1. Antibiotic Resistant Escherichia Coli in Non-Tidal Wetlands of Maryland Speakers: Neil Agarwal & Robert Vocke III University of Maryland, College Park

2. Establishing a Baseline MDE Clean Water Act Antibiotics

3. Site Overview Non-tidal, mitigation sites 3 plots per site

4. Data Collection Vegetation Soils Land Use Water Quality Antibiotic Resistance

5. Our focus today… Water quality data Why Antibiotic Resistance? The Concept of Measuring Resistance Preliminary Results

6. Water Quality Results Nitrate (mg/L)

7. Water Quality (continued) Ammonia (mg/L)

8. Water Quality (continued) Total N (mg/L)

9. Water Quality (continued) Total P (mg/L)

10. Why look at antibiotic resistance? The pharmaceutical problem Antibiotics and resistance Testing for antibiotic resistance is cost effective Questions to be asked: Prevalence and temporal changes Correlation to other aspects of health Resistance ‘sinks’ Nutrient Cycling

11. Disk Diffusion: The Concept Image courtesy of http://biology.clc.uc.edu/fankhauser/Labs/Microbiology Image courtesy of http://jyi.org/articleimages/555/

12. Disk Diffusion: The Concept Image courtesy of http://www.wings-china.com/showinfo.asp?typeid=24&xxid=554&Language=En

13. Fleming’s Discovery Image courtesy of http://www.accessexcellence.org/AE/AEC/CC/s5.html

14. Disk Diffusion: The Procedure Image courtesy of http://jyi.org/articleimages/555/

15. Disk Diffusion: The Procedure Image courtesy of http://jyi.org/articleimages/555/

16. Disk Diffusion: The Procedure Image courtesy of http://jyi.org/articleimages/555/

17. Disk Diffusion: The Procedure Image courtesy of http://jyi.org/articleimages/555/

18. Disk Diffusion: The Procedure Image courtesy of http://jyi.org/articleimages/555/

19. Disk Diffusion: The Procedure Image courtesy of http://jyi.org/articleimages/555/

20. Our specific procedure Collected surface samples Serial dilutions Culture on ColiBlue

21. ColiBlue media Red = ColiformsBlue = E. Coli

22. Our specific procedure Collected surface samples Serial dilutions Culture on ColiBlue Isolate a single E. coli colony Disk diffusion using 5 antibiotics: Ampicillin Erythromycin Tetracycline Sulfisoxazole Ciprofloxacin Negative Control: Vancomycin

23. Converting Inhibition Zones CLSI standard Example: Ampicillin with 7mm zone of inhibition 17 mm = Susceptible

24. Converting Inhibition Zones CLSI standard Example: Ampicillin with 7mm zone of inhibition 17 mm = Susceptible

25. Results of Testing AmpicillinTetracyclineCiprofloxacinSulfisoxazole Susceptible123211 Intermediate513212 Resistant2718011 Total33 34 % Resistant82%55%0%32%

26. What’s next? Find correlations to health Test upstream & downstream Effect on nutrient cycling

27. Antibiotic Resistant Escherichia Coli in Non-Tidal Wetlands of Maryland Speakers: Neil Agarwal & Robert Vocke III University of Maryland, College Park