Does Estrogen Affect Escherichia coli

Survivorship?

Robert McKinstry

Central Catholic High School

Aquatic Pollution Problems

Run-off: Debris Dirt Chemicals

Sewage and other components

Cleaning chemicals

Pharmaceuticals

Pharmaceutical Contamination

Recent India Study

Drugs: ciprofloxacin, treatments for hypertension, heart disease, chronic liver ailments, depression, gonorrhea, ulcers and other ailments

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.

Estrogen As a Pharmaceutical

Treatments: OsteoporosisSymptoms of menopause

Used in hormone replacement therapy and birth control pills.

How Estrogen Enters Water

a) Flushed down drains in the form of urine

b) The discarding of estrogen pills

c) Accidently dumped by industries

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).”

Escherichia coli

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.

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?

Purpose

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

vitro.

Hypotheses

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

Alternative hypothesis: Estrogen does affect E. coli survivorship in vitro.

• 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

Materials

• 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

Procedure

1. E. coli was grown overnight in sterile LB media.

2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask.

3. 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.

4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.

5. An estrogen suspension was mixed with the appropriate amount of SDF to create estrogen concentrations of 1%, 0.1%, and 0.01%.

Procedure6. 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.

7. The solutions were vortexed and allowed to sit at room temperature for 15 minutes.

8. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB plates.

9. The plates were incubated at 37 C for 24 hours.

10. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

Chart of Concentration

0% Concentration

0.01% Concentration

0.1% Concentration

1% Concentration

Microbe 0.1mL 0.1 mL 0.1 mL 0.1 mL

SDF 9.9mL 9.899 9.89mL 9.8mL

Estrogen Solution

0mL 0.001mL 0.01mL 0.1mL

Total 10mL 10mL 10mL 10mL

102 cells103 cells/mL

105 cells/mL with estrogen

108 cells/mL (E. coli)

100 uL100 uL

105 cells/mL (E. coli)

100 uL

Estrogen Effects on E. coli Survivorship

0

10

20

30

40

50

60

70

80

90

100

Concentrations of Estrogen

P > .05

P > .05

P < .05

Co

lon

y C

ou

nt

0% 0.01% 0.1% 1%

P-value

2.59E-08

Variable Concentration

T value Interpretation

0.01% Estrogen 1.50 Not Significant

0.1% Estrogen 1.19 Not Significant

1% Estrogen 6.19 Significant

Dunnett’s Test Analysis

T Critical = 2.76 (significant)

Alpha = .05

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.

Limitations, Extensions, and Future Studies

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.

Limitations

Extensions and Future Studies

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 experimenthttp://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

Results

0% Estrogen 0.01% Estrogen 0.1% Estrogen 1% Estrogen

56 80 104 4

86 80 70 18

86 94 54 4

76 104 70 38

86 98 52 44

100 92 72 6

68 94 74 100

86 94 68 28

70 90 70 32

74 80 60 28

Colony Counts

Estrogen Effects on E. Coli Suriviorship

0

10

20

30

40

50

60

70

80

90

100

Concentration of Estrogen

Co

lon

y C

ou

nt

P < .05

P > .05

P > .05

0% 0.01% 0.1% 1%