Transcript Slide 1

Healthy Gums
Moderate Periodontitis
Gingivitis
Advanced Periodontitis
Mouthwash Use
• $2 Billion a Year on Dental Products
– Toothpaste
– Mouthwash
– Dental Floss/Toothbrushes
• $885 Million - total annual spending
on oral antiseptics/rinses
(ACNielsen, 2002)*
Purpose of Mouthwash
• “Kills germs that cause bad breath”
– Halitosis
– Volatile Sulfur Compounds (VSC’s)
• Anaerobic, Sulfur Producing
• Fight Plaque & Gingivitis
– Bacteria Feed on Sugars
– Produce Acid
Antimicrobial Overuse
• We Overuse Antibiotics
• FDA calls it “one of the world's most pressing
public health problems” (www.fda.gov)
• 2 Million People Acquire Infections in
Hospital
• ½ Caused by Antibiotic Resistant Bacteria
• Kills 90,000+ People Annually
Antimicrobial Resistance
• Bacteria Change
–Reduces/Eliminates Effectiveness
of Antimicrobials
• Why is this a problem?
–Longer Illness
–Need for More Antibiotics
–Death
How does this happen?
Drugs place stress on environment:
killing defenseless bacteria.
How does this happen?
“Selecting” those that can
overcome the stress
How does this happen?
Resistant bacteria multiply and
become dominant strain
What does this have to do with
Mouthwash?
• Mouthwash & Antibiotics are
Antimicrobials
–Kill or Inhibit Growth
Overall Problem:
Does daily exposure to
mouthwash induce
antimicrobial resistance in
native mouth bacteria?
Alison Burchett
Centralia High School
Overall Hypothesis:
Regular exposure to
mouthwash may induce
antimicrobial resistance in
native mouth bacteria.
Test II: Affect of Mouthwash on
Mouth Bacteria
• Question: Which mouthwash(es) inhibit
the growth of most but not all mouth
bacteria?
• Hypothesis:
– Listerine
• Setup:
– Mouth Bacteria
– Petri Plates (Tooth/Tongue Surface)
Listerine (Cool Mint)
ACT
Listerine
R=0.89
(Natural
Citrus)
R=1.59
Crest Pro
R=1.48
R=0.695
Health
Safeway
Antiseptic
Mouthwash
R=1.56
Listerine
Tom’s of
Scope (Cool
Peppermint)
R=0.805
(Fresh Burst)
Maine
R=0.76
R=0.0
20.6% Alcohol
Solution
1.5% Hydrogen
Peroxide Solution
R=3.38
R=0.0
Listerine
Scope (Mint)
(Original)
R=1.29
R=0.84
Conclusions
• Listerine
– Least Effective in Preventing Growth
• (Of Successful Mouthwashes)
Test III: Effect of Mouthwash
Concentration
• Question: What concentration of Listerine will
kill/inhibit growth of most but not all bacteria?
• Hypothesis:
– 40% Most
– 50% All
•
Test III: Effect of Mouthwash
Concentration
Setup:
– Listerine
• Applied Listerine to bacterial solution
– 0%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
• 30 seconds
• Mediums:
– Blood Agar
– Bacto Agar
– LB Agar
0% Listerine
20% Listerine
Bacto Agar
Blood Agar
30% Listerine
LB Agar
Conclusions
• Mediums
– All Mediums are Appropriate
• Somewhere Between 20%-30% Kills
Most
– Above 30% Bacteria Can’t Grow
Test IV: Antimicrobial Resistance of
Native Mouth Bacteria
• Question: Does daily exposure to
mouthwash induce bacterial resistance?
• Hypothesis: Samples of exposed
bacteria will show signs of resistance
(have more bacterial growth)
• Setup:
– Growth Rates
– Optical Density
• % Transmittance and Absorbance
• 600 nm
– LB Broth
– Listerine
• 10 Applications, 1 every hour
• Ending Concentration = 14%
• 12 hour growth period
% Transmittance of bacterial growth
Sample
Non-mouthwash Users
Without
Non-mouthwash Users
Mouthwash
Non-mouthwash Users
Δ%T Mean
14.9
63
Mouthwash Users
88.8
85.4
93.9
Mouthwash Users
89.5
92
Non-mouthwash Users
21.8
18
Non-mouthwash Users
Mouthwash Users
11.5
21.6
75.7
Mouthwash Users
63.8
70
With
Mouthwash Non-mouthwash Users
Test IV: Antimicrobial Resistance of
Native Mouth Bacteria-Trial II
• Repeated Trial
• More Samples
• Application
–More Mouthwash Per Application
–Every Half Hour
–Ending Concentration = 9.1%
% Transmittance of Bacterial
Growth
Sample
Without
Mouthwash
Stress
With
Mouthwash
Stress
% of
Potential
MW User 1
79.9
81.9
97.6%
MW User 2
MW User 3
MW User 4
MW User 5
Non-User 6
Non-User 7
Non-User 8
Non-User 9
Non-User 10
Average
Standard
Deviation
% Transmittance of Bacterial
Growth
Sample
MW User 1
MW User 2
MW User 3
MW User 4
MW User 5
Non-User 6
Non-User 7
Non-User 8
Non-User 9
Non-User 10
Without
Mouthwash
Stress
With
Mouthwash
Stress
% of
Potential
79.9
92.6
86.5
9.5
53
73
17.9
77.2
3.4
22.1
81.9
90.7
87.7
20.4
41.2
66
82
69
20.9
60.1
97.6%
102.1%
98.6%
46.6%
128.6%
110.6%
21.8%
111.9%
16.3%
36.8%
Average
Standard
Deviation
94.7%
30%
59.5%
48%
Discussion
• Growth Rate
– Time Constraints
• Plaque Formation vs. Cloudy Growth
– Hard to Get Consistent Readings
• Bio Mass
Questions
• Is the bacteria resistant to just some or all
of the active ingredients?
• Are there different types of resistance?
Test V: Resistance to Individual
Active Ingredients
• Question: Do mouth bacteria become
more resistant to some antimicrobial
ingredients than others?
• Hypothesis: Exposed samples will show
resistance to some but not all active
ingredients. (Listerine)
• Setup:
– Individual Ingredients
• Thymol 0.064%
• Eucalyptol 0.092%
• Methyl Salicylate 0.060%
• and Menthol 0.042.%
– 1.7 mL Eppi Tubes
• 1 mL LB Broth
• Start Aerobic, End Anaerobic
– Took Optical Density
%T and % of Potential Growth
Individual Active Ingredients
Sample:
Application
LB Broth
Water
Listerine
Menthol
Methyl Salicylate
Eucalyptol
Thymol
Alcohol
Mouthwash User 1 Mouthwash User 2
Δ%T
72.1
57.4
%PG
Δ%T
%PG
Non-User
Δ%T
%PG
0
80%
14.3
27.7 194%
25.6
31.8 124%
18.2 25%
78.4 109%
74.9 104%
17.4 122%
2.7 19%
1.9 13%
17.4
1.5
0
68%
6%
0%
0
0
0
0%
0%
0%
76.1 106%
69.7 97%
71.9 100%
1.3
0
0
9%
0%
0%
%T and % of Potential Growth
Individual Active Ingredients
Sample:
Application
LB Broth
Water
Listerine
Menthol
Methyl Salicylate
Eucalyptol
Thymol
Alcohol
Mouthwash User 1 Mouthwash User 2
Δ%T
%PG
Δ%T
%PG
Non-User
Δ%T
%PG
72.1
57.4
0
80%
14.3
27.7 194%
25.6
31.8 124%
18.2
78.4
74.9
76.1
69.7
71.9
25%
109%
104%
106%
97%
100%
17.4 122%
2.7 19%
1.9 13%
1.3
9%
0
0%
0
0%
17.4
1.5
0
0
0
0
68%
6%
0%
0%
0%
0%
Analysis
• Possible Difference
–Menthol-Most Resistance
• Need To Do Follow Up Tests
Discussion
• Samples Showed Different Types of Resistance
– Dichotomy vs. Spectrum
• Treat Mouth as System
– Multiple Occurrences
• Other Ingredients
– Benzoic Acid
– Sodium Benzoate
• Known Antimicrobials
• Gum Chewing
– Xylitol
Limitations
• Samples
– Different People, Different Bacteria
– Oral Hygiene Habits
• Poorly-Controlled Variables
– Inoculation
• Confidence
Future Tests
• Relationship Between Sample Growth and Time
of Sample Collection
– Brushing
– Eating
• Xylitol
• Toothpaste With Mouthwash
• Genetically Modified “Good” Bacteria
– Resistant to Mouthwash
Conclusion
Bacteria exposed to mouthwash
on a daily basis showed signs
of resistance.
What does this mean?
Mouthwashes may become less
effective; no longer work.
Acknowledgements
•
•
•
•
•
My Parents, Tom and Patty Burchett
Travis Lankow
Centralia School District
Henri Weeks
Mike Stratton
– For his never-ending enthusiasm and ability to
always make science exciting.
Questions?
• Bacterial Swapping
(Kissing)
• Microbial Resistance
• Bacterial Strains
• Obsolete Dentists
• Environmental Stress
• Topic Evolution
• Listerine
• Alternative Resistance
Tests
• Hourly Applications
• Inoculation
• Spectrometer
• %Transmittance
• t-Tests
• Xylitol
Test I: Effectiveness of Mouthwash
• Controlled Variables:
– One Bacterial Strain
• E.coli DH5
– Growth Medium
• LB Broth Solution
– 12 Different Mouthwashes
– Positive and Negative Controls
• Hypothesis:
– Mouthwash = Less Growth
– More Alcohol = More Effective
• Listerine
Tissue culture flasks with 5mL LB Broth, DH5α, & Mouthwash
Control (no mouthwash)
Crest Pro Health
Scope (mint)
20.6% Alcohol Solution
Listerine (Original)
1.5% Hydrogen Peroxide
Solution
Listerine (Fresh Burst)
Tom’s of Maine
ACT
Listerine (Cool Mint)
Listerine (Natural Citrus)
Scope (Cool Peppermint)
Safeway Antiseptic Mouth
Rinse
Conclusions
• Alcohol
– Didn’t seem to work
– Didn’t support hypothesis
• Cetylpridinium Chloride
– Scope
– Crest Pro Health
• Hydrogen Peroxide
• Flavor and coloring had no effect
My Theory on Resistance and Kissing
Bobby’s Mouth
Cindy’s Mouth
My Theory on Resistance and Kissing
Bobby’s Mouth
Cindy’s Mouth
My Theory on Resistance and Kissing
Bobby’s Mouth
Cindy’s Mouth
My Theory on Resistance and Kissing
Bobby’s Mouth
Cindy’s Mouth
Types of Resistance
Genetic Mutation
•Drug Resistant TB
DNA Changes
Types of Resistance
Transformation (Microbial Sex)
•Penicillin Resistant Gonorrhea
Types of Resistance
Sharing of a Plasmid
•Guatemala Epidemic
•12,500 Died of Diarrhea
Sources
•
•
•
•
•
http://www.dentalgentlecare.com
http://www.enexus.com/gumdisease/
http://www.fda.gov
http://www.cdc.gov
Burdon, Kenneth. Text Book of Microbiology. 4th ed. New
York: Macmillan, .
• Et. Al, . Microbiology. 2nd ed. Hagerstown: Harper &
Row, .
• Et. Al, . Microbiology. 2nd ed. New York: Holt, Rinehart,
and Winston,
• Et. Al, . Antibiotics. Philadelphia: JB Lippincott Co., . .
Listerine
• $350 Million in Yearly Sales
• Most Commonly Used in the World
• 1921-First Over-The-Counter Oral Rinse
– Invented Term “Halitosis”
New Experimental Setup
• At what concentration of mouthwash is all
bacteria killed?
• Plate Dilution
– Apply Mouthwash?
– Select One Colony
• Inoculate Only With Inoculating Loop