Bacterial Source Tracking by Multiple Antibiotic Resistance Analysis of Escherichia coli

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Bacterial Source Tracking by Multiple Antibiotic
Resistance Analysis of Escherichia coli
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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

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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.)

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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

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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

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Sources of Contamination

• Treatment plants
• Septic systems
• Improper disposal from boats
• Agricultural runoff
• Pets
• Wildlife

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Elevated levels of E. coli

• Indicator of potential pathogens
• Other bacteria
• Viruses
• O157H7
• Beach closings
• Shellfish bed closings

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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

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Total Maximum Daily Load (TMDL)

• TMDLs required by 303(d) of the 1972 Clean
  Water Act (CWA)
• Must submit TMDLs to EPA for approval
• Under court order to complete TMDLs by 2008

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TMDL Calculation

• TMDL Point Sources Nonpoint Sources
  Projected Growth Margin of Safety   
• TMDL - sum of allowed pollutant loads for point
  sources, non-point sources, projected growth and
  a margin of safety  

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Key TMDL Components

• Describe Impairment 
• Identify Cause of Impairment 
• Set Quantitative Goal or Endpoint 
• Identify Pollutant Sources 
• Determine Allowable Load and Allocation 
• Determine Margin of Safety 
• Reasonable Assurance of Implementability   

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2002 TMDL Project Bacterial TMDL

• Salisbury University
• Biology (Mark Frana)
• Environmental Health Science (Elichia Venso)
• Maryland Department of the Environment
• TARSA
• Shellfish waters
• Nontidal waters

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Project Overview

• Isolate E. coli
• from known sources
• from water v
• verify as E. coli
• Determine concentrations of E. coli in water
• MAR analysis of E. coli from known sources
• Predict sources of water E. coli from known
  source profiles
• Submit results as input for TMDL calculation

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MAR Analysis

• Unknown Sources (water)
• Membrane filtration
• Colony enumeration
• Inoculation of unknown sources onto
  TSA/antibiotic plates
• Known sources
• Inoculation of known sources onto
• TSA/antibiotic plates
• Read and record resistance profiles
• Discriminant Analysis

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Membrane Filtration and Colony Enumeration

• Sterile filters, funnels, vacuum filtration
• Filter 3, 10, 30, 100 (2), 200 ml aliquots
• Transfer filters onto mTEC agar plates
• Chromogen (5-bromo-6-chloro-3-indolyl-D-glucuronid
  e)
• Catabolized to glucuronic acid red/magenta
  compound by E. coli
• Incubate at 35 degrees C for 2 h
• Incubate in Whirl-Pak bags in 44.5 degree C
  waterbath for 22-24 h
• Count colonies (20-80 range/plate)

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Transfer to Whirl-Pacs and Incubation in Water
Bath
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Antibiotic Resistance Analysis

• 28 concentrations of 7 antibiotics
• Trypticase Soy Agar (TSA) plates
• Inoculate TSA plates from 96-well plates
• 48-prong replica plater
• Incubate for 24 h at 35 degrees C
• Read plates
• 1isolate grew (resistant)
• 0isolate did not grow (susceptible)

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Antibiotics and Concentrations Used in Analysis

• Cephalothin 15, 25, 35 ug/ml
• Erythromycin 60, 70, 90, 100 ug/ml
• Neomycin 2.5, 5, 10 ug/ml
• Oxytetracycline 2.5, 5, 7.5, 10, 15 ug/ml
• Rifampicin 60, 75, 90 ug/ml
• Streptomycin 2.5, 5, 7.5, 10, 15 ug/ml
• Tetracycline 2.5, 5, 7.5, 10, 15 ug/ml

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Laboratory Activity

• MAR
• 2,425 Profiles
• DNA
• 52.6 Gels
• Statistical
• gt63,000 data entries

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Statistical Analysis

• Discriminant analysis of known source resistance
  profiles
• 2-way (human vs nonhuman)
• 3-way (human vs livestock vs wildlife)
• 5-way (human vs livestock vs wildlife vs domestic
  vs birds)
• Discriminant analysis of unknown sources

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Known Source Isolates

• Total isolates 403
• Black Vulture 10
• Cow 60
• Deer 62
• Dog 30
• Fox 77

• Goose 14
• Horse 26
• Human 64
• Rabbit 10
• Raccoon 40
• Swan 10

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Antibiotics and Concentrations Used in Analysis

• Cephalothin 15, 25, 35 ug/ml
• Erythromycin 60, 70, 90, 100 ug/ml
• Neomycin 2.5, 5, 10 ug/ml
• Oxytetracycline 2.5, 5, 7.5, 10, 15 ug/ml
• Rifampicin 60, 75, 90 ug/ml
• Streptomycin 2.5, 5, 7.5, 10, 15 ug/ml
• Tetracycline 2.5, 5, 7.5, 10, 15 ug/ml

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Students/Technical Assistance

• Current student laboratory assistants
• Natasha Patterson
• Gayle Raynor
• Rebecca Scott
• Gry Wesenberg
• Current laboratory technician
• Lesley Frana

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Acknowledgements

• This project was funded by Maryland Department of
  the Environment.
• We gratefully acknowledge the support of the
  Technical and Regulatory Services Administration
  of MDE and the Richard A. Henson School of
  Science and Technology for its support