Bacterial Source Tracking by Multiple Antibiotic Resistance Analysis of Escherichia coli

1. Bacterial Source Tracking by Multiple Antibiotic Resistance Analysis of Escherichia coli

2. Bacterial Source Tracking (BST) • Determination of the sources of fecal bacteria from an environmental water sample (1995) • Several different techniques are currently being used for BST

3. BST Technology • Genotypic • Pulsed-Field Gel Electrophoresis (Simmons/Herbein/Hagedorn – Va. Tech.) • Ribotyping (Samadpour – U. of Wash.) • Randomly Amplified Polymorphic DNA (RAPD)/PCR (Sadowsky – U. of Minn.)

4. BST Technology • Biochemical (Phenotypic) • Multiple Antibiotic Resistance (MAR) (Hagedorn - Va. Tech/ Kator – VIMS/ Wiggins – James Madison) • Coliphage (Geoff Scott – NOAA) • Carbon Source Profiles (Hagedorn – Va. Tech) • Sterols or Fatty Acid Analysis • Chemical (Non-Bacterial) • Optical Brighteners • Caffeine Detection

5. Fecal Indicators (Microbial) • Fecal Coliforms • Facultative, Gram negative, non-spore forming, rod –shaped, ferment lactose producing gas within 48 hours. Sources: animals, soil (44.50 C is selective) • Fecal Streptococci (Enterococci) • Facultative, Gram positive, tolerate high salt and are used as indicators particularly in brackish or marine waters. Sources: warm-blooded animals • Escherichia coli: • A sub-set of the fecal coliform. Sources: mammals and birds

6. Sources of Contamination • Treatment plants • Septic systems • Improper disposal from boats • Agricultural runoff • Pets • Wildlife

7. Elevated levels of E. coli • Indicator of potential pathogens • Other bacteria • Viruses • O157:H7 • Beach closings • Shellfish bed closings

8. Fecal Coliform Standards • Recreational Water • < 200 MPN/100 ml • Shellfish Beds • < 14 MPN/100 ml • Public Drinking Water Supply • < 20 MPN/100 ml • Treated Drinking Water • < 1% positive/month

9. Total Maximum Daily Load (TMDL) • TMDLs – required by §303(d) of the 1972 Clean Water Act (CWA) • Must submit TMDLs to EPA for approval • Under court order to complete TMDLs by 2008

10. TMDL Calculation TMDL = Point Sources + Nonpoint Sources + Projected Growth + Margin of Safety    TMDL - sum of allowed pollutant loads for point sources, non-point sources, projected growth and a margin of safety  

11. Key TMDL Components • Describe Impairment  • Identify Cause of Impairment  • Set Quantitative Goal or Endpoint  • Identify Pollutant Sources  • Determine Allowable Load and Allocation  • Determine Margin of Safety  • Reasonable Assurance of Implementability   

12. 2002 TMDL Project – Bacterial TMDL • Salisbury University • Biology(Mark Frana) • Environmental Health Science(Elichia Venso) • Maryland Department of the Environment • TARSA • Shellfish waters • Nontidal waters

13. Project Overview • Isolate E. coli • from known sources • from water v • verify as E. coli • Determine concentrations of E. coli in water • MAR analysis of E. coli from known sources • Predict sources of water E. coli from known source profiles • Submit results as input for TMDL calculation

14. MAR Analysis • Unknown Sources (water) • Membrane filtration • Colony enumeration • Inoculation of unknown sources onto TSA/antibiotic plates • Known sources • Inoculation of known sources onto TSA/antibiotic plates • Read and record resistance profiles • Discriminant Analysis

15. Membrane Filtration and Colony Enumeration • Sterile filters, funnels, vacuum filtration • Filter 3, 10, 30, 100 (2), 200 ml aliquots • Transfer filters onto mTEC agar plates • Chromogen (5-bromo-6-chloro-3-indolyl-D-glucuronide) • Catabolized to glucuronic acid + red/magenta compound by E. coli • Incubate at 35 degrees C for 2 h • Incubate in Whirl-Pak bags in 44.5 degree C waterbath for 22-24 h • Count colonies (20-80 range/plate)

16. Transfer to Whirl-Pacs and Incubation in Water Bath

18. 28 concentrations of 7 antibiotics Trypticase Soy Agar (TSA) plates Inoculate TSA plates from 96-well plates 48-prong replica plater Incubate for 24 h at 35 degrees C Read plates 1=isolate grew (resistant) 0=isolate did not grow (susceptible) Antibiotic Resistance Analysis

22. Antibiotics and Concentrations Used in Analysis • Cephalothin: 15, 25, 35 ug/ml • Erythromycin: 60, 70, 90, 100 ug/ml • Neomycin: 2.5, 5, 10 ug/ml • Oxytetracycline: 2.5, 5, 7.5, 10, 15 ug/ml • Rifampicin: 60, 75, 90 ug/ml • Streptomycin: 2.5, 5, 7.5, 10, 15 ug/ml • Tetracycline: 2.5, 5, 7.5, 10, 15 ug/ml

23. Laboratory Activity • MAR • 2,425 Profiles • DNA • 52.6 Gels • Statistical • >63,000 data entries

24. Statistical Analysis • Discriminant analysis of known source resistance profiles • 2-way (human vs nonhuman) • 3-way (human vs livestock vs wildlife) • 5-way (human vs livestock vs wildlife vs domestic vs birds) • Discriminant analysis of unknown sources

25. Total isolates 403 Black Vulture 10 Cow 60 Deer 62 Dog 30 Fox 77 Goose 14 Horse 26 Human 64 Rabbit 10 Raccoon 40 Swan 10 Known Source Isolates

26. Antibiotics and Concentrations Used in Analysis • Cephalothin: 15, 25, 35 ug/ml • Erythromycin: 60, 70, 90, 100 ug/ml • Neomycin: 2.5, 5, 10 ug/ml • Oxytetracycline: 2.5, 5, 7.5, 10, 15 ug/ml • Rifampicin: 60, 75, 90 ug/ml • Streptomycin: 2.5, 5, 7.5, 10, 15 ug/ml • Tetracycline: 2.5, 5, 7.5, 10, 15 ug/ml

27. Students/Technical Assistance Current student laboratory assistants: Natasha Patterson Gayle Raynor Rebecca Scott Gry Wesenberg Current laboratory technician: Lesley Frana

28. Acknowledgements This project was funded by Maryland Department of the Environment. We gratefully acknowledge the support of the Technical and Regulatory Services Administration of MDE and the Richard A. Henson School of Science and Technology for its support