating diseases

has been beyond belief. It is estimated that local communities have used about 10% of

all flowering plants on Earth to treat various infections, although only 1% have gained

recognition by modern scientists (Kafaru, 1994). Owing to their popular use as

remedies for many infectious diseases, searches for plants containing antimicrobial

substances are frequent (Betoni et al., 2006). Plants are rich in a wide variety of

secondary metabolites such as tannins, alkaloids and flavonoids, which have been

found in vitro to have antimicrobial properties (Lewis and Ausbel, 2006). A number

of phytotherapy manuals have mentioned various medicinal plants for treating

infectious diseases due to their availability, fewer side effects and reduced toxicity

(Lee et al., 2007). There are several reports on the antimicrobial activity of different

herbal extracts (Islam B et al., 2008; de Boer et al., 2005; Bonjar, 2004). Many plants

have been found to cure urinary tract infections, gastrointestinal disorders, respiratory


34

diseases and cutaneous infections (Somchit et al., 2003; Brantner and Grein, 1994).

Cytotoxic compounds have been isolated from the species of Vismia (Hussein et al.,

2003). Antibacterial activity of the essential oil as well as eugenol purified from

Ocimum gratissimum to treat pneumonia, diarrhea and conjunctivitis has also been

reported earlier (Nakamura et al., 1999). According to the WHO, medicinal plants

would be the best source for obtaining variety of drugs (Santos et al., 1995). These

evidences contribute to support and quantify the importance of screening natural

products. Medicinal plants are natural resources, to obtain valuable products which

can be used in the treatment of various ailments. Plant materials remain an important

resource for combating illnesses, including infectious diseases, and many of the plants

have been investigated for novel drugs for the development of new therapeutic agents.

Thus, the emergence of multiple drug resistance of pathogenic organisms has

necessitated a search for new antimicrobial substances from other sources including

plants (Betoni et al., 2006).

1.5.3.8.1 Traditional Plant-Based Medicines

Medicinal plants have been used as traditional treatments for numerous human

diseases for thousands of years and in many parts of the world. In rural areas of the

developing countries, they continue to be used as the primary source of medicine.

About 80% of the people in developing countries use traditional medicines for their

health care (Kim, 2005). The natural products derived from medicinal plants have

proven to be an abundant source of biologically active compounds, many of which

have been the basis for the development of new lead chemicals for pharmaceuticals.

With respect to diseases caused by microorganisms, the increasing resistance in many

common pathogens to currently used therapeutic agents, such as antibiotics and


35

antiviral agents, has led to renewed interest in the discovery of novel anti-infective

compounds. As there are approximately 500 000 plant species occurring worldwide,

of which only 1% has been phytochemically investigated, there is great potential for

discovering novel bioactive compounds. There have been numerous reports of the use

of traditional plants and natural products for the treatment of oral diseases. Many

plant-derived medicines used in traditional medicinal systems have been recorded in

pharmacopeias as agents used to treat infections and a number of these have been

recently investigated for their efficacy against oral microbial pathogens. The general

antimicrobial activities of medicinal plants and plant products, such as essential oils,

have been reviewed previously (Kalemba and Kunicka, 2003; Cowan, 1999).

Therefore, the purpose of this review is to present some recent examples from the

literature of studies that have served to validate the traditional use of medicinal plants

with specific biological activity. In particular, traditional medicinal plant extracts or

phytochemicals that have been shown to inhibit the growth of oral pathogens, reduce

the development of dental plaque, influence the adhesion of bacteria to surfaces and

reduce the symptoms of oral diseases will be discussed subsequently. In addition,

clinical studies that have investigated the safety and efficacy of such plant-derived

medicines will be described.

1.5.3.8.2 Identifying and evaluating medicinal plant products used to treat or

prevent oral diseases

Of all oral diseases, the incidence of those that have a microbial aetiology is

greatest in all parts of the world. A number of traditional medicinal plants have been

evaluated for their potential application in the prevention or treatment of oral diseases

(Table 1.1). Numerous studies have investigated the activity of plant extracts and


36

products against specific oral pathogens, while others have focused on the ability of

the products to inhibit the formation of dental biofilms by reducing the adhesion of

microbial pathogens to the tooth surface, which is a primary event in the formation of

dental plaque and the progression to tooth decay and periodontal diseases (Steinberg

et al., 2000).

1.6 Expression of biofilm associated genes of Streptococcus mutans

Streptococcus mutans is a bacterium that has evolved to depend on a biofilm

lifestyle for survival and persistence in its natural ecosystem. S. mutans is assembled

as communities attached to dental surfaces and forms matrix-embedded biofilms

(Marsh, 2005). Such biological organization provides a sheltered microenvironment

for the immobilized bacteria (Hall-Stoodley et al., 2004; Bowden and Hamilton,

1998). Adhesion is the initial step in the formation of biofilm communities. As a

primary bacterial agent of dental caries, the mechanisms by which S. mutans adheres

to tooth surfaces are important potential targets for anti-cariogenic intervention.

Sucrose-dependent mechanisms of adherence, as mediated by extracellular enzymes

[glucosyltransferases (GTFs) and fructosyltransferases (FTFs)] and glucan-binding

proteins (GBPs), have well-established roles in the virulence of S. mutans (Banas &

Vickerman, 2003; Kuramitsu, 2001, 1993; Steinberg, 2000). Sucrose-independent

mechanisms can also foster microbial colonization by providing binding sites for

bacteria (Shemesh and Steinberg, 2006; Lee et al., 1989). Beyond initial adherence, a

variety of genes are required for the adaptation of S. mutans and other oral

streptococci in biofilms (Whiteley et al., 2001; Costerton, 1987). These include

genes associated with intercellular communication systems and environmental sensing

systems, regulators of carbohydrate metabolism, and adhesion-promoting genes


37

Table 1.1: Medicinal plants used against Streptococcus mutans in the treatment of

dental caries.

Name B o t a n i c a l Part Used Name

B o t a n i c a l Part Used

1. Acacia leucophloea Bark 23. Glycyrrhiza glabra Root

2. Albizia lebbeck Bark 24. Ginkgo biloba Whole plant 3. Abies canadensis Whole plant 25. Juniperus virginiana Whole plant 4. Aristolochia cymbifera Whole plant 26. Kaemperia pandurata

Dried rhizomes

5. Annona senegalensis Whole plant 27. Legenaria sicerania Leaves 6. Albizia julibrissin Whole plant 28. Mentha arvensis Leaves

7. Allium Sativum Bulbs 29. Mikania lavigata Aerial parts 8. Anacyclus pyrethrum Root 30. Melissa officinalis Whole plant

9. Areca catechu Nuts 31. Magnolia grandiflora Whole plant 10. Breynia nivosus Whole plant 32. Melissa officinalis Whole plant

11. Citrus medica Roots 33. Magnolia grandiflora Whole plant 12. Coptidis rhizoma Whole plant 34. Nicotiana tabacum leaves 13. Caesalpinia martius Fruits 35. Physalis angulata Flower 14. Cocos nucifera Whole plant 36. Pinus virginiana Whole plant 15. Caesalpinia pyramidalis Whole plant

37. Polygonum cuspidatum Root

16. Chelidonium majus Whole plant 38. Rheedia brasiliensis Fruit 17. Drosera peltata Whole plant 39. Rhus corriaria Whole plant

18. Embelia ribes Fruit 40. Rosmarinus officinalis Whole plant 19. Erythrina variegata Root 41. Rhus corriaria Whole plant 20. Euclea natalensis Whole plant 42. Syzygium cumini Bark 21. Fiscus microcarpa Aerial part 43. Sassafras albidum Whole plant 22. Gymnema Sylvester Leaves,Roots

44. Solanum xathaocarpum Whole plant


38

(Shemesh et al., 2007; Senadheera et al., 2005; Lemos and Burne, 2002). The genes

include brpA (lytR) and vicR encoding regulatory proteins (Chatfield et al., 2005;

Senadheera et al., 2005; Li et al., 2002; Wen and Burne, 2002), the adhesion-

promoting genes gbpB and spaP (Jakubovics et al., 2005; Banas and Vickerman,

2003), and also the genes encoding polysaccharide synthesizing enzymes, including

gtfBC and ftf (Shemesh et al., 2006; Steinberg, 2000). Moreover genes such as relA is

required by S. mutans to form stable biofilms and tolerate acid stress (Lemos et al.,

2004), and smu630 is important in both sucrose-dependent and sucrose-independent

biofilm formation (Brown et al., 2005). A number of studies have indicated that

expression of the genes responsible for biofilm formation is dependent on

environmental conditions (Li and Burne, 2001; Kiska and Macrina, 1994; Hudson and

Curtiss, 1990), and is also genetically regulated (Lee et al., 2004; Kiska and Macrina,

1994).

1.7 Herbal extracts: Scope and significance as therapeutic agents

India has been traditionally a country known for its treasure of herbs, present

in the Himalayan region, the North East areas and also in its Southern part. Although

‘Ayurveda’ & ‘Unani’ systems of treatment used these herbs, but due to lack of

proper identification and characterization, the active compounds responsible for the

healing properties of these herbs, still remain largely unexplored. Plants have

traditionally provided a source of hope for novel drug compounds, as plant herbal

mixtures have made large contributions to human health and well-being (Iwu et al.,

1999). Recently, herbal medicines have increasingly been used to treat many diseases

including several infections. There exists vast literature on the antiviral,

anticariogenic, anthelmintic, antibacterial, antifungal, anti-inflammatory and


39

antimolluscal properties of different plants parts (Javed et al., 2011; Tomczyk et al.,

2010; Aremu et al., 2010; Sireeratawong et al., 2010; Khan et al., 2009; Abdel et al.,

2005). There uses as remedies for many infectious diseases, searches for substances

with antimicrobial activity in plants are frequent (Khan et al., 2010). Plants are rich in

a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and

flavonoids, which have been found in vitro to have antimicrobial properties (Sastraruji

et al., 2010). Many of the plants have been investigated for development of novel

drugs with therapeutic properties (Rogozhin et al., 2011).

1.8 Antibiotic resistance

‘Selective pressure’ refers to the environmental conditions that allow

organisms with novel mutations or newly acquired characteristics to survive and

proliferate. Mutations that increase an organism’s resistance to antimicrobial agents

occur naturally in bacteria. Exposure to a stimulus that inhibits or kills the susceptible

majority of a bacterial population allows a resistant subset of strains to grow at the

expense of susceptible organisms. A minority of strains present in a given setting may

be resistant to the antibiotic being used. The selective factor is the antibiotic (usually)

to which the sub-population is resistant. Hence, the phenomenon of antibiotic

resistance is based on selection for organisms that have enhanced ability to survive

doses of antibiotics that would have previously been lethal.

1.8.1 Multidrug resistance (MDR)

We have come a long way using antibiotics after the landmark discovery of

‘Penicillin’ in 1928. Microbes have also got them selected with harder resistance

mechanisms. A phenomenon of great concern in the medical community is the rise in


40

multi-drug resistant organisms, defined as microbes with simultaneous resistance to

more than one class of antibiotics (Guyot et al., 1999). Patients infected with such

organisms experience significantly higher degrees of treatment failure, prolonged

antibiotic usage and morbidity associated with infection (Sobel and Kaye, 2004;

Gupta et al., 2001). Among the wide array of antibiotics, β-lactams are the most

varied and widely used agents in use (Greenwood, 2000). The most common cause of

bacterial resistance to β-lactam antibiotics is the production of β-lactamases. Bacterial

resistance to β-lactam antibiotics has been attributed to the spread of plasmid-

mediated extended spectrum β-lactamases (ESBLs) (Normark and Normark, 2002).

1.9 Herbal extracts versus antibiotics

An antibiotic is a drug used to treat infections caused by bacteria and other

microorganisms. Originally, antibiotic was defined as a substance produced by one

microorganism that selectively inhibited the growth of another. Synthetic antibiotics,

usually chemically related to natural antibiotics, have since been produced that

accomplish comparable tasks. As opposed to the synthetic drugs, antimicrobials of

plant origin (Table 1.2) are not associated with many side effects and have an

enormous therapeutic potential to heal many infectious diseases. Pharmaceutical

antibiotics are usually made from one chemical that fights bacteria (Eisenberg et al.,

1993). Herbs contain hundreds of compounds. Since it's easier to develop a resistance

to one chemical than many, resistance against medicinal herbs is less likely to occur.

In recent years, patients in the United States and Europe have been increasingly

turning to unconventional medications as an alternative therapy. However, extensive

scientific data regarding their clinical effectiveness is lacking.


41

Table 1.2: Plants containing antimicrobial activity.

Common name Scientific name Activity Ashwagandha Withania somniferum Bacteria, fungi

Aveloz Euphorbia tirucalli S. aureus Bael tree Aegle marmelos Fungi Barberry Berberis vulgaris Bacteria, protozoa

Basil Ocimum basilicum Salmonella, bacteria Bay Laurus nobilis Bacteria, fungi

Betel pepper Piper betel General

Black pepper Piper nigrum Lactobacillus,Micrococcus, E. coli, E.

faecalis Blueberry Vaccinium spp E. coli Burdock Arctium lappa Bacteria, fungi, viruses Caraway Carum carvi Bacteria, fungi, viruses Cashew Anacardium pulsatilla P. acnes, Bacteria, fungi

Castor bean Ricinus communis General

Chamomile Matricaria chamomilla

M. tuberculosis, S. typhimurium, S. aureus, helminths

Chapparal Larrea tridentate Skin bacteria Clove Syzygium aromaticum General Coca Erythroxylum coca Gram-negative and –positive cocci

Cockle Agrostemma githago General Coltsfoot Tussilago farfara General

Coriander, cilantro Coriandrum sativum Bacteria, fungi Cranberry Vaccinium spp. Bacteria Dandelion Taraxacum officinale C. albicans, S. cerevisiae

Dill Anethum graveolens Bacteria Eucalyptus Eucalyptus globulus Bacteria, viruses Fava bean Vicia faba Bacteria

Garlic Allium sativum General

Ginseng Panax notoginseng E. coli, Sporothrix schenckii, Staphylococcus,

Trichophyton Goldenseal Hydrastis canadensis Bacteria, Giardia duodenale, trypanosomes Gotu kola Centella asiatica M. leprae

Horseradish Armoracia rusticana GeneralLemon balm Lemon verbena Aloysia triphylla Ascaris, E. coli, M. tuberculosis, S. aureus

Marigold Calendula officinalis Bacteria Onion Allium cepa Bacteria, Candida

Orange peel Citrus sinensis Fungi Quinine Cinchona sp. Plasmodium spp. Sainfoin Onobrychis viciifolia Ruminal bacteria

Tarragon Artemisia

dracunculus Viruses, helminths Turmeric Curcuma longa Bacteria, protozoa

Yellow dock Rumex crispus E. coli, Salmonella, Staphylococcus


42

1.10 Plants used in the present study

Trachyspermum ammi belongs to the family Umbelliferae and is known as a

popular aromatic herb and spice. Its fruit has been used as medicine, cooking and is

used to primarily control indigestion and flatulence. It is prescribed for colic, diarrhea,

antibacterial and other bowel disorders, and in the treatment of asthma (Nair and

Chanda, 2006). Prosopis is a genus of flowering plants in the pea family,

Leguminosae. It contains around 45 species of spiny trees and shrubs found in

subtropical and tropical regions of the Americas, Africa, Western Asia, and South

Asia. They often thrive in arid soil and are resistant to drought. Their fruits are pods

and may contain large amounts of sugar. Prosopis spicigera from the family

Leguminosae is not an extensively studied plant as not much literature is available. It

is known to possess anti-inflammatory property (Madan et al., 1972).

Ginger is a tuber that is consumed whole as a delicacy, medicine, or spice. It is

the rhizome of the plant Zingiber officinale. Ginger is on the FDA's "generally

recognized as safe" list. It lends its name to its genus and family (Zingiberaceae). It is

traditionally used for the treatment of rheumatism, nervous diseases, gingivitis,

toothache, asthma, stoke constipation, diabetes and arthritis (Chang et al., 1995). It

has phytoconstituents that have anti inflammatory, anti-oxidant and anti-cancer effects

(Isa et al., 2008; Ippoushi et al., 2003). Ginger contains up to three percent of a

fragrant essential oil whose main constituents are sesquiterpenoids, with (-)-

zingiberene as the main component. Smaller amounts of other sesquiterpenoids

(β-sesquiphellandrene, bisabolene and farnesene) and a small monoterpenoid fraction

(β-phelladrene, cineol, and citral) have also been identified. Ginger compounds are

active against a form of diarrhea which is the leading cause of infant death in


43

developing countries. Zingerone is likely to be the active constituent against

enterotoxigenic Escherichia coli heat-labile enterotoxin-induced diarrhea (Chen et al.,

2007).

Acacia nilotica is reported to have antimicrobial, antihyperglycemic and

antiplasmodial properties (Meena et al., 2006; El-Tahir et al., 1999; Sotohy et al.,

1995). Cinnamum zeylanicum and Syzygium aromaticum are known to possess

antipyretic activity (Lopez et al., 2005; Kurokawa et al., 1998) and essential oils from

these two species have been shown to possess antibacterial activities

(Prabuseenivasan et al., 2006). Eugenol, a compound found in S. aromaticum, is

reported to have strong antifungal (Chamin et al., 2004) and anti-inflammatory

activities (Dip et al., 2004) and has been investigated for its potential anticarcinogenic

effect (Dorai and Aggarwal, 2004). The essential oil from C. zeylanicum shows

antioxidant (Dhuley, 1999), antibacterial and antifungal activities (Wang et al., 2005).

Terminalia arjuna, a well known herbal cardiac tonic, is also known to possess

antimicrobial activity (Rani and Khullar, 2004; Miller, 1998).

Eucalyptus globules, traditionally used to treat diabetes (Duke, 1985). T.

arjuna contains ellagic acid, ethyl gallate, gallic acid and luteolin that exhibits

antimutagenic property (Kandil et al., 1998; Kaur et al., 1997). It also possesses a

significant antioxidant effect, comparable with vitamin E (Gupta et al., 2001). Plants

of the genus Eucalyptus have been shown to produce a number of phloroglucinol

sesquiterpene- or monoterpene-coupled compounds, namely, the macrocarpals and

euglobals. Their biological activities such as HIV-RTase inhibition, granulation

inhibition and antiviral effects have been reported (Nishizawa et al., 2001; Yamakoshi


44

et al., 1992). Globulol isolated from the fruit of this plant has been shown to be the

major source of its antimicrobial activity (Tan et al., 2008).

1.11 Objectives of the study

In view of the present background we initiated our study with the following

objectives:

To evaluate the effect of the selected plants extract and their active fractions on

the virulence properties of Streptococcus mutans; in vitro as well as in vivo.

To identify the gene expression pattern of genes involved in biofilm formation

in the presence of medicinal plant extracts.

To isolate and identify novel compound from plant source using spectroscopic

techniques; analyze its properties and assessment of application possibilities of

partially purified extracts containing these compounds against the virulence

factors of S. mutans.

To evaluate the antimicrobial activity of the selected plant extracts and their

solvent fractions against multidrug resistant bacteria and fungus.

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