Microbial infections in oral cavity Wenyuan Shi

1
Microbial infections in oral cavityWenyuan Shi
UCLA School of Dentistry Molecular Biology
Institute Microbiology, Immunology and Molecular
Genetics
2
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 .
3
(No Transcript)
4
Scanning EM image of oral microflora
5
1
2
3
4
5
6
From Marsh, 1999
6
(No Transcript)
7
Microbial Infections in Oral Cavity
G bacteria G- bacteria
yeast
Dental Caries Periodontitis Oral
candidiasis 79 population 45 population
80 millions
8
Dental caries
What causes dental caries?
9
DECAY HISTORY OF MAN
(NEOLITHIC TO MODERN ERA)
COMMON ERA
ROMAN ERA
IRON
NEOLITHIC
DMT
Sugar connection!
10
Salivary glands
Smith and Karst 2000
1
0.01-2.3mls/min/gland
2
3
0.02-1.3mls/min
11
The first medical approach to dentistry
Miller, 1890

Chemoparasitic Theory Plaque (a bacterial
mixture) is odontopathic
12
W.D. Miller and his "chemico-parasitic" theory
SUSCEPTIBLE HOST
PLAQUE
FERMENTABLE CARBOHYDRATE
DEMINERALIZATION
ACID PRODUCTION
13
What causes dental caries?

• Diet, especially sugar consumption
• Salivary flow
• Dental plaque
• Infection by a specific set of cariogenic
  bacteria within dental plaque

14
The first isolation of cariogenic bacteria
Clark, 1924

Isolation of cariogenic bacteria from caries
lesions Discovery of Mutans streptococci
15
Two major achievements in 40s and early 50s

• 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

16
Keyes, 1959Dental caries is a transmittable
infectious disease

• Two genetically distinct hamster families
  (caries-resistant and caries-susceptible)
  were used for the study
• When resistant litter members were caged with
  members of the same litter, they remained
  caries-free When resistant litter members
  were caged with members of caries-active
  litter, they develop active caries.

17
Keyes, 1962Searching for the infectious elements
that transmit dental caries
Results of litters

• The experiment
• Caries-inactive dams caged with one another
• Caries-inactive dams caged with caries-active
  dams
• Caries-inactive dams inoculated with feces (or
  plaque) from caries-active animals

Conclusion
Caries is transmitted through feces or plaque
18
Keyes, 1962Searching for the infectious elements
that transmit dental caries
Results of litters
Addition of antibiotics

• 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

Caries- inactive
Caries- inactive
Caries- inactive
Caries- inactive
Conclusion
Bacteria are the cause of caries
19
Keyes and Fitzgerald, 1960sRe-isolation of
Mutans streptococci

• Streptococcus mutans (human) (same species Clark
  isolated in England in 1924)
• Streptococcus sobrinus (human)
• Streptococcus rattus (rats)
• Streptococcus cricetus
• Streptococcus ferus
• Streptococcus macacae
• Streptococcus downeii

20
Cariogenic Bacteria

• Mutans streptococci
• S. mutans, S. sobrinus
• Lactobacilli
• L. acidophilus, L. oris, L. salivarius
• Actinomyces
• A. naeslundii, A. viscosus

21
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

22
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

23
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

24
Dental Caries

• Carbohydrates (Sucrose)
• Cariogenic bacteria such as S. mutans
• Glucans/Levans Acids

Plaque formation
Demineralization
25
The current approach to diagnose dental
cariesNaked eyesMechanical probingX-ray
26
Gross Visual Examination
27
Mechanical Probing and X-ray
28
The current approach to treat dental
cariesSurgical repairFluoride treatment
Mechanical removal
29
(No Transcript)
30
The limited efficacy of mechanical removal
31
How modern biology could impact dentistry?
32
How to detect oral pathogens in saliva or dental
plaque?
Bacteria in saliva Bacteria in
plaque
33
Current diagnostic methodsfor detecting oral
pathogens

• Cell growth based detection
• Selective cultures
• DNA based detection
• PCR, Southern blotting, FISH,
• Protein based detection
• Monoclonal antibodies

34
Detecting S. mutans in dental plaque
35
3D imaging of S. mutans in dental plaque
Green S. mutans cells
36
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
37
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

38
MAb-based chairside test for S. mutans
39
Microelectromechanical/Nanoelectromechanical
systems for detection of biomarkers in oral
fluids
40
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 doctors office!!!

41
Combined NMR/confocal microscopyfor in situ
detection of acid production
42
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 Ca2 sensors
• Monitor the pH profile of the plaque with
  built-in pH sensors

43
E-tooth
Polyaniline pH electrode response to S. mutans
biofilm growth
8
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
140
120
7
100
6
80
Potential Difference (mV)
Calculated pH
60
5
40
4
20
Reference electrode was electrodeposited Ag/AgCl
film
0
3
0
2
4
6
8
10
12
Time (hr)
44
The treatment of dental caries What to do when
someone has a high level of cariogenic bacteria?
45
Current methods for treating oral microbial
infections
Mechanical removal Antibiotic treatment
46
Problems with antibiotic treatments
Antibiotics (such as chlorhexidine) General
killing Disruption of the normal
microflora Re-dominance of cariogenic bacteria
47
Antibody assisted treatment

• Guy hospital
• Express anti-S. mutans antibody in plants
• Express anti-S. mutans antibody in non-
• harmful bacteria
• Provide external antibodies for treatment

48
Bacterial replacement

• Jeff Hillman
• Genetic engineering a S. mutans strain
• Dont produce acids
• Have growth advantages
• Replace the bad bacteria

49
Bacterial counter-attack

• Robert Burne
• Genetic engineering base-producing bacteria
• Neutralize acids produced by S. mutans
• Balance beneficial bacterial population

50
A smart bomb approach
51
Making a smart bomb
Targeting molecule
Killing molecule
52
The killing molecule antimicrobial peptide
53
Full Antibody Based Smart Bomb
Antimicrobial peptide
54
Conversion of a bad plaque to a good plaque
Smart bombs against S. mutans
55
Playing a genetic game with bacteria Quorum
sensing in dental plaque
S. mutans quorum sensing mutant
Wildtype
56
Playing a genetic game with bacteria Overdose S.
mutans with quorum sensing signal
Untreated S. mutans Overdosed S.
mutans
57
A microbiologists vision about dentistry
Diagnosis
Treatment/prevention