Healthy Gums Gingivitis

Moderate Periodontitis 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 BurchettCentralia 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)

Tom’s of

Maine

R=0.0

1.5% Hydrogen Peroxide Solution

R=3.38

20.6% Alcohol Solution

R=0.0

Crest Pro

Health

R=1.56

Listerine (Cool Mint)

R=0.89ACT

R=1.59

Listerine

(Natural Citrus)

R=0.695

Listerine

(Fresh Burst)

R=0.76

Listerine

(Original)

R=0.84

Scope (Mint)

R=1.29

Safeway Antiseptic

Mouthwash

R=0.805

Scope (Cool Peppermint)

R=1.48

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

• 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

Test III: Effect of Mouthwash Concentration

Bacto Agar

Blood Agar

LB Agar

0% Listerine 20% Listerine

30% Listerine

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

Sample Δ%T Mean

Non-mouthwash Users 14.9

Non-mouthwash Users 88.8 63

Non-mouthwash Users 85.4

Mouthwash Users 93.9

92Mouthwash Users 89.5

Non-mouthwash Users 21.8

Non-mouthwash Users 11.5 18

Non-mouthwash Users 21.6

Mouthwash Users 75.7

70Mouthwash Users 63.8

% Transmittance of bacterial growth

Without Mouthwash

With Mouthwash

•Repeated Trial•More Samples•Application

–More Mouthwash Per Application

–Every Half Hour–Ending Concentration = 9.1%

Test IV: Antimicrobial Resistance of Native Mouth

Bacteria-Trial II

% Transmittance of Bacterial Growth

Sample

Without Mouthwash

Stress

With Mouthwash

Stress% of

Potential AverageStandard 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

% Transmittance of Bacterial Growth

Sample

Without Mouthwash

Stress

With Mouthwash

Stress% of

Potential AverageStandard 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%

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: Mouthwash User 1 Mouthwash User 2 Non-User

Application Δ%T %PG Δ%T %PG Δ%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%

%T and % of Potential Growth

Individual Active Ingredients Sample: Mouthwash User 1 Mouthwash User 2 Non-User

Application Δ%T %PG Δ%T %PG Δ%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%

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 Str

ess• 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)

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

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

DNA Changes•Drug Resistant TB

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