microbial source tracking in lake michigan erika jensen, m.s. great lakes water institute university...
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Microbial Source Tracking in Lake Michigan
Erika Jensen, M.S.
Great Lakes WATER InstituteUniversity 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
What we cannot do:
Sample 10-20 E. coli and estimate host diversity in environment
Microbial Source Tracking
Gulls
Human
Dog
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 defined2. Genetic diversity between hosts/geographical range
are unknown 3. Population genetics not determined
Repetitive Element Anchored PCR REP and ERIC PCR
5’ NNNNNNNNNNN CGNCTTATCNGGCCTAC3’3’ CGGNCTCNGCNGCNNNNNNNNNNN5’
REP REP
Human Strain
Gull Strain
PCR products
PCR products
DNA Fingerprints of E. coli Isolated from Sewage Treatment Plant Influent
ERIC PCRSimilarity Range: 58-100%
REP PCRSimilarity Range: 50-100%
Complexity of E.coli Patterns
Universal Bands
Relative Genetic Diversity of E. coli Host Strains
Host Group No. of IsolatesRange 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%
Correct Classification Rates for E. coli
% of Isolates in Assigned Group
Gulls Sewage Cows Out GroupGulls 75 25 1 0Sewage 17 82 4 0Cows 6 17 76 0Out Group 6 0 0 94
PFGE Analysis of E. coli Isolates from Beach Water
Clonal Pattern Indicating Growth in the Environment
1 2 7 8 9 104 5 63
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
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 specificBacteroides
Total Bacteroides
Gull isolates on LB Gull isolates on LB plate with Ampicillinplate with Ampicillin
(20 (20 gg/ml)/ml)
SewageSewage isolates on LB isolates on LB plate with Ampicillinplate with Ampicillin
(20 (20 gg/ml/ml))
StormwaterStormwater isolates on isolates on LB plate with AmpicillinLB plate with Ampicillin
(20 (20 gg/ml/ml))
Examples of Ampicillin Plates from Different HostsExamples of Ampicillin Plates from Different Hosts
Antibiotic Resistance ArraysAntibiotic Resistance Arrays
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 – humanIV - animal
Source: U.S. Department of the Interior, U.S. Geological Survey - Ohio, 2002
EPA Method 1602
DNA FingerprintingPro – 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
VirusesPro – Useful for determining human vs. nonCon – Relationship between indicator bacteria
and viruses is not well understood
Conclusions:
ResearchersAnnette DanielsAlissa SalmoreCaitlin ScopelMichelle LuebkePat BowerOla Olapade
StudentsMagnolia TulodJosh HarrisElissa LewisEmerson LeeJennifer LeeAndrew HollandBecky KirbyHilary StreetBen WestonMorgan DepasMeredith Van Dyke
Graduate StudentsMarcia SilvaSachie OwagaHeidi PirkovLiang PengSukpreet Kaur
Great Lakes WATER InstitutePI, Dr. Sandra McLellan
Funding kindly provided by:Milwaukee Metropolitan Sewage DistrictWisconsin DNRNational Institute of HealthNOAA Sea GrantSC JohnsonWisconsin Coastal Management Program