Matthew Pilewski Grade 9 Central Catholic High School

Transcription

1 Matthew Pilewski Grade 9 Central Catholic High School

2 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?

3 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)

4 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

5 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.

6 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

7 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

8 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

9 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

10 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

11 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

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

13 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.

14 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

15 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.

16 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%

17 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.

18 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 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 µl of CONTROL cell suspension was spread onto the plates. 2. The plates were incubated overnight and resulting colonies counted.

19 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

20 Average Colonies per Plate P-value:1E-06 P-value: 8.46E P-value: P-value: Control 0.01x 0.1x Benzoyl Peroxide Clindamycin Phosphate

21 Average Colonies per Plate P-value: P-value: 3.07E-12 P-value: P-value: 1.19E Control 0.01x 0.1x Benzoyl Peroxide Clindamycin Phosphate

22 Average Colonies per Plate P-value: 7.07E-07 P-value: 1.96E P-value: P-value: Control 0.1x x Benzoyl Peroxide Clindamycin Phosphate

23 Average Colonies per Plate P-value:2.81E-12 P-value: 7.63E P-value: 7.5E Control 0.1x x Benzoyl Peroxide Clindamycin Phosphate

24 Average Colonies per Plate 600 P-value: P-value: P-value: Control Benzoyl Peroxide x Clindamycin Phosphate

25 Average Colonies per Plate P-value: 7.17E-07 P-value: 7.17E-07 0 Control x Benzoyl Peroxide Clindamycin Phosphate

26 T-Crit: 3.03 Variable Concentration T Value Interpretation Liquid Exposure 0.1x 0.01x Benzoyl Peroxide Clindamycin Phosphate Benzoyl Peroxide Clindamycin Phosphate Significant Significant Significant Significant Infusion x 0.1x Benzoyl Peroxide Clindamycin Phosphate Benzoyl Peroxide Clindamycin Phosphate Significant Significant Significant Significant

27 T-Crit: 2.75 Variable Concentration T Value Interpretation Liquid Exposure 0.1x 0.01x Benzoyl Peroxide Clindamycin Phosphate Benzoyl Peroxide Clindamycin Phosphate Significant Significant Insignificant Insignificant Infusion x 0.1x Benzoyl Peroxide Clindamycin Phosphate Benzoyl Peroxide Clindamycin Phosphate Significant Significant Significant Significant

28 Average Colony Count Approximate LD50: 0.034% Concentration of Clindamycin (%) E. coli S. epidermidis

29 Average Colony Count Approximate LD50: 0.12% Approximate LD50: 0.56% Concentration of Benzoyl Peroxide (%) E. coli S. epidermidis

30 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%.

31 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.

32 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

33 Benzoyl Peroxide. ChemistryDaily.com. Web. 24 Dec CLINDAMYCIN PHOSPHATE lotion CLINDAMYCIN PHOSPHATE solution CLINDAMYCIN PHOSPHATE gel Daily Med Current Medication Information. Web. 24 Dec Fey, Paul D, and Michael E Olson. Current concepts in biofilm formation of Staphylococcus epidermidis. Future MicroBiology Web. 24 Dec Fredrick, Kurt, Shinichiro Shoji, and Sarah E. Walker. Ribosomal Translocation: One Step Closer to the Molecular Mechanism. ACS Chem Biol Web. 24 Dec Guay, David RP. Topical clindamycin in the management of acne vulgaris. Web. 24 Dec 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 Web. 24 Dec 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 Owens, CD, K Stoessel. Surgical site infections: epidemiology, microbiology and prevention. Journal of Hospital Infection Web. 24 Dec 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

34 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.

35 SUMMARY Groups Count Sum Average Variance Control x x ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

36 SUMMARY Groups Count Sum Average Variance Control x x ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

37 SUMMARY Groups Count Sum Average Variance Control x x ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

38 SUMMARY Groups Count Sum Average Variance Control x x ANOVA Source of Variation SS df MS F P-value F crit Between Groups Within Groups Total

39 SUMMARY Groups Count Sum Average Variance Control x x ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

40 SUMMARY Groups Count Sum Average Variance Control x x ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

41 SUMMARY Groups Count Sum Average Variance Control x x ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

42 SUMMARY Groups Count Sum Average Variance Control x x ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

43 SUMMARY Groups Count Sum Average Variance Control x ANOVA Source of Variation SS df MS F P-value F crit Between Groups Within Groups Total

44 SUMMARY Groups Count Sum Average Variance Control x ANOVA Source of Variation SS df MS F P-value F crit Between Groups Within Groups Total

45 SUMMARY Groups Count Sum Average Variance Control x ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

46 SUMMARY Groups Count Sum Average Variance Control x ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total