Current Technologies for Identification and Measurement of fecal Pollution in Water

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Current Technologies for Identification and
Measurement of fecal Pollution in Water

• C.A. Carson
• University of Missouri

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EXAMPLES OF URGENCY OF FECAL POLLUTION PROBLEM

• Increased numbers of impaired waterways
• Closure of coastal shellfish beds
• Swimming beach closures
• Impact on public health, revenue, commerce

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FECAL BACTERIA

• Intestinal tracts are anaerobic
• Normal flora are anaerobic or facultative
  anaerobes
• Anaerobes are in larger numbers Bacteroides,
  Faecalibacterium, Clostridium
• Facultative anaerobes examples are Escherichia
  and Klebsiella thousand fold (plus) fewer

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COLIFORMS

• Gram (-), lactose fermenting, enteric rods
  normal gut flora
• Long used as indicators of drinking water safety
• Aerobes/facultative anaerobes examples are
  Escherichia (eg. E. coli), Enterobacter,
  Klebsiella
• Coliforms (now E. coli) EPA indicator for fresh
  water

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WATER-BORNE FECAL PATHOGENS (Examples)
Bacteria Viruses Protozoa
Escherichia Coxsackievirus
Crystosporidium Salmonella
Poliovirus Giardia Campylobacter
Hepatitis Entamoeba Yersinia
Adenovirus Toxoplasma
Aeromonas Reovirus Clostridium
Calicivirus Norovirus
Coronavirus
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HUMAN HEALTH RISK

• EPA standards for recreational waters based on
  1984 fecal coliform/bather illness correlation
• Total fecal coliforms NMT 200/100 ml
  (fresh)
• E. coli NMT 126/100 ml (fresh) 8
  ill/1000 bathers
• Enterococci NMT 33/100 ml (marine) 16
  ill/1000
• Correlation of indicator organisms with presence
  of pathogens and health risk (?)
• Question of relative risk of host source of
  pollution human vs cattle vs geese FC counts
  not indicate hosts of origin
• Current studies and surveys by SCCWRP

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EXAMPLES OF POTENTIAL SOURCES OF
FECAL POLLUTION

• Human sewage treatment systems - private,
  collective (aging urban utilities)
• CAFO s
• Pastured animals
• Pet animals
• Migratory birds
• Wild animals

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TARGETING FECAL POLLUTION

• Non-pathogenic bacteria-large numbers of harmless
  bacteria usually present for normal intestinal
  function
• Pathogenic (disease-producing) bacteria-normally
  absent or in low numbers
• Looking for pathogens (the real concern) in water
  samples resembles looking for a needle in a
  haystack
• Finding the haystack is easier
• Fecal coliforms E. coli Enterococcus are
  common/plentiful useful as indicators

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PROKARYOTIC BACTERIAL CELL
From Principles of Microbiology by Atlas. W.C.
Brown Co. 2nd Ed. 1997.
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GENERAL APPROACH TO MST

• Routine water sampling shows unacceptable levels
  of sentinel indicator(s) indicative of fecal
  pollution
• MST test(s) are chosen to provide evidence of
  host source(s)
• An associated intensified (serial) water sampling
  plan is formulated data will be derived-
  followed by conclusions and action

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TIERED CONCERNS

• Human vs. nonhuman sources (public health risk)
• Identification of human and various nonhuman
  sources via E. coli or other common indicator
  organisms

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BASIS OF MST METHODS

• Particular strains of enteric bacteria (eg. E.
  coli) inhabit intestinal tracks of humans,
  animals and birds
• These various host-specific strains can be
  distinguished by their different functions /
  biochemistry (phenotype) or different genetic/DNA
  structure (genotype)
• MST can be performed using either of these
  qualities

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Fecal Source Tracking MethodsCulture,
Phenotypic, Genotypic, Reference Library,
Non-Library

• Antibiotic resistance profiles specificity
• Carbon source utilization specificity
• Ribotyping based on rRNA gene probe specific
  requires 8 days
• rep-PCR position of repeat elements specific
  somewhat more rapid
• PFGE highly specific (excessive)
• All above require culture and libraries

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DATABASE / REFERENCE LIBRARY

• Representative of environmental bacteria
• Subject to temporal and geographic variation
• Watershed specific
• Library size-(?) 200 isolates / host class
• Expensive - cost / fingerprint pattern

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METHODS (Cont.)

• Host-specific PCR Bacteroides/Prevotella
  species culture-and library independent
• F phage typing accurate may not be present
• Enterovirus/adenovirus accurate may not be
  present
• Pathogen ID eg. Pathogenic E. coli, Salmonella,
  hepatitis virus pathogens scarce in nature
• Chemical methods caffeine, laundry detergents,
  etc. inexpensive may not be present

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COMPARISON OF SELECTED MST METHODS
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BACTERIAL FINGERPRINTING / rep-PCR(First Example)

• Multiple copies of target repeat elements per E.
  coli genome
• Repeat numbers and locations vary per bacterial
  strain
• Primers amplify segments of DNA between
  repeats/signature of strain
• E. coli strains generally associated with gut
  flora of particular host species (human, nonhuman)

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rep PCR TEST BASED ON LOCATION OF TARGET GENE
IN E. coli
E. coli
rep genes
DNA chromosome
1
1
2
1
2
3
2
3
3
Human
Cow
Dog
PCR Multiply 1,2,3
PCR Multiply 1,2,3
PCR Multiply 1,2,3
Different DNA Fingerprint patterns
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MATERIALS AND METHODS
rep PCR
PCR BOX A1R primer
Lyse cells
Select/Grow pure fecal E. coli isolates

• Bionumerics software
• Similarity coefficients of patterns calculated by
  dice method with fuzzy logic option.
• Discriminant analysis via cross validation of
  database

Pattern analysis by computer program
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FECAL E. coli ISOLATES FROM TWO INDIVIDUAL HUMAN
SAMPLES
(Bp)
(Bp)
10000
5000
3000
2000
1500
1000
800
600
400
200
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E. coli ISOLATES FROM TWO CATTLE FECAL SAMPLES
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FECAL E. coli ISOLATES FROM ONE LITTLE SAC RIVER
WATER SAMPLE
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PATTERN ANALYSIS

• Fecal E. coli isolates compiled in known-host
  database/library (human and non-human hosts)
• Environmental (water) E. coli isolates
  host-associated by comparison with database
  isolates maximum similarity with particular
  library pattern
• Arbitrary cutoff for unknown patterns at
  least 80 similarity with library pattern A-C
  quality factor

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Human E. coli Isolates
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Cattle E. coli Isolates
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Cattle and Human E. coli Isolates
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SECOND EXAMPLE METHODHost Specific/Gene Specific
Targeting Non library-based Procedure

• Bacteroides are most numerous human intestinal
  bacteria
• Different hosts have different species and strains

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MICROBIAL GENE - SPECIFIC/HOST SPECIFIC PCR
Target DNA
Microbe
Electrophoresis
PCR Multiply Target
Specific product size
___
___
Fecal pollution
No pollution
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GENE-SPECIFIC PCR INDICATIVE OF HOST SOURCE OF
FECAL POLLUTION
Unknown based on metagenomics
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FIELD APPLICATION OF MICROBIAL SOURCE TRACKING
METHODS UPPER SHOAL CREEK WATERSHED

• 3 county area in extreme SW part of MO Newton,
  McDonald, Barry Counties
• One of most agriculturally productive areas in MO
• 91,000 acres in the watershed 90 is pasture
  land grazed by over 300,000 head of cattle and
  fertilized by spreading poultry litter
• 50-80 million poultry produced here yearly
• 13 miles of Shoal Creek are designated as
  impaired due to high fecal coliform (FC) levels

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SHOAL CREEK MST DATA
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Seasonal Fecal E.Coli Sources (Average
Contribution)
SUMMER
WINTER
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Fecal E. Coli Sources and Flow
SUMMER STORM FLOWS
SUMMER BASE FLOWS
Wildlife
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STUDY CONCLUSIONS

• Cattle (particularly in streams) contribute
  substantially to water pollution
• Waste from pastured animals and spread poultry
  litter also contribute via runoff to streams
• There are multiple host sources of feces that
  combine for the total contribution
• Studies usually reveal multiple host sources,
  rather than a single host source
• Results from routine water quality monitoring,
  fecal source tracking and visual inspection can
  all combine to analyze problems and suggest
  solutions

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BOTTOM LINE

• MST methods are powerful tools to resolve
  questions of host sources of fecal pollution and
  associated high bacterial counts in water
• Current consensus is to use a combination of
  methods with different targets
• Results must be interpreted carefully, combined
  with local observations and based on multiple
  samples collected over a period of time

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ACKNOWLEDGMENT OF TEAM
MEMBERS

• Food and Agricultural Policy Research Institute
• U.S. Environmental Protection Agency
• U.S. Geological Survey
• College of Agriculture and Natural Resources
• Department of Agriculture Engineering
• Department of Agriculture Economics
• College of Veterinary Medicine
• U.S. Department of Agriculture
• Missouri Department of Natural Resources
• University of Missouri Extension Services