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 Bacteroides Host Specific Species - human marker positive in sewage, need field testing and quantification Antibiotic Resistance Testing - looks promising for course assessment, don’t know the resolution

Microbial Source Tracking What we cannot do: Sample 10-20 E. coli and estimate host diversity in environment Human Dog Need a huge genetic library to make accurate assessments. ASK SANDRA!!!!!!!!!!!!!!!!!!!! Too much diversity in the natural environment to estimate relative loading? 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 Are E. coli strains specific to a host Host Factors: Diet, Body Temperature, etc. 2. Are E. coli genetically related according to: a. Host species? b. Geographical location? (due to clonal expansion) c. Selection?

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 Similarity Range: 50-100% ERIC PCR Similarity Range: 58-100% REP PCR fingerprints; lanes 1, 10, 19 and 28 contain a 1 kb molecular weight marker and the remaining lanes are E. coli isolates. Pairwise similarity scores range from 50% to 100%. (B) ERIC PCR fingerprints; lanes 1, 10, 19 and 28 contain a 1 kb molecular weight marker and the remaining lanes are E. coli isolates. Pairwise similarity scores range from 58% to 100%.

Complexity of E.coli Patterns Despite distinct groupings still the chance that a gull can match up to a sewage isolate, cows with gulls, etc.

Universal Bands Universal bands found throughout various hosts make it more difficult to further classify hosts from water samples that share these banding patterns.

Relative Genetic Diversity of E. coli Host Strains Host Group 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 99.5 to 41.0% Pelican 13 99.5 to 31.7% 1 Percent similarity of rep PCR fingerprint patterns calculated by the Pearson coefficient 2 Outgroup

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

Clonal Pattern Indicating Growth in the Environment 1 2 7 8 9 10 4 5 6 3 Lanes 2, 6, 7, 8, 9, demonstrate a clonal pattern, indicating growth in the environment. We should not see identical strains in water, where we would expect that many different animals are contributing to pollution. We found that DNA fingerprinting is actually very useful for finding clonal populations (identical guys that have divided from the same cell); this would indicate replication in the environment. If your indicator organism replicates outside of the host, it diminishes it’s usefulness as an indicator of pollution; now you are just observing the “ecology of E. coli”, not necessarily pollution. 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 Field’s, OSU, PCR technique - Rapidly detects fecal contamination in water samples - Distinguishes source (human & cattle) Found in GI tract of mammals (including humans) – better measure of fecal pollution in recreational waters. Anaerobes comprise the majority of bacteria in the human colon – good indicator of human fecal contamination Technique - DNA methods necessary

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

E. coli Human specific Bacteroides Total Bacteroides

Antibiotic Resistance Arrays Examples of Ampicillin Plates from Different Hosts Gull isolates on LB plate with Ampicillin (20 g/ml) Sewage isolates on LB plate with Ampicillin (20 g/ml) Stormwater isolates on LB plate with Ampicillin (20 g/ml)

Antibiotic Resistance Frequency of Beaches and Host Samples Antibiotic/(g/ml) Sewage (n=1042) Gull (n=475) Other (n=285) Beach 4 (n=961) Beach 2 (n=1044) Beach 5 (n=960) Creek 5 ( n=933) Beach 1 Beach 3 (n=288) Ampicillin (20) 63 9 1 14 12 49 Chlorotetracycline (25) 26 13 2 6 8 21 Kanamycin (50) 11 <0.5 5 4 3 Nalidixic Acid (25) <1 Neomycin (50) 10 Oxytetracycline (25) 29 18 19 15 Penicillin G (90) 30 16 20 22 Streptomycin (12.5) Sulfathiazole (1000) Tetracycline (25) 28

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 Negative- is that the relationship between the viruses and fecal indicator bacteria is not well understood

EPA Method 1602 Single layer method – enumerative method – which is used for water samples Two step enrichment method shows presence and absence – used for testing sewage effluent 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 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