Healthy%20Gums

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Healthy Gums
Gingivitis
Moderate Periodontitis
Advanced Periodontitis
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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)

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Purpose of Mouthwash

• Kills germs that cause bad breath
• Halitosis
• Volatile Sulfur Compounds (VSCs)
• Anaerobic, Sulfur Producing
• Fight Plaque Gingivitis
• Bacteria Feed on Sugars
• Produce Acid

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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

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Antimicrobial Resistance

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

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How does this happen?
Drugs place stress on environment killing
defenseless bacteria.
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How does this happen?
Selecting those that can overcome the stress
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How does this happen?
Resistant bacteria multiply and become dominant
strain
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What does this have to do with Mouthwash?

• Mouthwash Antibiotics are Antimicrobials
• Kill or Inhibit Growth

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Overall Problem

• Does daily exposure to mouthwash induce
  antimicrobial resistance in native mouth bacteria?

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Antimicrobial Resistance of Native Mouth Bacteria
Antimicrobial Resistance of Native Mouth Bacteria

• Alison Burchett
• Centralia High School

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Overall Hypothesis

• Regular exposure to mouthwash may induce
  antimicrobial resistance in native mouth
  bacteria.

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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)

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Listerine (Cool Mint) R0.89
Listerine (Natural Citrus) R0.695
Scope (Cool Peppermint) R1.48
ACT R1.59
Crest Pro Health R1.56
Safeway Antiseptic Mouthwash R0.805
Listerine (Fresh Burst) R0.76
Toms of Maine R0.0
20.6 Alcohol Solution R0.0
Scope (Mint) R1.29
Listerine (Original) R0.84
1.5 Hydrogen Peroxide Solution R3.38
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Conclusions

• Listerine
• Least Effective in Preventing Growth
• (Of Successful Mouthwashes)

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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

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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

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0 Listerine 20 Listerine
Bacto Agar Blood Agar LB Agar

30 Listerine
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Conclusions

• Mediums
• All Mediums are Appropriate
• Somewhere Between 20-30 Kills Most
• Above 30 Bacteria Cant Grow

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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)

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• 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

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

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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
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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
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Discussion

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

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Questions

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

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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)

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• 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

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T and of Potential GrowthIndividual Active
Ingredients
Sample Mouthwash User 1 Mouthwash User 2
Non-User Application ?T PG
?T PG ?T PG
LB Broth 72.1 0 14.3 25.6
Water 57.4 80 27.7 194 31.8 124
Listerine 18.2 25 17.4 122 17.4 68
Menthol 78.4 109 2.7 19 1.5 6
Methyl Salicylate 74.9 104 1.9 13 0 0
Eucalyptol 76.1 106 1.3 9 0 0
Thymol 69.7 97 0 0 0 0
Alcohol 71.9 100 0 0 0 0
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T and of Potential GrowthIndividual Active
Ingredients
Sample Mouthwash User 1 Mouthwash User 2
Non-User Application ?T PG
?T PG ?T PG
LB Broth 72.1 0 14.3 25.6
Water 57.4 80 27.7 194 31.8 124
Listerine 18.2 25 17.4 122 17.4 68
Menthol 78.4 109 2.7 19 1.5 6
Methyl Salicylate 74.9 104 1.9 13 0 0
Eucalyptol 76.1 106 1.3 9 0 0
Thymol 69.7 97 0 0 0 0
Alcohol 71.9 100 0 0 0 0
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Analysis

• Possible Difference
• Menthol-Most Resistance
• Need To Do Follow Up Tests

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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

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Limitations

• Samples
• Different People, Different Bacteria
• Oral Hygiene Habits
• Poorly-Controlled Variables
• Inoculation
• Confidence

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Future Tests

• Relationship Between Sample Growth and Time of
  Sample Collection
• Brushing
• Eating
• Xylitol
• Toothpaste With Mouthwash
• Genetically Modified Good Bacteria
• Resistant to Mouthwash

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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.

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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.

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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

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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

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Tissue culture flasks with 5mL LB Broth, DH5a,
Mouthwash
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Conclusions

• Alcohol
• Didnt seem to work
• Didnt support hypothesis
• Cetylpridinium Chloride
• Scope
• Crest Pro Health
• Hydrogen Peroxide
• Flavor and coloring had no effect

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My Theory on Resistance and Kissing
Bobbys Mouth
Cindys Mouth
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My Theory on Resistance and Kissing
Bobbys Mouth
Cindys Mouth
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My Theory on Resistance and Kissing
Bobbys Mouth
Cindys Mouth
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My Theory on Resistance and Kissing
Bobbys Mouth
Cindys Mouth
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Types of Resistance
Genetic Mutation
DNA Changes

• Drug Resistant TB

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Types of Resistance
Transformation (Microbial Sex)

• Penicillin Resistant Gonorrhea

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Types of Resistance
Sharing of a Plasmid

• Guatemala Epidemic
• 12,500 Died of Diarrhea

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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., . .

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Listerine

• 350 Million in Yearly Sales
• Most Commonly Used in the World
• 1921-First Over-The-Counter Oral Rinse
• Invented Term Halitosis

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New Experimental Setup

• At what concentration of mouthwash is all
  bacteria killed?
• Plate Dilution
• Apply Mouthwash?
• Select One Colony
• Inoculate Only With Inoculating Loop