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

Thesis Question: n Can we identify sources of E. coli in Conesus Lake sub-watersheds? –Isolate E. coli from controls and stream samples. –Obtain PCR amplified fragments (genetic fingerprints). –Compare fingerprints from controls with fingerprints from samples to identify each sample isolate with a particular control group.

Thesis Question: n Is it possible to track reductions in E.coli coming from different source groups (cattle)? –Combine qualitative identification with quantification for % relative abundance of sources in stream samples seasonally and annually. –Done on a per sample basis.

Conesus Lake n Three major E. coli sources: –Wildlife n Geese and ducks at Graywood outlet to lake, on Maxwell’s farm, Long Point Park. –Humans n Septic leaks-reported twice at Graywood –4026 W. Lake Road septic fixed on 6/24/03 –Soil & Water & Severn Trent Services performing a repair on a sewer line, bypassing the manhole. V. Puffer said water running into stream was potable. 11/5/02 –Domestic/Agriculture n Cattle are only domestic animal with significant numbers on farms associated with experimental watersheds.

Methods-Isolation and Preservation of Controls n Fecal material is collected in sterile tubes. n ~1 gram of fecal material is combined with 10 ml of buffered water. n E. coli isolated using serial dilutions and membrane filtration onto plates containing mColiBlue 24 broth. n Check membrane technique with Eosin Methylene Blue agar. n E. coli grown in pure culture in Luria Bertani broth for 24hrs. n Centrifuge 1.5 ml of cells in LB and wash pellet with 1 ml of 1M NaCl. n Use 1  l of NaCl wash as template in PCR reaction.

Methods-PCR and Electrophoresis n Polymerase Chain Reaction (PCR) amplifies a given region of DNA using a pair of oligonucleotide primers and taq DNA polymerase. n Gel Electrophoresis separates DNA fragments amplified in PCR by applying a charge to DNA loaded into an agarose gel.

Methods-Experimental Design 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

Methods-Experimental Design n 100 isolates from each control group will be used to identify the most common fingerprints in each control group. –Holstein cattle –Canada geese –Humans n E. coli isolates will be analyzed from each stream in each season. n PCR reactions are run on a gel for ~18hrs. at 70V and a banding pattern is established.

Methods-Experimental Design n PCR amplified fragments will be run on a gel and digital images will be taken using a Kodak ID Icon and Capture IS 140 computer software. n Fingerprints from controls will be compared with those from sample isolates using Capture IS 140 software. –Jaccard Similarity Coefficient –Dendogram Construction –MANOVA

Results n A total of 200 E. coli colonies have been isolated from Holstein cattle and Canada geese (100 from each organism). –Preserved in glycerol n ~250 E. coli colonies have been isolated from stream samples. –Preserved in glycerol n 100 E. coli colonies from humans have yet to be isolated... (any volunteers ?!)

Results n PCR and electrophoresis are now producing repeatable fingerprint patterns from cattle and Canada goose E. coli isolates. n Digital imaging methods must be worked out further to produce comparable fingerprint patterns from different gels. n Preliminary work with digital imaging instrument and software seems promising.

Conclusion n Establish a library of genetic fingerprints from major source groups. n Experiment is expected to show changing patterns of E. coli sources seasonally and annually. n Looking for reductions in cattle E. coli on a % relative frequency basis.