2. Moderate Periodontitis Advanced Periodontitis
Healthy Gums
Gingivitis
Moderate Periodontitis
Advanced Periodontitis

3. 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)*
Ramifications of being obsessed ..spend a lot of money on it

4. 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

5. Antimicrobial Overuse
We Overuse Antibiotics
FDA calls it “one of the world's most pressing public health problems” (
2 Million People Acquire Infections in Hospital
½ Caused by Antibiotic Resistant Bacteria
Kills 90,000+ People Annually

6. Antimicrobial Resistance
Bacteria Change
Reduces/Eliminates Effectiveness of Antimicrobials
Why is this a problem?
Longer Illness
Need for More Antibiotics
Death

7. Drugs place stress on environment: killing defenseless bacteria.
How does this happen?
Drugs place stress on environment: killing defenseless bacteria.

8. “Selecting” those that can overcome the stress
How does this happen?
“Selecting” those that can overcome the stress

9. Resistant bacteria multiply and become dominant strain
How does this happen?
Resistant bacteria multiply and become dominant strain

10. What does this have to do with Mouthwash?
Mouthwash & Antibiotics are Antimicrobials
Kill or Inhibit Growth
So if antimicrobial resistance occurs in our bodies when we overuse antibiotics, couldn’t it be occurring in our mouths when we over use mouthwash?

11. Overall Problem:
Does daily exposure to mouthwash induce antimicrobial resistance in native mouth bacteria?
If over exposure to antbiotics can cause antimicrobail resistance,

12. Alison Burchett Centralia High School
Antimicrobial Resistance of
Native Mouth Bacteria
Antimicrobial Resistance of
Native Mouth Bacteria
Alison Burchett
Centralia High School

13. Overall Hypothesis:
Regular exposure to mouthwash may induce antimicrobial resistance in native mouth bacteria.

14. 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)

15. Scope (Cool Peppermint) R=1.48 ACT R=1.59
Listerine (Cool Mint)
R=0.89
Listerine
(Natural Citrus)
R=0.695
Scope (Cool Peppermint)
R=1.48
ACT
R=1.59
Crest Pro
Health
R=1.56
Safeway Antiseptic Mouthwash
R=0.805
Listerine
(Fresh Burst)
R=0.76
Tom’s of
Maine
R=0.0
20.6% Alcohol Solution
R=0.0
Scope (Mint)
R=1.29
Listerine
(Original)
R=0.84
1.5% Hydrogen Peroxide Solution
R=3.38

16. Conclusions Listerine Least Effective in Preventing Growth
(Of Successful Mouthwashes)

17. 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

18. 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

19. 0% Listerine 20% Listerine
Bacto Agar
Blood Agar
LB Agar
30% Listerine

20. Conclusions Mediums Somewhere Between 20%-30% Kills Most
All Mediums are Appropriate
Somewhere Between 20%-30% Kills Most
Above 30% Bacteria Can’t Grow

21. 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)

22. Setup: Growth Rates Optical Density LB Broth Listerine
% Transmittance and Absorbance
600 nm
LB Broth
Listerine
10 Applications, 1 every hour
Ending Concentration = 14%
12 hour growth period

24. % Transmittance of bacterial growth
Sample
Δ%T
Mean
Non-mouthwash Users
14.9
88.8 63
85.4
Mouthwash Users
93.9 92
89.5
21.8
11.5 18
21.6
75.7 70
63.8
Without Mouthwash
With Mouthwash

25. 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%

26. % Transmittance of Bacterial Growth
Sample
Without Mouthwash Stress
With Mouthwash Stress
% of Potential
Average
Standard Deviation
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
Talk about %T…lower value means more bacteria

27. % Transmittance of Bacterial Growth
Sample
Without Mouthwash Stress
With Mouthwash Stress
% of Potential
Average
Standard Deviation
MW User 1
79.9
81.9
97.6%
94.7%
30%
MW User 2
92.6
90.7
102.1%
MW User 3
86.5
87.7
98.6%
MW User 4
9.5
20.4
46.6%
MW User 5 53
41.2
128.6%
Non-User 6 73 66
110.6%
59.5%
48%
Non-User 7
17.9 82
21.8%
Non-User 8
77.2 69
111.9%
Non-User 9
3.4
20.9
16.3%
Non-User 10
22.1
60.1
36.8%
Talk about %T…lower value means more bacteria

28. Discussion Growth Rate Plaque Formation vs. Cloudy Growth
Time Constraints
Plaque Formation vs. Cloudy Growth
Hard to Get Consistent Readings
Bio Mass

29. Questions
Is the bacteria resistant to just some or all of the active ingredients?
Are there different types of resistance?

30. 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)

31. Setup: Individual Ingredients 1.7 mL Eppi Tubes 1 mL LB Broth
Thymol 0.064%
Eucalyptol 0.092%
Methyl Salicylate 0.060%
and Menthol %
1.7 mL Eppi Tubes
1 mL LB Broth
Start Aerobic, End Anaerobic
Took Optical Density

32. %T and % of Potential Growth Individual Active Ingredients
Sample: Mouthwash User 1 Mouthwash User Non-User
Application Δ%T %PG Δ%T %PG Δ%T %PG
LB Broth
72.1
14.3
25.6
Water
57.4
80%
27.7
194%
31.8
124%
Listerine
18.2
25%
17.4
122%
68%
Menthol
78.4
109%
2.7
19%
1.5 6%
Methyl Salicylate
74.9
104%
1.9
13% 0%
Eucalyptol
76.1
106%
1.3 9%
Thymol
69.7
97%
Alcohol
71.9
100%

33. %T and % of Potential Growth Individual Active Ingredients
Sample: Mouthwash User 1 Mouthwash User Non-User
Application Δ%T %PG Δ%T %PG Δ%T %PG
LB Broth
72.1
14.3
25.6
Water
57.4
80%
27.7
194%
31.8
124%
Listerine
18.2
25%
17.4
122%
68%
Menthol
78.4
109%
2.7
19%
1.5 6%
Methyl Salicylate
74.9
104%
1.9
13% 0%
Eucalyptol
76.1
106%
1.3 9%
Thymol
69.7
97%
Alcohol
71.9
100%

34. Analysis Possible Difference Need To Do Follow Up Tests
Menthol-Most Resistance
Need To Do Follow Up Tests

35. 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

36. Limitations Samples Poorly-Controlled Variables Confidence
Different People, Different Bacteria
Oral Hygiene Habits
Poorly-Controlled Variables
Inoculation
Confidence

37. Future Tests
Relationship Between Sample Growth and Time of Sample Collection
Brushing
Eating
Xylitol
Toothpaste With Mouthwash
Genetically Modified “Good” Bacteria
Resistant to Mouthwash

38. Mouthwashes may become less effective; no longer work.
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.

39. 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.

40. 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

41. 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

42. Tissue culture flasks with 5mL LB Broth, DH5α, & Mouthwash

43. Control (no mouthwash)
20.6% Alcohol Solution
1.5% Hydrogen Peroxide Solution
Tom’s of Maine
ACT
Listerine (Cool Mint)
Crest Pro Health
Scope (mint)
Listerine (Original)
Listerine (Fresh Burst)
Listerine (Natural Citrus)
Scope (Cool Peppermint)
Safeway Antiseptic Mouth Rinse

44. Conclusions Alcohol Cetylpridinium Chloride Hydrogen Peroxide
Didn’t seem to work
Didn’t support hypothesis
Cetylpridinium Chloride
Scope
Crest Pro Health
Hydrogen Peroxide
Flavor and coloring had no effect

45. My Theory on Resistance and Kissing
Bobby’s Mouth
Cindy’s Mouth

46. My Theory on Resistance and Kissing
Bobby’s Mouth
Cindy’s Mouth

47. My Theory on Resistance and Kissing
Bobby’s Mouth
Cindy’s Mouth

48. My Theory on Resistance and Kissing
Bobby’s Mouth
Cindy’s Mouth

49. Types of Resistance
Genetic Mutation
DNA Changes
Drug Resistant TB

50. Transformation (Microbial Sex)
Types of Resistance
Transformation (Microbial Sex)
Penicillin Resistant Gonorrhea

51. Sharing of a Plasmid Types of Resistance Guatemala Epidemic
12,500 Died of Diarrhea

52. Sources http://www.dentalgentlecare.com
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., . .

53. Listerine $350 Million in Yearly Sales Most Commonly Used in the World
1921-First Over-The-Counter Oral Rinse
Invented Term “Halitosis”

54. New Experimental Setup
At what concentration of mouthwash is all bacteria killed?
Plate Dilution
Apply Mouthwash?
Select One Colony
Inoculate Only With Inoculating Loop