Microbiology of Enamel Caries

Introduction
The process of enamel caries begins when acidogenic bacteria (such as streptococci and
lactobacilli) are exposed to glucose/sucrose in the dental biofilm. There are more than one
type of bacteria involved in the initiation and progression of enamel caries such as mutans
streptococci and non-mutans streptococci. These bacteria take in the sucrose and in turn
produce acids, resulting in a increase in concentration of hydroxyl ions, thus decreasing the
pH. The pH rapidly decreases from a resting pH of 7.0 to a pH of less than 5.0 within the
biofilm fluid and along the interface between the biofilm and enamel surface. When the
critical pH (about pH 5.5) is reached, demineralization of the enamel begins.
Formation of Enamel caries
Enamel is a unique hard tissue that is 95% mineralized (minimal protein content). It is a
cellular and non-vital (cannot repair itself)tissue, but at the same time, it is permeable
(water and small molecules) and always in a dynamic chemical equilibrium with the oral
environment.
Enamel caries is primarily a chemical process that is a result of an increase in proportions of
aciduric and acidogenic bacteria, especially mutans streptococci (eg. S. mutans and S.
sobrinus) and lactobacilli have been proven clinically to be responsible for most caries
formation as they are able to demineralize the enamel through the rapid metabolic activity
of utilizing sugars to produce acids. The acid produced provides a low pH which allows an
optimal environment for the aciduric bacteria to proliferate. [Marsh PD, 2006]
The pH of a tooth surface is highly dependent on three factors:
sites with large amount of dental plaque,
easily fermentable/metabolized carbohydrate sources and
low rates of diffusion of substrates and metabolites into and out of the plaque
are the susceptible to caries. This explains why pits and fissures have the highest
occurrence for enamel caries. Studies have shown a strong correlation between plaque
levels of mutans streptococci and caries at these sites. The deepest parts of these surfaces
are also often not accessible to toothbrushes, resulting in high availability of
substrate(carbohydrates) for the respective bacteria, leading to more production of acid,
thus further demineralization. However, recent studies have shown that mutans
streptococci were only minor components of plaque at these carious sites and these sites
had relatively high levels of lactobacilli, suggesting that lactobacilli might be responsible
for the demineralization.
*Additional research results have shown that mutans streptococci have a strong
relationship with initial caries formation, and lactobacilli were strongly associated with sites
that required restoration.
When there is a drastic change in oral environment (eg. reduced salivary flow rates),
rampant caries might occur. Further studies involving patients undergoing radiation
treatment have shown a large increase in the proportions of mutans streptococci and
lactobacilli in plaque and saliva. Large percentage of data collected have shown a strong
positive association between increased levels of mutans streptococci and
lactobacilli. [Fejerskov O. & Kidd E., 2008]
Bacteria involved in caries formation
1. Mutans streptococci (MS)
Evidence of presence of mutans streptococci:
Mutans streptococci is a group of bacteria that is commonly found at cavitated caries
lesions and have been proven to cause caries formation in animals that have a sucrose-rich
diet. It is able to synthesis water-insoluble glucan that makes the tooth surface more
susceptible to bacteria adhesion. [Hamada S, Slade HD, 1980]
Characteristics of mutans streptococci:
Mutans streptococci are non-mobile, catalase-negative, gram-positive cocci and form
short/medium chains. They are capable of rapid transport and fermentation of dietary
carbohydrates into lactic, formic, acetic and proprionic acids. These bacteria can undergo
both extracellular and intracellular polysaccharide synthesis even under adverse
environmental stress. However, the two most important characteristics of mutans
streptococci that closely relates it to the formation of caries is that it
is acidogenic (produce organic acid) and aciduric (can survive in acidic environments).
Action of mutans streptococci:
Mutans streptococci rapidly synthesize insoluble polysaccharides from sucrose. They
colonize on tooth surfaces and they are homo-fermentative lactic acid formers. When
excess sugar is available, streptococci are able to store these sugar as intracellular
polysaccharides (IPS) which can then be used as an energy source in between meals to
produce acid [Hamilton, 1976; Van Houte et al., 1970]
*Both non-mutans streptococci and mutans streptococci metabolize sugar to produce
acid. [Langlais RP, et al, 1994]
Other theories regarding mutans streptococci and other bacteria involved in caries
formation:
Studies have shown that the presence of high concentrations of mutans streptococci does
not necessarily equate to white lesion formation on tooth surface, and certain caries can
form even without a trace of mutan streptococci present [Nyvad, 1993].
Caries can be initiated by non-mutans streptococci (non-MS) and Actinomyces [van Houte et
al., 1996; Sansone et al., 1993]

Other bacteria such as A.gernesceriae, A. naeslundii, and A. israelli might also be

responsible for caries formation [Becker et al., 2002; Aas et al., 2008] In other words, any
bacteria species that are aciduric and dominant can be responsible for caries formation.
Further studies have also proven that dental biofilm also contains bacteria such as
Veillonella, which can metabolize lactic acid produced by the acidogenic
bacteria. Streptococcus salivarius and Streptococcus sanguinis synthesize arginine
deaminase and urease which are able to create urea and ammonia compounds that can
increase the pH and neutralize the acid produced by the acidogenic bacteria [Gracia & Hicks,
2008]

2. Non-Mutans streptococci (non-MS)

Non-MS refers to:
mitis streptococci
viridan streptococci (excluding the mutans group).
Recent studies show that non-MS also play an important role in caries formation.
Evidence of presence of non-MS
Non-MS have adhesins which adhere to proteins and sugar chains of acquired pellicles
coating the tooth surface. Non-MS have a variety of extracellular glycosidases that can
liberate sugars and amino-sugars from glycoproteins such as the mucin contained in saliva.
In addition, most non-MS can utilize arginine or arginine-containing peptides available in
saliva through the arginine deaminase system, which degrades the arginine molecule to
ammonia and carbon dioxide with production of ATP.
In summary, non-MS have diverse physiological activities, suggesting that they are versatile
enough to adapt to various conditions in supragingival biofilm. On the other hand, MS are
aciduric specialists in sugar metabolism and acid production, which make them less
competitive in clinically sound supragingival environments. [Takahashi & Nyvad, 2008]
Sansone et al proved non-MS were dominant at clinically healthy sites and white spot
lesions while MS were present at low and similar levels at both sites. However, the ability
of plaque to reduce pH in vitro was significantly greater at white spot lesions (pH 4.13)
that at clinically healthy sites (pH 4.29). These results suggest that MS are neither a unique
causative agent for white spot lesions, nor a main determinant of the acidogenicity of
plaque.[Sansone C, 1993]
Characteristics of non-MS
Sansone et al and Svenster G et al found that non-MS were heterogeneous for
acidogencity: some strains lowered the culture pH to below 4.4, a pH comparable to that
produced by MS, whereas for other strains the pH-lowering capacity was less pronounced.
In addition, the proportion of acidogenic non-MS was higher at white spot lesions than
clinically healthy sites[Sansone C, 1993] [Svenster G, 2003]. The acidogenic non-MS,
identified as S. gordonii, S. oralis, S. mitis and S. anginosus, were subsequently designated
as 'low-pH' non-MS.
Non-MS are not only genotypically heterogenous, but they are also able to change their
physiological characteristics adaptively. Moreover, they were reported to be able to
increase their acidurance adaptively. In the oral cavity, acidification of the biofilm due to
frequent sugar intake or poor salivary secretion can be a driving force to enhance the
acidogenicity and acidurance of the non-MS, resulting in establishment of a more acidic
environment. 'Low-pH' non-MS will increase selectively in this environment. This will cause
a shift in the composition and acidogenic potential of the biofilm, which, provided the
demineralization/remineralization balance is disturbed over an extended period of time,
leads to dental caries. [Takahashi & Nyvad, 2008]
Although 'low-pH' non-MS can adaptively increase their acidurance and acidogenicity, and
take over the position in supragingival plaque, MS are more competitive under severely
acidic conditions.[Takahashi & Nyvad, 2008]
Distribution of MS and Non-mutans bacteria in carious lesions [Takahashi & Nyvad, 2008]
Initial colonization: The initial colonizers of newly cleaned tooth surfaces constitute a
highly selected part of the oral microflora, mainly S. sanguinis, S. oralisand S. mitis. These
three species, which belong to 'mitis group' , may account for 95% of the streptococci and
56% of the total initial microflora. However, MS comprise only 2% or less of the initial
streptococci population. Mitis group bacteria as well as other viridans group streptococci,
except for the MS, are often referred to as the non-MS. The predominant species in mature
smooth surface plaque belong to non-MS. MS are found in very low numbers.
White spot lesions: The proportion of MS in plaque covering white spot lesions in enamel
is often higher than at clinically healthy sites, although still rather low, ranging between
0.001 and 10%. Meanwhile, non-MS still remain major bacterial groups in enamel lesions. It
has been shown that in the absence of MS and lactobacilli, the initial dissolution of enamel
can be induced by members of the early microflora, exclusively.
Caries lesions: In caries lesions, MS constitute about 30% of the total flora, indicating that
these species are associated with progressive stages of caries.
3. Lactobacilli
Evidence of presence of Lactobacilli
Recent findings have proven that other than non-mutans streptococci are present in dental
biofilms covering white spot lesions, including Lactobacilli [van Ruyven et al.,
2000] .Lactobacilli are not involved in the initiation of plaque due to their inability to adhere
to the enamel surface without the presence of other bacteria. However, after initial caries is
formed, lactobacilli is proven to play an important role in the progression of dental caries,
as it is shown that the amount of mutans streptococci decreases when a low pH is
available, whereas the amount of lactobacilli increases. The reason behind such action is
that lactobacilli is dependent on extracellular polysaccharide (EPS) produced by mainly
streptococci before it can colonize the tooth. Once lesions are established, lactobacillus
will then contribute to the demineralization of the teeth [Tanzer et al, 2001]
4. Actinomyces
Most of our knowledge about the role of Actinomyces in caries stems from studies of root
surface caries. However, there is no evidence that Actinomyces spp. have a specific role in
root caries. The basic patterns of microbial colonization are identical on enamel and root
surfaces, structurally as well as microbiologically. MS comprise only a small proportion of
the microflora of caries lesions, while non-MS and Actinomyces spp. were dominant in
dental plaque.
Actinomyces have adhesin-mediated adhesion to tooth surfaces, produce acids from various
sugars, and synthesize intracellular and extracellular polysaccharides. They have the similar
microbial acid adaptation and acid selection processes as the non-MS.
An extended caries ecological hypothesis
The extended caries ecological hypothesis explains the degree of involvement of different
bacteria at different stages of enamel caries formation.
In this hypothesis, dental plaque is a dynamic microbial ecosystem in which non-mutans
bacteria such as non-MS and Actinomyces are the key players for maintaining dynamic
stability. These bacteria can produce acids and the resulting acids can demineralize the
enamel. However, it is easily returned to neutral level by homeostatic mechanisms. When
there is abundant substrates (carbohydrates) or reduced salivary flow in the oral cavity,
this dynamic equilibrium gets disrupted. The pH decreases in the plaque may enchance the
acidogenicity and acidurance of the non-mutans bacteria adaptively. The population of nonMS and Actinomyces increases via acid selection, which alters the microbial environment to
an acidic one. When this acidic microflora is maintained for an extended period, the original
net gain of minerals on the enamel surface will change into a net loss of minerals due to
the demineralization of the acids produced. At this initial stage of demineralization, the
microflora could still be reversed and the net loss of mineralscould be gained back. If this
acidic microflora continues, more aciduric bacteria such as MS and lactobacilli may replace
the 'low-pH' non-mutans bacteria. They further accelerate the caries process by sustaining
an environment characterized by 'net mineral loss'. [Takahashi & Nyvad, 2008]
Although mutans streptococci are usually present in large amounts in carious enamel and
dentin, they seem to only play a minor role in white spot lesion formation on sound enamel
surfaces. From the initiation stage till the formation of white spot lesion, non-MS and
Actinomyces plays a more important role as compared to mutans streptococci. The major
role of mutans streptococci only begins when there is already caries on the enamel surface.
Gram-positive anaerobic rods and filaments, specifically lactobacilli, predominate in the
microbiota of deep dentinal caries. Therefore, dental caries in humans is caused by more
than one type of microorganism, depending on the many different factors that may
influence the formation, composition, and metabolism of the dental plaque.