&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
-------
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
-------
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
-------
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
-------
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)
-------
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
-------
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
-------
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
-------
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
-------
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.
i 10
(Adjusted C
Secon
3 IO 4k at CO
* ,
-
\
-
n
1
-'*!°!
JS
CD
1 1 Peptone Diluted ml TSB
1 1 Peptone Diluted wV
Peptone
• MH Diluted wf MH
DMH Diluted w) Peptone
Serial Dilution 10A
IBfc
-------
Comparison of Incubation Times
I
E. co\\ C3000 Grown in Mueller Hinton Broth
TJ *
a> *" inn -
— m
5 1°-
-
Hank t
•
I
~h
r
•* :c
t
r
I
FU.'m
1
I r
'•4 •:
L
1
I r
1
L
"2 1
"L
I
II
n
r. R^p 1
• Rep 3
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
j •
^^~~~ *""~"\
^ "\
~Z \~
•/ • N>
20C 4CO SCD BOP] IQQj I2DO
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
(saane)
| Ab+EDC/Sll]foNHs]
[Glass | > -NHj group > Covalently immobilized Ab
3. Protein G+ IgG antibody
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Time, min
of coomus non-comclementary brands Analytical ChemeHy 2007, under review
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
0
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|| 1 "s 1 s = s "i
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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
g 1
0
-100
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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
£•
1
£
C
C
s.
Four vims strains
A
/ TA,^'-^/-v^^rA-^^A«^'^/
. .-, ; ,
A
/ V\/-Arv^,^u^^\'A^-^lA
— \ .
' •* ^-^-^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
| Hollow FiborMorctoiicOOkDi) I
|
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1 <
tiMj.ln::.! [ law [ Itrar.™ ir.ll C™. •inirat™ ,.| Srr,.-il ]
Total Volume of ^^
-200-300 ml
t.
1
Butioo of Hollow Fiber
Collection of Total Gwiccn[r»!c
1
Vti-:iJ.ir\ i.V-i^L-r.irj-.r .-L.-t SinrnI,
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fatty microbe i utiog cnokculii dciecEionicctuiiqucs
AiJiy mitrclMi uiing e.miiiog culture meihed*
Add Blocking Reagent
,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
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1
Coccoid Cooling Tower Isolate
In HeLa Cell Nuclei (48h)
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Percent Identity
123
1 CC99
2 HT99
85.9 | 3 L. pneumophila X73402
4 C. burnetii AY342037
Percent Divergence
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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)
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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
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Waters
Acknowledgement
Waters
Detection of Various Freshwater
Cyanobacterial Toxins using Ultra-
Performance Liquid Chromatography-
Tandem Mass Spectrometry
N
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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"
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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
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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
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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
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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
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