Matthew Pilewski Grade 9 Central Catholic High School

Matthew Pilewski Grade 9 Central Catholic High School

Many medications are used to kill the main bacterial cause of acne, the anaerobic bacteria Propionibacterium acnes. Do these acne medications have any effect on other human microbial flora, even if the bacteria are aerobic?

Prokaryotic microorganisms Often the cause of human and animal disease Found almost everywhere 4 Basic shapes 1. Cocci (sphere) 2. Bacilli (rods) 3. Vibrios (bent rod) 4. Spirilla (spirals) 1. Can be aerobic (require O2) or anaerobic (cannot live in the presence of O2)

Cell walls contains an extra layer of lipopolysaccharides for extra protection. Outer membrane protects bacteria from several antibiotics Most pathogenic bacteria in humans are Gram (+) organisms. Simple cell wall Some antibiotics work against the formation of the cell wall

The microorganisms that reside in or on the surface of the body. (ex. gut, skin, oral, etc.) Includes bacteria, fungi, and archea Subject bacteria: Can cause disease if growing in uncommon areas or have numbers that exceed their typical range. Most studies are of ingested materials on human cells, BUT humans are a collection of human cells and symbionts. An estimated 10% of body mass is symbionts.

One of the most common forms of bacteria found in many environments Gram negative bacilli Part of the human flora; found in the human colon and digestive tract Reproduces rapidly, often within thirty minutes Most are non-pathogenic Aerobic

Common surface symbiont in many mammals (humans) Gram positive cocci Most forms are considered non-pathogenic Part of human skin flora Forms biofilms Along with P. acnes, is often attributed to formation of acne Aerobic

Common human skin disease Areas of skin with seborrhea (scaly red skin), comedones (blackheads and whiteheads), pimples, and possible scarring Excess amount of sebum and dead skin cells: bacteria can thrive Caused by bacteria, hormones, diet, stress, and medications Anaerobic bacteria P. acnes, found in sebaceous glands, attributed to acne Treated by over-the-counter and prescription medications in the form of a pill or wash

Chemical in the peroxide family that consists of two benzoyl groups joined by a peroxide group Used in low doses as a topical treatment for acne Often used in 1% and 10% concentrations Comes as lotion, cream, gel, wash or cleansing bar Effectively treats acne by exposing anaerobic P. acnes to oxidative free radicals *10% cream used for stock in experimentation

Antibiotic synthesized from naturally occurring lincomycin Deters reproduction of certain bacteria by preventing protein synthesis from occurring Used in oral or topical treatments Topical treatments can be found in gel, cream, or lotion Topical clindamycin phosphate hydrolyzes into the active form of clindamycin when added to moisture of the skin *1% cream used as stock in experimentation

Study on excised human skin showed that only a small percentage of benzoyl peroxide is absorbed into the skin (4.5% of 10% solution) Study on clindamycin phosphate suggested that the medication is more effective when phosphatase is present, which removes the phosphate group from the clindamycin

To determine the effects of clindamycin phosphate and benzoyl peroxide on S. epidermidis and E. coli survivorship.

Null Hypothesis: Benzoyl peroxide and clindamycin phosphate do not reduce the survivorship of S. epidermidis or E. coli. Alternative Hypothesis: Benzoyl peroxide and clindamycin phosphate do reduce the survivorship of S. epidermidis and E. coli.

Escherichia coli bacteria Staphylococcus epidermidis bacteria Clindamycin Phosphate Benzoyl Peroxide Sterile micro and macro pipettes and tips Bacterial spreader, ethanol, burner, turn table LB agar plates (1% Tryptone, 0.5% Yeast Extract, 1% NaCl) Beaker Tube racks Vortex Incubator Sterile tubes & sterile dilution fluid (SDF) Klett Spectrophotometer Gram-scale Cotton balls and forceps

A. Liquid Exposure 1. Bacteria (E. coli and S. epidermidis) were grown overnight in sterile LB media. 2. Samples of the overnight culture for E. coli and Staph were added to fresh media in a sterile sidearm flask. 3. The cultures were placed in an incubator (37 C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8 cells/ml. The cultures were then diluted in SDF to approximately 10 5 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.

0% Stock (Control) 0.1x Stock 0.01x Stock Microbe 0.1 ml 0.1 ml 0.1 ml SDF 9.9 ml 8.9 ml 8.9 ml *0.1x Stock used Variable 0 ml 1 ml 1 ml* Total 10 ml 10 ml 10 ml FINAL CONCENTRATIONS OF MEDICATION STOCK 0.1x 0.01x Benzoyl Peroxide 10% 1% 0.1% Clindamycin Phosphate 1% 0.1% 0.01%

5. The microbe was then added to the test tubes to yield a total of 10 ml in each test tube. 6. The tubes were allowed to sit for 15 minutes; 100 µl aliquots were then spread onto LB agar plates. 7. Plates were incubated at 37ºC and resulting colonies counted.

B. Agar Infusion The medication was infused into the agar by spreading 200 mg of stock or 200 µl of.1x dilution onto the agar. Plates were inverted and incubated at 37ºC for 2 hours to allow infusion. 1. 100 µl of CONTROL cell suspension was spread onto the infused plates. 2. The plates were incubated overnight and resulting colonies counted. C. Realistic Medication Application Sterile cotton ball was saturated in stock solution of medication using forceps and spread on the plates to imitate realistic application of the medication. Plates were incubated for 10 minutes to allow medication to infuse. 1. 100 µl of CONTROL cell suspension was spread onto the plates. 2. The plates were incubated overnight and resulting colonies counted.

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 an experimental group directly to the control group Identifies that the experimental group is significantly different than the control if t-stat > t-crit

Average Colonies per Plate 700 600 500 P-value:1E-06 P-value: 8.46E-06 400 P-value: 0.134 300 P-value: 0.29 200 100 0 Control 0.01x 0.1x Benzoyl Peroxide Clindamycin Phosphate

Average Colonies per Plate 1200 1000 800 600 P-value: 0.096 P-value: 3.07E-12 P-value: 0.002005 P-value: 1.19E-06 400 200 0 Control 0.01x 0.1x Benzoyl Peroxide Clindamycin Phosphate

Average Colonies per Plate 700 600 P-value: 7.07E-07 P-value: 1.96E-07 500 400 P-value: 0.475 300 P-value: 0.011 200 100 0 Control 0.1x x Benzoyl Peroxide Clindamycin Phosphate

Average Colonies per Plate 900 800 700 P-value:2.81E-12 P-value: 7.63E-10 600 500 P-value: 7.5E-07 400 300 200 100 0 Control 0.1x x Benzoyl Peroxide Clindamycin Phosphate

Average Colonies per Plate 600 P-value: 0.002351 P-value: 0.009247 500 400 P-value: 0.274 300 200 100 0 Control Benzoyl Peroxide x Clindamycin Phosphate

Average Colonies per Plate 500 450 400 350 300 250 200 150 100 50 P-value: 7.17E-07 P-value: 7.17E-07 0 Control x Benzoyl Peroxide Clindamycin Phosphate

T-Crit: 3.03 Variable Concentration T Value Interpretation Liquid Exposure 0.1x 0.01x Benzoyl Peroxide Clindamycin Phosphate Benzoyl Peroxide Clindamycin Phosphate 8.5761 Significant 8.467 Significant 8.9237 Significant 4.7658 Significant Infusion x 0.1x Benzoyl Peroxide Clindamycin Phosphate Benzoyl Peroxide Clindamycin Phosphate 10.708 Significant 11.83 Significant 8.812 Significant 7.345 Significant

T-Crit: 2.75 Variable Concentration T Value Interpretation Liquid Exposure 0.1x 0.01x Benzoyl Peroxide Clindamycin Phosphate Benzoyl Peroxide Clindamycin Phosphate 18.4595 Significant 3.9014 Significant 2.0399 Insignificant 0.3221 Insignificant Infusion x 0.1x Benzoyl Peroxide Clindamycin Phosphate Benzoyl Peroxide Clindamycin Phosphate 27.727 Significant 22.0008 Significant 26.9813 Significant 8.944 Significant

Average Colony Count 1000 900 800 700 600 500 400 300 200 100 0 Approximate LD50: 0.034% 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 Concentration of Clindamycin (%) E. coli S. epidermidis

Average Colony Count 1000 900 800 700 600 500 400 300 200 100 0 Approximate LD50: 0.12% Approximate LD50: 0.56% 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Concentration of Benzoyl Peroxide (%) E. coli S. epidermidis

Null Hypothesis: Rejected Alternative Hypothesis: Accepted Benzoyl peroxide: significantly reduced survivorship of E. coli at concentrations of 10%, 1%, and 0.1% Significantly reduced survivorship of S. epidermidis at concentrations of 10% and 1% Clindamycin phosphate: significantly reduced survivorship of E. coli at concentrations of 1%, 0.1%, and 0.01% Significantly reduced survivorship of S. epidermidis at concentrations of 1% and 0.1%.

There was a lag time when plating the cells. Exposure times to the medications varied slightly. Stock medications were slightly viscous, which prevented an exact amount of stock to be pipetted for each dilution. Pipettes were fluxed in the dilution after pipetting so that variation between dilutions was minimized. During infusion, the medication did not completely absorb into the agar before microbe was plated.

More trials Different microbes Different concentrations of medication Medications used together in different combinations to determine if any synergistic effects Test dependency of clindamycin phosphate on phosphatases in killing gram-positive and gram-negative bacteria Use higher concentrations of drugs to determine all LD50s Determine if the medications effect the growth rate of the bacteria in addition to survivorship

Benzoyl Peroxide. ChemistryDaily.com. Web. 24 Dec. 2012. CLINDAMYCIN PHOSPHATE lotion CLINDAMYCIN PHOSPHATE solution CLINDAMYCIN PHOSPHATE gel Daily Med Current Medication Information. Web. 24 Dec. 2012. Fey, Paul D, and Michael E Olson. Current concepts in biofilm formation of Staphylococcus epidermidis. Future MicroBiology. 917-933. Web. 24 Dec. 2012. Fredrick, Kurt, Shinichiro Shoji, and Sarah E. Walker. Ribosomal Translocation: One Step Closer to the Molecular Mechanism. ACS Chem Biol. 93-107. Web. 24 Dec. 2012 Guay, David RP. Topical clindamycin in the management of acne vulgaris. Web. 24 Dec. 2012. Kishi, Kenji, and Kazyhiro Hirai. Clindamycin Suppresses Endotoxin Released by Ceftazidime-Treated Escherichia coli O55:B5 and Subsequent Production of Tumor Necrosis Factor Alpha and Interleukin-1 β. Antimicrobial Agents and Chemotherapy. 616-622. Web. 24 Dec. 2012 Nishijima, S, I Kurokawa, N Katoh, and K Watanabe. The bacteriology of acne vulgaris and antimicrobial susceptibility of Propionibacterium acnes and Staphylococcus epidermidis isolated from acne lesions. n. p. Web. 24 Dec. 2012. Owens, CD, K Stoessel. Surgical site infections: epidemiology, microbiology and prevention. Journal of Hospital Infection 2008. Web. 24 Dec. 2012. Sagransky, Matt, Brad A Yentzer, and Steven R Feldman. Benzoyl peroxide: a review of its current use in the treatment of acne vulgaris. Web. 24 Dec. 2012.

The independent variables in this experiment were concentrations of benzoyl peroxide and clindamycin phosphate. The dependent variable in this experiment was the survivorship of the E. coli and S. epidermidis.

SUMMARY Groups Count Sum Average Variance Control 5 3162 632.4 4654.3 0.01x 6 1941 323.5 3507.1 0.1x 5 1536 307.2 2644.7 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 344651.4 2 172325.7 47.93842 1E-06 3.805565 Within Groups 46731.5 13 3594.731 Total 391382.9 15

SUMMARY Groups Count Sum Average Variance Control 5 3162 632.4 4654.3 0.01x 5 2043 408.6 4578.8 0.1x 5 1174 234.8 7305.2 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 397297.7 2 198648.9 36.03433 8.46E-06 3.885294 Within Groups 66153.2 12 5512.767 Total 463450.9 14

SUMMARY Groups Count Sum Average Variance Control 7 6717 959.5714 22301.29 0.01x 5 4161 832.2 7419.2 0.1x 8 387 48.375 314.8393 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 3581935 2 1790968 183.7573 3.07E-12 3.591531 Within Groups 165688.4 17 9746.376 Total 3747624 19

SUMMARY Groups Count Sum Average Variance Control 7 6717 959.5714 22301.29 0.01x 6 5585 930.8333 7952.167 0.1x 7 4461 637.2857 38739.24 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 437168.6 2 218584.3 9.152454 0.002005 3.591531 Within Groups 406004 17 23882.59 Total 843172.6 19

SUMMARY Groups Count Sum Average Variance Control 5 3162 632.4 4654.3 0.1x 5 1790 358 4466 x 5 694 138.8 6816.7 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 611641.6 2 305820.8 57.56807 7.07E-07 3.885294 Within Groups 63748 12 5312.333 Total 675389.6 14

SUMMARY Groups Count Sum Average Variance Control 5 3162 632.4 4654.3 0.1x 5 1925 385 2047 x 5 1320 264 572 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 352610.5 2 176305.3 72.7202 1.96E-07 3.885294 Within Groups 29093.2 12 2424.433 Total 381703.7 14

SUMMARY Groups Count Sum Average Variance Control 5 4275 855 7066 0.1x 5 115 23 65.5 x 5 0 0 0 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 2372963 2 1186482 499.1159 2.81E-12 3.885294 Within Groups 28526 12 2377.167 Total 2401489 14

SUMMARY Groups Count Sum Average Variance Control 5 4275 855 7066 0.1x 4 2030 507.5 4413.667 x 5 0 0 0 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 1845848 2 922924.1 244.601 7.63E-10 3.982298 Within Groups 41505 11 3773.182 Total 1887353 13

SUMMARY Groups Count Sum Average Variance Control 4 2031 507.75 2684.25 x 4 1371 342.75 1598.917 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 54450 1 54450 25.42511 0.002351 5.987378 Within Groups 12849.5 6 2141.583 Total 67299.5 7

SUMMARY Groups Count Sum Average Variance Control 4 2031 507.75 2684.25 x 4 1515 378.75 1989.583 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 33282 1 33282 14.24184 0.009247 5.987378 Within Groups 14021.5 6 2336.917 Total 47303.5 7

SUMMARY Groups Count Sum Average Variance Control 4 1801 450.25 1802.917 x 4 0 0 0 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 405450.1 1 405450.1 449.7713 7.17E-07 5.987378 Within Groups 5408.75 6 901.4583 Total 410858.9 7

SUMMARY Groups Count Sum Average Variance Control 4 1801 450.25 1802.917 x 4 0 0 0 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 405450.1 1 405450.1 449.7713 7.17E-07 5.987378 Within Groups 5408.75 6 901.4583 Total 410858.9 7