Chuck Somerville Marshall University Huntington, WV

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?

Why Study Antibiotic Resistance? “A growing threat” 70 percent “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.” untreatable “Unless antibiotic resistance problems are detected as they emerge... the world could be faced with previously treatable diseases that have again become untreatable....”

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 not Does not monitor water supplies

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 RM 307

Experimental Questions... Are antibiotic resistant bacteria present in detectable numbers in the Ohio River? If so, how are they distributed?

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.

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

Methods Mid-channel, sub-surface water samples 5 mile intervals in mainstem All major tributaries

Methods R2A agar + antibiotic for cultivable resistant cells Fungizone (375 ng/ml; used in all plates) Ampicillin (50  g/ml) Ciprofloxacin (4  g/ml) Erythromycin (8  g/ml) Streptomycin (25  g/ml) Sulfamethizole (128  g/ml) Tetracycline (12.5  g/ml) Virginiamycin (16  g/ml)

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)

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

Results 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

Ciprofloxacin

Tetracycline

Virginiamycin

VirginiamycinCiprofloxacin

VirginiamycinCiprofloxacinTetracycline

Virginiamycin

Results 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

- - - coliforms E. coli

- - - coliforms E. coli

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

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.

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

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.

Implications & new questions 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? CFU/ml Flow Volume (liters) Total bacteria Amp-R bacteria

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