Identification of E. coli Sources in the Conesus Lake Watershed Using PCR Jason Somarelli Advisor: Dr. Joseph Makarewicz SUNY Brockport Department of Environmental Science and Biology 1/15/03

Question: n Can we identify and quantify the E. coli in Conesus Lake sub-watersheds coming from farms? –Isolate E. coli coming from cattle –Separate cattle E. coli from other sources n Can we track reductions in E. coli coming from farms? n Grant Question: Will Best Management Practices reduce E. coli levels in sub- watersheds?

The problem: E. coli n E. coli contamination is a well documented problem around Conesus Lake n DEC reports of septic systems failing around Conesus Lake n Beach closings in 1996 and 1999 n Conesus Lake provides drinking water for: –Avon –Geneseo –York –Lakeshore property owners

Importance of E. coli as an Environmental Contaminant n Enhanced levels often stem from fecal contamination. n E. coli strain O157:H7 itself is potentially fatal. n The presence of E. coli is often indicative of : –Salmonella spp. –Shigella spp. –Hepatitis A virus –Norwalk group viruses

Conesus Lake n Three major E. coli sources: –Wildlife-geese –Humans n Septic leaks n Sewage treatment overflow –Domestic/Agriculture-almost all from cattle n Manure spreading in winter

Conesus Lake n Three major E. coli sources –Wildlife-geese –Humans n Septic Leaks n Sewage Treatment Overflow –Domestic/Agricultural-almost all from cattle n Manure spreading in winter n Cattle in and around stream beds

Identifying and Tracking E. coli n We know the sources of E. coli n We do NOT know how much E. coli comes from each source n Research: –Identify and separate E. coli by source –Combine ID with quantitative data to track changes in E. coli over time.

Bacterial Source Tracking (BST) n Use BST to identify E. coli sources n BST allows us to determine sources of E. coli and other fecal coliform bacteria –Pulsed-field Gel Electrophoresis –Antibiotic Resistance –PCR

Bacterial Source Tracking (BST) n Allows us to say where E. coli is coming from. n For the grant: Even if E. coli increases over time from humans or wildlife, we can eliminate these sources in our calculations.

Thesis Question n Usually, BST is used to target sites of remediation. n In this experiment: –Identify sources of E. coli. –Quantify E. coli in Conesus Lake sub- watersheds. n Can we identify and quantify decreases in E. coli coming from farms over time?

Design of Experiment-Lake and Watershed n Design is parallel with the grant n Control vs. Experimental Streams –Control Streams: n Long Point n Sutton Point n North McMillan –Experimental Streams: n Graywood n Sand Point n Cottonwood n Southwest Creek

Design-Sampling and Enumeration n Samples will be collected weekly from all seven streams n Quantification of E. coli will be performed by Dr. Simon of SUNY Geneseo using serial dilution techniques n Identification of E. coli colonies will be performed using PCR n Combining these will yield relative abundances of E. coli from each source

Methods-Isolation and Preservation n E. coli isolated using serial dilutions and mColiBlue plates. n E. coli colonies from Dr. Simon streaked on EMB plates. n E. coli grown in pure culture in LB for 24h. n Glycerol stocks prepared

PCR-Polymerase Chain Reaction n Amplifies a desired region of DNA, producing a unique genetic fingerprint. Plate PCR MachineFingerprint

Expected Results n PCR yields unique fingerprints for each isolate per source n Establish a library database of E. coli coming from known sources –Geese –Humans –Cattle n Genetic Fingerprints from known sources will be compared to fingerprints from unknown sources

Expected Results n Decreased levels of total E. coli in experimental streams. n Most Important: Decreased relative abundances of E. coli from domestic sources (cattle).

Future Research n Expand database to include wildlife, pets, more farm animals and repeat experiment. n Compare strains from our database with strains from other databases and bacterial libraries.

Aknowledgments n Dombek, P. E. et. al. Use of repetitive DNA sequences and the PCR to differentiate E. coli isolates from human and animal sources. Applied and Env. Micro. June 2000, p n Madigan, M. T. et. al. Brock Biology of Microorganisms. 9th ed Prentice Hall, NJ. n Winfrey, M. R. et. Al. Unraveling DNA: Molecular Biology for the Laboratory Prentice Hall, NJ.