Microbial Source Tracking in Lake Michigan - PowerPoint PPT Presentation

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Microbial Source Tracking in Lake Michigan

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  1. Microbial Source Tracking in Lake Michigan Erika Jensen, M.S. Great Lakes WATER Institute University of Wisconsin-Milwaukee April 14, 2005

  2. Microbial Source Tracking(MST) Tracking the origin of fecal pollution using a variety of methods including: Microbial Genotypic Phenotypic Chemical

  3. 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

  4. Microbial Source Tracking What we cannot do: Sample 10-20 E. coli and estimate host diversity in environment Human Dog Gulls

  5. 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

  6. 5’ NNNNNNNNNNN CGNCTTATCNGGCCTAC3’ 3’ CGGNCTCNGCNGCNNNNNNNNNNN5’ Repetitive Element Anchored PCR REP and ERIC PCR REP REP Human Strain PCR products Gull Strain PCR products

  7. REP PCR Similarity Range: 50-100% ERIC PCR Similarity Range: 58-100% DNA Fingerprints of E. coli Isolated from Sewage Treatment Plant Influent

  8. Complexity of E.coli Patterns

  9. Universal Bands

  10. Relative Genetic Diversity of E. coli Host Strains

  11. Correct Classification Rates for E. coli % of Isolates in Assigned Group GullsSewageCowsOut Group Gulls 75 25 1 0 Sewage 17 82 4 0 Cows 6 17 76 0 Out Group 6 0 0 94

  12. 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

  13. Bacteroides spp. • Found in GI tract of all mammals • Fecal anaerobe • Bacteroides - predominant bacteria in the colon Bacteroides 1000x E. coli • Kate Field’s, OSU, PCR technique • - Rapidly detects fecal contamination in water samples • - Distinguishes source (human & cattle)

  14. Bacteroides spp. Pros Detected Everywhere Human specific markers + for sewage Cattle specific markers + for cattle Cons Detected Everywhere Not culturable Obligate anaerobe PCR method required

  15. E. coli Human specific Bacteroides Total Bacteroides

  16. Antibiotic Resistance Arrays Examples of Ampicillin Plates from Different Hosts Gull isolates on LB plate with Ampicillin (20 g/ml) Sewageisolates on LB plate with Ampicillin (20 g/ml) Stormwaterisolates on LB plate with Ampicillin (20 g/ml)

  17. AntibioticResistance Frequency of Beaches and Host Samples

  18. 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

  19. EPA Method 1602 Source: U.S. Department of the Interior, U.S. Geological Survey - Ohio, 2002

  20. 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. nonCon –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

  21. 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 Students Magnolia Tulod Josh Harris Elissa Lewis Emerson Lee Jennifer Lee Andrew Holland Becky Kirby Hilary Street Ben Weston Morgan Depas Meredith Van Dyke Funding kindly provided by: Milwaukee Metropolitan Sewage District Wisconsin DNR National Institute of Health NOAA Sea Grant SC Johnson Wisconsin Coastal Management Program