1. Analysis of Bacterial Resistance Henry R. Walther Grade 11 Pittsburgh Central Catholic High School

2. Bacteria Resistance Disease-causing microbes that have become resistant to drug therapy Tuberculosis, gonorrhea, malaria, and childhood ear infections are just a few of the diseases

3. Causes of Bacteria Resistance horizontal gene transfer unlinked point mutations environmental pressure pass this trait to their offspring

4. Common Antibacterials Dirithromycin Erythromycin Troleandomycin Cefixime Cefotaxime Cefpodoxime Ceftazidime Triclosan

5. Triclosan A potent antibacterial and antifungal agent. Increasing in US consumer products (soaps, textiles, toys, kitchen utensils) Binds to ENR enzyme in cell, which increases affinity for NAD+, a stable conformation of the 3 chemicals which inhibits fatty acid synthesis ( inculding synthesis of cAMP)

6. Relationship The August 6, 1998 issue of Nature warned that triclosan's overuse could cause resistant strains of bacteria to develop, in much the same way that antibiotic-resistant bacterial strains are emerging, based on speculation that triclosan behaved like an antibiotic. The 2003 Sunday Herald newspaper reported that some UK supermarkets and other retailers were considering phasing out products containing triclosan.

7. E. coli One of the most common forms of bacteria, free living, symbiotic or pathogenic. Has been utilized as the most studied prokaryote. There are many of different strains of E. coli, most of which are non- pathogenic. However, there are strains which can produce fatal disease.

8. Purpose to examine the potential resistance of E.coli to the antibiotic ingredient triclosan

9. Null Hypothesis Triclosan’s effect on the survivorship of exposed, assumed resistant, bacteria and the unexposed bacteria will not differ outside of chance.

10. Materials Triclosan 60 sterile LB dishes E. Coli DH5 α Micro Pipette Sterile Micro tubes Spreader bar Ethanol Scale Incubator (37°C) Sterile 250mL Sidearm Flask Fine tip permanent marker Klett Spectrophotometer Weigh Boat Vortex Tube Rack SDF media LB media LB Media Tryptone (1%) Yeast Extract (0.5%) NaCl (1%) NaOH SDF Media 100mM KH 2 PO 4 100mM K 2 HPO 4 10mM MgSO 4 100mM NaCL (5%)

11. Procedure 1. A culture of E Coli DH5α was grown overnight in a sterile 250mL sidearm flask in LB media at 37°C. 2. The culture was left to grow until it reached an absorbance reading of about 50 Kletts, which represents a cell density of about 10 8 cells/mL. 3. The culture was serially diluted in SDF to a concentration of approximately 10 3 cells/mL. 4. The triclosan solution was prepared. 0.075 grams of triclosan were weighed out and poured it into 5mls of ethanol. This created a 0.15% concentration of triclosan. 5. The following ingredients were mixed to attain the proper concentrations; 0.15%0.015%0% E. coli0.01 ml Triclosan0.1 ml0.01 ml0 ml SDF Media0.89 ml0.98 ml0.99 ml

12. Procedure (cont.) 6. Two plates were spread from each different concentration and let grow over night in an incubator. 7. One bacteria colony was cultured from one of the 0% plates and one colony was cultured from each variable plate (assumed resistance). 8. Repeated step 1. 9. For each batch of bacteria repeat steps 2 through 5, but used 6 plates for each different concentration. 10. Record results.

13. P value=0.075904 P value = 0.84567

14. Conclusions The null hypothesis can be accepted because the p-value in the ANOVA is above the confidence cut off of.05. Exposing E. coli to triclosan did not appear increase the survivorship in 0.01X and 1X concentrations of triclosan. No evidence of resistance was found in this study.

15. Extensions/Improvements Use different species of bacteria Use different antibacterial agents Grow the bacteria with agent over more generations Use different concentrations of triclosan Infuse the agar with triclosan for prolonged exposure time.

16. References http://en.wikipedia.org/wiki/Escherichia_c oli http://en.wikipedia.org/wiki/Triclosan www.beyondpesticides.org/pesticides/factshee ts/Triclosan%20cited.pdf en.wikipedia.org/wiki/Antibiotic_resistance www.fda.gov/Fdac/features/795_antibio.html