MICROBIOLOGY RESEARCH AT SOUTHERN CALIFORNIA COASTAL WATER RESEARCH PROJECT SCCWRP

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MICROBIOLOGY RESEARCH ATSOUTHERN CALIFORNIA
COASTAL WATER RESEARCH PROJECT(SCCWRP)
Stephen B. WeisbergExecutive Director
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WHAT IS SCCWRP?

• Joint Powers Agency founded in 1969
• Initiated to address regional monitoring and
  research needs
• Cumulative regional assessments
• Methods development
• Data integration
• Member organizations include city, county, state,
  and federal agencies
• Unique combination of regulators and regulated

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MEMBER ORGANIZATIONS

• City of San Diego
• City of Los Angeles
• Ventura County Flood Control District
• Los Angeles County Department of Public Works
• Los Angeles County Sanitation Districts
• Orange County Sanitation District
• San Diego Regional Water Quality Board
• Santa Ana Regional Water Quality Board
• Los Angeles Regional Water Quality Board
• State Water Resources Control Board
• U.S Environmental Protection Agency

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SOME DIFFICULTIES WITH BACTERIAL MONITORING
SYSTEMS

• Laboratory processing is slow
• You should not have been swimming yesterday
• Relationship to health risk is not well
  established
• Santa Monica Bay Epidemiology study is the only
  one to evaluate urban runoff
• Poor relationship between bacteria and pathogens
• Hard to fix if we dont know the source
• Difficult to define appropriate clean-up levels
• Background is not zero

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SOME DIFFICULTIES WITH BACTERIAL MONITORING
SYSTEMS

• Laboratory processing is slow
• You should not have been swimming yesterday
• Relationship to health risk is not well
  established
• Santa Monica Bay Epidemiology study is the only
  one to evaluate urban runoff
• Poor relationship between bacteria and pathogens
• Hard to fix problems if we dont know the source
• Difficult to define appropriate clean-up levels
• Background is not zero

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SCCWRPs MICROBIOLOGICAL RESEARCH

• Rapid microbiological measurement methods
  development
• Mission Bay epidemiology study
• Microbiological source tracking evaluation
• Natural sources loading

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NEED FOR RAPID MEASUREMENT METHODS

• Present methods are slow
• Culture-based
• Take 24-96 hours
• Slow speed compromises the warning system
• Exposure occurs during sample processing
• Most events last less than 24 hours
• Slow speed also compromises upstream tracking
• Need a Geiger counter

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Surface Proteins
Small molecules
ATP
Nucleic Acids
Molecules released into medium
Things to Measure in a Bacterium
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CONCENTRATION TECHNIQUES IMMUNOMAGNETIC
SEPARATION (IMS)
Magnetic Beads
Antibody
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Magnetic Bead
Y Antibody
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MIX BEADS WITH ENVIRONMENTAL SAMPLE
Y
silica
Clay
Algae
Bacterium
Clay
Clay
Algae
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EXPOSE MIXTURE TO MAGNET SYSTEM FOR SEPARATION
(E.G. MILTENYI, IMMUNICON OR DYNAL)
Add Buffer Dye
Place tube in field
Collect Bacteria
30 min.
Remove Non-magnetic Debris
Bacteria move to tube walls
S
N
N
S
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FLOW CYTOMETRY OPTICS
Labeled bacterium
Scatter Detector
Scatter signal
Fluorescence Detector
Laser excitation
Fluorescence signal
High performance filters
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Near Real Time Microbial Analysis
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APPROACH

• Adapt technology from other industries
• Drinking water
• Hospital testing
• Food service
• Counter bioterrorism
• Partner with specialists in these areas
• Held a workshop in Monterey in May
• Shared developments to date
• Defined method evaluation protocols
• Will conduct blind testing this summer

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MISSION BAY EPIDEMIOLOGY STUDY

• Is there a health risk of swimming in Mission
  Bay?
• Comparison of swimmers and non-swimmers
• Can we relate to bacterial indicator
  concentration?
• Comparison among swimmers at different times and
  locations
• Can we relate health risk to non-traditional
  microbiological indicators?
• Virus
• Phage
• Bacteroides

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SAMPLING DESIGN

• Six beaches
• 32 sampling days
• Between 2 and 5 sampling sites per beach
• Dependent on beach length and swimmer density
• Sampled hourly from 1230 to 330
• Total and fecal coliforms by MF
• Enterococcus by Idexx
• Single beach composite at 1230
• Phage, virus, Bacteroides
• Total and fecal by Idexx
• Enterococcus by MF

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ENROLLMENT ACTUAL VS. GOAL
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DEMOGRAPHICS OF STUDY POPULATIONRACE AND GENDER
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PRIOR LARGE RECREATIONAL WATER STUDIES
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SCCWRPs MICROBIOLOGICAL RESEARCH

• Rapid microbiological measurement methods
  development
• Mission Bay epidemiology study
• Microbiological source tracking evaluation
• Natural sources loading

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MICROBIOLOGICAL SOURCE TRACKING

• MST tools potentially allow managers to
  discriminate between human and non-human sources
• Some even discriminate among non-human sources
• There are more than a dozen proposed MST methods
• Most have had limited testing
• No marine testing
• Which methods are most cost-effective?
• How reliable are the results?

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STUDY DESIGN

• Characterize five sources
• Human (direct samples)
• Human (sewage influent)
• Dog
• Cow
• Seagull
• Place these sources in combination into blind
  water matrix samples
• Three matrices
• Freshwater
• Saltwater
• Freshwater with humic acid addition

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EXAMPLE TEST MATRIX
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STUDY CONCLUSIONS

• No method predicted source material perfectly
• Genetic techniques did best
• Identified dominant sources in about 75 of
  samples
• Phenotypic techniques produced many false
  positives
• Host-specific PCR showed the most promise
• Not yet quantitative
• Only developed for two sources so far
• Viral measures accurately identified sewage, but
  did not identify individual human sources

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NATURAL LOADING PROJECT

• Determine properties of waterbodies in
  undeveloped watersheds
• Measure a wide array of constituents
• Bacteria
• Metals
• Nutrients
• Provide perspective for various activities
• 303D listing
• Model calibration
• TMDL development

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SAMPLING DESIGN

• 12 sampling sites
• Stratified based on geology and land cover
• Dry season sampling
• Twice per year (fall and spring)
• Two years (2004-2005)
• Wet season sampling
• Two three storms at each site
• Multiple samples over duration of the storm
• Sampled burned vs. unburned catchments twice this
  winter

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Questions or Comments?
Steve Weisberg Southern Ca. Coastal Water
Research Project 714-372-9203 stevew_at_sccwrp.org
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SOME TOUGH ISSUES

• Measuring low concentrations
• Many detectors are based on 1 ml sample
• State standard is 104 bacteria per 100 ml
• Dead or alive?
• Do our standards apply when measuring fragments?
• Intercalibration tests are necessary
• How do we use real-time information in a patchy
  environment?
• Rapid processing allows adaptive sampling
• Lesser processing cost allows more samples
• Capital equipment and training costs
• Many technologies require up-front investment
• Some require specialized training
• Per sample cost is typically lower

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REPORTED ILLNESSES
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COMPARISON TO THRESHOLDS
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POTENTIAL TOOLSNATURAL SOURCES IDENTIFICATION

• Microbial source tracking bacteria
• Iron-normalization metals
• Chiral chemistry organics
• Compound specific istotope analysis - organics

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IRON NORMALIZATION
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CHIRAL ENVIRONMENTAL CHEMISTRYENANTIOMERS
MIRROR IMAGES
The Ultimate in Pollutant Speciation
R-(-) o,p-DDT
S-() o,p-DDT
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COMPOUND SPECIFIC ISOTOPE ANALYSIS

• Elements occur in various isotope ratios (e.g.
  12C/13C)
• IRs are affected by many natural and
  anthropogenic processes
• Can be extremely effective for source
  identification

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OUR APPROACH

• Held a workshop jointly with USEPA to develop
  method evaluation protocols
• Defined four phases of testing
• Sequentially increasing level of complexity
• Conducted an evaluation test last year
• 22 research labs
• Tested 12 different MST methods

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EXAMPLE MST METHODS

• Phenotypic
• Antibiotic resistance profiling
• Carbon source utilization
• Genotypic
• Ribotyping
• Pulsed field gel electrophoresis
• Polymerase chain reaction
• Library-independent genotypic
• Terminal restriction fragment length polymorphism
• Host-specific PCR
• Toxin genes
• Direct pathogen measurement
• Adenovirus
• Enterovirus
• Phage

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EVALUATION CRITERIA

• Distinguish presence/absence of human source
• Correctly identify all sources
• Accurately determine relative contribution from
  each source
• Stability across matrices