Chuck Somerville Marshall University Huntington, WV

1. MU ORRC Ohio River Research Center Are Current Bacteriological Assessment Practices Sufficient for Water Resource Management in the Ohio River Basin? Chuck Somerville Marshall University Huntington, WV

2. MU ORRC Ohio River Research Center In other words . . . • Are fecal indicators (coliforms, fecal coliforms, E. coli) sufficient for determining the safety of water resources? • Specifically, do they predict the numbers and distribution of antibiotic resistant bacteria in surface waters?

3. MU ORRC Ohio River Research Center Why Study Antibiotic Resistance? • “A growing threat” • “About 70 percent of bacteria that cause infections in hospitals are resistant to at least one of the drugs most commonly used to treat infections.” • “Unless antibiotic resistance problems are detected as they emerge . . . the world could be faced with previously treatable diseases that have again become untreatable . . . .” http://www.fda.gov/oc/opacom/hottopics/antiresist_facts.html

4. MU ORRC Ohio River Research Center Why Study Antibiotic Resistance? • NARMS - the National Antimicrobial Resistance Monitoring System • USFDA, USDA & CDC collaboration formed in 1996 • Monitors resistance of isolates from humans, animals, raw food products & retail meats • Does not monitor water supplies http://www.fda.gov/cvm/narms_pg.html

5. MU ORRC Ohio River Research Center Why Study the Ohio River? • One of ten largest river systems in US • Provides drinking water for over 3 million people • Basin is home to nearly 10% of US population • No. 1 inland port & major recreation destination • Location of Marshall University @ RM 307

6. MU ORRC Ohio River Research Center Experimental Questions . . . • Are antibiotic resistant bacteria present in detectable numbers in the Ohio River? • If so, how are they distributed?

7. MU ORRC Ohio River Research Center Testing a common assumption . . . • The use of antibiotics in medicine and agriculture selects for resistant strains in the animal gut. • Failing or absent septic systems causes the distribution of resistant bacteria to the environment.  Antibiotic resistant bacteria should be a subset of fecal-derived bacteria in environmental samples.

8. Total cultivable bacteria Total Coliforms Fecal Coliforms Escherichia coli Antibiotic resistant bacteria

9. MU ORRC Ohio River Research Center Methods • Mid-channel, sub-surface water samples • 5 mile intervals in mainstem • All major tributaries

10. MU ORRC Ohio River Research Center Methods • R2A agar + antibiotic for cultivable resistant cells • Fungizone (375 ng/ml; used in all plates) • Ampicillin (50 mg/ml) • Ciprofloxacin (4 mg/ml) • Erythromycin (8 mg/ml) • Streptomycin (25 mg/ml) • Sulfamethizole (128 mg/ml) • Tetracycline (12.5 mg/ml) • Virginiamycin (16 mg/ml) http://www.nsri.upd.edu.ph/mrsl/img/services1.jpg

11. MU ORRC Ohio River Research Center Methods • m-FC at 44.5˚C for fecal coliforms (2001 – 2003) • IDEXX Colilert QuantiTray/2000 for total coliforms and Escherichia coli (2004 – present) • QT/2K + antibiotic for resistant coliforms and E. coli (2005 – present) http://ceeserver3.mit.edu/~Nepal/Mic_mFC.jpg

12. MU ORRC Ohio River Research Center Methods • Concurrent determination of: • pH, DO, turbidity, conductivity • Chlorophyll (UC) • Total N & P (UC) • Particulate N & P (UC) • Phytoplankton (NKU) • Zebra mussel veligers (TMC) • Land use patterns

13. MU ORRC Ohio River Research Center Results - 2006 • Two, 100-mile reaches of OR with complete cultivable counts (CFU/ml) and coliform data (MPN/ml) • Compared numbers of resistant cultivable cells to numbers of E. coli and resistant coliforms

14. MU ORRC Ohio River Research Center Results - 2006 Ciprofloxacin

15. MU ORRC Ohio River Research Center Results - 2006 Tetracycline

16. MU ORRC Ohio River Research Center Results - 2006 Virginiamycin

17. MU ORRC Ohio River Research Center Results - 2006 Virginiamycin Ciprofloxacin

18. MU ORRC Ohio River Research Center Results - 2006 Virginiamycin Ciprofloxacin Tetracycline

19. MU ORRC Ohio River Research Center Results - 2006 Virginiamycin

20. MU ORRC Ohio River Research Center Results - 2006 • Two, 100-mile reaches of OR with complete cultivable counts (CFU/ml) and coliform data (MPN/ml) • Compared distribution of resistant cultivable cells to distribution of E. coli and resistant coliforms

21. - - - coliforms E. coli

22. - - - coliforms E. coli

23. Total cultivable bacteria Total Coliforms Fecal Coliforms Escherichia coli Antibiotic resistant bacteria

24. MU ORRC Ohio River Research Center Conclusions • The Ohio River and major tributaries are significant reservoirs for antibiotic resistant bacteria. • Antibiotic resistant bacteria are present in much greater numbers than fecal indicator bacteria.

25. MU ORRC Ohio River Research Center Conclusions • The distribution of antibiotic resistant bacteria is distinct from that of fecal indicator bacteria.  Antibiotic resistant bacteria are not a subset of fecal indicator bacteria

26. MU ORRC Ohio River Research Center Conclusions • Antibiotic resistant bacteria in surface waters may represent a significant public health issue. • Their concentration and distribution can not be predicted by monitoring fecal-indicator organisms. • Additional work is needed to determine how resistant cells are distributed and their impacts on health.

27. CFU/ml Flow Volume (liters) MU ORRC Ohio River Research Center Implications & new questions Total bacteria • Tracking known sources of fecal bacteria does not reliably track ARB. So what? • Can resistance genes be transmitted on sand bed filters? • What types of genetic elements are involved in resistance? Amp-R bacteria

28. Thanks . . . • MU Students/Staff Lisa Smith Andy Johnson Tim Dotson Robert Nagy • Funding NSF-EPSCoR MU Graduate College Ms. Shelba Pew • Collaborators Miriam Kannan (NKU) Rebecca Evans-Kelly (NKU) Michael Miller (UC) John Hageman (TMC)