Microbiology of dental caries

Question 1

What things in the oral environment are relevant to caries?

Answer 1

-Tooth surface
- around 700 species of bacteria
- Saliva
- Gingival crevicular fluid
- Periodic availability of nutrients from host diet

Question 2

what factors are required for the development of dental caries?

Answer 2

• teeth
  -time
  -bacteria in plaque
  -Fermentable carbohydrate in diet

Question 3

Are some bacteria more important than others in caries

Answer 3

• Clarke (1924) grew Streptococcus mutans from advanced carious lesions
• 1960s – organisms responsible for “infectious” dental caries in rodent models identified as member of mutans streptococci (MS) group and lactobacilli
• MS in humans – S. mutans and S. sobrinus. Other mammals have their own version, e.g. S. rattus in rats; S. cricetus in hamsters

Question 4

Evidence implicating
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 (>106/ml) than subjects without active caries (<103/ml)

Question 5

What features of S. mutans makes it easier for it to survive?

Answer 5

It’s acidogenic & aciduric- produces high levels of acid & can survive & flourish in low pH conditions

Question 6

What virulence factors are present in S. mutans that may enable it to contribute to the caries process?

Answer 6

-Produces Antigen I/II so adherence to surfaces

-Glucosyl transferases- produce glucan polymers from sucrose

-Glucan binding proteins- attachment of cells to glucans

Question 7

Have any vaccines been developed for these virulence factors of s.mutans?

Answer 7

Vaccines have been developed (active and passive) against all three factors but no commercial vaccine yet available

Question 8

is lactobacillus associated with caries?

Answer 8

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.

Question 9

What kind of organism is lactobacillus?

Answer 9

It appears to be an opportunist organism that requires a low pH habitat so lactobacilli don’t initiate caries but will colonise existing lesions.
Doesn’t only appear in the mouth but also in the body and fermented food e.g yogurt

slide 8

Question 10

what kind of bacteria is veillonella

Answer 10

Gram negative anaerobic cocci that is associated with caries.

Question 11

Describe the importance of Veillonella in caries.

Answer 11

-Requires lactate as substrate for growth

-Lactate is produced by both streptococci and lactobacilli

-May be present in carious lesions because high levels of lactate are present.

-It has been suggested that veillonella may be beneficial to caries by reducing lactate levels but little evidence to support this.

Question 12

What was the consensus reached about the microbiology of caries from recent culture & DNA-based studies?

Answer 12

-There’s a complex bacterial community in carious lesions

-S. mutans is frequently not present

-Some samples from caries-free sites have high levels of S.

mutans

Other acidogenic species are significantly associated with carious lesions, including Scardovia wiggsiae, lactobacilli etc.

Question 13

What laboratory experiment did Bradshaw & Marsh carry out which forms the basis of much of our current thinking about the role of bacteria in caries?

Answer 13

-9 oral bacteria species growing together

-Glucose pulsed into them for 10 days.

-Fall in pH seen

-Increase in s.mutans and lactobacillus

-If pH was held at 7, no change in microbiota

Therefore, change in microbiota was in response to change in environment and not its cause

Question 14

What is the extended ecological plaque hypothesis (Takahashi & Nyved 2008)?

Answer 14

Dynamic stability stage: associated with health; only mild or infrequent acidification

Acidogenic stage: arises if there is frequent/moderate acidification

Aciduric stage: so much carbohydrate is being consumed so regularly that the pH of the whole mouth falls.

look at slide 14 and 15

Question 15

What was the background information that Takahashi & Nyved used to form their plaque hypothesis?

Answer 15

-S. mutans and lactobacilli are highly aciduric
-Mutans streptococci only a minor component of healthy plaque

look at slide 14 and 15

Question 16

Describe the dynamic stability stage.

Answer 16

1.Acid produced from sugars by range of bacteria lowers pH of plaque

2.Followed by alkalinisation phase, caused by:
-diffusion of acid
-buffering by plaque constituents and saliva
-production of alkali by bacteria

3. Production of alkali:
   -ureolysis – ammonia production from urea by urease and reduction of nitrate
   -arginine deiminase – dietary arginine catabolised to ammonia

Question 17

Describe the acidogenic stage.

Answer 17

1.Initiated by:
-Repeated, raised levels of sugar intake
-Reduced salivary flow
-Poor oral hygiene

2. Microbiota typically dominated by non-mutans streptococci and Actinomyces

Question 18

Describe the aciduric stage.

Answer 18

1. Occurs 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 Bifidobacterium and Propionibacterium species
2. 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

Question 19

What is a significant problem since more older people are retaining their teeth?

Answer 19

Caries affecting the exposed roots

Oral hygiene is key because there’s a strong correlation with the amount of plaque on the root surface and root caries

Question 20

What did microbial analysis find in relation to root caries lesions?

Answer 20

S. mutans and lactobacilli found, together with Actinomyces species & bifidobacteria.

Question 21

Components of saliva important in plaque and caries

Answer 21

Amylase - 38 (mg/ml)
Lysozyme - 22
IgA - 19
Phosphate - 17
Calcium - 6

Question 22

What does alpha amylase do?

Answer 22

Hydrolyses starch to glucose & maltose, thus making it available for fermentation

It also binds with high affinity to Streptococci and retains activity when bound,

so it’s involved in bacterial adherence to the pellicle

Question 23

In what ways can sugar become available to oral bacteria for fermentation?

Answer 23

Can be present in diet & directly obtained from that route

Can be broken down from starch by amylase

Can be derived from salivary glycoproteins by bacteria themselves

Question 24

How are the sugars then transported to the interior of the bacterial cell?

Answer 24

PTS (phosphotransferase system) & other systems

Question 25

What happens once the sugars have entered the bacterial cell?

Answer 25

They enter glycolysis at different stages of the pathway, but all sugars eventually give rise to pyruvate which is a key metabolic intermediate

Question 26

Where does the ATP for the PTS system in the bacterial cell come from?

Answer 26

The conversion of phosphoenolpyruvate to pyruvate.

look at slide 24 and 25

Question 27

What are the major pathways of glucose fermentation?

Answer 27

pyruvate can be converted into lactate or acetate.

Question 28

What does the bacteria in plaque do to sucrose?

Answer 28

Metabolises it to synthesise extracellular polysaccharides (EPS) which constitute the plaque matrix

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

Question 29

What are EPS (extracellular polysaccharides) and what do they contribute to?

Answer 29

“Sticky” polymers & they contribute to the adherence of bacteria to the pellicle and plaque

They also make bacteria more resistant to being washed away by dietary food & drink & salivary flow

It can also be used as a source of sugars for metabolism & growth when nutrients are limited

Question 30

What are the enzymes that form EPS called?

Answer 30

Glucosyl transferases

Question 31

How many glucosyl transferases does S. mutans produce?

Answer 31

3: GTFB, GTFC & GTFD (B,C,D form)

GTFB & GTFC (B and C) make water-insoluble glucans, rich in alpha-1,3-glucosidic linkages

GTFD makes water-soluble glucans rich in alpha-1,6-glucosidic linkages

Question 32

Why are insoluble glucans more problematic?

Answer 32

Because they, in particular, give plaque bulk & form a sticky mass which aids adhesion

Question 33

What do glucan binding proteins (GBPs) mediate

Answer 33

The specific attachment of bacterial cells to glucan

Question 34

Describe the process of glucan & fructan synthesis

Answer 34

Glucan synthesis:
Sucrose + (G)n primer –>
Glucan (G)n+1 + fructose

Enzyme - glucosyl transferase (GTF; “dextran sucrase”)

Fructan synthesis:
Sucrose + (F)n primer –>
Fructan (F)n+1 + glucose

Enzyme - fructosyl transferase (FTF; “levan sucrase”)

Question 35

Describe the process of glucan & fructan hydrolysis.

Answer 35

Glucan hydrolysis:
Glucan (G)n + water –> Glucan (G)n-1 + glucose

Enzyme - glucanase (“dextranase”)

Fructan hydrolysis:
Fructan (F)n + water –> Fructan (F)n-1 + fructose

Enzyme – fructanase (“levanase”)

Question 36

What is enolase?

Answer 36

A key regulatory enzyme of the glycolytic pathway

mediating the conversion of 2-phosphoglycerate to phosphoenolpyruvate

slide 32

Question 37

What inhibits the action of enolase?

Answer 37

Fluoride

Question 38

What are the effects of fluoride inhibition of enolase?

Answer 38

Reduced sugar transport because PEP is a key component of PTS system

Reduced acid production because pyruvate production is reduced

Question 39

What is xylitol?

Answer 39

Sweetening agent

Question 40

Describe the study carried out with xylitol and the findings.

Answer 40

In 2-year study (Turku), when sucrose was replaced in diet by xylitol, the mean increment of decayed, missing and filled teeth was 0.0, compared to 7.2 in sucrose control group

Partly due to lack of sucrose but also few oral bacteria produce acid from xylitol

Xylitol inhibits growth of S. mutans in vitro

Question 41

What is the primary mode of action of xylitol thought to be?

Answer 41

Passive; in that if the diet is thought to be rich in xylitol, it’s likely to be lower in sugar

Question 42

What direct effect does xylitol have on S. mutans?

Answer 42

It’s transported into S. mutans cells by fructose-PTS system but enters “futile cycle” of phosphorylation, dephosphorylation & export from cell

The futile cycle uses energy but produces none for the cell

Question 43

Is xylitol of high cariogenicity?

Answer 43

No, it’s of low cariogenicity & inhibits glycosyl transferase in cariogenic bacteria

Question 44

how could Xylitiol be delivered to caries?

Answer 44

Xylitol frequently delivered in chewing gum
Reduced caries for xylitol gum compared to sugared gum

Question 45

can Probiotics be used for oral diseases?

Answer 45

-Number of probiotic preparations now available for oral diseases

-Aim is to inhibit disease-associated oral bacteria

-Mainly re-badged “gut” probiotics

-Potential concern that many are lactobacilli – caries associated

-Evidence for efficacy against caries is lacking

Question 46

What is the aim of replacement therapy for caries?

Answer 46

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 strains. Large scale clinical trials awaited

Question 47

what are the Microbiological tests for caries prediction?

Answer 47

-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 multifactorial disease – individual bacteria are not primary cause
-Not widely used except for research