Dental Caries Student Project

Dental Caries - A Student Project Sharan Bisla 2000 The information on these pages is provided as an instructional tool for general biology and introductory microbiology classes. It is not intended as a diagnostic aid, nor should it replace consultation with medical professionals.

What is dental caries? There are relatively few infectious diseases of the upper alimentary canal but one common disease is dental caries. Dental caries, a pathological process of destruction of tooth structure by microorganisms, is also known as tooth decay and is commonly called "cavities." Dental plaque, which leads to caries, is the oral flora which adhere to teeth and break down tooth enamel.

History of dental caries Prehuman skeletons more than 500,000 years old indicate that very little caries was prevalent. In the In Europe during the Medieval period less than 10% of human teeth had cavities and they were mostly on parts of the teeth exposed by the receding gums of older people. During the 17th century there was an increase in the incidence of tooth decay and location of cavities changed to include the sides and biting surfaces of teeth. Young people were also starting to get tooth decay along with older people. Then in the European diet flour and sugar (sucrose) were introduced and this lead to an increase in dental caries. (1) Archaeological evidence from preColumbian Mesoamerica indicates that dental caries became prevalent among people at about the time the agriculture shifted to the triumvirate of maize, beans, and squash, all of which have high sugar contents.

A number of factors are necessary for caries to occur. These factors include the interaction of the host, oral flora, and the substrate which is primarily sucrose (sugar). In order for tooth destruction to occur, there must be a susceptible host, cariogenic microorganisms and a suitable substrate present for a sufficient length of time. Tooth brushing can reduce the likelihood of caries by reducing number of cariogenic organisms and removing the substrate. (2)

How dental caries occurs and the cariogenic bacteria involved Organisms predominant in plaque are of the genus Streptococcus, with Streptococcus mutans being the species most important in the formation of dental caries. According to the Bergey's Manual of Systematic Bacteriology (3), S. mutans are non-motile Gram positive cocci. They are 0.5-0.75 micrometers in diameter, occurring in pairs, short medium length chains, without capsules. S. mutans have several properties that are important in the carious process. First, they colonize on tooth surfaces. Second, they synthesize insoluble polysaccharides from sucrose. This allows adhesion to smooth surfaces and appears to be important in the formation of smooth surface caries. Third, they ferment sucrose to form lactic acid. Another organism that is important in the development of caries is the Lactobacillus. Lactobacillus are gram positive, non-spore forming rods. They normally constitute only a small fraction of plaque flora when compared to S. mutans. Lactobacillus species are not important in the initiation of caries but in the continuation.

Variety of microorganisms in the oral flora We streaked samples of oral flora on blood agar plates in order to see the variety of oral microorganisms. The teeth were brushed a couple hours before taking samples.

The blood agar plates were incubated aerobically at 37C, temperature of the mouth. These blood agar plates were incubated for two days and we noted that white-gray colonies grew on the surface of the agar. This gave us an idea of the various kinds of colonies that were present in dental plaque. We saw a large variety of colonies varying in type, shape, size and color.

This semester Dr. Jacobs and I made Gram stains of our own dental plaque from different regions of the teeth. This Gram staining was performed not only to reveal the size and shape of the various bacteria, but also tell us some general information about their basic chemistry. These Gram stains were viewed through the microscope and we observed there was great diversity in the oral flora found in the dental plaque.

This picture shows the diversity in the oral flora found in dental plaque. The spheres (cocci) represent the Streptococcus and the rods show us Lactobacillus. These two types of bacteria were the most predominant in dental plaque.

This picture illustrates a cheek cell in the center covered by various microorganisms present in dental plaque.

There were Gram positive and Gram negative rods (bacilli), spheres (cocci), and spirals (spirillae). There were differences in bacteria present in different samples of the mouth. We found that even in a "clean" mouth (the teeth had recently been brushed) there were still millions of bacteria in the dental plaque.

Corrosion chemistry of oral bacteria Lactic acid is a strong acid that is effective in demineralization of tooth structure. S. mutans are present in the mouth only when a solid surface such as teeth is present. Salivary concentrations of S. mutans range from undetectable to 10,000,000 colony forming units/ml, with a mean concentration of about 100,000 CFU/ml. (4) Streptococcus mutans and Lactobacillus are acidogenic and aciduric meaning that they can produce acids which can dissolve the tooth substance (calcium phosphate in the form of hydroxyapatite crystals) and that they can survive and even produce acids in a low pH environment. Compared to other oral flora, Streptococcus mutans and Lactobacillus are cariogenic and have enzymes with greater resistance to acid. They also have greater capacities of the cells to extrude protons from the cytoplasm. The most important enzyme for extrusion of protons is ATPase. This enzyme is found in higher amounts and is working at lower pH with S. mutans and Lactobacillus. Cariogenic bacteria produce lactic acid when they are exposed to high concentrations of sugar. Lactic acid has a pK of 3.8, while other carboxylic acids have a pK of 4.8. (The lower the pK number, stronger the acid and weaker the base.) Lactic acid can chelate the calcium phosphate and forces the chemical equilibrium to shift so the saliva, which is supersaturated with calcium phosphate ions, can not replace the calcium phosphate as rapidly as it is being pulled away from the teeth. Dental caries is essentially corrosion of teeth enamel. The bacterial cell membrane is permeable to undissociated carboxylic acids. These undissociated acids act as carriers to bring protons back into the cytoplasm. Bacteria that make lactic acid as a fermentation product can grow and produce acids at one pH unit lower than bacteria that make acetic, propionic, or butyric acid as a fermentation product. For this reason, these bacteria are especially important in formation of dental caries. (5)

Experiments we conducted confirm corrosion chemistry of dental caries During this semester we conducted an experiment to see how certain oral flora can destroy the tooth enamel which is made of calcium phosphate. We produced a minimal salts medium which consisted of calcium phosphate, sucrose, agar, water and other chemicals. After creating this minimal salts medium it was poured into sterile plates and solidified. Then samples of the different colonies that were grown on the blood agar plates were taken and smeared on the solidified calcium phosphate medium

plates. These plates were also incubated at 37C, again the temperature of the mouth for about three weeks and were observed twice a week, at the beginning and middle of the week. As time progressed I observed there was a clearing effect being produced around certain colonies of bacteria because of acid production.

We concluded from these experiments certain bacteria can ferment the sucrose into organic acids such as lactic acid. The lactic acid lowers the pH enough to dissolve the calcium phosphate in the medium as it would with tooth enamel (see pictures of results from the experiment).

Prevention Dental caries is a complex multifactoral disease. One of the most important factors in the etiology of dental caries is the patient's level of oral hygiene. If thorough hygiene is maintained there will not be a build up of plaque and bacterial colonization of tooth structure is not initiated. Maintenance of good oral hygiene requires education, motivation and reinforcement if compliance is to be achieved. The dietary profile also plays a important role in dental caries. The amount of sucrose consumed, the consistency of the food, and the frequency of feeding all affect the level of caries activity. The availability of fluoride in water will also decrease dental caries. Fluoride is incorporated into the calcium phosphate matrix of enamel and reduces the occurrence of dental caries.

What did I learn?

Before doing this directed study I was unaware of the vast diversity in bacteria that is present in the mouth. Each human has its own unique oral flora and the composition of the oral flora determines the susceptibility to tooth decay. The microorganisms that adhere to the tooth surface are called dental plaque. The dental plaque causes tooth decay by fermenting sucrose to lactic acid. The most interesting part of the study was creating a calcium phosphate medium which represented tooth enamel and growing colonies of bacteria to confirm corrosion chemistry of dental caries. It was interesting to look at dental caries from both microbiological and chemical perspectives and how they combined to cause tooth decay.

References (1) Nester, E. W., C. Roberts, N. Pearsall, D. Anderson, and M. Nester. 1998. Microbiology: A Human Perspective. WCB McGraw-Hill , Boston. (2) http://sdm.uchc.edu/ProjectD/Knowledge/1.Texts/4.Disease/DentalCaries.HTML Back to text (Dental school website at the University of Connecticut) (3) Holt, J. G., N. R. Kreig, P. Sneath, J. Staley, and S. Williams. 1994.Bergey's Manual of Determinative Bacteriology Ninth Edition. Williams&Wilkins, Baltimore. (4) http://sdm.uchc.edu/ProjectD/Knowledge/1.Texts/4.Disease/DentalCaries.HTML (5) http://www.odont.lu.se/mutans/mutgen.html Back to text