&EPA
   United States
   Environmental Protection
   Agency
                Proceedings of the
                2009 U.S. Environmental
                Protection Agency Workshop
                on Innovative Approaches for
                Detecting Microorganisms and
                Cyanotoxins  in Water
                MAY 20-21,2009
                REGION 3 OFFICES
                PHILADELPHIA, PA
   Office of Research and Development
   National Center for Environmental Research

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           The 2009 U.S. Environmental Protection Agency Workshop on
 Innovative Approaches for Detecting Microorganisms and Cyanotoxins in Water

                                    May 20 - 21, 2009

                                  EPA Region 3 Offices
                                Shenandoah Room, #104
                                    1650 Arch Street
                                    Philadelphia, PA


Workshop Objectives

   •  Provide a forum to discuss proposed solutions to the methodological challenges in the search
      for better methods of detection and assessment of waterborne microbial contaminants.
   •  Facilitate collaboration and cooperation among scientists and policy-makers from research
      entities, EPA, states, local agencies, and stakeholders.
   •  Assist EPA in identifying what research or technologies are needed to better inform decisions
      and/or policies associated with the assessment of microorganisms in water.
   •  Give STAR grantees of the past two solicitations regarding "Development and Evaluation of
      Innovative Approaches for the Quantitative Assessment of Pathogens and Cyanobacteria and
      Their Toxins in Drinking Water" the opportunity to present their latest findings.  Summaries of
      the grantees' projects can be found at:
      http://epa.gov/ncer/rfa/2005/2005jathogens drinking water.html and
      http://cfpub.epa.gov/ncer_abstracts/index.cfm/fuseaction/recipients.display/rfa_id/456/records_
      per page/ALL
Wednesday, May 20, 2009
1:00 p.m.
1:25 p.m.



1:55 p.m.


2:15 p.m.
Welcome and Overview of EPA's Office of Research and Development and
the Science To Achieve Results (STAR) Program
Barbara Klieforth, EPA, Office of Research and Development, National Center
for Environmental Research

OGWDW Microbial Research Needs from a Regulatory Perspective
Sandhya Parshionikar, Team Leader, Microbiology Technical Support Center
Office of Ground Water and Drinking Water

Overview Presentation From EPA Region 3
Victoria P. Binetti, EPA, Region 3

Crypto and Molecular Methods Work Being Done With EPA Regions 2 and 3
Eric Villegas, EPA, National Exposure Research Laboratory, Microbiological
and Chemical Exposure Assessment Research Division

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Wednesday, May 20, 2009 (continued)
2:35 p.m.
2:55 p.m.

3:15 p.m.



3:35 p.m.



3:55 p.m.



4:15 p.m.



4:35 p.m.


5:00 p.m.
Development of a Universal Microbial Collector (UMC) for Enteric
Pathogens in Water and Its Application for the Detection of Contaminant
Candidate List Organisms in Water
Kelly R. Bright, University of Arizona

Break

Development and Evaluation of an Innovative System for the Concentration
and Quantitative Detection of CCL Pathogens in Drinking Water
Saul  Tzipori, Tufts University

On-Chip PCR, Nanoparticles, and Virulence/Marker Genes for
Simultaneous Detection of 20 Waterborne Pathogens
Syed Hashsham, Michigan State University

Rapid and Quantitative Detection of Helicobacter pylori and Escherichia coli
O157 in Well Water Using a Nano-Wired Biosensor and QPCR
Evangelyn C. Alocilja, Michigan State University

Assessment of Microbial Pathogens in Drinking Water  Using Molecular
Methods Coupled With Solid-Phase Cytometry
Barry Pyle, Montana State University

Detecting Pathogens in Water by Ultrafiltration and Microarray Analysis
Anthea K. Lee, Metro Water District of Southern California

Adjourn
Thursday, May 21, 2009
8:30-9:00 a.m.
9:00-9:20 a.m.
9:20-9:40 a.m.
Robust Piezoelectric-Excited Millimeter-Sized Cantilever Sensors for
Detecting Pathogens in Drinking Water at 1 Cell/Liter
Raj Mutharasan, Drexel University

National Risk Management Research Laboratory (NRMRL) Microbial
Research
Jorge Santo Domingo, EPA, NRMRL, Water Supply and Water Resources
Division, Microbial Contaminants Control Branch

Rapid Concentration, Detection, and Quantification of Pathogens in
Drinking Water
Zhiqiang Hu, University of Missouri

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Thursday, May 21, 2009, (continued)
9:40 - 10:10 a.m.   Simultaneous Concentration and Real-Time Detection of Multiple Classes of
                  Microbial Pathogens From Drinking Water
                  Mark D. Sobsey, University of North Carolina at Chapel Hill

10:10-10:30 a.m.  Break

10:30 - 10:50 a.m.  Quantitative Assessment of Pathogens in Drinking Water
                  Kellogg Schwab, Johns Hopkins University

10:50 - 11:40 a.m.  Discussion on the Next Generation of Methods and Research Needs

11:40- noon       Development and Application of a Fiber Optic Array System for Detection
                  and Enumeration of Potentially  Toxic Cyanobacteria
                  Donald Anderson, Woods Hole Oceanographic Institute

12:00-1:10 p.m.   Lunch

1:10 - 1:30 p.m.    Development of High-Throughput and Real-Time Methods for the Detection
                  of Infective Enteric Viruses
                  Jason Cantera, University of California at Riverside

1:30- 1:50 p.m.    New Electropositive Filter for Concentrating Enterovirus and  Norovirus
                  From Large Volumes of Water
                  Mohammad Karim,  Oak Ridge Institute for Science and Education Research
                  Fellow, EPA

1:50-2:10 p.m.    Automated Methods for the Quantification and Infectivity of Human
                  Noroviruses in Water
                  Timothy Straub, Batelle Pacific Northwest Division

2:10 - 2:30 p.m.    Characterization of Naturally Occurring Amoeba-Resistant Bacteria From
                  Water Samples
                  Sharon Berk, Mid-Tennessee State University

2:30-2:50 p.m.    Break

2:50 - 3:10  p.m.    Analysis of Various Toxins Produced by Cyanobacteria Using
                  Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry
                  (UPLC/MS/MS)
                  Stuart Oehrle, Northern Kentucky University

3:10-3:20 p.m.    Development of Sensitive Immunoassay Formats for Algal Toxin Detection
                  Fernando Rubio, Abraxis LLC

3:20 - 4:00 p.m.    Wrap-up & Adjournment

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       The 2009 U.S. Environmental Protection Agency Workshop on
           Innovative Approaches for Detecting  Microorganisms
                          and Cyanotoxins in Water

                              May 20-21, 2009

                            EPA Region 3 Offices
                          Shenandoah  Room, #104
                              1650 Arch Street
                              Philadelphia, PA

This workshop was intended to facilitate progress on the quantitative assessment of
microbial agents in water and bring research scientists together with policy makers.
EPA's success is dependent, in large part, on its ability to make credible environmental
decisions based on solid scientific information and technical methodologies. Reliable,
sensitive, robust, and versatile detection and monitoring  tools are needed to address
the risk assessment and management of known and emerging  microbial contaminants
in source water, treated water, and/or distribution systems. The goal of this workshop
was to foster discussion on the development of cost-effective, timely, and innovative
technology solutions in assessing and managing environmental risks to human health.

Workshop Objectives

•  Provide a forum to discuss proposed solutions to the methodological challenges in
   the search for better methods of detection and assessment  of waterborne microbial
   contaminants.

•  Facilitate collaboration and cooperation among scientists and policy makers from
   research entities, EPA, states, local agencies, and other stakeholders.

•  Assist EPA in identifying what research or technologies are  needed to better inform
   decisions and/or policies associated with the assessment of microorganisms in
   water.

•  Give Science To Achieve Results (STAR) grantees of the past two solicitations
   regarding "Development and Evaluation of Innovative Approaches for the
   Quantitative Assessment of Pathogens and Cyanobacteria and Their Toxins in
   Drinking Water" an opportunity to present their latest  findings. Abstracts of the
   grantees' projects can be found at:
   http://epa.gov/ncer/rfa/2005/2005 pathogens drinking  water.html and
   http://cfpub.epa.gov/ncer abstracts/index.cfm/fuseaction/recipients.displav/rfa  id/45
   6/records  per  page/ALL

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                                                                                                   EPA Organization
                                                                                       Mission: To protect public health and to safeguard and improve the
                                                                                       natural environment - air, water, and land - upon which life depends
               Support for EPA's Mission
                               EPA Mission:
                         ! rcifcU i'.'".-.;n i'livih and s s :...-.-i-~\
                         the natural environment - air, water, land --
                         upon which life depends
                                 A
                                                REGIONAL OFFICES
                                                    Primary
                                               Interface with States and Tribes
                               OFFICE OF
                             RESEARCH AND
                              DEVELOPMENT

                                Scientific
                               Foundation
                                           //     Implem
        NCER's Extramural Programs
  Science To Achieve Results (STAR)
     Targeted Research Grants through RFAs
     Exploratory/Futures Grants
     Graduate Fellowships
     Competed Centers
     Greater Research Opportunities
  Earmarks
  Small Business Innovation Research (SBIR)
  Contracts
  Experimental Program to Stimulate Competitive
  Research (EPSCoR)
     Grantees and fellows are among the top scientists in the country
Science To Achieve Results (STAR) Program
           Established in 1995 as part of the overall
           reorganization of ORD
           Mission: include this country's universities and
           nonprofit groups in EPA's research program and
           ensure the best possible quality of science in
           areas of highest risk and greatest importance to
           the Agency
           Issue approximately 20-25 RFAs each year
           Each year: receive 2500-3200 grant applications
           Award about 250-300 new STAR grants,
           fellowships & SBIR contracts per year
           Manage about 1000 active research grants and
           fellowships
                                                                             Science To Achieve Results (STAR) Program
•  Goal-directed solicitation planning
•  Significant cross-agency and interagency involvement with
  solicitation planning, writing, and review
•  Competitive solicitations
•  Joint Solicitations with other Agencies
•  External peer review
•  Internal relevancy review: program office and regional input
•  Fund highest priority projects
   • Grantees and fellows are among the top scientists in the country
•  Communicate research results through website, ORD
  laboratories, program office and regional meetings, and
  publications (www.epa.gov/ncer)

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      STAR Results in Action: Regulations and
       Results from the Marshfield Clinic Research Foundation's
       research led to major, statewide changes (e.g., UV disinfection) in
       treatment of water from groundwater sources (Borchardt)
       UNC at Chapel Hill examined impacts of water distribution
       systems in contributing to Gl illness, results are included in
       considerations for updating the Total Coliform Rule (Tolbert)
       Results used by EPA's Office of Water in preparing: "Economic,
       Environmental, and Benefits Analysis of the proposed Metal
       Products & Machinery Rule" (Herriges)
       University of Iowa findings on mechanisms and kinetics of
       chloramine loss & byproduct formation in distribution systems
       used in the Stage 2 Disinfectants and Disinfection Byproducts
       (DBP)  rule published in 2006
       STAR  research results on "integrated pest management" used by
       cities & states to reduce childrens' exposures to pest allergens
       STAR  research findings led to voluntary industry action -
       protective clothing and hand-washing facilities for agricultural
       workers expected to reduce "take home" pesticide exposures
                                                                   STAR Results in Action:

                                                                 Tools and Methods for Decision Making

                                                        University of Maryland's Center for Marine Biotechnology's 1st of its kind
                                                        PCR technique that rapidly detects Helicobacterpylori in environmental
                                                        samples.  H. pylori had previously been extremely difficult to detect
                                                        because of its ability to transform into a non-culturable form.
                                                        STAR researchers developed molecular detection techniques for
                                                        pfisteria - used by states and CDC for real time monitoring of pfisteria
                                                        events
                                                        STAR research developed promising method for assessing pesticide
                                                        concentrations in saliva - accurate & less invasive method to quantify
                                                        exposure & dose
                                                        Rapid assessment protocol for stream  biomass developed - used in
                                                        OW guidance document and by states
                                                        Research played a key role in the preparation of a manual on economic
                                                        valuation for the British Department of Environment, Regions, and
                                                        Transport (Carson)
          STAR & SBIR Results in Action:
                    Practical Applications
   Tufts' U. alternative method (portable continuous flow centrifuge) for
   concentrating low numbers protozoa from large volumes of water aoc
   as an alternative concentration method by EPA (Tzipori)
   Soybean oil plastics being usei
   ;Wool)
id to manufacture tractor parts for John Deere
So:

Developed a benign catalyst to replace chlorine in oxidation processes
(Collins)
Developed a substitute for lead solder now used broadly in the electronics
industry (Wong)
STAR-supported grant research has led to new, environmentally friendly
packaging manufactured by Cargill-Natureworks and used by the Wal-Mart
Corporation Advanced Technology Materials, Inc developed dry scrubber
using deposition for semiconductor industry.  Business grew from five
partners to 1100 employees and sales over $250 million (NASDAQ : ATMI)
       STAR Results in Action:

                  Education

New course in green engineering
Fellows are now professors in many, major
universities
Fellows are working in government agencies
Fellows elected to 36 scientific panels and/or
advisory committees
Sustainability curricula expanded in many
universities as a direct result of P3
Four new small businesses created because of
P3
Science To Achieve Results (STAR) Program





   •  Program begun in  FY 1996                       \  __^ ^


   •  Funding levels historically between $2.5-5.0 M/yr


   •  NCER has been funded research in a wide variety of
      areas


   •  Research completed 3-4 years after award


   •  Solicitation preparation and Programmatic Reviews
      have extensive participation from OW, ORD, and
      Regional Offices
                                                               Drinking Water (& Water Quality)


                                                          Current components

                                                           • Identifying and quantifying microbes in water

                                                           • Decision making for water infrastructure sustainability

                                                           • Source water/aquifer protection from potential impacts
                                                             of geologic sequestration of carbon dioxide

                                                          Recent solicitations
                                                           •  Integrated Design, Modeling, and Monitoring of Geologic Sequestration of
                                                             Anthropogenic Carbon Dioxide to Safeguard Sources of Drinking Water
                                                           •  Development and Evaluation of Innovative Approaches for the Quantitative
                                                             Assessment of Pathogens and Cyanobacteria and Their Toxins in Drinking
                                                             Water
                                                           •  Innovative and Integrative Approaches for Advancing Public Health
                                                             Protection Through Water Infra structure Sustainability

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  History of STAR Drinking Water Projects
            http://epa.gov/ncer/science/dhnkingwater/recipients.html
•Crypto
SHuCV
(4)
•DBFs
(10)
•Arsenic
(3)
•CCL
bugs
(10)
•DBPs
(10)
•Crypto
(1)
•Tx-
Crypto&
DBPs(1)
•Bank
Filtration
(4)
• Crypto
(1)
•CCL
chemicals
(3)
•CCL
pathogens
(2)
•Epi
Studies (6)
Pathogens
inDW
(10)
       •Detection
       of Microbes
                     cyanotoxins
                     (6)
• Impacts to
U SOW from
Geologic
Sequestration
ofC02 (7)
• Infra-
structure RFA
Science To Achieve Results (STAR) Program

            Other Water-related RfAs

         Some examples:
         •  Forecasting Ecosystem Services from
           Wetland Condition Analyses (2008)
         •  Enhancing Ecosystem Services from
           Agricultural Lands (2009)
         •  Watershed Classification (2002, 2003)
         •  Ecological Thresholds (2004)
         •  EcoHABs

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  OGWDW Microbial Researc
      Needs from a Regulatory
                Perspective
         The U.S. Environmental Protection Agency
                    Workshop on
   Innovative Approaches for Detecting Microorganisms and
                 Cyanotoxins in Water
                    May 20-21, 2009
              Sandhya Parshionikar, PhD
               Technical Support Center
         Office of Ground Water and Drinking
                                                                                     Overview
     The SDWA requirements and regulatory process.
     Research input in Drinking water regulations
     Sources of data used
     Research Needs
     — General
     — Specific issue
     — Total Coliform Rule
         • Revisions
         • Research and Information Collection Partnership
     — Long term
       Safe Drinking Water Act
SDWA requires regulation of contaminants that:
                    May have an adverse health effect
                     must consider sensitive sub-populations of
                     infants, children, pregnant women, elderly,
                      individuals with history of serious illness
                   Occur or are likely to occur in PWSs
                     (considering frequency and level)
                   Present a meaningful opportunity for
                           health risk reduction
                     based on best available science and data
             Safe Drinking Water Act
                   Requirements
       EPA must publish Maximum Contaminant Level Goals
       (MCLGs)
       - Must set levels at which no health effects occur and which
         allows for adequate margin of safety
         Required EPA to regulate specific microbial contaminants
         (viruses, Giardia, Legionella, total coliforms, heterotrophic
         bacteria)

       EPA must promulgate MCLs or treatment technique
       requirement as close to the MCLG as is "feasible"
       (taking costs into consideration)
       - Required EPA to set treatment technique requirements for
         surface and ground water systems to protect for pathogens
         Safe  Drinking Water Act
                Requirements
EPA must develop Contaminant Candidate List (CCL) for
unregulated contaminants every 5 years
 -  Establish criteria for a program (UCMR) to monitor unregulated
   contaminants, and to identify no more than 30 contaminants to be
   monitored, every five years.
 -  Perform regulatory determination on five of CCL contaminants every
   five years

Requiring the Agency to review and revise, as appropriate,
each National Primary Drinking Water Regulation no less
often than  every 6 years
 -  Revisions must assure public health protection (the net effect of the
   rule must be to maintain or improve public health protection)
               Generalized Flow of
              Regulatory Processes
—I  UCMR Momtonn;
 I    •"
                                             (Six Year Review of
                                             Existing NPDWRs
  At each stage, need increased specificity and confidence in the type
  of supporting data used (e.g. health, occurrence and treatment).
                                                                               Office of Ground Water and Drinking Wat

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         Research Inputs into  DW
                   Regulations
                        MCLG
                        MCLor
                  Treatment Technique
     Control Measures
     - Efficacy data
Feasibility of treatment technologies
- Analysis of potential side-effects
      - Indicators
 Economic Considerations
   - Cost analyses
   - Benefit analyses
- System impact assessment
                                        Sources of Research Data Used
EPA Office of Research and Development
 — In house research
 - STAR grants
Regions
Water Research Foundation (formerly AwwaRF)
Contracts with Universities and research institutions
Interagency agreements
Co-operative agreements
Other published, peer reviewed literature
        Regulatory Drivers: Some Near
               Term Examples
  CCL4
  UCMR4
  Regulatory Determinations 3
  DS information collection
  6 year review
                                          Research Needs: General
           Office of Ground Water and Drinking Water
                                   Exposure Data
                                    - Analytical Methods
                                       • Innovative approaches to measurement
                                       • Practical implementable technologies
                                    - Occurrence data
                                       • Outbreak analyses
                                       • Endemic prevalence
                                    - Epidemiological studies

                                   Health effects
                                    - Dose response
                                    - Subpopulations affected
                                    - Host factors involved
                                                                                Office of Ground Water and Drinking Water
         Research Needs: General
  Treatment
   -  Behavior of pathogens under different types of treatment
     conditions
   -  Novel strategies for contamination mitigation
  Other research
   -  Pathogen virulence
   -  Role of host factors in infectivity
   -  Fate and transport of pathogens under environmental conditions
                                                Research Needs:
                                         Examples of Specific Issues
                                  Methods that detect pathogen infectivity/viability/strain
                                  identification
                                  Exposure to pathogens from drinking water contamination
                                  events

                                  Role of Biofilms in pathogen exposure and their impact on
                                  chlorine residuals
                                 Survival of nucleic acids under various treatments

                                 Innovative approaches for sampling and detection

                                 Research in Support of Revised TCR/DS
           Office of Ground Water and Drinking Water
                                                                                Office of Ground Water and Drinking Wat

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          Total Coliform Rule  (TCR)
   2000 - Stage 2 Federal Advisory Committee (FAC)
   Agreement In Principle (AIP) suggested review of
   distribution system issues with the 6-year review of the
   TCR


   2003 - Six year review of existing drinking water
   regulations -> TCRshouldbe revised

   2007 - Federal Advisory Committee convened to provide
   recommendations on

      •how EPA should revise the TCR, and

      •what research and information collection should be
      conducted to better inform distribution system risk
    Total Coliform Rule  Revisions
The Advisory Committee developed an AIP to be the
  foundation for the proposed rule

   - A more proactive approach to public health protection

   - Use of monitoring results shift from informing public
     notification to informing investigation and corrective action

          2010: Propose rule revisions

          2012:  Final rule

          2015:  compliance starts

   - Includes recommendations for distribution system research
     and information collection and the formation of a Research
     and Information Collection Partnership
   Research and Information Collection
               Partnership  (RICP)
Recommended by TCR Federal Advisory Committee to:
•  Inform and support the drinking water community to develop future
  risk management decisions regarding drinking water distribution
  systems

•  Partnership formed January 29, 2009 between EPA and Water
  Research Foundation

•  Steering Committee provides input on research and information
  collection priorities
   - 3 members from EPA
   - 3 members from water utilities
   - 3 additional members
   - Public health
   - Environmental
   - State Regulator
            Office of Ground Water and Drinking Water
              and Information Collection
             Partnership  (RICP)
Develop a research agenda to identify decision relevant
research and information collection needs or priorities
    • Biofilms
    • Nitrification
    • Intrusion
    •Storage
•Contaminant Accumulation
• Main Repair
•Cross Connection Control
    -First Draft Research Agenda - September 2009
    -Initial priorities for research and information
      collection identified - 2010
                                                                                        Office of Ground Water and Drinking Water
         Long Term  Research  Needs
  Online monitoring/Rapid results

   - Perturbations in water quality

   - Outbreak analysis
      • Quantitative
      • Genotyping/Strain identification
      • Sensitivity
  High through put detection

  Universal detection of all classes of pathogens

  Miniaturization of technology

   - Use in field

  Genomics/Proteomics
                New STAR RFA
EPA seeking new and innovative research applications that link
opportunities to advance public health protection with improvements
in the condition and function of the water infrastructure.


 The focus on improving the effectiveness of the water infrastructure
for protecting public health.

Should clearly demonstrate an integrated, multi-disciplinary approach
that leads to advances in design, operation, and management of the
water infrastructure and should directly tie those advances to public
health protection in conjunction with improving water efficiency and
reducing energy requirements.

http://www.epa.gov/ncer/rfa/2009/2009_star_water_infrastructure.ht
ml.
            Office of Ground Water and Drinking Water
                                                                                        Office of Ground Water and Drinking Wat

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           Region 3 Overview
                Victoria P. Binetti
                 US Environmental
                 Protection Agency
Workshop on Innovative Approaches
For Detecting Microorganisms
And Cyanotoxins in Water
Philadelphia, PA
  May 20, 2009
We are employing a "Healthy Waters"
strategy to restore and protect our waters
by
•  Protecting four water uses
   • Aquatic life
    Recreation
    Fish consumption
   • Drinking water health

   deducing causes of impairment
   • Nutrients
   • Sediments
   • Toxics
   • Pathogens
                                  .and by
                                  By addressing contaminant sources
                                  • Agriculture
                                  • Developed/Developing lands
                                  • Mining
                                  • Transportation

                                  Using approaches like
                                  • Wholesale solutions
                                  • Prevention partnerships
                                  • Integrated strategies
                                  • Green solutions
              Number of Systems vs. Population Served

-------
   Some observations on drinking water

   program implementation in Region 3


   Many public water systems are small, unde
   resourced, and have limited technical capacity
   Greatest number of violations overall are relat
   to monitoring
   Most frequent health-based violations relate t(
   pathogen regulations: Total Coliform Rule,
   Surface Water Treatment Rules
   Newer  regulations requiring source water
   sampling are challenging
                                   Implementing a multi-barrier

                                approach to safe  drinking water:

                              r Prevent/Reduce pathogens in source
                                waters

                                Eliminate/Inactivate pathogens through
                                treatment
                              • Assess/Monitor to detect pathogen
                                occurrence in finished water
                              » Assess exposure, health effects
  Comprehensive Source Water Protection
    I  MULTIPLE RISK REQUIRE MULTIPLE BARRIERS  \


   SDWA PROTECTING AMERICA'S PUBLIC HEALTH
              RISK          RISK      RISK     RISK
PROTECTION    RISK        RISK

 BARRIERS   PREVENTION  MANAGEMENT
   RISK

MONITORING/

COMPLIANCE
INDIVIDUAL
 ACTION
                               Needs today from the field include:

                              • Monitoring & quantification methods -
                                Cryptosporidium, bacteria, viruses
                                 • Low-cost, reliable
                              • Tools for viability assessment, speciation
                              • Pathogen indicators
                              • Real-time E. coliidentification
                              • Efficacy of best management practices for
                                nutrient & sediment control, in prevention of
                                pathogen contamination
                                Efficacy of best management practices used for
                                protection of surface waters, in protection of
                                ground water
      Issues for the Research Agenda

    Distribution system is the next frontier
    Aging, deteriorating infrastructure increases
    pathogen exposure risk
    Longer-lived, healthier—but more vulnerable—
    population?
    Impacts of population growth, climate change
    and patterns of development on water use and
    technology - e.g., water efficiency, water reuse,
    aquifer storage & recovery, etc.
    Climate change will affect pathogen
    distributions, geographically andseasonalli
    Water security concerns will remain—detection,
    response, recovery

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       Giardia & Cryptosporidium
                                                                         SERA
                                                                                                Overview
1.   Brief introduction to waterborne Cryptosporidium
     • Biology and diversity of Cryptosporidium species
     • Current detection methodologies
                                                                         2.   US EPA-NERL's waterborne protozoan research program
                                                                              • Building a "Protozoan Detection Toolbox"
                                                                         3.   Perspectives on the future of the "Protozoan Detection Toolbox"
                                                                              • Future directions and considerations
SERA
               Cryptosporidium species
SERA     Cryptosporidium Species Infecting
              Humans and Selected Animals
SERA                  Method 1622/1623:
             Detection of Cryptosporidium and Giardia
                         Does not differentiate human infectious vs
                         animal forms
                         No live vs. dead discrimination
SERA      Challenges for the 21st Century
                "Water Quality Tricorder"
                                                                          Protozoan Detection Svstems:
                                                                          1.  Fast and user friendly
                                                                          2.  Sensitive and quantitative
                                                                          3.  Species/genotype specific
                                                                          4.  Live vs. dead

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 vvEPA
               Question Driven Research
 1.  What are the total levels of Cryptosporidium In the watershed?

 2.  How complex Is the Cryptosporidium species diversity In the
    watershed?

 3.  What are the total levels of pathogenic Cryptosporidium In the
    watershed?

 4.  Are the Cryptosporidium oocysts In the watershed
    viable/Infectious?
 5.  Other questions..
SERA    Tracking Sources of Contamination
                       in a Watershed
                      Methodology
            Collection of 20-L water samples (93 samples)
Method 1623
                    Filtration of two 10-L
                        samples
         certified laborato
                                 One filter to CDC
                                    laboratory
                                 Immunomagnetic
                                   separation of
                                     oocysts
                               PCR, DMA sequencing
                 Species and Genotypes Found
                                                                             If.,.—
                                                                              it* [I
                                                                              i*-*r*
                                   iftTiiM mtA.M  AM «»« Cinn >n«
                                    ?C^.»'M"
                                            i' i« Sl-p
                                                    -

                                                                                                                        98. Applied and EnuironnEntal M
                   Summary and  Impact:
   Pathogenic C. hominis and C. pan/urn were not detected in all 93 samples analyzed

   Only minor species/genotypes infecting humans were detected (10 samples)
   Molecular-based detection technique used in this project proves to be sensitive to
   detect and genotype oocysts in source waters
      ad Utilities and Region 3 understand that oocysts in the surrounding county's
      :e water are predominantly non-pathogenic
  Utilities are setting out to work with the agricultural community by encouraging and

             What Lies Ahead for the
   Waterborne Cryptosporidium Research
                      Program?
                                                                                      Multiple Pathogen Detection Systems

-------
vvEPA
 Quantitative PCR-Based Detection of
—       Cryptosporidium spp.
• Many species and genotypes found in source water
• Most quantitative PCR published have varying degn
• Development of multiplex qPCR assays
                                   - Purified genomic DMA from CDC
                 Acknowledgements
SERA     Molecular Detection Technologies:
                      A Perspective
                                                                      is in its infancy

                                                                   2.  A better understanding of the differences
                                                                      CryptosporidiumX

                                                                   3.  Advances in the "Protozoan Detection
                                                                      Toolbox" will improve our understanding of
                                                                      these parasites and their relationship to public
                                                                   •SEPA
                                                                   Questions?
                                                                           (513)569-7017
                                                                         villegas.eric@epa.gov

-------
       Universal Microbial
 Concentrator Requirements

 •  Simple, easy to operate
 •  High capacity
 •  High flow rate
 •  Low cost
 •  Concentrates diverse microorganisms
 •  Elution efficiencies similar to existing
    methods
 •  Limit interfering substances
Identification:
     Culture methods (bacteria)
     Microscopy (parasites)
     PCR/cell culture (viruses)

-------
                                                             Argonide NanoCeram® Virus
                                                                      Sampler Filters
                                                                  Inexpensive: $40/filter
                                                                         (1MDS: $175/filter)
                                                                  High flow rates (up to 19 L/min)
    NanoCeram® Virus Filters

Alumina nanofibers [A1(OH)2] on
microglass fiber matrix
Electropositive, non-woven,
pleated, average pore size = 0.2|am
Pre-sterilized                  "~
Effective for fresh, brackish,
seawater
pH 5-10; Temps. 4-50°C
     Experimental Protocol
Test organism added to dechlorinated tap water at
Pressure applied (~ 2 p.s.i.) = flow rate of 2.0 L/min.
Effluent samples collected to determine capture
efficiency.
450 ml of eluting solution added to the filter housing
(30 min hold).
Eluting solution back flushed through the filter and
collected (pH adjusted to 7.5).
Eluent back flushed a second time.
Eluent assayed for virus recovery.

-------
           Elution Methods
Hydrophobic interactions:
•   Surfactants (Tween 80)
    Secondary Concentration Step
    Volume reduction - centrifuge tube
    ultrafiltration (Vivaspin concentrator)

    Reduces volume -1000-fold
    (from 150 ml to-150^
                                                               Method Advantages
                                                           Much lower cost ($40 for NanoCeram® filters vs.
                                                           $175 for 1MDS filters)

                                                           No organics used in the elution step

                                                           Reduced volume (~ 200 |il vs. ~ 20 ml)

                                                           Higher efficiencies than those reported for some
                                                           enteric viruses.

-------
             Future Work
Comparison to existing methods in a field study
collecting surface water samples in Arizona, Michigan,
and Mexico:

  - 1MDS filters, ultrafiltration
  - Adenoviruses, enteroviruses
  - cell culture, polymerase chain reaction

Evaluate physical methods for recovery of parasites
(Microsporidia) from NanoCeram® filters.

-------
Development and Evaluation of an Innovative System for

  the Concentration and Quantitative Detection of CCL

           Pathogens in Drinking Water
                   SaulTzipori
                   David Walt
                  Udi Zuckerman

 Tufts University Cummings School of Veterinary Medicine
              Grafton, Massachusetts
                 Overview

 Milestones of the Continuous Flow Centrifugation
 methodology (CFC) developed at Tufts
 Objectives of the current STAR award 2006 - 2009
 Progress: new automated method/equipment for
 multiple waterbome pathogens
 Future tasks
 Acknowledgements
                Acceptable Range of   Recovery (%)
                Mean Recovery (%)
                21-100             42.5
Source Water
Cryptosporidium
Giardia
                                                              2005 - the CFC 200 and 625B bowl
                                                              became commercially available
                                                              2007 - Under the second EPA STAR award
                                                                                                 1st automated CFC prototype

-------
            Objectives for 2006-2009


  Simultaneous concentration of representative
  microorganisms from each group of the CCL list


  Validation of the concentration methodology


  Detection and quantitative identification of the
  CCL list using multiplex miniaturized fiber optic
  bead microarrays coupled with a compact scanner


  Side by side comparison of this detection
  methodology with EPA standard methods
Expanding the CFC methodology beyond protozoa
                  concentration

• Design of a new multiple pathogens bowl
• Design of a portable computerized
  concentration/elution equipment
• Design of a disposable tubing kit
• Choosing the programming software
~ Testing variable operating protocols
                 How does it work?

Filtration components are based on size exclusion which is
prone to clogging and the overall procedure is labor intensive
and expensive


The new automated CFC methodology employs centrifugal
force to sediment the protozoa and bacteria inside the bowl with
minimal clogging problems.


The modified bowl allows the "particle-free sample" to flow
through the positive charged component in the core and the
viruses are adsorbed by the positive electrostatic forces


Elution buffers are injected sequentially where the t
protozoa/bacteria first, then the viruses second, are dislodged
and the concentrates are delivered to two separate sterile bags.

-------
    Recovery efficiency of the automated CFC with 10 L tap
      water samples spiked with multiple microorganisms
     C. parvum were spiked and the oocysts detected
     from the concentrate usina: method 1623
    MS2 bacteriophages (ATCC 15597-B1) were
    spiked and detected from the concentrate using
    the agar overlay method (the host was E. coli 1559)


    B. anthracis spores (kanamycin resistant strain,
    sterne) detected by MF
      Recovery of C. parvum oocysts, B. anthracis., and MS2
bacteriophages from 10L tap water samples using an automated CFC
        and a modified bowl (9,000rpm & 0.5 liter/min)
    Detection and integration

We have concentrated on the detection of
DNA isolated from E. coli as a model
system.  We have demonstrated the
detection of PCR amplicons from three
virulence genes using multiplexed bead-
based microarrays.
We expanding the protocol and microarray
to include all bacteria and viruses listed as
CCL3 candidates as listed

-------
            CCL3 candidates
Caliciviruses
Campylobacter jejuni
Entamoeba histolytica
Escherichia coli (0157)
Helicobacter pylori
Hepatitis A virus
Legionella pneumophila
Naegleria fowleri
Salmonella enterica
Shigella sonnei
Vibrio cholerae
                The next phase
  Walt's lab is currently working on the bioinformatix of the
  CCL list for the microarray detection: this will be
  completed over the next 12 months
  Once the detection platform is complete, the automated
  CFC spiked concentrates will be applied and qunatitated
  The detection will be compared with currently approved
  standard methods
  Ideally this approach should be evaluated by water testing
  labs — filed testing, as was done for C. parviun and Giardia
  Evaluate the technology as a continuous monitoring system
                                                       Acknowledgements
                                           EPASTAR program (RD 83300301) which is
                                           funding this work

                                           Haemonetics for technical and material
                                           support over the past 10 years
                                           Staff of the Division of Infectious Diseases
                                           for technical support

-------
                    MICHIGAN STATE
                    U  N  I V E R S I T 1

On-chip PCR, Nanoparticles, and Virulence/Marker
      Genes for Simultaneous Detection of 20
               Waterborne Pathogens
      U.S. EPA Workshop on  Innovative Approaches for Detecting
             Microorganisms and Cyanotoxins in Water
                      Philadelphia, PA
                     May 20, 2009 3:15 PM
                  Objectives
                     Syed A. Hashsham
       Volodymyr Tarabara
                                       James M. Tiedie
1.  Reducing the Time to Detect Growth using Dye-doped Nanoparticles

2.  On-chip PCR based Detection of 20 Pathogens

3.  Enhancement in Sample Concentration by Cross-flow Filtration
                                                                           A rapid btoasviy for single bacterial cell quantisation
                                                                           using bioconjugated n.inopjttklesi
                                                           Growth curve by plate count, real time PCR, absorbance and dye doped NP assay
                                                                                                                          Time taken to determine the increase in growth by various methods
                                                                                Contact the presenters
                                                                                                                                             Contact the presenters

-------

-------
Larger difference at lower copy numbers
                                                          Hand-held Gene Analyzer
                                                               Contact the presenters
                  Starting Copies

-------
  3. Enhancement in Sample Concentration by Cross-flow Filtration


   •Goals: Increasing
       1)  Rate of concentration (Jpermeate)
       2)  Recovery
       3)  Reproducibility
    •Approach:
    > Hydraulic management (Goals 1 & 2)
       - AP7P^Jp6rm6at67P
       - Jrf 71 -> Jp6rm6at6 71 and recovery 71

    > Preparation of reproducible non-adhesive membrane
     (Goals 2 and 3):
       - Non adhesive surface -^ recovery 71
       - Controlled approach to membrane blocking -> reproducibilitv 71
 Pump Evaluation: Reduction in Cross-flow over Time
        Rate of Sample Concentration
Influence of AP and Jcrossfiowon Bacteriophage Recovery
                                                                          Design of non-adhesive surface
                      Contact the presenters
                                                                                      Contact the presenters
* Amount of water filtered in 30 min normalized to 1 m2 of membrane surface area
                                                                                                                                      Protein Blocking of the membrane:
                                                                                                                                       May not always be "appropriate or practical due to concern related to the
                                                                                                                                         amount of time needed (...land potential for microbial contamination"
                                                                                                                                                                                  Hill et al. 2005
                                                                                                                                      New approach to membrane blocking
                                                                                   - reproducible, non-adhesive coatings based
                                                                                   on multilayer polyelectrolyte films
                                                                                   -fast and and straightforward coating procedure
                                                                                   - design flexibility (charge, hydrophilicity)
                                                                                   - have been shown to reduce adhesion of
                                                                                   bacteria, mammalian cell and proteins
                                                                                   - recoverable coating

-------
                                                                                                                                             Summary
                                                                                                                       1.  NP-based assay faster but expect to be busy

                                                                                                                       2.  On-chip PCR: efficient screening tool, for samples that

                                                                                                                           will result in 10 copies

                                                                                                                       3.  Sample concentration speed can be considerably

                                                                                                                           improved with higher pressure (8 fold to 150 L/30 min-m2)

                                                                                                                       4.  Improvements in blocking the filters: ongoing
        Acknowledgements
Michigan bconomic Development Corporation's 21st (
 I candidates:
  Robert Stedtfeld
  Elodie Pasco
  Tiffany Stedtfeld
  Dieter Tourlousse
  Farhan Ahmad
  Yu Yang
Most chip related experiments
Membrane Filtration studies
Validation
Sample Processing/DMA Biochip
micro-PCR Image Analysis
Nanoparticf
  Syed Hashsham, Volodymyr Tarabara, and James Tiedje

-------
       Rapid and Quantitative Detection of
     Helicobacter pylori and E. coli O157 in
            Well Water Using a IMano-Wired
                          Biosensor and QPCR
    2009 U.S. Environmental Protection Agency Workshop on Innovative
    Approaches for Detecting Microorganisms and Cyanotoxins in Water
                            May 20-21, 2009, Philadelphia, PA
Outline
  Hypothesis
  Results by objectives
  Summary of results
  Future work
Hypothesis
  A disposable biosensor and qPCR can be combined
  seamlessly to develop a unique biosensor-qPCR as a
  tool for near real-time determination of contaminant
  occurrence in drinking water.
                            i McGraw, Michelle Packard, Jongseol Yi
Objectives
  Develop a protocol for processing water samples for the
  biosensor and qPCR.
  Assess the performance of the biosensor and qPCR for
  sensitivity, specificity, recovery, and false
  positives/negatives of detection and enumeration for E. coli
  Oi57:H7 and H. pylori in groundwater samples from the
  field.
  Develop a method for detecting and enumerating E. coli
  Oi57:H7 and H. pylori by qPCR using bacteria isolated and
  screened by the biosensor system.
  S^alidate a method for testing viability ofE. coli 0157:!^
Highlight of  Results
  Developed a novel target extraction system using an
  electrically active magnetic nanoparticles.
  Developed a protocol for use of automated DNA extraction
  and evaluated it in difficult samples.
  Developed a data base on CPU vs qPCR units for E.coli and
  Enterococci, and will be adding in the data from each
  sample for the 0157.
  E. coli OlsyiHy biosensor has been tested in pure and
  seeded water samples.
  Viability test has been developed; sensitivity and specificity
  /vere evaluated.
Flowchart  of Research Plan
      Membrane filtration of water sample - 8 liters at source
         Extract!on/elution of cells at water source    j
                     T
     Biosensor for qualitative field screening ofmicrobial contaminants
| qPCR for quantitative enumeration
| Viability lest for E. coli O157:H7  |
                                                                                                                                                                               IBfc

-------
     Biosensor
ANALYTE   BIORECEPTOR  TRANSDUCER
                                      SIGNAL PROCESSING
w,
Antibodies
Nucleic Acids
Aptamers
Enzymes
Whole cells

Nanoscale materials


                                                   Data
                                                  Acquisition
  Advantages:
   • Rapid detection time
   • High sensitivity and specificity *
   « Compatible with data
    processing technologies
   • Can be ruggedized

  Real-Time  Quantitative  PCR  (qPCR)

•  Detects PCR product fluorescently in each well plate.

•  Fast PCR screening without gels.

•  Quantifies amount of PCR product at each cycle.

•  Detects presence or quantify fraction of sample made up
by particular species using species specific primers.

 •Uses threshold detection for relative abundance.
  Results  By Objectives

    Develop a protocol for processing water samples for
    the biosensor and qPCR.
  • Assess the performance of the biosensor and qPCR for
    sensitivity, specificity, recovery, and false
    positives/negatives of detection and enumeration for E. coli
    OlsyiHy and H. pylori in groundwater samples from the
    field.
  * Develop a method for detecting and enumerating E. coli
    0157:1-17 and H. pylori by qPCR using bacteria isolated and
    screened by the biosensor system.
    ^alidate a method for testing viability ofE. coli O!57:IJZj,,
   Membrane Filtration (MF) & Enrichment
                        Enterocccci cdunes cofilter [CFU/filterj
                     20      40      GD       60

— ff\

Im

i to
s-
« 20
0

1 -




jQ%
J
	 1 "J'~"

	 1 67%
»* m


J,



-— '

47%
^,--

n














^







—
"



_. 	 -
" sen









                                                                                         1-30    31-40 SI40 51-60 61.70 71-30 81-80 91-100 101
                                                                                         [6]      (5)  (11) (15) (11)  |fi)   91  (8)  (17)
                                                                                              Enteracocci crtorcC5 onfflter [CRJflltet)
Using EAM Nanoparticles for Target Extraction
                                                                                                                                              ANALYTE  BIORECEPTOR TRANSDUCER
Antibodies
Nanoparticles
                                                                                                                                                                                SIGNAL PROCESSING


                                                                                                                                                                                      >=>
                                                                                                                                              Electrically active magnetic nanoparticles (EAM) ftmctionalized with antibodie
                                                                                                                                                      100 ml water sample at the source
                                                                     | Electrically active magnetic nanoparticles for separation and concentration
                                                                                            1
                                                                                                                                                Biosensor for qualitative field screening ofmicrobial contaminants
                                                                                                                                                                          |viabililyleslforE.coliO157:H7 |

-------
 Iron oxide-polyaniline core/shell -> EAM
Unique electronic structure and
flexible electrical properties of
protonated polyaniline
Magnetic properties from the core
Simple and low cost preparation
Excellent environmental stability
                                       •
                              TEM images of (left) unmodified Fe2O3
                              NPs and (right) electrically active
                              magnetic NPs.
                                                                       Characterization of EAM
                                                                                   Scanning Electron Microscopy Images
                                                                     Characterization of EAM
                                                                     Transmission Electron Microscopy and Electron Diffraction Images
EAM  Nanoparticles (1:0.4)
                               Iron oxide Nanoparticles
                                                                                                                                                       1:0.1 EAM NPs                 1:0.4 EAM NPs
                                                                                                                                                          XRD shows EAM is crystalline.
Magnetic Measurement of EAM
                            Field (kOe)

v-Fe2O3 nanoparticles: aniline monomer weight ratio was varied as 1:0.1, 1:0.4, 1:0.6, and 1:0.8.
                                                                           lagnetic characterization
                                                                         Electrical conductivity of EAM
Y-Fe203:
Aniline Wt.
Ratio
1:0.1
1:0.4
1:0.6
1:0.8
Coercivity
(300K)
Oe
180
180
180
180
Retentivity
(300 K)
emu/g
15.3
9.57
9.48
9.18
Saturation
Magnetization
(emu/g)
61.1
40.3
St.?
33.5
                                                                                                                                                        Fe!Os: Aniline WtR,
                                                                        Low coercivity and retentivity values -> EAMs are in the ferromagnetic regime.
                                                                                                                                                                               Four point probe measurements
                                                                                                                                                                               in compressed pellets of 2000
                                                                                                                                                                               microns in thickness.
Y-FezO3: Aniline
Wt. Ratio
1:0.1
1:0.4
1:0.6
1:0.8
1:0.0
Conductivity
(S cm-1)
0.092
0.768
1.129
2.436
0.000017

-------
Energy dispersive spectroscopy
Element
CK
NK
OK
C1K
FeK
Total
Weight%
28.49
6.72
29.09
3.87
31.82
100.00
Atomic%
44.34
8.97
33.99
2.04
10.65

          it  1   it
                            a   i   u  *   it
                  1:0.6 EAM Nanoparticle
                                               IBt
                                                               Electrical Characterization of EAM
                                                                 EAM follows ohmic behavior.
                                                                 Ohm's law:
                                                                 I=V/R
                                                                                        -0.6  -0.4  -0.2   0.0  02
                                                                                                Voltage (V)
                                                                 Ab-EAM for Cell Capture
                                                                  Method: Physical adsorption (Muhammad Tahi,a.ai^o
                                                                   • Combine EAM nanoparticles with monoclonal
                                                                    antibodies to target cell in PBS solution
                                                                  Conditions:
                                                                   • Room temperature incubation
                                                                   • Time: 45 min
                                                                                                                                                YYY
                                                                                                 Y
                                                                                                lEAMj
                                                                                                                                                                      Antibody structure
                                                                                                                                              Monoclonal antibody to
                                                                                                                                                 Target cell
                                                                                                                                                                Antibody Modified EAMs
Immuno-EAM Bacterial Separation
   Monoclonal antibody specific to Escherichia coli O157:H7 conjugated to EAM nanoparticles
   Bound bacteria separated using a magnetic separator and resuspended in deionized water
                            BlMrtll
                       TEM image of polyaniline conjugated with
                       antibodies (labeled with gold nanoparticles to
                       confirm binding of Ab on polymer surface).
Experiments for immuno-EAM capture
-for!06cfu/ml
  Incubation time: 15, 30, 60 min
   • -> 30 min had most cell capture

  Antibody concentration: o.i, 0.25, 0.5, i.o mg/ml
   • -> 0.5 mg/ml had most cell capture

  EAM concentration: 10, 20 25 mg.ml
   • -> 10 mg/ml had the most cell capture
Capture efficiency for E. coli 0157:H7
                                                                                                                                  Cell capture was confirmed by plating:
Solution
10-5 dilution of
pure culture
(104CFU/ml)
10-8 dilution,
cell conjugate
(102CFU/ml)
Count of
Captured Cells
10,880 CFU/ml
(104CFU/ml)
10 CFU/ml
(101 CFU/ml)
Cell Count in Original
Culture
1.088x10' CFU/ml
4.0x10" CFU/ml
                                                                    Observation: Capture process decreased cell count by less than a
                                                                    factor of 10.

-------
 Results By Objectives
   Develop a protocol for processing water samples for the
   biosensor and QPCR.
   Assess the performance of the biosensor and qPCR for
   sensitivity, specificity, recovery, and false
   positives/negatives of detection and enumeration for
   E. coli Oi57:Hy and H. pylori in groundwater samples
   from the field.
      qPCR
   Develop a method for detecting and enumerating E. coli
   OlsyiHy and H. pylori by QPCR using bacteria isolated and
   screened by the biosensor system.
   ^/alidate a method for testing viability of E. coli OisyiHy.
Primers and probes for the qPCR assays
Standard curve for E.  coli assay
                     5'CAATGGTGATGTCAGCGTTS'  Developed by this study
                     5'ACACTCTGTCCGG CTTTTG3'
                     HEX-
                     CAACTGG ACAAGG GG CACCA
                     GC--BBQ
                     5'CAATGGTGATGTCAGCGTTS'  Developed by this study
                     5'ACACTCTGTCCGG CTTTTG3'
                     6FAM-
                     TTGCAACTGG ACAAGG CACCA
                     GC--BBQ
                     AGAAATTCCAAACGAACTTG  Frahmefa/, 2002
                     GAG TGC TCT ACC TCC ATC
                     ATT
                     FAMb-TGG TTC TCT CCG AAA
                     TAGCTTTAG GGCTA-TAMRAc
                                                                      Standard curve for 10-fold serial dilutions of generic E.cotfuidAgene. Linear
                                                                       jgression analysis shows an R2 of 0.995, a slope of -3.22 and an intercept of 38.439
Standard curve for E.  coli 0157  assay
Standard curve for Enterococci assay
    Standard curve for 10-fold serial dilutions of E.coli O157 uidA gene. Linear regression
    analysis shows an R2 of 0.99, a slope of-3.39 and an intercept of 39.121
   Standard curve for 10-fold serial dilutions of generic enterococci 23SrDNA. gene.
  ^Linear regression analysis shows an R2 of 0.992, a slope of -3.34 and an intercept of
   B.574
                                                                                                                                                                                          Bfc

-------
qPCR-
for
Enterococci
                             Analysis of VacA gene of H. pylori Vs Samples
No. ofH.
pylori/ 50 ml
samples
                                            1 S 1  * 1
                                  Raw water from waste water treatment plant at different period of time
                                                                                            Key Results
                                                                                                                                   Rapid qPCR methods have been developed for two
                                                                                                                                   fecal indicators E.coli and Enterococci and two
                                                                                                                                   pathogens Helicobacter and E.coli o^yHy.
                                                                                                                                   qPCR has been used to detect Helicobacter in sewage
                                                                                                                                   and detects what is likely the viable non-cultivable
                                                                                                                                   state (previous report and publication).
                                                                                                                                   qPCR is highly correlated to E.coli and Enterococci in
                                                                                                                                   Sewage but this same assay does not detect all of the
                                                                                                                                   species present in manure, either due to interferences
                                                                                                                                   or more likely due to specificity of the primers.
Results By Objectives

• Develop a protocol for processing water samples for the
  biosensor and QPCR.
  Assess the performance of the biosensor and qPCR for
  sensitivity, specificity, recovery, and false
  positives/negatives of detection and enumeration for
  E. coli Oi57:Hy and H. pylori in groundwater samples
  from the field.
    Biosensor
* Develop a method for detecting and enumerating E. coli
  OlsyiHy and H. pylori by QPCR using bacteria isolated and
  screened by the biosensor system.
  ^/alidate a method for testing viability off. coli Oi57:H7.
                                             *J/OO L.
                               Performance of biosensor
                             ANALYTE  BIORECEPTOR TRANSDUCER
L,
                          Lateral flow direct-charge transfer biosensor

                          Application Pad: Cellulose acetate membrane
                          Capture Pad: Nitrocellulose
                          Absorption Pad: Cellulose a
                          Electrode: Silver
                          Type: Disposable
                                                       Overall Dimension: 60mm * 5 mm
                                                       Electrode Gap: 0.5 mm
                                                                                            Sample  Preparation
                                                                                                                    Sample
                                                                      ° Antigen                  IT Antibody
                                                                    | JfConjugate Antibody/Pani/Magnetic nanoparticle

-------
 o  o0
°0°0°
     I
  Signal Measurement
                        Sandwich Complex
Discard Supernatant
    and Wash
                                                   Concentrated Sample
'YYYY
                                                Capture Pad Cross-section
              = Non-target  A = Antibody modified EAM V = Polyclonal antibody to  |
              '        "^ nanoarticles         B. anthracis      ' m
Antibodies and Bacterial  Isolates
   Antibodies
    •  Purified mouse monoclonal anti-E.coli OisyiHy (OEM
      Concepts)
    «  Purified goat polyclonal anti-E.coli OisyiHy (Kirkegaard
      & Perry Laboratories Inc.)
   Bacterial Isolate
    •  E.coli OlsyiHy 03000
Normalized Results of kOhm Output fitter 6min.
1 2 7.0%

£ 0.8 -

o °'4
o 0.2 -
1 0

cutoff
r


/
— " —
^^^ t n
= = =0= = = -F if
Blank 10^9 8 7 6 5 4 3 2 1
cfu/ml
   Data can be analyzed as a positive or negative detection based on concentration
   averages or individual readings
   Negatives seen at ioA6 and ioA2 cfu/ml; has fewer recorded data points
                                                                                      Seeded Water SampTeT
                                                                                      Cell Concentrations
Cumulative Total




c



•


* *
• * * ,,.»***'




1.00E+00 1.00E+02 1.00E+04 1.00E+06 1.00E+08 1.00E+10
cfu/ml
                                 27 positive samples ranging in concentration of id4-10^ cfu/ml
                                 70% cut-off
                                 89% true positive; 11% false negative
                                 Cumulative signal taken 2, 4, & 6 min after sample application
                                                                                                             Seeded Water Samples'
                                                                                                             Cell Concentrations
                                       ith Various
Cumulative 6 min



E 0.800 •
w 0.600





* ,
* *!*•'••**/** *
*



1.00E+00 1.00E+02 1.00E+04 1.00E+06 1.00E+08 1.00E+10
cfu/ml
        27 positive samples ranging in concentration of id4-10^ ctu/ml
        70% cut-off
        81% true positive; 19% false negative
        Detection signal taken 6 min after sample application

-------
Key Results
                            Proposed Alternative Design:
                            Screen-printed carbon electrode (SPCE) biosensor
  Sensitivity studies need to be continued.
  Can not currently quantify the concentration of
  bacteria in the sample because of observed hook
  effect due to cell crowding and variances between
  testing.
  The overall time interval from obtaining a sample
  to readout with the biosensor is < 20 minutes.
  Biosensor design and parameters need to be
  modified/improved to minimize false negative.
                                Results  By Objectives

                                » Develop a protocol for processing water samples for the
                                  biosensor and QPCR.
                                • Assess the performance of the biosensor and qPCR for
                                  sensitivity, specificity, recovery, and false
                                  positives/negatives of detection and enumeration forE. coli
                                  OlsytHy and H. pylori in groundwater samples from the
                                  field.
                                * Develop a method for detecting and enumerating E. coli
                                  OyyiHy and H. pylori by QPCR using bacteria isolated and
                                  screened by the biosensor system.
                                  Validate a method for testing viability of E. coli
BacTiter-Glo™ Microbial Cell Viability Assay
                                                                    Comparison of Noise Levels as a Result of Diluents
  Concentration of £ coli Cjooo (ATCC
  #15597) by centrifugation


  Antibody separation
   •  Goat-derived, polyclonal, biotinylated
     antibody (Meridian Life Sciences, Cat#
     6651096)
   •  Magna-Sphere streptavidin-coated
     magnetic beads (Promega Cat # 75481),


  The BacTiterTM Microbial Cell Viability
  Assay (Promega Cat#C8z3o)


  Greater numbers of positive results
  compared to the standard methods
     Likely due low specificity
.-.„,.»,. •
                                         Serial Dilution 1DA
nTSB Diluted w/TSB


• TSB Diluted ill Peptone


a Peptone Diluted w,< TSB


 i Peptone Diluted w/
 Peptone

• li'H Diluted «; MH


a MH Diluted w/ Peptone
                             TSB resulted in a loss of detection at a dilution of 1O8, while E. coli in both
                             MH broth and peptone water were significantly positive when compared to
                            Blanks.
                                                                                                                                        Comparison of Peptone and MH Broth as Diluents
a.
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(Adjusted C
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                                                                                                            Serial Dilution 10A
                                                                                                                                                                                          IBfc

-------
 Comparison of Incubation Times
               I
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        Focfuction oflvfagrie
     Separation to Assay
Sensitivity and specificity were insignificantly affected by time
  The BacTiter-GloTM assay reliably detected live E coli cells at
  D nee ntrat ions as low as 101 cfu/ml
                                                                                                                                         Although a slight decrease in sensitivity; a detection as low as 1.37x 103 cfu/ml
                                                                                                                                         remained possible with magnetic separation
Use of Portable Centrifuge and Luminometer
   Results revealed sensitivity levels in the range at the low 102 cfu/ml
Testing of Environmental Samples
                                                                       Sixty river surface water samples obtained from Ingham County Health
                                                                       Department
                                                                        •  Thirty sites
                                                                        •  Two sampling dates July 28, 2008 (week 1) and August 4, 2008
                                                                          (week 2)
                                                                        •  Both sample cohorts were cultured immediately after collection.

                                                                       Gold standard:
                                                                        •  Samples less than 300 cfu/mL considered negative
                                                                        •  Equal to or greater than 300 were positive

                                                                       Receiver operator curve (ROC) analysis performed using the gold
                                                                       standard as determined by Ingham County Health Department Data.
Surface Water Samples Week 1: One Week Refrigeration
                                                                                                                                                                    1 week refr;jcr,Viion
                                                                                                     ,-f $  $>  i?
                                                                                                                                                                                       • CPU

-------
 Surface Water Samples Week 2: One Day Refrigeration

--,---                          I rUv,' refi >'I^I'.VJI;ILI	
  Receiver Operating Characteristic (ROC) Curve Using Cutoff of
  300cfu/mL
                                                                                                  ROC Cane
                                                                                                                      	No Diagnostic Value
                                                                                                                     	Poly. (Weei 2 DGta)
                                                                                                                                                  Effect of Gold Standard Cutoff on  ROC








g '


Effect of True Value Cutoff on ROC
y = -2E-06/ + O.QG2X+ 0.1026
R2 = 0.7046

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True Value Cutoff
The resulting ROC curve (x axis=FPR; y axis=TPR) shows viability assay has little value
since the area under the curve is only 0.36. (FPR=false positive rate; TPR=true positive rate)
                                                                                                                                                   The greatest diagnostic value of the assay is noted when the gold standard cutoff is s
                                                                                                                                                   to approximately 600 cfu/mL; the area under the curve has approximately 0.76
Adoption of Gold Standard Set to 600 cfu/mL

   Cutoff level of 300 cfu/mL
    • 6 sampling sites with contradicting results
    • Contradiction between sampling location (left, center or
      right)
   Cutoff level of 600 cfu/mL
    • Decreased number of contradicting sites to one
    • Previously positive results now negative
                                                                                                                                                   Specificity Testing: E.  colivs. Salmonella
                                                                            Comparison of current and proposed cutoff levels on week 2 results: Red star indicates
                                                                            sites with contradicting results using the 300 cfu/mL cutoff; green star indicates
                                                                             intradiction with the 600 cfu/mL cutoff.
SA-PMPs Viability Trial #3
djusted CPS
s ;
01
o

nH n
§ g s
E. cdj C 3000
n-
1 i -
Salmonella

D SandanJ
1_ Vbgnstic Beads

CFU/ml

-------
Specificity Testing: E. coli vs. Salmonella, S. Aureus, and
Entemcoccus
             11
•   I	
Alternate Approach  to Viability Test
     A: Chemical-grade ATP:
                       Hexokmase
     Glucose + ATP   •      —>  Glucose 6-Phosphate + ADP + H~
     (Detectable change amperage increase)


     B: Hexokiiiase-bouud £. coli O157:H7;
                       Hexokmase
     Glucose + ATP   •      	*•  Glucose 6-Phosphate - ADP -i- H~
     (Detectable change amperage increase)
                                                                                    C: Negative Controls

                                                                                    Glucose + ATP  	
                                                                                    (No amperage increase)
                                                                                  Glucose - ATP
Output:  Papers  and Thesis
  Peer-reviewed Publications:
   • Yuk, J.S., Jin, J.H., Alocilja, E.G., and Rose, J.B. 2009. Performance
     enhancement of polyaniline-based polymeric wire biosensor.
     Biosensors and Bioelectronics Journal 24(5): 1348-1352 (available
     online at                                     in 2008).
   « Yuk, J.S. and Alocilja, E.G. 2009. Electrical characterization of
     magnetic polyaniline and bio-conjugated magnetic as molecular
     biowires. Sensors & Actuators: B. Chemical (in review).
  Thesis:
   • Arun Nayak, MS 2008; Stability And Quantitative Surveillance Of
     Helicobacter pylori And Campylobacter jejuni In Environmental Waters By
     Real Time qPCR.
 Output:  Presentations
     Nayak, A. Helic, ba, tei oylon VBNC in •,-••. :•&. Pi fsented in The 13* International Symposium on
     Health Related Water Microbiology Conference at Swansea, UK. Sept 4-9, 2005
     Sangeetha Srinivasan, Shannon McGraw, Lauren Bull, Evaiigeiyu Almjtlja, Erin Dreelin &Joan B.
     Rose. Detection of waterborne pathogens using Real Time PCR and Biosensor methods.


     Sangeetha Srinivasan, Marc P. Verhougstreate feloanB. Rose. Evaluation of Bacteroides, a new
     alternative indicator for fecal contamination. MI-ASM Branch Spring 2008 meeting at Central
     Michigan University, April n-i2, 2008.

     industry. Michigan Section, AWWA 7Oth Annual Conference. Kalamazoo, Michigan, September
     9-12, 2008
                                                                                                                               Acknowledgment
                                                                                                                                  Funding sources for outputs of this project:
                                                                                                                                   » US Environmental Protection Agency
                                                                                                                                   • Department of Homeland Security through the National Center for Food
                                                                                                                                     Protection and Defense
                                                                                                                                   • Michigan Department of Environmental Quality
                                                                                                                                  Graduate students working on this project:
                                                                                                                                   • Shannon McGraw, Michelle Packard, Sangeetha Srinivasan
                                                                                                                                  Undergraduate students working on this project:
                                                                                                                                   • Lauren Bui, Teresa Brinks
                                                                                                                                  Postdoc working on this project:
                                                                                                                                   • JongseolYuk
                                                                                                                                  Other students who are members of the Alocilja Research Group

-------
Any Question?

-------
Assessment of Microbial
Pathogens in Drinking Water
using Molecular Methods
Coupled with Solid Phase
Cytometry
     Barry H. Pyle, Associate Research Professor
  Department of Microbiology, Montana  State University
   U.S. Environmental Protection Agency Workshop on
 Innovative Approaches for Detecting Microorganisms and
      Cyanotoxins in Water, May 20-21 2009
             Philadelphia, PA    • • •    •
COLLABORATORS
Anne Camper
Susan Broadaway
Al Parker
Jo-An Lindstrom
    Montana State University
    Bozeman, MT
Tim Ford
    University of New England
    Biddeford, ME          •••
Overall Objective
   To develop and evaluate
   innovative approaches for
   quantitative assessment of
   pathogens
Target Microbial Pathogens

• Escherichia coli 0157:H7
  Helicobacter pylori
  Legionella pneumophila
  Mycobacterium avium
  Aeromonas hydrophila
  Giardia lamblia
  Cryptosporidium parvum
Procedures
•  Fluorescent in situ hybridization (FISH)
   Enhance with tyramide amplification
   Use polyamide nucleic acid (PNA) probes
  In situ nucleic acid amplification
   Specific target genes inside individual cells
   (Hodsonetal, 1995)
   Improved methods, e.g. (Notomi et al, 2000;
   Maruyama et al, 2003 & 2005)
  Membrane filtration
  Solid Phase Laser Cytometry
Solid phase laser cytometry
• Scan a 25 mm diameter  membrane
 filter in 3-4 minutes
 Detect individual fluorescent
 particles
• Discriminate between cells &  debris
 Locate particles on microscope
• Validate bacteria, eliminate other
 particles

-------
 Solid Phase Laser Cytometer

                        ChemScan
                        RDI
                        (AES-
                        Chemunex)
      «^»'-n -       ^^^^^^^^
    "^ssr   .___    |B|


    ~~
      Range of Cell Labels
 •Total Cell Count
   • Sybr Green
  Total Viable Count
    ChemChrome
    Enzyme activity
    Membrane integrity
  Identification Tests
    Antibodies
    Specific enzymes
    Nucleic acid probes
    FISH
  Dual Labeling
    Fab-CTC
    ChemChrome-Fab
    Viability substrate
    Free fluorochrome

    Specific su bstrate
Enzyme
                                                                 Nucleic acid probe  Antibody
                                                  DVC-FISH (Baudart et al, 2002)
 E. c0//O157:H7,1 mm u no-
 magnetic Beads, CTC,  FITC
   Pyleetal.,1999
CHEMCHROME V3-LABELED
Bacillus cereus
                                             Pyle et al., 2000
 B. cereus -  B183 ANTIBODY WITH
 ANTI-MOUSE TRITC LABEL
Pyle etal., 2000
B. cereus - CHEMCHROME WITH
B183ANTIBODY-TRITC
                                             Pyle et al., 2000

-------
E. coli SYBR Green vs FISH
  SYBR Green Stained     FISH with ECO-Alexa

        Images captured at same camera settings
E
pifluorescent Microscopy
SYBR Green
Log CFU/ml
5.87


6.09
FISH Eco Alexa
Log CFU/ml
5.78
6.38
6.01
6.06





Mean
• • •
•
•
•
• •
SYBR Green vs FISH Tyramide
   SYBR Green Stained
                   FlSH-HRPwithFlTC
                   Tyramide Amplification
          Images captured at same camera settings
Goal Performance Characteristics
  Detection of different target bacteria with
  specific probes
• Detection of low numbers of pathogens
  Includes VBNC bacteria
  Can include infectivity and/or virulence
  Viable or active cells
  Single cell enumeration
• Sensitivity - 1  cell per filterable volume
  Rapid - Results within 6-8 hours
Scope of  Project

  Drinking water and source waters
  Native American students at
  Little Big Horn College and
  Montana State University-Bozeman
 to participate
ACKNOWLEDGMENTS
 • U.S. Environmental Protection Agency
     Barbara Klieforth, Project Officer
  NIH Environmental Health Sciences
  NASA
  DoD - U.S. Army
  AES-Chemunex, Inc.
  LigoCyte Pharmaceuticals, Inc.,
     Bozeman
  Montana State University

-------
References
Baudart, J., J. Coallier, P. Laurent, and M. Prevost 2002 Rapid
   and sensitive enumeration of viable diluted cells of members of the
   family Enterobacteriaceae in freshwater and drinking water. Appl.
   Environ. Microbiol. 68:5057-5063.
Broadaway, S.C., S.A. Barton, and B.H. Pyle. 2003. Rapid staining
   and enumeration small numbers of total bacteria in water by solid-
   phase laser cytometry. Appl. Environ. Microbiol. 69:4272-4273.
Hodson, R. E., W. A. Dustman, R. P. Garg, and M. A. Moran 1995
   In situ PCR for visualization of microscale distribution of specific
   genes and gene products in prokaryotic communities. Appl.
   Environ. Microbiol. 61:4074-4082.
Maruyama, F., T. Kenzaka, N. Yamaguchi, K. Tani, and M. Nasu
   2003. Detection of bacteria carrying the stx2 gene by in situ loop-
   mediated isothermal amplification. Appl. Environ.  Microbiol.
   69:5023-5028.
 References  (continued)
Maruyama, F., T. Kenzaka, N. Yamaguchi, K. Tani, and M. Nasu
  2005. Visualization and enumeration of bacteria carrying a specific
  gene sequence by in situ rolling circle amplification. Appl. Environ.
  Microbiol. 71:7933-7940.
Notomi, T., H. Okayama, H. Masubuchi, T. Yonekawa, K.
  Watanabe, N. Amino, and T. Hase. 2000 Loop-mediated
  isothermal amplification of DMA. Nucleic Acids Research 28(12):i-
  vii.
Pyle, B.H., S.C. Broadaway, and G.A. McFeters. 1999. Sensitive
  detection of Escherichia coli O157:H7 in food and water by
  immunomagnetic separation and solid-phase laser cytometry.
  Appl. Environ. Microbiol. 65:1966-1972.
Pyle, B.H., S.C. Broadaway, J.T. Lisle, and G.A. McFeters. 2000.
  Improved detection of viable bacterial spores. Abstract Q-360,
  100th Annual  Meeting, American Society for Microbiology, Los
  Angeles, CA, May 21-25, 2000. (Poster). P. 624.

-------
Detecting Pathogens in Water by
  Ultrafiltration and Microarray
            Analysis

          Anthea K. Lee
    Metropolitan Water District of
        Southern California      -*•
                                                    Metropolitan Water District of
                                                     Southern California (MWD)
Consortium of 26 cities and water
districts
Provide water for >18 million people
in Southern California; 5200 square
mile service area
Delivers an average of 1.7 billion
gallons of water daily
                                                            MWD System

-------

-------
WGA for 10 ng starting material
Kit
REPLI-gUltrafastMini
(Qiagen)
NlustraGenomiphiV2
(GE Healthcare)
GenomePlex Complete
(Sigma)
expected yield*
(ug/mL)
350-500
200-350
40-93
actual yield** (ug/mL)
357
644

30
none detected
  DOP-PCR
   (Roche)
                not specified
    *Need 1-5 u.g per microarray
              WGA Results
           post-ultrafiltration
Cryptosporidium    MoBio      1.95-8.39
         ultraclean soil kit

 Adenovirus    Invitrogen    Not done yet
          Purelink Viral
          RNA/DNA kit
        10" inoculum
        17 fg
        DNA/bacterial
        cell
        Starting
        material -0.01
        ng DNA/10,000
        cells
        Scaling up
        using Midi kit
   £. co/i K12 microarray to test
     integrity of WGA products


  40 bp probes
  every 800 bp
  ~5800 probes
  cognate mismatch
  for each probe
  factory  standard
  positive and
  negative controls
          Target Preparation
       \VGApnxhM
with 
-------
        :uture Directions

Optimize UF for Adenovirus
Optimize larger scale WGA
Optimize microarray parameters
Finish infectivity studies
Design custom microarray
                                                                                                        4

-------
        Robust PEMC Sensors for Detecting
    Pathogens in Drinking Water at 1 Cell/Liter
                           Raj Mutharasan
                   Sen Xu (PhD) Yanjung Ding (PDF)
                Kishan Rijal (PhD), Gossett Campbell (PhD)
            Department of Chemical and Biological Engineering
                           Drexel University
        Innovative Approaches for Detecting Microorganisms in Water
                    Philadelphia, PA. May 20th, 2009
                                                        R833829
                                                                                 Research Objectives
  1.  Explore and establish experimentally piezoelectric-actuated millimeter-
     sized cantilever sensors suitable for detecting one pathogen in one liter
     of water using new cantilever oscillation and measurement modalities

  2.  Develop flow cell-PEMC sensor detection assembly for large sample
     volume

  3.  PEMC sensor for confirming pathogen identity by DNA signature

                         Motivation
I                                   Model parasites: Cryptosporidium parvum oocysts,
                                             Giardia lamhlia cysts
                                   Surrogates : £. co//O157:H7, £. co//JM101	
 Progress
   1.  Sensitive mode established; flow cell (version 4 designed &
      tested) model experiments with E. c«/r'O157:H7, Crypto
      and Giardia show detection limit ~ 10 — 50

   2.  Successful 1 liter samples completed using modified flow
      cell; 1 cell/mL completed

   3.  DNA-based detection of E. co//O157:H7 (rtx2gene) at
      —700 cells without amplification demonstrated in buffer
In Progress
    1.  Version-5 flow cell design and fabrication; river water
        Crypto at 10 and 100 liters

    2.  DNA-based detection of Crypto and Giardia,
Piezoelectric-Excited Millimeter-sized Cantilever (PEMC) Sensors
  Cantilever dynamics
                                                                                 Resonant frequency of Cantilever in air:
                                                                                  In liquid:
                                                                                  When analyte of mass Am binds:
           ' "*\Me+mae
                                                                                                          Am
                                                                                                               0)
                                                                                                                     PZT: Lead zirconate titanate
                                                                                                               Effective added oscillating liquid
                                                                                                                  mass
                                                                                                                  (3)
                                                                                                                                      /// r
                                                                                                                       stagnant       /// ; •
                                                                                                                       or flow liquid    ^;./.k)j^

-------
Dominant Higher modes= PEMC innovation



  Higher frequency modes are more sensitive
                „   ...    Am   kv" 1
                Sensitivity =	= —=—-
                          Af   27i2 f3
    0   100  200  300  400   500  600  700  800  900  1000 •


                     Frequency (kHz)
                                                                 Experimental Apparatus
Interface Chemistry

       APTES

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-------
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                                                                                      Coniirmation, Repeatability and quantitation S,

                                                                                                     Au-Protem G-Ab
                                                                                        - control: EL coh absent, Ab present    |
                                                                                        + control: H, coh present, Ab absent
E. coli O157:H7 in water [APTES-Immobilization]
Cryptospordium oocyst in tap water

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-------
 Confirmations w/ release & SEM imaging
                   G. lamblia cysts

Cyst.+ Glutaraldehyde + mono IgG
                                                                           Sample preparation
                                                                                  Centrifuge at 10,000 g for 5 min
                                                                                 Pellet + 50 ML 1% Triton X-100
                                                                                   10 mins in boiling water,
                                                                                   cool at 2-3 °C for 15 mins
                                                                                          £
                                                                                   Centrifuge at 10,000 g for 3 mins
                                                                                 Dilute supernatant in 10 mL TE buffer
                                                                                                                    Stock 1.5 ng/mL genomic
                                                                                                                       DNA (measured)
                                                                                                                      Diluted to 23 pg/mL
                                                                      1 mL- Shear 25X with 30-
                                                                        gauge 1/2" needle
                                                                       23 pg/mL genomic DNA
      obe based detection— Prep I  (buffer)
                  Samples


                  Sample-I
      Sensor Response
gDNA   to Hybridization  Hybridization
(pg'mL)     (Hz)     rate (min"1)
Detection of Microcystin LR in DtltCn mode
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-------
              Jorge Santo Domingo
                  US EPA
            NRMRL/WSWRD/MCCB
                Cincinnati, OH
                                                                    itoring Fecal  Pollution
                                                                       al Indicators
                  Microbial "
                     • Indicate presence of fecal pollution
       Microbial Source Tracking or
         Fecal Microbial Forensics
Use of detectable molecular variations between
related fecal microbial strains to infer the origin
 of pollution sources in afecally contaminated
                watershed
              (or food supply).
                                                           Adapted from Cindy Nakatsu; EPA's MST guide
    Library-dependent methods
1
                         to this
                                     FromDombek
                                     md others., 2000

-------
                                                         Host-Snscific PCR Asscivs
                                                              lulture-independent
                                                              Jbrary -independent
                                                             Rapid detection
                                                             Sensitive
                                                             Defined target
                                                             Automated analysis
                                                              oteutial for multiple assays
                                                              'oteutial for really cheap assays
                               16SrRNAof
                                  \m horseii

           650         660         670
             I           I           I
S. moo/7       GCU GAGU U  G AGAGG   GGUAGAAUUC
B. horseii      GCUAGAGUAU GGGAGAGGAU GGUAGAAUUC
                                                               Steps for assay development
                                                        • DNA extract from feces
                                                        • PCR amplification w/16 rDNA primers
                                                        • Cloning, sequencing, blast, and
                                                        phylogenetic analysis
                                                        • Rare groups used for assay development

-------
                                     (WWW GTC Wfl' (A8008314)
                                           ATCC
                                             ootis NCFB 2890' {XS97AS)
                                                       NCFB »75T [KTOM71
                                          ftomvai CCUG M*53T (V 107721
                                          DSU 238 1T (X87 1 M I
                                        M58tr (Y17300)
                                     *v*y«n* NCDO 2763' t XM270 j
                                          NCFB Z76&1 (X&UT1 )
                                             CCtXJ 33394^ (VIB097)
                                   fluvuft* CCUG 3270* T (Y!B098)
                                          JCM 5603r (A8012212)
                                   mtxavmui* CCM «S56' (AF 286*31
                                           LUG 13
                                               ATCC 43076
                                         ATCC 51!KT (AF06IQ07)
Multiple genes — Multiple bacterial groups
_

-------
Lu et al. Water Research 2007
                                                                                        Next steps
                                                                        Fragments are cloned and sequenced
                                                                        Sequences are classified by function and potential
                                                                        bacterial host
                                                                        Sequences associated with host-microbial
                                                                        interactions are used to develop PCR assays
                                                                        Assays are tested for host-specificity, host-
                                                                        distribution, and detection limits (both in fecal
                                                                        sources and water samples)
                                                                                            Site 1 -B-Sle2 -4-Site 3
              Lessons learned

  Detection limits can vary dramatically per fecal
  sample, host, water sample

  Host distribution can also vary considerably

  Preferential distribution and secondary habitats
  issues like E. coli

  Different markers for different sources of the same
  fecal sources

  Combination of assays best approach to enhance
  confidence levels
            Lessons learned
The more (markers) the merrier; you never know which marker will work


Survival of the targeted population is rather important

Feces might not always be the best starting point for assay development
There is unknown bacterial groups that might be used for assay
development

Abundance of host-specific populations can vary

-------
           Regional projects

RARE Project - Evaluate MST assays in
         tropical inland waters
          Acknowledgements
USEPA Computational Toxicology Grants
USEPAWSWRD

Jingrang Lu - NRC Award
Regina Lamendella, Daniel Oerther — UC
Rod Mackie, Tony Yanarell - UIUC
George DiGiovanni - UT El Paso
Stephen Hill, Tom Edge — Environment Canada

-------
 Rapid Concentration, Detection, and
 Quantification of Pathogens in Drinking
 Water
  ZhiqiangHu, Department of Civil and Environmental Engineering
  LelaK. Riley, Department of Veterinary Pathology
  MengshiLin, Department of Food Systems & Bioengineering

  University of Missouri, Columbia MO 65211
                                                                                          Outline
Lanthanum-Based Concentration and
Microrespirometric Detection of Microbes in
Water
 a Turbidity-based and Fluorescence-based
   microrespirometry to enumerate microbes and
   determine microbial activity in water
 a Lanthanum-based microbial concentration
Rapid detection and quantification of water-borne
pathogens by SERS coupled with nanosubstrates
        Part I. Lanthanum-Based
      Concentration and Detection
               Introduction


Rapid detection of potential pathogens in water is crucial to
drinking water supplies.
 o The numbers of microorganisms in water samples are often too low to
   be detected.
Coagulation/flocculation coupled with filtration is an attractive
method for concentration.
LaCl3 is a flocculant that can  concentrate microbes by strong
electrostatic  interaction.
 o Compared  with traditional flocculants (e.g., alum  and ferric salts),
   LaCl3 only hydrolyzes slightly  in the water so that it minimize  the
   impact on microbial properties.
               Introduction

Traditional assays enumerate microbes by measuring
the turbidity of the organisms.
Oxygen-based microrespirometry, however, can
enumerate the live microbes by measuring oxygen
consumption and determine microbial activities at the
same time.
Lanthanum chloride was used to concentrate the
microbes in water before they were detected and
quantified by microrespirometry.
                                                                                Materials and  Methods
Bacterial Strain used: E. call (ATCC 47076)
Floccurants/Coagulants: LaCl3, FeCl3 and A12(SO4)3 (final concentrations = 0.2
mM).
Concentration procedures
a Mixed at 200 rpm for 1 min, followed by slowly mixing at 30 rpm for 20 min.
a The samples were allowed to settle for 1 hour.
a The supernatant fluids (75mL) were carefully removed without disturbing the floes.

-------
       Microrespirometric Detection
    Composition in micro wells
    o For every flocculant treatment, aliquots (20 uL) of supernatant or
      sediment samples were taken and added to the microplate wells
      followed by the addition of 180uL BBL medium.
    Turbidimetric assay
    o The microtiter plate was read at 600 nm.
    Microrespirometric dectection
    o Oxygen probe and mineral oil were added.
    o Time-resolved fluorescence measurements were recorded with 340 nm
      excitation and 642 nm emission
  Time profiles ofE.coli growth at
  different initial cell concentrations
                                                                   Turbidimetric assay
                                                                                           Microrespirometric assay
 Strong Correlation between Bacterial
 Concentration and Time to Threshold
Concentration study Using turbidimetiic
                     assay
                                                                                              i
                                   ii
                                                                                          Concentration efficiencies and recovery rates of
                                                                                                different treatments
                                                                 Time profiles of absorbance of different samples
       Concentration Study based on
          microrespirometiic assay
Time profiles of oxygen probes signals of differ
                                     ^OHt^lf^lmt
                                     it]
                                    i     i
                               nitration efficiencies and rec<
                                   different treatments
       Bacterial Distribution Using
            Different Flocculants
                                                                      Turbidimetric assay
                                                                                             Microrespirometric assay

-------
Effect of flocculants (LaCl3, FeCl3 and
A12(SO4)3) on E. coll bacterial growth
| Microscopic (ESEM) Examination of
 Floes with Different Chemical Treatment
                 Summary
  Compared with traditional flocculants, LaCl3 has the
  highest  relative   concentration   and   recovery
  efficiencies. The  lanthanum-based method coupled
  with  ultraiiltration provides  a promising  pathogen
  concentration method for water utilities.
        Part II. Rapid detection and
        quantification of water-borne
        pathogens by SERS coupled
             with nanosubstrates
    Surface enhanced Raman spectroscopy
                  (SERS)
  When analyte molecules are adsorbed on metal surface with
  nanoscale roughness, Raman signal can be tremendously
  enhanced due to spatially localized surface plasmon
  resonance (SPR) from the "hot spots" where huge local
  enhancements of electromagnetic field are obtained.
  The enhancement factor can be more than 106. Limit of
  detection can reach the parts per billion (ppb) level or possibly
  a single molecule

                                                                          , .^j.-.-.1 .V1--J. ..-
      Renishaw RM 1000
                             Klarite™ substrates
    A Renishaw RM1000 Raman spectrometer system with 785 nm near-
    infrared diode laser source;
    Gold substrate (Klarite): fabricated on silicon wafers coated with gold,
    nanotextured pyramidal subunits.

-------
Objective
  To develop and validate SERS-based method
  for pathogen detection and quantification.

• Several species representing the major
  categories of pathogens in drinking water
  were chosen for SERS testing:
   a Enterococcus faecalis
   a Helicobacter pylori
   a Human adenovirus
   a Calicivirus
   a Encephalitozoon cuniculi
   a Eco//0157:H7
 —o-Cryptosporidium parvwn—
r

=>
i
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1
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Four vims strains
A
/ TA,^'-^/-v^^rA-^^A«^'^/
. .-, ; ,
A
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— \ .

' •* ^-^-^x.^^ s*~-—~ -^ ^' -A— - -^
— 	 -^ — —
AAxA -JVAj-w^J
Wavenumber (cm-1)
N orwal k virus
MMV4
j/Wr^^.

MAD1
/*v~.

SA11
— ,/V 	 ^---

^~H™
_
• SERS spectra of four virus strains show "fingerprint-like" spectral
patterns that can be used to classify and identify these strains; gold
nanosubstrateswere used in measurement.
                Bacteria
 <

 t
 I
                                    Enlerococcus
                                    faecafis
                  Raman Shift (cm*1)
     Distinctive SERS spectral patterns were observed between
     three waterborne bacteria
          Bacteriophage MS2
                                                                                                      —•-^H
t '^S^\J\*^**<*t»*<^^
                                                                    We also collected SERS spectra of
                                                                    bacteriophage MS2 on gold nanosubstrates
       400 600  800   1000  1200 1400 1600 1800
                Wavenumber (cm'1)

    Three bacterial pathogens exhibit different SERS
    spectra that can be used to identify them
                                                                    Principle Component Analysis (PCA)
                                                                            of Cryptosporidium spp.

  Non-viable C. parvum
Clear data segregations
were obtained between C.
muris and C. parvum; and
viable and non-viable C.
parvum.

These results indicate that
SERS can be used to
identity and discriminate
between different
Cryptosporidium oocysts
as well as viable or not
based on their unique and
distinct vibrational spectral
information.

-------
    PCA of GD-7 (Picornavirus) and

           MNV-4 (Norovirus)
                              •  PCA was able
                   MNV-4         to classify two
                   .,-	••••,      virus stains:
                /""       \     GD-7
               / •         ,/     (Picornavirus)
               >'          /      and MNV-4
               ..        ,/       (Norovirus)
GD-7

Band assignment of Raman peaks in the
range
Raimii Still1 i .-11
- 340
-620
-540
-665
-126
-783
-853
-936
-977
-1005
-1035
-1101
-1128
-1252
-1340
-1453
-1577
-1665
-1735

of 3 00 -2200 cm-1
laagmnent
COC gl>cosi die ring def
Anino acids (Phe)
Nudetc acids £1)
Nucleic acids (G)
Nucleic adds (A)
Nucleic acids (C,T)
TViosine
DMA bad±x>ne
Lipid (C=C deformation)
Phenvlalanine
Carbohvdrates(C-C deformation)
DNA CO-P-Q stretching)
C-N stretching
Amde IE
Nucleic acids (A, G)
Lipid (C-H; deformation)
Nudeic acids (A, G)
Amide I
>C=0 eser str
(Maquelin and others 2002)

                 Summary
SERS coupled with nanosubstrates and statistical
tools shows great potential to rapidly detect and
identify different water-borne pathogens.
                                                       Acknowledgements
                                                                   Funded by EPA STAR Program
                                                                            (#83384001)
     The effect of flocculation on pH
   Before
   flocculation
   After      La31
   flocculation
   0.2mM
   1mM
   5mM
     7.03
     6.62

     5.43
Fe3*


7.00
6.54

2.44
Al3*


7.05
6.63

4.19

-------
  Simultaneous Concentration and Real-
   time Detection of Multiple Classes of
 Microbial Pathogens from Drinking Water
            Prof. Mark D. Sobsey
Department of Environmental Sciences and Engineering
      Gillings School of Global Public Health
          University of North Carolina
          Chapel Hill, NC 27599-7341
            Objective 1

Refine and validate new and improved,
rapid hollow fiber ultrafiltration
methods to concentrate viruses and
cellular pathogens (bacteria and protozoan
parasites) from waters of variable quality
-Particles
- Dissolved organic matter
Compare to existing virus concentration
methods (1MDS VIRADEL)
             Objective 2

 Fabricate (or identify) and evaluate
 improved and cost-effective
 electropositive filters to rapidly and
 efficiently concentrate enteric viruses from
 waters of different quality by adsorption to
 and elution
 - Nanoceram cartridge filter (Argonide)
 Compare to existing virus concentration
 methods (1MDS VIRADEL)
            Objective 3
Improve and evaluate post primary concentration sample
preparation techniques:
- Rapid PEG precipitation
- Post PEG precipitation treatments to improve virus
  detection by quantitative real-time (RT-)PCR
- Large volume nucleic acid extraction
Further concentrate viruses
Remove inhibitors
Facilitate efficient, specific, and sensitive real-time,
molecular detection of viral nucleic acids
- Human adenoviruses
- Human enteroviruses
- Human noroviruses
             Objective 4

 Improve and optimize direct detection of
 viral RNA/DNA by real-time molecular
 methods for rapid and efficient detection
 of low numbers of target viruses
 - Sample volume per (RT-)PCR reaction
 -Additives to (RY-)PCR mixtures
            Objective 5
 -develop complete protocols of the methods
  and provide them to a select number of other
  water virology laboratories to conduct a
  collaborative (round-robin) test of the methods
  that characterizes their performance; and

-------
        Concentration of Adenoviruses,

  Noroviruses and Echoviruses from Water
 • Primary concentration
    - Recirulating flow hollow fiber ultrafiltration
       • 2 brands of filters
       • Modified endcaps to increase flow rate/flux
       • Alternative beef extract elution solutions
       • Performance in waters of different quality (source and treated)
    - Once-through, gravity-flow hollow fiber ultrafiltration
    - Nanoceram electropositive adsorbent filter
       • Nano alumina (AIOOH) fibers
       • Virus concentration from seawater
 • Secondary concentration
    - Polyethylene glycol precipitation
       • Effect of PEG and NaCI concentrations
                                         Recirculating HFUF Methods and Materials

                                         Hollow-fiber ultrafilters (HFUF):
                                            - Fresenius F80A
                                               • (Fresenius Medical Care, Lexington, MA)
                                            - Hemocor HPH
                                               • (Minntech Corporation, Minneapolis, MN)
                                         HFUF flow modifications:
                                            - Modified end caps with larger diameter openings
                                            - Increased flux for more rapid sample processing
 Recirculating  HFUF Methods and Materials

>  Test water: > 10-liter volumes of untreated source and de-chlorinated
  finished waters (SFPUC: San Francisco Public Utility Commission)
•  HFUF units: ca. 75,000 MWCO, designed for kidney dialysis
>  Peristaltic (flexible tubing roller) pump to re-circulate water through
  the unit
>  As water re-circulates, permeate is separated from retained particles,
  concentrating particles, including microorganisms, to <300 ml volume
Recovery of Adenovirus 2
• Hollow Fiber Ultrafiltration
-Virus assay by cell culture infectivity





140% -
120% -
100% -
80% -
60% -
40% -
20% -


T-


i

pAdV 2 source (inf.;n=3) BAdV 2 tap (inf;n=6)






   HFUF Recovery of Adenovirus 41
   Eluting solution comparison for Ad41 recovery from
   HFUF primary concentrates
Eluting Solution 1 (Standard)
1 L Phosphate-buffered Saline (PBS)
10 g laureth-12
50 |iL antifoam-A

Eluting Solution 2
1LPBS
10 g laureth-12
1 g NaPP
50 uL antifoam-A

Eluting Solution 3
1 L reagent water
52.7 g L-Arginine (A-5131) (0.25 M)
45.65g L-Lysine (L-5826) (0.25 M)
10 g laureth-12
50 |iL antifoam-A
eluting. solution eluting solution eluting solution
    1        2        3
                                           HFUF Recovery of Adenovirus 41

                                           Lower spike virus concentration (105/10L) (Left)
                                           Recovery from large volume (100L) (Right)

-------
HFUF Recovery of Pathogenic Microbe Suite
•— "S3S
£co// 01 57
Salmonella
Aeromonas
Echovirus-12
Cryptosporidium
Giardia
500
500
500
2000
20
20
Source Water
Average
Trials (N) Recov.
3 52±6
3 85±13
3 11 ±3
3 49±45
3 29±11
3 9±3
Drinking Water
Trials Average
(N) R^V'
3 44±12
3 117±27
3 7±5
3 ND
3 28±6
3 15±8
ND = No Data (eluting solution 2 with NaPP was toxic to cell cultures)
Bacteria, Virus and Spore Recovery from Treated OWASA Water
(10L) by Conventional & Modified Fresenius F200A HFUFs
Organism
E. coll KOI 1
Coliitapc
MS-2
Bacillus
(itmplineus
NoSlp.ififaiil
Bacillus alrop
O
Flowratc
(L/min)
0.17±0.02j
iiivenlioi
Trials
(N)
6
6
5
d
Avcrara
Recovery
(%)
112136
109tlS
71±19

Flownuc
(L/min)


Modified
Trials
(N)
13
13
13
Average
Recovery {%)
60121
85±12
57±L3
Difference by Mum Whitney Tesl for E, coli. coliphage MS-2 and
meia; p values of 0.0874, 0.5789, and 0.5663, respectively
Flow rate was significantly greater for HFUFs with modified endcaps
(Mann Whitney Test; p value <. 0001)
    Microbe Recovery from Water

 using Once-through Gravity HFUF

•  Gravity flow HFUF, ca. 30 cm long, 2 cm
  diameter, 20 nm pore size filter
•  10 L volumes of dechlorinated drinking water
•  Spike with high concentrations of E. coli K011
  (bacterium), coliphage PRD-1 (indicator virus),
  and spores of Bacillus atrophius (protozoan
  surrogate)
•  Filter by gravity flow (1 meter head) or with a
  peristaltic pump
•  Recover test microbes from filter by backflushing
  with buffered elution solution
  - Used two successive flushes of ca. 250 ml each
 Microbial Recoveries from  10L Volumes

    of Water by Once-through HFUF

• Average recoveries by gravity flow:

  -E. CO//K011 =90%,

  -PRD-1 -100%

  - Bacillus atrophius spores = 74%

• Recoveries using a peristaltic pump:

  -E. CO//K011 =48%,

  -PRD-1 =~100%

  - Bacillus atrophius spores = 52%
 PEG (Polyethylene Glycol) Precipitation of

        Viruses in HFUF Retentates

• Widely used for virus concentration
  - Protein precipitation
• Minimal virus inactivation; no extreme pH changes
• Secondary virus concentration methods need to be
  compatible with detection by both molecular and
  infectivity methods
• PEG precipitation has not been adequately
  evaluated or optimized for Adenoviruses,
  Noroviruses and Echoviruses
  - Evaluate effects of PEG and NaCI concentrations
    on method recovery of these viruses from  HFUF
    retentates and adsorbent filter eluates
Effects of PEG & NaCI Concentrations on Adenovirus
Recovery from Treated and Source Water Retentates
^^^^Hree
6%
9%
^^^Hl2*
^^^Hm
PEG
B*
9%
12%
15*
NV:
Q1M
0.1U
L. 1M
0.1M
D.3U
0.1M
C »1
-.,:
i"
<"
Ad 41
N Pefel suptmalart
2 6H16 1t>*1
107183 12ii
loataa 12*5
92i3 fl±8
19172 19±?3
Sill fi±S
2 818 «45
ii; i'
N PMM M^wmMMl
2 1417 7li
2 12S1J5 22*21
1U 2 411 2t2
3M 2 50±2I Itl
1U 2 7*8 Itl
3U 2 55*32 OtO
iu 2 :n oto
3M 1 2 2U21 OtO
M2
N pBH *jO*rfillfl(lt
2 3*130 MI 3 Treated
I 63166 7l \A/o-.-^r
i t..n « WatCr
; at?; tt \
2 SUS-i 11
I S1S7 3±
i »iu si
? 3ttK it 1
iu
N paw HfmtUH
i «n i«n Source
i wia «o wn+rr
» HIM vvaicr
2 5711D MO
2 ailM OtO
2 28111 OtO
2 SHU OH)
2 1H1S MO

-------
    Echovirus 12 and MS2 Recovery (%)
  by Different PEG Precipitation Conditions
  Conclusions for PEG Precipitation
        from HFUF  Retentates
  Effective for secondary virus concentration
  Higher virus concentratons in PEG pellets than in
  supernatants after centrifugation
  PEG-cpncentrated PEG samples were compatible
  with virus detection by both molecular and cell
  culture infectivity methods
  Overall, 9% or 12% PEG with either 0.1 or 0.3 M
  NaCI are effective conditions;
  - 0.3 M NaCI better than 0.1 M for Ad 41 in  source water
  Virus recoveries by PEG precipitation were more
  variable from source water retentates compared
  to those from drinking water retentates
 Argonide  Nanoceram Electropositive Filter

 •  Nanoceram filter (Argonide Corporation, Sanford, FL)
 •  Recently developed electropositive filter
 •  Reportedly unaffected by pH and salinity of water
 •  Made from nano alumina (AIOOH) fibers, 2 nm diam. 8 0.3 urn long;
   grafted to microglass fibers; made like paper; 5" pleated cartridge
 •  External surface area about 500 m2 per gram of material to provides
   a large area for adsorption of electronegative particles
Nanoceram alumina fibers
     Filter and  Water Sources

  Nanoceram filter and filter housing
  Challenge with 40 L of viruses-seeded water
  1010 PCR units of adenovirus
  1010 RT-PCR units of coliphage Qp
  106 RT-PCR units of Norovirus Gil.4
  106 murine norovirus
  Source and finished water from drinking water
  treatment plant in Carrboro, NC.
  Finished water dechlorinated with sodium thiosulfate
  Filter at 25 L/min
  Beef Extract Elution of Adsorbed Viruses

 • Elution medium: 3% BE (Powder, Becton-
   Dickinson and Company, Sparks, MD), 0.1
   M glycine and with the pH adjusted to 9.5.
 • A 500 mL volume was recirculated through
   the cartridge filter using a peristaltic pump
   at a flow rate  of 1.25-2.75 L per minute
 • Flow direction changed every 5 min
 • pH  monitored
 • Final eluent adjusted to pH 7.3
 Viral  Nucleic Acid Extraction

•  Chemical extraction from 100 |iL sample
  volumes
•  Guanidinium thiocyanate (GuSCN) extraction via
  Boomet al. (1990).
•  Extract applied to a HiBind RNA minicolumn
  (OMEGA Bio-Tek, Doraville, GA) and
  centrifuged at 16,000 x g for 1 minute.
•  Columns with nucleic acid washed 2X with 75%
  ethanol
p  Nucleic acids eluted from column with nuclease
  free water
•  Stored at -80° C until analysis.

-------
 Virus Quantification by  Real-Time PCR

 Previously described real-time PCR quantification:
  - adenovirus 41 (Jothikumar et al, 2005)
  - norovirus (Jothikumar et al, 2005)
  - murine norovirus (Bae and Schwab. 2008)
  - coliphageQp (Kirs and Smith. 2001)
 Quantitech probe PCR & RT-PCR kits (Qiagen, Valencia, CA)
  - Reaction volume = 25 u,L; 2 u,L of extracted viral nucleic acid.
 Smart Cycler thermocycler (v. 2.0c, Cepheid, Sunnyvale, CA).
 Calibration curve used to calculate virus particles (VP) based on cycle threshold
 value (Ct) created from ten-fold serial dilutions of viral stocks
  - Adenovirus: VP/2u,L = 10(-0.2814 * Ct value + 12.256) (R2 = 0.9986)
  - Norovirus: VP/2u,L =10(-0.2726 x Ct value + 10.362) (R2 = 0.9988)
  - Murine norovirus: VP/2jiL = 10(-0.239 x Ct value + 10.41) (R2 = 0.990)
  - QfJ: VP/2u,L = 10 (-0.306 x Ct value + 13.266) (R2 = 0.996)
 Total VP calculation: Total VP =VP/2u,L x 250 x vol. of spike, filtrate or BE solution
 (in ml)
 Adsorption efficiency: [1-(total VP in the filtrate/total VP in the spike)]*100
 Elution recovery: (total VP in eluentftotal VP in spike)*100
                                                   Virus  Recovery from Source Water
                                                            using Nanoceram Filter
Virus
Adenovirus 41
QpColiphage
Murine
Norovirus
% Ads.
81% (± 2.4%)
53% (± 29%)
74% (± 18%)
% Recovery
2.4% (±0.48%)
10% (± 2.8%)
9.8% (±3.3%)
# Trials
4
4
3

Virus Recovery from Finished
Water using Nanoceram Filter
Virus
Ad 41
Qp coliphage
Norovirus
% Ads.
97% (± 2.1%)
95% (± 0.86%)
ND
% Rec.
1.4% (±0.59%)
36% (±20%)
26.8%
# Trials
8
8
2


                                                                             Effect of Tween 80 on  BE Elution of Norovirus
                                                                                  Gil.4 Adsorbed to Nanoceram Filters


                                                                           Elution of noro GII.4 using 3% beef extracts and a peristaltic pump
                                                                                         Estimated
                                                                                       norovirus input
                                                                             Elution replicates
                                                                               % recovered
                                                                                    Average %
                                                                                    norovirus
                                                                                    recovered
                                                                 3.5X106     86%  88%  133% 139%   111% (±29%)


                                                                 3.5X106     95% 140%  99%  141%   119% (±26%)
                                                                              3% BE,
                                                                           0.1% Tween 80
                                                                              3% BE,
                                                                           0.01% Tween 80
                                                                 3.5x10s
                                                                          99%  53%  103%  98%    88% (± 24%)
   Ad41 and Norovirus GII.4 Recovery by PEG
   Precipitation from Nanoceram Filter Eluates

 Mean % recovery of Ad 41 and noro GII.4 from eluates by PEG precipitation
                           (n=3)
              6% PEG
              0.1 M NaCI
 6% PEG
0.3 M NaCI
 9% PEG
0.3 M NaCI
 9% PEG
0.3 M NaCI
Adenovirus 41   1.7% (±0.14%)  2.9% (±1.0%)   36% (±2.3%)   39% (± 6.6'

Norovirus GII.4   5.6% (±1.1%)   5.4% |± 0.46%|'^52% |± 7.8%)   59% |± 4.8%^
  Higher mean % recoveries of both viruses using 9% instead of 6% PEG
  (unpaired (-test, p < 0.05)
  Mean % recoveries not significantly different between 0.1 M and 0.3 M NaCI
  for Ad41 (unpaired (-test, p = 0.078) or Noro GII.4 (unpaired (-test, p = 0.122)
(RT-PCR)  Inhibitor Removal and
   Control in  PEG Concentrates

Substances in virus concentrates inhibit PCR
 - Humic and fulvic acids
 - Other organic compounds
    •  proteins, polysaccharides, polyphenols, glycoproteins, etc.
 - Metals
 - etc.
Quantitative real-time PCR is especially
sensitive to such inhibition
Various methods are  available to separate
viruses and viral  nucleic acids from inhibitors

-------
 Sample Processing Steps at which to
   Remove/Separate/Block Inhibitors
  \1
                                    qPCRrmx

        • Prior to nucleic acid extraction

        • During nucleic acid extraction

        • After nucleic acid extraction

        • During nucleic acid (RT-)PCR amplification
    PEG Samples and Viruses

  PEG concentrates from 40-L water samples processed by
  Nanoceram filter adsorption-elution (beef extract)
  3 mL of composite concentrate, added 10 |jL of adenovirus,
  norovirus, and MS-2 stocks
  - virus levels: 9.2x108, 2.8x104 and 5.2x108 PCR units
  Viruses also spiked into 3 mL of PCR grade deionized (Dl)
  water. (Dracor) as a inhibitor-free control sample
  Both PEC concentrate and Dl control processed
  qPCR CT values of PEG and Dl control samples were
  compared to calculate ACt values
  - ACt = CTSamp,e - CTD|Contr0|
  - Smaller ACt: less inhibition
  - Larger ACt: more inhibition
 Treatments before NA Extraction with GuSCN

• Sephadex G-200 column chromatography
  - High salt TE buffer to prepare columns
  - Biospin polypropylene columns
     • Bio-Rad Cat. #732-6204, 3 cm, 0.8 ml capacity
  - 1 mL polypropylene syringe column (BD) with
    sterile glass wool (Supelco)
• Chelex 100 + Sephadex G-200 columns
  - Chelex in bottom half; G-200 in top half
 Modifications during nucleic acid extraction

• GuSCN extraction of different sample volumes
  -400, 300, 200, 100, and 50 ul samples
• Chloroform extraction of 300 uL & 100 uL
  sample volumes
  - 1:1 volume ratio
• Polyvinylpyrrolidone (PVP)-GuSCN extraction
  - 1% final concentration of PVP in sample-GuSCN mix
  Post-extraction Modifications

  Isopropanol precipitation of NA Extract
  - Sample NA extract supplemented with Na
    acetate and isopropanol; centrifuged; NA ppt.
    washed with 70% EtOH; centrifuged; NA ppt.
    dried, then resuspended in water
            qPCR Methods
Adenovirus: JTVXF primer, JTVXR primer, JTVXP
  probe
  - Jothikumarand Cromeans (2005).
• Norovirus: JJGII primer, COG2R primer, Ring2-
  TP probe
  - Jothikumar and Lowther (2005)
• MS-2: ms2ks2 primer, ms2ks1 primer, ms2ks3
  probe
  - Bae and Schwab (2008)
• Smart Cycler (Cepheid)

-------
   Modifications to qPCR Mix
 Add PVP
 Add PVP and glycerol
 Add Bovine Serum Albumin (BSA)
                                                      Fluorescence Spectre-photometry:
  Fluorescent excitation emissions matrix
  (EEM) to quantify dissolved organic matter
  Detects and differentiates humic acids,
  fulvic acids, tryptophan and other potential
  organic inhibitors
  Sample run included quinine hemisulfate
  stock solutions for calibration and reagent
  grade water for comparison and
  background subtraction
EEM Peak Regions, Based on Excitation (Y-
 axis) and Emission Wavelengths (X-axis)
   StU  33J  340  3W  J8t'  -KM  43)  +4P 4«U 48U 5(10  5U.I  54V
 Treatments for qPCR Inhibitors

• No treatment before, during, or after extraction
  of viruses concentrated from water samples
  improved viral detection by qPCR with the same
  effectiveness for adenovirus, norovirus, and MS-
  2 in PEG concentrates of surface water samples
• Different methods or treatments may be needed
  for each type of water sample and virus.
• Specific treatments were more effective in
  lowering delta ct values for qPCR detection of
  viruses in many of the samples.
qPCR detection of three viruses in different water
sample volumes subjected to chloroform extraction
relative to detection in reagent water

Modification
CHCI3300
CHCI3100
Adenovirus
AW
value
7.23
2J6

AW
stdev
0.539
0.309

P
<0.01
<0.01
Norovirus
AW
value
8.62
3.34

AW
s*
dev
0.992
0.479

P
<0.01
<0.01

AW
value
5.50
5J5

MS-2
AW
s*
dev
0.334
0.715
P
<0.01
<0.01

Comparison of different surface water sample volumes subjected
GuSCN extraction for differences in qPCR detection of adenovirus
and norovirus relative to detection in reagent water


Sample
Volume
•WOfji.
300 fji.
200 jiL
100 iiL
SOfji.
Quasi-Point Source-Impacted Water
Adenovirus
ACt
value
584
480
496
533
437
ACt
stdev
050
068
082
085
178
P
<001
<001
<001
<001
00116
Norovirus
ACt
value
757
791
652
15i
ML
ACt
stdev
0268
206
0759
07CO
0770
P
<001
<001
<001
QQ64Q8
<001
Non-point Source-Impacted Water
Adenovirus
ACt
value
571
779
510
500
418
ACt
stdev
057
279
034
935'
062
P
<001
00113
<001
01280
<001
Norovirus
ACt
value
757
303
268
132
010
ACt
stdev
028
052
086
260
021
P
<001
<001
<001
Q55QQ
06465


-------
 Most Effective Sample Treatments

•  Sephadex G-200 followed by chloroform extraction
  - Best for adenovirus in NPS water sample
  - Best for MS-2 in quasi-PS water sample
•  Chloroform extraction alone
  - Good for norovirus in NPS water sample.
  - Best for MS-2 in NPS water sample
•  GuSCN extraction of smaller sample volume
  - Best for norovirus in both samples
•  Sephadex G-200 and Chelex 100 treatment
  - Best for adenovirus in quasi-PS water sample
                                             Overall Summary

                                    Primary virus concentration by improved
                                    recirculating UFUF is effective and rapid
                                    Primary virus concentration by once-though
                                    HFUF shows promise
                                    Primary virus concentration by Nanoceram filters
                                    is effective and very rapid but less effective than
                                    desired for adenoviruses
                                    PEG precipitation is effective for 2nd step virus
                                    concentration
                                    PEG sample treatments prior to nucleic acid
                                    extraction reduce sample inhibition and improve
                                    virus detection by qPCR
              Thank-you!
  Questions? Comments? Suggestions?
   Collaborators:
   Erik Andersen
   Lisa Casanova
   Christopher Gibbons
   HeeSukLee
   David Love
   Roberto Rodriguez
   O.D. Chip Simmons III
   Lauren Thie
   Jan Vinje
   Jianyong Wu
   MingJingWu
Additional $ Support:

AWWARF

NOAA-CICEET; NERRS

NWRI

SCCWRP

UNC Sea Grant
                                                                                                                   8

-------
    I KKX1MWNC1
      Quantitative Assessment of
      Pathogens in Drinking Water

          Kellogg J. Schwab Ph.D.
          Johns Hopkins University
          Bloomberg School of Public Health
          Department of Environmental Health Sciences
Microorganisms in Source and Finished Water

 Microbial contaminants can be divided into 3 categories:
        1.   Parasites
        2.   Viruses
        3.   Bacteria

 KEY concepts to keep in mind
        1.   Size of the microorganism
              Parasites > Bacteria » Viruses
        2.   Resistance to environmental degradation and chemical
           inactivation
              Parasites > Viruses » Bacteria
Waterborne Pathogens and Gastroenteritis
 Etiologies of Waterborne Outbreaks, 1991-2002
                                •On average, between 1991 and
                                2002, 17 waterborne disease
                                outbreaks (WBDOs) were reported
                                annually.

                                •38% of outbreaks had an unidentified
                                etiology

                                •WBDOs were primarily associated
                                with inadequately treated water
                                systems and contamination issues
                                related to aging distribution systems

                                •In some instances, the water systems
                                were in compliance with current
                                water quality standards
 Waterborne Pathogens and Gastroenteritis
    •  Multiple Factors Influence Reporting of AGI
       - Public awareness of waterborne illnesses
       - Local requirements for reporting cases of particular diseases
       - The surveillance and investigative activities of state and local public
         health and environmental agencies
       - Availability of and extent of laboratory facilities


    •  Current waterborne disease surveillance system is passive
       - Waterborne disease outbreaks are likely to be under reported
       - Endemic waterborne disease risk in the United States is not well
         understood
   Why  is  all of this of  interest?


  One of the major limiting factors in  assessing
  microbial loads in source and treated drinking
  water has been the lack of an effective microbial
  collection method capable of efficiently and
  simultaneously recovering low levels of
  bacteria, viruses and protozoa, which then can be
  identified and quantified rapidly with or without
  cultivation.

-------
           Research  Objective

Develop rapid, sensitive recovery and
detection  methods for the quantitative
assessment of pathogenic microorganisms
present in drinking water.
  Microbial Recovery
   Develop and optimize sensitive concentration and
   isolation methods utilizing filtration technology
   capable of simultaneously recovering low levels of
   protozoa, viruses, and bacteria from large volumes of
   water.

   • Demonstrate ability of tangential flow filtration (IFF) to
    efficiently recover/concentrate intact microorganisms from water

   • Determine lower limit of detection for each class of microorganism
Steps for Procc
I X
jssing 1 to 1,000 L Water Samples
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,0 microbial


                                                                    Microbial Surrogates Utilized in Method Evaluation


                                                                     •Vegetative Bacteria
                                                                       - Escherichia coli CN-13

                                                                       - Enterococcus faecalis

                                                                     •Spore-forming bacteria
                                                                       - Clostridium perfringens

                                                                     • Bacteriophage
                                                                       - MS2

                                                                       - PRD1

                                                                     • Viruses
                                                                       - Murine norovirus (MNV-1)
  Pathogen Detection

  Develop rapid, quantitative molecular detection
  techniques for the identification of target pathogens
  including direct comparison with existing traditional
  culture methods.

   • Optimized FISH methods for the identification of protozoa.

   • Developed mass spectrometry (MS) methods for the identification of select
    microorganisms.

   • Refined qPCR and qRT-PCR assays for the detection of select
    microorganisms.

   • Developed loop-mediated isothermal amplification (LAMP and RT-LAMP)
   for the detection of select microorganisms.

   • Em ployed the use of internal standard controls for the detection of PCR
    inhibition caused by molecular inhibitors present in water samples.
Fluorescent In Situ Hybridization (FISH)

  • Employs a fluorescently labeled oligonucleotide probe
   targeting species-specific sequences of 16S rRNA

  •rRNA
     - Exists in multiple copies
        • Present in high copy numbers
        in viable cells
     - Single-stranded regions allow easy
     access for the probe and natural signal amplification

  • Hybridization
     - Probes recognized by fluorescent antibodies

  • Observed under epifluorescence microscope

-------
   Detection using Mass Spectrometry (MS)
Detect capsid protein
 -  Multi-copy
 -  Uniquely identifiable
Digest with protease (trypsin)
 -  Spike peptide standards


Chromatographically separate
peptides and then determine
amino acid sequence


Search masses against genome
databases (e.g. NCBI)


Assess confidence-based score


Quantification
                                                                           MS Key Findings - Norovirus
                                                                             The NV capsid protein is detectable in the clinical range
                                                                             using MALDI-TOF MS

                                                                             Clinical sample complexity requires a more nuanced
                                                                             approach (ESI-MS/MS)

                                                                             Using additional sample processing,  MS/MS methods
                                                                             can improve sensitivity by 2-3 orders of magnitude

                                                                             AQUA peptides allow for the quantification of peptides
                                                                             from capsid protein of norovirus
Molecular Methods:  Real Time PCR
            T  >•-
           * *»
                                DNA is amplified by a cycling of
                                steps:
                                 - Denaturation
                                 - Primer annealing
                                 - Primer extension

                                TaqMan™ probe technology
                                allows for real time quantification
                                of target RNA/DNA
                                 - Fluorescent probe is cleaved
                                   during extension
                                 - Target is quantified in the
                                   form of a cycle threshold (C,)
                                   value
                                                                                  Molecular Methods:  LAMP

                                                                            Loop-mediated isothermal amplification (LAMP) is a novel detection
                                                                            method which relies on auto-cycling strand displacement DMA synthesis.
                                                                            - RT may be used in conjunction for detection of RNA viruses

                                                                            Increased sensitivity and specificity compared with conventional PCR
                                                                            - Multiple primers must recognize several distinct regions on the target RNA/DNA

                                                                            Products can be analyzed in real time by measuring the increase in
                                                                           turbidity during DMA amplification.
                                                                            - Allows for real time quantification
              Field Application
 Apply tangential flow ultrafiltration and quantitative
 molecular detection to large-volume, water samples for the
 analysis of microorganisms.


• Spike environmental water samples with microbial surrogates to
  evaluate the efficiency of recovery and detection methods.

• Apply complete concentration, recovery, and detection process to
  a variety of water samples including ground water, surface water,
  and finished drinking water.

• Compare newly developed technologies for the recovery and
  detection of microorganisms in water to existing US EPA methods.

• Identify viruses that are endemic and stable in the environment and
  investigate their use as traceable markers of fecal contamination
                                                                                 Collection  of Environmental Samples
                                                                             Water sampling in Lower
                                                                             Yakima Valley, WA
                                                                              - Sampled surface water
                                                                                and ground water
                                                                                impacted by surrounding
                                                                                dairy industry
                                                                              - Application of optimized
                                                                                TFU method for
                                                                                concentration of 100L
                                                                                water samples

-------
Water Sampling in Yakima Valley, WA
   Processing
    - Applied optimized TFU in the field
    - Seeded each sample with known
      concentration of MNV-1 to evaluate
      recovery efficiency
   Parameters
    - Utilized a Multi parameter Water
      Quality Sonde
        • Temperature, turbidity, pH,


   Analysis
    - IDEXX Most Probable Number
      (MPN) method


    - HSIMFA, qPCR/RT-PCR
                                        Surface Water (n=ll)
                                        Groundwater (n=10)
 Acknowledgements
 Collaborators
     Drs Rolf Halden, Thaddeus Graczyk
     Students- Kristen Gibson, David Colquhoun
 Funding - EPA STAR R833002
         Public Health  Implications

Developing a universal method for the recovery of microorganisms will enable
water utilities and regulatory agencies to better address problems within
source waters and public water systems.
The utilization of molecular detection techniques will provide increased
confidence in the sensitivity, specificity, and inhibition detection/control critical
for estimating levels of risk.
A more comprehensive understanding of the microbial contamination of water
sources will allow for exposure risk assessments to be generated for individual
microorganisms
Future applications of this method:
 - Further the development of the usefulness of host-specific viruses in
   microbial source tracking efforts
     • Currently limited by lacking concentration and detection methods
 - Assist in the formulation of effective control measures for the reduction of
   water-related transmission of pathogenic microorganisms

-------
 Development and application of a fiber optic
 array system for detection and enumeration
        of potentially toxic cyanobacteria
               Donald M. Anderson
 Woods Hole Oceanographic Institution, Woods Hole, MA
                                                                     The problems:
                                                                       Many cyanobacteria produce potent toxins that threaten human
                                                                       health

                                                                       CyanoIIABs can take multiple forms, ranging from dense surface
                                                                       scums to dilute suspensions that can still cause harm.

                                                                       Many different species and strains co-occur, and strains of the
                                                                       same species can be toxic or non-toxic, or can vary dramatically
                                                                       the amount of toxin produced under different conditions.

                                                                       Distinguishing characteristics can be difficult to discern under the
                                                                       light microscope, yet such fine levels of discrimination are not
                                                                       feasible in monitoring programs that generate large numbers of
                                                                       samples
The overall project goal is to adapt and validate a rapid
and accurate optical fiber-based technology for cyanoHAB
cell detection and enumeration in both laboratory and
field settings

Specific objectives are to:
 1) Design rRNA signal and capture probes for the three most
   important toxic cyanobacteria (Microcystis aeruginosa,
   Cylindrospermopsis raciborskii, andAnabaenaflos-aquae);
 2) Design and test a second probe pair for each species, to
   incorporate redundancy into the array;
 3) Test these probes in the fiber-optic array format and determine
   detection limits, specificity, and dynamic range;
 4) Refine hybridization conditions to reduce processing time;
 5) Develop procedures to analyze multiple cyanoHAB species
   simultaneously using a single fiber bundle in a multiplexed format

-------

-------

-------
Detection of A. fundyense cells in natural seawater
M
1 400.00
EJ>
"1 300.00
S 200.00
| 100.00
asurement of signal with various volumes of sample
with 1000 cells of A fundyense
3 X SD of background = 27





O.I 0.25 0.5
(L)
Volume of seawater


1.0

-------
                      Methods
Signal and capture probe design
 — 16S rRNA gene sequences compiled from GenBank for target
   cyanoHAB taxa: Cylindrospermopsis raciborskii, Microcystis a,
   and Anabaena flos-aquae
 — Probe identification performed using sequence alignments of
   target/non-target species
 - Included published probes for Microcystis, Anabaena/Aphanizomenon,
   and "Nostocgroup" (Nostoc/Anabaena/Aphanizomenon)
Probes tested against target and non-target species usin;
fluorescent in situ hybridization (FISH) to determine efficacy
and assess cross-reactivity; probes that exhibit cross-reactivity
require re-design
Probes successfully tested for cross-reactivity are then
transitioned to fiber-optic microarray format and tested
against synthetic target and cell lysates from target species

-------
                                                                        Twelve probes tested for cross-reactivitj

                                                                                           (in progress)

                                                                         - Microcystis probes (3)
                                                                             • Tested against 18 cultures (in progress)
                                                                             • All designed (3) and published (2) probes exhibit cross-reactivity
                                                                              with Oscillatoria; redesign in progress
                                                                         - Cylindrospermopsis probes (2)
                                                                             • Tested against 18 cultures
                                                                             • One probe transitioned to fiber optic microarray format
                                                                             • Second probe exhibited cross-reactivity with Anabaenopsis;
                                                                              redesign in progress
                                                                         -  Anabaena probes (5)
                                                                              All designed (3) and published (2) probes either exhibited cross-
                                                                              reactivity or failed to detect target species
                                                                              Taxonomy of Anabaena problematic (not monophyletic); redesig
                                                                              efforts needed to develop probe for Anabaena/Aphanizomenon or
                                                                              "Nostoc group"
          Microarray testing


  Capture probe performance tested using
  Cylindrospermopsis probe #1 (CYL1) coupled to
  activated microbeads and against a synthetic
  target
     Single bead array exposed to Cy5-labeled synthetic
     targets with sequences complementary to the capture
     probe
     Hybridization was performed at room temperature
     using 100 ul of synthetic target solution (100 uM) and a
     hybridization time of 10 minutes
            Future directions

Probe redesign and testing
Transition additional probes to microarray format (sin  '
bead arrays) and assess performance using synthetic targets
and cell lysates (assess detection limits, specificity, and
dynamic range)
Assess performance of multiplexed array using single and
multiple species
 - single species and mixed cultures
 - spiked/unspiked field samples (2009 field sample collections include
   lakes in OR, MA, MD, CA, FL and Great Lakes)
Explore application of the microarray technique on
a portable instrument

Explore remote deployment of the microarray
technique on a robotic, in situ instrument
embedded System Control

-------
              Acknowledgements
Woods Hole Oceanopranhic Institution
      Mindy Richlen
      Dave Kulis
      Rob Arnold
                                    Tufts TJniversitv
David Walt
Ryan Hayman
Shonda Gaylord
                           U.S.EPA -Science To Achieve
                           Results (STAR) Program
                           Grant #
                                    RD-83382801-OI

-------
                      Development of high-
                      throughput and real-time
                      methods for the
                      detection of infectious
                      enteric viruses
                    JJLCantera, H-Y Yeh, A Mulchandani,
                    Chen& MVYates
                    UNIVERSITY OF CALIFORNIA. RIVERSIDE
                                                                               lllbjlld k'^b''
                                                                 s  Enterics: adenoviruses, enteroviruses, noroviruses,
                                                                    rotaviruses
                                                                      Enteroviruses: coxsackievirus, hepatitis A virus, echovirus & poliovirus


                                                                 >  Can cause serious diseases when ingested
                                                                      e.g. gastroenteritis, meningitis, hepatitis, myocarditis, paralysis

                                                                 j  Stable in aquatic environments

                                                                 >  Transmitted by fecal-oral route

                                                                 >  Low infectious dose
Human Enteric Viruses
 Reovirus
 Ratavirus
 Mastadenc
 Calicivirus
 Parvovirus
 Coronavirus
             Ftaliovirus        Paralysis, meningitis, fever
             Coxsackievirus A, B  MejTJngitis, fever, respiratory disease, hand-foot-and-
                                       rditis, heart anomalies, rush
                                             ;ase, rush, gastroenteritis
             Hi
Human parvovirus   Gastroenteritis
Human coronavirus  Gastroenteritis, respiratory disease
Human torovirus   Gastroenteritis
                                :.'..•
Detection Methods , , UCR

Principle of the
assay
Visualization of
viral particles
Detection of viral
proteins or
antibodies
Detection of viral
genome

Detection of
cytophatic effect

Example
EM

ELISA
Probe
hybridization
RT-PCR
Plaque assay

Infect ivity
test
No

No
No
No
Yes
Koop
Detection limit
(particles/ml)
105 to 106

105
10"
lO'tolO3
10° to 101
ansS Duizer(2004] IntJ Food
Duration
<24hr

<2hr
<2hr
<8hr
2 to 14
days
Mi . :•'.)! 90:23-11
                                                                            Poliovirus: Life cycle
  To develop methods for high-throughput and real-
  time detection of infective enteric viruses

  Part 1: Genetically engineered reporter cells
       Viral protease-sensitive fluorescent substrate
       Detects viral protease
       Flow cytometry-based assay for detection of PV in
       wastewater

  Part 2: Nuclease-resistant molecular beacons (MBs)
       Detects viral genome
       Modified MB for visualizing the dynamics of viral
       replication in living cells

-------
(-1 RNA   y    T_


(+) RNA   5'UTR  «.
                                    \   V
                                          RNA-dependent RNA pol.

                                     non-structure
genome
                                      1
poiypeptide      I.'.'.'.'.'.'.'.'.I
                              t               t

                                      1                1
                              rnrzBii   zc    i
       • rter cells:  Microscopic examination
                                     3hpi
                                                         Shpi
                                                                                     Reporter cells:
                                                                                                             Fluorescence Activated
                                                                                               BGM-PV
                                                                                           Infection with PV1
                                                                                           Infected BGM-PV
                                                                                             FACS analysis
                                                                                                                                  FACS dot plots

                                                                                                                       '--


Reporter cells:  FACS results
                                                C

                                               '-I
                             CFP intensity
Increase in CFP intensity in PV-infected cells vs uninfected cells

->   FACS can distinguish infected cells from non-infected cells
->   FACS can detect quantitative differences in the number of infected cells in the sample
                                                                                     Reporter cells: FACS resets
                                                                                                       0  2  4  6  8  10 12 14 16  18  20  22  24
                                                                                                                     Time (hpi)

                                                                                        An increasing number of infected cells was detected as PV1 infection progressed

                                                                                        ->  FACS assay is suitable for following the kinetics of poliovirus infection
                                                                                        ->  FACS can detect infected cells as early as 5 hpi I	

-------
     £    150
     u- jjf
     o
     a
y = 0.1366x + 30.573
Rz = 0.9857
              0   100  200  300  400  500  600  700 800  900  1000

                                   PFU

FACS detected cells infected with 50 PFU of PV1 after 8 hpi... positive signals from
cells infected with 1 PFU of PV1 were detected after 12 hpi!

-^ FACS is a sensitive and rapid method for PV1 detection
                                                                                          When tested in waste water spiked with PV1,
                                                                             PFU (Plaque assay)
                                                No significant difference between FACS and plaque assay results

                                                -> FACS is a reliable method for PV1 detection in waste water
 ;   Using FACS on fluorescent reporter cells:


      distinguished infected from uninfected cells

      detected PV-infected cells as early as 5 hpi (at high
      infective dose)

      detected 1 PFU of PV after 12 hpi

      Good correlation  between FACS-based and plaque assays
      when tested on wastewater spiked with PV1
                                                                                       Molecular BeaCOnS: Structure and principl,
                                                                                                    Principle
                                                               3UTH
                                                       PNAS
                                                                                         Intracellular delivery of MB CBV6-Tat-target hybrids (A) or MB without Tat modification or without
                                                                                                                        targets (B)
                                                                                                       PNAS

-------
   >  Modified molecular beacon

        Nuclease-resistant MB with TAT peptide was designed

        Detected as few as 1 PFU during the early stage of viral
        replication

        Fluorescence assay was comparable with the plaque
        assay

        Used to monitor the dynamics of viral replication during a
        12-h infection period
                                                                                                    )ectives
>  Developed methods for detecting infective viruses


  Sensitivity could reach 1 PFU at shorter incubation time than
  conventional plaque assay


  Reliable for viral quantitation


         > Detection of other epidemiologically important viruses
              e.g., hepatitis virus, adenovirus, norovirus


         > High throughput screening for viral protease inhibitors


         > Development of other FRET-based sensors
Acknowledgment
     Drs. Marylynn Yates and Wilfred Chen

  >  Members of Yates and Chen Laboratories

  >  B. Walters (UCR Institute for Integrative Genome
     Biology)

  >  U.S. Environmental Protection Agency
   -   The

-------
SEPA
    New Electropositive Filter for Concentrating
       Enterovirus and Norovirus from Large
                  Volumes of Water
    Mohammad R. Karim, Eric R. Rhodes, Nichole Brinkman, Larry Wymer,
                     and G. Shay Fout
-&EPA
                  Presentation Outline
     Human enteric viruses
     Why should we be concerned about viruses in
     water
     General method for virus detection
     Research need for virus sample collection
     Evaluation of a new filter for concentrating
     viruses from water
     Conclusions
•
:PA pj
Human Enteric Viruses k
Genus
Enterovirus
Hepatovirus
Reovirus
Rotavirus
Mastadenovirus
Norovirus
Astrovirus
Coronavirus
Popular Name/Species
Human EnterovirusA
(CAV2-8, 10, 12, 14,16; EV71, 76, 89, 91)
Human Enterovirus B
(CAV9, CBV1-6;E1-7,9, 11-21,24-27,29-33;
EV69,EV73-75, EW7-8, EV79-88, EV100-101
Human Enterovirus C
CAV1, 11, 13, 17, 19-22, PV1-3
Human Enterovirus D
EV68, 70
Hepatitis A
Human reovirus
Humanrotavirus
Human adenovirus
Noroviruses
Human astrovirus
Human coronavirus
Disease caused
Paralysis, aseptic meningitis,
encephalitis, myocarditis, fever,
respiratory disease,
gastroenteritis, etc.
Hepatitis
Unknown
Gastroenteritis
Gastroenteritis, respiratory
disease, conjunctivitis
Gastroenteritis
Gastroenteritis
Gastroenteritis, respiratory
1
Bosch,1998. Int. Microbiol. 1 :191-196, and Khetsuriani et al., 2006. MMWRSurveill. Summ.55(8):1-20


Branch


                                                                      •SEPA
                                                                          Why should we be concerned about viruses in
                                                                          water?
     Number of Waterborne Disease Outbreaks Associated with
           Drinking Water, by Year and Etiologic Agent
                  — United States, 1971-2006
      Total 814 outbreaks and 575,819 cases of illness were reported
      Overall, 8% of outbreaks were caused by viruses
       Yoder et al., 2008. MMWRSurveill. Summ., CDC. 57(9):39-62
                                               J»-
SEF
p«


^B ,™
A
rcentage of Waterborne-disease Outbreaks Associated w
Drinking water, by Illness and Etiology -
United States, 2005 - 2006
Afl rflncss (n - Wf AGl orty (n rtneC
H«' 232*
50%
' A f ngmrjintf.f '4^t
' Hopavnn. auapaetod tinaJ ypan nciAito^ period, svrngriwm and A^attwi of (Sn*w
*Crn? i>ut»»^ tfud nvolnd bacterial tatd -.Fi*J aywili
Yoder et al., 2008. MMWR Surveill. Summ., CDC. 57(9):39-62

th

•x


-------
SEPA
            Routes of Enteric Viruses Transmission
       jsch,1998. Int. Microbiol. 1:191-1£
-&EPA
         Drinking Water Contaminant Candidate List 2
                            (CCL 2)
                                                                                  Caliciviruses
                                                                                  Coxsackieviruses
                                                                                  Echovi ruses
                                                                                  Adenoviruses
SEPA
     How do we detect viruses in water?
•SEPA
                                                                                     Virus Methods in General
                                                                              Sample collection

                                                                              Elution
                                                                                Beef Extract

                                                                              Reconcentration
                                                                                Organic Flocculation
                                                                                Celite Concentration
                                                                                Polyethylene Glycol
                                                                              Virus assay by cell culture or
                                                                              molecular methods (RT-PCR,
                                                                              qRT-PCR)
      Virus Methods: sample collection apparatus
                               0"	Water Source
                                   	*- Water Discharg
                          Discharge
                           Module
             Virus Cartridge       Injector Module
             Housing Module
                                                                            SEPA
                                                                                  Pore Size of Filter Medium and Size of Microbial Particles
                                                                                                           j
                                                                                                          » !,

-------
vvEPA
          Types of Filters Commonly Used  in Virus
                   Concentration Procedures

     Negatively charge Filters
        Requires conditioning the water prior to filtration
        pH adjustment to 3.5
        Addition of multivalent cations

     Positively charged filters
        1MDS electropositive filters (Cuno, Meriden, CT) are commonly used
        for environmental water sampling
        Does not require conditioning the water.
        However, requires pH adjustment for waters with pH values
        exceeding 8.0
-&EPA
   Research need for virus sample collection
                        Virosorb®1MDS Filter
        Recommended by ICR Method
        Charge-modified, glass and cellule
        Available in 25.4 c
        Cost > $200
        These filters are not cost-effective for routine viral monitoring
                                                                                           •SEFA
                                                                                                             NanoCeram  Filter Characteristics
                                                                                                  \annrrrtaaKfdnfprat.trnnplFrfs'')
                                                                                                                                 The active ingredient of the filter
                                                                                                                                 media is nano alumina (AIOOH) fiber.

                                                                                                                                 The fibers are only 2 nanometers in diameter
                                                                                                                                 and 0.3 |jm long and have a surface area of
                                                                                                                                 500-600 m2/g.
                                      In NanoCeram cartridge filters, these fibers a
                                      dispersed throughout a microglass fiber mati

                                      Size: 12.7 cm X 6.35 cm ; total surface area



                                     • Cost approximately $40
                          NanoCeram  Filter
                                                                                                         Virus Sample Collection
                                                                                                  200-1500LofWat

-------
Retention of Poliovirus •
No. of replication Seed titer3 (PFU) T
1 5.1 x 105
2 9.4 X 105
3 5.4 X 105
4 7.7 X 105
5 7.6 X 105
6 3.7 X 105
7 2.4 X 105
8 5.0 X 105
9 4.0 X 105
10 6.0 X105
Mean
Range
Poliovirus was seeded in 100 L of deionized water
'Total virus PFU in 100 liters of deionized water
» Detection limit
by NanoCeram® Filters
iter in the filtrate
(PFU)
5.0 x 10"
1.1 x 105
8.0 x 10"
6.0 X 10"
1.8X105
9.0 X 10"
7.0 X 10"
1.0X105
EPA
                                                                                                       Virus methods: elution

                                                                                        Elution Scheme
                                                                                        > Single elution9
                                                                                          Double elution with 1 min contact times"
                                                                                          Double elution with 1 min, then overnight, contact times'
                                                                                        > Triple elution"
                                                                                        1 Sobsey, HI D. and A. R. Mickey. 1985. Appl. Environ. Hicrobiol. 49:259-264.
                                                                                        1 USEPAICR method
                                                                                        : Dahllng, D. R. 2002. Water Environ. Res. 74:564-568.
                                                                                        i Dahllng, D. R., and B. A. Wright. 1984. Appl. Environ. Hicrobiol. 47:1272-1276.
Poliovirus Recovery by NanoCeram® Filters Using Six Different
                    Elution Procedures.
       1st elution
       2nd elution for 120 min
48 (±16)
28 (±17)
77 (±16)

50 (±15)
23 (±11)
73 (±18)

48 (±8)
21 (±11)
70 (±18)

39 (±3)
21 (±9)
60 (±13)

32 (±5)
11 (±2)
43 (±4)
        seeded in 100 L of deionized water and filtered through NanoCeram© filter
                                    >=,EPA

                                            Poliovirus  Recovery at Different pH of Water
                                                                                                            rh
 Poliovirus Recovery at Different Flow Rate
    WaterpH = 7;P=0.08
Recovery of Poliovirus 1, Coxsackievirus B5,
From Tap Water Using NanoCeram
Virus Elutions Mean
Poliovirus 1 1st elution
2nd elution for 15 min
Combined percent recovery
Coxsackie B5 1st elution
2nd elution for 1 5 min
Combined percent recovery
Echovirus 7 1st elution
2nd elution for 1 5 min
Combined percent recovery

^•eionaza-dAssessmen, Research B-an.h
and Echovirus 7
® Filter
percent recovery
35 (±9)
19(±5)
54 (± 8)
18 (±12)
9 (±6)
27 (±17)
14 (±6)
18 (±9)
32 ±8)



-------
            Ohio River Water Characteristics
Event
During 100 liter spiking
experiments
During 10 liter spiking
experiments
pH (range)
7.7 (7.6-7.8)
7.7 (7.6-7.8)
Turbidity (range)
NTU
41 (26-90)
1.2(0.17-2.75)
                                                                                        v>EPA
                                                                                               Comparison of Poliovirus Recovery by NanoCeram® and
                                                                                                   1MDS Filters From Seeded Tap and River Water
Type of
filter
Elution
Mean virus recovery (%)
100 L sample
water | water
10 L sample
water | water
                                                                                             Nanoceram" 1*elution                23±14   21 ±18   182±42   30±16
                                                                                                       2"'elution               28 ±13   16 ±15   95 ±64   25 ±15
                                                                                                       Combined percent recovery   51 ±26   38 ±35   277 ±22   65 ±22

                                                                                             1MDS      1* elution                39 ±4   25 ±20   31 ±14   13±4
                                                                                                       2"'elution               28 ±6   11 ±4   13±13   17±9
                                                                                            	Combined percent recovery   67±6   36 ±21    44±9    30 ±11
                                                                                            For 100 L samples p=>0.05; For 10 L samples, tap water p=<0.001, river water p=0.015
*=,EPA
    Comparison of Norovirus Recovery by NanoCeram®
    and 1MDS Filters From Seeded Tap and River Water
Type of filter
NanoCeram®
1MDS
Mean virus recovery (%)
Tap water
3.6 ± 0.6
1.2 ± 1.4
River water
12.2 ±16.3
0.4 ± 1.8
    Norwalk virus was seeded in 10 L of dechlorinated tap water or
    river water and filtered through NanoCeram® or 1MDS filters
                                                                                            Comparison of RT-PCR Reaction Inhibition For Norwalk Virus
                                                                                            and Poliovirus in NanoCeram® and IMDS Filters Concentrates
Type of
filter

Nanoceram161



IMDS

Elution

1 fl elution
2ml elution


1 fl elution
2ml elution
RT-PCR inhibition for norwalk virus/poliovirus
Tap water
Triall
+/ND
+/+


+/ND
+/+
Trial 2
+/ND
+/+


+/ND
+/+
Trial 3
+/ND
+/+


+/+
+/+
River water
Triall
+/+
+/+


+/+
+/+
Trial 2
+/+
+/+


+/+
+/+
Trial 3
+/+
+/+


+/+
+/+
                                                                                           ND = not done
                                                                                           '+' indicates spiked sample
                                                                                                               e not inhibitory for RT-PCR reactiot
S.EPA




     RT-PCR Detection of Poliovirus From Seeded Tap and River
                          Water Samples


   Type of filter   Elution3    RT-PCR detection of poliovirus in 100 L seeded water samples"

                               Tap water               River Water

                        Trial 1    Trial 2   Trial 3   Trial 1   Trial 2   Trial 3
  aFirst elution was done for one minute and 2nd elution was done for 15 n

  b"+" indicates RT-PCR positive and"-" indicates RT-PCR negative
                      Conclusions

 The mean retention of poliovirus by NanoCeram® filters was 84 percent.


 The highestvirus recovery (77%) was obtained by immersing the filters in
 beef extract for 1 min during the first elution and 15 minutes during the
 second elution.


 The recovery efficiencies of poliovirus, coxsackie B5, and echovirus 7
 were 54%, 27%, and 32%, respectively.


* There was no significant difference in poliovirus recovery at tap water pH
 range of 6 to  9.5


 There was no significant difference in virus recovery over a water flow
 rates of 5.5 L/min to 20 L/min.

-------
vvEPA

                           Conclusions


     NanoCeram® filters were comparable or better than the 1MDS filters.

     Cost approximately one-sixth of 1MDS filter, thus can be used for routine
     viral monitoring of water.



    Published in Applied and Environmental Microbiology.
-&EPA
                          Future Directions
     This work has paved the way toward a validation project aimed at
     replacing the 1 MDS filter with the Nanoceram® filter in an enterovirus
     detection method.
     If this validation goes as expected, this new method will be considered
     forUCMRS.

-------
Automated Methods for the
Quantification and Infectivity of
Human Noroviruses in Water

Timothy Straub, PI, timothy.straub@pnl.gov.
 Richard Ozanich, Co-Pi, Richard.Ozanich@pnl.gov.
• Rachel Bartholomew, Co-Pi, Rachel.Bartholomew@pnl.gov.
Cindy Bruckner-Lea, Co-Pi, Cindy.Bruckner-Lea@pnl.gov
                                                                    Project Overall Aims
            Alms: Preeu* I
         lor primary and MCOndary concentration
               and purification
                     c*ptuf* and purification or
                     viruses that will allow
                     anaiym is by PCR and cntl
                                       quantitative PCR
Methods for Capturing Pathogens from
Large Volumes of Water- Aim 1

  Need: Ability to efficiently capture and concentrate
  viruses, bacteria, and protozoa from large volumes of
  water
   « Pathogen concentrations in water are often very low (<1/100 ml
     for bacteria to <1/1,000 L for viruses)
  Methods we are investigating are mostly off the shelf
  technology
   • Hollow fiber filtration:  Large volumes require large columns, high
     flow rates can be problematic
   • Sodocalcic glass wool: Very cheap, and may have great
     potential for viruses - investigating this summer DOE FaST team
  Modified system (next slide) may allow flow rates up to 15
  L per minute
  Large Scale System, Adapted from Vince Hill
                                           Challenge:
                                           Automate to deliver
                                           concentrated
                                           samples for further
                                           processing
Secondary Concentration:
The Major Bottleneck - Aim 2
  For water we get to a primary filtrate and then:
     Centrifugation will concentrate bacteria and protozoa, but it is a
     manual process.
   • Viruses are left in the supernatant and still need to be
     concentrated.
   • Or we use single-plex immunomagnetic separation: e.g. the
     "disease of the day" approach, and we lose information about
     other pathogens.
  BEADS: Bridging the Gap between
  Large Volume Concentration and Detection
  Sample:
Large volume,
 matrix high,
 pathogens
    low)
  BEADS = Biodetection enabling analyte
          delivery system
For secondary concentration and purification
                                   Detector:
                                    Clean,
                                   tiny volume
                                    (ML-mL)

-------
  Biodetection  Enabling Analyte
  Delivery System (BEADS)
Guiding principles

1.  Analytical separations can
   be performed on an
   interactive surface like a
   derivatized bead

2.  Analytes of interest (cells,
   DMA, proteins, etc) are
   perfused over a column of
   beads and captured

3.  Matrix materials are
   washed away, leaving
   purified analytes
        *    Perfuse and capture
                     0
Wash away matrix
 General Approach:

 Renewable Surfaces


  Interactive surface on beads is
  delivered fresh for each sample
  Compatible with users' pathogen
  detection requirements
     Nucleic acid techniques
     Cell culture
   • Immunodiagnostic "sandwich
     assays"
^ Operates within a scaleable fluidics
  architecture
     From uL to 10 L volumes
     Architecture allows us to handle
     samples that are high in
     particulate matter and/or soluble
     inhibitors
                                                                                     '00
                                Beads are flushed to waste
                            or sent downstream for further analysis
  Renewable Separation
  Columns (RSC) used in BEADS
                 + 10-150 in caiDOH
                 * prtymcr. tytogd. glau «:
          ' Automated capture and release of particles '
          • Disposable mtcmcohtmns. -1 tit volume
            hM rite eorcrd
          1C|rn magnetic parMsi
   Renewable separation columns are the defining feature of the BEADS platform.
   Depending on the user's needs, any one of these columns, and any type of separation
   media can be used.
                                                Parallel Research Tracks Include
                                                Automation and Reagent Development
BEADS Scale up
 • Need to process large
   volumes (1 -10 Lor greater)
   From a primary concentrate.
   Baseline experiments need to
   show that we can capture and
   release pathogens as
   efficiently as our small
   systems.
   Possible secondary
   concentration issues to
   achieve overall 104-105
   concentration factor.
Multi-agent capture
   Default:  Use multiplexed
   preparations of commercially
   (and custom) available IMS
   antibodies
   New generation methods:
   broad spectrum capture
   reagents for protozoa,
   bacteria, and viruses.
   How will either cell capture
   approach fare when
   challenged with low target
   organisms and high
   background flora?
   Batch Trials with  Lectins: Reagent
   Development for BEADS
  Combinations of biotin
  labeled lectins were first
  mixed with bacteria, and then
  captured on streptavidin
  magnetic beads (indirect
  capture)
     Loss of CPU indicates better
     capture results
     Demonstrated capture of
     vegetative cells and spores.
> Challenge: direct capture.
   • Lectins conjugated to the beads
     do not work as well.
^ Viral capture has not shown
  as much promise

                                                Reverse transcription real-time PCR - Aim 3
                                                  For human noroviruses, there is not much choice for the
                                                  development of better primers and probes
                                                     Variations within the ORF1-ORF2 junction - most conserved to
                                                     detect the most known strains.
                                                  "Fast" vs. Slow real-time PCR
                                                     Newer real-time platforms allow PCR to be completed within 40
                                                     minutes.  HOWEVER
                                                   • Still need to perform reverse transcription, and that is still
                                                     relatively slow
                                                     Your assay must be optimized for this platform... ORF1-ORF2 is
                                                     not a good place to do this (secondary structure).
                                                  For the purposes of this project, we are using the
                                                  standard thermal cycling conditions.

-------
Infectivity Assays for Human Noroviruses
Aim 4
 Our original work investigated the INT407 cell line grown as 3-D cell cultures.
 While we see evidence of infectivity, we are not observing significantviral
 replication. Investigations with 3-D Caco-2 cells has revealed interesting
 results
                                                                                 Observational differences between Uninfected
                                                                                 and hNoV Infected 3-D Caco-2 cells
                                                                               Phase
                                                                               contrast
                                                                               4-3
                                                                                                                          •
                                                                                                                                   Cells challenged
                                                                                                                                   with hNoV
                                                                                                                                   negative stool
                                                                                                                                   samples (4-1
                                                                                                                                   patient sample)
Cells challenged
with hNoV
positive stool
samples (4-3
patient sample)
                                                                                              Only by TEM were differences between uninfected
                                                                                                   and infected cells easier to visualize
Real-time PCR observations indicate viral
RNA replication in Caco-2 and INT407 Cells
Cell line Virus
sample
••••••
Caco-2 1G (GU)
386 (Gil)
4-3 (Gl)
INT407 MG(GII)
386 (Gil)
Predicted Observed
copies copies in
applied to cells 1 hr
529+59
|41 +7
6,390 +
681
529 + 59 [
41 +7

Observed
copies in
cells 48
1 1 (No sd)
Not
detected
36,206 +
6,244
Not done
88 + 77
(2/3
detect)
Observed
copies in
cells 72
2,324 *
180
9,375 +
1048
132,919 +
37,863
5,370 +
992
74 +.128
(1/3
detect)
MA
MM
Observed
copies in
cells 1
IS63 +
329
Not
detected
Not done
4,800 +
316
429 + 363
(3/3
detect)
SSSSSxr
                                                                                 Possible investigation of the role of STAT-1
                                                                                 in controlling viral replication
                                                                               STAT-1 Expression in hNoV Infected 3-D   STAT-1 Expression in hNoV Infected 3-D
                                                                                          INT 407 cells                        Caco-2 cells
                                                                               RNAi experiments targeting suppression of STAT-1 may help us
                                                                               understand its role in hNoV replication in human cells and may confirm
                                                                               findings about its role in limiting disease in the murine NoV model.
Research Summary
   Fluidic architecture is currently being constructed to
   process large volumes of water.
^ Secondary capture reagents being investigated at the
   bench
   •  Testing this summer: DOE Faculty and Student Team (FaST) will
      allow us test both the large volume systems and perform batch
      capture experiments for secondary concentration - No charge to
      EPA STAR
>• Further investigation of Caco-2 cell line for hNoV
   infectivity.
   •  Results have been very promising, and if there is an underlying
      genetic mechanism inhibiting viral replication, this could provide
      new insights to develop better infectivity assays.
                                                                                 Acknowledgements
                                                                                 A portion of this research was performed using EMSL, a national scientific user
                                                                                 facility sponsored by the Department of Energy's Office of Biological and
                                                                                 Environmental Research located at Pacific Northwest National Laboratory. Funding
                                                                                 for this work is provided by the United States Environmental Protection Agency
                                                                                 STAR Grant Program (Grant # R833831010). The norovirus infectivity assay is
                                                                                 jointly provided by NIAID under the Food and Waterborne Integrated Research
                                                                                 Network Program (Contract number NO1-AI-30055) and the STAR Grant Program

-------
             Recent EPA Study
                                                                          Environmental Parameters
Examined 40 natural water samples:
 (lakes, rivers, ponds, wetlands, etc.)

Examined 40 cooling tower samples

Also examined 20 other industrial: chillers, hot tubs,
hot water taps/tanks, etc.

Designed a protocol to screen for infected amoebae
Temperature, pH, dissolved organic carbon (DOC),
total nitrogen (N) and total bacteria per ml
Logistic regression analyses were performed to find any
parameter or set of parameters that were good predictors
of the occurrence of infected amoebae

-------
1
                        Coccoid Cooling Tower Isolate
                        In HeLa Cell Nuclei (48h)

-------
Percent Identity
  123
                          1  CC99
                          2  HT99
                   85.9 |  3  L. pneumophila X73402
                          4  C. burnetii AY342037
Percent Divergence

-------
                  Results
     22 of 40 cooling tower samples were positive

     3 of 40 natural samples were positive

     2 of 20 other industrial samples were positive
     (hot tubs)
   Odds ratio of finding infected amoebae in cooling
   towers vs natural environments is 16,
   i.e., 16 times more likely to find them in CTs
   (based on the way we look for them)
 5 novel strains were identified, related to Legionella

Only 2 of the 22 infections were from L. pneumophila
And 1 of the hot tub infections was from L. pneumophila

 Several have not yet been isolated or identified

 Of those that are culturable, at least 3 tested so far
 appear to infect human macrophages
 Two non-culturable strains also infect macrophages
    No environmental parameter was a significant
    predictor of occurrence of infected amoebae
    when cooling tower data were used alone

    When data from 90 combined samples were used,
    pH and DOC were significant predictors

    BUT cooling towers have higher pH values than
    almost all natural samples, and also have a higher
    range of DOC
     Therefore it appears to be pH and DOC, but
     it may be something else specific to CTs
     that were not measured in this study
         Summary/Conclusion
 Occurrence of infected amoebae was significantly higher
 in cooling towers than in nature (16:1 odds ratio)

Non-Legionella were more common than Legionella,
and half or more of these were not culturable

7 novel sequences were found,
with several yet to be sequenced

Environmental parameters?? Possibly pH and DOC
                                                                                  Update
   Several other infected amoeba specimens have
   been observed in the past year—

             Meat industry (3)

          Eyewash station (TTU)

        Fish tank in public pet store

         Distribution pipes (MTSU)

-------
            Acknowledgments
  • Center for the Management, Utilization and Protection
  of Water Resources, Tennessee Technological Univ.
  • Middle Tennessee State University
   Faculty/Associates: Dr. Mary Farone, Dr. Anthony Farone,
           Dr. John Gunderson, Dr. Anthony Newsome,
           Dr. Nizam Uddin
Numerous students: Witold Skolasinski, Kate Redding,
Jennifer Skimmyhom, Elizabeth Williams, Maryam Farsian,
Josh Currie, James Ventrice, Chanson Boman, Allison Reid,
Marya Fisher, Jon Thomas

-------
                                                       Waters

                                                                                  Acknowledgement
                                                   Waters
           Detection of Various  Freshwater
          Cyanobacterial Toxins using Ultra-
        Performance Liquid Chromatography-
              Tandem Mass  Spectrometry
N
-------
Various Micrdcystins and others
                                                 Waters

                       Factors affecting cyanobacterial
                       bloom formation:
Waters
                                                                                                 Pathway/Route of Exposure
                                                                               Moderate to high levels of essential inorganic nutrients
                                                                               (nitrogen and phosphorus)
                                                                               some are nitrogen-fixing
                                                                               water temperature 10° to 30°C
                                                                               pH levels between 6 and 9
                                                                               low flow and low turbidity
                                                                               light is not a large factor - phycobilin
                                                                                                    Recreational waters - dermal, inhalation, and ingestion
                                                                                                    Drinking water - ingestion, dermal, ingestion.
                                                                                                    Dietary Supplements - ingestion
                                                                                                    Vegetables and Fruits - ingestion
                                                                                                                     This is a "Global Challenge"
                                                 w
   Enzyme-linked Immunosorbent Assay (ELISA)
   — Uses polyclonal antibodies against different microcystin variants.
   — Samples are read spectrophotometrically to determine microcystin
     concentration.
     Detection limit in low ppb
   — Cloudy or Murky samples pose a challenge
   High-Performance LC
   — Powerful separation capability
   — UV detection (not sensitive w/o SPE)
   LC and Mass Spectrometry
     Offers specificity and sensitivity
bters
                                                                           Microcystin LR  Mass Spectrum
Waters
Final Separation using Traditi'
HPLC/MS/MS
Waters
     JIT'S POSEIBLE.-
                                                                                                                                                       1=Cylindrospermopsin, 2=Anatoxin-a, 5= Microcystin RR, 6=Nodularin, 7=Microcystin
                                                                                                                                                       YR, 8=Microcystin LR, 9=Microcystin LA, 11=Microcystin LW, and 12=Microcystin LF

                                                                                                                                                        2.1X150mm Atlantis dC18 (3.5iim)@30°C-0.29mL/min

-------
                                                 Waters

Small Particle (sub 2um)
Higher separation power
Higher tensile strength
r-«^.


                                             Acouity
                                               W^WftrnvauL/
                                          UPLC™/M|/MS,Separation
                                          8.5  minutes
Waters
                                        1=Cylindrospermopsin, 2=Anatoxin-a, 3=Cyclo (Arg-Ala-Asp-D-Phe-Val) (IStd), 4=[Leu5]-
                                        Enkephalin (IStd), 5= Microcystin RR, 6=Nodularin, 7=Microcystin YR, 8=Microcystin LR,
                                        9=Microcystin LA, 10=Microcystin LY, 11=Microcystin LW, and 12=Microcystin LF
                                                                                                                                                         Possible Internal Standards
                                                                                                                  Molecular Formula: CMHS7NSO7
                                                                                                                                                                     ,
                                                                                                                                                           -O     CHjCH -C
                                                                                                                                                                           CH, NH O

                                                                                                                                                                                  C CM
                                                                                                                                                                                                            .hJH  |sJ

                                                                                                                                                                                                             1
                                                                                                                                                                                                HO.C—-"  >	NH
                                                                           Detection Lii
                                                                          0Jr—!-A^V^A^-A^
                                                                                                                              Waters
                                                                                                                      Water Samples—Filter Only
                                                                                                                      (spiked  at 4ppb)
                                                                             Waters
                                                                                                                                                                                               Cylindrospermopsin

-------
   Water Samples—Filter Only
   Cspiked atUpptt)
Waters
I
                                     LR-Confirmatory ioi
Interesting Peak in Some Water
Samples at mass of anatoxln-a
Waters
•
-
••
•


A Anatoxin-a (1 Oppb Spike)
SmlMt 2*3) 3 WfMtf 3 Cwn« ES-
23P l£t!3 • 149 14 prataaHCU1
?m|t*: 2t3l 2 UfaUOtnmnES*
; « l« *3 * « D> i*na»or. 10,
?30 J*0 ttc :•
=.- y ;oi ~ >rn if iniai^m
J3f


.



1 i • -
Anatoxin-a(IOppb)
JL J Spiked with Phe-Ala
339 '« !3*M9«£AMIIW»|CIH
i^
JM JJD 3so :to ??n :;;:• : •••
239 I46U .J)03j«-JKHft(Q)
(tSmiMn.jai ; MMioUCrDrnwEV
Iff
li

                                                                                                 Why MS/MS is useful
Waters
                                                                                                                                                 Ohio River Sample
                                                                                                                                                                               Ohio River Sample (CYN Spiked)

                                                                                                                              1
                                                                                                                              1
                                                                                                                              1

                                                                                                                                                                                                no   200   :TO
                                                                                                                                                                                                 ) ww » ? C'anw ES
                                                                                                                                                                                                      •
                                                                                                                                                                                                    1
                                                                                                                                                                                                    I
Extreme Watfr Sgfmple
                                                Waters
  2=Anatoxin-a, 2a=Phe-Ala, 5= Microcystin RR, 7=Microcystin YR, 8=Microcystin LR,
  9=Microcystin LA, 10=Microcystin LY, 11=Microcystin LW, and 12=Microcystin LF
                        Solid Phase Extraction (SPE) for
                        Water Samples
                                                                Waters
                                                                           Current methodology exist for common microcystins
                                                                           using CIS based SPE
                                                                           Anatoxin and cylindrospermopsin add challenges to
                                                                           existing SPE protocols
                                                                           VERY Preliminary work has begun on using a
                                                                           multimodal  SPE protocol* (2 multimodal cartridges for
                                                                           different analytes from a single water sample)
                                                                              Load water onto 2 cartridges in series, than separate and
                                                                              process each separately for the different analyte sets
                                                                            - - Run 2 injections per sample (one for Cylindro, the other
                                                                              foranatoxin and microcystins)
                                                                          ^Patent applied for
                                                                          Preliminai
                                                                          Lake Water-S
                                                                                                              ylindrospe'rmop
                                                                         Waters
                                                                     mi snsra.,m,™, __f
                                                                                                                                                       1 Cartridge (CYN only)
                                                                                                             Lake Water Spiked (approx 0.8ppb)-SPE

-------
K» Lake Water-
                                                   Waters

         2 Cartridge (Anatoxin and Microcystins)
                           Lake Water Spiked (approx 0.1 ppb Anatoxin-a)-SPE
               LakeWater-SPE
                                                                           Conclusions
                                              Waters
Separation of all main Microcystins, Anatoxin-a, and
cylindrospermopsin is possible in under 10 minutes using
UPLC as the separation device (versus 40 Minutes by HPLC)

MS/MS offers enhanced selectivity and sensitivity

Combined with new SPE method, one can easily go to sub
ppb levels
                                                                                                                                                      This work was supported, in part, from the following grant
                                                                                                                                                       — U.S. Environmental Protection Agency Grant (RD-83322301)

                                                                                                                                                      Waters Corporation


                                                                                                                                                      Lake Superior State University
   Thank You for Attending
                                                   w

-------