Microbiology of dental caries

Question 1

What are the 4 factors needed to cause caries?

Answer 1

Tooth surface
Bacteria in plaque
Time
Fermentable carbohydrate in diet

Question 2

How is the oral environment relevant to caries?

Answer 2

• Tooth surface
• Around 700 species of bacteria
• Saliva
• Gingival Crevicular fluid
• Periodic availability of nutrients from host diet

Question 3

What bacteria in the past have been considered to be more important than others in caries?

Answer 3

• Clarke grew Streptococcus mutant from advanced carious lesion
• 1960s organisms responsible for infectious dental caries recognised as metans streptococci and lactobacilli

Question 4

What evidence links Streptococcus mutans in caries?

Answer 4

• Streptococcus mutans found in high numbers in carious lesions
• Subjects with active caries have higher salivary counts of S.mutans than subjects without active caries
• S mutans acidogenic and aciduric - producing high levels of acid and can flourish in this environment

Question 5

What are the virulence factors of strep mutans?

Answer 5

• Antigen 1/2 - adherence to surfaces
• Glucosyl transferases to produce gluten polymers from sucrose
• Glucan binding proteins - attachment of cells to glucans

Question 6

Why can we not vaccinate against strep mutans currently?

Answer 6

Originally through active vaccines could be produced but problem as some streptococci have antigens that react with cardiac tissue so not possible to take the risk of the vaccine affecting this tissue.

Question 7

Give some details on lactobacillus bacteria

Answer 7

• Earliest studies associated lactobacilli with caries
• Very low levels in individuals without caries
• Many different species found in caries and thought to be of equal importance
• Most oral bacteria are only found in the mouth but oral Lacktobacillus spices are found elsewhere in the body and in fermented foods e.g. yogurt
• Appear to be opportunist organisms that require low pH habitat
• Lactobacilli do not initiate caries but will colonise existing lesions

Question 8

Veillonella are found in high quantities in caries.

Give some details on this bacteria

Answer 8

• Gram negative anaerobic cocci
• Associated with caries
• Requite lactate as substrate for growth
• Use lactate produced by streptococci and lactobacilli
• May present in carious lesions as high levels of lactate present
• Could be beneficial in caries as it will reduce lactate level and the lactate causes demineralisation

Question 9

Caries in the microbiome era:

Give some explanation of this

Answer 9

Advances in DNA sequencing and analysis have transformed our understanding of the human-associated microbiota.

Each site in the body has its own characteristic microbiota.

Individuals have their own, relatively stable, characteristic microbiota which is far more diverse than previously realised.

Question 10

What has been concluded about the microbiology of caries?

Answer 10

• Complex bacterial community in carious lesions
• S.mutans frequently not present
• Some samples form caries-free sites have high levels of S.mutans

Question 11

What is the role of pH in caries ecology?

Answer 11

Laboratory model containing 9 oral bacteria species growing together.
Pulsed with glucose on 10 consecutive days - fall in pH and increase in S.mutans and Lactobacillus.

If pH was held at 7, no change in microbiota was seen.
Change in microbiota was response to change in environment, not its cause.

(Lab experiment:
9 bacterial species grown in continuers culture (free in nutrients and remove waste products)
Added glucose on 10 consecutive days. This causes a fall in pH and an increase in step nuts and lactobacillus.
In the next experiment, the pH was maintained at 7, there was no change in microbiota.
This shows that the change in microbiota was a response to change in environment.)

Question 12

Give some background on extended ecological plaque hypothesis

Answer 12

Mutans Streptococci only minor component of healthy plaque and do not adhere well to teeth.

White spot lesions - some strep mutans found. Lesions can form without MS.

Cavitated lesions in dentine - often dominated by MS and lactobacilli.

S.mutans and other streptococci make acid from sugars and produce final pH of 4-5, below 5.5, necessary for demineralisation of enamel.

S.mutans and lactobacilli are highly acidic - able to grow and continue to produce acid at low pH.

(Lesions can form without strep mutans.
Sometimes there were no mutans in cavitated dentine lesions.
Found that many bacteria can lower acid below 5.5 so demineralisation of enamel starts.
The bacteria were highly aciduric)

Question 13

Summarise the dynamic, acidogenic and aciduric stage during caries microbiology

Answer 13

Dynamic = only mild and infrequent acidification

Acidogenic = moderate or frequent acidification (snaking, large amounts of carbohydrate)

Aciduric = buffering capacity is broken down, pH of mouth falls, due to large number of carbs frequently)

Question 14

Give lots of detail on the dynamic stability stage

Answer 14

• Acid produced from sugars by range of bacteria lowers pH of plaque
• Followed by alkalisation phase caused by:
  Diffusion of acid
  Buffering of plaque constituents and saliva
  Production of alkali by bacteria
• Production of alkali:
  Ureolysis (ammonia production from urea by urease and reduction of nitrate)
  Arginine deaminase (dietary arginine catabolised to ammonia)

Question 15

Give details on the acidogenic stage

Answer 15

Initiated by:

• Repeated, raised levels of sugar intake
• Reduce salivary flow
• Poor oral hygiene

Microbiota typically dominated by non-mutant streptococci and Actinomyces.

(Repeated sugar intake and reduced salivary flow (dry mouth) and poor oral hygiene less to this.

Dry mouth - carbohydrate is not washed away in saliva and acid produced for a longer period.
Poor oral hygiene - increases amount of plaque present
Tends to be dominated by other bacteria that can produce acid but can’t tolerate acid conditions)

Question 16

Give details on the aciduric stage

Answer 16

After prolonged acidogenic stage, the buffering capacity of saliva is lost and bacterial community changes with:
- Selection of aciduric bacteria, particularly S.mutans and lactobacilli and some others

Thus, the ecological change drives the change in the composition of the microbiota and not vice versa, but the presence of aciduric bacteria compounds the disease state.

(High level acid for a long period of time,
Erodes buffering capacity of saliva.
Acidiuric bacteria grow here e.g. strep mutan group)

Question 17

Root caries?

Who is is more of a problem in?

Answer 17

Older people due to retaining teeth affected exposed roots.

Strong correlation with amount of plaque on root surface, reinforcing need for good oral hygiene.

Microbial analysis has found S.mutans and lactobacilli in root caries lesions.

Question 18

What components of saliva are important in plaque and caries and give details on the most important?

Answer 18

Amylase
Lysozyme
IgA
Phosphate 
Calcium

Amylase:
Hydrolyses starch into glucose and maltose, thus making it available for fermentation (simple sugars available to bacteria).
Binds with high affinity to oral streptococci and retains activity when bound. This may be evolutionary are strep mutant can obtain glucose by having amylase bound to their surface.
Involved in bacterial adherence to pellicle.

Question 19

Give details on sugar transport into bacteria and entry into glycolysis

Answer 19

Sugars can be derived from host diet or salivary components e.g. sucrose, maltose.

sugars are transported to the interior of cell during the phosphotransferase system (PTS) and other systems.

Following transport, sugars enter glycolysis at different stages of the pathway.

All sugars eventually give rise to pyruvate, which is a key metabolic intermediate.

Question 20

What are the 3 ways that sugars is available to bacteria?

Answer 20

Ways sugar is available to bacteria:
Present in diet
Broken down from starch
Derived from salivary glycoproteins

Question 21

Give brief details on the glycolic pathway

Answer 21

Glucose is phophorylated with 2 molecules of ATP. This forms fructose,1,6,bisphosphate which is split into 2. These molecules are then dephosphorylated with the generation of 2 ATP molecules for each molecule.
Net gain of of 2 ATP molecules.

Question 22

How does the phosphotransferase system work?

Answer 22

ATP made in glycolysis is used in this system for sugar transport.

Energy comes for the liberation of ATP when pyruvate is formed.

Glucose binds to transporter system in cell wall of bacterial cell. Phosphate is used to convert glucose and bring it into cell as glucose 6 phosphate.
Uptake of sugar is driven by glycolytic pathway itself.

Question 23

What are the main products formed in glucose fermentation?

Answer 23

Lactate
Propionate 
Butyrate
Acetate
Formate

These are are short chain simple sugars.

Question 24

Is sucrose more important than other sugars?

Answer 24

There is a wide range of sugar transporters and metabolic pathways for acid production from sugars among oral bacteria.

Some bacteria have ability to form polymers from sucrose specifically.

Question 25

Extracellular polysaccharides:

Where do they come from?
What are they and what do they do?

Answer 25

Bacteria in plaque metabolise sucrose to synthesise extracellular polysaccharides which constitute the plaque matrix.

Some bacteria e.g. S.mutans also from intracellular polysaccharides.

EPS are sticky polymers and contribute to the adherence of bacteria to the pellicle and plaque.

EPS make bacteria more resistant to being washed away by dietary food and drink and salivary flow.

EPS can also be used as a source of sugars for metabolism and growth when nutrients are limited.

(EPS from bacteria is a component of plaque matrix.
Strep mutans can make intracellular polysaccharides which are storage molecules for later use.

EPS - sticky to help bacteria be maintained and form bulk of plaque to make it difficult to remove by oral hygiene or mastication forces.
EPS stores sugars for metabolism so bacteria can use this for growth between meals)

Question 26

What is the importance of glucosyl transferase?

Answer 26

S.mutans produces three glycosyl transferases (GTFB, GTFC and GTFD).

B and C make water-soluble glucans rich in alpha 1,3 glycosidic linkages.

Insoluble glucans, in particular, give plaque bulk and formed a sticky mass which aids adhesion.

Specific attachment of bacterial cells to glucan is mediated by glucan binding proteins.

(Enzymes that form extra cellular polysaccharides.
Strep mutans produce B C and D form. B and C enzymes makes insoluble glucan where as D enzymes makes water soluble glucan.
Insoluble glucan which are important in giving plaque bulk and forming large sticky mass to prevent removal.
Bacteria produce glucan bidding proteins to help attachment of bacterial cells to glucan mass.)

Question 27

What are the EPS synthesis equations?

Answer 27

Glucan synthesis:
Sucrose + primer = glucan + fructose
Enzyme is glucosyl transferase.

Fructan synthesis
Sucrose + primer = fructan + glucose
Enzyme is fructose transferase

(Enzyme recruits sucrose to add to glucan primer molecule (a polymer in process of enlogating). The glucose residue is extracted from glucose and added to polymer and a fructose molecule is liberated.

Similar process in fructan synthesis but in this case, the fructose reside is incorporated into growing polymer and free glucose in liberated. The enzyme in this cause is fluctosyl transferase.)

Question 28

Give the hydrolysis equation for EPS

Answer 28

Glucan hydrolysis:
Glucan + water = glucan (n-1) + glucose

Fructan hydrolysis
Fructan + water = fructan (n-1) + glucose

(We can also get hydrolysis of EPS. Glucanase or dextronase can use a molecule of water to bind with the glucans to reduce the glucan polymer by one residue to liberate free glucose.
Similar for fructan and fructose is liberated from the fructan polymer)

Question 29

What is enolase?
What inhibits enolase?
What consequences does inhibition of enolase have?

Answer 29

Enolase is the key regulatory enzyme of the glycolytic pathway. (Last step of glycolysis is the conversion of 2-phosphoglycerate to phosphoenolypyruvate)

Fluoride inhibits enolase.

Inhibition of enolase has 2 important consequences:

• Reduces sugar transport as phosphoenolypyruvate is a key component of the phophotransferase system
• Reduced acid production because private production is reduced

Question 30

How can probiotics be used in controlling caries?

Answer 30

Prebiotics - sugar alcohols

Xyltiol is the most commonly used.

In one study, sucrose was replaced in diet by xylitol, a massive reduction in caries indices was seen.
Thought that this was due to lack of sucrose but in addition its is the case that few bacteria can produce acid from xylitol.

Xylitol inhibits growth of strep mutans in lab.

Mode of action of Xylitol:

• Passive effect due to reduced sucrose consumption
• Transported into S.mutan cells by fructose-PTS system but enters futile cycle of phosphorylation, dephosphorylation and export from cell
• Futile cycle used energy but produces none of the cell

(Xylitol has a mainly passive action. If a diet is rich in it, it will be lower In sugar most likely,
It does have a direct effect in strep mutans. As sucrose is transported into cell by a fructose PTS system, it enters a futile site where it is phosphorylated, dephosphorylated and then exported from the cell.
The futile cycle requires energy but makes no energy)

Question 31

How is xylitol used to treat caries?

Answer 31

Frequently delivered in chewing gum.

Reduced caries for xylitol gum compared to sugared gum.

Xylitol is of low cariogenicity inhibits glycosyl transferase in cariogenic bacteria and some evidence of in-vivo benefits.

(In chewing gums, evidence that this causes reduce level of caries.
Xylitol - low cariogenicity and can inhibit glycosyl transferase in cariogenic bacteria.
Some evidence of in-vivo benefits.)

Question 32

How can probiotics treat oral disease?

Answer 32

Aim is to inhibit disease-associated oral bacteria.

Mainly re-badged ‘gut’ probiotics.

Potential concern that many lactobacilli which is caries associated.

(Public shown amenable to this concept. Idea of drinking good bacteria in yogurt to strengthen gut mucosa and promoting recovery from infectious GI disease.

Number of oral probiotics are marketed.
Majority of them are rebadged gut biotics. Many are lactobaccil, this is associated with caries so this may not be a good idea to use it in probiotics.
No evidence that using probiotics for caries is beneficial.)

Question 33

What is replacement therapy for caries?

Answer 33

• Aim is to replace S.mutans with other strains/species
• Attempted with other Streptococcus species that are less acidogenic and aciduric but were found to compete poorly with S.mutans
• Alternative is to modify S.mutans strains so that they produce less acid. Strategy then is to use antimicrobials to reduce plaque levels and then re-seed with avirulent stains.

(Another possible treatment:
Replace strep mutans with an inactivated strep mutans or another species of bacteria.
Replacing them = found it impossible to remove strep mutans as it has a niche in oral microbiome and it is difficult to dislodge it.

Inactivate strep mutans by removing them, genetically modifying to produce less acid. Replace inactive strep mutans to allow it to occupy niche in cavity)

Question 34

Microbiological tests for caries prediction?

Answer 34

Commercial test kits are available.
Test for numbers of S.mutans and some also for lactobacilli.
Effective for populations, less so for individuals.
Caries is a multifactorial disease - individual bacteria are not primary cause.

(This is disease activity prediction.
Test for numbers of strep mutans,
Problem = tends to work for populations but less reliable for individuals due to having different bacteria.
Bacteria individually are not the primary cause, so microbiology tests are not widely used.)

Question 35

Summarise microbiology of dental caries

Answer 35

• Many oral bacterial species are acidogenic
• Frequent consumption of fermentable carbohydrates is principal risk for caries
• S.mutans and other aciduric bacteria are selected for by acid environment and availability for sucrose
• Aciduric bacteria continue to produce acid under acid conditions and lesion progress
• EPS increases plaque formation
• Prebiotics such as sugar alcohols may have anti-caries properties
• Potential for probiotics but yet proven