Cariology Exam One [Oral Microbiota and Caries Part 1 & 2: Banas]

In oral health there is a mutually beneficial symbiosis between the

host and oral bacteria

______ is one of the most prevalent infectious disease known

Dental caries

Dr. Willoughby D. Miller's experiment:

1980s

Incubated: Bread + (diluted) saliva + tooth (extracted)

What happens and why

The tooth eventually began to dissolve

- Carbohydrate is the substrate

- Bacteria came from saliva

- Bacteria produce acid which dissolves the tooth

- If the bacteria was heated prior to incubation, then the tooth survived

Attempts to correlate specific bacterial species with decay failed when most isolates tested were capable of fermenting carbohydrates to acid. This would become known as the

Non-specific plaque hypothesis

Concluded that caries was initiated due to the collective acidogenic properties of plaque bacteria. This would become known as the

Acidogenic theory

Competing hypotheses early on? Which is considered more correct?

Non-specific plaque hypothesis and Acidogenic theory

- Acidogenic theory is technically more correct

F.J. Orland et al experiment

- 1954

Gave conventional animals and germ-free animals identical diets and access to water with 5% sucrose for a period of several months

What Caries rates do you expect in the germ free rats?

1. No caries

2. Same as in conventional rats

3. Less than in conventional rats

4. More than in conventional rats

3. Less than in conventional rats

- If you do not have bacteria you will not have caries

- Nothing to produce acid

- All conventional rats developed carious lesions, whereas there was not even microscopic decay on the teeth of the germ-free rats

- Bacteria are necessary for decay to occur

Dr Robert Stephan

- Early 20th century

Demonstrated that plaque pH varies depending on diet and depending on its microbial composition

Take home message of Dr. Stephan and the Stephan curves

- Something fundamentally difference of the composition of bacteria from the caries lesion and the composition of bacteria from a healthy site such that you get more acid production at the caries site.

- There must be something different in the bacteria

Critical pH below which demineralization of enamel is favored is pH

5.4

Israel J Kligler argued that species that stood out with respect to producing acid and surviving at low pH would be likely

etiologic agents of caries

Kligler found that _______ dominated in advanced (dental) lesions.

lactobacilli

Using low pH media selective for lactobacilli, it was shown that their numbers typically

Increased as the health of a tooth site deteriorated

Lactobacilli are still a contributor of caries but we know know

They tend not to be in more amounts later in the caries process rather than early on in the caries process

- Don't seem to initiate but definite take advantage later on (greedy bastards)

J. Kilian Clark isolated a bacterial species from a carious lesion in 1924 that he called ___________

Streptococcus mutans

How did Streptococcus mutans get its name

Because individual coccal cells appeared elongated, like a mutant form of a coccus

Clark showed that this organism could be isolated from

early carious lesions at a higher frequency than could lactobacilli.

Clack also noted that S. mutans formed a

sticky slime layer that adhered to teeth whereas the lactobacilli did not

S. mutans was the most __________ when tested in an animal model

cariogenic species

Unlike other cariogenic species S mutans could cause

smooth surface decay

S mutans produced more acid at a _________ than most other species of oral strep in in vitro tests

faster rates

Human cross-sectional and prospective studies found that the presence of high proportions of S mutans correlated with

decay

Levels of S. mutans increased during transition from

health to disease

S mutans formed a highly adherent ____ when grown in the presence of sucrose

biofilm

Proposed that specific species in dental plaque were more responsible than others for promoting the caries process

Specific Plaque Hypothesis

Limits of the case for Streptococcus mutans

- Only a small percentage of plaque species have been tested in an animal model

- Occlusal decay is the most common

- Only tested against a subset of other oral streptococci

- Even in carious lesions the total percentage of S mutans is small (debatable)

- S mutans cannot be detected in some carious lesions

- Plaque levels of S mutans do not predict caries as well as caries history

When starch-rich plants foods are a small part of diet

low caries rates are observed

Cereal agriculture is linked to a

rise in caries, mainly root surface and CEJ, and initiated in adulthood

When sucrose is introduced into the diet there is an increase in

fissure and approximal caries and initiation in childhood

One must also consider that eating habits change

Greater wealth and food availability eventually lead to eating for pleasure as well as sustenance

Although the specific plaque hypothesis always included the possibility of other cariogenic species, along with the influence of the entire plaque ecology for some people it became synonymous with

S mutans as the cause of dental caries

Marsh et al. in the 1990s put forth the

Ecological Plaque Hypothesis

The ecological plaque hypothesis allows for the role of specific cariogenic species, but puts a greater emphasis on the

global composition of the plaque

The EPH: In this scenario, a change in the homeostatic balance of the resident microbiota is responsible for

conditions that promote the growth of pathogens

The change in balance may be initiated by

frequent eating/snacking so that the resident microbiota adapts to the low pH conditions which in turn further promotes an environment conductive to growth of S mutans and lactobacilli

Specific Plaque hypothesis vs Ecological plaque hypothesis: prevention

Specific Plaque hypothesis

- Prevention/Treatment: Target the pathogen

Ecological plaque hypothesis

- Prevention/Treatment: Target the dysbiosis

4 main types of plaque architecture

Rotund-shaped

Corncob

Hedgehog

Seaweed

The ________ shaped pattern of plaque is associated with caries and has the highest proportion of

rotund, S mutans

Caries experiences are sometimes categorized based on

behaviors age or site

Nursing-bottle or Infant caries

associated with prolonged breastfeeding or excessive use of a bottle of carbohydrate containing liquid

Early childhood caries (ECC)

The presence of 1 or more decayed, missing, or filled tooth surfaces in any primary tooth in a child 71 months of age or younger.

Severe early childhood caries (SECC)

Any smooth-surface caries in a child younger than 3 years of age. From ages 3-5 or more cavitated, missing, or filled smooth surfaces in primary maxillary anterior teeth or a decayed, missing, or filled score of ≥ 4 (age3), ≥ 5 (age 4), or ≥ 6 (age 5)

Fissure caries

most common site of decay

Smooth surface caries

caries on non-retentive sites

- Requires strong adhesion

Secondary caries

Caries around a previous restoration

Root surface caries

Caries of exposed root surfaces

Do all Caries types have similar etologies

Probably. S mutans may have the biggest role in smooth surface caries and there may be contributions from proteolytic strains for root surface caries

- Can have any in the absence of S mutans as well though

There is not a single explanation for all caries

This is true

Possible that individuals who consume high amounts of sucrose favor the colonization and accumulation of

S mutans

- More likely to develop frequent and severe caries

Individuals who consume lesser amounts of sucrose may be more likely to experience

infrequent caries that are largely driven by plaque species other than S mutans

Possible that it is less about WHO is there than the ACID METABOLISM of who is there so that there are many different...

microbial profiles that can lead to a similar outcome

STUDY UOI:

Looked at children who were caries free at a baseline visit and took a plaque sample

enrolled 130 children in a study lasting 18 months

control and gender match children without decay and look at difference in microbiology

Results: FLIP CARD

- 18 children developed decay

Minus 18 months:

- Low proportions of acid-buffering strep

- Presence of Candida sp

- High proportions of actinomyces, Prevotella, kingella

- Lower proportions of Haemophilus, Neisseria

Minus 12 months:

- Same as above; add more S mutans

Minus 6 months:

- Same as above; add overall streptococcal acidogenicity

Take away

- See beginning of a dysbiosis in some children as long as 18 months ahead of a diagnosis

- At 12 months ahead of a diagnosis start seeing differential of population including S mutans

- At 6 month ahead of a diagnosis you have a more acidogenic population

Individual Microbial Markers of the UOI study:

- 72% of subjects had high levels of S. mutans ahead of, or at the time of, diagnosis

- 67% of subjects had a buffering imbalance among streptococci and Actinomyces

- 61% had elevated levels of Actinomyces

- 44% had elevated levels of Candida

- The Only microbial indicator for one subject was S mutans

- One subject had NO microbial indicators ahead of diagnosis

- Elevated levels of Lactobacillus, Scardovia and other genera may occur in more advanced caries

Takeaway

- Multiple pathways to caries experience

QUESTION: An observation that a certain species of bacteria is more numerous at site of dental caries than at healthy sites establishes the fact that the species is capable of causing caries

1. True

2. False

False

QUESTION: The observation that caries occurs in individuals from whom S. mutans cannot be isolated is good evidence that other bacteria species are etiological agents of caries

1. True

2. False

True

SUMMARY: The consensus hypothesis for caries etiology has become the

Ecological plaque hypothesis. The role of S. mutans within this framework is still debated

SUMMARY: Plaque bacteria are necessary for caries to develop

This is correct

SUMMARY: The microbial etiology of caries is not identical in all instances but levels of _____ have been correlated with all types of caries. Still caries may occur in the absence of detectable levels of ____

S. mutans (same answer)

SUMMARY: Dietary carbohydrates are correlated with ___ but ____ consumption has been linked to the very high rates of caries development

caries rates, but sucrose

SUMMARY: The ecological balance of the metabolic activities of all plaque inhabitants likely influences caries susceptibility, at least to some extent. Host factors may

affect caries risk with or without influencing the microbial ecological balance

END PART ONE

END PART ONE

This Flashcard is pointless

This is true

START PART TWO

START PART TWO

There is accumulating evidence that _____________ play a role in the development or severity of dental decay

Host matrix metalloproteases (MMP)

MMPs are involved in

Proper tooth formation

MMP-2 and MMP-9 have been shown to be involved in

dentin collagen breakdown in caries lesions

Certain mutant alleles for MMP13 were associated with

significantly decreased risk of caries

MMP inhibitors in rats resulted in

reduced dentinal progression of lesions

There is something different about the composition of the plaque microbiota in caries such that it is more highly __________ and when coupled with other host factors or behaviors the demineralization of tooth structure is favored

acidogenic

- May be due to increased proportion of strong cariogenic species

- Or it may be due to an overall imbalance of plaque acidogens

Window of Infectivity

Prime time after eruption of teeth in children were they are particularly susceptible in acquiring organisms that are associated with caries

- Acquisition of S. Mutans usually occurs following tooth eruption

Caufield et al (1993) followed 46 mother-infant pairs and found that most infants acquired S mutans during a "window" from

19-31 months

- Other studies have documented earlier colonization times but are consistent with a window of opportunity

Why is the Window of Infectivity so important?

S mutans colonzied of tooth fissures or colonization with other highly acidogenic species as they first become accessible may leave the tooth primed for future decay

There is evidence for an inverse relationship between plaque levels of S mutans and levels of S sanguinis. The former is associated with tooth decay whereas the latter is associated with a healthy plaque

There is also evidence that the longer acquisition of S mutans is delayed the more likely the individual will remain caries free

Many now omit or downplay the window of infectivity because it

was based on the acquisition of a single species (S mutans) and suggests that caries is a transmissible infection, a concept that is no longer accepted

A sucrose-rich diet can lead to

development of a cariogenic plaque

Based on the Stephen curve, the figure shows the relative pH drop and recovery in cariogenic and non cariogenic plaque.

- Note the effect of snacks as well as the effect of the plaque ecology

- Snacking favors demineralization because it is keeping you at a time where you are at or worse than critical pH

- If you are always presenting carbohydrates then you are keeping your plaque pH perpetually at an acid level

- Timing is also important (siping pop over hours versus chug and done)

Host defense can become compromised. In some instances composition of the plaque microbiota may be a result of host factors other than dietary choices and oral hygiene or host may be uniquely susceptible to any sort of plaque composition

OOOOOOOH ya this is a true statement

Factors beyond control: Host anatomy

Deeper pits and fissures, retentive areas; host enamel acid sensitivity

Factors beyond control: Genetics as described for MMPs

Correlated a single amino acid change in lactotransferrin with caries risk. In one form the LTF kills S. mutans, in other form it does not

Factors beyond control: Low salivary flow

Decrease host defenses in saliva. Can be secondary to radiation or medicine

There are plenty of plaque species that are statistically correlated with decay. But S mutans is a species for which prospective causal mechanisms exists that links its

metabolism of sucrose and acid properties with the development of caries

S mutans is a member of the ____________, a group of species with similar cariogenic properties for humans or animals.

mutans streptococci

The most common mutans streptococci in humans are

S mutans, S sobrinus

Three virulence traits of S mutans

1. Adherence Mechanisms

2. Acidogenicity

3. Aciduricity/ Acid tolerance

Factors effecting Adhesion of S mutans

1. A newly erupted tooth surface is ideal, especially if the organism gets stuffed into a relatively unoccupied fissure. Doesn't have to rely on efficiency of adhesion mechanisms

2. Sucrose-independent adhesion. Modestly efficient. Won't lead to large proportions

3. Sucrose-dependent adhesion= very efficient

Sucrose- Independent adhesion

Thought to be salivary proteins that are part of the acquired enamel pellicle. however, this mechanism is not as efficient as it is for primary plaque colonizers. Consequently, S mutans is usually not represented in high proportions based on sucrose- independent adhesion

- Surcrose-independent adhesion is mediated by AgI/II (we learned it as SpaP in micro if that helps), a protein expressed on the surface of S. mutans

Sucrose-Dependent Adhesion

Main basis for sucrose dependent adhesion is glucan synthesis catalyzed by glucosyltransferases (GTFs)

- GTFs split sucrose and take glucose part and make chain polymer (glucan) that can take many forms

- Sucrose specific (can't just do it with glucose need sucrose)

- Glucan can be water soluble or insoluble depending on linkage between the glucose molecules

- Water insoluble version is most common is S mutans and is really responsible for its adherence to surfaces

S mutans possess 3 GTFs

gtfB encodes a GTF-I

gtfC encodes a GTF-SI

gtfD encodes a GTF-S

S. sobrinus possesses 4 GTFs

gtfI encodes a GTF-I

gtfS encodes GTF-SI

gtfT encodes GTF-S2

gtfU encodes GTF-S3

Water-soluble glucan is primarily alpha-1,6 linked with __ branch points than water-insoluble glucan.

fewer

Commercial preparations of linear alpha 1,6 linked dextran are used experimentally to stimulate

water-soluble glucans

The strains that make the most water insoluble glucans tend to be the most

Cariogenic

Water-insoluble glucan is primarily alpha-1,3-linked with more branching. It is also called

mutan

Why is glucan effective in promoting adhesion and accumulation

polymers such as glucan have the opportunity to engage in weak-force interactions in proportion to the size of the polymer. The collective result of many weak-force interactions is a strong interaction that precludes the need for specific ligand receptor binding.

It is likely that the -OH groups in glucan probably have the opportunity for extensive hydrogen bonding, and probably divalent cation interactions that help it stick tenaciously to tooth surfaces

S mutans Ferments many

carbohydrates

- A **** ton of them:

--

When abundent nutrient is present what does S mutans form

- If abundant then primarily Lactate

- Acetate

- Formate

- Ethanol

Experiment:

- Take S mutans grown at pH 7 and expose them to pH 2

- Many more will die than if you took S mutans grown at pH 5 and exposed them to pH 2

THUS

The organism has the ability to adapt to low pH

- If it has the time to adapt to a slower pH drop it will survive

The properties of aciduricity and acid tolerance result from a

global response by the bacteria

There are many many changes that together comprise acid adaptation or the

Acid tolerance response (ATR)

What kind of proteins are represented in the ATR

- Stress proteins such as chaperones, proteases, etc

- Membrane proteins

- Metabolic proteins involved in glycolysis, alternative acid production, and branched-chain amino acid synthesis

Intracellular pH is maintained by

- Pumping protons out of the cell

This is accomplished by an ATPase called the F-ATPase or H-ATPase and by a change in membrane fatty acids that are less permeable to protons

- By virtue of being able to carry out glycolysis at low pH, the organism generates ATP that can be spent for pumping out protons

- The dlt operon is involved in glycogen-like storage of glucose and may provide a source of glucose to drive glycolysis when necessary in order to generate ATP to maintain intracellular pH

Acid adapted bacteria have higher levels of

F-ATPase