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