Fecal Coliform Source Tracking using Antibiotic Resistance Patterns and DFA

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Fecal Coliform Source Tracking using Antibiotic
Resistance Patterns and DFA

• Cheryl Bettis
• Clayton College and State University
• Summer 2004

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Goals

• Track the sources of fecal coliforms using water
  samples from Jesters Creek
• Compare the water isolates Antibiotic Resistance
  Patterns (ARP) to a database of known values to
  help classify them as either
• Sewage Wild
• Horse Dog

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Location of Sampling

• Jesters Creek, Morrow Georgia
• Part of Reynolds Nature Preserve
• 3 Sample Dates
• 2nd Sample needed to be re-done
• Heavy Rainfall
• Too much contamination to yield isolated (pure)
  colonies

Clayton County
http//a-s.clayton.edu/Daughtry/jesterscreek.htm
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Possible Culprits
www.google.com
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Introduction

• Fecal contamination of water
• EPA 5 cells/ml (500 CFU/100ml) contaminated
• 2nd sample
• Too numerous to count on 1ml samples
• 3rd sample
• Average count was 33 cells/ml (3300 cells/100ml)
• Majority of bacteria were fecal coliforms
• Non-Point Source Contamination

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Materials and Methods

• Sample Collection
• Filtration
• Incubation on mFC (with rosolic acid) media at 44
  º C water bath

Filtration Apparatus
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Materials and Methods

• Selected blue isolated colonies
• Incubated in microwells containing EC MUG media
• MUG Fluorescence
• Frozen Stock Cultures made after adding glycerol
  (-80 º C)

http//www.millipore.com/catalogue.nsf
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Materials and Methods

• Inoculation and Incubation working culture
• Dilution
• Stamping plates using replicator
• MH agar antibiotic plates
• Lowest antibiotic concentration to highest

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Antibiotic Selection

• Ampicillin
• Cephalothin
• Chlortetracycline
• Doxycycline
• Erythromycin
• Oxytetracycline
• Penicillin G
• Polymixin B
• Rifampin
• Streptomycin
• Trimethoprim
• Total 11

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Plate Growth
Originally had 291 isolates each subjected to
11 antibiotics at 3 different concentrations 33
characteristics for each isolate
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Score Data

• Database
• 1 growth () resistant
• 0 no growth (-) susceptible
• Determined against the control plate
• No growth on control deleted out of database
• Each individual colony is scored as or - not
  plate as a whole

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Database Snapshot
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Discriminant Function Analysis

• SPSS Software
• Compared my unknown creek water isolates to a
  database of known samples
• Gives us an idea of how similar the ARPs of my
  isolates are to the ones in the database
• Attempts to put each isolate in a class
• Sewage Wild
• Horse Dog

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Results (n)

• 40.6 Sewage (97)
• 10.9 Wild (26)
• 29.7 Horse (71)
• 18.8 Dog (45)
• N239

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The Mysterious Purple Colony

• Classified as sewage
• Possibly Human

• Not sure why the colony produced a purple pigment
  on its own and remained that way throughout the
  course of plating
• New strain of fecal coliforms?

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Problems

• Sample collection and rainfall
• 2nd sampling Heavy contamination
• Seal tape used to cover plates
• Better foil lids that Maggie invented

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Conclusions

• Need a better way to help distinguish classes
• Sewage and Wild
• Real human samples
• More known isolates from other sources (cat,
  squirrel, bats etc...)
• More testing is needed to look at fecal
  contamination over a longer time period or
  compare different weather patterns

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Discussion

• More work needed in the area to help control
  bacterial numbers
• Rock burms and silt-fences have already been
  placed by the creek bed
• Still loaded with bacteria after rainfall
• Non-point source
• Hard to control bacterial numbers
• Horse farm nearby linked to a significant amount
  of contamination after rainfall

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Jesters Creek
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Special Thanks

• Dr. Michelle Furlong
• Maggie Mills
• Dave Stuchkus
• Dr. Valerie Harwood (protocol)
• Dr. Steve Burnett (DFA)