Transcript Antibiotic resistance of bacteria found in York County Watersheds

Antibiotic resistance of bacteria found in York County Watersheds
Liz Knisley
Department of Biology, York College of Pennsylvania
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The main cause of antibiotic resistant bacteria is caused
by public overuse of antibiotics (Baquero et al 2008)
Cases have already seen deaths due to antibiotics not
working against serious bacterial infections such as
cases related to Staphylococcus aureus (Stachowiak et al
2010)
Since 1990 an increase of about ten times has been
seen in the amount of beta-lactamases that inactivate
beta-lactam class antibiotics (Laxinarayan et al 2013)
Resistance is rapidly spreading around the world due to
an increase of travel and trade worldwide (Laxinarayan
et al 2013)
Although techniques are used to disinfect waste water,
leftover bacteria is free to transfer resistance genes
through horizontal gene transfer (Baquero et al 2008)
Antibiotics used for disease control and growth
promotion agriculturally also contribute to surface
runoff into watersheds (Laxinarayan et al 2013)
S usquehanna
C o d o ro u s C re e k
P in c h o t
1000
R L R e s e r v o ir
D a ir y F a r m
*
L o g 1 0 ( n u m b e r o f c o lo n ie s )
Introduction
100
*
Figure 4. Electrophoresis gel of PCR products for the TetW gene. Lane 1
shows a 500 base pair ladder and lanes 2-4 show positive bands for the TetW
gene (1,000 base pairs) in all three water samples of the Garber Dairy Farm
Pond water source.
10
1
A m p ic illin
Figure 1. Log10(number of colonies) grown on a concentration of 5 ug/mL antibiotic
treated agar for different water systems in York County (n=5). Water samples were
collected in mid-September on the same day at three sites and kept over a period of
four months at 4 degrees Celsius for testing. Plates that grew lawns of 300+ colonies
were listed as too numerous to count and graphed at 300. Error bars are shown as the
mean +/- standard deviation. Garber Dairy Farm Pond and Pinchot Lake are shown as
significantly different for the Ampicillin treated agar.
S usquehanna
C o d o ro u s C re e k
H1: The Red Lion reservoir water will have the
largest amount of antibiotic resistant bacteria
Methods
S3
S4
S5
P in c h o t
1000
R L R e s e r v o ir
D a ir y F a r m
L o g 1 0 ( n u m b e r o f c o lo n ie s )
H2: Viable bacteria will show a higher
resistance to beta-lactam class antibiotics
S2
A m o x ic illin
A n tib io tic
Hypotheses
S1
C e p h a le x in
Figure 5. Nutrient agar plates treated with different Amoxicillin concentrations
after incubation from site 1 of the Codorous Creek and Pinchot Lake
100
Conclusion
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Collection and Storage
0 .1
A m p ic illin
C e p h a le x in
A m o x ic illin
A n tib io tic
Culture + Abx*
PCR
Primers used:
16S rRNA, BlaIMP,
MecA, and TetW
Concentrations:
No Abx, 5 μg/mL, and 100 μg/mL
Watersheds used
(3 Samples per site)
•Susquehanna River
•Codorous Creek
•Pinchot Lake
•Red Lion Reservoir
•Garber Dairy Farm Pond
Figure 2. Log10(number of colonies) grown on a concentration of 100 ug/mL antibiotic treated agar for
different water systems in York County (n=5). Water samples were collected in mid-September on the
same day at three sites and kept over a period of four months at 4 degrees Celsius for testing. Plates
that grew lawns of 300+ colonies were listed as too numerous to count and graphed at 300. Error bars
are shown as the median with range.
Agarose Gel Analysis
(0.5% 150V)
Antibiotics Used
•Amoxicillin
•Ampicillin
•Cephalexin
Coding Information
TetW
Tetracycline
resistance gene
BlaIMP
Metallo-betalactamase
resistance gene
MecA
Methicillin
resistance gene
16S rRNA
Universal
prokaryotic 16S
Figure 3. Electrophoresis gel of PCR products for 16s ribosomal gene with an annealing
temperature of 60⁰C. The lane to the left of the sample lanes shows a 500 base pair
ladder. Lanes 1-3 are water samples from sites 1-3 of Garber Dairy Farm Pond, 4-6 are
samples from sites 1-3 of the Susquehanna River, lanes 7-9 are samples from sites 1-3 of
the Codorous Creek, lanes 10-12 are samples from sites 1-3 of Red Lion Reservoir, and
lanes 13-15 are samples from sites 1-3 of Pinchot Lake. Bands are shown in lanes 1-3, 1011, and 13-15 at 1,500 base pairs.
•There is no difference in the amount of bacteria grown
on antibiotic treated plates based on waterway
•There is no difference between the amount of
resistance between beta-lactam antibiotics and other
types of antibiotics
•Without knowing the actual CFU/mL of each sample
hard to determine significance
Future Directions
•Collect water samples over yearly months to determine how
different times of the year may affect bacterial growth
•Test different types of bacteria in local waterways against
antibiotics other than Gram positive bacteria
•Focus directly on the Garber Dairy Farm Pond and compare
bacteria resistance to antibiotics farmers may be using
Literature Cited
Baquero, F., Martinez, J., and Canton, R. 2008. Antibiotics and antibiotic
resistance in water environments. Current Opinion in Biotechnology [serial
online] 19: 260-265.
Laxminarayan, R., Duse, A., Wattal, C., Zaidi, A.K.M., Wertheim, H.F.L.,
Sumpradit, N., Vlieghe, E., Hara, G.L., Gould, I.M., Goosens, C.G., So, A.D.,
Bigdeli, M., Tomson, G., Woodhouse, W., Ombaka, E., Peralta, A.Q., Qamar, F.N.,
Mir, F., Kariuki, S., Bhutta, Z.A., Coates, A., Bergstorm, R., Wright, G.D., Brown,
E.D., and Cars, O. 2013. Antibiotic Resistance-the need for global solutions. The
Lancet Infectious Disease Commission [serial online] 13: 1057-1098.
Stachowiak. M, Clark, S.E., Templin, R.E., and Baker, K.H. 2010. TetracyclineResistant Eshcerichia coli in a Small Stream Receiving Fish Hatchery Effluent.
Water Air Soil Pollut. [serial online] 211:251-259.
Acknowledgments
I would like to thank my mentor, Dr. David Singleton for all his help with my research. I would also like
to thank Dr. Bridgette Hagerty for her help with data analysis and Dr. Brian Gray for his guidance
through senior thesis. Special thanks to the Biology Department for all my opportunities and
experiences throughout my college career.