6. Biofilms and Plaques

Question 1

I think dental caries is very complex in a sense that it involves many different factors. You have socioeconomic and behavioral factors, but also factors that are more associated with oral biology (see list below):
• \_\_\_\_
• tooth surface
• \_\_\_\_
• diet
• \_\_\_\_

Answer 1

saliva
bacteria
time

Question 2

Biofilm and Pathogenesis of Dental Caries Disease

Dental caries results from complex interactions of ____ bacteria (and their products), host ____ and dietary ____ (e.g. sucrose) leading to establishment of ____ biofilm on the susceptible tooth surface.

Answer 2

specific
saliva
carbohydrates
virulent-cariogenic

Question 3

Dental Caries: An observation from ancient times…

This observation is that the interaction between diet, the microbiota, and biofilms is actually not new. This is a very interesting paper where they sequenced ancient dental plaque from mesolithic to the modern times, and they found consistently that oral microbiota changed with changes in ____ shifts, e.g. in the neolithic period, a ____ diet was introduced, and there was a dramatic change in the oral microbiota. For example, ____, which is a known cariogenic microorganism, started to appear, and it became more dominant and frequently isolated, especially when ____ was introduced in modern times. With that, we observe development of dental caries, and cavitation associated with this microbiota shift due to dietary changes.

Answer 3

dietary
farming-based
Strep mutans
processed sugar

Question 4

Dental caries continues to be the single most prevalent and costly ____
disease affecting both adults and children in the US and worldwide. The public in the
US spends close to ____ billion to treat the ravages of this disease or its consequences.

Unfortunately, dental caries continues to be a big problem nowadays, particularly, though, in the specific subpopulations such as low ____ populations, where some behavior (e.g. sugar intake and also some microbiota differences) causes an interesting distribution.
There are still worldwide regions where dental caries has a prevalence of 80% or more. This is true even in our country where you see pockets of this, even in Philadelphia. In the clinic, you will see a large group of the population affected by dental caries.

Answer 4

oral infectious
50
socioeconomic

Question 5

Complex Microbiology: Polymicrobial interactions

We need to do better to improve oral health and prevent this disease. Over the past decade, the concept has been introduced that we are dealing with a very complex microbiology, so it’s not only bacteria. You have many different species in the oral cavity, and that entails different interactions with different micrograms.

This paper shows the following:
• (talking about L picture): There are a variety of different species, and as the disease progresses from healthy to
cavitated, you have changes not only in the ____ but also in terms of the ____. The graph here clearly shows the diversity being dramatically reduced as the disease progresses, and some species (e.g. S. mutans and other Strep species) become more dominant in this microbiota.

• (talking about the R picture): Some species have been used to analyze the pattern of the ____ and proportions to predict the ____ of dental caries based on this list of species (middle list) and on microbiome data that we have been gathering in the 10-15 years. Today, there’s a lot of focus on the microbiology of dental caries, but rather, it’s a different perspective that can be equally important and actually work in parallel with these microbiological changes.

Answer 5

proportions
diversity

microbial compoistion
onset

Question 6

Here are some clinical observations that I made myself when I was in the clinic. When you have a healthy condition (top two pictures), you actually have a very ____ number of bacteria attached to the dental pellicle.

When there is a transition to the plaque that causes dental caries (bottom pictures), there’s another observation - there are changes in the ____/____/____ characteristics of this plaque.

Point: over the course of the transition, you have a big change in the number of microbial species
but also in the characteristics (physical/chemical/biochemical) of these species.
Plaque = gelatinous/gooey consistency

Answer 6

limited
physical
chemical
biochemical

Question 7

If you observe more closely, you can see that microorganisms are not alone in this plaque. This micrograph is of plaque collected from caries-active children.

**Bacteria are surrounded by extracellular ____, helping the bacteria to stick together and
form a
highly ____ aggregate structure that is ____ attached to the tooth surface.*

Answer 7

polysaccharides
cohesive
firmly

Question 8

Biofilms

Biofilms are highly structured microbial communities attached to a surface and enmeshed in an extracellular polymeric matrix.

Biofilm:
• not just associated with ____ disease, but also causes many different infections across the human body
• not just a bunch of cells on top of each other; very ____ communities that are first and foremost firmly
attached to a surface (e.g. tooth surface, implant, dental materials)
• communities are usually enmeshed in an extracellular ____ matrix

L pic: Imagine the different bacterial species as the brick, and the mortar is the matrix, the material that helped you build this microbial habitat.

R pic: biofilm in cystic fibrosis. See bacteria that are embedded in the extracellular matrix - they are together; you cannot ____ them. This is a characteristic of the biofilm associated with dental caries.

Answer 8

dental
structured
polymeric

separate

Question 9

Exopolysaccharides (EPS) are key components of the ____ in cariogenic oral biofilms,
and are recognized ____ involved in the pathogenesis of dental caries.

Point of picture: shows bacteria enmeshed in polysaccharide matrix

What makes the matrix help form this cohesive aggregate that is very firmly attached to the tooth surface? (See next slide - the suspense is killing you, I know.)

Answer 9

matrix

virulence factors

Question 10

How does the matrix help form the aggregate that is firmly attached to the tooth surface?

This polysaccharide, polymer-like material, is produced primarily by extracellular enzymes called ____, e.g. ____ and ____ (Gtfs and Ftfs). Once these enzymes are secreted by microorganisms, they utilize ____ and ____ (sucrose is the main substrate for them). They break down the sucrose molecule, take up the ____ moiety, and start to build a glucose polymer called ____. There are different kinds - you don’t need to know the details of the chemistry of this for the exam - just have the idea that there are two types:

1) insoluble, which is rich in ____-linked glucose
2) soluble, which has mostly ____-linked glucose and branches of ____-linked glucose

The bulk of the plaque matrix of the biofilm is made by the ____ group - makes sense because you can see that plaque is not soluble in water; it’s highly insoluble. There are also different polysaccharides like Ftfs that help to build the biofilm matrix.

Answer 10

exoenzymes
glycosyltransferases
fructosyltransferases
starch
sucrose
glucose
glucan
alpha-1,3
alpha-1,6
alpha-1,4

insoluble

Question 11

S. mutans is an important microorganism within the complex microbiome and has been linked with dental caries not only because it produces acid, but also because it produces several of those ____ that I mentioned earlier (e.g. Gtfs). It produces ____ different Gtfs and ____ Ftf that help to produce those polysaccharide materials. Interestingly, this microorganism also expresses ____ glucan- binding proteins.

**Interesting mechanism here: this microorganism helps not only to produce the glue-like material,
but also has ways to bind very effectively (aka it produces ____ proteins) to self-produced polysaccharide, which allows it to bind ____ to build up cariogenic plaque when you provide the fuel (____), which is why diet (namely, sugar) is very closely linked to dental caries and the formation of cariogenic biofilms.

Answer 11

exoenzymes
three
one
four

glucan-binding proteins
sugar

Question 12

Glucosyltransferases (Gtfs)

• produced and secreted chiefly by oral ____ - catalyze the synthesis of glucan from dietary ____ (glucose transfer)

I’m going to talk a little bit about what Gtfs are, but there’s no need to get too caught up in the details. I just want you guys to have an idea of the concept of this. reads slide

Luckily, now we have the full understanding of how this structure looks, including the 3D crystal structure of the enzymes, and in this specific enzyme, in addition to the ____ and ____ terminals and ____ domain, there is a ____ domain, which helps the enzyme produce the polysaccharide.

Answer 12

streptococci (streptococcus mutans)

amino
C
catalytic
glucan-binding

Question 13

Here, you have sucrose (made of glucose and fructose moieties) that approaches the enzymes in the catalytic core, and hydrolysis occurs.

What is interesting about sucrose (compared to other sugars like maltose) and how does it relate to hydrolysis?
Answer:
- what’s interesting: lots of ____ here; all of the energy is released as the hydrolysis occurs - ok but why do we care, dude?: energy released is important because the enzymes need that energy to transfer the ____ moiety to build up the polysaccharide and cause polymerization into ____ –> this is why ____ = unique molecule to build up glucan

Answer 13

energy
glucose
glucan
sucrose

Question 14

Gtfs

• present in human whole ____ and in the ____ as well as bound to ____ membranes.

Remember that the salivary pellicle coats tooth surfaces.
**The third bullet point kinda covers the green bar, but it says “present in human whole ____ and in the pellicle as well as bound to bacterial membranes”
• **It’s covering the labels for the domains of the protein; the one in the middle is the catalytic domain, and
the one on the end is the glucan-binding domain (GBD).

Answer 14

saliva
pellicle
bacterial
saliva

Question 15

This classic study demonstrated that these enzymes, once secreted, can bind to the pellicle. The top picture is a very close-up view of saliva-coated hydroxyapatite (sHA). Once the enzyme is adsorbed to the tooth surface and once you provide sucrose, the enzyme (Gtf) produces polysaccharides directly on the surface of ____ (remember that HA is similar to tooth enamel).

Talking about graph: Here, we have a typical way to determine the optimal pH for the enzyme activity.
• “soluble” curve: this is the enzyme in the ____ phase
• “PSHA” curve: this is the enzyme once it is ____ to the tooth surface

Can somebody tell me what this type of graph tells me re: enzyme activity?

Once the enzyme is adsorbed onto the tooth surface, the curve becomes almost a ____ line, which tells us that no matter what pH there is, the enzyme is highly ____. This is important because caries occurs when the ____ is very active (wasn’t sure what he meant by this). These enzymes, even when there are active caries, are still catalytically active to produce these polysaccharides.

Answer 15

HA
solution
adsorbed

straight
active

pH

Question 16

S. mutans expresses several membrane-associated Glucan binding proteins (gbpA, gbpB, gbpC, gbpD)

Why are these polysaccharides (aka EPS) so important?
Follow-up study that showed that once you have this glucan-coated surface and you add bacteria (e.g. S. mutans), it acts like a ____ and you get a huge number of bacteria coated to the surface.

Comparing glucan-coated HA surfaces and those HA surfaces without glucan coating: ____ increase in the number of bacteria that attach to the glucan-coated HA surface.
- One of the reasons that S. mutants can bind so avidly to the glucan-coated HA surface is that it has ____ glucan-binding proteins that help it attach to the surface.

Answer 16

magnet
7-8 fold
four

Question 17

Gtf-derived Exopolysaccharides (EPS)

Glucan formed
on bacterial surface (____-produced)

Glucan formed on sHA surface
(____-produced)

Glucans formed in ____ by surface-adsorbed Gtfs.

These enzymes that are secreted by the bacteria can bind to the HA surface and produce polysaccharides right onto the tooth surface (pellicle). Didn’t have a chance to show this but the enzymes can attach to the bacterial surface and produce polysaccharides on the bacterial membrane as well. See that when the bacteria become so firmly attached to the HA surface, you cannot remove this by swishing food in your mouth; you need a ____ action to remove it.

In clinic, you’re going to see a lot of kids with caries/plaque. Touch the plaque with your instrument and see how sticky it is; it’s difficult to get off the instrument, and a lot of that property has to do with these ____.

Answer 17

GtfB
GtfC
in situ

mechanical

polysaccharides

Question 18

Ecological Biofilm Concept

The most well-accepted model for the development of dental caries/building of this biofilm is the ____ model, which has to do with different bacterial species, but also ____ produced in situ. In a healthy condition, you have a complex microbiotic composition, and they are constantly interacting with the pellicle or saliva-coated surfaces, including the tooth surface.

Microorganisms that have high affinity for the pellicle are called ____ (____ organisms that can bind pretty avidly to tooth surface; have several mechanisms to do so). Usually in this initial colonized community, you have very few ____ bacteria (including not much S. mutans), so it is called a non-cariogenic type of situation.

Answer 18

ecological
EPS
early colonizers
cariogenic

Question 19

Ecological Biofilm Concept (cont.)

However, when you have changes in environment, e.g. you’re under a lot of stress and you start intaking more sugar, that will provide the kind of fuel that will help to produce this EPS.

____ in saliva, bacteria, and on tooth surface will help to start the production of this EPS and if you continue with this protracted sugar eating, you will favor the building up of this biofilm that not only contains a lot of cariogenic bacteria but also the matrix that helps you form this ____ unit.

This is a reality for a lot of people suffering from early childhood caries. Most of these kids have a very sugar-rich diet; you’re going to hear about ____ caries - the kids have a bottle that they stick in their mouths and sometimes go to sleep with, which creates a constant flow of sugar, helping create a massive accumulation of plaque that’s rich in EPS.

Answer 19

Gtfs
cohesive
bottle

Question 20

Acid Production and Acidic pH

Most of the bacteria (e.g. S. mutans, which is just the example he’s giving but he says there are obviously other ones) that produce acids are entrapped in this matrix, and these types of microorganisms can do two things:
◦ Transport ____ (sugars, etc.) through multiple effective mechanisms like ____ transport
systems and ____ (PTS allows transportation of ____ sugars, from glucose to sucrose to lactose), which
is important bc these organisms can rapidly transport any sugar directly to the intracellular environment ◦ Actively produce ____ once the sugars enter the intracellular environment
‣ Because of being really effective at producing acid, they’re considered ____ bacteria

Answer 20

multiple carbs
sugar ABC
PTS
multiple

acid
acidogenic

Question 21

Acid Production and Acidic pH

This is a two-way approach: if bacteria cannot effectively transport the ____ to the intracellular environment, they cannot produce ____.

Simple experiment that you can do in your lab (since we all have our own labs. ok.): make a buffer with the bacteria in it; put a few drops of glucose or sucrose in it, and within 15 min, the pH goes from 7 to about ____.

Answer 21

sugar

4.3

Question 22

Ecological Biofilm Concept

If the bacteria that are trapped in the biofilm produce acid (remember that you have constant supply of sugar), you have ____ of the enviornment

Answer 22

acidification

Question 23

Surviving at Acidic pH

Increased activity of proton-translocating ATPase in response to low pH:
The main role of this enzyme is to pump ____ out of the cell in order to protect intracellular pH from acidification, which is critical for the optimum function of enzymes of the glycolytic pathway.

Low pH induction of DNA repair:
A DNA repair system is activated at ____ pH to protect from damaging effects of acids.

Changes in the membrane fatty acid composition:
May affect the ____ of the membrane to protons

◦ Problem: The bacteria that can tolerate acid (acid-tolerant) are the very same organisms that can
produce acid (\_\_\_\_). It's like survival of the fittest - they produce acid, acidify the environment, kill the other \_\_\_\_ bacteria that cannot survive in that environment, and all of a sudden, you have a \_\_\_\_ of diversity; you have much less number of microorganisms that are present. Those that remain are highly pathogenic, because they can produce AND survive in acid, and they have at least three different mechanisms to do so (listed on the slide).

Answer 23

protons (H+)
low
permeability

acidogenic

commensal
reduction

Question 24

Ecological Biofilm Concept

Now you have this really terrible combination where you have ____ of the microenvironment, reduction of ____, and increase in the proportion of ____ (and also highly acidogenic) pathogenic organisms in the biomass. This is what we have in the transition from what was non-cariogenic to highly ____ biofilms.

Answer 24

acidification
diversity
acid-tolerant
virulent/cariogenic

Question 25

Acid Production and Acidic pH

The acid production by S. mutans and other acidogenic bacteria combined with their aciduricity (acid-tolerance) results in low pH values (____) within plaque/biofilm, which can cause demineralization of ____

Talking about graph: The ____ curve was a way to determine how the pH goes down in the oral cavity when saliva spreads, bc saliva has a very powerful ____ capacity (and can neutralize acid), which is one of its most important protective mechanisms.

When you ask a patient to rinse with a solution of glucose or sucrose and you stick an electrode in the plaque, you’re going to see the pH dropping very quickly, but it will not stay there, because the saliva will help to bring the pH back to neutral.

However, there is a period below the critical pH (critical pH = between 5 and 5.5) where the enamel starts to ____, starting at the microscopic level.

However, usually because the pH goes back up quickly enough, there isn’t enough time to develop ____ of the enamel surface.

Answer 25

5-5.5
enamel

stephan
buffering

demineralize

destruction

Question 26

Acid Production and Acidic pH

Frequent ____ consumption/acid production and prolonged periods of ____ eventually leads to the development of carious lesions.

But if you provide sugar constantly, what happens?
• Going back to the e.g. of the kid who’s constantly exposed to sugar/sucrose: there’s no time
for the ____ step, so the pH is constantly kept at or below the critical pH level. So, you go from a microscopic level of demineralization to seeing these white spots (bottom L picture of tooth), which is ____, but not statically, and which you will see a lot of in clinic.

Once you get to more severe cases (bottom R picture), there is cavitation of the enamel surface, and you have to use restorations. Think of it as surgically removing the infected teeth tissue, which you want to avoid.

Answer 26

sugar
enamel dimineralization

pH neutralization
reversible

Question 27

Ecological Biofilm Concept

(1) How the ____-matrix modulates the formation of a spatially organized 3D biofilm architecture, and
(2) why ____ accumulate within the EPS-rich milieu remain intriguing mysteries…

… especially when there is an abundance of buffering ____ that surrounds the teeth, which would be capable of rapidly neutralizing these acids

These microbiological/biochemical characteristics will help to maintain this highly acidified environment, which will lead to the clinical onset of ____. What we need to do is prevent this acidification of the environment before you get to demineralization of the enamel (even at the microscopic level).

Answer 27

EPS
acids
saliva
cavitation

Question 28

Fluorescence imaging of intact EPS-matrix

Alexa-Fluor 647 dextran (for ____ labeling) and pH indicator Lysosensor (for in situ ____ measurements).

The fluorescent probes can be incorporated during ____ assembly.

Laser scanning ____ fluorescence and computational analysis

R picture: This is what polysaccharides look like if you’re just looking into a regular microscope. See that it’s ____ and ____.

This particular polysaccharide was labeled with the probe, and we triggered the enzymes to use their natural ability to take up these fluorescent probes and incorporate them into the glucan structure. I’m not going to go into detail about this.

Result: labeling of the PS using fluorescent imaging, which helps show the structure in more detail. We used this method to see how the bacteria and PS are ____ with respect to each other, how they ____ up the structure, etc.

Answer 28

EPS
pH

biofilm matrix
confocal

insoluble
branched

positioned
build

Question 29

We used a saliva-coated HA model. These are discs of HA that mimic the tooth enamel. We position them ____ because most of the surfaces where plaque accumulates are in vertical, not horizontal, position. Most plaque is in the ____ surfaces.

This (small box that’s expanded from the larger white box below it) is the typical biofilm that starts to form after a few days, and we can reconstruct this in 3D. We observed that this bacteria that formed in this cariogenic biofilm tends to form these ____ (it’s the middle left picture on his slide, but it’s green (not green and red) because the probe is recognizing just the bacteria; he changes it to the mixed red and green version later), and because the probe can see the matrix, we can also recreate the matrix of the biofilm (right picture). We can merge them together to see how the biofilm will look with the bacteria embedded in the matrix (picture with mixed green and red).

Bottom R three pictures: see the bacteria (green) meshed in this matrix (red) that helps to form these little clusters (R-most picture with mixed green and red).

Bottom L three pictures: in many cases, it looks like an organ; you have these bacterial clusters (green) that are surrounded by this matrix (red) that helps to keep this particular shape (mixed green and red), forming a compartmentalized structure.

Answer 29

vertically
free smooth
clusters

Question 30

The EPS matrix assembly during the development of mixed-species biofilms

We can do a time-lapse to see how the biofilm forms over time. Remember that red: matrix and green: biofilm. As biofilm starts to develop (top row of pictures), we see ____, not a carpet, of bacteria forming on the surface, usually associated with EPS. If you go back to the original microscopy, you see the bacteria bound to and covered by the EPS. This (as in the pictures on this slide) is a more advanced stage, a little later in the process - what you see is this ____ helping to form the clusters. As the biofilm evolves (second row), we see this island-like shape of clusters that are very associated with the matrix - called a ____ associated with EPS. As the biofilm grows/ expands (bottom row), you can see how the microcolonies become larger and the EPS more complex re: surrounding these structures.

What happens if you don’t provide sucrose? (L column; top picture) - very ____ clusters/accumulation of bacteria

What happens if you provide sugar but not sucrose? (L column; bottom picture) - Remember the biochem aspects of this. Difference b/w sucrose and glucose is the energy in the linkage. You provide a similar amount of sugar, and although you have more growth than if you provided no sugar, you don’t see the same ____ in the biofilm that has the matrix caused by sucrose being the fuel. Closer look: sugar without sucrose example has cells that are sparsely ____ and don’t form the clusters that form when the matrix is there.

Answer 30

clusters
EPS
microcolony
small
architecture
distributed

Question 31

The EPS matrix assembly during the development of mixed-species biofilms

See the polysaccharides on the HA surface and on the microbial surface; they help with initial ____, and as this process moves on, you see a more complex biofilm being formed (time increases as you go down the column).
We know that S. mutans produces these two Gtfs (in the R picture).

Answer 31

adhesion

Question 32

Role of GtfB and GtfC in the assembly of 3D matrix and microcolonies

What happens then if we still provide the sugar but we delete those two genes that are responsible for the production of those polysaccharides?

• We don’t see that ____ again. They can ____ to the surface but they cannot form those structures.

What happens then if you use enzymes that can digest the matrix (e.g. mutanase, which can break down all alpha-1,3-linked glucose)? (See next slide.)

Answer 32

structure

bind

Question 33

Role of GtfB and GtfC in the assembly of 3D matrix and microcolonies

If you use enzymes like ____ that can digest the matrix, what happens is shown in the second column of pictures on the white background - after incubating the structure with mutanase, the matrix was totally ____ and the whole structure ____ in two hours. This illustrates the importance of the matrix in building up this particular structure.

Answer 33

mutanase
degraded
collapsed

Question 34

Dynamics of EPS-modulated microcolony assembly and structural organization

Pictures: changing bacterial characteristics over time are in green and changing EPS characteristics are shown in red. What our grad student saw is the initial ____ (first column) and the formation of these really densely packed ____ (2nd column), which grow in size and ____ with each other (3rd column).

In every single step, she saw the ____ bridging these structures and forming a highly structured matrix to form this compartmentalized architecture (4th).
(Talking about row of red pictures) Speaking about the biochem: you see the evolution from a very ____ structure to a very ____ that helps structure the biofilms.

Answer 34

clustering
microcolonies
merge

EPS

amorphous
organized scaffold

Question 35

Relevance of Biofilm-Matrix Architecture in Virulence

Buffering Saliva
In the context of virulence, acid production alone may not be the key factor, but rather ____ and ____ acidic microenvironments
are formed within the 3D biofilm architecture

What is the relevance of having this architecture in the context of dental caries, particularly when you have saliva surrounding the surface? (aka why does this architecture help in development of caries even when tons of saliva is around?)
reads slide
Remember that we are dealing with a 3D, not flat, structure, and that it’s surrounded by one of the most perfect buffering solutions made by the body (saliva).

Answer 35

how

where

Question 36

Creating structured and localized acidic microenvironments

The EPS-matrix acts as a
____ barrier, which impedes rapid neutralization by the pH 7.0 buffer -> ____ environment

Map on the L (wasn’t in the copy of the slides he gave us): can take any different section and using retrometric analysis, you can measure the pH at different heights or thicknesses of the biofilm. You can see that areas close to the HA interface (bottom are of the graph on the R of the pic) are very ____ (pH between 4.5 and 5.5; boxed in red on height graph) but some areas in the middle (aka in the middle of the biofilm, but also in the middle of the graph) are areas where the pH is close to ____ (boxed in green on height graph).

Observe that the ____ organization allows the bacteria producing the acid inside this biofilm to be accumulating in these specific locations despite the presence of the buffer, and that’s what forms these acidic pockets.

Answer 36

diffusion-modifying
acidic
acidic
neutral
structural

Question 37

Certain ways to disrupt the matrix include using enzymes that can degrade the matrix, like mutanase, which can digest the ____-linkages, and dextranase, which can digest all the ____ polysaccharides.

See the acidic environment on the inside of these biofilms. What happens if we add the enzymes to this solution? Within 30 min, you have almost full ____ of the interior of the biofilm, indicating that the matrix not only now helps to form these clusters and make the architecture but also acts as a diffusion-limiting barrier, which impedes rapid neutralization, even at pH 7.

This could explain why bacteria associated with dental caries help to maintain this acidic microenvironment in the interior of this plaque/biofilm despite our mouth having so much saliva.

Answer 37

alpha-1,3
alpha-1,6
neutralization

Question 38

This diffusion-limiting barrier property is important. See the no acidification vs. acidification chart above. In this model of a biofilm, we have we three different species - we have S. mutans (pathogenic), S. oralis, and A. naeslundii (the last two are ____, or those not directly associated with dental caries; he says that A.naeslundii might be associated with ____ caries).

If you don’t have an acidified environment within the biofilm, all the bacteria reach ____; they can live in equilibrium in this environment, as seen in the L graph. If the biofilm is allowed to acidify, forming this compartmentalized acidic microenvironment, what happens? The commensal bacteria like S. oralis, which are very dominant in the beginning, rapidly ____; A. naeslundii also ____.

Those that are able to survive and produce acid (S. mutans in this example) survive - what happens? They become the ____ species.

Now I’m trying to converge the ____ concept of these biofilms with the ____ perspective of how the matrix helps to form these clusters, create this acidic microenvironment, and therefore, coordinate these changes in the microbiota.

Answer 38

commensal
root

homeostasis
die
dies

dominant

ecological
biochemical

Question 39

Creating structured and localized acidic microenvironments

Enamel acid-dissolution at microscopic level… to clinical onset of ____ areas of dimeralization

There is an accumulation of ____ areas of highly acidic microenvironments, and that is problematic, because if you have an acidified environment in this localized area, you start to have dissolution/ ____ of enamel.

Dental caries is not erosion; erosion = ____decalcification of enamel. Caries, however, is ____ onset of these areas of demineralization, which is problematic because of the risk of cavitation, and once you create cavitation, there’s no returning. You have to surgically remove tooth tissue in order to repair it.

The ____ organization of the biofilm and the ____ formation of this acidic microenvironment might explain why clinically you don’t have an erosion; you have localized onset of caries instead. Our goal is to stop the caries from getting to this point. Hopefully through better understanding of this process, we can intervene more effectively.

Answer 39

localized
localized
demineralization
homogenous
localized
structural
localized

Question 40

Although the immediate cause of enamel dissolution is certainly acid production, the absence of the ____ effects of the biofilm matrix would minimize the ability of acids to demineralize in the presence of (buffering) saliva……which could explain why S. mutans-derived ____ and their ____ products
have been linked with the disease of dental caries (as demonstrated both in
animal and in clinical studies).

TL;DR: He says caries production is not just about bacteria producing acid. You also have to have the following:

1. structure that is ____ to remove
2. ____ barrier –> maintains ____ acidic microenvironment –> causes ____ of enamel

Answer 40

"cloistering"
Gtfs
EPS
tough
diffusion-limiting
localized
demineralization

Question 41

Key Concepts

Dental caries continues to be the single most common and costly ____ diseases (which affects both children and adults) in the US and worldwide.

Dental caries is a complex and ____ disease, which involves personal, environmental, socio-economical and biological (biochemical) factors among others.

At biological level, the disease results from interactions of pathogenic bacteria such as ____ (and their products), host ____ and dietary ____ (e.g. sucrose) leading to establishment of ____ plaque-biofilms

Caries is caused by an interplay of four different things:
• ____ (amount, composition, frequency) – (he again stresses the importance of diet)
• ____
• ____
• ____ in biofilm
• (not mentioned in the blue but he also reiterates the importance of ____)

Answer 41

oral infectious
multifactorial
S. mutans
saliva
carbohydrates
virulent-cariogenic

diet
tooth
time
bacteria
saliva

Question 42

Key Concepts

At biochemical level, the assembly of an exopolysaccharides matrix via enzymatic reactions (mediated between ____ and ____) is a key virulence determinant in the pathogenesis of dental caries.

The concerted actions of S. mutans-derived GtfB and GtfC (in the presence of sucrose) form ____ buildup of EPS on saliva-coated HA and on microbial surfaces, which promotes ____ accumulation of bacteria.

The continuous production of EPS on sHA and bacterial surfaces enmeshes and bridges the cells together, forming densely packet bacterial islets (EPS- ____).

Answer 42

Gtfs
sucrose
heterogenous
local
microcoony complexes

Question 43

Biofilm Assembly and Dental Caries Pathogenesis

Bacteria attach to the tooth surface. Without ____, the commensal bacteria will prevail because they can attach better to the surface to begin with.

However, when you change the environment by providing sugar, especially ____, now you are giving the proper substrate to produce the EPS, both on the tooth surface directly, but also on the surface of the bacteria, facilitating this accumulation.

We were able to see this phenomenon using microscopy; you can see the bacterial ____, associated with EPS, forming the little ____. If the process continues, the structure will become more complex, the EPS starts being produced in higher quantities, larger ____ are formed, and over time, you have this highly ____ structure.

Answer 43

sugar
sucrose
clusters
aggregates
microcolonies
compartmentalized

Question 44

Key Concepts

The sequential accumulation of EPS in situ assembles an intricate and ____ 3D matrix scaffold that impedes rapid neutralization by buffer ____.

The presence of acidogenic-aciduric bacteria that are enmeshed and surrounded by the matrix facilitates the creation of ____ pH microenvironments (____) within biofilms

At the same time, highly ____ regions are formed at the plaque-biofilm/teeth interface, which could eventually lead to ____ of the enamel surface.

Highly acidic microenvironments allow the ____ bacteria (the acid-tolerant bacteria) to prevail, and over time, lead to these acidic regions, which cause dental caries.

Answer 44

diffusion-limiting
locally

acidic
niches

acidic
demineralization

acidogenic

Question 45

Biofilm Assembly and Dental Caries Pathogenesis

Just remember the distribution of the ____, the formation of those acidic pockets, and the maintenance of ____ pH (____ regions on the pH map) at the interface, which over time will lead to the cavitation of the enamel.

Answer 45

pH
low
darker

Question 46

The biochemical perspective (which was his focus today) and the microbe perspective go hand in hand in many ways.

Biochemical perspective: ____ matrix will facilitate structure in the microenvironment, which in turn might explain some of the microbiome-based findings, where the ____ gradually decreases as the disease progresses, while specific acidogenic/acid-tolerant bacteria become the more ____ species.

I cannot go into detail today about different species that have been found, but I think it’s a very interesting topic re: how these microorganisms interact and how they work together to facilitate this development of the biofilms and vice versa.

Answer 46

EPS
diversity
dominant