Oral Microbiology Midterm

What is metagenomics?

Describes the study of microbial populations through analyses of the total genomes present.

Which metagenomic techniques do we need to know?

- 16S
- Whole Genome Shotgun (WGS)
- Metatranscriptomics
- Proteomics
- Metabolomics

What are the 6 features of the 16S ribosomal RNA gene?

· Present in all organisms
· Essential cellular function
· Essential regions cannot contain mutations; lethal to cell
· Variable regions reflect evolution
· Gene length is approximately 1500bp
· >100,000 rRNA gene sequences in databases

Whole Genome Shotgun (WGS)

dna

Metatranscriptomics

study of messenger RNA transcripts

Proteomics

Proteins measured by mass spect.

Metabolomics

identification of all metabolites by mass spectrometry or NMR associated with a microbial population

Why is 16S rRNA gene sequencing used in Phylogenetics?

- allows differentiation between organisms
- everyone has it so you can use it to compare
- hypervariable regions are what are used to determine the differences between species.

Greatest risk of transmissible disease observed in dentistry

HBV

HBV

hepatitis B virus

Average Saliva Production for Adults

- average 1.5 L
- produce less when you age/ take meds

Average Saliva Production for Children

average 2.0 L

Lysozyme fn

β(1-4) breaks down bond between N-acetylmuramic acid and N-acetylglucosamine (NAM-NAG complex) in peptidoglycan

Lactoferin fn

Inhibits bacterial growth by binding and sequestering Fe2+ ions.*

Target of Epigallocatechin gallate (EGCG) and mechanism in Streptococcus mutans

Competes for binding on the salivary pellicle

EGCG: benefits seen from a study

biggest component in green tea leaves that promoted oral health seen in tea workers.

Epigallocatechin gallate (EGCG) mechanism in Streptococcus mutans

- Inhibit normal function of glucosyltransferases; gtf (B,C,D)
- Alter bacterial cell walls and/or cell membrane: causes bacteria to starve to death

glucosyltransferases

- forms glucans outside the cell
- energy for the cell
-EGCG blocks it

Aciduricity

ability of an organism to withstand acidic conditions

What is the effect of organic acids on the environment?

Organic acids decrease the pH of the environment, making it more acidic.

How does Streptococcus mutans (Sm) deal with acid stress?

- modification of its' membrane
- pumps out protons to maintain internal pH balance

What role does modifying the cell membrane play in acid stress resistance?

- decreases proton permeability, helping to protect the cell from acidic conditions.

What protein is most important in increasing membrane rigidity to reduce proton permeability?

fabM

What proteins are involved in the biogenesis and maintenance of the cell membrane in response to acid stress?

- Dcp
- Ffh
- Dgk

How does S. mutans manage its internal pH in an acidic environment?

- pumps out excess protons from inside the cell to maintain a neutral or less acidic internal pH

Which enzyme complex helps S. mutans pump protons out of the cell?

F1F0 ATPase

What is the cost of pumping protons out of the cell in S. mutans?

energy

Acidogenicity

ability of an organism, such as Streptococcus mutans (Sm), to produce acids from fermenting carbohydrates like glucose and sucrose

How does S. mutans generate acids?

S. mutans uses glycolytic fermentation to generate acids. This process converts glucose or sucrose into pyruvate via glycolysis.

What is the first step in acid production by S. mutans?

The first step is glycolysis, where glucose or sucrose is converted into pyruvate.

What happens to pyruvate after glycolysis in S. mutans?

After glycolysis, pyruvate is shuttled into various metabolic pathways that lead to the production of organic acids

What does xylitol inhibit in S. mutans?

DexA

What role does DexA (Dextransucrase A) play in S. mutans?

enzyme involved in the production of dextran, a polysaccharide that helps the bacteria adhere to surfaces like tooth enamel and contributes to plaque formation.

What is xylitol, and can it be metabolized by bacteria?

- sugar alcohol
- same sweetness as regular sugar
- bacteria can't metabolize it

Critical pH of Hydroxyapatite

around 5.5

Critical pH of Fluorapatite

4.5

As pH increases, _____ occurs.

Remineralization
- usually only occurs if pH is over 5.5

As pH decreases, ______ occurs.

Demineralization

What helps maintain enamel equilibrium by buffering acids and providing calcium and phosphate ions to support remineralization?

Saliva

FA vs. HA: which is more prone to demineralization

HA

FA vs. HA: which provides greater protection against tooth decay?

FA

Major target of Fluoride

inhibits ATR (acid tolerance) mechanisms:
- Arginine Deiminase System (ADS)
- Ammonia Synthesis
- inhibits F0F1 ATPase

primary effect of fluoride:

- prevents demineralization of the tooth
- inhibits F0F1 ATPase

secondary effects of fluoride:

- below 4.5 pH, fluoride will also become demineralized

What is ADS?

- metabolizes arginine into ammonia and citrulline.
- the ammonia raises the pH which allows bacteria to survive in acidic environments
- binds protons and neutralizes them

What is F0F1 ATPase?

- bacterial enzyme complex involved in proton pumping to maintain pH balance
- maintains intracellular pH

What is the "zombie effect"?

Bacteria that are killed by SDF further act as a carrier for silver ions, which is lethal to other bacteria that come in contact with it.

What is SDF?

silver diamine fluoride

What does SDF target?

Antimicrobial effects:

- disrupts peptidoglycan cell walls

- lyse cell membranes

- denature ribosomes

- bind DNA directly bc there's no nucleus

Alpha diversity: what does it measure?

diversity within a single sample or site
- unique number of species w/in a sample

Beta Diversity: what does it measure?

similarity or differences between two or more samples/sites

Alpha diversity example in the oral microbiome and insights

- examining the bacterial species on one person's tongue
- shows variety in microbes

Beta diversity example in the oral microbiome and insights

- Comparing the bacteria in your supragingival plaque to your own tongue or to someone else's supragingival plaque

- Bacteria at the same site (e.g., plaque) on different people tend to be more similar than bacteria at different sites on one person

What is the chain of infection starting with the source?

-> Reservoir (microbe persists naturally)
-> Mode of transmission
-> Portal of entry
-> Susceptible host
-> Pathogenic source

Composition of Dental Plaque

1. salivary components like mucin
2. desquamated epithelial cells
3. microorganisms
• all embedded in extracellular matrix
- bacteria
- debri
- food

What is EPS?

extracellular polysaccharides

Fn of EPS?

- protects against phagocytosis
- aids in biofilm development (acts like glue)

Mutation _____ genetic variation

increases

What can mutations lead to?

Increased Virulence

Gene Deletion

loss of function

Alterations/mutations of genes we need to know

- Gene Deletion
- Gene duplication
- Gene shuffling

Gene duplication

additional copy of a gene

Gene shuffling

change of position on chromosome

Salivary Pellicle

protective film that coats the surfaces of the mouth and throat

Acquired pellicle

when the salivary pellicle coats a hard surface. It is referred to as an acquired pellicle

Glucosyltransferase

- (GTFs) for glucose to glucans

Role of Glucosyltransferase w/in Streptococcus mutans

- Produce adhesive glucans (water-insoluble)
- Promotes Bacterial Adhesion and biofilm formation

Saliva as a diagnostic tool

pH level, flow rate
(need to review lecture)

Agglutination

the clumping of particles:
- Antigen/Antibody
- Mucin/Surface Protein
swallowing clears this

S-IgA

- Immunoglobulin
- Dimeric
- Joined by (J) chain
- Secretory component (Facilitates trans-cellular transportation)

mucins

- agglutinins

- long oligosaccharide chains on it

- trap for bacteria

Immobilized S-IgA/ mucins embedded in the acquired pellicle result in ?

"fixed" agglutination on the tooth surface

Dr. McKay's 21 City Study Results

- As you increase fluoride concentration they saw a decrease in dental caries in kids
- optimal dose was about 1.0-1.2 ppm