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
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

4. Microbial Source Tracking
What we cannot do:
Sample 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

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
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?

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

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

8. 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.

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

10. 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

11. Correct Classification Rates for E. coli
% of Isolates in Assigned Group
Gulls Sewage Cows Out Group
Gulls
Sewage
Cows
Out Group

12. 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

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)
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

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)
Sewage isolates on LB plate with Ampicillin
(20 g/ml)
Stormwater isolates on LB plate with Ampicillin
(20 g/ml)

17. 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

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

19. 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

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. 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

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