1. 1
Introduction
One of the most common bacterial infectious diseases in humans is dental caries.
Since the human diet has changed to that of high carbohydrates, dental caries have been
always making people suffer because oral bacteria are always living inside the mouth,
and their nutrition comes from the host’s food, so it is highly unlikely to prevent dental
caries in any generation. The history of dental caries has been found even on ancient
humans’ teeth. There were many historical evidence showing how much people were
trying to fight their dental problems. Especially before people discovered correct
treatments and analgesic medications, oral problems were directly connected to a
decreased maintenance of nutrition. People have been researching the cause of dental
problems and how to prevent them. People found out the main oral bacteria in humans’
mouths that cause dental caries are Streptococcus mutans. These bacteria mainly produce
biofilms, make acidic conditions in a mouth’s environment that cause the enamel of the
tooth surface to become weak, and they create dental calculus gradually. When people
get this bacteria, it is very unlikely to kill off all of them from the mouth even with
cleaning the mouth every day. People inherit this bacteria mainly from their close family
members, such as their mothers. It is important for people to know how to prevent
spreading of their bacteria to their children. Some scientists found out that ancient people
had different oral bacterial conditions compared with the modern mouth environment,
and they created probiotic treatments based on those researches. Probiotic treatment is
safe for anyone in any generation to help prevent future creation of dental caries and
problems.

2. 2
The History of S. mutans
Clarke (1924) used Robert Koch’s disease-causing germs research technique to
discover bacteria that live on the teeth. He cut off the root of an extracted tooth with low
levels of caries, and dug into the pulp of the tooth using a chisel to expose the carious
lesion, and whittled away at the diseased tissue with a small drill, then he cultured the
tissue in a glucose agar flat board with a 7.0pH (Figure 1). The colonies were elevated,
grayish white, and opaque. This bacteria grew to a medium size coccus form without
having a capsule in neutral or alkaline condition. He cultured other colonies from 50
different caries teeth and found they had homogeneous properties with the first colonies’
bacteria, so he decided those belonged to the same species. He compared the species
with other Streptococcus groups based on their biological properties and morphological
characteristics, but it did not fit with any others, so he named this bacteria Streptococcus
mutans.

3. 3
Figure 1. Colonies of S. mutans after 24 hour incubation on glucose agar plates (Clarke,
1924).
After the discovery of S. mutans, the Chicago University Zollar research team and
Notordam University Lobund research team studied how oral bacteria affects the human
mouth in 1955, as reported by Onisi (1971). They fed a caries induction diet to 22
germfree rats and 39 commercial rats. The results showed the 39 commercial rats that
had oral bacteria developed caries. On the other hand, the 22 germfree rats did not
develop any caries even though they had been eating the same type of food. This
research provided the evidence that oral caries is an infectious disorder caused by oral
bacteria. Then, they fed the caries induction diet with various bacteria that had
previously succeeded in causing caries to 13 germfree rats. The bacteria that succeeded

4. 4
in causing caries outbreaks were combinations of enterococcus, a protein lytic bacterium
and anaerobic polymorphic bacteria (Onisi, 1971).
Properties of S. mutans
The morphology of S. mutans is: gram positive, with neutral alkalinity. When S.
mutans grows in liquid nutrient mediums, it makes a medium or long chain of spherical
cells, and in acidic conditions, it becomes bacillary. When it grows on an agar dish, the
cell body gets longer, and the color of colonies is grayish white or light yellow. Its
nucleoid region is spherical when observed by cytological staining. The best growth
condition is a pH of 7.0, and it cannot grow under the pH 5.6. The production of the acid
is quick, and within the first 24 hours, it makes its surrounding environment pH 4.2,
which is the strongest acidic pH level it can create. Then, it dies within the next 48 to 72
hours. The carbohydrates that S. mutans decomposes to produce acids are: glucose,
lactose, raffinose, mannitol, inulin, salicin, sucrose, maltose, levulose, galactose,
mannose, melibiose, cellobiose, sorbitol, trehalose, esculin, and dulcitol. In contrast, S.
mutans do not produce acid from: glycerol, arabinose, xylose, rhamnose, inositol,
melezitose, and sorbose (Onisi, 1971).
The Main Issue: Dental Caries
Dental caries is one of the most prominent diseases people experience around the
world (Larson, Shavit, and Griffin, 1991). It is defined as the destruction of enamel and
dentin tissue that results in tooth decay (Medical Dictionary, 2015 a). Caries are also
known as dental cavities. While caries are a growing concern for modern days, they have

5. 5
not been as much of a major issue for humans in the past. Ever since the first tooth has
been painful, people have been trying to find ways to fix these types of problems. The
results have varied from country to country, and different concepts that were successful
have quite easily spread internationally. This paper will provide insights to the
development of dental caries and the expansion of knowledge to combat one of the
world's leading diseases.
Caries are caused by the byproducts of many oral bacteria. The two main
components of caries are Streptococcus mutans and Lactobacillus acidophilus (Larson et
al., 1991). S. mutans is a non-motile, gram positive bacteria that is mainly responsible for
the initiation of caries formation (Bisla, 2000). S. mutans is one of the few bacteria that
is capable of binding to the smooth, flat surface of teeth (Bisla, 2000). It functions by
breaking down different types of carbohydrates into various products, depending on what
type of sugar was used. The main form of sugar used by this bacteria is sucrose, which is
catabolized into energy for the bacterium, while the byproduct is lactic acid. If the sugar
present in the surrounding environment is not sucrose, it is broken down into a form of
sticky, pasty substance that is used for the formation of biofilms (Medical Dictionary,
2015 b).
The Origins of Bacterial Oral Flora
These bacteria have been present in humans since the evolution of the first
homosapiens 100,000-200,000 years ago (Flores, 2007). The presence of caries, however,
has been very low overall. If these bacteria have been present from such a long time ago,
then why is it that caries have not been as prevalent as it is today? Just to make a point,

6. 6
caries have still been present in prehistoric times, but it was quite rare during those times.
It is important to note that the earliest of humans had been primarily carnivores with very
little use of vegetation. The main diets of our early ancestors was fish and other seafood,
while hunting on land was used later. Just hundreds of years ago when agriculture
became a prominent part of human society is when caries became a strongly growing
issue.
Larson et al. (1991) named 4 categories for classifying the evolution of human
dietary habits: precontact preagriculture (1000BCE- 1150AD), Precontact agriculture
(1150- 1550AD), early contact (1607- 1680AD), and late contact (1686- 1702AD). In
the study performed by Larson et al. (1991) just under 900 skulls of various ages and
genders were evaluated from the south-east Atlantic coast known as the Georgia Bight.
This area was chosen for study because it is abundant in multiple resources necessary to
sustain a social group of individuals. The tooth decay for this study was measured by the
amount of lesions found per type of tooth, how much damage they caused, and other
structures of the jaw that were affected because of caries. Larson et al. (1991) also took
into consideration the different genders and age groups, especially considering that the
young had not been given as much time to develop caries as their older counterparts.
They had also considered the amount of natural damage applied to teeth over time
through the natural grinding process performed when chewing foods.
The results of the study had supported the idea that dental caries are directly
related to the advancement of agriculture. The results were separated into categories
based on age, gender, type of tooth/teeth affected and a combination of the three. Overall
it was found that the precontact preagricultural societies experienced the fewest caries.

7. 7
Once people were introduced to the idea of agriculture in the precontact period the
amount of caries vastly increased. This trend increased for men into the early contact
period, but interestingly enough the amount of caries slightly decreased for both women
and young adults, seen in Figure 2. This difference provided an explanation as to the
location for the majority of samples collected for the precontact agriculture period. The
skulls collected and studied were from a population in which agriculture played a
particularly significant importance in the inhabitants of that area when compared to the
rest of the coastal locations. There was also a large increase in caries during the late
contact period for all groups studied.
Figure 2. A Comparison of tooth decay across genders, age, and time periods (Larson et
al., 1991).

8. 8
The same trend can be seen in the different types of teeth that were studied. All
teeth had an overall increase from the precontact preagricultural period to the late contact
period (Figure 3). The teeth most affected by the increase of agriculture were the molars.
This is because molars are the main teeth used in grinding food once in has entered the
mouth. Therefore, they come in most contact with the foods we eat, and stay in contact
for the longest amounts of time when compared to the other teeth. The molars also have
the least smooth surface, which allows far more bacteria to stick to them easier, as well as
have food particles remain trapped on their surface.
Figure 3. Comparison of tooth type and caries formation at different time periods
(Larson et al., 1991).

9. 9
The same type of trend can be seen in the Japanese culture. Around 900 AD
Japanese people began agriculture with their major harvest of rice, which led to a drastic
increase in the prevalence of caries. There was a great decrease in the number of people
with caries around World War II because of the lack of resources available to the citizens
at that time. There is a very interesting story associated with Japan and America after the
war was finished. After the war American soldiers were sent to Japan to make a peace
offering by bringing many new types of foods, particularly chocolate. The Japanese
children were thrilled by this new food and quickly learned to associate the words “give
me chocolate” with the action of receiving the food— even though they did not
understand what the words had meant. This introduction of foods also led to what can be
considered the Japanese industrial revolution in which the supply of food was greatly
increased for all citizens, marking the return of caries, with an increased amount even
when compared to the previous agricultural boom.
A recent study performed by Adler, Dobney, Weyrich, Laidonis, Walker, and
Haak (2013) supports the idea that the oral bacteria strains have been decreasing in
variety after two major points of human technological evolution have occurred: the
switch from hunter-gatherer to farming, and the industrial revolution. Their method of
research was to study the buildup of calculus on the teeth of individuals that lived from
the pre-mesolithic period to the medieval period in Europe. They took the plaque
samples from roughly 34 skeletal remains and isolated DNA from each sample. The
DNA was then sequenced into cDNA libraries that had three variable regions for
bacterial identification. The lab used primers specific for S. mutans strains and
Porphyromonas gingivalis as well as other bacteria found in the modern oral cavity.

10. 10
It was found that the bacteria collected from the ancient samples had quite a bit different
composition than bacteria found in the environment, but they were quite similar to the
overall composition of modern dental bacteria strains. The ancient samples also had a
much higher concentration of Actinobacteria compared to modern samples. It was
discovered that the biodiversity of bacteria from the pre-mesolithic samples was much
larger than modern samples, and there was a much stronger balance between benign and
harmful bacteria than in the samples from the medieval period and modern samples. It is
also interesting that the composition of oral bacteria from the medieval time was very
similar to modern oral bacteria, while the main food source has not changed much from
the medieval times to now. With less diversity, humans are now more susceptible to
invasive bacterial species and other harmful pathogens.
Risk Factors and S. mutans Proliferation
The transfer of oral bacteria has already been researched extensively. According
to the American Association of Pediatric Dentistry strains of S. mutans are spread from
parent to child through a passing of salivary fluids. This type of horizontal bacterial
transmission depends strongly on the amount and quality of saliva an adult produces, and
the activity the adult does that involves sharing said saliva with the infant. In many cases,
the bacteria are passed on to the infant even when they have no teeth and are hosted
around the tongue until teeth are protruded. In many other cases the bacteria is passed on
once the child has a couple of teeth already erupted from the gum line. The passing of
these bacteria strongly depends on how the adult interacts with the child, which means
that even an act as simple as kissing the child on or near the lips before they sleep could

11. 11
have negative consequences. When feeding a child it is most common to pass on these
bacteria because the adult will often chew larger foods to make it easier for the child to
continue the digestion process with already softened food. Another variable for
transmission includes the activities of slightly older children at day care centers. Since
children have a natural tendency to place objects in their mouths the passing of bacteria
between children is relatively easy if they are not under strict supervision of the caretaker.
Those with the highest risk of bacterial transfer are often of low socioeconomic
status because of a number of different reasons, with the most prominent being little oral
health care education, and the consumption of high carbohydrate snacks (AAPD, 2014).
Little or no education in oral health care means less time brushing teeth and not using
floss or mouthwash are great ways to make sustain a nurturing environment for the
cariogenic bacteria. Prevention is always possible if adults are willing to make the effort
to do so. Of the prevention methods available, the most important is proper oral health
care education. This is because it allows parents to change their own habits early in the
child's life so that the child may copy these better habits and have it more permanently
engrained in their memories. Another method of prevention is having professional dental
work performed on teeth to maintain a healthy mouth and remove any harmful bacteria
that can be passed on as well as cause caries in the parent. One of the more important
recent discoveries is the use of fluoride in mouthwashes and in toothpaste since it is one
of the essential minerals used in tooth formation and it helps to remineralize the teeth
while reducing the effects of cariogenic bacteria.

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A Brief History of Dental Care
Let us now look at evidence of the first forms of technology developed in fighting
dental caries. Tooth pain has been an issue that came up occasionally for people, and the
cause was not very well known. In a review article by Forrai, it was written that the only
knowledge people had was that if the tooth had been damaged enough to break open, the
inside pulp would come out in an almost stringy substance that was very painful when
touched. Therefore, people used to say that the teeth were always infected with “tooth
worms,” as seen in Figure 4.
Figure 4. Depictions of the Tooth Worm. Mythological Destruction (left) and a swollen
nerve chord (right) (Forai, 2006).
Ancient Egyptians wrote their concerns and possible remedies for tooth infections
in their book called the Papyrus Ebers. Their book covered various cures to ailments like
honey or onions to help toothache, and cinnamon with myrrh to fight halitosis.

13. 13
The earliest records of Egyptian dentists date back to approximately 2650 BCE. Their
main focus was to help prevent tooth problems rather than to fix the problems that
already existed. Most of the treatments created resulted from trial-and-error methods that
were then recorded in the Papyrus Ebers. One of their beliefs led Egyptians to use
dismembered mice as a quick pain reliever since they thought mice were protected by the
sun and were able to fend off death. In order to cure their pain, they had to take half a
mouse that was freshly killed and apply its warm body to the painful area within the
mouth. Knowledge of abscesses was also quite advanced, and the method used to treat
them is surprisingly similar to modern day dentistry. If an abscess was detected, the
Egyptian dentist would drill a hole in the infected area to allow the built-up pus to ooze
out of the wound while also applying antiseptic herbs to the area, such as myrrh, to allow
it to heal properly. Other forms of treatment discovered in Egyptian tombs included
metal wiring around adjacent teeth to support loose teeth, wooden hand drills, and
hieroglyphics with carved figures of the various equipment used by the dentists. It is also
possible that they have made cement-like mixtures to help fill the holes in teeth caused by
caries.
One of the leading advancements in dental technology is the invention of the
tooth brush. The habit is thought to be traced back to Indian origins (Tooth Museum of
Japan, 2014 a). The Guatama Buddha taught people of his religion that it is important to
be clean when you communicate with your gods. This meant that it is important to wash
your hands, then use a branch from the neem tree to scrub your teeth in preparation of
praying. Also, the Buddha said that removing foul smells from your mouth would
improve the ability to eat, to help remove mucous, and other such benefits.

14. 14
A Chinese monk visiting India had acquired this new knowledge and spread the concept
throughout his country when he returned. In addition, the concept of brushing teeth
spread to Japan with the introduction of Buddhism. This idea became common
knowledge among people of all social classes in Japan by the time between 794 and 1185
AD. The main concept was that cleanliness is very important, not just for when praying
to the gods. The concept of brushing teeth in Europe also has its origins tracing back to
Buddhism. Even now, the Neem sticks are being sold in India as a symbol of tradition.
Japanese monks are now still using this method of brushing their teeth with wooden
sticks before they pray to gods. Japanese temples and shrines also still have water
fountains with wooden scoops used for washing the hands and mouth before entering.
European and Roman culture was quite different in their approach to brushing
teeth. Instead of using wooden sticks, they would take crushed animal bones and egg
shells and burn them into ash. Those ashes were then applied to the teeth by hand and
scrubbed using their fingers (Fukugawa, 2008). In the year 959AD a toothbrush was
already created using a wooden stick with a horse's main as its bristles. The idea was
recorded by a visiting Japanese monk and the fully functional toothbrush was introduced
to Japan. So, in Europe the higher social class used toothbrushes made in the same
manner as the Chinese brushes. During the 17th
century the technology of the toothbrush
finally spread out among the common people of Europe.
There is evidence that dental bridges were already being used in ancient Lebanon
around 500 BCE. There were remains of bridges in cemeteries that were uncovered in
archaeological studies, and it is possibly one of the oldest recorded findings of major
dental bridgework seen in Figure 5 (Tooth Museum of Japan, 2014 b). Its main purpose

15. 15
was to support loose or weak teeth by binding them together with the stronger stable teeth
adjacent to it. These bridges were made of pure gold. Also, it was found that the bridges
sometimes contained substitute teeth for the areas where the original ones were lost.
There were also bridges found in Egypt near the pyramids of Giza that were made about
5000 years ago.
Figure 5. Various images of archaic bridgework around the world (Tooth Museum of
Japan, 2014 b).
Another important advancement in dental technology was the creation of dentures.
The dentures made before the 19th
century in Europe were purely aesthetic and not
functional in any way (Fukugawa, 2008). These dentures were made from either bone or
ivory for teeth. They were unable to move with the shape of the mouth, so they couldn't
be used for chewing or talking a lot, and they would often smell very bad even after a
single day's use. People who used those dentures were often higher class members of
society and they would be used for rare occasions, such as social parties. The people who
used them would have to eat at home before they attended the party. These dentures
were very expensive because they were lined with gold and they needed to be molded to

16. 16
each person that was to wear them. For example, George Washington's dentures were
found with gold that was plated to shape the roof and floor of his mouth with ivory
chunks used for teeth, so it stayed white even after being buried for hundreds of years,
which is shown in Figure 6. They actually included some of his real teeth that had fallen
out previously, so not all the teeth in the denture were pure white. His dentures were
made around 1789.
Figure 6. Wooden dentures of a Japanese monk from 1538 (Tooth Museum of Japan,
2014 c).
The first functional dentures were found in Japan that were used by a woman
monk in 1538 (Fukugawa, 2008). They were made out of a sturdy wood, unlike that of
the Europeans. The dentures were shaped to fit the exact locations of missing teeth so the
bite pattern of the mouth would be similar to the original. Proof of the use of these
dentures for eating comes from the scratches and extra stains found on the surfaces that
often come in contact with food. The base of the denture was sculpted to fit very tightly
with the mouth so it could be used for chewing and speech. The molding for the denture
was made from taking a large clump of warm beeswax and attaching it to the roof or
floor of the mouth and letting it dry in the shape of the mouth. It would then be removed

17. 17
and used for the sculpting of the wooden denture. The beeswax would be applied to the
same area of the mouth multiple times to ensure the best possible fit for the soon to be
made denture. These dentures were made by carpenters who were hired mainly to make
statues of gods or Buddha. Then, around 1600 this specialized job had already been
established to create dentures. Closer to 1700 the idea was greatly accepted and dentures
were made cheaper and quicker than previous times. The technology and methods of
creation had made a more stable job market for those interested.
Currently research is being done in Japan regarding eating capabilities and the
number of teeth a person has (Fumiyo and Nishiwaki, 2006). This was research
performed by the Japanese government in 1999. The study found those around 50 years
old would typically have lost an average of 4.9 teeth. By the age of 60 they lost an
average of 10.5 teeth, at 70 it was 16.6 teeth, and at 80 it was an average of 24.5 teeth,
with an adult mouth capable of sustaining 32 permanent teeth, including the 4 wisdom
teeth. For this reason about half the people at the age of 60 use either full or partial
dentures, while half of those around 80 years old would use a complete denture.
Comparing the average chewing capability of a fully toothed person with
someone who has lost even a single tooth, the person who lost a tooth has only half the
chewing capacity without any assistance from false teeth. If a person has lost 2-7 teeth
they will decrease their chewing capacity by a total of 70% compared to a normal mouth.
Any amount of teeth lost beyond 7 will render a person nearly unable to chew anything.
Adding in dentures (either full or partial), however, will return their chewing capacity to
slightly more than a person who has lost somewhere between 2-7 teeth. While people
can typically eat normal foods losing anywhere up to 7 teeth, they significantly lose the

18. 18
ability to eat many types of food at the loss of their 8th
tooth. For example, many types of
vegetables and meats are impossible to eat when the 8th
tooth is lost. Based on this
research, we can clearly see how difficult it is to preserve a good quality of life and health.
Tooth Anatomy
Oral bacteria create various communities with plaque, which become ecosystems
in the human mouth. Each tooth consists of two main parts: the crown which is the top
part sticking out from the gums, and the root portion hidden inside the gum line (Figure
7). Each tooth consists of 4 types of dental tissue. Those tissues are separated between
hard tissues, which are enamel, dentin and cementum, and the soft tissue in the center of
the tooth that includes connective tissue, nerves, and blood vessels, all of which are
known as the dental pulp. The hard tissues protect the sensitive inside soft parts of tooth.
The hardest and outermost tissue is enamel, which is calcified tissue. The enamel is the
cover coating the surface of the crown to protect inside tooth tissue from damage. When
the enamel gets damaged, it cannot be fixed because the enamel does not have any living
cells. The second layer under the enamel is dentin. The main part of the dentin is made
out of microscopic tubules that are canals or small hollow tubes. If enamel get damaged,
the outside stimulus, such as foods, heat, cold, or pH difference get in to the tubules and
cause sensitivity. The root portion of the hard tissue is cementum that covers the surface
of the tooth’s roots to help attach the tooth to the periodontal ligament. The soft tissue,
pulp chamber contains the most sensitive parts of the tooth, nerves and blood vessels.
The nerves and blood vessels are in the center of the tooth and supply nutrients to each
tooth. The root and part of the crown of each tooth is protected by the soft tissues,

19. 19
gingiva, which has the common name of “gums”. It mainly covers and protects teeth
without the support of the enamel, while it also stabilizes the structure of the teeth
strongly attached to the jawbone, which is surrounding the roots of the teeth (Mouth
Healthy, 2014).
Figure 7. The Anatomy of a Tooth (Mouth Healthy, 2014).

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Biofilms
Biofilms are coaggregations of bacteria that function as a community and have
such sophisticated levels of communication they act as a single organism unit (Medical
Dictionary, 2015 b). The biofilm forms what is commonly known as plaque. Plaque is a
softened sticky coating that forms ideal conditions for bacteria to proliferate in. The
plaque advances to a stage known as calculus in which the biofilm forms a hardened
crystallized structure around the teeth. During each of these stages the bacteria catabolize
various sugars and produce lactic acid. Lactic acid works to increase the acidity of the
oral cavity (the area within the mouth) by reacting with water in the saliva. It is
converted into lactate, while it releases hydrogen ions measured by the pH using a
negative logarithmic scale. The lower acidity then reacts with the enamel coating the
outermost layer of the teeth and begins to demineralize it. This process removes different
minerals from the teeth, such as calcium and phosphate, which makes them more
susceptible to other forms of damage. If enough of the enamel is removed, the damage
spreads to the dentin tissue of the teeth. If the mouth is not remineralized during this time
period, the bacteria will spread into the inner layers of the tooth where they will continue
to produce acids. Pain is not normally felt when the enamel is damaged as there are no
nerves within this layer of the teeth. It is a protective coating harder than bone. When
the enamel becomes thin, however, the tooth starts to become sensitive to different
temperatures in the mouth. As infection spreads to dentin pain is felt since the nerves are
much closer to this layer of the tooth. If the infection spreads far enough it will enter the
dental pulp, which is the major source of blood and nutrients to the teeth, as well as the
center of nerve bundles. If an infection spreads to the pulp, there is a high risk of the

21. 21
infection spreading to any other part of the body, with the most concerning being the
brain. Bacteria can cause damage to surrounding cells and form pockets between the
cells called abscesses. When the bacteria reach the brain they can cause abscesses and
turn off many functions of the brain by preventing signal transduction between the cells.
This quickly leads to death if not immediately treated.
There has been a lot of mention of S. mutans but not so much about L. acidophilus.
This is because the latter has been less researched than the former due to the fact this
bacteria only functions malevolently in the presence of an already sustained biofilm. L.
acidophilus is unable to adhere to the smooth surfaces of the teeth. This would indicate
the bacteria is able to stick to moist, sticky surfaces, such as biofilms. This is also
supported by the fact that these bacteria are naturally found in the gastrointestinal tract of
mammals, which is covered in thick concentrations of mucous (NIH, 2015).
Lactobacillus strains are considered to be opportunistic pathogens since their main
byproducts are actually neutral or beneficial to the host, while they can be harmful when
the correct conditions are met. They are able to help digest different forms of
carbohydrates in the intestines and any of the nutrients they do not use are absorbed by
the intestinal epithelial cells. The lactic acid they produce as a byproduct is also able to
hinder the growth of invasive bacteria in the intestines (University of Maryland Medical
Center, 2015). Both bacterial strains are facultative anaerobes, meaning they function
best when there is little or no oxygen present, but are able to sustain function in its
presence, and their main form of energy production is through fermentation (Metwalli,
Khan, Krom, and Jabra-Rizk, 2006).

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In the human mouth, there are more than 700 different types of bacteria living in
various places, such as the tooth surface, gingival sulcus, and dorsum of tongue and
create flora while are affected by saliva/sputum (Kolenbrander and London, 1993).
Biofilm in the mouth are normally called dental plaque, and they are involved main
dental diseases; dental caries and periodontal disease, so these diseases are considered as
a biofilm infectious disease (McNab, Ford, El-Sabaeny, Barbieri, Cook, and Lamont,
2003). A biofilm is an immobile and adhered social group. Biofilms can grow on almost
all surfaces of living and nonliving materials within environment with running water.
The bacteria creating biofilms also emit exopolysaccharides that can allow many
different kinds of microorganisms to be embedded and become dense aggregates. The
exopolysaccharide plays an important part in the environment and nature of the biofilm.
Exopolysaccharides are used for: pathogenicity, bacterial adhesion, immune system
resistance, resistance to dry environments, resistance to antibiotics and disinfectants,
resistance to heavy metals, protection from organic solvents, protection from
bacteriolysis of bacteriophages, and protection from protistan phagocytosis (Yoshida,
2010).
Biofilms show resistance to antibacterial, antibiotics, and immune systems from
the outside world because the biofilm is being occupied with an adhesive high matrix
exopolysaccharide. For example, antibacterial solutions can kill most free-living
planktonic cells, but have a much lower effect on those microorganisms living in
biofilms. For one reason, the matrix can cover all bacteria within the biofilm and protect
against those harmful medicines and other outside negative factors. Moreover, biofilms
are environments that lower the metabolic activity of microorganisms instead of just

23. 23
suspending them in the matrix. The bacteria use a self-induced signal for their
communication to create biofilms, toxins, and the “immune system” against
antimicrobials. This shows a biofilm-caused infectious disease can easily become
chronic and obstinate (Costerton, 2002).
In the mouth the tooth, which is a stereome, provides the solid-phase aspect used
in biofilm formation. Then, the various oral bacteria and microorganisms stick to a
pellicle, or thick piece of skin, also known as an acquisition film, and they stick to saliva
proteins. Those biofilms are called dental plaque, and are the main cause of dental
problems, such as dental caries. Those diseases of the mouth are regarded as infectious
diseases by biofilms (Shigeyuki and Takashi, 2006). Dental plaque is made out of 70-
80% of water, and rest of the 20-30% are composed of various bacteria, microorganisms,
and chemical substances. The constitution of a biofilm is strongly influenced by the
adhesion part of the plaque on a tooth and its overall maturity. The matrix of the biofilm
is mainly made out of protein and carbohydrates. It has strong adhesive properties to a
tooth surface, and it keeps a high density of lactic acid and another bacterial nourishment
sources. Therefore, the matrix is one of the factors used to decide the pathogenicity of
the plaque. The stroma of the plaque is gel-like and takes on an electrolytic property, so
penetration and diffusion of water, nutrients, and materials that have electric charge
characteristics and macromolecular to the plaque are normally difficult to diffuse. On the
other hand, the penetration and the diffusion speed of non-electrically charged related
materials, such as glucose, is faster. For these reasons the bacteria, living inside the
plaque, digest fermentable carbohydrates, such as glucose, to create organic acids with a
side product of mainly lactic acid. This brings a sudden fall of pH. When the surface of

24. 24
the tooth’s pH becomes equal to or less than pH 5.5, the critical pH, decalcification starts.
Furthermore, the intercellular matrix of water-soluble glucan and fructan or
polysaccharides like amylopectin work as a storehouse of energy, and it is digested at the
time of starvation to sustain acid production. Also, the intercellular matrix has insoluble
glucan created by Streptococcus mutans, which works as a barrier to disturb diffusion and
causes the disappearance of organic acids in the oral cavity, while it helps promote acid
accumulation inside the plaque. Therefore, the essential factors of caries pathogenicity of
the plaque is dependent on the production of organic acids and a change of diffusion rates
(Yoshida, 2010).
Creation of Biofilms by S. mutans and the Role of Glucosyltransferase
S. mutans creates glucan, a polymer of fructose, through the use of
glucosyltransferase (GTF) from sucrose. Sucrose is a disaccharide made out of a
heterodimer of glucose and fructose. GTF conducts hydrolytic activity on sucrose to
cleave it into glucose and fructose, and connects the produced glucose residues to create
the glucan polymer. S. mutans produces three kinds of GTF responsible for the main
cause of human caries. The GTF creates soluble and water-insoluble glucan from
sucrose. When GTF composes glucan, the activity increases when there are low
concentrations of molecular oligosaccharides and polysaccharides available to become
primers. This is called primer dependence. Table 1 shows different kinds of GTF and
primer dependence for each GTF. Each GTF has different properties of extrapolymeric
substances (EPS) found in glucan, so they have different roles as pathogenic factors
(Yoshida, 2010).

25. 25
Table 1. Glucosyltransferase properties of S.mutans (Yoshida, 2010).
Name of
enzyme
Gene Localization
Primer
dependence
Solubility of
glucan
Characteristic
GTF-B gtfB
Microbial
cell-binding - insoluble
Produce
voluminous
water-insoluble
glucan
GTF-C gtfC
Microbial
cell-binding - insoluble
Important for
adherence
GTF-D gtfD
Culture
supernatant + soluble
Produce
adherent glucan
with GTF-C
Figure 8 shows the relationship between GTF with S.mutans during adherance to
a tooth surface. At first, the insoluble glucan of GTF-C is formed in the presence of
GTF-D, which has adhesiveness, so it becomes the basis for S. mutans to stick to a tooth.
Then, GTF-B produce a voluminous insoluble glucan to strengthen adhesion of S.
mutans, and it creates plaque by combining with neighboring microorganisms (Shigeyuki
and Takashi, 2006).

26. 26
Figure 8. Functions of Glucosyltransferase in S. mutans (Shigeyuki and Takashi, 2006).
GTF plays an important role for biofilm formation. The expression of the GTF-B
gene, essential for the creation of insoluble glucan by S.mutants in the biofilm, did not
become clear yet before this research. Therefore, Yoshida and Kuramitsu (2002 a)
investigated gene expression profiling of GTF-B using a reporter gene for a promoter of
the gene with a green fluorescent protein (GFP). They cultured the colonies of S. mutans
854S mutants that transduced plasmids having the fusion gene of GFP with the promoter
of GTF-B. Then, they produced biofilm using a carbon source of 0.5% sucrose on a
polystyrene plate using the colonies to analyze the expression of GTF-B gene under a
confocal laser scanning microscope. They found the GTF-B gene of S. mutans strongly
emerged at an initial stage of biofilm formation, particularly during the microcolonization
stage (Figure 9). They compared the expression of GTF-B gene of S. mutans 854S
floating within the nutrient medium and the state of the biofilm using flow cytometry,
Production of Plaque
Sucrose
Insoluble
glucan
Gl
Adherent
glucan
Soluble
glucan Adhesion of Bacteria
Voluminous insoluble glucan
GTF-B
GTF-C
GTF-D

27. 27
and the biofilm had nearly 5 times more reinforced expression than in the floating state.
They analyzed the basis of these results using real-time polymerase chain reaction (PCR),
the expression of the GTF-B gene in the biofilm was reinforced around four times more
than that of the floating bacteria (Yoshida, 2002 a).
Figure 9. The biofilm of S. mutans 854S colonies. A: Sagittal plane and top view. B: 3D
image (Yoshida, 2002 a).
A Biofilm-related Gene with Sucrose Independence in S.mutans
It became clear S. mutans have sucrose dependence genes to create biofilm, but it
did not yet become clear about biofilms’ connection to sucrose independence genes
within S. mutans, so Yoshida and Kuramitsu (2002 b) analyzed the gene clusters involved
with sucrose independence of S. mutans. At first they made a mutant library with random
insertional inactivation gene chromosomes with an erythromycin tolerance gene in

28. 28
colonies of S. mutans GS5. They analyzed the mutant colonies that had decreased the
ability to form biofilms using a carbon source, such as glucose, from the library. Those
mutant colonies had an inactivated comB gene, which is one of the gene clusters involved
in hereditary transformation ability. Therefore, they produced other mutant colonies of
comA, comC, comD, and comE genes that are com control systems related genes
containing an erythromycin tolerance gene, and analyzed the biofilm formation ability of
these bacteria under the same glucose conditions. A decrease of biofilm formation ability
was found similar to the case using comB, seen in Figure 10 (Yoshida and Kuramitsu,
2002 b).

29. 29
Figure 10. Concentrations of biofilm formation by mutant colonies of the comB control
gene system (Yoshida and Kuramitsu, 2002 b).
The signal transmission mechanism of the peptide inducer is by this com system.
The peptide inducer precursor is translated from comC genes received and processed by
an ABC transporter encoded by comAB, and is drained as a Competence Stimulating
Peptide (CSP) outside of the cell body. The CSP is made from the molecules that
conduct signal transmission between bacteria. The signal peptide discharged by the
outside of the cell body is sensed by the sensor kinase of two important adjustment
factors, which are encoded by comD connecting to the cell membrane. It phosphorylates
the regulator the comE gene encodes, and the phosphorylated comE gene provides

30. 30
transcription instructions for comAB and comCDE operons. The result developed stating
that Quorum Sensing, through the peptide inducer, participated in the biofilm formation
in S. mutans, which can be seen in Figure 11 (Yoshida and Kuramitsu, 2002 b).
Figure 11. Com system regulation and enzymatic control in S. mutans (Yoshida and
Kuramitsu, 2002 b).
Quorum Sensing and Biofilm Formation of S. mutans
Quorum sensing is a form of communication between bacteria that developed
through peptide-related signals used in the biofilm formation by S. mutans. The signal
transduction system between bacteria is shown in the Table 2. According to Table 2, S.
mutans has a signal transduction system by non-acyl-homoserine lactone molecules,

31. 31
called autoinducer-2 (AI-2) (Merritt, Qi, Goodman, Anderson, and Shi, 2003). Yoshida,
Ansai, Takehara, and Kuramitsu (2005) analyzed the Quorum sensing system through the
use of AI-2 of S. mutans. LuxS is an enzyme acting on the catabolism of S-
adenosylmethionine, and produces homocysteine and AI-2 precursors from ribose
homocysteine. Therefore, they also analyzed its role in biofilm formation using deletion
stocks of the luxS gene in S. mutans. When the luxS genetic deletion stock was cultured
with glucose as a carbon source, the biofilm formation ability was almost indifferent from
the wild-type stock. On the other hand, when they cultured the mutant with sucrose as a
carbon source, the biofilm formation ability became clearly decreased (Yoshida et al.,
2005) (Figure 12).
Table 2. The model of signal transduction systems between bacteria (Merritt et al.,
2003).
Type of Bacteria Signal type
Gram Negative Bacteria
Allogenic communication by acylated
homoserine lactone (AHL)
Gram Positive Bacteria Allogenic communication by peptidic signal
Gram Negative and Gram Positive
Bacteria
Allogenic and/or xenogeneic communication by
Autoinducer-2 (AI-2)

32. 32
Figure 12. Biofilm formation of S. mutans luxS deletion stock using sucrose as a carbon
source. A. Quantity of biofilm formation: the white space is quantity of biofilm
formation, and the black space is growth. B. The left is GS5 stock, the right is luxS
deletion stock. Both are the biofilm of S. mutans dyed with crystal violet. (Yoshida et al.,
2005).

33. 33
The properties of biofilms were very different between luxS genetic deletion stock
and the wild-type stock culture when both used sucrose as their carbon source. Yoshida
et al. (2005) also found the expression of the GTF-B and GTF-C genes of the luxS
genetic-deficiency stock increased in a Middle logarithmic growth phase (Figure 13). In
the luxS genetic-deficiency stock with sucrose, the expression of the GTF-BC gene is
reinforced, so the bacterial mass was produced during the comparatively-early stages of
biofilm formation. The bacterial mass of luxS genetic deficiency stock exists in two
types: one type sticks to a solid surface, while the other is washed away by solutions.
The results of this research showed there was a decrease in biofilm formation at later
stages of development. Furthermore, they analyzed bacterial influence over other oral
cavities on the quantity of biofilm formation of the luxS mutant stock by co-cultivating
the luxS mutant stock and other oral cavity-causing bacteria. Then, they found that the
oral streptococci, such as: Streptococcus gordonii DL1, Streptococcus sobrinus MT8145,
and Streptococcus anginosus FW73, compensate for the lack of biofilm formation of S.
mutans luxS mutants by building biofilms to the same level as the wild-type stock culture
of S. mutans (Figure 14).

34. 34
Figure 13. S. mutans GTF-B, C, and D gene expression analysis with real-time PCR
methods (Yoshida et al., 2005).
Figure 14. The biofilm formation of S. mutans luxS mutants that are co-cultured with
other oral cavity-causing bacteria (Yoshida et al., 2005).

35. 35
Oral Diseases: the Power of S. mutans
It is well known that S. mutans is the main source of caries causing agents in the
mouth. It is also known when plaque becomes too excessive it has the chance to enter the
bloodstream if an opportunity presents itself, such as deep enough demineralization of the
tooth to the dental pulp, or through improper brushing of the teeth leads to gingival
bleeding. The types of diseases that can occur from biofilm formation are still being
studied while new discoveries are being made frequently. Kojima, Nakano, Wada,
Takahashi, Katayama, Yoneda, Higurashi, Nomura, Hokamura, Muranaka, Matsuhashi,
Umemura, Kamisaki, Nakajima, and Ooshima (2012) performed a study of S. mutans and
its effects on the formation of ulcerative colitis (UC).
Kojima et al. (2012) began the research by looking into the effects of various
strains of S. mutans on the body. It is stated that pathogenic oral bacteria are classified in
two categories: one form is used in the formation of dental caries, while the other form is
used in the creation and agitation of periodontitis. Studies have shown there are 4
different serotypes, known as c, e, f, and k, for the S. mutans bacteria found in the mouth.
Serotype “c” is the most prevalent form of these bacteria found in the mouth, while
serotype “e” makes approximately 1/5 of the overall strains, and the “f” and “k”
serotypes make up the smallest group. This is fortunate because serotypes “f” and “k”
were also found to be the most dangerous to a person’s health if those bacteria enter the
bloodstream. This is because these forms of bacteria have collagen-binding proteins that
allow them to stick very well to cells and are highly resistant to phagocytosis. The ability
to bind collagen also makes them more important in inflammatory diseases.

36. 36
In addition to this information, it was discovered that patients who had ulcerative colitis
also commonly had higher concentrations of S. mutans in the bloodstream.
In the study, health mice were infected with colitis by providing them water
contaminated with dextran sodium sulfate (DSS). Once the mice began expressing
symptoms of irritable bowel syndrome and colitis, they were then provided with either
one of the strains of S. mutans or the placebo. Bacteria was given using IV injections and
the overall effects on their health was closely observed. Within a few days of injection
the mice injected with the serotype “k,” also known as the TW295 strain, showed an
increase in weight loss compared to the others, an increased disease activity index, and
they also had higher mortality rates than the other mice infected with serotype “c,”
known as the MT8148 strain (Figure 15).

37. 37
Figure 15. Survival rates of mice with induced ulcerative colitis with and without
inoculation of S. mutans strains (Kojima et al., 2012).
After this discovery, Kojima et al. (2012) wanted to find the minimum
concentration of the serotype “k” bacteria necessary to cause the colitis to become more
sensitive. The bacterial inoculations were tested at various strengths on the mice and a
concentration of 105
cells were determined to be the minimum amount to cause an
aggravation effect. This concentration is relatively small and has been determined to be
relatively easy to attain in the bloodstream when passed from the mouth. Since the
concentrations can be easily passed through the blood to organs in the body it was

38. 38
determined S. mutans can be naturally passed to the organs and cause aggravation if they
have the opportunity to find a cut in the mucous membranes of the mouth that allows
them to enter the bloodstream. This is also important in showing ulcerative colitis is
triggered by an upset in the blood surrounding the digestive tract, rather than being
caused by something within it.
The next step in the study was to find the location of infection within the body.
To do so, cells were extracted from areas that experienced inflammation caused by an
immune response and test those cells for the presence of a DNA sequence found in the
serotype “k” specific bacteria. Initially the researchers tested the small intestine and
colon as they were the main focus of this study, but they were very surprised when the
results from the gel were negative for any signs of bacterial colonization. In response to
this discovery, the researchers then decided to take a look at the liver since it partially
regulates digestion in the small intestine. As seen in Figure 16, the liver presented very
strong bands of DNA in the electrophoresis indicating a large quantity of the serotype “k”
bacteria were present in the liver, while there were no bands present for the small
intestine and the colon. A PCR and gel electrophoresis were also prepared for testing the
presence of the main type S. mutans of the mouth, but all results came back negative.

39. 39
Figure 16. DNA analysis using gel electrophoresis for the detection of serotype “k”
strains of S. mutans with cell extraction locations (Kojima et al.,2012).
Once the liver was verified as the target organ for these bacteria additional
research was necessary to find the specific types of cells within the liver that were being
infected. The cells were coated with anti-serotype “k” antibody and placed under UV
light (Figure 17). Prior to infection, the bacteria was transformed to produce green
fluorescent protein (GFP) in addition to its normal functions. While the liver cells were
placed under the UV light the bacteria were shown to infect just the hepatocytes, or liver
cells, and not the skin cells lining the liver, as seen in Figure 17. While the mice infected
with this bacteria died at an earlier age than the negative control group, it is still unknown
what causes the death to occur initially, especially since the colonization in the liver
reaches a peak at 3 hours after introduction then stops within 3 additional hours. It has
been proposed that the influx of bacteria may cause the liver to trigger apoptosis
signaling within most of its cells when it comes in contact with the bacteria.

40. 40
Figure 17. Immunofluorescence of GFP-induced serotype “k” S. mutans in extracted
liver cells stained blue (Kojima et al., 2012).
To test for the viability of the serotype “k” bacteria, Kojima et al. (2012) took a
closer look at the glucose side chains of the peripheral proteoglycans. They found the
structure in the TW295 bacteria had a significant difference in the structure, which
allowed the bacteria to be resistant to phagocytosis. To test this theory, a wild-type
MT8148 bacterial colony was modified to produce surface proteoglycans closely
resembling that of the TW295, and TW295CND mutants were created with surface
sugars very similar to the wild-type. Looking at Figure 18 it can be seen that the bacteria
expressing proteoglycans similar to the wild-type bacteria were much more susceptible to
endocytosis than those expressing the sugars similar to the TW295 strain. It was also
found that the bacteria with similar proteoglycans to the TW295 had much better
collagen binding capability suggesting these type of bacteria are much more likely to
attach to cell surfaces. To further support this idea cell adhesion rates were measured for
each type of bacteria, and the results had shown bacteria with the TW295 proteoglycan

41. 41
had much better binding affinity. It was also interesting that the wild-type bacteria with
transformed surface sugars had similar capability to aggravate colitis, but they don’t
normally have the chance to since they are quickly endocytosed before they can reach the
liver.
Figure 18. Phagocytosis rates and total collagen binding estimates of wild-type, serotype
“k” (TW295), mutant MT8148GD, and mutant TW295CND S. mutans strains (Kojima et
al., 2012).

42. 42
Once the serotype “k” strain proteoglycan was determined to be the cause of
inflammatory response, the liver cells were then checked for the signaling molecule
released in response to the bacteria. Figure 19 shows both normal cells and colitis-
induced cells gave the same response of a release of Interferon gamma (INF-gamma)
when presented with this particular bacteria, while the wild-type (vehicle) strain produced
no such response, which was the same as the TW295CND mutant. It was then concluded
this molecule is responsible for the initiation of the signaling pathway that causes
irritation of the intestinal tract. To test this theory an anti-INF-gamma antibody was
created and administered to the mice infected with colitis, and the mice had significant
reduction in the symptoms present.
Figure 19. Cellular production of Interferon gamma in response to contact with wild-type,
serotype “k,” and serotype “k” mutants of S. mutans strains (Kojima et al., 2012).

43. 43
A brief study of human oral flora was performed following the mice studies.
Saliva samples from healthy individuals and those with irritable bowel syndrome were
collected and screened for the various types of S. mutans each had. The healthy
individuals had the expected levels of serotypes “k” and “f,” while those who had IBS
produced higher concentrations of these particular strains. Taking a look at the
information collected from this study, it has been made clear that S. mutans has the
potential to cause serious health diseases if it is allowed to travel throughout the body via
the bloodstream.
Heritability Factors of Dental Caries
Biofilm formation is extremely important for the creation and continuation of
dental caries. It is known that the risk of caries varies from person to person, and the
severity of cavity formation occurs in a wide range. Wang, Shaffer, Weyant, Cuenco,
DeSensi, Crout, McNeil, and Marazita (2010) published a study they had performed
based on the correlation between the shape and type of tooth with the ability of oral flora
to attach to the tooth surfaces. The study was performed in the north eastern United
States and was performed on patients who had a family with at least one child at or below
the age of 18 years old. The study looked to involve the entire family of the patient to
allow a proper evaluation of the transmission of S. mutans and other oral bacteria
between family members. Assessment of caries levels were made from the patients and
their families after drying the teeth with gauze and using light. During the process of
inspection hand tools were not used unless the researcher was unable to determine the

44. 44
possibility of caries. All tooth surfaces were recorded following the World Health
Organization’s Decayed Missing and Filled Tooth (DMFT) scale. All teeth classified as
decayed were rated between 1 and 4, with 4 being the most decayed. Heritability of
bacteria was determined using a complex mathematical model that took into
consideration gender, individual additive polygenic effect, and individual residual error
factors.
It was found that the average number of teeth with heritable decay was most high
in the primary teeth, as well as this category of the DMFT being the most prominent in
the type of heritable traits, as seen in Figure 20. In most cases the primary teeth were the
most affected by heritability while the permanent teeth were not usually as affected. The
age and gender of the patients studied had very little effect on the outcome of primary
teeth health, while the permanent teeth had quite a unique trend. It was found that in the
patients aged 18 years and younger, in every case, the males would have relatively higher
rates of dental caries on average compared to females, even when the female population
sizes were slightly higher than males. There was a significant correlation found between
the heritability of caries in primary teeth and the heritability of caries in permanent teeth.
In addition about 18% of all the genes involved in the likelihood of caries are commonly
found active in both primary and permanent teeth. Wang et al. (2010) had also confirmed
missing and filled teeth were harder to judge properly using the DMFT scale because it
cannot take into account the access to health care each patient possibly has, which turns
the missing and filled teeth into non-genetic factors for the study.

45. 45
Figure 20. Heritability estimates for each type of damage to teeth that are primary (grey)
or permanent (black) (Wang et al., 2010).
In addition to tooth surface structure, other factors have to be taken into account
to identify the most likely factors that influence caries heritability. Bretz, Corby, Melo,
Coelho, Costa, Robinson, Schork, Drewnowski, and Hart (2006) worked together to find
a link between the likeliness of dental caries heritance, and the preference each patient
had for the sweetness of sucrose. The patients for this experiment were all twins between
4 to 7 years old, either monozygotic or dizygotic, and were all from low socio-
economical families living in Brazil. The status of the twins was confirmed prior to the
caries investigation by using blood samples collected from the family members to verify
the relationships within the families by looking at single nucleotide polymorphisms

46. 46
(SNPs) shared between the twins. The study looked at a total of 115 pairs of twins, with
more of them being dizygotic in their relationships.
Prior to the full examination provided by the researchers each patient had a
professional dental hygienist cleaning to remove surface plaque and allow the examiners
to see locations of damage or decay more easily. The researchers came up with a system
to rate the severity of caries damage on a scale from 1 to 4, with 1 being the least harmful,
and 4 being damage that is approximately 2-3mm deep within the dentin layer. Overall
caries prevalence was calculated using the lesion severity index (LSI), which used the
formula: (N1+ 2N2 + 3N3+ 4N4)/ (the total number of surfaces present), and each of the N
numbers represents the caries damage for each tooth measured within a single mouth.
Once the caries prevalence was calculated, the patients were treated to grape juice
solutions with varying levels of sucrose concentrations inside each cup.
Grape juice solutions were provided one at a time to each of the patients and their
facial expressions were recorded as either a frown, neutral, or a smile. The scores
assigned ranged from 0 (frown) to 2 (smile) and these numbers were used to further
calculate the sucrose sweetness preference scores (SSPS). Most of the children followed
the trend of liking higher concentrations of sucrose in the grape juice, while most of them
did not like the taste of lower concentration juices, as seen in Figure 21. Following the
same trends of previous research on this subject children usually preferred to have juice
concentrations with much higher sugar content. Bretz et al. (2006) had also realized even
though twins may have had the same sugar preferences, each of the members had
variations in the total amount of caries present in their mouths when compared with other
family members. Therefore, they had concluded the structure of the tooth enamel is

47. 47
controlled by genes that have been activated in various amounts. The genes responsible
for controlling tooth enamel strength has not yet been discovered. They had also
concluded sweetness preference may not always be genetically inherited as culture plays
an important role in food preference, especially for children. It was also mentioned taste
preference changes with age and gender maturity, so most of the research performed in
this study was only able to support the concept that taste preference heritability has a
stronger influence on young children. Since taste preference has variations, it can be
concluded it is influenced by both genetic heritability and epigenetic (cultural) factors. In
the end, it was determined preference for higher concentrations appears to have no
significant impact on the amount of caries that can be inherited.
Figure 21. The sucrose sweetness preference observed in each patient using the SSPS
(Bretz et al., 2006).

48. 48
The Probiotic Controversy
Since dental caries is such a big issue for many people, it would make sense that
researchers are currently looking for a way to fight dental caries without resorting to
powerful drugs. Since caries are caused by bacteria, any form of drug strong enough to
kill them off would also kill our cells, and many drugs will only kill off some of the
bacteria, thus causing some of them to form a drug resistance. Montalto, Vastola, Marigo,
Covino, Graziosetto, Curigliano, Santoro, Cuoco, Manna, and Gasbarrini (2003)
performed a study on the effects of probiotic treatment using a mixture of 4 different
strains of lactobacillus, following the recent trends in society stating probiotics have
beneficial effects on our health. Montalto et al. (2003) chose to study the effects of
lactobacillus strains on the oral flora were provided in both capsule and liquid form. For
the study 35 healthy volunteers were chosen with no prior history of dental health
problems. Each of the volunteers were provided either a placebo, or probiotics with a
mixture of L. sporogens, L. bifidum, L. bulgaricus, L. thermophiles, L. acidophilus, L.
casei, and L. rhamnosus, with each in nearly equal proportions. The patients were
instructed to take the probiotic or placebo every day for 45 days. Prior to beginning
treatment saliva samples were collected from each of the volunteers and the oral bacterial
colonies were grown on petri dishes to determine the initial concentrations of bacteria in
each person. Effectiveness of the probiotic treatment was measured by the comparison of
overall levels of S. mutans present in each of the volunteers. The plates used for bacterial
growth had medium that was selective for either lactobacillus strains, or for streptococcus
strains for easier measurements.

49. 49
Figure 22 shows the growth rates of the two studied bacterial strains in the
volunteers provided with a probiotic capsule and liquid placebo. The purpose of
providing just a probiotic capsule or just a liquid form is to measure the effects the
probiotic support of bacterial growth in the mouth either by having direct contact with the
surface of interest, or by having some nutrients absorbed by the body to support bacterial
growth. The results supported the concept that a probiotic solution provided enough
support for the growth of the natural oral flora of lactobacillus strains using either method
of probiotic application. It was found even the placebo had some effect on the overall
growth of lactobacillus and streptococcus strains. The experiment had shown probiotics
had an effect to help increase the population density of lactobacillus strains in the mouth
regardless of which method was used, and even though lactobacillus strains had grown
more in the mouth the streptococcus strains were still present in the same concentrations
as before the experiment began. These results support the conclusion that the
lactobacillus strains used in this experiment have no harmful effects on streptococcal
strains. It is still unknown how these bacteria are able to proliferate when provided by
capsule because the bacteria should theoretically only come in contact with the cells in
the body after being dissolved in stomach acid.

50. 50
Figure 22. Growth rates of lactobacillus and streptococcus strains in volunteers receiving
a probiotic capsule with placebo liquid. Bacterial colonies at T0 are colony
concentrations prior to experimentation with T1 revealing concentrations after probiotic
application (Montalto et al., 2003).
Research performed by Okamoto (2013) studied the effects of probiotic
treatments applied to humans and other animals. The research shows even chimpanzees
have Streptococcus mutans and other forms of Streptococcus, while they also have
similar eating habits to humans. This means they have the highest chance of developing
caries compared to any other primate group. They do not get caries, however, nearly as

51. 51
often as humans. When comparing human oral bacteria diversity to chimpanzees' the
human bacterial population is roughly a couple thousand species, while the chimpanzee
has over 10,000 different species. Okamoto also found the chimpanzee has many strains
of bacteria considered to be probiotic that humans lack. Based on this researchers believe
using probiotics is part of the future of successfully preventing caries.
For example, some medical offices in Japan have already begun treatment using
probiotic supplements. The Parksite Dental Clinic (2013) has explained on their web
page the importance of balancing the beneficial bacteria with the harmful ones within the
body. Keeping the correct balance helps you stay healthy, not only with oral health, but
with other types throughout the body. The clinic states L. reuteri bacteria— naturally
found in the human body— has healing properties that will not harm a patient in any way.
They found a Japanese research group had been adding supplements containing these
bacteria to milk that was being fed to babies. This milk helped reduce the incidence of
many health problems, with the most prominent being fevers, colds, and digestion issues.
Since so many strains of lactobacillus are useful in promoting dental caries
formations, it should be believed that research in this particular area of probiotics would
be finished. This was not the case with researchers in Japan. Nikawa, Makihira,
Fukushima, Nishimura, Ozaki, Ishida, Darmawan, Hamada, Hara, Matsumoto, Takemoto,
and Aimi (2004) led an investigation on the effects of L. reurteri on the formation of
dental caries and the overall growth of S. mutans along with other cariogenic bacteria. L.
reuteri was chosen for this study because it is naturally found in the gastrointestinal tract
of humans and it provides many antimicrobial effects to the surrounding environment,
while it is also resistant to both lipolytic and proteolytic enzymes. Nikawa et al. (2004)

52. 52
also lead this research because of previous studies in which L. reuteri was used as the
main anaerobic fermentator in milk and the children who drank the milk were found to
have reduced levels of caries compared to others.
Prior to testing probiotics on people, both L. reuteri and S. mutans were grown in
separate plates for a number of days before the primary experimentation began. In this
first stage of the experiment samples of L. reuteri were collected during the exponential
growth phase and were combined with samples of S. mutans in microfuge tubes using a
variety of concentrations of each type of bacteria. It was found that the larger the
concentration of L. reuteri, the less likely the streptococcus strains were to survive, as
seen in Figure 23. The second part of the study was performed to verify the results from
the first part. In the second part yogurt products were collected from various locations in
Japan and each of these products were used like antibiotics to evaluate the antimicrobial
properties they possessed. The method of study was a classic Kirby-Bauer test in which
flat circular sterilized paper discs were submerged in each of the various types of yogurt
for approximately 20 seconds and were placed on plates inoculated with S. mutans
(Nikawa et al., 2004). After incubation for 48 hours the total inhibition effects of each of
the yogurts was measured. Of all the types of yogurt used in this part of the experiment,
none had provided any inhibitory effects, except for the Reuteri yogurt containing L.
reuteri that provided significant reduction of S. mutans growth rates.

53. 53
Figure 23. The inhibitory effects of L. reuteri on the survival rates of S. mutans grown in
vitro (Nikawa et al., 2004).
The third part of the experiment involved using the Reuteri yogurt on the test
subjects, who were randomly chosen from the female dental hygienist student population.
There were a total of 40 students used for this experiment with each group consuming a
placebo for 2 weeks and the Reuteri yogurt for 2 weeks. Either the placebo was taken
first, then followed by the Reuteri yogurt, or the Reuteri yogurt was taken first, followed
by the placebo yogurt. In the experiment the students had saliva samples collected each
day before consuming the yogurt, and a few minutes after consuming the yogurt. The
saliva samples were serially diluted using water as the diluent, and were grown on
Mannitol Salts agar for 48 hours. The samples were then tested further by placing them
in multi-well plates with a hydroxyapatite (HAP) bead placed in the bottom of each well,
and HAP degradation was measured.

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L. reuteri is an incredible bacteria found within the body. It is useful for its
ability to prevent harmful cariogenic bacteria from proliferating while it also is able to
resist common functions the body goes through that prevent pathogenic bacteria from
growing, especially when considering the enzymes involved in bacterial cell destruction.
This experiment supports the idea that L. reuteri is useful as a probiotic inhibitor of S.
mutans. As seen in Figure 24 Reuteri yogurt had always produced a significant decrease
in the concentration of S. mutans in both groups studied in this experiment. HAP
degradation was studied to see how strong of an acidic environment would each sample
produce after incubation at different time increments. For the first 24 hours of incubation
with the HAP beads, there were no differences in Ca2+
release between the wells
containing just streptococcus strains and the wells containing just lactobacillus strains.
At any point in time after that there was a drastic increase in the amount of calcium
released by streptococcus strains, which indicates the level of acidity that particular
bacteria produces, while the lactobacillus strain had no production of acidity. Based on
these results, it would be safe to encourage the use of probiotics containing L. reuteri as
the main bacterial strain for its use in fighting off high concentrations of S. mutans and
for a small reduction in the likelihood of forming oral biofilms.

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Figure 24. The inhibitory effects of Reuteri yogurt and placebo based on a daily intake of
yogurt for a total of 4 weeks. Group 1 received Reuteri yogurt for 2 weeks prior to the
placebo, while group 2 was the opposite (Nikawa et al., 2004).
Immunizations Against Dental Caries
After the introduction of the technology for vaccinations people have always been
eager to find new ways to use this technology. It was rather unfortunate dentistry didn’t
have as much use for this type of medicine since the bacteria that are most harmful in the
mouth are found on tooth surfaces, while the beneficial bacteria are found within the skin
and on the surface of the skin. Saito, Otake, Ohmura, Hirasawa, Takada, Mega,
Takahashi, Kiyono, McGhee, Takeda, and Yamamoto (2001) researched the effects of a
mutant form of cholera toxin that would theoretically prevent S. mutans from
proliferating. For this study they took lab mice and immunized them with 10 microliters
total of a vaccine made from the mutated cholera toxin and applied the vaccine using a

56. 56
nasal spray of 5 microliters to each nostril, and the process was repeated once a week for
2 weeks.
The cholera mutant protein mCT E112K works together with PAc protein as a
vaccine by binding to surface receptors of S. mutans. When activated the surface
receptors will trigger a strong immunoglobulin A (IgA) response from the body. All cells
that could form antibodies were detected using ELISA plating methods. These cells were
extracted from the Cervical Lymph Nodes (CLNs) and separated using a magnetized
system. Once separated, the cells were incubated with 1 microgram per milliliter of PAc
for 4 days and were measured for cell proliferation and CD4 activation in response to the
poison. After confirmation that CD4 receptors responded well to PAc an immunization
solution was created using the mutant mCT E112K protein. The new vaccine was tested
on mice over a brief period of time and saliva samples were collected occasionally
throughout the experiment.
It was found that vaccination using just a solution of PAc or just a solution of the
mutant protein mCT E112K alone had no effect on the overall growth of S. mutans, while
a solution which mixed the two proteins together displayed inhibitory effects. To
confirm the safety of the vaccine, CLN cells of these mice were extracted and inoculated
in media. PAc was introduced to each of the different forms of vaccinated cells, and it
was found only the cells vaccinated with both PAc and mCT E112K created a strong
immune response, while all the other forms of vaccinated cells gave no immune response.
To test the inhibition effects of the vaccine on S. mutans a mutant strain was introduced
to the mice that was resistant to streptomycin for a few days, then the mice were fed
streptomycin to remove the original strains from their mouths. After being given the

57. 57
vaccination the mice were provided normal diets for the remainder of the experiment.
The saliva samples collected verified the thought that the vaccine was effective when
combined with both types of proteins, as seen in Figure 25.
Figure 25. S. mutans isolates from oral cavity growth after exposure to different
combinations of vaccines (Saito et al., 2001).
The mechanism for how mCT E112K works together with PAc is not well known.
When giving vaccination using a nasal spray the PAc doesn’t adhere well to the mucous
membranes within the nasal passage, and this causes a poor response from those cells
affected to create IgA. The protein mCT E112K, however, acts as a mucosal adjuvant,
meaning it assists the joining of a protein to the sticky cellular membranes in mucous.

58. 58
This research supports the concept that a vaccine can be created to prevent the spread of
dental caries. This method works to prevent the formation of biofilms and plaque not
because of an interaction with the food the S. mutans uses for metabolism, but it works by
effectively poisoning the bacteria and preventing them from being able to replicate. This
is an interesting approach because it eliminates the source of the caries even though it
cannot fix the damage that has already happened. By using biotechnology it is possible
to achieve many great goals to help make oral health care better for everyone.
Bacteria Replacement
New technology is being produced every day to fight off oral bacteria-based
diseases. It is extremely important because oral health controls, to an extent, a person’s
entire social life as well as having major effects on their overall wellbeing. Recognizing
the importance of finding new ways to treat these problems Tagg and Dierksen (2003)
searched for ways to use replacement therapy to help promote a healthier mouth. They
sought a way to treat many oral health problems without the use of antibiotics, despite
what many other people were using at the time. They realized antibiotics are only useful
for a limited amount of time before a resistance is created in the bacteria and the drugs
lose all possible effectiveness.
Before talking about the experiment, it is important to first explain what bacterial
replacement therapy means, and how it is supposed to benefit the patient receiving it.
Tagg and Dierksen state the human microbial flora is determined very soon after birth
and competition between the organisms determines where each will be most successful

59. 59
upon any easily accessible epithelial surface. Smaller concentrations of these bacteria,
usually controlled by environmental factors, are typically beneficial creating a mutualistic
relationship between the host body and the bacteria. Most of these bacteria, however, are
opportunistic pathogens and can pose great harm to the body if they are allowed to
proliferate beyond a certain threshold level. Replacement therapy is a method of
introducing bacteria normally native to the area it is being applied to when other forms
have become too prominent and begin causing sicknesses. By introducing other bacteria
scientists hope to allow for natural healthy competition between the colonies that can
then control the amount of bacteria being too aggressive, which can be seen in Figure 26.
Figure 26. A culture of Micrococcus luteus inoculated with various competitors to show
the effectiveness of bacterial replacement therapy in vitro (Tagg and Dierksen, 2003).

60. 60
Previously researchers had attempted to find ways to use bacterial replacement
therapy to help prevent the growth of S. mutans strains from causing too much damage to
the teeth. It was found, however, the only bacteria capable of preventing the overgrowth
of S. mutans were a few other forms of streptococci and enterococci. With this discovery,
research still continued using the non-pathogenic strains of streptococci as a possible
inhibitor to S. mutans since they should share very similar characteristics and qualities. It
is already known that introduction of a new colony of bacteria to already established
environments usually results in the new bacteria being outcompeted by the natives.
Therefore, it is quite difficult to perform bacterial replacement therapy and produce the
desired results for more than a few months. This is the same reason why horizontal
bacterial transfer between even blood relatives or spouses is not successful after even just
a couple of months. Therefore, bacterial replacement therapy uses not only strains
extremely similar to the native species that is attempted to be replaced, but the new strain
must also be highly competitive, which will allow it to establish itself in the oral cavity as
well as other native strains.
In the case of bacterial replacement therapy for S. mutans strains that are highly
cariogenic, not many alternatives exist while offering better solutions. The best
competitors of S. mutans are also highly cariogenic, defeating the purpose of replacing
the native species in the mouth, while the species that possess little cariogenicity are the
least competitive, which leads to their inability to proliferate in the oral cavity.

61. 61
Conclusions
Dental caries have been a major problem ever since the advancement of
technology in agriculture. Since the proliferation of the human species thousands of
years ago, the diversity of the oral bacteria has greatly decreased, leading to the
dominance of harmful strains. These fermentative bacteria produce acids capable of
eating away at the protective layers of the teeth, causing permanent damage that, left
untreated, can lead to other very serious consequences. Since the start of tooth ailments,
people have been attempting to find ways to handle these troubles. The original thoughts
of the tooth worm led to an increased interest in ways to fight off this mythical beast.
This led to the discovery of many different medicinal herbs with natural painkilling
properties used both strictly as medicine, and recreational for pain prevention.
Knowledge of oral health care quickly spread with the use of bridges to help stabilize
loosened teeth using the support of the other teeth nearby. The toothbrush that originally
began with religious implications was found to be a great source of fighting cariogenic
oral bacteria strains and was adapted worldwide as one of the main sources of fighting off
such disease.

62. 62
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