Use%20of%20New%20Tools%20for%20Managing%20Risks%20Microbial%20Source%20Tracking

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Use of New Tools for Managing Risks Microbial
Source Tracking
Joan B. Rose (rosejo_at_msu.edu) Department of
Fisheries and Wildlife
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Microbial Source Tracking

• Tools are now available to determine the
  molecular fingerprint of the fecal pollution.
• Health risks
• Remediation
• Prioritization
• Responsibility

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Microbial Source Tracking

• Indicator bacteria E.coli dont provide source
  of pollution
• MST methods have developed using genetic
  approaches to identify source.
• Can be library dependent or library
  independent/host specific

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Why host specific ?

• Host specific method is library independent
• For Library-dependent, DNA libraries are built
  using isolates from animals human sources in
  the area
• Library dependent methods use these libraries to
  match to the unknown environmental sample
• Host specific method requires no library, the
  marker is specific to the host, and has fewer
  false(-) and false() the library methods

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Muck in Saginaw Bay

• Algae, detritus, muck - not a new development
• Documented problem in Saginaw Bay since the
  1960s
• Problem in other areas of the Great Lakes

Bay City State Park in 1929
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The Problem

• Perceived increase in duration and spatial
  distribution compared to past years
• Degradation of the aesthetic value of beaches
• Public and landowner concerns
• Detection of fecal indicators
• Potential human health implications

Photo by Dr. Juli Dyble NOAA
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The water/ muck interface notice lots of
aquatic vegetation both in water and dried on
shore Photos by Dr. Juli Dyble NOAA
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Looking out toward the water very turbid Photos
by Dr. Juli Dyble NOAA
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Digging in the muck at the water line reveals
lots of vegetation holding it all together Photos
by Dr. Juli Dyble NOAA
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Moving a little further away from the waterline
you sink right in! Photos by Dr. Juli Dyble NOAA
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Sinking even deeper in muck Photos by Dr. Juli
Dyble NOAA
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Closer to the water, if you dig into the muck,
you find more vegetation
Further up, if you wipe off the top layer, it is
green beneath A muck mat? Photos by Dr. Juli
Dyble NOAA
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Most abundant Filamentous diatom
Second most abundant A tightly coiled Spirogyra?
2 representative pennate diatoms
Another common diatom
Spirogyra saw some, but not abundant
Cyanobacteria Anabaena only saw a couple
Cyanobacteria Microcystis
Cyanobacteria Oscillatoria or Lyngbya?
All pics are 40X except Spirogyra (20X), bright
field Photos by Dr. Juli Dyble NOAA
Cladophora?
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What are Fecal Indicators?

• A microorganism that
• is present when feces and presumably pathogens
  are present (health risk)
• occurs in high concentrations in polluted waters
  and has a relationship to the degree of pollution
• is easy to cultivate and identify

Adapted from Bonde 1966
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E. coli

• Part of the fecal indicator bacteria which are
  generally harmless themselves
• Found in high numbers in the gut of humans and
  other warm blooded animals, including birds
• Excreted daily in the feces of people and mammals
• Used to indicate the potential presence of
  pathogens, microorganisms that come from the gut
  and cause diseases such as diarrhea

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Where are fecal indicators found?

• Sewage (even treated)
• Septic tank effluent (the liquid from the septic
  tank)
• Septage (solids from the septic tank)
• Manure and animal waste lagoons
• Bird and other animal droppings
• Rainfall can wash the fecal wastes and associated
  indicator bacteria into nearby water bodies
• Almost all surface waters have some background of
  fecal indicators due to wildlife, with the levels
  increasing the closer you get to sewage/feces

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Water quality Standards/Criteria For
Recreational Water
Indicator Geometric Means Michigan EPA

E.Coli CFU lt130/100ml lt126cfu/100ml
Enterococci CFU None lt33cfu/100ml
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Our Approach host-specific markers

• Use multiple source tracking markers
• Host specific genetic marker (esp)
• Differentiate Human/non human
  pollution
• Traditional MF method and PCR used to detect the
  gene. A culture dependent library independent
  method
• Bacteriodes use PCR

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What is PCR?

• Enzymatic reaction that makes many copies of DNA
  from single molecule
• 2n copies of DNA from single molecule where n
  No. of cycles
• So, 35 cycles of PCR would yield 235 copies of DNA

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Polymerase Chain Reaction (PCR)

• Developed in 1985 by Kary Mullis
• Dr. Mullis received the Nobel Prize in Chemistry
  in 1993
• PCR is considered as one of the most important
  discoveries in molecular biology

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PCR Machine
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Enterococci

• EPA recommended
• Esp (Enterococcus surface protein) as human
  marker
• Only found in human feces
• Cause of urinary tract and gastrointestinal
  infection
• 
  

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Enterococcus membrane filtration (MF)
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Human Pollution Marker Validation
Source esp marker
Sewage 67/67
Septic tank 14/16
Poultry 0/8
Swine 0/12
Cattle 0/35
Canada Geese 0/12
Seagull 0/28
Pelican 0/7
Wild Birds 0/10
Scott et al., 2005. Env. Sci. Technol., 39
283-287
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BACTERIODES

• Bacteroides are specialists in the intestinal
  environment of animals.
• Are the most numerous types of bacteria
  approximately 30 of what is excreted.
• These bacteria are anaerobes and can not grow in
  the presence of oxygen.
• Specific genetic targets have been identified in
  cattle, humans and swine.
• 
  

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Host Specific Markers are Key to Source Tracking
Future

• Bacteroides (genetic approaches PCR)
• 4/4 sewage 4/4 human 4/5 cow (lowest
  concentration missed) 4/4 dogs however no marker
  for Birds Missed 2 samples with dog and 2 with
  cow that were mixed.
• E.coli Toxin genes able to detect sewage (4/4).
• Enteroviruses and Adenoviruses found in 3 of 4
  sewage samples.
• Enterococci ESP marker found in 100 of 109 human
  sewage water samples and zero of 80 animal
  samples.

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Prevalence of Human and Bovine Markers
HUMAN
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Prevalence Of esp Human marker
Silver Beach At Lake Michigan
At Lake Michigan
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Prevalence Of Human And Bovine Markers
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Conclusions

• New tools allow for the detection of multiple
  sources of pollution using a tool box approach.
• This will help address the types of pathogens of
  concern.
• This will also help to determine type of future
  controls on land that will protect water quality.
• More samples need to be collected and assayed in
  the future.

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• THANK YOU