Does Estrogen Affect Escherichia coli Survivorship?

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

2. Run-off • A common problem in urban regions is run-off. • Rain water can pick up debris, dirt, pollutants, and chemicals resulting in aquatic pollution. • This can cause problems such as eutrophication, toxicity, or other disturbances.

3. Pharmaceutical Run-off • One study found 21 pharmaceuticals that were dumped into a stream in India. • The drugs were used in treatments for hypertension, heart disease, chronic liver ailments, depression, gonorrhea, ulcers and other ailments. • Enough ciprofloxacin, a powerful antibiotic, was dumped into an Indian stream each day to treat 90,000 people.

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 • Found in medications for postmenopausal women to prevent osteoporosis as well as treat the symptoms of menopause. • Used in hormone replacement therapy and birth control pills.

6. Estrogen Drain Run-off • High levels of estrogen have been found in waterways across the world due to improper disposal. • It is near-impossible for the estrogen to be removed. • A website run by the Florida Museum of Natural History states, “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).”

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

8. E.coli Escherichiacoli is a large and diverse group of gram (-) bacteria. Most strains of E.coli are harmless, others can cause sickness. Estimated to cause infection in more than 70,000 patients a year in the United States. Reported to cause both large outbreaks as well as isolated sporadic infections in small numbers of individuals. 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. It is not known if estrogen affects the growth and therefore the survivorship of E. coli.

10. Purpose To determine if estrogen has an effect 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 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 • Micropipettes • Micro rack • Ten micro tubes • LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)

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 degrees 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 P < .05 0% 0.01% 0.1% 1%

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

19. P > .05 P > .05 Colonies P < .05

20. 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.

21. 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

22. References • Estrogen Makes Fish Too Feminine To Reproduce http://www.flmnh.ufl.edu/fish/InNews/estrogen2007.html • Short Term Exposure to Estrogen Cuts Fish Fertility http://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

23. Results Colony Counts