Acne Medication Antimicrobial Effects Fiorente Pampena Pittsburgh Central Catholic High School Grade 9

Acne Vulgaris Common human skin disease Caused by bacteria, hormones, diet, steroids Bacteria thrives with excess sebum and dead skin cells Whiteheads, blackheads, nodules, pustules, papules Treated by over-the-counter and prescription medicines Pills, washes, creams

Clearasil Stubborn Acne Control Pads Cotton pads soaked in medication Active ingredient is salicylic acid Salicylic acid helps correct abnormal shedding of cells Cleans outer layer of skin by unclogging pores and preventing lesions 1.9% salicylic acid by volume

Witch Hazel Hamamelis, genus of deciduous shrubs and trees Medical astringent Cuts, scrapes, bug bites, acne Constricts body tissue Minimizes pores and dries oil Contains tannins, which have antioxidant properties Medication used 86% by volume

Human Microbial Flora The microorganisms inside or on the surface of the human body Includes bacteria, fungi, and archaea Can cause disease if growing in uncommon areas or large numbers An estimated 10% of the body mass is symbionts Do acne medications interfere with human microflora?

Gram (-) vs. Gram (+) Bacteria Gram-Negative Gram-Positive Single layer of peptidoglycan Cell wall contains extra layer of lipopolysaccharides For outer protection Protects bacteria from antibiotics Several layers of peptidoglycan Antibiotics work against formation of cell wall Most pathogenic bacteria in humans are gram-positive

Cutibacterium acnes Gram-positive Part of human microflora Live deep within follicles and pores Attributed to acne vulgaris Secrete digestive enzymes Destabilize the cells that form the walls of the follicle Rapid growth caused by cellular damage in follicles triggers inflammation

Staphylococcus epidermidis Common surface symbiont in many mammals Gram-positive Most forms considered non-pathogenic Part of human microflora Attributed to acne vulgaris along with C. acnes Used as the microflora model in this experiment

Alternative Hypothesis Hypotheses Null Hypothesis Alternative Hypothesis There will be no difference in the survivorship of Staphylococcus epidermidis bacteria in the plates containing witch hazel or Clearasil. There will be a difference in the survivorship of Staphylococcus epidermidis bacteria in the plates containing witch hazel or Clearasil.

Materials Staphylococcus epidermidis bacteria Clearasil Stubborn Acne Control Pads Witch hazel Sterile micro/macro pipettes and tips Bacterial spreader, ethanol, burner, turn table LB agar plates (1% Tryptone, 0.5% Yeast Extract, 1% NaCl) Beaker Test tubes Test tube racks Vortex Incubator (37°C) Sterile dilution fluid (SDF) Sterile disks and forceps Sterile water

Purpose To determine which of the two acne medications (witch hazel and Clearasil) work best at killing and preventing bacteria when applied to human skin.

Procedure A. Liquid Exposure 1. Bacteria (S. epidermidis) were grown overnight in sterile LB media. 2. Sample of the overnight culture of S. epidermidis were added to fresh media in a sterile sidearm flask. 3. 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 10⁸ cells/mL. The culture was then diluted in SDF to approximately 10⁵ cells/mL. 4. Medications were diluted with SDF in test tubes into two concentrations with a third tube containing 0% of the medications to be used as a control. 5. The microbe was then added to the test tubes to yield a total volume of 10 mL in each tube. 6. The tubes were allowed to sit for 5 minutes; 100μl aliquots were then spread onto LB agar plates. 7. Plates were incubated at 37°C overnight and resulting colonies counted.

Concentrations (mL) of Liquid Pulse Exposure 0% Stock (Control) 0.1% Stock 1% Stock Microbe (S. epidermidis) 0.1 mL Sterile Dilution Fluid (SDF) 9.9 mL 8.9 mL 7.9 mL Acne Medication 0 mL 1 mL 2 mL Total 10mL

Procedure cont. B. Agar Infusion Transferred 200μl of medications and sterile water onto LB agar plates. Transferred 180μl of sterile water and 20μl of medications onto other plates. Spread liquid evenly on every plate, getting a new sterile spreader for each plate. Incubated plates in a 37°C incubator until solutions were absorbed into agar. Spread S. epidermidis evenly on every plate, getting a new sterile spreader for each plate. Plates were incubated at 37°C overnight and resulting colonies counted.

Procedure cont. C. Zones of Inhibition Spread S. epidermis evenly on LB agar plates with a sterile spreader and incubated in a 37°C incubator. Dipped sterile disk in acne medications (100% concentration) using sterile forceps. Placed disks with medications around perimeter of plate. (4 disks on each plate) Dipped sterile disk in a dilution of medications (10% concentration) using sterile forceps. Placed disks with 10% dilution of medications around perimeter of plate. Plates were incubated at 37°C overnight and resulting colonies counted.

Acne Medication Effects on Staph Survivorship (Liquid Pulse Exposure) P-value: 3.09E-22

Liquid Pulse Dunnett's Test Acne Medication Concentrations T-value Conclusion Clearasil 1% 47.44 Significant Clearasil 10% 47.66 Witch Hazel 1% 13.96 Significant  Witch Hazel 10% 19.52

Acne Medication Effects on Staph Survivorship (Agar Infusion) P-value: 1.52371E-22

Agar Infusion Dunnett's Test Acne Medication T-value Conclusion Clearasil 200μl 34.26 Significant Clerasil 20μl + 180μl Sterile Water Witch Hazel 200μl  19.22 Witch Hazel 20μl + 180μl Sterile Water 14.11

Acne Medication Effects on Staph Survivorship (Zones of Inhibition) P-value: 2.0108E-15

Zones of Inhibition Dunnett's Test Acne Medication Concentrations T-value Conclusion Clearasil 100% 37.88 Significant Clearasil 10% 29.97 Witch Hazel 100% 14.26 Witch Hazel 10% 10.7

Conclusion All concentrations of both acne medications significantly reduced survivorship of Staphylococcus epidermidis bacteria. Null hypothesis is rejected because the Clearasil acne medication had significantly higher T-values than the witch hazel in all Dunnett's tests.

Limitations There was lag time when plating the microbe Exposure times to the medications varied slightly Study does not reveal other effects on bacterial health Disks were left opened for a short period of time Only addresses survivorship Limited range of exposures Limited exposure time Only 1 species used

Extensions More trials Different microbes Different concentrations of medication Medications used together in different combinations to determine if any synergistic effects Determine if the medications effect the growth rate of the bacteria in addition to survivorship

References AR, Shalita. “Department of Dermatology, SUNY Downstate Medical Center, Brooklyn, NY 11203, USA. Wpbowe@Gmail.com.” Addiction & Health, StatPearls Publishing, europepmc.org/abstract/med/18786494. Toombs, Ella. “Cosmetics in the Treatment of Acne Vulgaris.” NeuroImage, Academic Press, 21 July 2005, www.sciencedirect.com/science Otto, Michael. “Staphylococcus Epidermidis - the 'Accidental' Pathogen.” Nature News, Nature Publishing Group, 1 Aug. 2009, www.nature.com Reuter, Juliane, et al. “Which Plant for Which Skin Disease? Part 1: Atopic Dermatitis, Psoriasis, Acne, Condyloma and Herpes Simplex.” The Canadian Journal of Chemical Engineering, Wiley-Blackwell, 5 Aug. 2010, onlinelibrary.wiley.com Scalbert, Augustin. “Antimicrobial Properties of Tannins.” NeuroImage, Academic Press, 15 May 2001, www.sciencedirect.com/science Gollnick, H., and M. Schramm. “Topical Drug Treatment in Acne.” Dermatopathology, Karger Publishers, 13 Mar. 1998, www.karger.com

Statistical Analyses ANOVA: Single Factor Dunnett's Test Analysis of Variation Statistical test that compares the means of multiple groups P-value from ANOVA testing identifies significance if < 0.05 Statistical test that compares experimental group directly to control group Identifies that experimental group is significantly different than the control if t-stat > t-crit

Liquid Pulse ANOVA

Agar Infusion ANOVA

Zones of Inhibition ANOVA