1) plaque begins to release enzymes and acid into underlying enamel DEMIN
2) The enamel UNDER-NEATH the plaque demins first bc it reflects light differently, this looks like a chalky white spot (remains UNCAVITATED)
3) Upon application of MI paste or CPP-ACF, Ca2+ and HPO4 are driven back into the enamel and it will begin to heal.
4) Bottom Line: we can remineralize teeth and change the makeup of plaque!!!
Quick Note about White Lesions: as long as lesions remain UNCAVITATED, they can be healed with CPP-ACF. That’s partially why a sharp explorer is no longer used to check for caries; you may inadvertently cavitate (make a hole thru) a soft white lesion and prevent any chance of healing it “naturally”.
Caries multi-factorial disease:
2. TIME - driven by the FREQUENCY of the eating (shifts the demineralization <> remin balance)
3. DIET -dependent on DIETARY SUCROSE
4. MICROFLORA - a BACTERIAL DISEASE: S. mutans, lactobacilli, A. ciscosus, S. sobrinus (all acidogenic, acid tolerant)
Caries is modified by?
FLUORIDE, CALCIUM, AND PHOSPHATE
i. Harder tooth structure
ii. Inhibits acid production by bacteria
ii. Demin remin balance
iii. Low flow of saliva = high risk
a. Treat the caries before it even starts (a carious lesion is really the END-STAGE of disease)
b. Medical management of caries
Caries Management by Risk Assessment
a. The idea is to TILT the balance toward NO CARIES by decreasing risk and disease.
b. Assessment Determine Risk Status
a. Based on how many disease factors you have as well as disease indicators, each person gets labeled at different RISK LEVELS for caries:
c. Make changes to “Our Present Understanding of Caries”:
a. Caries is a BACTERIAL DISEASE Change the microflora with topical chlorhexidine, fluoride, and povidone iodine, Dr. Shi’s cavity-fighting lollipop (kills S. mutans)
b. Caries is dependent on DIETARY SUCROSE reduce dietary sucrose and ADD XYLITOL (a sugar that is metabolized differently by bacteria and doesn’t contribute to biofilm)
c. Caries is driven by the FREQUENCY decrease the frequency of eating (CHUG vs SIP), avoid sodas with high acidity (critical pH for acid challenge = 5.5)
d. Caries is modified by SALIVA increase salivary flow by mechanical stimulation/vigorous chewing and changing drugs with reduce flow
e. Caries is modified by FLUORIDE, CALCIUM, AND PHOSPHATE add fluoride, Ca2+, & P8
a. Causes: Medication Side effects, stress, dehydration, salivary gland dysfunction, disease, hormonal imbalance, smokine
b. Signs: difficulty eating or swallowing, tongue sticking to the roof of your mouth, changes in taste, inadequate denture retention, soft tissue trauma
i. Stimulated Saliva Flow
1. Normal: >1 ml/min
2. Low: 0.7 -1 ml/min
3. Very Low: <0.7 ml/min
ii. Unstimulated Saliva Flow
1. Normal: >0.25 ml/min
2. Low: 0.1 – 0.25 ml/min
3. Very low: <0.25 ml/min
How does a decrease in salivary flow influence the risk for dental caries?
Saliva acts as a buffer; reducing saliva prolongs the affects of acid
During the formation of hydroxyapatite, what element may occur in the voids created by the disordering of the hydroxyl groups of the hydroxyapatite crystal?
In the delicate balance between remineralization and demineralization, what is the critical pH for most individuals thought to be?
<5.5 demineralization occurs
In our contemporary concept of dental caries, what set of parameters need to be present in order for dental caries to occur?
Nutrition, Bacteria, Time, Host
What immunoglobulin is associated with salivary secretions?
What is the general formula for the calcified matrix of enamel?
Who proposed the Chemo-parasitic Theory?
What are the reparative mechanisms for an acellular tissue such as tooth enamel?
There are no repair mechanisms for enamel.
What is the most acidic organic acid produced through glycolysis by plaque bacteria?
What is the approximate percentage of inorganic material in enamel and dentin?
96% in Enamel, 70% in Dentin
Under normal physiologic conditions what ion is the saliva saturated with?
What enzyme is responsible for the conversion of pyruvate to lactate during bacterial glycolysis?
Which dental plaque organism is thought to be a key bacterium in the induction of pit and fissure caries?
After a short sucrose exposure the plaque pH will fall below the critical pH for approximately how long?
Which of the glucans are thought to be the most caries promoting?
Branched (insoluble) Glucans
How are the bacteria that cause dental caries transmitted from one individual to another?
From care-giver to child
Treatment of Xerostomia
i. Increased water intake
ii. Change medications
iii. Saliva substitutes
iv. Lubricating getl intraorally, Vaseline on lips
v. Toothpaste without additives
vi. DO NOT USE lemon and glycerine swabs/toothettes turns to alcohol
vii. DO NOT UES alcohol containing mouthwashes
Demineralization – Remineralization -> Healing WHITE SPOTS
a. Add mineral ions to the oral environment (see pictures of dentinal tubules below)
ii. Amorphous Calcium Phosphate (ACP)
iii. Recaldent (CPP-ACP)
CPP-ACPF truly heals the white spot! Remin thru body of lesion + re-mineralized enamel is more acid resistant (red = fluroroapatite)
a. If all else fails…too late to prevent? To remineralize?
b. The best restoration is to never to a restoration at all
c. Restorative dentistry is the END-STAGE treatment for dental caries
d. Glass Ionomer Restoratives: for transitional restorations
i. Can release fluoride and continue to uptake and release fluoride, also great at sealing margins
ii. Ex. Fuji IX GP Extra, Fuji II LC
e. Glass Ionomer “Sealant”
i. Protection for newly and partially erupted molars
ii. Remineralization of affected dentin
The Chemo-Parasitic Theory (W.D. Miller – 1890)
1. A unifying theory using an in-vitro caries model
2. Much of his work was based on the work of others:
a. Pasteur – discovered microorganisms could convert sugar lactic acid
b. Magitot (1867) – showed fermentation of sugars would dissolve tooth enamel
c. Underwood and Miles (1881) – observed bacteria in carious lesions; considered caries to be ABSOLUTELY DEPENDANT upon presence of bacteria
3. Miller tied in all these theories together – made association of role of bacteria with the importance of production of organic acid to the decomposition of teeth!
a. Acid present within deeper carious lesions
b. Different starchy foods could decalcify teeth when mixed with saliva
c. Several types of mouth bacteria could produce enough acid to cause caries
d. Lactic acid was identified product (LA is the most destructive and acidic organic acid)
e. Different microorganisms invade carious dentin
f. Acids produced from sugars found in the mouth could destroy the mineralized tooth.
i. Weakness: not able to define the importance of bacterial plaque biofilms and their relationship to the tooth surface.
the modern Concept of Dental Caries
1. Caries is a biofilm-associated disease which is primarily caused by the production of organic acids by the plaque bacteria.
2. The organic acids are result from the fermentation of specific carbohydrates thru glycolysis.
3. This is a MULTI-FACTORIAL DISEASE – only in interaction of biofilm(bacteria) + host(teeth) + presence of carbohydrate(substrate) will caries occur (REVERSIBLE)
a. If penetrate dentin, cannot be reversible - @ that point, would have to restore with mechanical means (filling)
b. Virulence comes from formation of BIOFILM
c. Closely related to the consumption of fermentable carbohydrates
d. Infectious and transmissible among animal species (often momchild)
e. A particular group of bacteria are most probable agents (Mutans sps)
Infectious Nature of Caries
a. Dental Plaque
b. Dental Biofilms: form on all exposed dental tissues
i. Bacterial colonization is NOT random – occurs thru a highly specific adhesion/receptor interaction
ii. Difficult to eliminate and alter
iii. Salivary Pellicle: an acellular film containing proteins and carbohydrates that are favorable for bacteria to grow and presents specific binding sites that allow bacteria to attach on (if you don’t have pellicle, there probably won’t be any caries formation
i. Bacteriological agents
ii. Genetic Component?: genetics play little part in caries but there are genetic symptoms that have associated malformaitons of teeth
iii. Very transmissible!
iv. Experiment: gnotobiotic “germ free” animals were given high carbohydrate diets no caries
1. But when inoculated with bacteria caries developed
2. Antibiotics could suppress caries activity
3. Bottom Line: showed you can have high carbohydrate diet but without bacteria, there is no caries!
Experiments of Keyes & Fitzgerald:
provided experimental evidence for the infectious nature of caries
i. Experiment 1: caged together hamsters with high caries prevalence with hamsters with low caries both groups got similar caries rates
ii. Experiment 2: took fecal pellets and oral plaque swabs and showed that caries-inactive group can become caries-active
iii. Experiment 3: caries-resistant offspring delivered by C-section would become caries active when raised by females of caries active group mom passed onto kids
1. Caries is caused by a microbial infectious agent.
2. This disease is NOT a classical genetic disease that is related to some genetic trait
3. Most likely infection is transmitted from mother to offspring early in life (only when teeth are beginning to develop…S. mutans cannot colonize when there are no teeth present)
MICROBIAL ECOLOGY OF BIOFILM – QUANTITATIVE and QUALITATIVE
features of dental biofilms contribute to caries induction and progression
a. Non-specific Plaque Hypothesis: Numbers important
b. Specific Plaque Hypothesis (what most people believe to be true now): a unique set of organisms need to be present and DO NOT have to be in high numbers
i. S. mutans – highly suspect induction of pit and fissure/smooth surface caries
ii. A. viscosus/naeslundii – root surface/cemental caries
iii. Lactobacillus sps – caries progression and dentinal caries
c. You don’t want to completely remove the biofilm because it protects you from more deadly diseases and keeps the other microflora in check.
CHEMISTRY OF TOOTH ENAMEL- General Characteristics
i. It’s a TISSUE!!! Hardest tissue in the body (acellular)
ii. Has great ability to absorb and reflect light
iii. Only formed in one point of your life and must survive over your lifetime in an environment that is not sterile and under great physical stress
iv. Unfortunately, it’s the bacterial surface of choice!
Chemical composition of teeth
Inorganic Salts 96 wt% 70 wt%
Organics 1 20
Water 3 10
mainly HYDROXYAPATITE, a hydrated calcium phosphate salt
2. This is NOT stoichiometrically balanced!!!
Organics (1-20 wt%)
1. Remnants of EMP (enamel matrix proteins)
2. Lipids (more so in dentin)
3. Since organic molecules have nitrogen, they can oxidize yellow color
Water (3-10 wt%)
1. Water content of enamel can vary resulting in changes in appearance of enamel
2. When teeth are dehydrated (like first generation bleaching use to cause), it leaves a white, chalky appearance.
Enamel: mineralized tissue composed of individual hydroxyapatite tightly packed together into prism-like structures
i. Enamel Rods: made of tightly packed, highly organized hydroxyapatite crystals
iii. Most important part of the crystal is the center – this is where the two hydroxyl groups reside
1. If the two hydroxyl groups come together unfavorably, then electrons cannot be equally paired will get void and eventually fill up with water
2. As you get more voids, the structure will become weaker and weaker
3. FLUORIDE ions fill in the voids and can participate in H-bonding theory on how fluoride treatments help prevent cavities
CHEMICAL BASIS FOR CARIES
a. Consists of balance of the following eqn: Ca10(PO4)6OH2 [H+] [Ca2+] + [PO4-3] + H2O
i. (acid catalyzed equilibrium reaction)
ii. If you lower pH, you will drive the equilibrium to ionized version of the salt
iii. Dentist job is to make sure there is not enough H+ around to make the acid catalyzed equilibrium ion
iv. @ neutral pH, the hydroxyapatite is in the form of the salt, BUT if you increase the acidity of the environment, then you drive the equilibrium towards the individual ions
v. Whether or not a solution is saturated with respect to HA can be determined by the solubility produce principle
1. This principle (derived from the Law of Mass Action) states “ that the velocity of a reaction is proportional to the product of the masses of the reacting substances, each raised to the power equal to the number of molecules taking part”
i. The solubility of hydroxyapatite and other calcium phosphates is greatly affected by pH
ii. The concentration of the surrounding solution (i.e. saturation) will also influence the solubility of the salt
iii. Artificially Demineralize teeth by:
1. Replacing all saliva with water (i.e. like in a xerostomic patient)
2. Consuming large volumes of carbonated, soft drinks
is NOT PURE!
i. Ion Substitutions
1. Carbonate anion replaces phosphate groups because of size and charge(carbonate makes the hydroxyapatite more acid labile – this is natural)
2. Fluoride anion substituting for hydroxyl (makes crystal more acid resistant)
i. Caries occurs when Demineralization dominates over remineralization
ii. Demin occurs from the production of organic acids
iii. Diffusion of acid into enamel rods
iv. Dissociation of hydroxyapatite
v. Graph: Stephan Curve – pH shift following acid production (same graph as on pg 2)
1. When you eat and expose the biofilm to carbohydrate, the production of acids begins in 2 mins and you see the pH drop from 6.8-7.2 (neutral) 5.3
2. RED LINE = CRITICAL pH = 5.5 below which the ionized form of the salt (hydroxyapatite) is the major form
3. Shows that you need to eat your sugars quickly so saliva can buffer the acids and you don’t remain below the red line for too long
i. Many caries-causing bacteria are homofermentative
ii. Produce lactic acid has highest pKa which means its very willing to give up its proton very very strong acid!
iii. The production of organic acids is a function of sugar availability
iv. Carbohydrate Transport – regular metabolism
1. Membrane bound complexes
a. Phosphoenol pyruvate-phosphate transferase complex (PTS-PEP): main transfer system can uptake carbs @ a fast rate
b. Permease complex
Production of water soluble glucans
1. If CARBS are present in HIGH CONCENTRATION, then bacteria also:
a. Makes internal dextrans to STORE the glucose in case of starvation later
b. Make EXTRACELLULAR GLUTANS – their own glue, contributes to biofilm
2. Glycosyl transferase (GTF) enzymes: capable of synthesizing long chain polymers when sucrose present make GLUCANS
a. Enzymatic hydrolysis of SUCROSE to FRUCTOSE AND GLUCANS
i. If carbohydrates are in excess, use transferase to make glucans
ii. BRANCHED (1-4 linkage) very water INSOLUBLE bacteria increase their BIOMASS (GLUE)
iii. UNBRANCHED (1-6 linkage) very water soluble
b. Mutans group: mainly synthesize branched water-insoluble glucans
3. Glucans: can be branched or unbranched depending upon the bacterial enzyme it’s formation is IRREVERSIBLE!!!
Bacterial Virulence Factors
1. Bacterial attachment
2. Bacterial acid production
3. Sugar consumption
4. Bacterial production of water insoluble extracellular glucans
vi. Bacterial Virulence Factors
1. Bacterial attachment
2. Bacterial acid production
3. Sugar consumption
4. Bacterial production of water insoluble extracellular glucans
Arch form and Tooth Morphology
Human Dentition; these are great bacteria breeding grounds:
1. Deep Grooves or Pits and Fissures
2. Interproximal contacts are great bacteria breeding grounds
3. Heights of contour on GINGIVAL THIRD are NON-Cleansing (a teepee formation, i.e. tip of cusp, is self-cleansing)
4. Crowding and narrow arch forms
Saliva: help prevent caries because of its contents but also helps start caries because of pellicle
1. Produced by major and minor exocrine glands in the mouth
a. Major: parotid, sublingual, submandibular mainly make the protein component of saliva
b. Minor: occupy most mucosal surfaces of the mouth more hydrating and mucin-contating (can trap water and keep tissues hydrated)
a. Assisting in food intake and taste if you are xerostomic, can only taste SALT and nothing else
b. Provide natural defense against host microbes (anti-fungal, bacterial, viral)
c. Provides protective functions for stability of mineralized tooth structures
Saliva for Food and Nutrition
a. PRPs(proline rich proteins) + Mucin provide lubrication for mucosal tissues
b. Bicarbonate anion provide buffering pH to facilitate taste receptor activity
c. Amylase, Lipase, Protease enzymes which help breakdown food
i. Dimeric immunoglobulin with a PROTEIN secretory component that helps stabilize the molecule
ii. Secreted into oral cavity thru the mucosal epi cells and salivary glands
iii. Is produced by small intestinal B cells which migrate to the mouth thru the circulatory system
iv. What happens? IgA will attach to bacteria and once swallowed and made its way into the small intestines, Peyer’s Patches will start to make antibody to the bacteria eventually these antibodies make their way back into the mouth via Bcells/circulatory system and it will attack platonic (free floating bacteria)
v. Advantages: modulates biofilm formation by inhibition of bacterial attachment
vi. Disadvantage: will NOT KILL, just attaches onto bacteria (signal!), DOES NOT activate complement proteins
Lactoperoxidase: efficiently convert HALIDES and PSUEDO-halides to highly reactive oxidizing molecules (“bleach” – HOCL)
i. Found in most exocrine glandular secretions
ii. Enzyme that is by-product of milk
iii. Lactoperoxidase + hydrogen peroxide + Cl- + OSCN- OCl+, OSCN+ react with –SH groups denaturing proteins
i. Cleaves 1-4 linkages of NAM-NAG of bacterial peptidoglycan
ii. Effective against gram positive bacteria
iii. Found in tears, nasal secretions, and exocrine glandular fluid
i. Binds to Iron, an important metabolic co-factor
ii. BACTERIOSTATIC properties
iii. Present in most exocrine gland secretions
Salivary Components that provide Stability of Enamel
i. Calcium binding proteins: this is primary factor
1. If a tooth is placed in a vial of free-floating calcium phosphate get PRECIPITATION of amorphous calcium phosphate CRYSTALS
2. Why doesn’t this happen in the mouth? Bc of CALCIUM BINDING PROTEINS
a. Because saliva contains high concentrations of Ca-binding protein which will SEQUESTER the free-Ca2+, allows us to maintain HIGH levels of Ca2+ without precipitating into crystals
b. Need to have saturation of calcium to maintain HA
3. Mechanism – the key is the trick the system into thinking you have an ORGANIC calcium so that it doesn’t precipitate out into crystal (Inorganic organic)
ii. Proline Rich Proteins
iii. Phosphate and Calcium
a. Caries Resistance is related to ALL the salivary factors acting upon the system.
b. Caries is a chemically-controlled acid driven dissociation of the inorganic salt known as Hydroxyapatite.
c. Caries is caused by the presence of biofilm-associated bacteria which produce organic acids with fermentation of sugars
d. Sucrose is required for the production of water soluble bacterial glucans which promote bacterial plaque formation and stability
e. Caries is caused by an infectious agent which is TRANSMISSIBLE
f. This disease IS NOT the result of a genetically inherited trait!
Why is microbiology relevant for Dentistry?
a. Acids cause enamel/dentin demineralization (caries)
b. Bacteria are the main acid producer in the mouth
Plaque-Host Substrate Theory
a. 3 Factors (can’t have one without the other) to make caries:
i. Host (Teeth and saliva)
ii. Substrate (Diet and Time)
iii. Plaque Bacteria
b. If you didn’t have plaque, you could eat all the sugar you wanted and never develop caries! Therefore, bacteria is required for caries development
Willoughby Dayton Miller + Experiments
a. Scientific pioneer in dentistry, worked with Koch
b. Proposed combination of specific microorganisms and carbohydrate consumption as cause for tooth decay
c. Tried to apply Koch’s postulates to dental caries
d. Published a famous book: Microorganisms of the Human Mouth
e. Miller tried to isolate the causative bacteria (according to Koch’s Postulates) but was not successful…instead he make recommendations for keeping the teeth clean which we still use today
i. “cleanse teeth often to remove plaque”
ii. “delimit intake of sugar”
iii. “use antimicrobials to treat”
a. Most popular Suspects: Lactic Acid Producing Bacteria
i. Mutans streptococci (caries initiation)
1. S. mutans
2. S. sobrinus
ii. Lactobacilli (caries progression)
1. L. acidophilus
2. L. rhamnosus
3. L. fermentum
iii. Actinomyces (early caries lesions and root caries)
1. A. neaslundii
2. A. odontolyticus
First Isolation of Cariogenic Bacterium
1. J.K. Clarke discovered S. mutans in 1924
2. Isolated form human cariogenic lesion able to produce caries
3. …and this discovery was forgotton until 1960’s Keyes & Fitzgerald (discovered that dental caries is TRANSMISSIBLE and an INFECTIOUS DISEASE)
Isolation of Lactobacilli
1. First implicated by Bunting
2. Rediscovered by Jay in 1947
3. Lactobacilli were found to colonize existing lesions but were absent during early lesion formation – opportunistic but NOT causative
Discovery of Actinomyces
1. Originally implicated in perio disease, associated with caries in 70s/80s
2. Can ferment carbohydrates and produce acids
3. Produce NH3+ to control environmental pH (you may think that producing base is caries-preventative but it’s not enough base to bring the pH above the critical level; it’s mainly for the bacteria to be able to survive in the mouth)
4. Linked to early stages of demineralization and root caries
Virulence Factors of Cariogenic Bacteria
3. Glucan Formation
i. Ability to lower pH below the critical pH for enamel/dentin demineralization
ii. Inhibits growth of many “commensal” non-acid producing bacteria
i. Allows cariogenic bacteria to thrive under acidic conditions
ii. Can result in dominance of cariogenic species in plaque
i. Long-chained, branched extracellular polysaccharide that is WATER INSOLUBLE
ii. Allows tight adherence of cariogenic bacteria to the tooth surface
iii. Enhances resistance to mechanical removal
iv. Enhances resistance to antimicrobial treatments
i. “Unspecific” surface adhesion: fimbraie/pilli
ii. Adhesion to specific surface molecules
1. Attachment of surfaces
2. Attachment to other bacterial species
The Evolution of Oral Microbiology Research 1950 – 1960’s
i. Culture based bacterial isolation
a. Plating of salivary and plaque samples
b. Development of selective plating methods
c. Biochemical characterization
d. Microscopic examination
2. Limitations: more than 700 oral bacterial species identified BUT only 100 can be cultivated why? Very complex organisms that often live off other species so can’t be grown in isolation
ii. Germ-free animal models for disease (“gnotobiotic “ – to show that the microorganisms could develop disease)
iii. Advanced microscopy techniques
1. Electron Microscopy Transmission: important invention so that we can see “inside” the cell by slicing slide by slide
2. Electron Microscopy Scanning : allows you to look at the surface structure
The Evolution of Oral Microbiology Research Molecular Approaches to Oral Biology
i. Checkerboard DNA-DNA hybridization
1. i.e. to test bacterial species against dental plaque samples
2. Run probed in one direction and then back and forth:
a. Whenever there is HYBRIDIZATION will light up
b. A DARKER spot indicates heavier load or better match
ii. Culture in-dependant approaches 16S rRNA gene sequencing
1. 16S is highly conserved and so you can ID bacteria based on this
2. When matching species, the more DIFFERENCES and FARTHER APART the genes are, the more unrelated they are
iii. Whole genome information:
1. scan for genes that encode for metabolic capacities, virulence features (in this way, scientists can study virulence without even growing the organism itself!)
2. lead to genetic manipulation
iv. Human Oral Microbiome currently underway, to sequence all plaque bacteria!
Caries in PRIMARY TEETH
i. S. mutans only predominates in subjects with caries in DENTIN
ii. Actinomyocese and Veilonella sps seemed to be the main culprit
Caries in PERMANENT TEETH
i. Streptococcus is the main player for Caries
ii. Veillionella sps is the most ABUNDANT species but its also abundant in healthy subjects
1. So is it Cariogenic?
2. NO, it’s ANTI-CARIOGENIC because it can metabolize lactic acid to CO2 and water!
e. Graph: Prevalence of Cariogenic Lesions in Different Lesion Types
f. Caries IS POSSIBLE WITHOUT S.MUTANS!!! (BUT FOR THE BOARDS, S. MUTANS IS STILL THE MAIN CARIOGENIC SPECIES!)
Specific Plaque Theory (Loesche 1976)
i. Within a diverse plaque community, only a few species actively contribute to disease
ii. Focused on controlling disease by targeting a limited number of organisms for txn
Non-specific Plaque Theory (Theilade 1986)
i. Considered that disease as the outcome of the overall activity of the total plaque microflora
ii. A heterogeneous mixture of “any” microorganisms could play a role in disease rather than specific species
Ecological Plaque Hypothesis (Marsh 2003)
i. Even in healthy plaque, you have a few cariogenic species that are kept in check by other bacteria
ii. It’s not until a major ecological pressure (i.e. no saliva flow) hits the community that you shift the balance towards disease (is REVERSIBLE!)
iii. Picture: the Ecological Development of Dental Caries
-Demineralization is reversible
- BUT if you keep on eating sugar, you never go back above the critical pH and you stay in the ionized calcium form
How does Dental Plaque Develop?
i. First Layer: Salivary pellicle (forms immediately after you’ve had cleaning)
1. Pellicle is a double-edged sword: need it to help the tissue stay moist and protect against acid but also provides area for bacteria to attach
2. Thin film formed by salivary components lubricates enamel/dentin tissue, protective against acids
3. Certain proteins in the pellicle can serve as receptors for bacterial attachment
a. Parotid Saliva Agglutinin (S. mutans)
b. Mucin MG2 (S. oralis and S. sanguinis)
ii. Primary Colonizer can directly attaché to surfaces
iii. Secondary Colonizer can attach to primary colonizers
Dental Plaque (Oral Biofilm) Formation Other Imaging Techniques
a. Confocal Laser Scanning Microscopy: allows to look INSIDE the biofilm
b. Species-Specific Labeling with Fluorescent In-Situ Hydridization (FISH): can use to compare Colocalization
Biofilm Development – Interspecies Interactions
a. Nutrition – Metabolic Interactions
i. Host Factors:
1. Sucrose (diet)
2. Proteins (Saliva, crevicular fluid)
ii. Cross-feeding between bacterial species one species will eat the metabolic by-product of another!
1. Lactic acid (streptococci and Veillonella)
3. Fatty Acids
1. Acid production by one organism inhibits growth of acid-sensitive species
2. Acid consumption of amnomia production as a counter measure to neutralize pH
ii. Chemical/Biological warfare
1. Hydrogen peroxide production
2. Bacteriocin production
a. Narrow spectrum (means only kills either gram + or – but not both) antimicrobial small proteins typically active against related organisms
b. Well-regulated production
c. Streptococci have greatest bacteriocin
Complexity of Interspecies Interactions between Streptococci
i. Competition between S.mutans (Sm) and S. sanguinis (Ss)
ii. S. mutans produces MUTACIN to kill Ss but Ss retaliates by producing HYDROGEN PEROXIDE
Sm -> Ss: Sm colonized first so it kills Ss
Ss -> Sm: Ss colonized first so it kills Sm
Sm + Ss: if colonize @ the same time, they decide to work together – how?
Maybe they talk together!
a. Cooperative metabolic interactions
b. Cooperative enzymatic actions
c. Cooperative integration into the biofilm community
Bacterial Interactions & Biofilm Architecture
a. Species distribution is NOT RANDOM
i. Metabolic interactions
ii. Specific interspecies coaggregation
1. Specific cell to cell recognition between species
2. Cells attach to each other via specific structures
iii. Intra/Interspecies signaling
1. Small proteins/peptides CSP (competence)
2. Quorum sensing via Al-2 (luxS) for biofilm formation, bacterioicin production
a. i.e. when you make mutants without this gene, biofilms have a hard time forming
b. Bacteria have the ability to “count” their population size!
iv. Interspecies Competition
Exchange of Genetic Material
a. The Evidence: discovered that many bacteria that were not related to each other had small stretches of DNA that were similar HORIZONTAL GENE TRANSFER
b. The Mechanisms
i. DNA availability
1. Cell lysis
2. Regulated DNA release?
3. Donor cell (conjugation)
ii. DNA Uptake
3. Transduction (phages)
c. Host Factors
i. Provision of nutrition via oral fluids: glycol and heme-containing proteins & minerals
ii. Salivary proteins as enabler of initial surface adhesion
iii. Defense responses: antibacterial peptides and immune responses (which are limited in saliva)
Modern Approaches in Dentistry
A. Disease PREVENTION!
a. Move from surgical to medical approach
b. From treatment to prevention
c. From professional care to self-care
B. Is there cure for Caries? Here are some theories, but they don’t all work………
a. Inhibition of Adherence this is difficult because by just having the pellicle on your tooth, you’re inviting the bacteria in
b. (Passive) Immunization saliva has a very poor antibody concentration; only has IgA and IgA can only attach/cover bacteria but doesn’t actually kill it
c. Replacement therapy patients are generally very resistant to having genetically altered bacteria placed into their mouth
d. Disruption of signaling
e. Complete Plaque Removal? This is not a perfect solution, only solves the problem short term. Even if you remove all the plaque, because the saliva provides a pellicle the plaque will eventually quickly come back!
f. Selective Pathogen Elimination
i. “Restoring Homeostasis” – first you remove the cariogenic pathogens AND the plaque the plaque that naturally regrows will NOT have the pathogen in it therefore, a healthy plaque that prevents other cariogenic species from growing is a good barrier (not all plaque is bad!)
ii. Targeted removal of S.mutans from biofilm using short antimicrobial peptides ( Dr. Shi’s Lab!)
i. CFU = colony forming units
ii. 2_1G2 and C16G2 = short antimicrobial peptides that target S. mutans
iii. Conclusion: S.mutans does grow back but it NEVER grows to the same levels as they were before keep the concentration down!
iii. Protective Biofilm Growth: the experiments below show that a BIOFILM IS ENOUGH to block S. mutans growth can be a mechanism for anti-caries campaign
iv. Interference with Quorum Sensing: by removing the ability of bacteria to sense each other, the colonizes first rapidly overgrow and in the quick depletion of nutrients, many die off
A Medical Approach to Dentistry:
based on assessment of what of bacteria are in your mouth, you can create tailored treatments for people who are at high, medium, and low risk for caries
a. Current Advances in Diagnostics
i. Species – Specific Detection of cariogenic species in saliva and plaque
1. Mono-clonal antibody (MAbs) bacterial detection techniques: for all of them, you can scan your sample with the antibody for a specific bacteria (in plaque and saliva!)
a. Linkage to fluorescent dyes FLUORSCENCE
b. Linkage to color particles or enzymes COLOR
c. Linkage to latex beads AGGLUTINATION
d. Limitations: unfortunately these procedures are not very efficient for chairside!
2. Specific DNA Probes
3. Instant Membrane Strip Test for S. mutans detection: indicates the levels of S. mutans in the mouth
b. The FUTURE: Dentist-Scientists who capitalize on research advances to revolutionize current dental practice!