Microbiology of Enamel Caries

-Lin Yi

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), 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 occurance 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 bateria, 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 is a nonmobile bacteria that is catalase-negative and gram-positive cocci in 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 homofermentative lactic acid formers. When excess sugar is available, streptococci is able to store these sugar as intracellular polysaccharides (IPS) which can then be used as an energy source in between our 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 bateria 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 synthesis 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-MS
Non-MS refers to:
1. mitis streptococci
2. 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 deiminase 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 generalists, 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 Svensäter 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] [Svensäter 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 ba 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]

3. 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. oralis and 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 predonimant species in mature somooth 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.

4. Lactobacilli

Evidence of presence of Lactobacilli

Recent findings have proven that non-mutans aciduric bacteria other than non-mutans streptococci are present in dental biofilms covering white spot lesions, including Lactobacilli [van Ruyven et al., 2000] .Lactobacilli is not involved in the initiation of plaque due to its inability in adhering to the enamel surface without the presence of other bacteria. However, after initial caries is formed, lactobacilli is proven to play a 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]

*Details on lactobacilli can be found in Specific Plaque Hypothesis

5. 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 deantal 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 non-MS 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 caies 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. [Langlais RP, et al, 1994]

References:

Becker MR (2002), Molecular analysis of bacterial species associated with childhood caries. J Clin Microbiol.40(3):1001-9.

Fejerskov O. and Kidd E. (2008). Dental Caries: the Disease and its Clinical Management (2nd Ed) (pg 179 to 180). Oxford: Blackwell Munksgaard Ltd.

Fejerskov O. and Kidd E. (2003). Dental caries the disease and its clinical management(1st edition) (pg 83, 85). Oxford: Blackwell Munksgaard Ltd.

Franklin Gracia-Godoy, M. John Hicks (2008), Maintaining the integrity of the enamel surface: the role of dental biofilm, saliva and preventive agents in enamel demineralization and remineralization. J Am Dent Assoc. 139 Suppl:25S-34S.

Hamada S, Slade HD (1980), Biology, immunology, and cariogenicity of Streptococci mutans. Microbiol Rev, 331-384

Hamilton IR (1976), Microbial aspects of dental caries, Pg 683 -701

Langlais RP, Langland OE, Nortje CJ. (1994). Diagnostic imaging of the jaws. Baltimore: Williams & Wilkins. Chapter 6 pg164

Marsh PD (2006), Dental plaque as a biofilm and a microbial community - implications for health and disease. BMC Oral Health.15;6 Suppl 1:S14

N. Takahashi, B Nyvad (2008), Ecology Revisited: Microbial Dynamics and the Caries Process. Caries Res ;42:409-418

Nyvad B. (1993). Microbial colonization of human tooth surfaces. APMIS Suppl. 1993;32:1-45.

Sansone C, van Houte J, Joshipura K, et al. (1993) The association of mutans streptococci and non-mutans streptococci capable of acidogenesis at a low pH with dental caries on enamel and root surfaces. J Dent Res 1993;72:508-516

Svensäter G, Borgström M, Bowden GH , et al (2003) The acid-tolerant microbiota associated with plaque from initial caries and healthy tooth surfaces. Caries Res. 37(6):395-403.

Tanzer JM, Livingston J, Thompson AM. (2001). The microbiology of primary dental caries in humans. J Dent Educ. 2001 Oct;65(10):1028-37.

Van Houte J, Lopman 1, Kent R (1996). The final pH of bacteria comprising the predominant flora on sound and carious human root and enamel surfaces. J Dent Res 75:1008-1014.

Van Houte J, Gibbonsr J. & Bangharts B. (1970). Adherence as a determinant of the presence of Streptococcus salivarius and Streptococcus sanguis on the human tooth surface. Archives of Oral Biology 15, 1025-1034.

van Ruyven FO, Lingström P, van Houte J, Kent R (2000), Relationship among mutan streptococci, 'low-pH' bacteria, and iodophilic polysaccharide-producing bacteria in dental plaque and early enamel caries in humans. J Dent Res 79(2):778-78A.




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