Tetracycline Resistance in Escherichia coli strains collected from North River and Stone Village Pond.

1. Tetracycline Resistance in Escherichia
coli strains collected from North River
and Stone Village Pond
By Sarah Autry, Anissa Boyers, Daniela
Espinoza-Hernandez, and Loren Smith
https://my.clevelandclinic.org/health/diseases/16638-e-coli-infection

2. Background
● Antibiotics
○ derived from live organisms, (fungi and bacteria)
○ Kill bacteria or inhibit growth
○ Resistance occurs when a bacterium with pre-existing
resistance is naturally selected for and replicates,
leading to a population of resistant bacteria.
● Tetracycline
○ Broad spectrum antibiotic
○ Acts on the 30S ribosomal subunit
○ Prevents tRNA from binding at the A site

3. Background continued
● Escherichia coli (Gram -)
○ Family Enterobacteriaceae
○ Rod shaped
● Tetracycline Resistance in Escherichia coli
○ Large plasmids with resistance genes

4. Experimental Questions
1.) Does Escherichia coli (E.coli) collected from the North
River and Stone village pond have tetracycline resistance?
2.) What is the effect of the concentration of Tetracycline on
the growth of E. coli?
3.) Do Escherichia coli strains that exhibit tetracycline
resistance (smaller clearing zones) have large plasmid DNA?

5. Hypotheses
● Null Hypothesis: Escherichia coli with higher levels of
tetracycline resistance will NOT have larger plasmid DNA
indicated by gel electrophoresis, which is correlated with
tetracycline resistant plasmids.
● Alternate Hypothesis: Escherichia coli with higher levels
of tetracycline resistance WILL have larger plasmid DNA
indicated by gel electrophoresis, which is correlated with
tetracycline resistant plasmids.

6. Outline of Experiment

7. Water Collection &
Coliscan Filtration
● Water samples were filtered by using
ColiscanⓇ Membrane Filter Kit
● It is placed onto a saturated pad of
Coliscan-MF liquid medium into each
petri dish.
● E. coli is indicated by a blue colony
Membrane Water Filter Test Kit E. Coli & Coliforms | Posts by Madeline Binder
(SciFairLady) | Bloglovin’

8. Isolation of
Escherichia coli
● Cultured on EMB plates
● Observed for green color which
indicates E. Coli colonies
● Subcultures transferred to broth
● Indole test to confirm E. coli (+)
from Enterobacter and Klebsiella
(-)

9. ● Filled each disc with a
different concentrations of
tetracycline clockwise
around the TSA plate 0.5µL,
1.0µL, 2.5µL, 5.0µL, 7.5µL,
10µL of the dilution onto
each disc
Testing Strains for
Tetracycline Resistance

10. ● The materials used were
resuspension buffer, lysis
solution, ammonium acetate,
chloroform and a precipitation
solution
● Removing the supernatant,then
added TE to prevent from
degrading
● Plasmids present without
chloroform
● Added gel loading dye and ready
for gel electrophoresis
Plasmid Extraction & Gel Electrophoresis

11. Zones of Inhibition
Observed
Results
15 E. coli Strains Isolated

12. Results - Grand Averages of Zones of
Inhibition
Figure 5. The grand
averages of the
zones of inhibition
(mm) in Escherichia
coli strains A, B, C,
D, E, F, G, H, I, J,
K, L, M, N, and O
when exposed to
various tetracycline
concentrations.

13. Results - Gel Electrophoresis of Plasmid
DNA
Figure 7. Gel electrophoresis of large plasmid DNA in Escherichia coli strains. The
gel was run on (0.7%) agarose for about an hour. The DNA ladder is located in lane
12 and 17. Plasmid DNA was found in lanes A, B, C, I, L, N, O.

14. Conclusions
Figure 5. The grand
averages of the
zones of inhibition
(mm) in Escherichia
coli strains A, B, C,
D, E, F, G, H, I, J,
K, L, M, N, and O
when exposed to
various tetracycline
concentrations.
Stars indicate: the
presence of plasmids

15. Future Work
PCR to detect Tet M and Tet B genes
● Tet B Codes for an efflux pump
If PCR indicated that these genes
were present, then this would correlate
with Tetracycline resistance.
https://ib.bioninja.com.au/standard-level/topic-
3-genetics/35-genetic-modification-and/pcr.html

16. References
Heringa, S., Monroe, J., & Herrick, J. (2007, October 24). A simple, rapid method
for extracting large plasmid DNA from bacteria. Nature Precedings. From
https://doi.org/10.1038/npre.2007.1249.1.
(n.d.). (2021, September 15). Resources, Micrology Labs. Micrology Labs. From
https://www.micrologylabs.com/resources-2/.

17. Questions?