Antibiotic resistance of bacteria found in York County WatershedsLiz Knisley

Department of Biology, York College of PennsylvaniaIntroduction

• 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)

HypothesesH1: The Red Lion reservoir water will have the largest amount of antibiotic resistant bacteria

H2: Viable bacteria will show a higher resistance to beta-lactam class antibiotics

Methods

S1 S2 S3 S4 S5

Collection and Storage

Culture + Abx* PCRPrimers used:

16S rRNA, BlaIMP, MecA, and TetW

Concentrations:No Abx, 5 μg/mL, and 100 μg/mL Agarose Gel Analysis

(0.5% 150V)

Watersheds used(3 Samples per site)• Susquehanna River

• Codorous Creek• Pinchot Lake

• Red Lion Reservoir•Garber Dairy Farm Pond

Antibiotics Used• Amoxicillin• Ampicillin• Cephalexin

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.

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, 10-11, and 13-15 at 1,500 base pairs.

Conclusion•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

AcknowledgmentsI 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.

Literature CitedBaquero, 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. Tetracycline-Resistant Eshcerichia coli in a Small Stream Receiving Fish Hatchery Effluent. Water Air Soil Pollut. [serial online] 211:251-259.

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.

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.

Figure 5. Nutrient agar plates treated with different Amoxicillin concentrations after incubation from site 1 of the Codorous Creek and Pinchot Lake

Coding InformationTetW BlaIMP MecA 16S rRNA

Tetracycline resistance gene

Metallo-beta-lactamase

resistance gene

Methicillin resistance gene

Universal prokaryotic 16S

Ampicillin Cephalexin Amoxicillin1

10

100

1000

Antibiotic

Lo

g1

0(n

um

ber

of

colo

nie

s)

SusquehannaCodorous CreekPinchotRL ReservoirDairy Farm

*

*

Ampicillin Cephalexin Amoxicillin0.1

1

10

100

1000

Antibiotic

Lo

g1

0(n

um

ber

of

colo

nie

s)SusquehannaCodorous CreekPinchotRL ReservoirDairy Farm