Does Estrogen Affect Escherichia coli Survivorship?

1. Does Estrogen Affect EscherichiacoliSurvivorship? Robert McKinstry Central Catholic High School

2. Aquatic Pollution Problems • Run-off: Debris Dirt Chemicals • Sewage and other components • Cleaning chemicals • Pharmaceuticals

3. Pharmaceutical Contamination • Recent India Study • Drugs: ciprofloxacin, treatments for hypertension, heart disease, chronic liver ailments, depression, gonorrhea, ulcers and other ailments

4. Estrogen • Chemical steroid. • Functions as the primary female sex hormone. • Promotes the growth of secondary female sex characteristics, accelerates metabolism, reduces muscle mass, and decelerates height growth.

5. Estrogen As a Pharmaceutical • Treatments: Osteoporosis Symptoms of menopause • Used in hormone replacement therapy and birth control pills.

6. How Estrogen Enters Water • Flushed down drains in the form of urine • The discarding of estrogen pills • Accidently dumped by industries

7. Estrogen Contamination • High levels – Improper disposal • Near-impossible to remove • Florida Museum: “When adult male fish are exposed to short term and low concentrations of a synthetic estrogen, their fertility can drop by as much as 50%, according to a study by scientists at the Department of Energy's Pacific Northwest National Laboratory (PNL).”

8. Escherichiacoli Large and diverse group of gram (-) bacteria Free living, symbionts, or pathogens U.S. infections > 70,000 per year Large outbreaks rare, sporadic food infestations Serves as a common prokaryotic cell model.

9. E.coli and Estrogen E.coli and other coliforms are common in rivers across the world. Estrogen is a growing problem in many of the same rivers and tributaries. Does estrogen affect E.coli populations?

10. Purpose To determine if estrogen has an affect on E.coli survivorship in vitro.

11. Hypotheses Null hypothesis: Estrogen does not affect E.coli survivorship in vitro. Alternative hypothesis: Estrogen does affect E.coli survivorship in vitro.

12. Estrogen (0.5 mg pills) Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl) Turntable 40 Luria Broth agar plates Bunsen burner Spread bar Incubator Klett Spectrophotometer Matches Materials • Labeling tape • Ethanol • Latex gloves • E.coli DH5 Alpha • Micropipettes • Micro rack • Ten micro tubes • LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride). • Mortar and Pestle

13. Procedure • E.coli was grown overnight in sterile LB media. • 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 108 cells/mL. • The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL. • An estrogen suspension was mixed with the appropriate amount of SDF to create estrogen concentrations of 1%, 0.1%, and 0.01%.

14. Procedure • 100 µL of cell culture was then added to the estrogen solutions, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL. • The solutions were vortexed and allowed to sit at room temperature for 15 minutes. • After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB plates. • The plates were incubated at 37 C for 24 hours. • The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

15. Chart of Concentration

16. 100 uL 100 uL 100 uL 108 cells/mL (E.coli) 105 cells/mL (E.coli) 105 cells/mL with estrogen 103 cells/mL 102 cells

17. P > .05 P > .05 Colony Count P < .05 0% 0.01% 0.1% 1%

18. Dunnett’s Test Analysis T Critical = 2.76 (significant) Alpha = .05

19. Conclusions • The null hypothesis that Estrogen does not affect E.coli survivorship in vitro was rejected for the concentration of 1% estrogen. • The null hypothesis could not be rejected for the lower concentrations of 0.01% and 0.1% estrogen. • The results indicate that 1% estrogen did negatively influence E.coli survivorship.

20. Limitations, Extensions, and Future Studies Limitations • The E.coli plating was not exactly synchronized, possibly contributing to varying colony counts. • The estrogen was quite insoluble, requiring the use of a suspension. • Larger sample sizes will be employed in the next round of testing. • Higher and lower concentrations of estrogen will be tested. • Directly infusing estrogen into the LB agar for prolonged cell exposure. Extensions and Future Studies

21. References • Estrogen Makes Fish Too Feminine To Reproduce http://www.flmnh.ufl.edu/fish/InNews/estrogen2007.html • Short Term Exposure to Estrogen Cuts Fish Fertilityhttp://www.flmnh.ufl.edu/fish/InNews/fishfert2003.htm • Estrogen knocks out fish in whole-lake experiment http://pubs.acs.org/subscribe/journals/esthag-w/2003/aug/science/jp_lake.html • http://www.ninemilerun.org/resources/scientific_reports/Bacteria%20&%20Detergent%20Results%20August%202006.pdf • http://www.nmenv.state.nm.us/swqb/wps/San_Juan/SanJuanRiver-2004BacteriaSamplingReport.pdf • http://www.cdc.gov/nczved/dfbmd/disease_listing/stec_gi.html

22. Results Colony Counts

23. P > .05 P > .05 P < .05 0% 0.01% 0.1% 1%