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Bacterial Source Tracking by DNA Fingerprinting of Escherichia coli

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... Resistance (MAR) (Hagedorn - Va. Tech/ Kator VIMS/ Wiggins James Madison) Coliphage (Geoff Scott NOAA) Carbon Source Profiles (Hagedorn Va. Tech) ... – PowerPoint PPT presentation

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Title: Bacterial Source Tracking by DNA Fingerprinting of Escherichia coli


1
Bacterial Source Tracking by DNAFingerprinting
of Escherichia coli
2
Bacterial Source Tracking (BST)
  • Determination of the sources of fecal bacteria
    from an environmental water sample (1995)
  • Several different techniques are currently being
    used for BST

3
BST Technology
  • Genotypic
  • Pulsed-Field Gel Electrophoresis
    (Simmons/Herbein/Hagedorn Va. Tech.)
  • Ribotyping (Samadpour U. of Wash.)
  • Randomly Amplified Polymorphic DNA (RAPD)/PCR
    (Sadowsky U. of Minn.)

4
BST Technology
  • Biochemical (Phenotypic)
  • Multiple Antibiotic Resistance (MAR) (Hagedorn -
    Va. Tech/ Kator VIMS/ Wiggins James Madison)
  • Coliphage (Geoff Scott NOAA)
  • Carbon Source Profiles (Hagedorn Va. Tech)
  • Sterols or Fatty Acid Analysis
  • Chemical (Non-Bacterial)
  • Optical Brighteners
  • Caffeine Detection

5
Fecal Indicators (Microbial)
  • Fecal Coliforms
  • Facultative, Gram negative, non-spore forming,
    rod shaped, ferment lactose producing gas within
    48 hours. Sources animals, soil (44.50 C is
    selective)
  • Fecal Streptococci (Enterococci)
  • Facultative, Gram positive, tolerate high salt
    and are used as indicators particularly in
    brackish or marine waters. Sources warm-blooded
    animals
  • Escherichia coli
  • A sub-set of the fecal coliform. Sources
    mammals and birds

6
Elevated levels of E. coli
  • Indicator of potential pathogens
  • Other bacteria
  • Viruses
  • O157H7
  • Beach closings
  • Shellfish bed closings

7
Fecal Coliform Standards
  • Recreational Water
  • lt 200 MPN/100 ml
  • Shellfish Beds
  • lt 14 MPN/100 ml
  • Public Drinking Water Supply
  • lt 20 MPN/100 ml
  • Treated Drinking Water
  • lt 1 positive/month

8
Sources of Contamination
  • Treatment plants
  • Septic systems
  • Improper disposal from boats
  • Agricultural runoff
  • Pets
  • Wildlife

9
Project Overview
  • Study site selection
  • Monitor study sites for elevated levels of E.
    coli
  • Isolate E. coli from known sources
  • Isolate E. coli from water
  • Verify as E. coli
  • DNA analysis (Pulsed-Field Gel Electrophoresis)
  • Match water sources to known sources
  • Possible remediation plan

10
Determining Fate and Transport Sample E. coli
levels monthly for one year Use GPS and GIS to
map and analyze levels Determine principal
inputs of contamination
11
Scat Collection
  • Locations within drainage area
  • Identifiable samples


12
Water Samples
  • Select rivulets or Drainage areas
  • Monitor E. coli levels by MPN
  • Monitor pH, dissolved oxygen, temp., salinity,
    conductivity
  • Focus on MPN hot spots for collecting isolates
    for DNA analysis

13
Isolation of E. coli
  • A-1 Broth with MUG (4-methylumbelliferyl-B-D-
    Glucuronide)
  • E. coli-specific enzyme glucuronidase
  • Fluorogenic metabolite
  • Presumptive E. coli
  • Turbidity
  • Gas
  • Fluorescence

14
Isolation of E. coli
  • API 20-E Strips (Biochemical tests for gram
    negative bacteria)
  • E. coli verified

15
DNA Sample Preparation Enzyme Digestions
  • Plugs restricted with Not I
  • Restriction sites recognized
  • DNA cut
  • Bands generated

5..GCGGCC GC..3 3..CG CCGGCG..5
16
Gel Preparation
  • Plugs glued to comb
  • Gel formed around DNA plugs

17
Pulsed-Field Gel Electrophoresis
  • Fragments separated by electric current
  • Multi-directional current over 20 hours
  • Downward net flow

18
Electrophoresis Chamber
19
Gel Analysis
  • Gels stained with Ethidium Bromide
  • Gels photographed under UV light
  • Banding patterns analyzed

20
Bacterial Source Identification
  • E.coli banding patterns from scat added to
    library
  • E. coli banding patterns from water matched to
    library

21
DNA Match Water Sample to Deer Scat Sample
Shiles Creek water sample Shiles
Creek deer scat sample
22
DNA Match Water Sample to Muskrat Scat Sample
Shiles Creek water sample Shiles Creek
muskrat scat sample
23
Conclusions
  • Molecular Biology advances provide a means to
    identify nonpoint sources of bacterial
    contamination.
  • Lab results identified sources consistent with
    rural and urban settings.
  • This technology adds to our knowledge base for
    better decision-making regarding potential
    remediation.

24
Students/Technical Help
  • The Described Project
  • Jon Neville, Amanda Zych, Julie Wood
  • Currently
  • Natasha Patterson, Gayle Raynor, Rebecca Scott,
    Gry Wesenberg
  • Current Technical Help
  • Lesley Frana
  • -

25
Acknowledgements
  • Maryland Department of Natural Resources
  • This project was funded in part by the U.S.
    Environmental Protection Agency Section 319
    Nonpoint Source Program through the Maryland
    Department of Natural Resources.
  • Maryland Department of the Environment
  • Technical and Regulatory Services Administration
  • R.A. Henson School of Science and Technology
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