Synergistic Effect of Ibuprofen and Alcohol on Flora Ryan McNelis Grade 11 Pittsburgh Central Catholic High School

Rationale for Experiment Commonly, ingested materials are investigated for effects on human cells. However, effects on the human flora may also be important.

Microbial Flora The internal and external flora has eukaryotic fungi, protists, and bacteria Not much is known about the association between humans and their flora Effects can be mutualistic, parasitic, pathogenic, and commensal Provides nutritional and digestive benefits, secrete vitamins, stimulate antibody production, and protect against pathogenic microbes Substance intakes by humans might have unintended effects on these important functions of the flora

Escherichia Coli (E. Coli) Large and diverse group of gram (-) bacteria Free living, symbiont, or pathogen Live in the intestinal tract of many mammals Most strains are not pathogenic Serve as a common prokaryotic cell model

Ibuprofen and Ethanol Ibuprofen: Anti-inflammatory drug Used to reduce hormones-used for inflammation or pain in the body-reduces fever, headaches, and injuries in the body. Ethanol: Primary alcohol found in beverages. Created by the fermentation of sugars by yeast.

Ibuprofen and Ethanol: Negative Effects Ibuprofen: associated with cardiovascular risks, skin blistering, nausea, headache, and dizziness Ethanol: causes lack of balance, gastrointestinal disease; over long term may act as a carcinogen and contribute to birth defects.

Brand Tested Children’s Dye Free Ibuprofen 100mg of Ibuprofen per 5 mL Inactive Ingredients: acesulfame potassium, anhydrous citric acid, D&C yellow no.10, FD&C red no.40, flavors, glycerin, polysorbate 80, pregelatinized starch, purified water, sodium benzoate, sucrose, xanthan gum

Problem Accompanied with drug side effects, combination of over the counter drugs and alcohol can have dire side effects Do a common daily over the counter drug (Ibuprofen) and ethanol have synergistic effects and reduce survivorship of human bacterial flora?

Purpose To investigate whether various concentrations of Ibuprofen and Ethanol have an adverse synergistic effect on microbial flora populations.

Hypotheses Null: The concentrations of Ibuprofen and ethanol will have no significant effect on E. Coli survivorship Alternative: The concentrations of Ibuprofen and ethanol will cause a significant decrease in the survivorship of E. Coli.

Materials E. coli LB media (Per Liter:1% Tryptone, 0.5% Yeast Extract, and 1% NaCl) Latex Gloves 2 syringe Sterile Filters Micropipettes BunsenBurners Micro tubes Spread Bar Children’s Ibuprofen Incubator ethanol Ethanol Sterile Dilution Fluid (SDF) (per 1 liter) (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl) Matches Vortex Klett Spectrophotometer 30 LB agar plates

Procedure E. coli was grown overnight in sterile LB agar. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 107 cells/mL. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL. Ibuprofen and ethanol was mixed with the appropriate amount of Sterile Dilution Fluid to create a 20% stock. Ibuprofen was used directly from the bottle, after sterile filtration. 100 μL of cell culture was then added to the solutions, yielding a final volume of 10 mL and a cell density of approximately 104 cells/mL. The following ingredients were mixed to create the desired experimental exposures:

Chart of Concentrations 0% Alcohol 0.1 % Alcohol 1% Alcohol 0% Ibuprofen 0, 0 0, Low 0, High 0.5% Ibuprofen Low, 0 Low, Low Low, High 5% Ibuprofen High, 0 High, Low High, High

Procedure 6. The solutions were vortexed and allowed to sit at room temperature for 15 minutes. 7. 100 μL aliquots were removed from the tubes and spread on LB plates. 8. The plates were incubated at 37 degrees for 24 hours. 9. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell. 10. The appropriate statistical analyses were performed to adequately assess the hypothesis.

Results 0% Alcohol 0.1 % Alcohol 1% Alcohol 0% Ibuprofen 165 140 151 160 193 171 190 146 151 159 106 108 159 121 136 147 174 93 0.5% Ibuprofen 191 145 122 129 121 183 155 143 151 160 144 168 123 138 184 123 5% Ibuprofen 189 177 232 171 178 155 167 157 158 161 165 204 219 180 226

Results P (Ethanol)= 0.0000265 P (Ibuprofen)= 0.345 P (interaction)= 0.031887

Statistical Analyses ANOVA: Single Factor Analysis of Variation Statistical test that compares the means of multiple groups P-value from ANOVA testing identifies significance if P-value is <0.05 Dunnett’s Test Statistical test that compares an experimental group directly to the control group Identifies that the experimental group is significantly different than the control if the t-value > t-crit Two Factor ANOVA Can reveal synergistic effects of two variables

Dunnett’s Test T-Crit=2.15 Concentration T-Value Interpretation L IBU, L Ethanol 1.59 Not Significant H IBU, L Ethanol 0.14 L IBU, H Ethanol 1.72 H IBU, H Ethanol 2.69 Significant

Conclusions The null hypothesis was rejected; the data supports a synergistic effect The null hypothesis was rejected for the following concentration of ibuprofen and ethanol: 5% Ibuprofen, 1% Ethanol (H, H) Ethanol appeared to have a significant effect, Ibuprofen alone did not

Limitations and Extensions The plating was not precisely synchronized, which could have resulted in extra bacterial reproduction in the tubes Only one type of exposure tested (liquid pulse) Only a few concentrations were tested Extensions Test a larger range of concentrations Test on other models (yeast) Other exposure methods Varying exposure times

References www.drugs.com/ibuprofen.html www.ncbi.nlm.nih.gov/pubmed/18461099 www.cdc.gov/ecoli/ www.mayoclinic.com/health/e-coli/DS01007

2 Factor ANOVA Count 18 Sum 3023 2811 2973 Average 167.9444 156.1667 Total   Count 18 Sum 3023 2811 2973 Average 167.9444 156.1667 165.1667 Variance 794.4085 618.5 1666.618 ANOVA Source of Variation SS df MS F P-value F crit Sample 16851.81 2 8425.907 13.44091 2.65E-05 3.204317 Columns 1364.593 682.2963 1.088391 0.345457 Interaction 7290.296 4 1822.574 2.907349 0.031887 2.578739 Within 28209.83 45 626.8852 53716.54 53

Single Factor ANOVA Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 6 1030 171.6667 279.8667 Column 2 900 150 1335.6 Column 3 830 138.3333 827.0667 Column 4 891 148.5 969.5 Column 5 894 149 57.2 Column 6 880 146.6667 818.2667 Column 7 1102 183.6667 684.6667 Column 8 1017 169.5 389.5 Column 9 1263 210.5 280.3 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 25506.7 8 3188.338 5.086 0.000154 2.152133 Within Groups 28209.83 45 626.8852 Total 53716.54 53