Transcript Document 7240837

Microbial infections in oral cavity
Wenyuan Shi
UCLA School of Dentistry
Molecular Biology Institute
Microbiology, Immunology and Molecular Genetics
The discovery of microorganisms by Antony van Leeuwenhoek
1683
When examining a dental plaque from the mouth of an old man, Leeuwenhoek
found "an unbelievably great company of living animalcules, a-swimming more
nimbly than any I had ever seen up to this time. The biggest sort . . . bent their
body into curves in going forwards . . . Moreover, the other animalcules were in
such enormous numbers, that all the water . . . seemed to be alive ….”
Scanning EM image of oral microflora
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From Marsh, 1999
S. sanguinis
S. gordonii
S.
S.
mitis oralis
Pioneer colonizers
S.
salivarius
Microbial Infections in Oral Cavity
G+ bacteria
G- bacteria
Dental Caries
Periodontitis
79% population
45% population
yeast
Oral candidiasis
80 millions
Dental caries
What causes dental caries?
DECAY HISTORY OF MAN
(NEOLITHIC TO MODERN ERA)
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COMMON
ERA
FRENCH
BRITISH
DANISH
20
15
ROMAN ERA
10
IRON
5
NEOLITHIC
3000 BCE
2000 BCE
1000 BCE
0
Sugar connection!
1000 CE
0
2000 CE
Salivary glands
Smith and Karst 2000
1
0.01-2.3mls/min/gland
2
0.02-1.3mls/min 3
The first medical approach to dentistry
Miller, 1890
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Chemoparasitic Theory
Plaque (a bacterial mixture) is odontopathic
W.D. Miller and his "chemico-parasitic" theory
SUSCEPTIBLE
HOST
PLAQUE
FERMENTABLE
CARBOHYDRATE
DEMINERALIZATION
ACID
PRODUCTION
What causes dental caries?
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Diet, especially sugar consumption
Salivary flow
Dental plaque
Infection by a specific set of cariogenic bacteria
within dental plaque
The first isolation of cariogenic bacteria
Clark, 1924
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Isolation of cariogenic bacteria from caries lesions
Discovery of Mutans streptococci
Two major achievements in 40’s and early 50’s
- 1943, Belding and Belding published a sketchy
description of rats developing caries as a result of
inoculation with human streptococci.
- 1954, Orland et al., demonstrated that germ-free rats
will not develop caries no matter how much sugar
they eat unless they are inoculated with Mutans
streptococci
Keyes, 1959
Dental caries is a transmittable infectious disease
Two genetically distinct hamster families (“cariesresistant” and “caries-susceptible”) were used for the
study:
- When “resistant” litter members were caged with
members of the same litter, they remained cariesfree; When “resistant” litter members were caged
with members of “caries-active” litter, they develop
active caries.
Keyes, 1962
Searching for the infectious elements that
transmit dental caries
- The experiment:
- Caries-inactive dams caged with one
another;
- Caries-inactive dams caged with cariesactive dams;
- Caries-inactive dams inoculated with feces
(or plaque) from caries-active animals;
Results of
litters
Cariesinactive
Cariesactive
Cariesactive
Conclusion: Caries is transmitted through feces or plaque
Keyes, 1962
Searching for the infectious elements that
transmit dental caries
Results of
litters
- Caries-inactive dams
caged with one another;
- Caries-inactive dams
caged with caries-active
dams;
- Caries-inactive dams
inoculated with feces from
caries-active animals;
Conclusion:
Cariesinactive
Cariesactive
Cariesactive
Addition of antibiotics
Cariesinactive
Cariesinactive
Cariesinactive
Bacteria are the cause of caries
Keyes and Fitzgerald, 1960’s
Re-isolation of “Mutans streptococci”:
• Streptococcus mutans (human) (same species Clark
isolated in England in 1924)
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Streptococcus sobrinus (human)
Streptococcus rattus (rats)
Streptococcus cricetus
Streptococcus ferus
Streptococcus macacae
Streptococcus downeii
Cariogenic Bacteria
• Mutans streptococci
– S. mutans, S. sobrinus
• Lactobacilli
– L. acidophilus, L. oris, L. salivarius
• Actinomyces
– A. naeslundii, A. viscosus
How cariogenic bacteria cause caries?
Acid production (acidogenicity)
• Lower the pH to below 5.5, the critical pH,
which drives the dissolution of calcium
phosphate (hydroxyapatite) of the tooth
enamel
• Inhibit the growth of beneficial bacteria.
• Further lower the pH, promote progression of
the carious lesion
How cariogenic bacteria cause caries?
Acid tolerance (aciduricity)
•Allows the cariogenic bacteria to thrive under acidic
conditions while other beneficial bacteria are inhibited.
•This results in dominance of the plaque by cariogenic
bacteria
How cariogenic bacteria cause caries?
Glucan formation
•Allows the cariogenic bacteria to stick onto the teeth
and form a biofilm
•Glucan mediated biofilms are more resistant to
mechanical removal
•Bacteria in these biofilms are more resistant to
antimicrobial treatments
Dental Caries
Carbohydrates (Sucrose)
Cariogenic bacteria such as S. mutans
Glucans/Levans
Plaque formation
Acids
Demineralization
The current approach to diagnose
dental caries
Naked eyes
Mechanical probing
X-ray
“Gross” Visual Examination
Mechanical Probing and X-ray
The current approach to treat
dental caries
Surgical repair
Fluoride treatment
Mechanical removal
The limited efficacy of
mechanical removal
How modern biology could impact
dentistry?
How to detect oral pathogens
in saliva or dental plaque?
Bacteria in saliva
Bacteria in plaque
Current diagnostic methods
for detecting oral pathogens
• Cell growth based detection
–Selective cultures
• DNA based detection
–PCR, Southern blotting, FISH,
• Protein based detection
–Monoclonal antibodies
Detecting S. mutans in dental plaque
3D imaging of S. mutans in dental plaque
Green: S. mutans cells
Detection of multiple cariogenic bacteria
with MAbs conjugated with different dyes
S. mutans labeled with BODIPY
conjugated SWLA1 antibody
A. naeslundii labeled with Alexa 488
conjugated SWLA4 antibody
L. casei labeled with Rhodamine
conjugated SWLA5 antibody
Co-localization of multiple cariogenic
bacteria in dental plaque
• Blue – S. mutans labeled with
BODIPY conjugated SWLA1
antibody
• Green - A. naeslundii labeled
with Alexa 488 conjugated
SWLA4 antibody
• Red – L. casei labeled with
Rhodamine conjugated
SWLA5 antibody
MAb-based chairside test for S. mutans
Microelectromechanical/Nanoelectromechanical systems
for detection of biomarkers in oral fluids
The Next Generation of
Microsensor
Go wireless!
Imagine a biosensor imbedded in the oral cavity that can send all
information about physical/chemical/biochemical parameters of
dental plaques in vivo to the dentist without the patient visiting the
doctor’s office!!!
Combined NMR/confocal microscopy
for in situ detection of acid production
The E-tooth
A unique adapting device for confocal microscope that can:
•Monitor oral pathogens with antibody-based confocal microscopy
•Monitor the demineralization hot spots with build-in Ca+2 sensors
•Monitor the pH profile of the plaque with built-in pH sensors
E-tooth
Polyaniline pH electrode response to S. mutans biofilm growth
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120
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100
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80
60
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40
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20
0
3
0
2
4
6
8
Time (hr)
10
12
Calculated pH
Potential Difference (mV)
140
S. mutans biofilm
was grown on top of
the polyaniline pH
electrode and the pH
within the biofilm was
measured over time.
The preliminary
results indicate that
E-tooth can measure
the dynamic pH
changes in S. mutans
biofilm
Reference electrode
was electrodeposited
Ag/AgCl film
The treatment of dental caries
What to do when someone has a high
level of cariogenic bacteria?
Current methods for treating oral
microbial infections
Mechanical removal
Antibiotic treatment
Problems with antibiotic treatments
Antibiotics (such as chlorhexidine)
General killing
Disruption of the normal microflora
Re-dominance of cariogenic bacteria
Antibody assisted treatment
•Guy hospital
–Express anti-S. mutans antibody in plants
–Express anti-S. mutans antibody in nonharmful bacteria
–Provide external antibodies for treatment
Bacterial replacement
•Jeff Hillman
–Genetic engineering a S. mutans strain
•Don’t produce acids
•Have growth advantages
•Replace the bad bacteria
Bacterial counter-attack
Robert Burne
–Genetic engineering base-producing bacteria
–Neutralize acids produced by S. mutans
–Balance beneficial bacterial population
A smart bomb approach
Making a smart bomb
Killing molecule
Targeting molecule
The killing molecule: antimicrobial peptide
Mode of Action of peptide antibiotic on
gram-positive bacteria
(pore formation)
Full Antibody Based Smart Bomb
Antimicrobial peptide
Conversion of a bad plaque to a good plaque
Smart bombs
against S. mutans
Playing a genetic game with bacteria
Quorum sensing in dental plaque
Wildtype
S. mutans quorum
sensing mutant
Playing a genetic game with bacteria
Overdose S. mutans with quorum sensing signal
Untreated S. mutans
Overdosed S. mutans
A microbiologist’s vision about dentistry
Diagnosis
Treatment/prevention