Transcript Document

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
Cows
Out Group
Gulls Sewage
75
25
17
82
6
17
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 Field’s, 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
(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
(n=961)
Beach 3
(n=288)
Ampicillin (20)
63
9
1
14
12
12
14
49
12
Chlorotetracycline
(25)
26
13
2
6
8
12
6
21
13
Kanamycin (50)
11
<0.5
0
5
4
5
5
9
3
Nalidixic Acid (25)
13
<1
1
0
<0.5
0
0
<0.5
<1
Neomycin (50)
10
<0.5
1
4
4
5
4
9
2
Oxytetracycline (25)
29
8
1
11
12
18
11
19
15
Penicillin G (90)
30
6
0
15
10
16
20
22
9
Streptomycin (12.5)
22
2
0
16
9
11
16
16
15
Sulfathiazole (1000)
19
2
3
5
4
5
5
9
8
Tetracycline (25)
28
5
2
8
8
14
8
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