6. Biofilms and Plaques Flashcards
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 • \_\_\_\_
saliva
bacteria
time
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.
specific
saliva
carbohydrates
virulent-cariogenic
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.
dietary
farming-based
Strep mutans
processed sugar
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.
oral infectious
50
socioeconomic
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.
proportions
diversity
microbial compoistion
onset
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
limited
physical
chemical
biochemical
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.*
polysaccharides
cohesive
firmly
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.
dental
structured
polymeric
separate
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.)
matrix
virulence factors
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.
exoenzymes glycosyltransferases fructosyltransferases starch sucrose glucose glucan alpha-1,3 alpha-1,6 alpha-1,4
insoluble
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.
exoenzymes
three
one
four
glucan-binding proteins
sugar
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.
streptococci (streptococcus mutans)
amino
C
catalytic
glucan-binding
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
energy
glucose
glucan
sucrose
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).
saliva
pellicle
bacterial
saliva
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.
HA
solution
adsorbed
straight
active
pH
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.
magnet
7-8 fold
four
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 ____.
GtfB
GtfC
in situ
mechanical
polysaccharides
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.
ecological
EPS
early colonizers
cariogenic
