Microbial Source Tracking in Lake Michigan Erika Jensen,
Microbial Source Tracking in Lake Michigan Erika Jensen, M.S. Great Lakes WATER Institute University of Wisconsin-Milwaukee April 14, 2005 Microbial Source Tracking (MST) Tracking the origin of fecal pollution using a variety of methods including: Microbial Genotypic Phenotypic Chemical Microbial Source Tracking What we can do: DNA Fingerprinting good for specific sites, e.g. flock of birds; VERY GOOD for determining growth in the environment Bacteroides Host Specific Species human marker + in sewage, cow marker + in manure Antibiotic Resistance Testing promising for course assessment
Viruses good for discriminating between human and non human contamination Microbial Source Tracking What we cannot do: Sample 10-20 E. coli and estimate host diversity in environment Human Dog Gulls Objective: Identify Sources of E. coli by Determining Host Specific Genetic Profiles Questions: 1. Are E. coli strains specific to a host? 2. Are E. coli genetically related according to: Host species, geographic location, selection? Challenges: 1. Genetic targets not defined 2. Genetic diversity between hosts/geographical range are unknown
3. Population genetics not determined Repetitive Element Anchored PCR REP and ERIC PCR REP REP 5 NNNNNNNNNNN CGNCTTATCNGGCCTAC3 3 CGGNCTCNGCNGCNNNNNNNNNNN5 Human Strain PCR products Gull Strain PCR products DNA Fingerprints of E. coli Isolated from Sewage Treatment Plant Influent REP PCR ERIC PCR Similarity Range: 50-100% Similarity Range: 58-100% Complexity of E.coli Patterns
Universal Bands Relative Genetic Diversity of E. coli Host Strains No. of Isolates Range of diversity1 Sewage 155 99.5 to 29.2% Gull 136 99.5 to 21.8% Cattle 111
99.5 to 41.9% Stormwater 155 99.5 to 41.0% Pelican 13 99.5 to 31.7% Host Group Correct Classification Rates for E. coli % of Isolates in Assigned Group Gulls Sewage Gulls 75 25 Sewage 17
82 Cows 6 17 Out Group 6 0 Cows 1 4 76 0 Out Group 0 0 0 94 Clonal Pattern Indicating Growth in the Environment 1 2
3 4 5 6 7 8 9 10 PFGE Analysis of E. coli Isolates from Beach Water Bacteroides spp. Found in GI tract of all mammals Fecal anaerobe Bacteroides - predominant bacteria in the colon Bacteroides 1000x E. coli Kate Fields, OSU, PCR technique
- Rapidly detects fecal contamination in water samples - Distinguishes source (human & cattle) Bacteroides spp. Pros Cons Detected Everywhere Detected Everywhere Human specific markers Not culturable + for sewage Cattle specific markers + for cattle Obligate anaerobe PCR method required E. coli
Human specific Bacteroides Total Bacteroides Antibiotic Resistance Arrays Examples of Ampicillin Plates from Different Hosts Gull isolates on LB plate with Ampicillin Sewage isolates on LB plate with Ampicillin Stormwater isolates on LB plate with Ampicillin (20 g/ml) (20 g/ml) (20 g/ml) Antibiotic Resistance Frequency of Beaches and Host Samples
16 14 Viruses Viruses are useful for discriminating between human & non human sources of fecal contamination Library independent method (host specific) Human human enterovirus Animal bovine enteroviruses Coliphages viruses that infect E. coli 4 subtypes of F+ RNA I human & animal II & III human IV - animal EPA Method 1602 Source: U.S. Department of the Interior, U.S. Geological Survey - Ohio, 2002 Conclusions: DNA Fingerprinting Pro Good for determining growth in envir. Con Requires extensive genetic library
Bacteroides Pro Detected everywhere Con Detected everywhere ARA Pro Useful for determining human vs. non Con Not able to pinpoint pollution source Viruses Pro Useful for determining human vs. non Con Relationship between indicator bacteria and viruses is not well understood Great Lakes WATER Institute PI, Dr. Sandra McLellan Researchers Annette Daniels Alissa Salmore Caitlin Scopel Michelle Luebke Pat Bower Ola Olapade Graduate Students Marcia Silva Sachie Owaga Heidi Pirkov
Liang Peng Sukpreet Kaur Funding kindly provided by: Milwaukee Metropolitan Sewage District Wisconsin DNR National Institute of Health NOAA Sea Grant SC Johnson Wisconsin Coastal Management Program Students Magnolia Tulod Josh Harris Elissa Lewis Emerson Lee Jennifer Lee Andrew Holland Becky Kirby Hilary Street Ben Weston Morgan Depas Meredith Van Dyke
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