Proceedings
The U.S. EPA's
Research on Microorganisms
in Drinking Water Workshop
August 5-7, 2003
Cincinnati, Ohio
I
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Research on Microorganisms in Drinking Water Progress Review Workshop
Table of Contents
Introduction
Overview of the U.S. EPA's Drinking Water Research Program
Fred S. Hauchman
Regional Concerns for Microorganisms in Drinking Water (See Appendix 1)
Bruce Macler
Topic Area 1: Research Supporting Office of Water's Ground Water/Source Water Regulatory
Activities
Presentation Abstracts
SDWA Requirements & Microbial Research Needs (Surface Water, Ground Water, & Distribution Systems)
(See Appendix 1)
Stig Regli
Microbial Dose-Response Modeling: A Predictive Bayesian Approach
James D. Englehardt, JeffSwartout
The Use of Randomized Trials of In-Home Drinking Water Treatment To Study Endemic Water Borne
Disease
Timothy J. Wade, Rebecca Calderon, John M. Colford, Jr.
Screening Models To Predict Probability of Contamination by Pathogenic Viruses to Drinking Water Aquifers
Bart Faulkner
Integrated Approach for the Control of Cryptosporidium parvum Oocysts and Disinfection By-Products
in Drinking Water Treated With Ozone and Chloramines
Jason L. Rennecker, Amy (Driedger) Samuelson, Benito Corona-Vasquez, JaehongKim, Hongxia Lei,
Roger A. Minear, Benito J. Marinas
Prevalence and Distribution of Genotypes of Cryptosporidium parvum in United States Feedlot Cattle
Robert Atwill
Poster Abstracts
Microbial Drinking Water Contaminants: Endemic and Epidemic Waterborne Gastrointestinal Disease
Risks in the United States
Rebecca L. Calderon, Gunther Craun
Evaluating Microbial Indicators and Health Risks Associated With Bank Filtration
Floyd J. Frost
A Prospective Epidemiological Study of Gastrointestinal Health Effects Associated With Consumption
of Conventionally Treated Groundwater
Christine Moe, Stuart Hooper, Deborah Moll, Debi Huffman, Ricardo Izurieta,
Renea Doughton-Johnson, Tatiana Ochoa, Jim Uber, Dominic Boccelli, Joan Rose,
Pierre Payment
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Using Neural Networks To Create New Indices and Classification Schemes
Gail Brian, Srini Lingireddy
Topic Area 2: Research Supporting Office of Water's Contaminant Candidate List (CCL)
Presentation Abstracts
The Contaminant Candidate List: Determining the Need for Future Drinking Water Standards (See
Appendix 1)
Tom Carpenter
The Roles of Pathogen Risk Assessment in the Contaminant Candidate List Process
Glenn Rice, Michael Wright, Brenda Boutin, Jeff Swartout, Michael Broder, Patricia Murphy, Jon Reid,
Lynn Papa
Overview: CCL Pathogens Research at NRMRL
Donald J. Reasoner
Topic Area 2.1: CCL Protozoa
Presentation Abstracts
Detection ofCyclospora cayetanensis and Microsporidial Species Using Quantitative Fluorogenic 5' Nuclease
PCR Assays
Frank W. Schaefer, III, JeffD. Hester, Manju Varma, Michael W. Ware, Barley D.A. Lindquist
Development of Detection and Viability Methods for Waterborne Microsporidia Species Known To Infect
Humans
Rebecca Hoffman, Marilyn Marshall, Mark Borchardt
Development and Evaluation of Procedures for Detection of Infectious Microsporidia in Source Waters
Paul A. Roche lie
Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/
Infectivity of Three Protozoa From Large Volume of Water
Saul Tzipori, Udi Zuckermann
Topic Area 2.2: CCL Viruses
Presentation Abstracts
Norwalk Virus Dose Response and Host Susceptibility
Christine Moe, Lisa Lindesmith, Ralph Baric, Jacques LePendu, Peter Tennis
Development of a Rapid, Quantitative Method for the Detection of Infective Coxsackie and Echo Viruses
in Drinking Water
Marylynn V. Yates, W. Chen, A. Mulchandani
Poster Abstracts
Dose-Response Assessments for NLV and Coxsackievirus in Drinking Water
Brenda Boutin, Jeff Swartout
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Methods Used To Analyze a Norovirus Outbreak
Jennifer L. Cashdollar, Sandhya U. Parshionikar, Christina M. Newport, Sandra Willian-True,
Daniel R. Dahling, G. Shay Font, Outbreak Investigation Team
Development of a Molecular Method To Identify Astrovirus in Water
Ann C. Grimm, Jennifer L. Cashdollar, Frederick P. Williams, G. Shay Font
Effectiveness of UV Irradiation for Pathogen Inactivation in Surface Waters
Karl Linden, Mark Sobsey, Gwy-Am Shin
Topic Area 2.3: CCL Bacteria
Presentation Abstracts
Disinfection of Helicobacterpylori wdAeromonas Species
Laura Boczek, Samuel L. Hayes, Clifford H. Johnson, Donald J. Reasoner, Eugene W. Rice,
Sashi Sabaratnam
Genomic and Physiological Diversity ofMycobacterium avium Complex
Gerard Cangelosi
Mycobacterium avium Complex (MAC) in Drinking Water: Detection, Distribution, and Routes
of Exposure
Phanida Prommasith, Timothy E. Ford
Poster Abstracts
Sensitivity of Three Encephalitozoon Species to Chlorine and Chloramine Treatment as Detected by
an In Vitro Microwell Plate Assay
Cliff H. Johnson, Marilyn M. Marshall, Jackie Moffet, Charles R. Sterling, Laura A. DeMaria,
Gene W. Rice
Inactivation ofAeromonas by Chlorine and Monochloramine
Laura A. Boczek, Cliff H. Johnson, Eugene W. Rice
Mycobacterium paratuberculosis and Nontuberculous Mycobacteria in Potable Water
Stacy Ffatter, Terry C. Covert
Detection of Helicobacter pylori Using a Highly Variable Locus Upstream of the 16S Ribosomal
RNA Gene
M. Shahamat, M.R. Alavi, J.E.M. Watts, K.R. Sowers, D. Maeder, F. Robb
Using Real-Time PCR To Detect Toxigenic Strains of Microcystis aeruginosa
Carrie Moulton
Role of Adaptive Response in the Kinetics of Mycobacterium avium Inactivation With Monochloramine
Ning Tong, Lutgarde Raskin, Benito J. Marinas
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Topic Area 3: Distribution Systems and Biofilms
Presentation Abstracts
The Effect of Chlorine, Chloramine, and Mixed Oxidants on Biofilms in a Simulated Water Distribution
System
Mark C. Meckes, Roy C. Naught, David W. Cmehil, Leslie Wilsong, Janet C. Blannon,
Mano Sivaganesan
Molecular Characterization of Drinking Water Microbial Communities
Jorge Santo Domingo, Mark C. Meckes, Catherine Kelty, Margaret Williams, Joyce M. Simpson,
Donald J. Reasoner
Poster Abstracts
Phylogenetic Analysis of Prokaryotic and Eukaryotic Microorganisms in a Drinking Water Distribution
System Simulator
Margaret M. Williams, Mark C. Meckes, Cathy A. Kelty, HildredS. Rochon, Jorge W. Santo Domingo
Identification and Characterization ofAeromonas Isolates From Drinking Water Distribution Systems
Jennifer Birkenhauer, M. Rodgers
Pathogenicity of Biofilm Bacteria
Dennis Lye
Topic Area 4: Cross-Cutting Research and Emerging Topics
Presentation Abstracts
The Application of Mass Spectrometry to the Study of Microorganisms
Jody A. Shoemaker, Susan T. Glassmeyer
Cyanobacteria and Their Toxins
Elizabeth D. Hilborn
Transport of Chemical and Microbial Contaminants From Known Wastewater Discharges: Potential
Chemical Indicators of Human Fecal Contamination
Susan T. Glassmeyer, Imma Ferrer, Edward T. Furlong, Jeffrey D. Cahill, Steven D. Zaugg,
Stephen L. Werner, Michael T. Meyer, Dana W. Kolpin, David D. Kryak
High Throughput DNA-Based Tools To Study Water Microbial Communities
Jorge Santo Domingo, Joyce Simpson, Margaret Williams, Catherine Kelty
Detection of Emerging Microbial Contaminants in Source and Finished Drinking Water
Using DNA Microarrays
Timothy M. Straub, Paul A. Rochelle, Ricardo DeLeon, DarrellP. Chandler
Mammalian Cell Response to Pathogens
Samuel L. Hayes
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Poster Abstracts
Effectiveness of UV Irradiation for Pathogen Inactivation in Surface Waters
Karl Linden, Mark Sobsey, Gwy-Am Shin
Survey of U.S. Public Health Laboratories: Microbial Pathogens on the Candidate Contaminant List
Elizabeth D. Hilborn, Michael O. Royster, Doug J. Drabkowski
Comparative Diversity of Fecal Bacteria in Agriculturally Significant Animals To Identify Alternative
Targets for Microbial Source Tracking
Joyce M. Simpson, Samuel P. Myoda, Donald J. Reasoner, Jorge W. Santo Domingo
Developing Dynamic Infection Transmission Models for Microbial Risk Assessment (MRA) Applications
Patricia Murphy, Brenda Boutin, JeffSwartout, Glenn Rice, Jon Reid, Michael Broder
Virulence Factors ofAeromonas: A Molecular Genetic Characterization
Keya Sen, Mark Rodgers
Effects of pH and Temperature on the Kinetics ofAeromonas hydrophila Inactivation With Combined
Chlorine
Kwanrawee Sirikanchana, Benito J. Marinas
Agenda
Poster Titles and Sessions List
Participants List (including Remote Participants)
Appendix 1: Presentations of Regional Research Needs and Office of Water Regulatory Activities and
Research Needs
Regional Concerns for Microorganisms in Drinking Water
Bruce Macler
SDWA Requirements & Microbial Research Needs (Surface Water, Ground Water, & Distribution Systems)
Stig Regli
The Contaminant Candidate List: Determining the Need for Future Drinking Water Standards
Tom Carpenter
Appendix 2: Additional NCER STAR Drinking Water Grant Microbial Research
Experimental Infection of Healthy Adults with a Cryptosporidium Genotype 1 Isolate (TU502)
Cynthia Chappell, P. Okhuysen, R. Langer, D. Akiyoshi, S. Tzipori
Experimental Challenge of Healthy Adult Volunteers With Cryptosporidium muris Oocysts
Cynthia Chappell, P. Okhuysen, R. Langer, S. Tzipori
Appendix 3: STAR Grant Presentation Abstracts and Agenda From the USEPA/USGS Meeting on
Cryptosporidium Removal by Bank Filtration, September 9-10, 2003
Abstract Not Provided
William Blanford
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Study of Particle and Pathogen Removal During Bank Filtration of River Waters
Edward J. Bouwer, Charles R. O'Melia, W. Joshua Weiss, Kellogg J. Schwab, Binh T. Le,
Ramon Aboytes
Evaluating Microbial Indicators and Health Risks Associated With Bank Filtration
Floyd J. Frost
Application of a Multipath Microsphere Tracer Test To Understanding Transport of Bacteria and Protozoa
at a Bank Filtration Site
Rick Langford, Dirk Schulze-Makuch, Suresh Pillai
Pathogenic Microbe Removal During Riverbank Filtration
Joseph N. Ryan, Yumiko Abe, Rula Abu-Dalo, Menachem Elimelech, GarrettMiller,
Zachary Kuznar, Ronald W. Harvey, David W. Metge
Event Description
Agenda
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Introduction
One of the high-priority research areas identified by the U.S. Environmental Protection Agency's (EPA)
Office of Research and Development (ORD) is drinking water. Although the United States has one of the saf-
est water supplies in the world, drinking water quality varies from place to place, depending on the condition
of the source water and the treatment it receives. In 1996, Congress amended the Safe Drinking Water Act to
emphasize sound science and risk-based standard setting.
Threats to drinking water safety come from the occurrence of chemical contaminants or pathogens in
drinking water, and research is needed in a variety of areas to improve the ability to assess and thereby reduce
the public health risks from America's public water systems. The continued occurrence of waterborne disease
outbreaks demonstrates that the safety of drinking water might be threatened by pathogenic microorganisms if
treatment is inadequate or if the quality of water in the distribution system is compromised.
ORD's research on microorganisms that may impact human health through drinking water is conducted
across its three national laboratories (National Health and Environmental Effects Research Laboratory,
[NHEERL]; National Exposure Research Laboratory [NERL]; and the National Risk Management Research
Laboratory [NRMRL]) and through two national centers (National Center for Environmental Assessment
[NCEA] and National Center for Environmental Research [NCER]). In addition, EPA's drinking water re-
search program may indirectly benefit from microbial research being conducted through its National Home-
land Security Research Center, although no individual research efforts from this center were presented at the
workshop. NCER is responsible for implementing the Science To Achieve Results (STAR) competitive grants
program; the remaining national laboratories and centers manage intramural research programs.
This workshop was organized by representatives from ORD's laboratories and centers to bring together
ORD's intramural and extramural scientists who are researching microorganisms in drinking water, staff from
the Office of Water (OW), and regional office representatives. The meeting was open to the public. The work-
shop provided a forum for the scientists to present their research, for OW to identify the research needs associ-
ated with their upcoming regulatory agenda, and for all participants to discuss applications of the research.
The EPA uses meetings like this one to discuss research progress on topics of major scientific interest to
the Agency. The research reported is of critical importance to EPA, as it has the potential to strengthen the sci-
entific basis for both assessing the risk from exposure to pathogenic microorganisms and developing appropri-
ate risk-management practices to mitigate their effects.
The meeting had both platform and poster sessions. Presentations were provided by ORD intramural scien-
tists, STAR grantees, and representatives from a regional office and OW. The abstracts in this report are organ-
ized by research topic area into platform presentations and poster presentations in the order of the Agenda or
poster listing. For the one regional office presentation and the two presentations by OW in which regulatory
agendas or research needs were identified, rather than an abstract, the full presentations are provided in Ap-
pendix 1, Presentations of Regional Research Needs and Office of Water Regulatory Activities and Research
Needs. In addition, one STAR grantee was unable to attend the meeting; the abstracts of her two research
grants are provided in Appendix 2, Additional NCER STAR Drinking Water Grant Microbial Research.
Finally, for this meeting EPA arranged for Web-broadcast of the presentations, enabling remote partici-
pants to hear and view the plenary presentations and discussions. Hence, you will see a remote participants list
included in this proceedings document.
Rather than participate in this meeting, STAR grantees doing riverbank filtration research were invited to
present their research at a separate meeting arranged by EPA/ORD/NCER and EPA/OW jointly with the U.S
Geological Survey (USGS), The USEPA/USGS Meeting on Cryptosporidium Removal by Bank Filtration.
This meeting was held on September 9-10, 2003, at the USGS facility in Reston, VA. The meeting consisted of
a series of plenary presentations provided by researchers from OW, NCER's STAR program, USGS, U.S. De-
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
partment of Agriculture, universities, and states. This meeting also was open to the public. No formal proceed-
ings document was prepared; however, the event description, agenda, and abstracts of the STAR research pre-
sented are included in Appendix 3, STAR Grant Presentation Abstracts and Agenda From the USEPA/USGS
Meeting on Cryptosporidium Removal by Bank Filtration, September 9-10, 2003. In addition, pdf versions of
the full presentations can be found at: http://es.epa.gov/ncer/publications/meetings/drinking_sept9_03.html.
For more information on ORD's drinking water research program, please contact the Acting National Pro-
gram Director for Drinking Water, Gregory Sayles, at 513-569-7607 (sayles.gregory@epa.gov). For more in-
formation about EPA's STAR drinking water research grants program, you may contact Cynthia Nolt-Helms
at 202-343-9693 (nolt-helms.cynthia@epa.gov) or Angela Page at 202-343-9826 (page.angelad@epa.gov). For
more information on EPA's ORD, please visit our homepage: http://www.epa.gov/ord.
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Overview of the U.S. EPA's Drinking Water Research Program
Fred S. Hauchman
National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC
Presentation Abstract
Drinking water is one of the highest priority research programs of the U.S. Environmental Protection
Agency's (EPA) Office of Research and Development (ORD). To address the wide range of issues relating to
waterborne contaminants, the U.S. EPA has established an integrated, multidisciplinary drinking water re-
search program that is closely linked to the Office of Water's (OW) regulatory activities and timelines. Drink-
ing water research is conducted or supported by ORD's six national laboratories and centers: the National
Exposure Research Laboratory, the National Health and Environmental Effects Research Laboratory, the Na-
tional Risk Management Research Laboratory, the National Center for Environmental Assessment, the Na-
tionational Center for Environmental Research, and the National Homeland Security Research Center. The
research program supports OW decisionmaking and the implementation of EPA rules at the state and local
level through the development of new scientific data, innovative methods, and cost-effective technologies for
improving the assessment and control of drinking water risks.
ORD's drinking water research program activities and plans for fiscal years 2003-2010 are described in a
new Drinking Water Research Multi-Year Plan (MYP). As described in the MYP, the Safe Drinking Water
Act (SDWA) provisions with the most significant implications for research on waterborne pathogens include
the Microbial/Disinfection Byproduct (M/DBP) set of rules, the Contaminant Candidate List (CCL) of unregu-
lated contaminants, distribution systems, and source water protection. Research to address key uncertainties
associated with the Source Water and Ground Water rules (part of the M/DBP cluster of rules) includes efforts
to: (1) improve methods to detect Cryptosporidium in water matrices; (2) assess risks associated with exposure
to protozoa and viruses; and (3) remove Cryptosporidium, particularly for small systems. Research on unregu-
lated pathogens is primarily focused on developing new or improved analytical detection methods, and on de-
termining the ability of conventional and advanced treatment to remove or inactivate microorganisms. Studies
also are being conducted on innovative molecular approaches to characterize and prioritize pathogens for pos-
sible listing on future CCLs. Distribution system studies include research on opportunistic pathogens in bio-
films, and on the relationship between Mycobacterium disease and isolates of this microorganism in the distri-
bution system. Finally, research on source water assessment methods, tools, and best management practices is
being conducted to support both SDWA and Clean Water Act provisions that relate to the protection of drink-
ing water sources. This presentation will provide an overview of current and planned research activities in each
of these areas.
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Regional Concerns for Microorganisms
in Drinking Water
Bruce Macler
U.S. Environmental Protection Agency, Region 9, San Francisco
The full presentation can be found in Appendix 1.
The Office of Research and Development's National Center for Environmental Research
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Topic Area 1: Research Supporting Office
of Water's Ground Water/Source Water
Regulatory Activities
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Research on Microorganisms in Drinking Water Progress Review Workshop
SDWA Requirements & Microbial Research Needs
(Surface Water, Ground Water, & Distribution Systems)
Stig Regli
U.S. Environmental Protection Agency, Office of Water/Office of Ground Water
and Drinking Water
The full presentation can be found in Appendix 1.
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Microbial Dose-Response Modeling: A Predictive
Bayesian Approach
James D. Englehardt1 and JeffSwartouf
University of Miami, Miami, FL; Office of Research and Development, National Center for Environ-
mental Assessment, U.S. Environmental Protection Agency, Cincinnati, OH
Presentation Abstract
Absolute dose-response assessments have not been possible for low doses of chemicals and microbes, due
to the infeasibility of direct testing of low-dose response. The problem may apply to high doses in microbial
risk assessment as well, because health effects of microbes may not generally extrapolate from animals to hu-
mans as readily as for chemicals. The objective of this project is to develop a robust dose-response model for
human population response across all doses following exposure to a pathogen. The goal is to derive an ap-
proach that is physically and biologically plausible, and that accounts for variability in human susceptibility,
variability in microbial (intra-species) strain virulence, variability in human-pathogen interaction, and variabil-
ity in the form and quantity of available information.
A predictive Bayesian approach has been selected as the most efficient means of integrating all the desired
factors into a framework that is independent of any arbitrarily chosen confidence limit. Recently, a predictive
Bayesian method for absolute dose-response assessment from limited information has been proposed. Informa-
tion types can be diverse, such as epidemiologic results, genetic prevalence data, cell culture data, and medical
judgment, as well as conventional dose-response data. All available information is integrated rigorously, and
the function narrows in response to information content. Response is measured in terms of believed risk, which
is slightly higher than the expected frequency of health response as would be estimated by traditional frequen-
tist methods, even with an allowance for uncertainty.
Predictive Bayesian models based on both the infection and illness endpoints are demonstrated in this pro-
ject. The exact form (based on the confluent hypergeometric function) of the beta-Poisson dose-response
model is used for modeling the infection endpoint. The model for the illness endpoint is derived from a self-
organized critical pattern of pathogenic illness severities and is demonstrated numerically. Results indicate that
self-organizing characteristics of pathogenesis result in a third parameter of the dose-response function for mi-
crobes corresponding to the assumed definition of illness (minimum severity). Information-limited predictive
Bayesian dose-response assessments, obtained for Cryptosporidium parvum infection and illness endpoints,
are compared and contrasted. The dose corresponding to 10" cases of waterborne cryptosporidiosis per capita-
year is estimated to be 0.002 oocysts per exposure. Figure 1 shows the predictive dose-response function for
Cryptosporidiosis based on the human response data for three C. parvum isolates, which also are shown in the
figure.
The findings are significant in that they incorporate varied information, rigorously accounting for limited
data in a manner that allows for straightforward updating with new information, without dependence on preset
confidence intervals. Follow-on activities include an analysis and integration of data for a new C. parvum iso-
late and application of the model to other pathogens. Investigation of more flexible, but still biologically rele-
vant models for better fitting of the entire dose-response curve is being pursued.
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
1
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Figure 1. Predictive dose-response function for C. parvum: illness endpoint.
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Microbial Dose-Response Modeling: A Predictive
Bayesian Approach
James D. Englehardt1 and JeffSwartouf
University of Miami, Miami, FL; Office of Research and Development, National Center for Environ-
mental Assessment, U.S. Environmental Protection Agency, Cincinnati, OH
Presentation Abstract
Absolute dose-response assessments have not been possible for low doses of chemicals and microbes, due
to the infeasibility of direct testing of low-dose response. The problem may apply to high doses in microbial
risk assessment as well, because health effects of microbes may not generally extrapolate from animals to hu-
mans as readily as for chemicals. The objective of this project is to develop a robust dose-response model for
human population response across all doses following exposure to a pathogen. The goal is to derive an ap-
proach that is physically and biologically plausible, and that accounts for variability in human susceptibility,
variability in microbial (intra-species) strain virulence, variability in human-pathogen interaction, and variabil-
ity in the form and quantity of available information.
A predictive Bayesian approach has been selected as the most efficient means of integrating all the desired
factors into a framework that is independent of any arbitrarily chosen confidence limit. Recently, a predictive
Bayesian method for absolute dose-response assessment from limited information has been proposed. Informa-
tion types can be diverse, such as epidemiologic results, genetic prevalence data, cell culture data, and medical
judgment, as well as conventional dose-response data. All available information is integrated rigorously, and
the function narrows in response to information content. Response is measured in terms of believed risk, which
is slightly higher than the expected frequency of health response as would be estimated by traditional frequen-
tist methods, even with an allowance for uncertainty.
Predictive Bayesian models based on both the infection and illness endpoints are demonstrated in this pro-
ject. The exact form (based on the confluent hypergeometric function) of the beta-Poisson dose-response
model is used for modeling the infection endpoint. The model for the illness endpoint is derived from a self-
organized critical pattern of pathogenic illness severities and is demonstrated numerically. Results indicate that
self-organizing characteristics of pathogenesis result in a third parameter of the dose-response function for mi-
crobes corresponding to the assumed definition of illness (minimum severity). Information-limited predictive
Bayesian dose-response assessments, obtained for Cryptosporidium parvum infection and illness endpoints,
are compared and contrasted. The dose corresponding to 10" cases of waterborne cryptosporidiosis per capita-
year is estimated to be 0.002 oocysts per exposure. Figure 1 shows the predictive dose-response function for
Cryptosporidiosis based on the human response data for three C. parvum isolates, which also are shown in the
figure.
The findings are significant in that they incorporate varied information, rigorously accounting for limited
data in a manner that allows for straightforward updating with new information, without dependence on preset
confidence intervals. Follow-on activities include an analysis and integration of data for a new C. parvum iso-
late and application of the model to other pathogens. Investigation of more flexible, but still biologically rele-
vant models for better fitting of the entire dose-response curve is being pursued.
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
1
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TAMU Pareto II fit
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Pred. dose-resp. function
Maximum (ex pos ure) risk
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Figure 1. Predictive dose-response function for C. parvum: illness endpoint.
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
The Use of Randomized Trials of In-Home Drinking Water Treatment
To Study Endemic Water Borne Disease
Timothy J. Wade1, Rebecca Colder on1, and John M. Coif or d, Jr.2
1 Human Studies Division, U.S. Environmental Protection Agency, Chapel Hill, NC; 2 School of Public
Health, University of California—Berkeley, Berkeley, CA
Presentation Abstract
Randomized trials of water treatment have demonstrated the ability of simple water treatments to signifi-
cantly reduce the incidence of gastrointestinal illnesses in developing countries, where drinking water is of
poor quality. Whether or not additional treatment at the tap reduces enteric illness in areas where water is
treated to a higher degree has not been fully resolved. Randomized trials of in-home water treatment have been
conducted to determine how much enteric illness, if any, is transmitted through a public water system and to
determine whether or not current water treatment practices adequately protect public health.
The goal of this project is to review randomized trials of in-home water treatment conducted in developed
countries, discuss key study design characteristics and sources of bias, and present preliminary data from sev-
eral ongoing trials. The advantages and limitations of this type of study design also will be presented. The ba-
sic study design is as follows: households are randomly assigned to receive a device that provides additional
treatment of pathogenic microorganisms in the household. The control group receives either no device, or re-
ceives an identical looking placebo device. Household members record occurrences of gastrointestinal symp-
toms. Any statistically significant difference between the rate of illness in the control group and the rate of ill-
ness in the active group is considered to be the amount of excess illness attribut- able to drinking regularly
treated tap water. Clinical specimens also may be collected and tested for common pathogenic microorgan-
isms.
Features to consider when designing or interpreting the results include: source water quality, distribution
system water quality, study location, study population, sample size, device design, use of a placebo, recruit-
ment methods, blinding of participants and investigators, randomization procedures, tracking water consump-
tion, collection of clinical specimens, collection of water samples, statistical analyses, and outcome measure-
ment and definition.
Four trials have been completed and published, two in Canada, one in Australia, and one small pilot study
in the United States (Walnut Creek, California). The two trials in Canada identified a significant excess risk of
illness in those receiving regular tap water. A blinded study in Australia, however, found no such increase in
illness and, unlike the Canadian studies, included a placebo device and a blinded control group. Several major
trials are ongoing or nearly completed in the United States. These include the full-scale followup to the pilot
study being conducted in Davenport Iowa. Results from a second trial in an HIV infected population also will
be published soon, and a large multiyear study in an elderly cohort is nearing completion. An additional study
is being conducted in a community with a groundwater drinking water supply.
Results of these ongoing trials may provide a more complete picture of the adequacy of current microbial
drinking water regulations. It is unlikely that trial data alone can provide a complete picture of the risks associ-
ated with tap water consumption. The applicability of trials in restricted geographic locations to the entire U.S.
population is questionable. Moreover, the interpretation of null results must be fully considered, and alterna-
tive hypotheses must be developed to explain such findings.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Screening Models To Predict Probability of Contamination
by Pathogenic Viruses to Drinking Water Aquifers
Bart Faulkner
Office of Research and Development, National Risk Management Research Laboratory,
U.S. Environmental Protection Agency, Ada, OK
Presentation Abstract
The purpose of this research is to develop simple, screening level models to predict the overall ability of
ground-water systems to attenuate viable viruses introduced to them. Compartmental modeling approaches are
used because of their simplicity. Stakeholders and decisionmakers often seek cursory screening level models to
identify at-risk drinking water supplies (see Figure 1). When applied under appropriate circumstances, com-
partmental models can allow prioritization of groundwater systems for further investigation, especially if such
models employ probabilistic methods in their development that can capture much of the uncertainty in input
parameters. The goals of this research are to develop such screening models and to perform sensitivity analyses
of the models, and to develop prior distributions that correspond to actual uncertainties in the domain of the
inputs.
The approach used is to identify components of groundwater systems that can be considered to behave as
compartments, contributing to a final catchment scale subsurface flow model. After these flow models are
developed, appropriate attenuation functions are derived and applied. In most cases, these are based on
physical, deterministic governing differential equations. By invoking certain assumptions such as steady state,
or well-mixed reactors, the equations can be solved to yield algebraic expressions. Uncertainty is captured in
final model estimates of attenuation by (1) error propagation methods, or (2) Monte Carlo methods. Bayesian
approaches will be used to specify prior distributions for parameters to allow for sparse data and robustness,
and to quantify uncertainty.
At present, a complete probabilistic modeling approach has been developed for homogeneous unsaturated
soils. Assuming gravity flow, a suite of prior distributions has been developed for each of the 12 U.S. Depart-
ment of Agriculture soil categories. Probabilistic outputs show that for one-half meter thick soils, most exhibit
probabilities of failure to attenuate most viruses to the 99.99 percent reduction demanded in many regulatory
compliance settings. Sensitivity analyses show that mean logio of saturated hydraulic conductivity and the wa-
ter-to-air mass transfer affected virus fate and transport about three times more than any other parameter, in-
cluding inactivation rate of percolating viruses. A user-friendly computer model with a graphical user interface
has been developed and released.
The sensitivity analyses suggest extreme infiltration events may play a predominant role in leaching of
viruses in soils, as such events could impact hydraulic conductivity. The water-air interface also plays a large
role. The current research issue of accurate estimation of the air-water interfacial area is an important one, not
only for modeling transport of contaminants subject to hydrophobicity effects, but also for unsaturated-zone
virus transport modeling.
Developing additional components of an overall catchment-scale groundwater model, including residence
time in the saturated zone, remains as an important next step. Challenges include developing appropriate as-
sumptions to make residence time prediction for spatially explicit virus loadings tractable, and yet still reason-
able for useful prediction.
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Public Water
Supply Well
^""/J.• IV'' :%• Proposed hydrogeologic barrier;
Figure 1. A user-friendly Monte Carlo-based model (Virulo) has been developed by the U.S. EPA. It can be
used to predict the probability of failure of a proposed hydrogeologic barrier to attenuate viruses to
a given level.
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Integrated Approach for the Control of Cryptosporidium parvum
Oocysts and Disinfection By-Products in Drinking Water Treated
With Ozone and Chloramines
Jason L. Rennecker, Amy (Driedger) Samuelson, Benito Corona-Vasquez, Jaehong Kim,
Hongxia Lei, Roger A. Minear, and Benito J. Marinas
Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign,
Vrbana, IL
Presentation Abstract
The overall goal of this project was to develop process design recommendations for the simultaneous con-
trol of Cryptosporidium parvum oocysts and disinfection by-products (DBFs) during ozone/chloramines se-
quential disinfection of natural waters. Because the main objective of the study was to develop an integral con-
trol strategy, the scope of work included investigating a limited number of DBFs (bromate, formaldehyde, and
cyanogen halides) associated with the ozone/chloramines sequential disinfection process. This presentation
will emphasize experimental findings on the inactivation of C. parvum oocyst with sequential ozone/chlor-
amines (as well as ozone/free chlorine) schemes. The DBF work of the study will be presented at a future
NCER STAR meeting focusing on DBF aspects.
C. parvum oocysts have emerged as the microbial water contaminant with greatest resistance to chemical
disinfectants. There is particular concern because both the free and combined forms of chlorine, most com-
monly used as primary inactivation agents, are considered practically ineffective in controlling C. parvum
oocysts under typical drinking water conditions. In contrast, ozone and chlorine dioxide are both considered
viable chemical disinfectants, but there is concern about potentially high disinfection requirements. A promis-
ing alternative for more efficient control of C. parvum oocysts is the sequential application of certain combina-
tions of disinfectants. The efficiency of C. parvum oocyst inactivation by combined as well as free chlorine can
be increased significantly after limited exposure to ozone.
There are various factors that could affect the overall efficiency of sequential ozone/monochloramine as
well as ozone/free chlorine disinfection processes. These include pH, temperature, ozone pretreatment level,
and oocyst resistance variability. Experimental results showing the role of these various parameters will be
presented.
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Prevalence and Distribution of Genotypes of Cryptosporidium parvum
in United States Feed lot Cattle
Robert Atwill
University of California at Davis, Davis, CA
Presentation Abstract
The overall goal for this research project is to characterize the ability of feedlot cattle in the United States
to load the environment with the protozoal parasite, Cryptosporidium parvum. Specific objectives are to estab-
lish the prevalence, intensity, and distribution of genotypes of C. parvum in populations of feedlot cattle and to
identify risk factors that are associated with feedlot cattle shedding C. parvum.
Fecal samples were collected from 5,274 cattle, whereby approximately 240 cattle from 22 different feed-
lots located in California, Washington, Texas, Oklahoma, Colorado, South Dakota, and Nebraska were sam-
pled. Within each feedlot, fecal samples were typically collected from sets of cattle who had just arrived, had
been at the feedlot 4 to 8 weeks, and from cattle several weeks prior to slaughter to generate a comprehensive
survey of fecal shedding of C. parvum.
Out of 5,274 fecal samples, only 9 (0.2%) had detectable levels of oocysts as measured by our standard
diagnostic test, direct immunofluorescent microscopy (see Table 1). This assay can reliably detect oocyst con-
centrations down to about 600 oocysts per gram of feces. To determine if a percentage of cattle were shedding
small numbers of oocysts, 10 negative cattle were retested from each feedlot using immunomagnetic separa-
tion of oocysts followed by direct immunofluorescence (IMS-DFA). Our IMS-DFA method can detect as few
as 1 oocyst per gram of bovine fecal material, arguably the most sensitive method published to date for detect-
ing C. parvum in bovine feces. Using this highly sensitive method, 2 out of 220 (0.9%) fecal samples con-
tained low levels of oocysts, indicating that false negatives were relatively uncommon in our data. This dataset
is being statistically modeled to estimate the environmental loading rate using methods developed in our labo-
ratory.1 Using a nested polymerase chain reaction technique associated with restriction fragment length poly-
morphism developed by Xiao et al. (1999) of the Centers for Disease Control and Prevention that targets the
18S rRNA gene, the genotype for these isolates was the bovine genotype A.
Although we are still in the process of calculating the environmental loading rate of C. parvum bovine
genotype A from feedlot cattle, these preliminary data suggest that feedlot cattle are not heavily infected with
C. parvum in middle and western United States. This lack of substantial shedding of C. parvum among feedlot
cattle is a positive finding, given the fact that feedlots are located throughout the United States, feedlots can
produce large amounts of fecal material on a per acre basis, and given the infectious potential of bovine C.
parvum for susceptible humans.
Reference
1. Atwill, E.R., et al. 2003. Improved quantitative estimates of low environmental loading and sporadic peri-
parturient shedding of Cryptosporidium parvum in adult beef cattle. Applied Environmental Microbiology
69:4604-4610.
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Table 1. Prevalence of Fecal Shedding of Cryptosporidium parvum in Feedlot Cattle in the United States.
California
Washington
Texas
Oklahoma
Colorado
South Dakota
Nebraska
TOTAL
4
1
3
3
4
4
3
22
0/960
2/240 (0.8%)
0/711
0/722
6/957 (0.6%)
1/964(0.1%)
0/720
9/5,274 (0.2%)
0/40
0/10
0/30
1/30
0/40
1/40
0/30
2/220 (0.9%)
1 Diagnostic method was direct immunofluorescent assay.
2 Diagnostic method was immunomagnetic separation of oocysts coupled with a direct immunofluorescent
assay, capable of detecting one oocyst per gram of feces.
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Microbial Drinking Water Contaminants: Endemic and Epidemic
Waterborne Gastrointestinal Disease Risks in the United States
Rebecca L. Calderon1 and Gunther Craun2
U.S. Environmental Protection Agency, Research Triangle Park, NC;
Gunther F. Craun Associates, Staunton, VA
Poster Abstract
In the United States, drinking water treatment is rightfully considered a major public health achievement
of the 20th century. However, a residual number of waterborne outbreaks continue to be reported when water
treatment systems fail or are poorly operated, and distribution systems or sources become contaminated. In
some reported outbreaks, current drinking water standards had not been exceeded. Recent epidemiologic stud-
ies also have suggested that cases of mild, unreported gastroenteritis may be associated with consumption of
tap water from systems where no outbreak was reported. The U.S. EPA has collaborated with the Centers for
Disease Control and Prevention for more than 30 years in the surveillance and investigation of waterborne dis-
ease outbreaks. The periodic analysis of waterborne outbreak statistics from 1971 to 2000 has assisted the U.S.
EPA in developing a research program and promulgating regulations for safe drinking water (e.g., the Surface
Water Treatment Rule, Ground Water Treatment Rule, Total Coliform Rule). The nature and magnitude of
endemic waterborne disease, however, is not well characterized in the United States. Recent studies conducted
in Canada suggest that in some communities that meet all current drinking water regulations, drinking water
could be a significant cause of gastrointestinal (GI) disease. Because persons with GI illnesses rarely visit a
physician for treatment, the causes of these illnesses in developed countries are difficult to study by traditional
observational epidemiologic designs.
EPA's Surface Water Treatment Rule (SWTR) of 1989 requires all communities that use surface water as
a source of their drinking water to filter their water, unless special criteria are met. The promulgation of this
rule provided an opportunity to use a natural experiment and a quasi-experimental epidemiologic design to
evaluate endemic waterborne illness risks. After surveying water utilities affected by the SWTR, 21 utilities
were found that would be good candidates for an epidemiologic study of waterborne disease. A pilot epidemi-
ologic study was conducted in one community to evaluate the endemic waterborne gastrointestinal illnesses
risks and obtain information for use in designing additional studies. This community intervention study col-
lected information about daily GI symptoms from families before and after filtration was added to the drinking
water treatment process Information was obtained during July through December in 1996 and 1997. As a re-
quirement for participation, families had to include one or more children in the study. Analyses of the pilot
study community indicate a significant reduction occurred in the rate of credible-gastrointestinal (CGI) illness
after filtration of drinking water. The relative risk of CGI before filtration was almost double that after filtra-
tion (RR=1.8, 95% CI=1.5-2.1). The attributable risk of CGI associated with unfiltered drinking water is esti-
mated to be 34 percent. We concluded that this is an efficient study design and have moved forward with two
other community intervention studies. The second and third communities will include an assessment of CGI in
families in a control community to control for community variability in the incidence of GI illness and help
interpret the observed waterborne contribution to illness risks. The second study in Washington State has fin-
ished data collection, and the third study in southwest Texas is in the middle of data collection.
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Evaluating Microbial Indicators and Health Risks
Associated With Bank Filtration
Floyd J. Frost
Lovelace Clinic Foundation, Albuquerque, NM
Poster Abstract
The purpose of the proposed project is to compare serological responses to Cryptosporidium antigens in
users of bank-filtered water (one community with only bank filtration and disinfection and one community
with bank filtration, conventional filtration, and disinfection) with the responses of similar people residing in
an area that uses disinfected but unfiltered high-quality groundwater. The hypothesis is that, if bank filtration
completely removes Cryptosporidium oocysts, the serological responses of the three populations should be
similar. The specific goals of the study are to: (1) identify approaches to collecting sera from similar popula-
tions in different geographic locations so that rates of serological responses can be compared; (2) pilot test the
approach in three different geographical locations by collecting sera from cities that use bank filtration and
nearby cities that use high-quality groundwater for a drinking water source; (3) analyze the sera for serological
responses to Cryptosporidium and Giardia antigens and compare the frequency and intensity of responses be-
tween the bank filtration cities and the groundwater cities; and (4) compare serological responses in the same
cities at times when bank filtration efficacy is likely to be optimal and when it is likely to be least effective.
Sera from 50 people from each of three communities (users of bank filtered and chlorinated, bank filtered
plus direct filtration plus ozonation, and chlorinated groundwater) will be collected at baseline and at five fol-
lowup blood draws. A questionnaire on risk factors will be collected at each blood draw. Sera will be tested for
the presence of antibody responses to two Cryptosporidium antigens (15/17-kDa and 27-kDa) and for serologi-
cal changes (seroconversion). The baseline level of serological responses as well as the rates of seroconversion
will be compared for each population (50 baseline and 250 periods for estimating rates of seroconversion).
Comparisons will adjust for collected risk factor data from each individual. For purposes of extrapolating these
results to other locations, a series of source and finished water quality indicators will be measured for each
water source.
No results are available at this time. Analysis of sera will take place once all sera are collected. Then, all
sera from a subject will be run on the same Western blot to reduce variations between blots. Blood draws will
continue every 4 months. Data entry protocols will be developed and implemented for data entry of the ques-
tionnaires. Sample analysis will commence once all of the samples are collected, because the analysis of each
subject's samples will be on the same Western blot. The distribution systems analysis also will commence.
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Research on Microorganisms in Drinking Water Progress Review Workshop
A Prospective Epidemiological Study of Gastrointestinal Health
Effects Associated With Consumption of Conventionally
Treated Groundwater
Christine Moe1, Stuart Hooper1, Deborah Moll2, Debi Huffman3, Ricardo Izurieta2'3,
Renea Doughton-Johnson , Tatiana Ochoa , Jim Uber , Dominic Boccelli' ,
Joan Rose , and Pierre Payment
Collins School of Public Health, Emory University, Atlanta, GA; 2 Centers for Disease
Control and Prevention, Atlanta, GA;3University of South Florida, St. Petersburg, FL;
4University of Cincinnati, Cincinnati, OH; 5Michigan State University, Lansing, MI;
6Universite du Quebec, Quebec, Canada
Poster Abstract
The overall goal of this study is to estimate the risks of endemic gastrointestinal (GI) illness associated
with the consumption of conventionally treated groundwater in the United States and determine the relative
contributions of source water quality, treatment efficacy, and distribution system vulnerability to endemic
waterborne disease.
In the United States, nearly 89 million people depend on community groundwater systems for drinking
water. Epidemiology studies in communities using surface water sources have suggested that 10-40 percent of
GI illness may be associated with drinking water. Recent national groundwater surveys have found significant
occurrence of microbial contamination in groundwater sources, and there is no information about the endemic
illness that may be associated with consumption of treated groundwater. Also, there is uncertainty about the
relative magnitudes of risks from problems with distribution systems and risks from treatment deficiencies.
The specific aims of this study are to: (1) compare GI illness rates in individuals drinking highly purified bot-
tled water to GI illness rates in individuals drinking conventionally treated groundwater bottled at the treatment
plant to determine the risk of GI illness associated with source water quality and treatment; and (2) compare GI
illness rates in individuals drinking bottled treatment plant water to GI illness rates in individuals drinking tap
water from selected areas of the distribution system to determine the risk of GI illness associated with distribu-
tion system vulnerability.
This study is a 12-month, double-blinded, randomized intervention trial of 900 households in a large
metropolitan area in the southeastern United States with a community groundwater system that uses con-
ventional treatment, meets current water quality standards, and has a well-characterized distribution sys-
tem with areas of vulnerability. Study households will be randomly divided into three groups of 300
households: Group 1 households will drink bottled water that has been treated with ozonation and reverse
osmosis (O3-RO bottled water), Group 2 households will drink bottled water collected at the water treat-
ment plant after treatment (WTP bottled water), and Group 3 households will drink tap water from their
home. Groups 1 and 2 will be blinded to their group assignment. One-half of the households within each
group will be recruited from vulnerable areas in the distribution system to examine the health risks from
distribution system intrusion. A summary of the experimental design is shown in Figure 1. Study partici-
pants will report GI illness symptoms and selected risk factors in a weekly health diary and biweekly
telephone interview. Samples will be routinely collected from raw source water, treated water, distribu-
tion system water, and bottled water and analyzed for microbial indicators of fecal contamination and in-
trusion. Data analyses will compare GI illness rates and water quality among the three study groups and
among study households in different parts of the distribution system.
Work to date has involved developing survey materials and obtaining Institutional Review Board ap-
proval, coordinating activities with the local utility, obtaining and analyzing historic and current water quality
data, and investigating areas of the distribution system that may be more vulnerable to intrusion. The vulner-
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ability assessment has been performed using hydraulic simulations of the distribution system, water quality
data, operations data (main breaks and repairs, customer complaints), and expert opinion provided by plant and
distribution system personnel.
This is the first study to measure the risk of GI illness associated with the consumption of conventionally
treated groundwater and to distinguish between the risk from source water and treatment vs. the risk from the
distribution system. The results of this study will provide valuable information on the magnitude of endemic
GI illness associated with drinking water in the United States. The next steps in this study will be to recruit and
enroll households and begin the intervention and data collection.
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Water Treatment Plant
Less-vulnerable distribution system
Vulnerable distribution system
300 Pure Bottled HH(A+B)
vs.
300 WTP Bottled HH(A+B)
Water Treatment P ant
Vulnerable distribution system
150 WTP Bottled HHs
150 Tap Water HHs
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Using Neural Networks To Create New Indices
and Classification Schemes
Gail Brian and Srini Lingireddy
Department of Civil Engineer ing, University of Kentucky, Lexington, KY
Poster Abstract
The hypothesis for this project is that shifts in indicator and indigenous bacterial populations can be relia-
bly and mathematically related by neural network models to the presence, concentration, age, and source of
microbial pathogens in river water. To test this hypothesis in the Kentucky River, tools must be developed that
provide early warning of potentially "risky" conditions in source waters for Water Treatment Plants. Specifi-
cally, the objectives are to: (1) collect and analyze surface water samples for a host of surrogate indices and
probable human pathogens over a multiyear period; (2) apply traditional modeling techniques and statistical
analysis to the database to find relationships between indices and pathogen presence; (3) define the correlation
between atypical coliform colonies and presence/ concentration of other surrogate indicators and pathogens;
(4) confirm the relationship between the ratio of atypical coliform colonies to total coliforms (AC/TC) with
time; and (5) apply neural network modeling (ANN) for identification of indices and combinations of indices
related to pathogen presence, concentration, and probable source as well as to gain insight into guidelines for
the application of ANNs for surface water quality modeling.
A multiparameter database will be created locally, while other databases are accumulated internationally.
ANNs will be applied to predict the presence of pathogens (enteric viruses, protozoa) from other water quality
parameters (i.e., turbidity, pH, alkalinity, indicator bacteria, indicator bacteriophage, fecal sterols, bacterial
ratios). ANN modeling has been applied to a dataset provided by multiple investigators from Europe for the
prediction of enteric viruses from shellfish samples. It has been shown that the ANN approach is superior to
that of logistic regression. Also, it has been demonstrated how ANNs can be used to determine which of the
variables are of significance. A case study related to the research objectives of this grant has shown that the
AC/TC ratio can find sources of human pollution in a creek where other proposed indicators cannot. Along the
waterway studied, levels of fecal coliforms and enterococci were statistically indeterminate. However, the
AC/TC ratio dropped whenever the waterway passed through inadequately sewered towns with statistical sig-
nificance.
Initial sampling of the Kentucky River has shown a trend between drops in the AC/TC ratio and the pres-
ence of fecal sterols, FRNA phage, protozoa, and enteric viruses. However, many more samples are required
before this noted trend can be statistically verified. ANN modeling has again shown itself to be superior to
other regression-based modeling methods for the precise prediction of pathogen presence. This provides the
basis for new monitoring and control models. If the AC/TC ratio is upheld as a valid means of assessing the
presence of fresh fecal contamination, these new indices could be applied for initial watershed sampling to find
"hot spots" for applying remediation. The ratio might provide the basis for new types of TMDL models that
consider the age and source of fecal material as well as load.
The next steps are to continue sampling on a weekly to biweekly basis and analyzing the in-house data-
bases accumulated, and to develop an LC/MS based methodology for fecal sterols.
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Topic Area 2: Research Supporting Office of
Water's Contaminant Candidate List (CCL)
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Research on Microorganisms in Drinking Water Progress Review Workshop
The Contaminant Candidate List: Determining the Need for Future
Drinking Water Standards
Tom Carpenter
U.S. Environmental Protection Agency, Office of Water/Office of Ground Water
and Drinking Water
The full presentation can be found in Appendix 1.
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The Roles of Pathogen Risk Assessment in the Contaminant
Candidate List Process
Glenn Rice, Michael Wright, Brenda Boutin, Jeff Swartout, Michael Broder, Patricia Murphy,
Jon Reid, and Lynn Papa
National Center for Environmental Assessment, U.S. Environmental Protection Agency, Cincinnati, OH
Presentation Abstract
Under the 1996 Amendments to the Safe Drinking Water Act, the U.S. EPA is required to make regular
determinations of whether or not to regulate contaminants on the Contaminant Candidate List (CCL). The pro-
vision of safe public drinking water includes ongoing monitoring for contaminants with the potential to cause
adverse health effects in humans through tap water exposures. The CCL includes 10 bacterial and viral patho-
gens. The U.S. EPA's Office of Research and Development (ORD) and Office of Water (OW) have published
a draft research strategy to identify and prioritize research for contaminants listed on the CCL. The CCL re-
search strategy highlights the need to conduct risk assessments at two different time points. The initial assess-
ment (see Figure 1) identifies and integrates all of the available information needed to estimate the risk posed
by a contaminant, and identifies and prioritizes the information gaps so that the most appropriate research can
be conducted. After the data identified in the initial assessment are collected, a second risk assessment is con-
ducted to provide a more accurate estimate of the risk posed by the contaminant than was determined by the
initial assessment. The OW can use these assessments to decide whether or not a contaminant should be regu-
lated. EPA's National Center for Environmental Assessment (NCEA) is developing pathogen assessments to
address this initial assessment phase. A recently completed NCEA assessment of exposure to Mycobacterium
avium Complex (MAC) via tap water ingestion will be presented as an example of this process. After evaluat-
ing the available information, the infection risk from MAC-contaminated tap water appears to be limited to a
few populations including people with AIDS, transplant recipients and others patients receiving immunosup-
pressive therapies, individuals with compromised pulmonary systems, and children. CD4+ cell counts are a
strong predictor of MAC infection risk in the AIDS population; therefore, the MAC assessment targeted the
fraction of the U.S. AIDS population having less than 100 CD4+ cells/mm3, the severely immunocompromised
fraction of the AIDS population. Members of this population would be expected to experience the most severe
responses to MAC infections. Two exposure assessments were developed to address MAC ingestion exposures
in this population (see Figures 2 and 3). Epidemiologic research designed to examine the association between
MAC infections in the severely immunocompromised fraction of the AIDS population and water-related
activities is needed. If water-related activities are risk factors for MAC infection, then research assessing
pathogen exposure-response relationships in the AIDS population and in other susceptible populations also is
needed.
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Are data available on
treatability by
generally available
technology?
LEGEND:
Activities
Scientific
Questions
Figure 1. CCL Phase I decision making process.
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CDC, 2002b
US AIDS Population
(mean =3 34,000)
Fraction of U.S. AIDS Population with
< 100 CD4+ cells/mm3 blood (20%)
Aragon et al., 2003; Kim et al., 1998
No Tap Water Consumptiorr^sfot At Risk (21-43%)
Always Consume Tap Water 1
(12%)
Some Tap Water Consumption J
(remaining fraction, 45-67%)
Eisenberg et al., 2002
Further Treat>Not At Risk
Tap Water (50%)
At Risk Population
(mean = 400 individuals with AIDS having
CD4+ < 100/mm3 ingest detectable levels of MAC/day)
Figure 2. Model 1: Severely immunocompromised AIDS population exposed to MAC via tap water
ingestion.
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CDC. 2002b
US AIDS Population
(mean = 334,000)
Fraction of AIDS population with
< 100 CD4+ cells/mm3 blood (20%)
Davis et al..
Percentage of Tap Water
fo/.
Consumed at Home
100
76-99
51-75
26-50
1-25
None
Fraction of HIV Population
18
28
16
26
At-Risk Population
(mean = 1500 individuals with AIDS having
CD4+ < 100/mm3 ingest detectable levels of MAC/day)
Figure 3. Model 2: Severely immunocompromised AIDS population exposed to MAC via tap water
ingestion.
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Overview: CCL Pathogens Research at NRMRL
Donald J. Reasoner
Microbial Contaminants Control Branch, Water Supply and Water Resources Division,
National Risk Management Research Laboratory, U.S. Environmental Protection Agency,
Cincinnati, OH
Presentation Abstract
The Microbial Contaminants Control Branch (MCCB), Water Supply and Water Resources Division, Na-
tional Risk Management Research Laboratory (NRMRL), conducts research on microbiological problems as-
sociated with source water quality, treatment processes, distribution, and storage of drinking water. MCCB's
work on Contaminant Candidate List (CCL) microorganisms was formally initiated in 1998 coincident with
the publication of the CCL. The CCL included 10 microorganisms: Acanthamoeba (Guidance), Adenoviruses,
Aeromonas hydrophila, Caliciviruses, Coxsackieviruses, Cyanobacteria (blue-green algae), Echoviruses, Heli-
cobacterpylori, Microsporidia, andMycobacterium avium intracellulare. MCCB has completed planned disin-
fection studies on A. hydrophila and will complete similar studies with H. pylori next year. Disinfection stud-
ies with Adenovirus were poised to begin shortly after September 11, 2001. Those studies were set aside to
conduct disinfection studies on surrogates for bioterrorism agents. When disinfection studies on Adenovirus
begin, they will be done as a collaborative effort between NRMRL and the National Environmental Research
Laboratory. In collaboration with the University of Arizona, limited studies on the disinfection of micro-
sporidia have been completed using chlorine and chloramine. As a result of the events of September 11, disin-
fection studies on Coxsackievirus, Echovirus, and M. avium intracellulare will be delayed for 2 to 3 years.
Work on inactivation of cyanobacteria toxins by drinking water disinfection treatment is ongoing with the
University of Wisconsin and the Wisconsin State Health Department.
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Topic Area 2.1: CCL Protozoa
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Research on Microorganisms in Drinking Water Progress Review Workshop
Detection of Cyclospora cayetanensis and Microsporidial Species
Using Quantitative Fluorogenic 5' Nuclease PCR Assays
Frank W. Schaefer, III, JeffD. Hester, Manju Varma, Michael W. Ware, and Harley D.A. Lindquist
National Exposure Research Laboratory, Cincinnati, OH
Presentation Abstract
Both Cyclospora cayetanensis, a coccidian parasite, and Encephalitozoon spp., a microsporidian parasite,
have a fecal-oral life cycle. These parasites can be transmitted as contaminants of either food or water. Tradi-
tional microscopic methods for detecting and identifying these organisms in water are tedious, time consum-
ing, and not always accurate. In the case of microsporidians, detection and identification of spores (see Figures
1 and 2) is usually done by transmission electron microscopy, which, although accurate, is not feasible for
rapid analysis of water samples. To address these shortcomings, we have developed molecular assays for these
organisms using 5' nuclease PCR that incorporate both a primer set and a dual labeled fluorogenic probe. For
C. cayetanensis, both a species-specific primer set and fluorescent labeled probe were designed based on the
uniqueness of the 18S ribosomal gene sequence of this parasite. For Encephalitozoon spp. (E. hellem, E. intes-
tinalis, and E. cuniculi), species-specific primer sets and a genus-specific fluorogenic probe designed to anneal
within the Encepahalitozoon 16S rRNA gene were used. Oocysts and spores were counted accurately on a
fluorescence activated cell sorter to determine the sensitivity of these assays. Results were that as few as 1 C.
cayetanensis oocyst and 1 Encephalitozoon spp. spore could be detected per 5 uL reaction volume. Utilizing
standard curves, the quantity of the parasites detected can be estimated with these assays. Specificity of these
molecular assays were tested against DNA isolated from numerous other related and unrelated protozoa, fresh
water algae, and bacteria. In no case were any cross-reactions detected. These assays, although sensitive and
specific, will not determine the viability or infectivity of the detected parasite. In addition, these are assays and
are not complete methods suitable for routine use with water samples. A complete method for analysis of water
samples will require insertion of both suitable water concentration steps as well as parasite purification steps
before the 5' nuclease PCR assay is utilized.
Figure 1. Transmission electron micrograph of Figure 2. Scanning electron micrograph
an Encephalitozoon spp. spore with its of an Encephalitozoon spp.
polar tube extended.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Development of Detection and Viability Methods for Waterborne
Microsporidia Species Known To Infect Humans
Rebecca Hoffman1, Marilyn Marshalf, and Mark Borchard/
WI State Laboratory of Hygiene, University of Wisconsin—Madison, Madison WI; Department
of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ; Marshfield
Medical Research Foundation, Marshfield, WI
Presentation Abstract
Microsporidia are obligate intracellular parasites capable of initiating disease in a host of vertebrate and
invertebrate species following ingestion of a small, environmentally resilient spore. Waterborne transmission
of this organism is suspected. However, methods to detect Microsporidia in the aquatic environment are de-
velopmental at best. The objective of this project is to develop a robust strategy for detection of waterborne
Microsporidia using seeded model and natural waters, and ultimately perform method validation in unseeded
natural waters.
Flow cytometry with cell sorting (FCCS) was used to generate precisely enumerated Encephalitozoon in-
testinalis seeding standards. Standards were spiked into 10 L of filtered tap water at a concentration of either
10 or 100 spores/L and concentrated using a modified continuous flow centrifuge (CFC). Retentate and rinse
volumes were further concentrated by centrifugation, dried on well slides, and examined microscopically for
the presence of spores. Recoveries ranged from 39 to 76 percent (n=24).
Sample purification studies included the evaluation of several direct and indirect immunomagnetic separa-
tion (IMS) products. In reagent water samples, indirect methods using beads directed against rabbit IgG were
shown most promising with recoveries of spores ranging from 73 to 95 percent. Flow cytometry with cell sort-
ing also was evaluated for the ability to isolate E. intestinalis spores from water concentrates. Approximately
80 percent of spores seeded into reagent water were recovered using FCCS with subsequent microscopic detec-
tion. Further experiments combined CFC concentration of seeded filtered tap water samples with isolation by
FCCS and microscopic detection. Spore recoveries ranging from 31 to 77 percent (n=23) were achieved using
this approach.
Molecular methods including polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR)
following cell culture were assessed for detection of Microsporidia and viability determination. Several primer
sets, including those directed against beta-tubulin, 16S rRNA, and hsp70, were shown capable of detecting
Microsporidia; however, their usefulness as viability indicators differed. More recently, real-time PCR using
primers directed against 16S rRNA has been shown promising for detecting low levels of spores seeded into
reagent and source water. Future studies will evaluate recoveries of spores seeded into natural water samples,
concentrated using the CFC, and detected using real-time PCR.
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Development and Evaluation of Procedures for Detection of Infectious
Microsporidia in Source Waters
Paul A. Rochelle
Metropolitan Water District of Southern California, La Verne, CA
Presentation Abstract
The Microsporidia group of protozoa, particularly Enterocytozoon bieneusi and Encephalitozoon spp., are re-
sponsible for a substantial human disease burden. Many animals can carry Microsporidia, so it is possible that
source waters may be contaminated and consequently serve as a route of transmission to humans. However,
there are no routine methods for detection of Microsporidia in water and very little is known about their occur-
rence. There is a critical need to determine the role that drinking water plays in the epidemiology of this group
of parasites. The overall objectives of this study are to develop methods to recover Microsporidia from water,
determine the viability and infectivity of detected spores, and use the methods to assess the occurrence of Mi-
crosporidia in untreated source waters.
The project involved: (1) evaluation of filtration methods for recovery of Microsporidia spores from environ-
mental water samples; (2) development and assessment of an immunomagnetic separation (IMS) procedure for
purification and further concentration of spores; (3) evaluation of molecular and microscopic methods for de-
tecting spores; and (4) development of an infectivity assay combining in vitro cell culture with a molecular
detection assay.
Recovery efficiencies for capsule filters ranged from an average of 13.2 percent for 0.8-1 um porosity filters
to 38 percent for 0.55 um porosity filters. Initial trials with centrifugal filters, consisting of a modified nylon
membrane in a centrifuge tube, resulted in recovery efficiencies of 52 percent, 22 percent, and 36 percent with
porosities of 0.2 jam, 0.3 jam, and 0.45 jam, respectively. A prototype ultrafiltration apparatus achieved up to
40 percent recovery using 0.05 um hollow fiber filters. Most antibody preparations demonstrated considerable
background staining, particularly with environmental samples. In addition, a significant number of spores did
not stain. Non-antibody based stains, such as a modified trichrome method and Calcofluor, were found to be
effective only in nonenvironmental samples. A quantitative molecular detection assay was developed for
E. intestinalis with a sensitivity of 10 spores. A cell culture-based infectivity assay also was developed for E.
intestinalis spores. Infection in RK13 cells was rapid and led to effective spore propagation. A confluent
monolayer of RK13 cells in a 75-cm flask produced more than 1 x 10 spores within 1 week of inoculation
with a low dose of E. intestinalis spores. The 50 percent infectious dose for this assay was 36 spores (see Fig-
ure 1). The infectivity assay was used to measure the efficacy of ultraviolet (UV) disinfection; at least 90 per-
cent inactivation of E. intestinalis spores was obtained with aUV dosage of 3.3 mJ/cm2.
This research project has made considerable progress in the development and evaluation of methods for detect-
ing environmental Microsporidia spores and measuring their infectivity. These techniques (once optimized)
can be used to assess the extent of Microsporidia contamination in water, which will allow the water industry
and public health officials to determine whether water represents a significant route of transmission for these
parasites.
An efficient recovery procedure will need effective antibodies with high avidity and specificity that can be
used for both IMS purification and immunofluorescent detection of spores. Consequently, a wider array of an-
tibodies should be evaluated. In addition, a wider range of polymerase chain reaction primers needs to be
screened for improved specificity. The cell culture-based infectivity assay can be used to assess spore survival
in the environment and the efficacy of disinfectants such as ultraviolet light, ozone, and chlorine dioxide for
inactivation of Microsporidia spores.
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Research on Microorganisms in Drinking Water Progress Review Workshop
2 •
o
to
c
o
Q.
D)
O
-1
-2 •
0.5
1 1.5 2
Spore dose (Log10)
Figure 1. Dose response curve for E. intestinalis in RK-13 cell culture.
2.5
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Research on Microorganisms in Drinking Water Progress Review Workshop
Development and Evaluation of Methods for the Concentration,
Separation, Detection, and Viability/lnfectivity of Three
Protozoa From Large Volume of Water
Saul Tzipori and Vdi Zuckermann
Division of Infectious Diseases, Tufts University School of Veterinary Medicine, North Grafton, MA
Presentation Abstract
The objective of this project was to evaluate and optimize a modified continuous flow centrifugation
(CFC) method for recovery of Cryptosporidium giardia and microsporidia from turbid and large volumes
of water. The CFC method allows for concentration of oocysts, cysts, and spores from large volumes of
water, and for continuous monitoring of their presence in water, as opposed to one-time sampling of exist-
ing methods. This method is efficient, portable, rapid, and easy to operate. The third phase of this project
included further optimization of the CFC method for recovery of microsporidia from 10 to 50 L of water,
simultaneous recovery of all three pathogens from volumes of 10, 50, and 1,000 L of water, and viability
testing after recovery to ascertain that the CFC method does not lead to parasite inactivation.
A large number of spiked experiments were conducted over the period of support, and the table below
illustrates the rate of recovery of the three protozoa from 50 L of simultaneously spiked tap water.
Table 1. Percent recoveries by CFC when 50 L of tap water are spiked with three
parasites (Giardia, C. parvum, E. intestinalis).
1
2
o
3
4
Average%±S.D
1,500
1,500
500
500
75
71
84
37.4
57.5±38.7
2,500
2,500
2,500
2,500
37.6
82.6
58.2
10.6
44.6±35
500
500
500
500
78.4
82.4
54
N.A.
53.6±46.5
Recoveries for C. parvum, Giardia lamblia, and E. intestinalis spiked various volumes of water and
turbidity were better than expected. The recoveries of all three pathogens were particularly impressive
after numerous repeated spiking experiments. The reproducibility and the consistency of this system also
were very impressive. In spiked experiments performed in parallel, the CFC outperformed other currently
used filtration methods in terms of efficiency of recovery, speed, and simplicity. The CFC method is cur-
rently undergoing validation by the U.S. EPA Office of Water in several water utilities. The results will
be presented at the meeting.
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Topic Area 2.2: CCL Viruses
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Research on Microorganisms in Drinking Water Progress Review Workshop
Norwalk Virus Dose Response and Host Susceptibility
Christine Moe1, Lisa Lindesmith2, Ralph Baric3, Jacques LePendu3, and Peter Teunis4
Rollins School of Public Health, Emory University, Atlanta, GA; Department of Epidemiology, University
of North Carolina, Chapel Hill, NC; Institute of Biology, Nantes, France; National Institute for Public
Health and Environment, Bilthoven, The Netherlands
Presentation Abstract
Human caliciviruses are a leading cause of epidemic and endemic acute gastroenteritis and are responsible for
numerous waterborne outbreaks. The overall objective of this research project is to develop our understanding of
the risks associated with exposure to waterborne human caliciviruses as a function of dose and host susceptibility.
The dose-response was examined for two important human caliciviruses (HuCVs), a prototype Genogroup I virus
(Norwalk virus (NV)) and a prototype Genogroup II viruses (Snow Mountain Agent (SMA)) to: (1) identify the
dose range of NV and SMA (IDi0, ID50, and ID90) in human volunteers; (2) examine the immune response (serum
and secretory antibodies) and determine the characteristics of volunteers that are susceptible to infection; and (3)
evaluate the fit of several mathematical models of dose-infectivity to our data. This presentation focuses on the
dose-response results of the NV study.
A double-blinded human challenge study was conducted to determine the dose-infectivity relationship for NV.
Subjects were given various doses of a suspension of NV, monitored in a clinical setting for gastrointestinal
symptoms for 5 days and returned for Day 8, 14, and 21 followup visits. Stool specimens were assayed for NV by
reverse transcription-polymerase chain reaction (RT-PCR). NV serum and salivary antibodies were measured by
enzyme immunoassay. Saliva samples were tested for secretor status presence of H type-1 antigen and FUT2 gene
as a marker of susceptibility. Infection was defined as excretion of NV or seroconversion.
A total of 75 subjects were challenged with NV, and 22 became infected. Twenty-two of the 75 subjects were
secretor negative, and none became infected. NV doses ranged from 1 x 10"1 to 1 x 107 PCR detectable units
(PDU). Approximately 68 percent of the infected subjects had gastrointestinal symptoms. Most subjects shed the
virus for at least 8 days postchallenge, and several continued to shed the virus for 18-23 days post-challenge. Sub-
jects with an early (< 5 days post-challenge) anti-NV salivary IgA response appeared to have protective immunity
to NV infection as compared to subjects with a late anti-NV salivary IgA response.
The infectivity response appeared not to be consistent with a simple dose-response model, indicating
considerable heterogeneity in susceptibility to infection among subjects. Susceptibility to infection with
Norwalk virus appeared to depend on a genetic marker (FUT2) for a probable virus receptor (H type-1 antigen)
on host epithelial cells in the gastrointestinal tract. The 22 subjects who did not have this gene appeared to be
completely resistant to infection, regardless of virus dose. When the analysis was restricted to the susceptible
fraction of the challenged volunteers (n=53), a dose-response effect was seen, although considerable
heterogeneity still remained. At low doses there was a considerable probability of infection, but it takes very
high doses to reach an infection probability near 1.
There seemed to be no indication of a dose response for illness among infected subjects. When pre-
challenge anti-NV serum IgG was used as an indicator of prior NV infection (assuming higher levels indicated
more recent infection or infection with a virus more closely related to NV), there appeared to be weak evidence
of a protective effect against infection among those with higher levels of anti-NV IgG.
When illness conditional on infection was considered, higher baseline IgG seemed to be associated with
slightly increased risk. This suggests that a subject with a high baseline anti-NV IgG level needs a higher dose
to become infected, but then has a slightly elevated risk of becoming ill.
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NV is highly infectious. The low infectious dose, mild illness or asymptomatic infections, and prolonged
shedding facilitate waterborne and secondary transmission of this virus. A genetic marker of host susceptibility
was identified that suggests that 80 percent of the general population is susceptible to NV infection. However,
some persons exhibit an early, protective, salivary IgA response. Quantitative models that describe the interaction
between virus inoculation and growth and observable immune variables may improve understanding of the
infection process and consequently improve risk predictions. The results of these studies are valuable for
estimating the risk of HuCV infection and gastroenteritis associated with exposure to contaminated water and to
establish safe exposure limits for HuCVs in water to reduce waterborne disease.
The next steps of the project include completion of the dose-response analyses for the SMA challenge study
and completion of the examination of the role of T-cell mediated immunity in NV and SMA challenge and infec-
tion.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Development of a Rapid, Quantitative Method for the Detection
of Infective Coxsackie and Echo Viruses in Drinking Water
Marylynn V. Yates, W. Chen, and A. Mulchandani
Department of Environmental Sciences and College of Engineering, University of California, Riverside, CA
Presentation Abstract
The objective of this research was to improve on the current analytical methods for quantitative detection
of infective nonpolio enteroviruses (NPEV) in drinking water. The specific objectives of this research were to:
(1) develop a molecular-beacon-based (MB) RT-PCR method to detect NPEV; (2) establish a potential correla-
tion between IMS-MB-RT-PCR detection and cell culture detection for infective viruses; (3) using the molecu-
lar beacon, develop and evaluate real-time monitoring of virus replication in cell culture; and (4) evaluate the
above methods to quantify the presence of infective NPEV in concentrated drinking water samples. A set of
primers was designed to amplify a 155-base pair section of RNA of Echovirus 11 (Echo 11), the virus chosen
to represent the enterovirus group. An antisense-MB also was designed to specifically recognize a 25-base pair
sequence within the 5 noncoding region of Echo 11. Viral RNA was reverse-transcribed and subsequently am-
plified by polymerase chain reaction (PCR). Using the MB-based reverse transcription-PCR (RT-PCR) assay,
detection and quantification of the virus was achieved. A detection limit of 0.1 plaque-forming units (PFU)
was obtained for Echo 11. Specificity testing positively identified other members of the enterovirus group
(Coxsackieviruses Bl, B3, and B6; Echoviruses 11, 17, and 19; Poliovirus 1); nonenteroviruses (Parechovirus
1; Adenoviruses 2 and 15; Rotavitus WA; hepatitis A virus; MS2 and phiX174 bacteriophages; E. coli
0157:H7 and Salmonella typhimurium) were not detected.
After development of the MB-based assay, IMS was added to the process to minimize the potential for the
amplification of noninfective viruses. Using the IMS-MB-RT-PCR assay, the detection limit for Echovirus 11
was 3 pfu (see Figure 1). The method was then tested using surface and groundwater concentrates that were
spiked with echoviruses. The method was able to detect the viruses at a concentration of 3 pfu in the surface
water and less than 1 pfu in groundwater. In parallel, the spontaneous hybridization between molecular bea-
cons and their target sequences was exploited as a means for real-time detection of virus replication in situ.
Newly synthesized viral RNA was used as an indicator for viral infection. A molecular beacon targeting a spe-
cific region of the enterovirus RNA was used for the initial demonstration. Buffalo green monkey kidney cell
cultures with or without infection with Echo 11 were collected at various time points, fixed, and permeablized.
After introduction of molecular beacons, whole fluorescence was monitored with a fluorescence microscopy.
The results demonstrated that only cells infected with Echo 11 were brightly fluorescent, and noninfected cells
were not fluorescent. These results could be achieved within a few hours post-infection. This result suggests
the possibility to provide real-time and quantitative determination of infective viruses without the need to per-
form a conventional plaque assay, which takes days to complete.
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Detection of echovirus 11 in spiked
surface water sample using IMS-MB-
RT-PCR
150
Cycles
Figure 1. Detection of echovirus 11 in spiked surface water sample using IMS-MB-RT-PCR.
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Dose-Response Assessments for NLV and Coxsackievirus
in Drinking Water
Brenda Boutin and JeffSwartout
National Center for Environmental Assessment, U.S. Environmental Protection Agency, Cincinnati, OH
Poster Abstract
This poster presents the choices made for surrogate pathogen dose-response selection and the outcome of
those choices. The objective of both the calicivirus and coxsackievirus assessments is to determine an estimate
of the dose-response based on data available for selected surrogate pathogens.
Dose-response data are limited or nonexistent for coxsackievirus and calicivirus. Drinking water exposure
is the only route presented in this poster. Potential surrogate pathogens that had available dose-response data
were considered for the coxsackievirus and calicivirus risk assessments. There is no formalized process or cri-
teria for selection of representative surrogates for Contaminant Candidate List pathogens. For the most part,
family/genus, relative infectivity, and similarity of disease endpoints may be the most data available, but these
data may not substantiate the selection of a surrogate. The National Center for Environmental Assessment
(NCEA) is now developing a white paper to recommend criteria and a selection process for surrogates.
Coxsackievirus and echovirus-12 are both single-stranded, non-enveloped RNA viruses in the Picornaviri-
dae family, Enterovirus. The probability of infection by coxsackievirus was based on a dose-response relation-
ship developed from echovirus-12 enterovirus because no data associated with the ingestion of coxsackievirus
were available from the published literature. Both viruses replicate in the gastrointestinal tract; infection is
mostly asymptomatic. However, disease endpoints for both viruses can range from mild unspecified febrile
illness to fatal central nervous system complications.1
The dose-response model for calicivirus was developed based on data for rotavirus because dose-response
data are not available for Norwalk Like Virus (NLV) or calicivirus. Calicivirus belongs to the Caliciviridae
family, of which human calicivirus/NLV are members. Rotavirus is an unrelated (family Reoviridae) but may
have similar infectivity, and serves as a conservative surrogate for the infectious calicivirus (attack rates in
drinking water outbreaks range from 31 to 87%). The use of the rotavirus dose-response data may represent a
conservative assessment for NLV infectivity in humans. All members of the population are at risk of infections
from exposure to either calicivirus or coxsackievirus.
A number of models are used in the literature to describe microbial dose-response data. This assessment
considers only the physically and biologically relevant dose-response models; these include the exponential
and beta-Poisson models. The Pareto II also is considered, as it closely approximates the beta-Poisson over a
wide range of parameter values and is much more analytically tractable than the beta-Poisson. The parameter
values of these models fitted to the rotavirus data are presented in Table 1.
In addition, the results of fitting these models to the echovirus-12 human-infectivity data, used as a surro-
gate for coxsackievirus, are shown in Table 1. The table shows the fitted model parameters and selected infec-
tious dose estimates. Uncertainty in low-dose response was estimated by bootstrapping the data set based on
the initial beta-Poisson fit. The results of the bootstrap simulation indicate about a five-fold span in the 95 per-
cent confidence interval on risk of infection at low doses (< 0.01 pfu). The upper 95 percent bound on low-
dose risk for echovirus-12 is still 260 times less than the maximum possible risk (where exposure to 1 pfu al-
ways results in infection). In contrast, a similar bootstrap simulation performed on the rotavirus data, used as a
surrogate for NLV, yields an upper 95 percent confidence bound on low-dose risk that is only 20 percent lower
than maximum possible risk.
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The next steps include recommendations for research on exposure and dose response for calicivirus, cox-
sackievirus, and NLV. NCEA is working to develop surrogate selection criteria and a selection process, as well
as secondary transmission models for person-to-person transmission of infectious virus such as calicivirus.
References
1. Embrey M. Coxsackievirus in drinking water (Literature Summary). Final Report. The George Washing-
ton University School of Public Health and Health Services, Department of Environmental and Occupa-
tional Health, 1999.
2. Embrey M., et al. Caliciviridae in drinking water. In: Handbook of CCL Microbes in Drinking Water.
American Water Works Association, 2002.
Table 1. Maximum likelihood parameter estimates, predicted IDS and goodness-of-fit statistics for
each model (Infectivity data, Echovirus 12).
Exponential
r = 0.000583
ID50 =1187
IDoi = 17.2
IDoooi =0.17
D=22.3 (df=3)
(p = 0.0002)
(significant lack of fit)
Pareto II
a = 1.06
P = 994
ID50 =918
IDoi = 9.5
IDoooi = 0.094
D=3.21 (df=2)
(p = 0.20)
Beta-Poisson
a = 1.06
P = 994
ID50 =918
IDoi = 9.5
IDoooi =0.094
D=3.21 (df=2)
(p = 0.20)
3 Subscript indicates cumulative response percentile at which the ID is calculated (based on MLE parameters).
b Plaque-forming units.
°D = deviance (-2 x maximum log-likelihood); df = degrees of freedom for X2 statistic; p = significance of fit,
where p < 0.05 indicates lack of fit.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Methods Used To Analyze a Norovirus Outbreak
Jennifer L. Cashdollar, Sandhya U. Parshionikar, Christina M. Newport, Sandra Willian-True,
Daniel R. Dahling, G. Shay Fout, and the Outbreak Investigation Team
U.S. Environmental Protection Agency, Cincinnati, OH
Poster Abstract
The goals and objectives of this project are to: (1) isolate and identify the viral agents in well water sam-
ples associated with two outbreaks of acute gastroenteritis reported to the Wyoming Department of Health in
February 2001 and October 2001; (2) isolate and identify the viral agents in patient stool samples; and (3) de-
termine the link between water consumption and illness.
The project had a three-way approach: (1) an epidemiological investigation was performed to identify any
common routes of exposure among those afflicted with gastroenteritis; (2) an environmental survey was done
of the premises involved in each outbreak to determine possible sources of contamination; and (3) laboratory
analysis was performed on well water samples for coliform and viral detection using reverse transcription po-
lymerase chain reaction (RT-PCR) and DNA sequencing. Stool samples also were analyzed for the presence of
noroviruses.
Epidemiological studies revealed a close association between water consumption and illness. Environ-
mental surveys in both outbreaks determined that the water supply was vulnerable to fecal contamination. Well
water samples in both cases were positive for coliforms, and RT-PCR and DNA sequencing revealed norovi-
ruses as the causative agents of acute gastroenteritis.
This investigation demonstrates that the U.S. EPA's viral concentration and molecular methods, in con-
junction with epidemiological and environmental analysis, are very useful in outbreak studies. The methods
used in this study can be performed in most laboratories with trained personnel and appropriate equipment,
which would allow for routine monitoring of enteric viruses in drinking water, thus preventing any future out-
breaks from occurring.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Development of a Molecular Method To Identify
Astrovirus in Water
Ann C. Grimm, Jennifer L. Cashdollar, Frederick P. Williams, and G. Shay Fout
Microbiological and Chemical Exposure Assessment Research Division, Biohazard Assessment Research
Branch, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH
Poster Abstract
Astrovirus is a common cause of gastroenteritis that has been determined to be responsible for several out-
breaks. Because astrovirus can be waterborne, there is interest in testing environmental water for astrovirus.
We have developed a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay (see Figure 1)
that is designed to detect all known astrovirus strains.
The assay was based on a primer set that contained multiple upper and lower primers as well as multiple
probes. This would allow for amplification of all of the known strains of astrovirus using a single reaction.
When tested, this assay was able to detect strains from all eight serotypes. In addition, an internal control was
developed, so that it will be possible to determine if the sample being tested contains PCR inhibitors. Most
probable number analysis determined that when amplified with the developed assay, a single DNA molecule of
the internal control could be detected if inhibitors were not present. The assay was successfully adapted to
real-time PCR, and this method was used for integrated cell culture/RT-PCR detection of infectious virus. The
methods were successfully used to detect astrovirus present in clinical samples and spiked water samples.
A simple, sensitive method for detecting all known astrovirus strains has been developed that can be used
to detect this virus in water. This assay will be field tested by analyzing environmental water samples.
AstUl-U4 AstPl-P4 AstLl-L2
RT-PCR product 0.2 kb
Figure 1. Assay design.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Effectiveness of UV Irradiation for Pathogen Inactivation
in Surface Waters
Karl Linden1, Mark Sobsey2, and Gwy-Am Shin2
1 Civil and Environmental Engineering, Duke University, Durham, NC; 2Environmental Sciences and
Engineering, University of North Carolina-Chapel Hill, Chapel Hill, NC
Poster Abstract
Ultraviolet (UV) irradiation is now recognized as an effective and cost-competitive measure to achieve
significant level of inactivation of Cryptosporidium while not producing appreciable level of harmful disinfec-
tion by-products (DBFs) at practical doses. However, the effectiveness of UV technology against new and
emerging pathogens and uncertainties in application of UV disinfection for unfiltered surface waters still needs
to be assessed before widespread use of this technology as a primary disinfectant in drinking water treatment
processes. The primary objectives of this research project are to evaluate the susceptibility (or resistance) and
repair potential of select Contaminant Candidate List (CCL) pathogens and indicator microorganisms to or
after UV disinfection from low- and medium-pressure (LP and MP) UV sources, and to investigate the extent
of microbial association with particles in unfiltered systems and the effects of this particle association and
other water quality parameters on UV disinfection potential.
Preliminary results indicate that both LP and MP UV irradiation are very effective against most of the in-
dicator and emerging microorganisms tested. However, some of the indicator microorganisms like coliphage
MS2, bacteriophage PRD-1, and Bacillus subtilis endospores as well as CCL and emerging pathogens like My-
cobacterium terriae (a substitute for Mycobacterium avium complex), adenovirus type 2, and Toxoplasma
gondii oocysts showed relatively high resistance against both UV irradiation. Although the effectiveness of LP
and MP UV appeared to be similar against most of the microorganisms tested, there was some remarkable dif-
ference between these two UV technologies in terms of their effectiveness against adenovirus 2. To determine
the level of particle association and its effect on UV disinfection, raw surface water samples have been col-
lected from various utilities across the United States, and the waters have been examined for particle associated
coliform and aerobic endospores using physical particle disruption techniques such as homogenization and
blending. However, the levels of the indigenous microbes in the raw waters were typically low (< 1,000/100
mL), so that it was not feasible to assess the degree of particle association in these waters based on those
physical methods.
Currently, the use of microscopic techniques (nucleic acid staining/probes along with confocal micros-
copy) are being investigated to determine the level of particle association in those raw waters. Regarding the
development of new assay systems for some of the CCL microorganisms, we have been successful in develop-
ing a new assay system (Long-template [LT] RT-PCR) for Norwalk virus and a new molecular biology assay
(RT-PCR) for adenovirus 40 or 41, which are being and will be used in the current and future inactivation
study on these viruses by LP and MP UV. In addition, a method has been established to perform wavelength
specific studies using a polychromatic UV light source (MP UV lamps) with a set of UV bandpass filters, and
this setup will be utilized to develop wavelength effectiveness information for select CCL microorganisms like
adenovirus 2, M. terriae, and T. gondii oocysts. Finally, protocols have been established to examine repair
phenomenon following UV disinfection in various conditions in real water treatment situations, and these pro-
tocols will be implemented to evaluate the presence and extent of repair after UV irradiation in the select CCL
and emerging microorganisms in the later phases of this research.
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Topic Area 2.3: CCL Bacteria
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Research on Microorganisms in Drinking Water Progress Review Workshop
Disinfection of Helicobacter pylori and Aeromonas Species
Laura Boczek, Samuel L. Hayes, Clifford H. Johnson, Donald J. Reasoner, Eugene W. Rice,
and Sashi Sabaratnam
Microbial Contaminants Control Branch, Water Supply and Water Resources Division,
National Risk Management Research Laboratory, U.S. Environmental Protection Agency,
Cincinnati, OH
Presentation Abstract
Helicobacter pylori and Aeromonas hydrophila are contaminants listed on the U.S. EPA's 1998 Contaminant
Candidate List (CCL). The sensitivity of H. pylori to chlorine and of Aeromonas spp. to inactivation by free
chlorine, chloramines, and ultraviolet (UV) was examined. Selective and nonselective monitoring media were
evaluated to assess recovery of chlorine or UV-stressed Aeromonas spp. Results of experiments using free
chlorine showed that the H. pylori and Aeromonas spp. were readily inactivated under all conditions studied.
H. pylori showed more than 3.5 orders of magnitude inactivation by 0.5 mg/L chlorine in 80 seconds at 5 °C.
The Aeromonas spp. were inactivated by more than 5 orders of magnitude within a 1-minute exposure to free
chlorine at pH 7 or 8, and at 5 °C or 25 °C. Reductions of the Aeromonas spp. with 2.0 mg/L of monochlor-
amine reached approximately 2 orders of magnitude at pH 8.0 for 4 minutes and greater than 5 orders of mag-
nitude inactivation after 8 minutes of exposure. Aeromonas spp. were found to be sensitive to UV irradiation,
with fluences of less than 7 mj/cm2, giving between 5-7 logio reductions. For free chlorine, there was no ob-
servable difference in recovery of chlorine-stressed Aeromonas spp. organisms between selective and nonse-
lective media. However, with UV disinfection, some Aeromonas spp. counts on nonselective media were sig-
nificantly higher than those obtained on selective agar. These findings suggest that selective agars may under-
estimate the number of viable Aeromonas recovered after exposure to UV irradiation.
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Genomic and Physiological Diversity
of Mycobacterium avium Complex
Gerard Cangelosi
Seattle Biomedical Research Institute, Seattle, WA
Presentation Abstract
The Mycobacterium avium complex (MAC) is an environmental pathogen of susceptible humans, espe-
cially AIDS patients, children, and the elderly. MAC infections are debilitating and very difficult to treat due
to intrinsic multi-drug resistance. Infections originating from potable water have been documented; however,
the occurrence of MAC in water does not always correlate with high rates of MAC disease. The ability to pre-
dict consequences of human exposure to MAC remains an elusive goal. One factor that may contribute to this
problem is the genetic and phenotypic heterogeneity of MAC isolates. Strains and colony types are thought to
vary with regard to infectivity, susceptibility to antibiotics, and ability to survive in various environments. The
goal of this research project is to understand the genomic and physiological bases for these phenotypes. Such
an understanding could lead to refined methods for detecting environmental MAC populations that are likely
to cause disease in humans.
Microarrays and restriction fragment length polymorphism are being used to quantify the genomic diver-
sity of MAC isolates from clinical and environmental sources. In addition, transposon mutagenesis and disease
models are being used to characterize the highly mutable properties of colony type, intrinsic drug resistance,
and virulence of MAC. By combining these two lines of investigation, we hope to identify genomic markers
that can be used to identify virulent strains of MAC in the environment.
Comparative genomic hybridization to a MAC genomic microarray has revealed extensive strain-to-strain
diversity within MAC. There also is extensive heterogeneity within individual isolates, as evident from RFLP
analysis and the appearance of multiple colony types on laboratory growth media. A novel colony type switch,
termed red-white, has been identified that affects virulence, drug susceptibility, and other phenotypes. To char-
acterize the genetic basis for these phenotypes, a transposon mutagenesis system has been developed with
which we have begun to identify MAC genes required for pathogenicity and intrinsic multi-drug resistance
(see Figure 1).
Microarray and mutational analysis will be expanded. Moreover, phenotypic and genomic diversity will be
examined among environmental isolates. Phenotypic diversity will be measured by using intracellular growth
assays, and genomic diversity by microarray analysis. Through this investigation, we hope to: (1) test the hy-
pothesis that some environmental strains of MAC are more virulent than others, and (2) identify genomic
markers that may be used to identify virulent strains.
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RW-E
RW-F
WR2.55
RW1,
RW2
RW-J
WR2.58
M. avium 104
5.48 mB
RRgi,
RRg2,
RRg6,
RRg-B,
RRg-D,
RRg-G,
WRg1,
WRg2
RRg4
Figure 1. Map ofMycobacterium avium strain 104 genome, with positions of transposon insertions that affect
colony morphotype and/or multi-drug resistance.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Mycobacterium avium Complex (MAC) in Drinking Water: Detection,
Distribution, and Routes of Exposure
Phanida Prommasith1 and Timothy E. Ford2
Harvard School of Public Health, Boston, MA; 2Montana State University, Bozeman, MT
Presentation Abstract
The objectives of this project are to: (1) develop techniques and methods for detecting MAC in both
drinking water and biofilm samples; (2) investigate the presence and distribution of MAC in water distribution
systems in four Massachusetts towns; and (3) examine possible routes of exposure for residential end-users. A
method was developed to detect MAC from drinking water, standard filtering techniques have been modified,
and percent recovery was compared with DAPI direct counts. The most efficient protocol was compared with
selective culture on paraffin slides. Both environmental water samples and MAC-spiked autoclaved water were
used in these tests. Environmental biofilm samples were collected from residential sites and from hot water by-
pass systems.
Two fluorescent oligonucleotide probes have now been designed and tested. The probes target the
16sRNA genes of Mycobacterium avium and M. intracellulare. PCR-restriction enzyme pattern analysis
(PRA) of the HSP65 gene also has been used to identify MAC. Identification of species has been confirmed by
comparison with the NCBI-GenBank database. To investigate the presence and distribution of MAC, potable
water samples have been systematically examined from four communities with different water supplies and
distribution characteristics. All samples have been analyzed for pH, temperature, free and total chlorine, alka-
linity, ammonia-N, nitrite-N, nitrate-N, total iron, and assimilable organic carbon, MAC, and heterotrophic
plate counts.
In addition, to investigate the possible routes of exposure, water samples have been collected from kitch-
ens and showers in the end-users' homes (cold and hot water) and analyzed for the presence of MAC. Cur-
rently, biofilms are being grown in end-user toilet cisterns. Any residential sites found to have MAC in water
samples have been subject to be more frequent sampling.
Small doses of antibiotics (nalidixic acid, ethambutol, ofloxacin) and anti-fungus (cyclohexamide) added
to 7H10 media were found to improve the detection of MAC. It is still inconclusive as to whether the paraffin
slide culture is a more sensitive technique. The FISH probe for the M. avium showed a positive hybridization
signal less than 35 percent FA stringency hybridization conditions on isolated cells. The probe forM intracel-
lulare needs to be redesigned and retested to ensure successful hybridization.
Approximately 20 percent (n=861) of water samples collected to date were positive for Mycobacteria,
with 8.36 percent positive for MAC. The household results ranged from 0 percent to more than 40 percent
positive for MAC; the distribution system results ranged from 0 percent to almost 30 percent positive for
MAC. Of the MAC positive plates, 11 percent were found to haveM avium (20-450 cfu/L), and 3.5 percent
wereM intracellulare (4-12 cfu/L). No significant differences were detected in the presence of MAC between
kitchen faucets and showerheads, although numbers of MAC were slightly higher in showerhead samples. Ap-
proximately 100 percent ofM. intracellulare were found in cold water (8-17 °C), whereas mostM avium (66
percent) were found in hot water (35-46 °C).
The next steps will include: (1) concluding the investigation of the use of paraffin slide cultures; (2) final-
izing FISH protocols to directly hybridize to biofilm samples; (3) improving M. intracellulare probe and pro-
tocols; (4) evaluating biofilms grown in toilet tanks for MAC; and (5) examining relations of MAC coloniza-
tion to water quality parameters.
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Sensitivity of Three Encephalitozoon Species to Chlorine
and Chloramine Treatment as Detected by an In Vitro
Microwell Plate Assay
Cliff H. Johnson1, Marilyn M. Marshall2, Jackie Moffet2, Charles R. Sterling2, Laura A. DeMaria1,
and Gene W. Rice1
1U.S. Environmental Protection Agency, Cincinnati, OH; 2 University of Arizona, Tucson, AZ
Poster Abstract
Microsporidia are obligate intracellular parasites that form environmentally resistant, infectious spores.
These parasites are ubiquitous in the environment, infecting members of almost every class of vertebrates and
invertebrates. At least 14 microsporidian species are known to infect humans. Of primary concern are the mi-
crosporidian species that infect the human gastrointestinal tract, Entercytozoon bieneusi and Encephalitozoon
intestinalis. Spores are typically able to survive and maintain their infectivity for weeks. A modified in vitro
cell culture assay was performed on three species of Encephalitozoon (E. intestinalis, E. helium, and E. cuni-
culi) after exposure to chlorine and chloramine at a concentration of 2 mg/L at 25EC, pH 7, and pH 8, respec-
tively. Spores were harvested from RK-13 cell monolayers and Percoll purified assayed using an in vitro mi-
crowell plate viability procedure. Ten-fold dilutions of chlorine and chloramine treated spores were inoculated
onto RK-13 cell monolayers grown on 15mm sterile Thermanox coverslips in 24 well plates. Five coverslips
were inoculated for each spore dilution. After incubation, the coverslips were fixed with methanol, stained
with Giemsa stain, and examined with a light microscope. The percentage of infectivity was calculated by di-
viding the number of positive (infected) wells by the total number of wells inoculated. Most probable number
determinations also were determined for each assay. For each treatment and time period, two replicates of 5
coverslips were performed. Varying log reductions were observed for the three Encephalitozoon species.
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Inactivation of Aeromonas by Chlorine and Monochloramine
Laura A. Boczek, Cliff H. Johnson, and Eugene W. Rice
U.S. Environmental Protection Agency, Cincinnati, OH
Poster Abstract
The Bacterial genus Aeromonas is currently listed on the U.S. EPA's Candidate Contaminant List (CCL).
Resistance to chemical disinfection is an essential aspect regarding all microbial groups listed on the CCL.
This study was designed to determine the inactivation kinetics of Aeromonas spp. for free available chlorine
and monochloramine. Three species, Aeromonas caviae, A. hydrophila, A. veronii, which are known to be as-
sociated with human infections, were studied in pure culture under oxidant demand-free conditions. Free chlo-
rine experiments were conducted at pH 7 and 8, at 5 °C and 22 °C. Experiments using preformed mono-
chloramine also were conducted at both temperatures and at pH 8. Three media were evaluated for their ability
to recover chlorine-stressed organisms [nutrient agar (Difco), ampicillin dextrose agar (Biolife), and Ryan agar
(oxoid)]. Experiments using free chlorine indicated that the Aeromonas spp. were readily inactivated under all
conditions studied. The organisms were inactivated by more than five orders of magnitude within a 1-minute
exposure to free chlorine at both temperatures. Inactivation kinetics were similar for other bacterial organisms,
with greater inactivation occurring at lower pH values and at higher temperatures. For free chlorine, there was
no observable difference in recovery of chlorine-stressed organisms on the three bacteriological media. Two
wells containing Aeromonas spp. (approximately 103 CFU/mL) have been located in a recent survey. Water
from these wells also was used in disinfection experiments.
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Mycobacterium paratuberculosis and Nontuberculous Mycobacteria
in Potable Water
Stacy Pfotter and Terry C. Covert
National Exposure Research Laboratory, U.S. Environmental Protection Agency,
Cincinnati, OH
Poster Abstract
Nontuberculous mycobacteria (NTM) include Mycobacterium species that are not members of the Myco-
bacterium tuberculosis Complex. Members of the NTM group are important causes of disease in birds and
mammals. Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium paratuberculosis are
NTM and members of the Mycobacterium avium Complex (MAC). These organisms are found in a variety of
environments, including soil and water, and are included on the Contaminant Candidate List (CCL). Earlier
exploratory occurrence studies suggest that NTM have widespread occurrence in potable water throughout the
United States. M. paratuberculosis is the causative agent for Johne's disease in cattle. In addition to well-
documented evidence ofM paratuberculosis as the causative agent of Johne's disease in cattle, there has been
evidence linking M. paratuberculosis with Crohn's disease, a chronic inflammatory disease of the intestinal
tract in humans. Transmission of M paratuberculosis via water contaminated with cattle feces may be one
route of infection.
Current NTM research focuses on three areas: (1) development of an improved cultural method for isola-
tion of NTM in drinking water, (2) development of a rapid polymerase chain reaction (PCR) multiplex method
for detection of MAC organisms in drinking water, and (3) development of a molecular method for detection
of M. paratuberculosis in water.
Improved Cultural Method
Current methods for isolating NTM from environmental samples require harsh decontamination tech-
niques to reduce the levels of background organisms often leading to loss of 50-70 percent of the target NTM.
The goal of this research is to develop improved selective method(s) that do not use classical decontamination
procedures. The use of antibiotics, dyes, detergents, and other growth inhibitors are being examined for their
ability to reduce background organisms and permit growth of NTM. A membrane filter method approach has
been selected. Screening studies with spiked drinking water samples comparing candidate methods to classical
decontamination techniques have been initiated. Candidate methods that permit better recovery of NTM and
better reduction of background organisms will be tested with additional recovery studies and analyses of drink-
ing water samples.
Various antibiotics, dyes, and detergents have been examined using a membrane filter cultural method
approach. Thus far, an oxidizer has shown promise for better recovery (80 percent) and reduction of back-
ground organisms than the standard accepted cultural method. An improved cultural method would lead to
better estimates of the occurrence of NTM, better estimates of the numbers of NTM in positive samples, and
the possibility of recovering NTM unusually sensitive to decontaminating agents. Future research will entail
additional NTM recovery studies, followed by comparison studies with the standard cultural approach and the
improved method with distribution samples.
PCR Multiplex Method
Current methods for detection of MAC organisms in drinking water typically take from 3 to 8 weeks for
completion of analyses, with additional time for identification of the organisms. The goal of this research is to
develop a rapid PCR multiplex method for detection of M. avium andM intracellulare. Drinking water sam-
ples (500 mL) are membrane filtered, and the filters are placed in modified 7H9 broth for 7-day enrichment.
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After enrichment, the cells are centrifuged and lysed to harvest the genomic DNA. The DNA is amplified
(PCR) using primers specific for M. avium and M. intracellulare and all Mycobacteria. The PCR product is
visualized by gel electrophoresis. Sixty samples (reservoir and drinking water) have been analyzed by the
standard culture method and the multiplex PCR method. Nine samples were positive by both methods, seven
were positive only by multiplex PCR, and three were positive only by the cultural method. The use of multi-
plex PCR significantly decreases the time for analyses for these organisms, and is able to detect MAC organ-
isms not detected by the culture method. The next steps include completion of detection limit studies and addi-
tional comparison studies with the standard culture technique using drinking water samples.
Method for Detection of M paratuberculosis
A new project in our laboratory involves the development of a molecular detection and quantification
method for M. paratuberculosis (MAP) in water. The method will be an important step in determining the sig-
nificance of exposure to MAP in contaminated water, and may help to establish the link between contaminated
water and Crohn's disease. Current methods of detection, which include culture-based methods, are inade-
quate. A 16- to 20-week incubation time is required to grow the organism, during which other microorganisms
overgrow the medium. Harsh decontamination procedures used to reduce background organisms also kill a
portion of MAP. This study proposes to develop a rapid molecular method to detect and quantify MAP in envi-
ronmental samples by targeting a genetic molecule specific to MAP. One potential target is the MAP-specific
insertion sequence IS900. The element is found only in MAP, and is present in 14 to 18 copies per cell. Other
possible targets include seven recently discovered MAP-specific gene segments. A quantitative PCR-based
method would significantly reduce detection times from approximately 16 weeks to a few hours.
Figure 1. Electrophoretic separation of multiplex PCR products obtained from drinking water isolates. Lanes
contain the following: lanes 1, 2, 3, 4, 5, 7, 8, 9, and 10, M avium isolates; lane 11,M avium posi-
tive control; lane 12, M. intracellulare positive control, and lane 6, cpX174 RF DNA/ Hae III mo-
lecular weight marker (Gibco-BRL) containing fragments of 1353, 1078, 872, 603, 310, 271, 234,
194, 118, and 72 base pairs.
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Detection of Helicobacter pylori Using a Highly Variable Locus
Upstream of the 16S Ribosomal RNA Gene
M. Shahamat, M.R. Alavi, J.E.M. Watts, K.R. Sowers, D. Maeder, and F. Robb
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD
Poster Abstract
Helicobacter pylori is a highly successful infectious agent, and it is the principal cause of gastritis and
peptic ulcer disease. Strong associations also have been found with gastric adenocarcinoma and lymphoma.
Although regional rates of infection vary, H. pylori is still a major public health concern in many parts of the
world. This highly infectious bacterium is able to use multiple routes of transmission to infect a susceptible
host. It is thought to be transmitted by the fecal-oral route but also may be transmitted directly through saliva
or from the environment; however, the precise mode of transmission is not well understood. H. pylori has been
demonstrated to change morphologically (see Figure 1) and enters nonculturable stages of survival in the envi-
ronment and still can become infective. Currently, it is difficult to elucidate the environmental route of H. py-
lori, as there is no reliable method for detecting and differentiating H. pylori strains from environmental
sources.
To overcome culturability limitations, a molecular approach was utilized consisting of designing primers
complementary to intergenic spicer region (ISR) between the 16S and 23S to detect H. pylori from water
sources. In the H. pylori genome, the 16S and 23S rRNA genes are not contiguous (see Figure 2). This study
found a highly conserved region followed by an intergenic spacer variable region upstream of the 16S rRNA
gene. Primers were designed for this conserved region and to the 5'-end of the 16S rRNA gene to amplify the
variable region in between. Each strain of H. pylori tested gave a positive amplification by polymerase chain
reaction (PCR), using the primers mentioned above to amplify the variable region. A number of other species
tested, such as Campylobacter, Escherichia coli, Salmonella, Shigella, Vibrio, and Bacillus species, resulted in
no amplification. Tests performed using laboratory constructed mixtures of different bacterial species gave
only positive results if H. pylori was present. The sensitivity and specificity of this PCR method for direct de-
tection of H. pylori in environmental samples has been determined and found to be optimal for water samples.
H. pylori strain differentation is possible using this molecular technique, and future studies will involve
terminal restriction fragment length polymorphism analysis to detect and differentiate strains of H. pylori. This
analysis will provide a rapid and reliable detection tool for H. pylori strains in both clinical and environmental
samples, deepening our understanding of the source of infection.
Figure 1. Helicobacter pylori Strain RSB6 in River water for 140 days.
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Typical organization of the ribosomal genes in microorganisms
Conserved Region
23S
tS
Inter Spaced Region (ISR; var iatole region)
Relative locations of the ribosomai RNA genes in H. pylori genome.
Genomic map of H. pyiori strain 26695
23S-5S-1
16S-1
16S-2
23S-5S-2
Figure 2. Relative location of ribosomal RNA in H. pylori and other bacteria.
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Using Real-Time PCR To Detect Toxigenic Strains
of Microcystis aeruginosa
Carrie Moulton
Office of Water, U.S. Environmental Protection Agency, Cincinnati, OH
Poster Abstract
Both toxigenic and nontoxigenic strains of Microcystis exist in nature with a patchy distribution. Only
about 50 percent of all Microcystis blooms tested for heptotoxicity are positive by the mouse bioassay. The
goal of this project is to develop the capability to discern between toxic and nontoxic blooms of Microcystis.
Polynucleotide sequences were used within the 16S ribosomal RNA gene and the mcyA gene in the micro-
cystin synthetase operon to develop primer/probe sets for a multiplex 5'-nuclease polymerase chain reaction
(PCR) assay. Presumably the primer/probe set developed for the mcyA gene will give positive results only with
strains that produce toxins.
Nineteen polynucleotide sequences for the mcyA gene and four sequences for the 16S gene were entered
into the Primer Express® software. Primer/probe sets were selected using two criteria: (1) the lowest probabil-
ity of meeting the designated parameters by random chance; and (2) the highest number of M. aeruginosa
strains that contained the selected sequences as found in GenBank for each primer and probe. Eleven different
strains of M. aeruginosa, six of which were previously documented to be toxigenic, were obtained from the
University of Texas Culture Collection and established in BG-11 media. These strains were originally isolated
from samples collected in Canada, Australia, South Africa, and the United States. PCR products for the 16S
gene found in the presence or absence of NMT products served as a positive control to show that PCR inhibi-
tors were not present and that analytical procedures were not compromised.
As predicted, only the strains determined to be toxigenic by high performance liquid chromatography and
enzyme-linked immunosorbent assay were positive for detection of the NMT region of the mcyA gene (see
Table 1). In addition, the NMT region of the mcyA gene has been detected in 18 toxigenic cultures and 2 non-
toxigenic cultures, but not in 17 other nontoxigenic cultures by other investigators. Using real-time PCR to
detect toxigenic strains of M aeruginosa was successful and indicates the potential for a highly sensitive and
precise assay. Further testing to assess the correlation between the presence of this genetic region and toxi-
genicity of environmental samples is needed.
Table 1. Multiplex PCR.
B2662
LB 2664
B2666
B2667
B2669
B2670
LB 2386
B2661
B2671
B2672
B2676
Negative control
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
18.43
18.33
23.21
18.27
19.34
17.44
18.15
18.64
17.33
21.91
18.11
Negative
18.88
19.17
23.78
18.4
19.93
17.92
Negative
Negative
Negative
Negative
Negative
Negative
*Cycle threshold, Ct, is the first thermocycle in which there is a
significant increase in fluorochrome emission from the probe.
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Role of Adaptive Response in the Kinetics of Mycobacterium avium
Inactivation With Monochloramine
Ning Tong, Lutgarde Raskin, and Benito J. Marinas
Department of Civil and Environmental Engineering, University of Illinois
at Vrbana-Champaign, Vrbana, IL
Poster Abstract
Mycobacterium avium is a waterborne opportunistic pathogen commonly detected in drinking water. The
persistence of M. avium in drinking water distribution systems has been associated to its presence in biofilms,
inside which these microorganisms appear to gain protection against disinfectant attack. It is also suspected
that M. avium detected in tap samples, also containing a measurable level of chlorine residual, survives em-
bedded inside suspended particles detached from biofilms. Because chlorine reacts with biofilm or particle
organic matter, the embedded cells get exposed to lower disinfectant concentrations. Furthermore, exposure to
low concentrations of the disinfectant hydrogen peroxide has been shown to trigger an adaptive response, re-
ferred to as the SOS response, with both A. hydrophila and Escherichia coli. The SOS response triggers the
synthesis of a number of proteins, some of which can repair DNA damage induced by the disinfectants. The
main objective of this study is to characterize the effects of disinfectant concentration, temperature, and pH on
the inactivation kinetics of M avium (ATCC 15769) with combined chlorine. The occurrence and impact of a
biochemical adaptive response during the inactivation of M avium with this disinfectant also has been as-
sessed. Experiments were performed in batch reactors with the temperature controlled at target values in the
range of 1-30 °C. The solution pH is maintained constant at target values in the range of 6-10 with phosphate
and borate buffers. Chlorine concentrations range from 0.01 to 10 mg/L as C12.
Resulting inactivation curves were characterized by pseudo-first order kinetics without the occurrence of a
lag phase. A single curve was obtained for relatively high concentrations (e.g., 5.0 mg/L and 10 mg/L at 20 °C
and pH 8) when plotting the natural logarithm of survival ratio versus CT. This finding confirmed the validity
of the CT concept (a fixed value of the product of the concentration and contact time resulted in a fixed degree
of inactivation at a given temperature and pH, independently of the disinfectant concentration used) at rela-
tively high concentrations. In contrast, the inactivation kinetics M. avium at low monochloramine concentra-
tion (e.g., <1 mg/L as C12 at 20 °C and pH 8) was only approximately 25 percent of that observed at high con-
centration at the same temperature and pH. The greater resistance to inactivation observed at the lower
monochloramine concentration was consistent with the occurrence of an adaptive response similar to that re-
ported for other bacteria. However, additional work at the molecular level is necessary to assess if the adaptive
response mechanism is the SOS response observed with other bacteria.
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Topic Area 3: Distribution Systems
and Biofilms
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Research on Microorganisms in Drinking Water Progress Review Workshop
The Effect of Chlorine, Chloramine, and Mixed Oxidants on Biofilms
in a Simulated Water Distribution System
Mark C. Meckes, Roy C. Haught, David W. Cmehil, Leslie Wilsong, Janet C. Blannon,
andMano Sivaganesan
National Risk Management Research Laboratory, U.S. Environmental Protection Agency,
Cincinnati, OH
Presentation Abstract
Throughout the world there are millions of miles of water distribution pipe lines that provide potable water
for use by individuals and industry. Some of these water distribution systems have been in service well over
100 years. Treated water moving through a distribution system comes into contact with a wide range of mate-
rials under a variety of conditions, which can affect water quality. Suspended solids in finished water can settle
out under low flow conditions and can be resuspended as flows increase. Disinfectants and water additives
react with organic and inorganic materials within the distribution system, producing by-product compounds
that may be undesirable in the water supply. Oxidant resistant microorganisms can colonize; pipe surfaces,
cracks, and crevices produce a complex microenvironment known as "biofilm." These biofilms can be highly
resistant to many disinfection methods and techniques. This resistance to disinfection can extend to the entire
colony of microbes, which can include microbial indicators of contamination such as coliform bacteria.
The extent of biofilm growth that can occur under conditions of limited nutrients and in the presence of
residual oxidizing agents was evaluated using U.S. EPA's water distribution system simulator (DSS). This
presentation describes the features of the DSS and how it was used to compare the effectiveness of three disin-
fecting agents on system biofilms. Results from this work suggest that chloramine and MIOX® were more
effective than free chlorine in reducing culturable drinking water biofilms within the DSS.
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Molecular Characterization of Drinking Water
Microbial Communities
Jorge Santo Domingo, Mark C. Meckes, Catherine Kelty, Margaret Williams, Joyce M. Simpson,
and Donald J. Reasoner
National Risk Management Research Laboratory, Water Supply and Water Resources Division,
Microbial Contaminant Control Branch, U.S. Environmental Protection Agency, Cincinnati, OH
Presentation Abstract
The objective of this study was to monitor the impact of chlorination and chloramination treatments on
heterotrophic bacteria (HB) and ammonia-oxidizing bacteria (AOB) inhabiting a water distribution system
simulator (see Figure 1). HB densities decreased while AOB densities increased when chloramine was added.
AOB densities decreased below detection limits after the disinfection treatment was switched back to chlorina-
tion. The presence of AOB was confirmed using a group-specific 16S rDNA-PCR method. 16S rDNA se-
quence analysis showed that most bacterial isolates from feed water, discharge water, and biofilm samples
were a-Proteo-bacteria or (3-Proteobacteria. The latter bacterial groups also were numerically dominant among
the sequences recovered from water and biofilm 16S rDNA clone libraries. The relative frequency of each cul-
turable bacterial group was different for each sample examined. Denaturing gradient gel electrophoresis analy-
sis of total community 16S rDNA genes showed notable differences between the microbial community struc-
ture of biofilm samples and feed water. The results of this study suggest that disinfection treatments could
influence the type of bacterial community inhabiting water distribution systems.
Phylogenetic analysis of natural microbial communities
Total DMA from environmental sample
PCR amplification ^T
Amplified rRNA_pr rDNA
Cloning
PCR clone library
Sequencing
L^ Direct cloning
Random clone library
Screening
rDNA containing clones
Sequencing
Phylogenetic identification of natural clones
Clones specific primers and probes
Hybridization with
fluorescent probes
In situ detection
PCR Amplification
^
Detection of cloned rDNA sequences
in environmental samples
Figure 1. Phylogenetic analysis of natural microbial communities.
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Phylogenetic Analysis of Prokaryotic and Eukaryotic Microorganisms
in a Drinking Water Distribution System Simulator
Margaret M. Williams1, Mark C. Meckes2, Cathy A Kelty2, Hildred S. Rochon3,
and Jorge W. Santo Domingo
National Research Council, Cincinnati, OH; U.S. Environmental Protection Agency, Cincinnati, OH;
3School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA
Poster Abstract
Within potable water distribution systems, opportunistic pathogens such as Legionella species infect pro-
tozoa, gaining protection from disinfectant residuals. Analyzing the prokaryotic and eukaryotic populations in
distribution system water provides a basis for understanding the interactions between these microorganisms.
Samples were obtained from the feed and discharge water of a ductile iron distribution system simulator that
receives drinking water containing a 0.5 ppm monochloramine (NH2C1) residual. Bacteria were isolated on
R2A agar, then identified by polymerase chain reaction amplification of the 16S rRNA gene (rDNA), followed
by sequence analysis. To determine a broader range of microbial populations present in the systems, clone li-
braries of 16S rDNA and 18S rDNA were made from sample water. The majority of the isolates were closely
related to bacteria belonging to the alpha proteobacteria, including Sphingomonas, Brevundimonas, and Cau-
lobacter species. Sequence analysis of the clones obtained from the pipe loop discharge water showed a mix-
ture of alpha and beta proteobacteria, as well as the presence ofNitrospira sp., which are nitrite oxidizers. Us-
ing genus-specific 16S rDNA primers, two Legionella-like species were identified: Tatlockia micdadei and
Legionella-Like Amoebal Pathogen 1 (LLAP1). A wide range of eukaryotic microorganisms, including
dinoflagellates such as Gymnodinium and Peridinium spp., have been identified from clones obtained using
universal 18S rDNA primers (see Figure 1). Determination of the predominant protozoan species within the
distribution water will allow the identification of possible hosts for Legionella species and other opportunistic
pathogens. Prokaryotic and eukaryotic community analysis is a first step in elucidating the relative activity and
survivability of each group of organisms.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Figure 1. Phylogenetic tree demonstrating the relationships among dinoflagellate sequences obtained from
the feed (L3F samples) and discharge (L3D samples) of the distribution system simulator (DSS).
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Research on Microorganisms in Drinking Water Progress Review Workshop
Identification and Characterization of Aeromonas Isolates
From Drinking Water Distribution Systems
Jennifer Birkenhauer1 andM. Rodgers2
1Oak Ridge Institute for Science and Education, U.S. Environmental Protection Agency,
Cincinnati, OH; 2U.S. Environmental Protection Agency, Cincinnati, OH
Poster Abstract
Members of the bacterial genus Aeromonas are commonly isolated from both fresh and salt waters world-
wide, and some are believed to cause infections in humans, including gastroenteritis and wound infections.
Currently, aeromonads are on the U.S. EPA's Contaminant Candidate List, and are suspected of contaminating
drinking water distribution systems. Identification of aeromonads to the species level is difficult as new spe-
cies, taxa, and biogroups continue to be proposed. In this study, both metabolic and genomic fingerprinting
identification methods were employed to obtain an understanding of the occurrence and types of aeromonads
in drinking water distribution systems in the United States.
Water samples were analyzed from 18 drinking water distribution systems across the United States, eight
of which were found to contain aeromonads. All colonies were isolated from ADA-V medium and were con-
firmed to be aeromonads as recommended in EPA Method 1605. Confirmed isolates, 212 in total, were then
subjected to both a Restriction Fragment Length Polymorphism (RFLP) analysis (Borrell, et al, 1997) and to a
carbon source utilization assay employing the BIOLOG microbial identification system.
The BIOLOG microbial identification system offers a straightforward approach to identifying environ-
mental microbes. However, we found that only after compiling our own database were we able to gain confi-
dence in the system's ability to correctly identify each isolate. The RFLP analysis, while requiring much more
time and technical skill, was able to give a more consistent identification of each isolate, with the exception to
certain biotypes.
Based on both the metabolic and genomic fingerprinting of these organisms, we were able to identify sev-
eral different biotypes, including A. hydrophila, A. bestiamm, and A. salmoncida from drinking water distribu-
tion systems. Because some of the species that were isolated have been implicated in human disease, the re-
sults from this study indicate that a more comprehensive survey of drinking water utilities is warranted to
determine if aeromonads in drinking water pose a threat to public health.
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Pathogenicity of Biofilm Bacteria
Dennis Lye
National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH
Poster Abstract
There is a paucity of information concerning any link between the microorganisms commonly found in
biofilms of drinking water systems (see Table 1) and their impacts on human health. For bacteria, culture-
based techniques detect only a limited number of the total microorganisms associated with biofilms. The pos-
sibility of unknown opportunistic pathogens occurring in potable water and biofilms within drinking water
systems still exists, but it is unlikely that pathogenic microorganisms will be found using individual in vivo
culture-based techniques or by screening large numbers of isolates using the currently available in vitro viru-
lence tests. A combination of molecular-based techniques and animal-exposure studies will provide the infor-
mation necessary to fully characterize the pathogenicity of microorganisms commonly associated with
biofilms.
Table 1. Opportunistic microbial pathogens that could be encountered in
biofilms of drinking water systems.
BACTERIA:
Helicobacter pylori
Escherichia coli
Mycobacterium avium complex
Legionella spp.
Pseudomonas aeruginosa
peptic ulcers
gastroenteritis
chronic diarrhea, lung disease
Legionnaires disease
burn infections
VIRUSES:
Polio
Coxsackie
Norwalk
Hepatitis A
poliomyelitis
upper respiratory
gastroenteritis
infectious hepatitis
PROTOZOA:
Cryptosporidium
Giardia
Entamoeba
Acanthamoeba
gastroenteritis
gastroenteritis
amoebic dysentery
eve infection
FUNGI:
Aspergillus
pulmonary disease
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Topic Area 4: Cross-Cutting Research
and Emerging Topics
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Research on Microorganisms in Drinking Water Progress Review Workshop
The Application of Mass Spectrometry to the Study
of Microorganisms
Jody A. Shoemaker and Susan T. Glassmeyer
National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH
Presentation Abstract
The purpose of this research project is to use state-of-the-art mass spectrometric techniques such as
electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry
(MS), to provide "protein mass fingerprinting" and protein sequencing information for microorgansims listed
on the 1998 Contaminant Candidate List (CCL) that cause waterborne disease. The responsibility of
characterizing and investigating microorganisms has traditionally fallen to microbiologists, but recent
advances in mass spectrometry have allowed analytical chemists also to enter the realm of microorganisms.
Protein mass fingerprinting libraries will be developed and evaluated to determine whether MS techniques
can identify protein fingerprints related to the infectivity/viability of selected microorganisms and whether they
can differentiate between species and strains of selected microorganisms. Sequence information for proteins
that are found to be specific or unique to species/strain and infectivity/viability also can be obtained with these
MS techniques.
This global proteomic project has a number of subtasks for which preliminary results have been obtained
on microorganisms such as coxsackievirus, Cryptosporidium parvum, and enterococci. Through the use of
mass spectrometry, a potential viral biomarker of coxsackievirus has been identified that may indicate whether
the virus is infectious. A unique mass spectral peak was observed in an infectious coxsackievirus, but was not
observed in a noninfectious coxsackievirus. This unique peak may be responsible for viral infectivity, thus, be
a potential biomarker.
In addition to viruses, initial experiments were performed to determine the ability of MALDI to analyze C.
parvum both in an intact form, as well as oocysts that have been rendered nonviable. MALDI analysis was
performed on several different harvests of the intact oocysts, as well as the separated cell walls and sporozoites
that make up the oocysts. The analysis of the oocysts walls was inconclusive due to lack of discernable mass
spectral peaks, but MALDI analysis of the sporozoites yielded reproducible mass spectra.
Whole enterococci cell protein profiles were evaluated using MALDI as a tool to identify seven different
enterococci species. Many mass spectral peaks were shared among the different enterococci species; however,
each species showed unique peaks, primarily in the 6,000 to 7,000 m/z region. When environmental isolates
were tested, the signature peaks were observed in many of the different isolates, suggesting that these peaks
could be used for species identification. Sequence analysis of the environmental isolates by 16S rDNA con-
firmed the identity of the strains tested, and matched the MALDI identity prediction in 75 percent of the sam-
ples. The results from this study indicate that the analysis of whole enterococci cells by MALDI generate
unique protein profiles, which can be used for the rapid identification of fecal enterococci environmental iso-
lates.
Although mass spectrometry currently is not sensitive enough to detect single cells in drinking water, the
basic proteomic information obtained with these mass spectrometric techniques can be used to develop more
sensitive and precise microbiological techniques that focus on these unique proteins in drinking water samples.
These conventional microbiological methods can then be used to gather the occurrence data that will be used to
create better U.S. EPA regulations for protecting humans from microbiological contaminants in U.S. drinking
water supplies.
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Cyanobacteria and Their Toxins
Elizabeth D. Hilborn
Office of Research and Development, National Health and Environmental Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Triangle Park, NC
Presentation Abstract
Cyanobacteria and their toxins are listed as microbial contaminants on the Candidate Contaminant List.
Increasingly, toxic cyanobacterial blooms are being reported in surface fresh water bodies worldwide. It is be-
lieved that both increased occurrence associated with eutrophication and climatic changes, and increased de-
tection due to improvements in scientific knowledge and methods to detect blooms are contributing to this
trend. Recent studies suggest that consumers of drinking water derived from surface sources in the United
States may be exposed episodically to low concentrations of these toxins. However, there is little documented
information about potential human health effects associated with exposure to these contaminants at ambient
concentrations. Characteristics of Cyanobacteria and their toxins, and recent U.S. EPA activities will be dis-
cussed.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Transport of Chemical and Microbial Contaminants From Known
Wastewater Discharges: Potential Chemical Indicators
of Human Fecal Contamination
Susan T. Glassmeyer1, Imma Ferrer2, Edward T. Furlong2, Jeffrey D. Cahilf, Steven D. Zaugg2,
Stephen L. Werner2, Michael T. Meyer3, Dana W. Kolpin4, and David D. Kryak5
3U.S. Environmental Protection Agency, Cincinnati, OH; 2U.S. Geological Survey, Denver, CO;
3U.S. Geological Survey, Ocala, FL; 4U.S. Geological Survey, Iowa City, IA;
U.S. Environmental Protection Agency, Research Triangle Park, NC
Presentation Abstract
The quality of drinking and recreational water is currently ascertained using indicator bacteria such as Es-
cherichia coli and fecal enterococci. However, the tests to analyze for these bacteria require 24 to 48 hours to
complete, and do not discriminate between human and animal fecal material sources. One solution to these
problems is to use chemicals that are commonly found in human wastewater as supplementary tracer com-
pounds. The chemicals have the advantage of requiring shorter analysis times (3-4 hours), and a suite of human
specific markers can be selected that are unique to human wastewater. For this project, compounds includes
those that are produced and excreted by humans (e.g., coprostanol), that are consumed and pass easily through
humans (e.g., pharmaceuticals and caffeine), and that are associated with humans and deposited into the com-
bined graywater/blackwater household septic waste stream (e.g., surfactants). At 10 wastewater treatment fa-
cilities, a treated effluent sample, as well as surface water samples from upstream and at two successive points
downstream from the facility were collected. This longitudinal sampling scheme was used to determine the
persistence of the target compounds in streams. Compounds that are quickly removed or degraded may not be
persistent enough to serve as tracers; those that are too recalcitrant would similarly not be suitable as they
would be present after the pathogens have been eliminated.
To estimate the environmental persistence of pathogens, the water samples were analyzed for E. coli and
fecal enterococci in addition to the suite of chemicals being measured. For chemical analysis, the water sam-
ples were extracted using either solid phase extraction (for the pharmaceuticals) or liquid-liquid extraction (for
the other wastewater contaminants) and were analyzed using either high-performance liquid chromatogra-
phy/mass spectrometry (HPLC/MS; pharmaceuticals) or gas chromatography/mass spectrometry (GC/MS;
other wastewater contaminants). The concentration of microbial indicators was determined using modified
mTEC (E. coli) or mEI (enterococci) media. Of the 114 chemical analytes investigated in this project, more
than 80 were found in at least one sample. Although most concentrations were in the range of 0.1 to 1.0 |_ig/L,
in some of the more highly contaminated samples, concentrations were in the range of 5-20 |J.g/L. The concen-
trations of the majority of the chemical compounds present in the samples generally followed the expected
trend: they were either nonexistent or at only trace levels in the upstream samples, had their maximum values
in the wastewater effluent samples, and then declined in the two downstream samples. However, at most loca-
tions, there were indicator bacteria in the upstream samples, illustrating some of the difficulty in using bacteria
to monitor water quality.
This work indicates that these human wastewater constituents do have utility as tracers of human wastewa-
ter discharge. However, until the behavior of these chemical analytes is evaluated in a rigorous epidemiologi-
cal study, their true potential as chemical indicators of human fecal contamination will not be determined. To
begin this assessment, samples are currently being analyzed as part of the National Epidemiological and Envi-
ronmental Assessment of Recreational Water Study, which should determine if there is a correlation between
concentration of any of the chemicals and incidence of illness.
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Research on Microorganisms in Drinking Water Progress Review Workshop
High Throughput DMA-Based Tools To Study Water
Microbial Communities
Jorge Santo Domingo, Joyce Simpson, Margaret Williams, and Catherine Kelty
National Risk Management Research Laboratory, Water Supply and Water Resources Division,
Microbial Contaminant Control Branch, U.S. Environmental Protection Agency, Cincinnati, OH
Presentation Abstract
The demands for water safe for human consumption and recreational activities have increased rapidly in
recent years due to human exponential growth. The impact of this growth has affected the performance of
wastewater treatment facilities and changed the biological and chemical stability of watersheds throughout the
nation. One of the most significant challenges facing the Nation is to meet the standards established by regula-
tory agencies, recognizing the shortcomings characteristic of current technologies used to monitor microbi-
ological water quality. One relevant shortcoming is the strict dependence on culturing techniques to determine
the presence and estimate densities of indicator bacteria and microbial pathogens. Culture-based methods tend
to underestimate the densities and diversity of microorganisms because they can only recover a small number
of organisms. Frequently, accurate identification of microorganisms can take a day or two to several weeks.
Nucleic acid-based approaches can circumvent many of the shortcomings of the culture-based methods. For
instance, the possibility of rapidly and simultaneously monitoring for the presence of hundreds of microorgan-
isms and genes relevant to public health is now becoming a reality in light of the recent advances in microarray
technology. This presentation will review recent technological developments in nucleic acid research that can
be used to assess the microbiological quality of water systems (see Figure 1). Examples will be provided to
illustrate the application of molecular tools to: (1) evaluate microbial changes in water quality of fecally im-
pacted watersheds; and (2) study microbial diversity during treatment, distribution, and storage of drinking
water. The importance of constructing comprehensive molecular databases for water distribution systems and
watersheds also will be discussed. These databases are necessary to optimize the various methods that will be
used in the years to come by environmental microbiologists. In summary, we believe that the use of rapid and
high-throughput methods will result in the development of risk management measures that are based on a
framework of sound science.
Figure 1. Microarray for water quality assessment.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Detection of Emerging Microbial Contaminants in Source
and Finished Drinking Water Using DMA Microarrays
Timothy M. Straub1, Paul A. Rochelle2, Ricardo DeLeon2, and Darrell P. Chandler3
Battelle Memorial Institute, Pacific Northwest, Richland, WA; Metropolitan Water District
of Southern California, Los Angeles, CA; Argonne National Laboratory, Argonne, IL
Presentation Abstract
DNA microarrays represent a potentially significant technology and analytical technique for the simulta-
neous detection of multiple pathogens in a single water sample, with the ability to incorporate live/dead dis-
crimination using messenger RNA expression. The objectives of this project are to develop and use DNA
arrays for detecting pathogens in natural, turbid, and processed water supplies. Cryptosporidium parvum, Es-
cherichia coli 0157:H7, and Helicobacterpylori serve as model organisms.
The genetic sequence differences for certain genes for strains of C. parvum and closely related nonpatho-
genic species are often single nucleotide substitutions at various locations within a gene. An array was de-
signed and tested to investigate these single nucleotide substitutions within the hsp70 (70 kilodalton heat shock
protein) gene for two species of Cryptosporidium. A similar array was constructed to investigate both single
nucleotide substitution and the presence or absence of a virulence gene for H. pylori. For E. coli 0157:H7,
certain strains lack either or both shiga-like toxin genes. An array was constructed for this pathogen and tested
with various strains of E. coli 0157:H7. For all arrays, polymerase chain reaction (PCR) using fluorescently
labeled primers for each organism was performed and then hybridized to the array. The resulting hybridization
patterns on the array were analyzed to determine if differences between species and strains could be elucidated.
For C. parvum, differences between two species (C. hominis and C. parvum) were easily differentiated by
both their pattern of hybridization to the array, and statistical discrimination of the data for single nucleotide
substitutions. Likewise, single nucleotide substitution also was possible for H. pylori. For this organism, the
difference between strains also was readily apparent by the absence of a virulence factor gene. For E. coli
0157:H7, discrimination between strains that lacked either or both shiga-like toxin genes was confirmed.
Specificity testing with nontarget organisms revealed extremely low false positive rates. Sensitivity of the ar-
rays was dependent, in part, by the PCR process that was used to generate the fluorescent probes to hybridize
to the array, but it was in the general range of 10 to 100 cells.
Microarray analysis of waterborne pathogens allows excellent discrimination between strains and closely
related species of organisms. In the context of these findings, the arrays can serve to potentially fingerprint
isolates in documented waterborne disease outbreaks. Also, the presence or absence of virulence factor genes
in certain isolates may render these organisms more or less pathogenic. In this case, microarray analysis may
aid epidemiologists to link mild cases of gastroenteritis due to consumption of contaminated drinking water to
less virulent forms of known waterborne pathogens.
The true ability of DNA microarrays for detection of any waterborne pathogen using just one assay
method has yet to be realized. This is due, in part, to generation of labeled probes using PCR. To realize this
goal, many different PCR primers, to cover known and emerging pathogens, must work within the same reac-
tion vessel. This is an extremely difficult task and will require next generation reagents and bioinformatics
software to design PCR primers that will work in a reaction of this type. Currently, this avenue is being inves-
tigated as well as an alternate, where messenger RNA is hybridized directly to the arrays. Using the multi-
plexed PCR approach, a 6-plex PCR has been performed successfully. With the RNA approach (see Figure 1),
the presence of 12 different gene sequences representing 8 unique genes for an E. coli 0157:H7 isolate that
lacked its shiga-like toxin 2 gene were detected simultaneously. The principle advantages of the RNA ap-
proach are the ease with which multiplexing can be achieved and the potential for live/dead discrimination.
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Research on Microorganisms in Drinking Water Progress Review Workshop
uidA1 OraGreen
sit1 lamB
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eaeA stxl iacZ
,; ^
1IIC hlyA uidA3
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Figure 1. Detection of virulence and marker genes for E. coli 0157:H7
ATCC 43890 using the direct mRNA hybridization approach.
This strain lacks the shiga-like toxin 2 gene, and this is
confirmed in this image.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Mammalian Cell Response to Pathogens
Samuel L. Hayes
U.S. Environmental Protection Agency, Cincinnati, OH
Presentation Abstract
The goal of microbiological water testing is to determine whether or not the consumption of and/or expo-
sure to a water sample will lead to health problems in an exposed population. Water utilities currently rely on
the detection of indicator bacteria as a measure of the potential presence of pathogenic microorganisms. Al-
though it is possible to directly analyze for the presence of specific pathogens, it is difficult to design assays
that detect the full range of potential human pathogens and assess the virulence of each pathogen detected. A
better approach might involve measuring the interaction between waterborne microorganisms (i.e., potential
pathogens) and human cells as a measure of exposure assessment.
Microbial pathogens have been shown elicit host responses resulting in shifts in cell metabolism and pro-
tein synthesis. These shifts are reflected in the messenger RNAs (mRNAs) produced by the responding cell or
tissue. Therefore, it should be possible to determine whether a disease response has occurred by monitoring the
expression of mRNA molecules. DNA microarrays are one way to monitor changes in mRNAs. The specific
goal of this project is to look at mammalian cellular response to pathogens, specifically assessing gene expres-
sion. The focus will be on pathogens that are known (or suspected) to cause disease from exposure to contami-
nated water. These pathogens typically cause gastrointestinal illness. Thus, experimentation will focus on dis-
ease mechanisms associated with intestinal infections. The experimental design is as follows:
Stage 1:
• Establish appropriate animal model for intestinal infection
• Establish corresponding animal intestinal cell line
• Grow pathogen and prepare inoculum at the desired density
• Infect animal model and cell lines
• Analyze mRNAs using species-specific cDNA microarrays
• Look for similar mRNA expressions between the animal model and tissue cell line.
Assuming similar gene expression patterns are found between the tissue culture and animal models, move to
Stage 2 testing.
Stage 2:
• Establish and infect human intestinal cell line
• Analyze mRNAs from human tissue cultures, look for similar patterns as seen in the animal model us-
ing human cDNA microarray.
The literature suggests that pathogens elicit characteristic sets of mRNAs in host tissues. These mRNAs
could be used to define the pathogen present. Alternatively, a common set of mRNAs may be produced in re-
sponse to many different pathogens. In either case, the identification of specific mRNAs in host tissue will in-
dicate significant risk. By establishing a relationship between animal models and tissue culture, the case will
be made that human tissue culture can serve as a model for human infection in terms of mRNA expression.
This addresses effects of exposure and identifies disease mechanisms from exposure. An important secondary
goal of the project is to eliminate using animal testing for pathogen exposure research.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Effectiveness of UV Irradiation for Pathogen Inactivation
in Surface Waters
Karl Linden1, Mark Sobsey2, and Gwy-Am Shin2
1 Civil and Environmental Engineering, Duke University, Durham, NC; 2Environmental Sciences and
Engineering, University of North Carolina-Chapel Hill, Chapel Hill, NC
Poster Abstract
Ultraviolet (UV) irradiation is now recognized as an effective and cost-competitive measure to achieve
significant level of inactivation of Cryptosporidium while not producing appreciable level of harmful disinfec-
tion by-products (DBFs) at practical doses. However, the effectiveness of UV technology against new and
emerging pathogens and uncertainties in application of UV disinfection for unfiltered surface waters still needs
to be assessed before widespread use of this technology as a primary disinfectant in drinking water treatment
processes. The primary objectives of this research project are to evaluate the susceptibility (or resistance) and
repair potential of select Contaminant Candidate List (CCL) pathogens and indicator microorganisms to or
after UV disinfection from low- and medium-pressure (LP and MP) UV sources, and to investigate the extent
of microbial association with particles in unfiltered systems and the effects of this particle association and
other water quality parameters on UV disinfection potential.
Preliminary results indicate that both LP and MP UV irradiation are very effective against most of the in-
dicator and emerging microorganisms tested. However, some of the indicator microorganisms like coliphage
MS2, bacteriophage PRD-1, and Bacillus subtilis endospores as well as CCL and emerging pathogens like My-
cobacterium terriae (a substitute for Mycobacterium avium complex), adenovirus type 2, and Toxoplasma
gondii oocysts showed relatively high resistance against both UV irradiation. Although the effectiveness of LP
and MP UV appeared to be similar against most of the microorganisms tested, there was some remarkable dif-
ference between these two UV technologies in terms of their effectiveness against adenovirus 2. To determine
the level of particle association and its effect on UV disinfection, raw surface water samples have been col-
lected from various utilities across the United States, and the waters have been examined for particle associated
coliform and aerobic endospores using physical particle disruption techniques such as homogenization and
blending. However, the levels of the indigenous microbes in the raw waters were typically low (< 1,000/100
mL), so that it was not feasible to assess the degree of particle association in these waters based on those
physical methods.
Currently, the use of microscopic techniques (nucleic acid staining/probes along with confocal micros-
copy) are being investigated to determine the level of particle association in those raw waters. Regarding the
development of new assay systems for some of the CCL microorganisms, we have been successful in develop-
ing a new assay system (Long-template [LT] RT-PCR) for Norwalk virus and a new molecular biology assay
(RT-PCR) for adenovirus 40 or 41, which are being and will be used in the current and future inactivation
study on these viruses by LP and MP UV. In addition, a method has been established to perform wavelength
specific studies using a polychromatic UV light source (MP UV lamps) with a set of UV bandpass filters, and
this setup will be utilized to develop wavelength effectiveness information for select CCL microorganisms like
adenovirus 2, M. terriae, and T. gondii oocysts. Finally, protocols have been established to examine repair
phenomenon following UV disinfection in various conditions in real water treatment situations, and these pro-
tocols will be implemented to evaluate the presence and extent of repair after UV irradiation in the select CCL
and emerging microorganisms in the later phases of this research.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Survey of U.S. Public Health Laboratories: Microbial Pathogens
on the Candidate Contaminant List
Elizabeth D. Hilborn1, Michael O. Royster2, and Doug J. Drabkowski3
Office of Research and Development, National Health and Environmental Effects
Research Laboratory, U.S. Environmental Protection Agency, Research Triangle
Park, NC; 2 Crater Health District, Petersburg, VA; 3Association of Public Health
Laboratories, Washington, DC
Poster Abstract
During March 1998, the U.S. EPA published the Candidate Contaminant List (CCL) of drinking water
contaminants; these chemical contaminants and microbial pathogens will be evaluated by the U.S. EPA for
potential regulation. Microbial pathogens contained in the list include: Aeromonas hydrophila, adenovirus,
calicivirus, coxsackie virus, echo virus, Helicobacter pylori, Microsporidia spp., and Mycobacterium avium-
intracellulare complex (MAIC). Because few of these pathogens are reportable and detection methods among
laboratories may vary, an estimate of the public health burden of illness is needed to prioritize pathogens for
regulatory action. State public health laboratories (SPHL) serve as reference laboratories in many states. If
SPHL are likely to receive requests to detect CCL pathogens in human clinical specimens, they may serve as
future active surveillance sites to help establish population-based estimates of illness with CCL pathogens.
During early 2000, a survey of SPHLs was performed by the Association of Public Health Laboratories.
The survey goal was to ascertain the number of clinical specimens submitted, the number of specimens in
which evidence of infection with a CCL pathogen was verified, and analytic methods used to detect evidence
of infection. Each state laboratory director was asked to report fiscal year 1999 (FY 99) data via a self-com-
pleted questionnaire.
Forty-seven of 50 (94 percent) SPHL representatives completed and returned questionnaires. During FY
99, the number of clinical specimen submissions, percent positivity, and analytic methods varied by CCL
pathogen. Number of submissions ranged from 1,009 for analysis of calicivirus, to 199,641 for analysis of
MAIC. Percent positivity ranged from less than 1 percent of specimens examined for evidence of A. hydro-
phila, coxsackie virus, and Microsporidia infection, to 40 percent of specimens examined for evidence of
calicivirus infection. Analytic methods used by SPHLs included: culture, immunologic and molecular assays,
and direct visualization of pathogens. SPHLs solely reported using polymerase chain reaction (PCR) to detect
calicivirus in clinical specimens; this technique resulted in the highest percent detection (40 percent as com-
pared to < 5 percent for all other pathogens).
This survey provided information about which CCL pathogens are currently detected at SPHL and analytic
methods used during 1999. SPHL may be useful in active surveillance systems for nontuberculous Mycobacte-
rium spp., adenovirus, and enteroviral (coxsackie virus, echo virus) infections. SPHL are least likely to be
good locations for surveillance ofH. pylori, Microsporidia spp., and calicivirus. The use of PCR to detect evi-
dence of calicivirus in clinical specimens resulted in the highest percent detection of calicivirus among all CCL
pathogens. However, PCR may be underutilized in SPHLs. Increased use of molecular techniques may in-
crease the diagnostic efficiency of CCL pathogens within SPHLs.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Comparative Diversity of Fecal Bacteria in Agriculturally Significant
Animals To Identify Alternative Targets for Microbial Source Tracking
Joyce M. Simpson1, Samuel P. Myoda2, Donald J. Reasoner1, and Jorge W. Santo Domingo1
1 Water Supply and Water Resources Division, National Risk Management Research Laboratory,
U.S. Environmental Protection Agency, Cincinnati, OH; 2Division of Water Resources, Delaware
State Department of Natural Resources and Environmental Control, Dover, DE
Poster Abstract
Animals of agricultural significance contribute a large percentage of fecal pollution to waterways via run-
off contamination. The premise of microbial source tracking is to utilize fecal bacteria to identify target popu-
lations that are directly correlated to specific animal feces, thus permitting identification of contamination
sources and implementation of remediation practices.
To identify alternative targets for source tracking studies, comparison of fecal bacterial populations was
performed using Denaturing Gradient Gel Electrophoresis (DGGE) targeting the V3 region of the 16S rDNA
gene. Fecal populations from individual horses, cattle, swine, sheep, and goats were compared (see Figure 1).
The greatest diversity was found in the ruminant animal species. Within the ruminants, between 40 and 51
percent of the bands within the fecal patterns were dominant populations (i.e., occurred in greater than 50 per-
cent of animals tested) and 7 percent were highly dominant (occurred in greater than 80 percent of animals
tested). Within the non-ruminants, only 14 to 18 percent of the bands were dominant, and 4 percent were
highly dominant. Eleven bands were common to all fecal samples, and eight bands were present in ruminants
only. Another eight bands were predominantly found in ruminants, and three bands were predominant in non-
ruminants. No bands specific to non-ruminants were found in any of the animals tested.
A comparison using Dice's similarity coefficient and Ward's dendrogram algorithm indicated that fecal
patterns tended to cluster according to digestive physiology (i.e., ruminants clustered with ruminants) rather
than by species. Non-ruminant species tended to cluster more closely within species than to each other and
were not as intermixed as ruminant results.
Phylogenetic examination of the common and divergent banding populations should provide information
to determine if there are suitable alternative organisms that may be used to track fecal pollution. Elucidation of
novel organisms related to fecal contamination would potentially increase the ability to identify sources more
accurately, thereby allowing the appropriate remediation response to be expeditiously selected.
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
COW ] I GOAT ] SHEEP
HORSE j [ PIG ]
1
H
l-r
Figure 1. DGGE gel representing typical fecal banding patterns observed for different animal species.
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Developing Dynamic Infection Transmission Models for Microbial
Risk Assessment (MRA) Applications
Patricia Murphy, Brenda Boutin, Jeff Swartout, Glenn Rice, Jon Reid, and Michael Broder
Office of Research and Development, National Center for Environmental Assessment,
U.S. Environmental Protection Agency, Edison, NJ
Poster Abstract
Drinking water MRAs generally are conducted with methods and assumptions that are analogous to exist-
ing chemical risk assessment methodologies. These assessments usually consider only a primary environ-
mental transmission route, such as drinking water, where a human host is exposed and infected solely through
this route. There are, however, several issues unique to infectious diseases that the traditional chemical frame-
work does not address, including: (1) the potential for secondary transmission (ST), where the infectious agent
is passed directly or indirectly from an infectious human to other susceptible humans; (2) acquired immunity to
the infectious agent, where previous exposures render the human host either completely or partially resistant,
and the duration of that immunity; and (3) the environmental population dynamics of pathogens, which are
living organisms.
To explore the influence of these additional, interdependent factors on traditional MRA approaches, the
National Center for Environmental Assessment initiated collaborative research with investigators at the Uni-
versity of California-Berkeley (UCB) and the University of Michigan (UM) to develop and apply infection
transmission models for waterborne pathogen exposures. The academic research teams extended existing ap-
proaches for compartmental modeling of dynamic infection transmission systems to include infection routes
appropriate for enteric waterborne pathogens, that is, exogenous sources, human-human contact, and human-
water-human pathogen circulation. Using data from the scientific literature, computer simulation approaches
were used to study how model output changed in relation to alternative assumptions for the studied exposure
scenarios, alternative values for the input variables, and alternative analytical forms of the model, that is, de-
terministic or stochastic.
This project has demonstrated that existing infection transmission models can be extended and modified to
integrate diverse and complex information on host, agent, and environmental characteristics that affect patho-
gen exposure and risk. Results show that ignoring or mis-specifying ST effects in the context of MRAs leads
to mis-characterization of individual- and population-level risks and mis-estimation of the health benefits at-
tributable to different drinking water treatment interventions.
Existing MRA methodologies for estimating health risks from waterborne exposure to pathogens with a
significant potential for human-human transmission require conceptual and analytical modifications to accu-
rately capture host-agent-environment interdependencies that determine human exposure and risk. The final
report from this research is under development. It will provide the focus for an upcoming expert workshop that
will develop specific recommendations on when and how ST effects should be modeled and incorporated into
assessments and will form part of the basis for a 2008 EPA guidance document on MRA tools.
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Virulence Factors of Aeromonas: A Molecular Genetic
Characterization
Key a Sen and Mark Rodgers
Office of Ground Water and Drinking Water, U.S. Environmental Protection Agency,
Cincinnati, OH
Poster Abstract
Surveys of finished drinking water from eight small systems (2001) and eight large systems (2000) con-
ducted by the U.S. EPA, revealed that 7 out of 16 water utilities encompassing several states (NY, KY, IA,
OH) were contaminated with Aeromonas species. Altogether 205 organisms were isolated by EPA Method
1605. The goal of this project was to determine whether the Aeromonas species isolated from these drinking
water utilities had the potential to be pathogenic.
A molecular genetic approach was chosen. Published literature was searched to identify the genes that
played a role in the pathogenesis of the organism. Only those genes were selected that definitively played a
role in the virulence of the species when tested in animal models or cell cultures. The isolates were tested for
the virulence factors elastase, lipase, flagella A and B genes, the cytotoxic enterotoxin, Act (achytoen), and the
cytotonic enterotoxins, Alt and Ast. Oligonucleotide primers were developed against these genes, and when
used in the polymerase chain reaction (PCR), a portion of the gene was amplified in the positive control. A
positive control was an Aeromonas species known to have the virulence gene being tested. PCR was per-
formed in three duplex assays with the 205 isolates using the following primer sets together: elastase (ahyB)
and lipase (pla); fla and alt; act and ast.
Preliminary findings showed that pla was present in 86 percent, act in 69 percent, fla in 55 percent, ahyB
in 40 percent, and alt and ast in 45 percent and 35 percent of the isolates, respectively. Only one isolate had all
six virulence genes. Multiple species were isolated from most of the utilities. Different combinations of viru-
lence factors also were observed, sometimes even in different strains of the same species. However, a domi-
nant strain having the same set of virulence factors was usually isolated from different rounds of sampling
from a single tap.
These results suggest the importance of examining as many Aeromonas isolates as possible from a water
sample, as within the same species the occurrence of certain virulence factors may vary. The results also sug-
gest that the Aeromonas strains isolated from water utilities have the potential to be pathogenic. However, ad-
ditional virulence factors, which have not yet been identified or characterized, may be needed to cause actual
disease.
Isolates having different combinations of virulence factors will be tested in animal models to determine
whether there are one or more combinations of virulence factors that are necessary for establishing diarrhea in
the models. Aeromonas isolates from the UCMR survey of finished drinking water, which is being conducted
in 2003, also will be tested for the above virulence factor genes.
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Effects of pH and Temperature on the Kinetics of Aeromonas
hydrophila Inactivation With Combined Chlorine
Kwanrawee Sirikanchana and Benito J. Marinas
Department of Civil and Environmental Engineering, University of Illinois
at Vrbana-Champaign, Vrbana, IL
Poster Abstract
Aeromonas hydrophila is a waterborne opportunistic pathogen commonly detected in natural water
sources (e.g., surface water and groundwater) and also in drinking water, even in the presence of detectable
levels of chlorine residual. There is a need to elucidate the mechanisms responsible for the survival of A. hy-
drophila during and/or after the disinfection process. Previous studies have shown that the inactivation kinetics
of A. hydrophila with monochloramine is independent of monochloramine concentration in the range of 0.01 -
10 mg/L as C12 at constant pH and constant temperature. However, water quality parameters including pH and
temperature could affect the inactivation kinetics of A. hydrophila. The main objective of this study is to char-
acterize the effects of pH and temperature on the inactivation kinetics of A. hydrophila (ATCC 7966) with
combined chlorine. Experiments are performed in batch reactors with the temperature controlled at target val-
ues in the range of 1-30 °C with a recirculating water bath. The solution pH is maintained constant at target
values in the range of 6-10 with phosphate and borate buffers. Disinfectant concentrations range from 0.01 to
10 mg/L.
The kinetics of A. hydrophila inactivation with monochloramine under all conditions investigated was
characterized by an initial lag phase followed by pseudo-first order inactivation. The temperature effect was
found to be consistent with Arrhenius law at each pH. Experiments performed at pH 6, 8, and 10 for each tem-
perature revealed strong pH dependence. As the pH decreased from pH 10 to 6, the inactivation kinetics was
faster due to both a shorter lag-phase and a faster rate of post-lag phase inactivation. In general, the concentra-
tion of monochloramine used did not affect the kinetics of A. hydrophila inactivation with this disinfectant,
thereby confirming the validity of the CT concept (a given value of the product of disinfectant concentration
and contact time resulted in a given degree of inactivation independently of the disinfectant concentration
used) at each pH and temperature tested for the concentration range investigated.
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
The U.S. Environmental Protection Agency's
Research on Microorganisms in Drinking Water Workshop
Marriott Kingsgate Conference Hotel
151 Goodman Drive
Cincinnati, OH 45219
August 5-7, 2003
AGENDA
Tuesday, August 5, 2003
10:00 - 10:10 a.m. Welcome
Hugh McKinnon, Director, National Risk Management Research Laboratory
10:10 - 10:25 a.m. Introductory Remarks
J. Paul Gilman, Assistant Administrator and Agency Science Advisor
Office of Research and Development
10:25 - 10:50 a.m. Overview of the U.S. EPA's Drinking Water Research Program
Fred Hauchman, Drinking Water National Program Manager, NHEERL
10:50 — 11:15 a.m. Overview Presentation From the U.S. EPA's Office of Ground Water and Drinking
Water
Gregory Carroll, Chief, Technical Support Center, OW/OGWDW
11:15 - 11:35 a.m. Overview of Water Security Research and Technical Support Activities
Jonathan Herrmann, Water Security Team Leader, and Hiba Shukairy, Technical
Support Center, OW/OGWDW
11:35 — 12:00 noon Overview of Regional Concerns for Microorganisms and Drinking Water
Bruce Macler, EPA Region 9 (San Francisco)
12:00-1:00 p.m. Lunch
1:00 - 4:15 p.m. Topic Area 1: Research Supporting Office of Water's Ground
Water/Source Water Regulatory Activities
Moderators: Stig Regli, OW/OGWDW, and Pat Murphy, NCEA
1:00 - 1:30 p.m. Safe Drinking Water Act (SDWA) Requirements and Microbial
Research Needs (Surface Water, Ground Water, and Distribu-
tion Systems)
Presented by Stig Regli, OW/OGWDW
1:30 — 1:50 p.m. Microbial Dose-Response Modeling: A Predictive Bayesian
Approach (EIMS #54468)
Presented by Jeff Swartout, NCEA
1:50 — 2:10 p.m. The Use of Randomized Trials of In-Home Drinking Water
Treatment To Study Endemic Water Borne Disease
Presented by Timothy J. Wade, NHEERL
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Research on Microorganisms in Drinking Water Progress Review Workshop
2:10-2:30 p.m. Break
2:30 - 2:50 p.m. Screening Models To Predict Probability of Contamination by
Pathogenic Viruses to Drinking Water Aquifers
Presented by Bart Faulkner, NRMRL
2:50 — 3:10 p.m. Integrated Approach for the Control of Cryptosporidiumpar-
vum Oocysts and Disinfection By-Products in Drinking Water
Treated With Ozone and Chloramines
Presented by Benito Marinas, University of Illinois at Urbana-
Champaign
3:10 — 3:30 p.m. Prevalence and Distribution of Genotypes of Cryptosporidium
parvum in United States Feedlot Cattle
Presented by Robert Atwill, University of California-Davis
3:30 - 4:15 p.m. Panel Discussion
4:15-5:45 p.m. Poster Session I
All posters will be set up during both Poster Sessions with one-half of the posters
staffed at each session.
5:45 p.m. Adjournment
Wednesday, August 6, 2003
8:15 - 5:40 p.m. Topic Area 2: Research Supporting Office of Water's Contaminant Candidate
List (CCL)
Moderator: Cynthia Nolt-Helms, NCER
8:15 — 8:50 a.m. CCL and National Drinking Water Advisory Council
(NDWAC) Process
Presented by Tom Carpenter, OW/OGWDW
8:50 - 9:15 a.m. The Roles of Pathogen Risk Assessment in the Contaminant
Candidate List Process (EIMS #22389)
Presented by Glenn Rice, NCEA
9:15 - 9:40 a.m. Overview: CCL Pathogens Research at NRMRL
Presented by Don Reasoner, NRMRL
9:40-10:00 a.m. Break
10:00-11:50 a.m. Topic Area 2.1: CCL Protozoa
Moderators: Carrie Moulton, OW/OGWDW, and Alan Lindquist, NERL
10:00 - 10:20 a.m. Detection of Cyclospora cayetanensis and Microsporidial
Species Using Quantitative Fluorogenic 5' Nuclease PCR
Assays (EIMS #56083)
Presented by Frank Schaefer, NERL
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
10:20-10:40 a.m. Development of Detection and Viability Methods for
Wateborne Microsporidia Species Known to Infect Humans
Presented by Rebecca Hoffman, University of Wisconsin at
Madison
10:40 — 11:00 a.m. Development and Evaluation of Procedures for Detection of
Infectious Microsporidia in Source Waters
Presented by Paul Rochelle, Metropolitan Water District of
Southern California
11:00 - 11:20 a.m. Development and Evaluation of Methods for the Concentration,
Separation, Detection, and Viability/Infectivity of Three
Protozoa From Large Volumes of Water
Presented by Saul Tzipori, Tufts University
11:20 - 11:50 a.m. Panel Discussion
11:50-12:50 p.m. Lunch
12:50 - 2:20 p.m. Poster Session II
All posters will be set up during both Poster Sessions with one-half of the posters
staffed at each session.
2:20-3:50 p.m. Topic Area 2.2: CCL Viruses
Moderators: Robin Oshiro, OW/OST, and Betsy Hilborn, NHEERL
2:20 - 2:40 p.m. Use of PCR-Based Methods for Virus Occurrence Studies
(EIMS #56084)
Presented by Shay Fout, NERL
2:40 - 3:00 p.m. Norwalk Virus Dose Response and Host Susceptibility
Presented by Peter Teunis, National Institute of Public Health
and Environment, The Netherlands
3:00 - 3:20 p.m. Development of a Rapid, Quantitative Method for the Detection
of Infective Coxsackie and Echo Viruses in Drinking Water
Presented by Marylynn Yates, University of California-
Riverside
3:20 - 3:50 p.m. Panel Discussion
3:50-4:10 p.m. Break
4:10-5:40 p.m. Topic Area 2.3: CCL Bacteria
Moderators: Jim Sinclair, OW/TSC, and Don Reasoner, NRMRL
4:10 — 4:30 p.m. Disinfection of Helicobacterpylori and Aeromonas Species
Presented by Don Reasoner, NRMRL
4:30 - 4:50 p.m. Genomic and Physiological Diversity ofMycobacterium avium
Complex
Presented by Gerard Cangelosi, Seattle Biomedical Research
Institute
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
4:50 — 5:10 p.m. Mycobacterium avium Complex (MAC) in Drinking Water:
Detection, Distribution, and Routes of Exposure
Presented by Phanida Prommasith, Harvard School of Public
Health
5:10-5:40 p.m. Panel Discussion
5:40 p.m. Adjournment
Thursday, August 7, 2003
8:00-9:00 a.m. Topic Area 3: Distribution Systems and Biofilms
Moderators: Lisa Almodovar, OW/OST, and Mark Meckes, NRMRL
8:00 — 8:20 a.m. The Effect of Chlorine, Chloramine, and Mixed Oxidants on
Biofilms in a Simulated Water Distribution System
Presented by Mark Meckes, NRMRL
8:20 - 8:40 a.m. Molecular Characterization of Drinking Water Microbial
Communities
Presented by Jorge Santo Domingo, NRMRL
8:40 - 9:00 a.m. Panel Discussion
9:00-9:20 a.m. Break
9:20 - 12:30 p.m. Topic Area 4: Cross-Cutting Research and Emerging Topics
Moderators: Keya Sen, OGWDW/TSC, and Rebecca Calderon, NHEERL
9:20 - 9:40 a.m. The Application of Mass Spectrometry to the Study of
Microorganisms (EIMS #18338)
Presented by Jody Shoemaker, NERL
9:40 - 10:00 a.m. Cyanobacteria and Their Toxins (EIMS #54617)
Presented by Elizabeth Hilborn, NHEERL
10:00 - 10:20 a.m. Transport of Chemical and Microbial Contaminants From
Known Wastewater Discharges: Potential Chemical Indicators
of Human Fecal Contamination (EIMS #18337)
Presented by Susan Glassmeyer, NERL
10:20 - 10:40 a.m. High Throughput DNA-Based Tools To Study Water Microbial
Communities
Presented by Jorge Santo Domingo, NRMRL
10:40 - 11:00 a.m. Break
11:00 — 11:20 a.m. Detection of Emerging Microbial Contaminants in Source
and Finished Drinking Water Using DNA Microarrays
Presented by Timothy Straub, Pacific Northwest National
Laboratory
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
11:20 — 11:40 a.m. Mammalian Cell Response to Pathogens
Presented by Sam Hayes, NRMRL
11:40-12:30 p.m. Panel Discussion
12:30 p.m. Adjournment of Public Workshop
12:20-1:30 p.m. Lunch
1:30 - 4:00 p.m. EPA-Only Discussion Session
4:00 p.m. Adjournment of EPA-Only Discussion Session
U.S. Environmental Protection Agency
Organization Abbreviations
ORD, Office of Research and Development
ORD Laboratories and Centers:
NHEERL - National Health and Environmental Effects Laboratory
NERL - National Exposure Research Laboratory
NCEA - National Center for Environmental Assessment
NRMRL - National Risk Management Research Laboratory
NCER - National Center for Environmental Research
OW. Office of Water
OW Offices:
OGWDW - Office of Ground Water and Drinking Water
OST - Office of Science and Technology
TSC - Technical Support Center
The Office of Research and Development's National Center for Environmental Research
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_ Research on Microorganisms in Drinking Water Progress Review Workshop _
The U.S. Environmental Protection Agency's
Research on Microorganisms in Drinking Water Workshop
Marriott Kingsgate Conference Hotel
151 Goodman Drive
Cincinnati, OH 45219
August 5-7, 2003
POSTER TITLES AND SESSIONS
Poster Session I: Tuesday, August 5, 4: 15 - 5:45 p.m.
Poster Session II: Wednesday, August 6, 12:50 - 2:20 p.m.
All posters will be set up during both poster sessions.
Poster Session Topic Area 1: Research Supporting Office of Water's Ground Water/Source Water
Regulatory Activities
I Microbial Drinking Water Contaminants: Endemic and Epidemic Waterborne
Gastrointestinal Disease Risks in the United States
Presented by Rebecca L. Calderon, NHEERL
II Evaluating Microbial Indicators and Health Risks Associated With Bank Filtration
Presented by Twila Kunde, Lovelace Clinic Foundation
I A Prospective Epidemiological Study of Gastrointestinal Health Effects Associated With
Consumption of Conventionally Treated Groundwater
Presented by Stuart Hooper, Emory University
II Using Neural Networks To Create New Indices and Classification Schemes
Presented by Gail Brion, University of Kentucky
Topic Area 2: Research Supporting Office of Water's Contaminant Candidate List
Topic Area 2.2: CCL Viruses
II Dose-Response Assessments for NLV and Coxsackievirus in Drinking Water (EIMS
#22389)
Presented by Brenda Boutin, NCEA
I Methods Used To Analyze a Norovirus Outbreak (EIMS #56084)
Presented by Jennifer Cashdollar, NERL
II Development of a Molecular Method To Identify Astrovirus in Water (EIMS #56080)
Presented by Ann C. Grimm, NERL
I Detection and Occurrence of Human Caliciviruses in Drinking Water
Presented by Gwy-Am Shin, University of North Carolina at Chapel Hill
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Topic Area 2.3: CCL Bacteria
I Sensitivity of Three Encephalitozoon Species to Chlorine and Chloramine Treatment as
Detected by an In Vitro Microwell Plate Assay
Presented by Cliff Johnson, NRMRL
II Inactivation ofAeromonas by Chlorine and Monochloramine
Presented by L.A. DeMaria (Laura Boczek), NRMRL
I Mycobacterium paratuberculosis and Nontuberculous Mycobacteria in Potable Water
(EIMS #18289 & #18341)
Presented by Terry Covert, NERL, and Stacy Pfaller, NERL
II Detection of Helicobacter pylori Using a Highly Variable Locus Upstream of the 16S
Ribosomal RNA Gene
Presented by Manoucher Shahamat, University of Maryland
I Using Real-Time PCR To Detect Toxigenic Strains ofMicrocystis aeruginosa
Presented by Carrie Moulton, Technical Support Center, OW/OGWDW
II Role of Adaptive Response in the Kinetics of Mycobacterium avium Inactivation With
Monochloramine
Presented by Benito Marinas, University of Illinois at Urbana-Champaign
Topic Area 3: Distribution Systems and Biofilms
II Phylogenetic Analysis of Prokaryotic and Eukaryotic Microorganisms in a Drinking
Water Distribution System Simulator
Presented by Margaret M. Williams, NRMRL
I Identification and Characterization ofAeromonas Isolates From Drinking Water Distribu-
tion Systems
Presented by Jennifer Birkenhauer, NERL
II Pathogenicity of Biofilm Bacteria (EIMS #18286)
Presented by Dennis Lye, NERL
Topic Area 4: Cross-Cutting Research and Emerging Topics
II Effectiveness of UV Irradiation for Pathogen Inactivation in Surface Waters
Presented by Gwy-Am Shin, University of North Carolina at Chapel Hill
I Survey of U.S. Public Health Laboratories: Microbial Pathogens on the Candidate
Contaminant List (EIMS #54616)
Presented by Elizabeth D. Hilborn, NHEERL
II Comparative Diversity of Fecal Bacteria in Agriculturally Significant Animals To
Identify Alternative Targets for Microbial Source Tracking
Presented by Joyce M. Simpson, NRMRL
I Developing Dynamic Infection Transmission Models for Microbial Risk Assessment
(MRA) Applications (EIMS #18473)
Presented by Pat Murphy, NCEA
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
II Virulence Factors ofAeromonas: A Molecular Genetic Characterization
Presented by Keya Sen, Technical Support Center, Office of Ground Water and Drinking
Water
I Effects of pH and Temperature on the Kinetics ofAeromonas hydrophila Inactivation
With Combined Chlorine
Presented by Benito Marinas, University of Illinois at Urbana-Champaign
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
The U.S. Environmental Protection Agency's
Research on Microorganisms in Drinking Water Workshop
Marriott Kingsgate Conference Hotel
151 Goodman Drive
Cincinnati, OH 45219
August 5-7, 2003
Participants List
Noreen Adcock
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Division
(MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7724
Fax: (513)569-7328
E-mail: adcock.noreen@epa.gov
Lisa Almodovar
U.S. Environmental Protection Agency
Office of Water
Ariel Rios Building (4304T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 566-1085
Fax: (202)566-1139
E-mail: almodovar.lisa@epa.gov
Rob Atwill
University of California, Davis
Department of Population Health
and Reproduction
School of Veterinary Medicine
18830 Road 112
Tulare, CA 93274
Telephone: (559)688-1731
Fax: (559)686-4231
E-mail: ratwill@vmtrc.ucdavis.edu
Tom Behymer
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-564)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7392
Fax: (513)569-7757
E-mail: behymer.thomas@epa.gov
Jennifer Birkenhauer
U.S. Environmental Protection Agency
Oak Ridge Institute for Science and Education
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7012
Fax: (513)569-7191
E-mail: birkenhauer.jennifer@epa.gov
Ben Blaney
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
Laboratory (MS-235)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7852
Fax: (513)569-7680
E-mail: blaney.ben@epa.gov
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Laura Boczek
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7282
Fax: (513)569-7328
E-mail: boczek.laura@epa.gov
Brenda Boutin
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
Assessment (MS-190)
26 W Martin Luther King Drive
Cincinnati, OH 45230
Telephone: (513)569-7532
Fax: (513)569-7916
E-mail: boutin.brenda@epa.gov
Susan Boutros
Environmental Associates Limited
24 Oak Brook Drive
Ithaca, NY 14850
Telephone: (607) 272-8902
Fax: (607)256-7092
E-mail: susanboutros@eal-labs.com
Nichole Brinkman
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-320)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7315
E-mail: brinkman.nichole@epa.gov
Gail Brion
University of Kentucky
Department of Civil Engineering
161 Raymond Building
Lexington, KY 40506-0281
Telephone: (859) 257-4467
Fax: (859)257-4404
E-mail: gbrion@engr.uky.edu
Rebecca Calderon
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental
Effects Research Laboratory
Human Studies Division (MS-58C)
Research Triangle Park, NC 27516
Telephone: (919) 966-6200
Fax: (919)966-6212
E-mail: calderon.rebecca@epa.gov
Gerard Cangelosi
Seattle Biomedical Research Institute
4 Nickerson Street
Seattle, WA 98117
Telephone: (206) 284-8846
Fax: (206)284-0313
E-mail: gcang@sbri.org
Tom Carpenter
U.S. Environmental Protection Agency
Office of Ground Water and Drinking
Water
Ariel Rios Building (4607M)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 564-4885
Fax: (202)564-3760
E-mail: carpenter.thomas@epa.gov
Gregory Carroll
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7948
Fax: (513)569-7191
E-mail: carroll.gregory@epa.gov
Jennifer Cashdollar
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Microbiological and Chemical Exposure
Assessment Research Division
Biohazard Assessment Research Branch (MS-320)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7142
Fax: (513)569-7117
E-mail: cashdollar.jennifer@epa.gov
The Office of Research and Development's National Center for Environmental Research
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Research on Microorganisms in Drinking Water Progress Review Workshop
Hongtu Chen
Harvard Medical School
Harvard School of Public Health
Building I, Room G.29
665 Huntington Avenue
Boston, MA 02115
Telephone: (617) 432-0738
Fax: (617)432-3349
E-mail: hongtuchen@hotmail.com
John Cicmanec
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
Laboratory (MS-G75)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7481
Fax: (513)569-7585
E-mail: cicmanec.john@epa.gov
Terry Covert
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-314)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7318
Fax: (513)487-2512
E-mail: covert.terry@epa.gov
Armah de la Cruz
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-314)
26 W Martin Luther King Drive
Cincinnati, OH 45268-1314
Telephone: (513)569-7224
Fax: (513)569-7170
E-mail: delacruz.armah@epa.gov
Clyde Dempsey
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
Laboratory (MS-235)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7842
E-mail: dempsey.clyde@epa.gov
Jorge Santo Domingo
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7085
Fax: (513)569-7328
E-mail: santodomingo.jorge@epa.gov
Vivian Doyle
U.S. Environmental Protection Agency
Environmental Accountability Division
Region 4
61 Forsyth Street, SW
Atlanta, GA 30303
Telephone: (404) 562-9942
Fax: (404)562-9439
E-mail: doyle.vivian@epa.gov
David Dziewulski
New York State Department of Health
Bureau of Water Supply Protection
Flanigan Square, Room 400
547 River Street
Troy, NY 12180
Telephone: (518)402-7650
Fax: (518)402-7659
E-mail: dmdl4@health.state.ny.us
Bart Faulkner
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Ecosystem and Subsurface Protection Branch
919 Kerr Research Drive
Ada, OK 74820
Telephone: (580) 436-8530
Fax: (580)436-8703
E-mail: faulkner.bart@epa.gov
Mary Ann Feige
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7944
Fax: (513)569-7191
E-mail: feige.maryann@epa.gov
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
Shay Fout
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Microbiological and Chemical Exposure
Assessment Research Division
Biohazard Assessment Research Branch (MS-320)
26 W Martin Luther King Drive
Cincinnati, OH 45268-1320
Telephone: (513)569-7387
Fax: (513)569-7117
E-mail: fout.shay@epa.gov
Christy Frietch
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Treatment Technology Evaluation Branch
(MS-B-24)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7001
Fax: (513)569-7172
E-mail: frietch.christy@epa.gov
Paul Gilman
U.S. Environmental Protection Agency
Office of Research and Development
Ariel Rios Building (8101R)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 564-6620
Fax: (202)565-2910
E-mail: gilman.paul@epa.gov
Susan Glassmeyer
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Microbiological and Chemical Exposure
Assessment Research Division (MS-564)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7526
Fax: (513)569-7757
E-mail: glassmeyer.susan@epa.gov
Ann Grimm
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-320)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7397
Fax: (513)569-7117
E-mail: grimm.ann@epa.gov
Sally Gutierrez
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
(MS-689)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7118
Fax: (513)569-7658
E-mail: gutierrez.sally@epa.gov
Jafrul Hasan
U.S. Environmental Protection Agency
Office of Water
Office of Science and Technology
Health and Ecological Criteria Division
Ariel Rios Building (4304T)
1200 Pennsylvania Avenue, NW, Room 7233Q
Washington, DC 20460
Telephone: (202) 566-1322
Fax: (202)566-1140
E-mail: hasan.jafrul@epa.gov
Fred Hauchman
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects
Research Laboratory (MS-B105-01)
Research Triangle Park, NC 27711
Telephone: (919) 541-3893
Fax: (919)685-3247
E-mail: hauchman.fred@epa.gov
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
Richard Haugland
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Microbiological and Chemical Exposure
Assessment Research Division
Microbial Exposure Research Branch (MS-314)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7135
Fax: (513)487-2512
E-mail: haugland.rich@epa.gov
Sam Hayes
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
Laboratory (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7514
Fax: (513)569-7328
E-mail: hayes.sam@epa.gov
Roland Hemmett
U.S. Environmental Protection Agency
Region 2 (MS-100)
8790 Woodbridge Avenue
Edison, NJ 08837
Telephone: (732) 321-6754
E-mail: hemmett.roland@epa.gov
Jonathan Herrmann
U.S. Environmental Protection Agency
National Homeland Security
Research Center (MS-163)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7839
Fax: (513)487-2555
E-mail: herrmann.jonathan@epa.gov
Elizabeth Hilborn
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects
Research Laboratory (MS-58C)
Research Triangle Park, NC 27711
Telephone: (919) 966-0658
Fax: (919)966-0655
E-mail: hilborn.e@epa.gov
Rebecca Hoffman
Wisconsin State Laboratory of Hygiene
Environmental Health Division
2601 Agriculture Drive
Madison, WI 53718
Telephone: (608) 224-6260
Fax: (608)224-6213
E-mail: beckyh@mail.slh.wisc.edu
Stuart Hooper
Emory University
Rollins School of Public Health
Department of International Health
1518 Clifton Road, NE, Room 762
Atlanta, GA 30322
Telephone: (404) 712-8355
Fax: (404)727-4590
E-mail: shooper@emory.edu
Fu-Chih Hsu
Scientific Methods, Inc.
12441 Beckley Street
Granger, IN 46530
Telephone: (574) 277-4078
E-mail: fuchih@scientificmethods.com
Jay Hua
U.S. Environmental Protection Agency
Region 7
Water, Wetlands, and Pesticides Division
Water Resource Protection Branch
90 IN 5th Street
Kansas City, KS 66101
Telephone: (913)551-7748
Fax: (913)551-9748
E-mail: hua.jay@epa.gov
Maggie Javdan
U.S. Environmental Protection Agency
Office of Research and Development
Office of Science Policy
Ariel Rios Building (8103R)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 564-5264
E-mail: javdan.maggie@epa.gov
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
Patrick Jjemba
University of Cincinnati
Department of Biological Sciences
PO Box ML006
Cincinnati, OH 45221-0006
Telephone: (513)521-9757
Fax: (513)556-5299
E-mail: jjembap@email.uc.edu
Cathy Kelty
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Division
(MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7080
Fax: (513)569-7328
E-mail: kelty.catherine@epa.gov
Twila Kunde
Lovelace Clinic Foundation
Environmental Health Research Division
2309 Renard Place, SE, Suite 103
Albuquerque, NM 87106
Telephone: (505) 262-3472
Fax: (505)262-7598
E-mail: twila@lcfresearch.org
Robert Lange
U.S. Environmental Protection Agency
Region 3
Office of Water
Safe Drinking Water Act Branch (MS-3WP32)
1650 Arch Street
Philadelphia, PA 19103-2029
Telephone: (215) 814-5459
Fax: (215)814-2302
E-mail: lange.robert@epa.gov
Jim Larkin
Scientific Methods, Inc.
12441 Beckley Street
Granger, IN 46530
Telephone: (574) 277-4078
Fax: (574)243-1148
E-mail: jim@scientificmethods.com
Alan Lindquist
U.S. Environmental Protection Agency
National Homeland Security Research
Center (MS-163)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7192
Fax: (513)487-2555
E-mail: lindquist.alan@epa.gov
Srinivasa Lingireddy
University of Kentucky
Department of Civil Engineering
354H, Raymond Building
Lexington, KY 40506
Telephone: (859) 257-5243
Fax: (859)257-4404
E-mail: lreddy@engr.uky.edu
Suzanne List
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7957
Fax: (513)569-7116
E-mail: list.suzanne@epa.gov
Dennis Lye
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-314)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7870
Fax: (513)569-7170
E-mail: lye.dennis@epa.gov
Benito Marinas
University of Illinois at Urbana-Champaign
Department of Civil and Environmental
Engineering
3230 Newmark Civil Engineering Laboratory
205 N Mathews Avenue
Urbana, IL 61801
Telephone: (217) 333-6961
Fax: (217)333-6968
E-mail: marinas@uiuc.edu
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
Dennis McChesney
U.S. Environmental Protection Agency
Region 2
Division of Environmental Science
and Assessment
Monitoring and Assessment Branch (MS-220)
2890 Woodbridge Avenue
Edison, NJ 08837-3602
Telephone: (732) 321-6729
Fax: (732)321-6616
E-mail: mcchesney.dennis@epa.gov
Hugh McKinnon
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
Laboratory (MS-235)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7418
E-mail: mckinnon.hugh@epa.gov
Mark Meckes
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7348
Fax: (513)569-7328
E-mail: meckes.mark@epa.gov
Bruce Mintz
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research
Laboratory (MS-D305-01)
109 TW Alexander Drive
Research Triangle Park, NC 27709
Telephone: (919) 541-0272
Fax: (919)541-7588
E-mail: mintz.bruce@epa.gov
Debbie Moll
Centers for Disease Control and Prevention
Health Studies Branch (MS-E-23)
1600 Clifton Road, NE
Atlanta, GA 30333
Telephone: (404) 498-1364
Fax: (404)498-1355
E-mail: dmoll@cdc.gov
Carrie Moulton
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7919
Fax: (513)569-7191
E-mail: moulton.carrie@epa.gov
Patricia Murphy
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
Assessment (MS-272)
2890 Woodbridge Avenue
Edison, NJ 08837
Telephone: (732) 906-6830
Fax: (732)906-6845
E-mail: murphy.patricia@epa.gov
Cynthia Nolt-Helms
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
Research
Ariel Rios Building (8722R)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 564-6763
Fax: (202)565-2446
E-mail: nolt-helms.cynthia@epa.gov
Nena Nwachuku
U.S. Environmental Protection Agency
Office of Water
Office of Science and Technology
Ariel Rios Building (4304T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202)566-1116
Fax: (202)566-1140
E-mail: nwachuku.nena@epa.gov
Robin Oshiro
U.S. Environmental Protection Agency
Office of Water
Ariel Rios Building (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 566-1075
Fax: (202)566-1053
E-mail: oshiro.robin@epa.gov
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
James Owens
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Microbiological and Chemical Exposure
Assessment Research Division (MS-593)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7235
Fax: (513)569-7464
E-mail: owens.jim@epa.gov
Angela Page
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
Research
Ariel Rios Building (8722R)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202)564-5172
Fax: (202)565-2446
E-mail: page.angelad@epa.gov
Linda Papa
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
Assessment (MS-190)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7587
Fax: (513)569-7916
E-mail: papa.lynn@epa.gov
Latisha Parker
U.S. Environmental Protection Agency
Office of Science and Technology
Ariel Rios Building (4304T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202)566-1118
Fax: (202)566-1139
E-mail: parker.latisha@epa.gov
W. Bruce Peirano
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources
Division (MS-690)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7540
E-mail: peirano.bruce@epa.gov
Joyce Perdek
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
Laboratory (MS-104)
2890 Woodbridge Avenue
Edison, NJ 08837
Telephone: (732) 321-4380
Fax: (732)321-6640
E-mail: perdek.joyce@epa.gov
Dan Petersen
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
Laboratory (MS-G75)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7831
E-mail: petersen.dan@epa.gov
Stacy Pfaller
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-314)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7893
Fax: (513)487-2512
E-mail: pfaller.stacy@epa.gov
Phanida Prommasith
Harvard School of Public Health
Department of Environmental Health
665 Huntington Avenue, HSPH 1, Room G-28
Boston, MA 02115
Telephone: (617) 432-3615
Fax: (617)432-3349
E-mail: pprommas@hsph.harvard.edu
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
Bebe Raupe
BNA, Inc.
PO Box 498769
Cincinnati, OH 45249
Telephone: (513)677-2870
Fax: (513)677-3278
E-mail: braupe@bna.com
Donald Reasoner
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch (MS-387)
26 W Martin Luther King Drive.
Cincinnati, OH 45268
Telephone: (513)569-7234
Fax: (513)569-7328
E-mail: reasoner.donald@epa.gov
Chrissy Reckelhoff
Association of Schools of Public Health
6368 Dry Ridge Road
Cincinnati, OH 45252
Telephone: (513)385-1050
Fax: (202)296-1252
E-mail: reckelhoff.chrissy@epa.gov
Stig Regli
U.S. Environmental Protection Agency
Office of Ground Water and Drinking
Water
Ariel Rios Building (4607M)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 564-5270
Fax: (202)564-3760
E-mail: regli.stig@epa.gov
Jon Reid
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
4556 Winton Road
Cincinnati, OH 45232
Telephone: (513)967-3231
E-mail: reid.jon@epa.gov
Randy Revetta
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch
(MS-G75)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7129
E-mail: revetta.randy@epa.gov
Glenn Rice
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
Assessment (MS-190)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7813
Fax: (513)569-7916
E-mail: rice.glenn@epa.gov
BK Robertson
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7763
E-mail: robertson.boakai@epa.gov
Paul Rochelle
Metropolitan Water District of Southern California
Water Quality Laboratory
700 Moreno Avenue
La Verne, CA 91750
Telephone: (909)392-5155
Fax: (909)392-5246
E-mail: prochelle@mwdh2o.com
Pam Rodgers
Battelle Memorial Institute
505 King Avenue
Columbus, OH 43201
Telephone: (614) 424-4624
Fax: (614)424-3667
E-mail: rodgersp@battelle.org
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
Mary Rothermich
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
Assessment (MS-190)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7869
Fax: (513)569-7916
E-mail: rothermich.mary@epamail.epa.gov
Frank Schaefer
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-320)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7222
Fax: (513)569-7117
E-mail: schaefer.frank@epa.gov
Rita Schoeny
U.S. Environmental Protection Agency
Office of Water
Ariel Rios Building (430IT)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202)566-1127
Fax: (202)566-0441
E-mail: schoeny.rita@epa.gov
Pati Schultz
U.S. Environmental Protection Agency
Office of External Affairs (MS-284)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7966
Fax: (513)569-7770
E-mail: schultz.patricia@epa.gov
Keya Sen
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7026
Fax: (513)569-7191
E-mail: sen.keya@epa.gov
Lois Shadix
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7864
E-mail: shadix.lois@epa.gov
Manou Shahamat
University of Maryland Biotechnology Institute
Center of Marine Biotechnology
70 IE Pratt Street
Baltimore, MD 21202
Telephone: (410)234-8881
Fax: (410)234-8896
E-mail: shahamat@umbi.umd.edu
Gwy-Am Shin
University of North Carolina, Chapel Hill
Department of Environmental Sciences
and Engineering
CB# 7431, ESE, SPH, UNC-CH
Chapel Hill, NC 27599-7431
Telephone: (919) 966-0793
Fax: (919)966-4711
E-mail: gwyam@isis.unc.edu
Jody Shoemaker
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Microbiological and Chemical Exposure
Assessment Research Division (MS-564)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7298
Fax: (513)569-7757
E-mail: shoemaker.jody@epa.gov
Hiba Shukairy
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7943
Fax: (513)569-7191
E-mail: shukairy.hiba@epa.gov
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
Joyce Simpson
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch
(MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7016
Fax: (513)569-7328
E-mail: simpson.joyce@epamail.epa.gov
Jim Sinclair
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7970
Fax: (513)569-7191
E-mail: sinclair.james@epa.gov
Mano Sivaganesan
U.S. Environmental Protection Agency
National Risk Management Research Laboratory
Water Supply and Water Resources
Division (MS-690)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7638
Fax: (513)569-7658
E-mail: sigaganesan.mano@epa.gov
Barbara Smith
U.S. Environmental Protection Agency
Region 9
75 Hawthorne Street (MS-PMD1)
San Francisco, CA 94105
Telephone: (415) 972-3735
Fax: (415)972-3735
E-mail: (415)947-3558
Stuart Smith
Ground Water Science
372 W Wyandot Avenue
Upper Sandusky, OH 43351
Telephone: (419) 209-0298
Fax: (419)209-0336
E-mail: stusmith@udata.com
Timothy Straub
Battelle Memorial Institute - Pacific Northwest
National Laboratory
Environmental Microbiology
PO Box 999, MSIN P7-50
902 Battelle Boulevard
Richland,WA 99352
Telephone: (509) 372-1953
Fax: (509)376-1321
E-mail: timothy.straub@pnl.gov
Jeff Swartout
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
Assessment (MS-190)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7811
Fax: (513)569-7916
E-mail: swartout.jeff@epa.gov
Ruth Sykes
U.S. Environmental Protection Agency
Region 2
Division of Environmental Science
and Assessment
Laboratory Branch (MS-230)
2890 Woodbridge Avenue
Edison, NJ 08837
Telephone: (732) 906-6961
Fax: (732)906-6165
E-mail: sykes.ruth@epa.gov
Peter Teunis
RIVM-IMA
National Institute of Public Health
and Environment
Antonie van Leeuwenhoeklaan 9
PO Box 1
Bilthoven, 3720BA
The Netherlands
Telephone: 31-30-274-2937
Fax: 31-30-274-4456
E-mail: peter.teunis@rivm.nl
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
Marilyn Thornton
U.S. Environmental Protection Agency
Region 4
Science and Ecosystem Support Division
980 College Station Road
Athens, GA 30605-2720
Telephone: (706) 355-8553
Fax: (706)355-8803
E-mail: thornton.marilyn@epa.gov
Saul Tzipori
Tufts University School of Veterinary Medicine
Department of Biomedical Sciences
200 Westboro Road, Building 20
North Grafton, MA 01536
Telephone: (508) 839-7955
Fax: (508)839-7911
E-mail: saul.tzipori@tufts.edu
Steve Via
American Water Works Association
1401 New York Avenue, NW, Suite 640
Washington, DC 20005
Telephone: (202) 628-8303
Fax: (202)628-2846
E-mail: svia@awwa.org
Tim Wade
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects
Research Laboratory
Human Studies Division
Epidemiology and Biomarkers Branch (MS-58C)
Research Triangle Park, NC 27711
Telephone: (919) 966-8900
Fax: (919)966-0655
E-mail: wade.tim@epa.gov
Barb Walton
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects
Research Laboratory (MS-B305-02)
Research Triangle Park, NC 27711
Telephone: (919) 541-7776
E-mail: walton.barb@epa.gov
Dan Williams
U.S. Environmental Protection Agency
Office of Research and Development
National Water Supply and Water Resources
Division
Treatment Technology Evaluation Branch
(MS-B24)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7237
Fax: (513)509-7172
E-mail: williams.daniel@epa.gov
Margaret Williams
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7243
Fax: (513)569-7328
E-mail: williams.margaret@epa.gov
Karen White
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7248
Fax: (513)569-7328
E-mail: white.karenm@epa.gov
Karen Wirth
U.S. Environmental Protection Agency
Office of Ground Water and Drinking
Water
Ariel Rios Building (4607M)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 564-5246
Fax: (202)564-3760
E-mail: wirth.karen@epa.gov
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
Michael Wright
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
Assessment (MS-190)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7922
Fax: (513)569-7916
E-mail: wright.michael@epa.gov
Marylynn Yates
University of California, Riverside
Associate Executive Vice Chancellor
4108HinderakerHall
Riverside, CA 92521
Telephone: (909) 787-2358
Fax: (909)787-4362
E-mail: marylynn.yates@ucr.edu
Remote Participants
Kerri Alderisio
New York City Department of Environmental
Protection
465 Columbus Avenue
Valhalla, NY 10595
Telephone: (914) 773-4423
E-mail:
kalderisio@dep.nyc.gov@westchestergov.com
Paul Berger
U.S. Environmental Protection Agency
Office of Water
Office of Ground Water and Drinking Water
Ariel Rios Building (4607M)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 564-5253
E-mail: berger.paul@epa.gov
Abel Euresti
U.S. Environmental Protection Agency
Region 6 Houston Laboratory
Environmental Services Branch
10625 Fallstone Road
Houston, TX 77099
Telephone: (281)983-2162
Fax: (281)983-2248
E-mail: euresti.abel@epa.gov
JeffGratz
U.S. Environmental Protection Agency
Region 2
290 Broadway, 28th Floor
New York, NY 10007-1866
Telephone: (212) 637-3554
E-mail: gratz.jeff@epa.gov
Ariel Iglesias
U.S.Environmental Protection Agency
Region 2
2890 Woodbridge Avenue (MS-215)
Edison, NJ 08837
Telephone: (732) 452-6426
E-mail: iglesias.ariel@epa.gov
Paul Kutzy
Westchester County Department of Health
145 Huguenot Street
New Rochelle, NY 10801
Telephone: (914)813-5156
E-mail: pjk3@westchestergov.com
Michael Lowy
U.S. Environmental Protection Agency
Region 2
290 Broadway, 24th Floor
New York, NY 10007-1866
Telephone: (212) 637-3830
E-mail: lowy.michael@epa.gov
Cindy Mack
U.S. Environmental Protection Agency
Office of Water
Office of Wastewater Management
Ariel Rios Building (4707M)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 564-6280
E-mail: mack.cindy-y@epa.gov
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
Bruce Macler
U.S. Environmental Protection Agency
Region 9
75 Hawthorne Street (WTR-6)
San Francisco, CA 94105
Telephone: (415) 972-3569
Fax: (415)947-3549
E-mail: macler.bruce@epa.gov
Cristina Maldonado
U.S. Environmental Protection Agency
Region 2, CEPD
CEPD Centre Europa 417
San Juan, PR 00907
Telephone: (787) 977-5827
Fax: (787)289-7102
E-mail: maldonado.cristina@epa.gov
Jorge Martinez
U.S. Environmental Protection Agency
Region 2, CEPD
Centro Europa 417
San Juan, PR 00907
Telephone: (787) 977-5827
Fax: (787)289-7102
E-mail: martinez.jorge@epa.gov
Yves Mikol
New York City Department of Environmental
Protection
465 Columbus Avenue
Valhalla, NY 10595
Telephone: (914) 773-4426
E-mail: ymikol@dep.nyc.gov
Renee Morris
U.S. Environmental Protection Agency
Office of Water
Office of Wastewater Management
Ariel Rios Building (4707M)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 564-8037
E-mail: morris.renee@epa.gov
Robert Poon
U.S. Environmental Protection Agency
Region 2
290 Broadway, 24th Floor
New York, NY 10007-1866
Telephone: (212) 637-3821
E-mail: poon.robert@epa.gov
Rebecca Quinones
U.S. Environmental Protection Agency
Region 6
Sample Management
10625 Fallstone Road
Houston, TX 77099
Telephone: (281)983-2168
Fax: (281)983-2248
E-mail: quinones.rebecca@epa.gov
Anne Seeley
New York City Department of Environmental
Protection
59-17 Junction Boulevard, 20th Floor
Flushing, NY 10595
Telephone: (718)595-5346
E-mail: aseeley@dep.nyc.gov
Erin Shutak
New York City Department of Health
2 Lafayette, 11th Floor CN 56
New York, NY 10007
Telephone: (212) 676-1542
E-mail: eshutak@health.nyc.gov
Sandra Spence
U.S. Environmental Protection Agency
Region 8 Laboratory
16194 W 45th Drive
Golden, CO 80403
Telephone: (303)312-7754
Fax: (303)312-7800
E-mail: spence.sandra@epa.gov
Alysia Tani
U.S. Environmental Protection Agency
Region 8 Laboratory
16194 W 45th Drive
Golden, CO 80403
Telephone: (303)312-7809
Fax: (303)312-7800
E-mail: tani.alysia@epa.gov
Paul Zambratto
U.S. Environmental Protection Agency
Region 2
290 Broadway, 28th Floor
New York, NY 10007-1866
Telephone: (212) 637-4012
E-mail: zambratto.paul@epa.gov
The Office of Research and Development's National Center for Environmental Research
-------
Appendix 1: Presentations of Regional Research
Needs and Office of Water Regulatory Activities
and Research Needs
-------
Regional Concerns foi
Microoganisms in Drinking Water
Bruce Macler
Drinking Water Office
Region 9
415 972-3569
Regions Mostly Need Help
With Applications
The here and now of regional operations
offices day-to-day activities require
• Information to prioritize work
• Tools to make decisions
• Tools for regulatory monitoring and compliance
Some of these are being worked on, some are
not
-------
Information to Prioritize Work
Regions and State counterparts are swamped
• Have to aggressively prioritize
• Ask: What is the magnitude of the problem to be
solved? (that is, beyond the political...)
For microbial pathogens, mostly epidemiology
• What is the extent of microbial disease from
drinking water?
• Do we have a microbial problem on beaches?
Microbial Epidemiology
Questions for Drinking Water
Do we have a national public health problem
from undisinfected wells?
• >50% of public wells not disinfected
• But, is there a problem?
Is there remaining microbial disease from
treated water?
Answers useful for reg implementation
-------
Microbial Epidemiology
Questions for Beaches
What is the extent of waterborne illness from
bathing exposures?
• Is freshwater exposure different from marine?
• Can we confirm the significance of bather-to-
bather contamination?
• Are some exposure situations more problematic?
Are the current beach criteria accurate?
Microbial Tools to Make
Decisions
Drinking water decisions primarily involve
determining "fecal contamination"
Beach decisions need information on sources
of fecal contamination
• Is it pathogenic to humans?
• Or, is it "false positive" and non-pathogenic?
-------
Drinking Water Tools
Need confidence in vigor of current fecal
indicators
• Do they adequately represent range of
pathogens? (apparently, no)
• Need an approach that is more definitive
Need suitable surrogates to determine
adequacy of disinfection treatment
• Chlorine, UV, ozone, etc
Beach Microbial Needs
Biggest problem is to determine when a
bathing beach may be contaminated with
microbials pathogenic to humans
Current fecal indicators can be positive for
apparently non-pathogenic situations
• Birds versus people
Indicators may not match risk
-------
Monitoring and Compliance
Needs
Cheaper
Easier
More definitive
For drinking water, Cryptosporidium parvum
For bathing beaches, approach for human
pathogens.
-------
SDWA Requirements &
Microbial Research Needs
(Surface Water, Ground Water,
& Distribution Systems)
Stig Regli
OGWDW/USEPA
8/5/2003
EPA Regulation Setting
Requirements Under SDWA
Must publish MCLGs for contaminants that
- may have adverse health effects
- occur in public water systems at frequencies &
levels of public health concern
- provide meaningful opportunity for health risk
reduction for persons served by PWS
MCLGs shall be set at levels at which no
known health effects occur and which
allows an adequate margin of safety
-------
EPA Requirements Under
SDWA (cont'd)
Must promulgate MCLs as close to the
MCLG as is "feasible"
- "feasible" means with use of "best available
technology" (taking costs into consideration)
Must promulgate treatment technique
requirement if not economically or
technically feasible to monitor
Must perform regulatory impact analysis
(RIA) for each regulation
SDWA - Risk Assessment
For each regulation specify to extent practicable:
- Estimates of public health effects for each population
- Expected, upper, and lower bound risk estimates for
each population
- Each significant uncertainty identified in risk
assessment
- Peer reviewed studies that support estimates above &
methodologies used to reconcile data inconsistencies
-------
SDWA - Health Risk Reduction
& Cost Analysis (HERCA)
1 Quantifiable & nonquanitifiable reduction of
health risk
- Above also for reductions in co-occurring contaminants
1 Quantifiable and nonquantifiable costs
• Incremental costs & benefits associated with each
alternative standard
• Effects of contaminant on general population and
groups within general population
- Children, pregnant women, elderly, individuals with
serious illness, or subpopulations at greater risk
• Any increase in health risk that may occur
SDWA - Feasible technologies
List feasible technologies, treatment
techniques, and other means for achieving
compliance
List any technology, treatment technique, or
other means affordable for small systems:
->3300 to 10,000
- >500 to 3300 people
- >25 to 500 people
-------
Research Inputs into DW
Regulations
Health Effects & Assessment
TIMCLGs
TIEpidemiology
Analytical Methods
71 Feasibility of monitoring
Drinking Water
Regulations
MCL/Treatment Technique
Occurrence/Exposure
Treatment Technologies
71 Effectiveness, costs
Long Term 2 Enhanced Surface
Water Treatment Rule (LT2)
SDWA requires EPA to promulgate LT2
with Stage 2 Disinfection Byproducts Rule
Goal: provide equivalent level of protection
for all systems using surface water
Covers: 5500 systems, 174 million people
Status: propose 2003, promulgate 2004
-------
LT2 Components
Systems monitor Cryptosporidium in source water
to determine if more treatment is needed
- Small systems monitor E.coli;if levels are low they can
avoid monitoring Cryptosporidium
Tool box of treatment options to achieve different
Cryptosporidium removal credits
Unfiltered systems must provide at least 2 log
inactivation of Cryptosporidium
Finished water reservoirs must be covered or
disinfected (4 log virus inactivation)
Source water treatment level reevaluated six years
after first round of monitoring
LT2 Research Issues
What treatment strategies are available for small
systems & how can they be evaluated?
What are appropriate indicators to determine
source water pathogen risk?
What are appropriate indicators for assessing
effectiveness of surface water treatment?
What proportion of the total waterborne pathogen
risk is linked to source/treatment issues?
Does control for Giardia & Cryptospridium
adequately control for other pathogens?
-------
Ground Water Rule (GWR)
SDWA requires EPA to promulgate GWR before
Stage 2 DBPR promulgation
GWR Goal: identify GW systems vulnerable to
fecal contamination & require remedial action for
such systems
Covers: 154,000 systems serving 118 million
people
Status: proposed FRN 5/10/00, promulgate 03/04
GWR Components
Periodic sanitary surveys
Hydrogeologic sensitivity assessments
Source water monitoring (E.coli, enterococci, or
coliphage) for systems if:
- Sensitive hydrogeologic setting
- Total coliform hit in distribution system
Corrective action if significant deficiency or fecal
indicator is positive
Compliance monitoring for disinfected systems
(show > 4 log virus inactivation)
-------
GWR Issues
What are the most appropriate indicator(s) for
vulnerability to fecal contamination?
What tools are available to make indicator
monitoring more cost effective (e.g, micro arrays,
molecular techniques)?
How does the sensitivity of naturally occurring vs.
lab-adapted viruses to different disinfectants
compare?
What proportion of the total waterborne pathogen
risk is linked to source/treatment related GW
issues?
Revised TCR & Distribution
System (DS) Requirements
SDWA requires 6 year review of all rules
including TCR
TCR revision to be coupled with
development of DS requirements
DS Goal: protect public health from
distribution system contamination
Status: now in problem definition phase;
proposed FRN anticipated 2006
7
-------
DS Issues
How do we assess public health risk associated
with: cross connections, intrusion, contamination
following repair or replacement, biofilms,
nitrification, uncovered storage & water age?
Which DS deficiencies pose greatest risk to public
health?
What are appropriate indicators of DS
deficiencies?
How effective is current technology for reducing
the most important potential DS health risks?
What proportion of the total waterborne pathogen
risk is linked to distribution system issues?
TCR Issues
What are the most appropriate monitoring
strategies for routine monitoring? After a TC-
positive?
- Location, frequency, sample volume
What control and prevention strategies are
effective?
What are the most appropriate approaches for
indicating distribution system risk?
- Microbes: TC, E.coli, etc.
- Non-microbial: hydraulics, disinfectant residual, etc.?
-------
National Estimate
SDWA requires EPA & CDC to develop
national estimate of waterborne disease
Status: Approaches for generating estimate
are in development (OGWDW, ORD, CDC)
- FRN indicating estimates & uncertainties using
different approaches to be published 2004
- Much more data will be needed to address
uncertainty of estimates (beyond 2004)
National Estimate - Issues
What percent of national incidence of GI illness is
associated with drinking water?
- Which estimation methodologies are most reliable?
What percent of waterborne illness is associated
with source/treatment issues versus DS issues?
Can ongoing estimates of national drinking
waterborne disease be used as a benchmark for
evaluating benefits of drinking water regulations?
-------
The Contaminant Candidate
List: Determining the Need
for Future Drinking Water
Standards
US Environmental Protection Agency
Research on Microorganisms in
Drinking Water Workshop
August 5-7, 2003
Tom Carpenter
CCL Team Lead
Office of Ground Water and
Drinking Water
Overview of Presentation
Statutory Requirements for the CCL
NRC recommendations for future CCLs
NDWAC Schedule
Overview of the Methods and Issues
Efforts Underway
-------
SDWA CCL Schedule
Research & Occurrence (UCMR) Data
Collection for CCL2 contaminants
CCL1
Feb
1998
Reg Determinations
2003
(Actual)
CCL3
Feb 08
t
02
04
06
Reg Determinations
Aug 2001 CCL2
Feb 03
08
Reg
Determ'
CCL2
8/06
10
Reg
Determ'
CCL3
8/11
Research & Occurrence
DataCollection for CCL3
Future CCL Development
Same SDWA requirements as CCL1 and similar
components needed to develop future CCLs
- Develop list
- Initiate the regulatory determination process
National Academy of Sciences Panel Reports
- Last report of trilogy recommends strategies for future CCLs
Evaluate NRC Report "Classifying Drinking Water
Contaminants for Regulatory Consideration"
Provides Recommendations
- Extensive process for identifying and narrowing contaminant
universe
- Recommendations for data quantity and quality
- Significant quantitative aspect
- Validation of recommended approach using case example
-------
NRC Recommendations for the CCL
"Universe"
(-100,000)
The universe of potential
drinking water
contaminants includes:
Naturally occurring
substances
Emerging waterborne
pathogens
Food-borne pathogens
Animal pathogens
Chemical agents
Byproducts and
degradates of chemical
agents
Radionuclides
Biological toxins
STEP ONE
Screening Criteria and
Expert Judgement
PCCL
(>1,000)
STEP TWO
Classification Tool and
Expert Judgement
PCCL Includes:
Contaminants that
occur, or have the
potential to occur in
drinking water AND
cause, or may cause,
adverse health effects
NRC Recommendations (cont'd)
Strongly recommends a classification approach that should
not sacrifice complexity for transparency
- allows for complex decision process that scores and weights classification attributes
of contaminants based on pattern recognition
- calibrate and validate using existing contaminants as training sets
Evaluate new molecular/genetic methods to identify
new/emerging microbiological contaminants as part of new
approach
- base evaluation of microbes on similarities of virulence, physical, and/or genetic
attributes (Virulence Factor Activity Relationships)
- relies on new genomic and molecular analytical methods and indicators
- WAR is long-term goal — need to identify interim products as proof of concept
-------
Methodology Issues NDWAC is discussing
Prototype Classification Approach
- How did NRC arrive at this approach
. evaluation of several prioritization approaches
. common characteristics were selection of contaminant pool,
determination of exposure and toxicity, what was the prioritization
method
. most examples used for chemicals not/pathogen
- NRC Panel started at the beginning
. had limited resources
. wrestled with how the attributes of exposure and toxicity inter-relate
to one another
. NRC recommendations are not a complete road map, there is more
work to do
Methodology issues discussions (cont'd)
The universe of potential contaminants
Occur in Drinking water
Adverse
Health Effects
Potential
Adverse
Health Effects
Potential to
Occur in
Drinking water
-------
Methodology issues discussions (cont'd)
. The universe to the PCCL
- Intersection of the major characteristics identify
contaminants to carry to the PCCL
- Seek expert judgement on the screening process
- Screening criteria need to be connected to the data
sources
- Identify data elements to capture and compile the
universe from these evaluations
- Develop screening criteria
. NDWAC discussing guidelines and concept
. generate from the data sources to identify potential and
known criteria
. apply "automated" screening
Virulence Factor Activity Relationships
Indicators
Genetic Elements
Surface Proteins
Attachment Factors
Metabolic Pathways
Other Virulence Attributes
Classification
Outcomes
Virulence
Potency
» Classifies pathogens
» Will not be fully developed for CCL2
• Relies on molecular technologies and gene sequencing
• Research and analytical capabilities are improving
• EPA research efforts are underway for microbes (i.e., Aeromonas)
» NRC strongly recommends interagency participation (e.g. participation
in National Science and Technology Council's Biotechnology Research Group)
» Next steps are to identify and coordinate range of research needs
-------
Methodology issues discussions (cont'd)
VFARs: A new process for pathogens
Genomics and proteomics are rapidly emerging
technologies and should provide new indicators
Identify pilot and prototype projects
. Literature reviews, State of the science, available data
sources
. Develop model systems to test "virulence" of the potential
pathogens
. Develop interagency partnerships
. Identify/develop/modify analytical methods for VFAR indicators
NDWAC Charge
Discuss, evaluate, and provide advice on
methodologies, activities, and analysis needed to
implement the NRCs recommendations on an
expanded approach for the CCL listing process. This
may include advice on:
- an overall implementation strategy
- classification attributes and criteria
- pilot projects to validate new classification approaches
- proof of concept activities to support VFAR analysis
- communication issues
- additional issues not addressed in the NRC report
-------
CCL NDWAC Work Group
Request NDWAC advice to assist EPA in developing
methodologies that can be used for future CCLs
NDWAC Work Group with 3 Activity Groups
- CCL may need parallel paths for pathogens and chemical
contaminants
- One microbiological/pathogen and one chemical subworkgroup
- Both technical activity group should include classification/information
technology expertise
Convened Work Group September 2002
6 plenary meetings to date
2 remaining meetings through the Fall of 2003
Novel approach: comparative genetics
Identify virulence genes from genomes by location
•Many virulence-associated genes cluster together (pathogenicity islands)
•Genes flanking virulence genes may be co-expressed and have related
functions www.tigr.org select genome browser
Identify virulence genes from genomes by expression
•Transcriptosomes (grouping of genes according to their transcription
regulation patterns) may identify virulence-associated genes
Identify virulence genes from genomes by primary sequence
•GC-content, flanking IS sequences or repeats, can identify recently acquired
genes. Surface-exposed genes are generally more AT-rich which makes
them prone to mutation www.cbs.dtu.dk/services/GenomeAtlas
Identify virulence genes by multiple sequences
•substitution rates (Ka/Ks) identify genes under strong selection
•polymorphisms within (surface expressed) genes indicate avoidance of
immune response
7
-------
Microarrays: promises, outcome
"Applications of functional genomics of food microorganisms:
novel risk assessment procedures" (Curr. Opin.Biotech. 1999,10:511)
Applications of MA to food pathogens (published as of May 2002)
Detection/
differentiation
Diversity/
conservation
Gene
regulation
•detection of bacterial virulence genes by microarray performs
better than PCR
•direct detection of £. co//on chicken carcasses
•differentiate Staph. spp. by low-density microarray of PCR products
•identification of diversity in gene content of C.jejuni
•identification of homologs conserved within pathogenic
Salmonellas
•gene regulation under Fe-limitation (in Pasteurella) proves complex
and pleiotrophic. How to differentiate primary from secondary,
down-stream effects?
•gene expression during acid-adaptation of E. coli was studied
•sarA and agrof S. aureus regulate known virulence genes, also
many others
Genomic Data Searches
VFAR Discovery Phases
Phase I September-October, 2002
41 Limited virulence factor keyword search of GenBank
41 Basic Local Alignment Search Tool (BLAST) alignments
Phase II October-November, 2002
41 Comprehensive keyword search of GenBank
4> Comparison of other available genomic databases
Phase III November-December 2002
4> Keyword search of whole genomes
4> Selective virulence factor sequence alignments against other
whole genomes
vp BLAST alignment of complete virus genomes
-------
Bioinformatics Pilot
Assessement of VFAR
Literature about E. coli
Select representative
genes
Select probes]
Alignment
[Primer design]
Check for sequence
similarity
Select representative
probes
Download sequences
Investigators:
Syed Hashham,
James Cloe
Joan Rose
Gene Combinations Associated with
Water-borne Pathogen Virulence
waterborn
properties
waterborn,
pathogenic
waterborn,
non-pathogenic
virulence
properties
Investigators
T Wassenaar
J. Gamieldien
Subtract: all genes from non-pathogenic, non-
i organisms (minimal gene set)
-------
For Additional Information or Questions
Tom Carpenter
202-564-4885
202-564-3760 (fax)
carpenter.thomas@epa.gov
Jitendra Saxena
202-564-5241
202-564-3760 (fax)
Saxena.Jitendra@epa.gov
10
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Appendix 2: Additional NCER STAR Drinking
Water Grant Microbial Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
Experimental Infection of Healthy Adults with a Cryptosporidium
Genotype 1 Isolate (TU502)
Cynthia Chappell1, P. Okhuysen2, R. Lunger1, D. Akiyoshi3, and S. Tzipori3
Center for Infectious Diseases, The University of Texas Health Science Center at Houston, School
of Public Health, Houston, TX; 2Department of Internal Medicine, The University of Texas Health
Science Center at Houston Medical School, Houston, TX; 3Division of Infectious Diseases,
School of Veterinary Medicine, Tufts University, North Grofton, MA
Cryptosporidium parvum causes diarrheal illness worldwide. Molecular studies have identified two dis-
tinct genotypes with different transmission cycles. Genotype 1 (Gl) strains are primarily a human-to-human
transmission, and genotype 2 (G2) strains are zoonotic. Previous dose-response studies in healthy adults em-
ployed five genotype 2 isolates, which varied widely in infectivity, yielding ID50's between 9 and 1042 oo-
cysts. This study is the first report of experimental Gl infections in healthy adults. The Gl isolate (TU502)
used in this study originated from a human case and was amplified in gnotobiotic piglets. A single dose (10,
30, 100, or 500 oocysts) of TU502 was administered to 16 volunteers, which were monitored for 6 weeks.
Results showed that the TU502 ID50 was similar to the most infectious of the G2 isolates. The onset of diarrhea
and oocyst shedding following TU502 challenge were similar to the G2 isolates; however, the duration of diar-
rhea and oocyst shedding showed important differences. The typical 4-7 days of diarrhea seen with G2 isolates
was prolonged in TU502 volunteers, lasting up to 22 days. Further, 83 percent of volunteers challenged with
the G2 isolates cleared their oocysts by <14 days as compared to 60 percent of volunteers receiving TU502.
Two subjects shed for 24 and 35 days, respectively. Total oocysts shed per person ranged from 5 X 106 to 1 X
1010, the latter occurring in the volunteer with the longest episode of diarrhea and oocyst shedding. These data
suggest that the Gl isolate, TU502, was highly infectious in healthy adults and was associated with a longer
diarrheal illness followed by an extended period of oocyst shedding. These characteristics suggest a high risk
of infection from environmental sources and a risk of secondary transmission from contact with symptomatic
and asymptomatic oocyst shedders. These findings are consistent with the high proportion of Gl isolates asso-
ciated with outbreaks of human cryptosporidiosis. This work was supported, in part, by EPA STAR Grant
#R-82918001 and NIH GCRC Grant #RR-02558.
The Office of Research and Development's National Center for Environmental Research
-------
Research on Microorganisms in Drinking Water Progress Review Workshop
Experimental Challenge of Healthy Adult Volunteers
With Cryptosporidium muris Oocysts
Cynthia Chappell, P. Okhuysen , R. Lunger , and S. Tzipori
1 Center for Infectious Diseases, The University of Texas Health Science Center at Houston, School
of Public Health, Houston, TX; 2Department of Internal Medicine, The University of Texas Health
Science Center at Houston Medical School, Houston, TX; 3Division of Infectious Diseases,
School of Veterinary Medicine, Tufts University, North Grofton, MA
Cryptosporidium muris has long thought to be a pathogen of animals, but not humans. However, a recent
study indicated that infections with C. muris might occasionally occur in immunocompromised persons. In
addition, probable C. muris cases were reported in two children but were not confirmed. Detection of cases is
complicated by the fact that monoclonal antibodies commonly used in Cryptosporidium assays do not recog-
nize C. muris and might yield false negative results. The purpose of this study was to determine if C. muris
oocysts are infectious for healthy adults. Six serologically negative, healthy volunteers were challenged with
105 C. muris oocysts and monitored for infection and illness for a minimum of 6 weeks. All six volunteers be-
came infected, but only one developed a diarrheal illness, which lasted for 4 days. In contrast, previous studies
have shown that C. parvum isolates were associated with illness attack rates of 52-86 percent. Interestingly, the
duration of C. muris oocyst shedding was longer (40-45 days or more) than with C. parvum (3-12 days, de-
pending on the isolate tested). In some volunteers, the study period was extended due to continued oocyst
shedding. These data indicate that healthy adults are susceptible to infection with C. muris oocysts and in some
cases (17 percent) might experience a diarrheal illness. Further, the longer period of oocyst shedding might be
important for secondary transmission. These findings support the need for dose-response studies to more fully
describe the risk of C muris infectivity and illness in the community setting.
The Office of Research and Development's National Center for Environmental Research
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Appendix 3: STAR Grant Presentation Abstracts
and Agenda From the USEPA/USGS Meeting
on Cryptosporidium Removal by Bank
Filtration, September 9-10, 2003
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William Blanford
(Abstract Not Provided)
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Research on Microorganisms in Drinking Water Progress Review Workshop
Study of Particle and Pathogen Removal During Bank Filtration
of River Waters
Edward J. Bouwer1, Charles R. O'Melia1, W. Joshua Weiss1, Kellogg J. Schwab2, Binh T. Le2,
and Ramon Aboytes3
1 Johns Hopkins University, Baltimore, MD; 2Johns Hopkins University Bloomberg School
of Public Health, Baltimore, MD;3American Water Belleville Laboratory, Belleville, IL
Project Goals and Objectives
The overall objective of this research project is to evaluate the merits of riverbank filtration (RBF) for re-
moving/controlling pathogens in drinking water and to investigate the potential for using removal of particles
and other water quality indicators as surrogates for pathogen removal in riverbank systems.
Approach
This research consists of: (1) field studies to document actual changes in pathogen and particle concentra-
tions from rivers of similar source quality in the context of variations in subsurface travel distances, pumping
rates, porous medium properties, and residence times; and (2) parallel laboratory column studies with aquifer
media to provide insights into process mechanisms and the relationship between pathogens and potential sur-
rogate parameters upon transport through riverbank media under a variety of physical and chemical conditions.
Preliminary Findings
Field monitoring results from the three study sites indicate that RBF serves as a consistently significant
barrier to the transport of microorganisms from the river water sources (see Table 1). Cryptosporidium oocysts
were detected in 11 out of 16 sampling rounds in the Ohio River, 12 out of 16 sampling rounds in the Wabash
River, and 8 out of 16 sampling rounds in the Missouri River, but never in any of the corresponding well wa-
ters. Similarly, Giardia cysts were detected in 4 of the sampling rounds in the Ohio River, 2 sampling rounds
in the Wabash River, and 3 sampling rounds in the Missouri River, but never in any of the corresponding well
waters. With the exception of Bacillus and bacteriophage 4>X174, all of the monitored organisms were always
below the detection limit in the well waters, with corresponding reductions of average concentrations ranging
from 0.8 logs to more than 6 logs.
Preliminary design of column experiments has been accomplished. Riverbed sediment was collected from
the Potomac River in western Maryland to serve as representative riverbank media for the column studies. The
sediment was dried and sieved to remove fine and coarse material but otherwise was not manipulated or
cleaned prior to packing into glass columns. Preliminary experiments used 2.5 cm diameter, 30 cm long col-
umns. Breakthrough curves for bacteriophage MS2, bacteriophage PRD1, and Polio virus indicate that under
several ionic strength conditions, polio virus is removed by a substantially greater extent than MS2, suggesting
that the phage might be useful as a conservative indicator for the transport of the human virus through river-
bank media.
Significance of Findings
The field monitoring data support the use of RBF at controlling the transport of potentially harmful micro-
organisms from river water sources. Reductions in average bacteria concentrations are often well in excess of 2
logs. This is significant because bacteria have been proposed as potential surrogate parameters for the protozo-
ans (which are more difficult to measure accurately in the field due to their low and variable concentrations in
natural systems).
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Research on Microorganisms in Drinking Water Progress Review Workshop
Next Steps
Future column studies are intended to examine the relationship between the protozoans and the other mi-
crobes in aquifer media, as well as other potential surrogate/indicator parameters (including latex micro-
spheres, natural river water particles, and turbidity measurements).
Table 1. Field monitoring results: January 2002 through July 2003 (log removals given in brackets). Aver-
ages were calculated as the sum of counts divided by the sum of volumes sampled over all sam-
pling rounds; detection limit was calculated as 1 divided by the sum of the volumes.
Ohio
River
Well #9
Well #2
8.7 xlO4
1.7 xlO2
[2.7]
8.0 xlO2
[2.0]
7.6 xlO2
<2.0
1.3 x 10b
<5.0
<5.0
1.5xl04
<1.0xl0
<1.0xl0
4.6x10'
<2.0
1.7x10
1.1
[3.2]
<2.0
3
2.7 x 10"2 7.3 x 10"2
<1.2 x 10"3 <1.2 x 10"3
<2.4 x 10"3 <2.4 x 10"3
Wabash 2.8 xlO5 2.2 x 103 6.1 x 106 5.7 x 105 3.6 xlO1 2.4 x 103 1.7 xlO"2 1.0 x 10"1
River
Collec- 3.5xl03 <1.1 <5.0 <5.0 <1.1 <1.1
torWell [1.9] [>3.3] [>6.1] [>5.1] [>1.5] [>3.3] [>1.1] [>1.9]
Well #3 <2.0xl02 <2.0 <5.0 <5.0 <2.0 <2.0 <2.5 x 10'3 <2.5 x 10'3
<1.3 x 10"3 <1.3 x 10"3
Missouri
River
Well #4
Well #5
4.2 xlO5
6.3 x 104
[0.8]
l.lxlO3
[2.6]
8.9 xlO2
<1.1
[2.9]
<2.5
[>2.6]
6.1 xlO5
<1.0xl01
<1.0xl01
[>4.8]
3.1xl04 3.4 xlO1
<1.0xl01 <1.1
<1.0xl01 <2.5
[>3.5] [>1.1]
2.6 x 103 2.4 x 1Q-2 6.3 x 10'2
<1.1 3.0] [>1.0] [>1.4]
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Research on Microorganisms in Drinking Water Progress Review Workshop
Evaluating Microbial Indicators and Health Risks
Associated With Bank Filtration
Floyd J. Frost
Lovelace Clinic Foundation, Albuquerque, NM
The purpose of the proposed project is to compare serological responses to Cryptosporidium antigens in
users of bank-filtered water (one community with only bank filtration and disinfection and one community
with bank filtration, conventional filtration, and disinfection) with the responses of similar people residing in
an area that uses disinfected but unfiltered high-quality groundwater. The hypothesis is that, if bank filtration
completely removes Cryptosporidium oocysts, the serological responses of the three populations should be
similar. The specific goals of the study are to: (1) identify approaches to collecting sera from similar popula-
tions in different geographic locations so that rates of serological responses can be compared; (2) pilot test the
approach in three different geographical locations by collecting sera from cities that use bank filtration and
nearby cities that use high-quality groundwater for a drinking water source; (3) analyze the sera for serological
responses to Cryptosporidium and Giardia antigens and compare the frequency and intensity of responses be-
tween the bank filtration cities and the groundwater cities; and (4) compare serological responses in the same
cities at times when bank filtration efficacy is likely to be optimal and when it is likely to be least effective.
Sera from 50 people from each of three communities (users of bank filtered and chlorinated, bank filtered
plus direct filtration plus ozonation, and chlorinated groundwater) will be collected at baseline and at five fol-
lowup blood draws. A questionnaire on risk factors will be collected at each blood draw. Sera will be tested for
the presence of antibody responses to two Cryptosporidium antigens (15/17-kDa and 27-kDa) and for serologi-
cal changes (seroconversion). The baseline level of serological responses as well as the rates of seroconversion
will be compared for each population (50 baseline and 250 periods for estimating rates of seroconversion).
Comparisons will adjust for collected risk factor data from each individual. For purposes of extrapolating these
results to other locations, a series of source and finished water quality indicators will be measured for each
water source.
No results are available at this time. Analysis of sera will take place once all sera are collected. Then, all
sera from a subject will be run on the same Western blot to reduce variations between blots. Blood draws will
continue every 4 months. Data entry protocols will be developed and implemented for data entry of the ques-
tionnaires. Sample analysis will commence once all of the samples are collected, because the analysis of each
subject's samples will be on the same Western blot. The distribution systems analysis also will commence.
This abstract also was presented as a poster at the Research on Microorganisms in Drinking Water Workshop
in Cincinnati, OH, August 5-7, 2003.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Application of a Multipath Microsphere Tracer Test To Understanding
Transport of Bacteria and Protozoa at a Bank Filtration Site
Rick Langford1, Dirk Schulze-Makuch1, and Suresh Pillai2
University of Texas, El Paso, TX; 2Texas A&M University, Kingsville, TX
The objective of this research study is to determine whether bank filtration is effective in removing micro-
bial pathogens in an arid environment. The study site uses the Rio Grande that experiences significant annual
fluctuations in both water quantity and quality. A well-characterized site with numerous monitoring wells has
been established. The pumping well is 17 m from the stream bank. The water table during the experiment was
2 m below the land and the stream surface. The aquifer is composed of medium and fine-grained sand compris-
ing two flow units. Observation wells are screened over 1 or 1.5 m intervals. The average hydraulic conductiv-
ity was about 2x10" m/s based on a test analysis. However, the responses indicated that sediment heteroge-
neities affected the hydraulic behavior at the field site.
A 427-hour tracer test using bromide and fluorescent microspheres provides initial results that are relevant
to the transport of pathogens through the subsurface under riverbank filtration conditions. Bromide was in-
jected into an observation well at the channel margin. Differently colored fluorescent microspheres (0.25 jam,
1 jam, 6 |_un, and 10 jam) were injected into the stream bottom and into two observation wells. Conclusions
from the tracer test include the following:
• Both bromide and microspheres continued to be observed throughout the 18 days of the experiment.
• The bromide recovery in the pumping well and in the deeper observation wells showed early and late
peaks with long tails indicating that the geological medium at the field site behaves like a double-porosity
medium, allowing the tracer to move relatively quickly through the higher conductivity units while being
significantly retarded in the low hydraulic conductivity units.
• Some wells showed consistently higher concentrations of bromide.
• The 1 |_m micospheres were abundant in the observation wells and allowed tracing of flowpaths. These
showed multiple peaks similar to the bromide results. This indicates highly preferential transport paths in
the sediment.
• Microspheres from the three injection sites had distinctly different transport paths and rates.
• Both bromide and microspheres appeared in the stream soon after injection, moving apparently against a
2 m head difference.
• The 6 |_m and 10 |_m microspheres were observed in low concentrations and were episodically detected in
the stream and in two widely spaced observation wells.
The significance of these results is that:
• Inorganic microspheres might mimic the episodic occurrence of microorganisms in wells.
• Even in this relatively homogeneous aquifer, preferential transport within the aquifer results in highly di-
vergent transport paths and rates. Microspheres from one of the injection sites traveled essentially perpen-
dicular to the expected transport direction.
• Even small variations in the sand grain size can effectively compartmentalize the aquifer.
The next steps of this project will include field studies to observe the migration and persistence of selected
organisms (Escherichia coli, enterococci, coliphages, cysts, oocysts, and enteroviruses) in the pumping well
and observation wells under different pumping rates. Continued combined chemical sampling, along with the
microbial sampling, will document whether the changes in water chemistry alter the behavior of the organisms.
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Research on Microorganisms in Drinking Water Progress Review Workshop
Pathogenic Microbe Removal During Riverbank Filtration
Joseph N. Ryan1, Yumiko Abe1, Rula Abu-Dalo1, Menachem Elimelech2, Garrett Miller2,
Zachary Kuznar2, Ronald W. Harvey3 and David W. Metge3
University of Colorado at Boulder, Boulder, CO; 2Yale University, New Haven, CT;
U.S. Geological Survey, Reston, VA
Project Goals and Objectives
Our incomplete understanding of processes and properties affecting the transport of pathogenic microbe
transport during riverbank filtration is currently limiting our ability to predict the effectiveness of this water
treatment option. We are conducting a series of fundamental experiments designed to better understand the
effects of microbe size, physical and geochemical heterogeneity of the porous media, and high pumping rates
on the transport of Cryptosporidium parvum oocysts in alluvial valley aquifers used for riverbank filtration.
Our major objective for this research is to develop a model of oocyst transport in porous media that can
accommodate the physical and geochemical heterogeneity present in alluvial valley aquifer used for riverbank
filtration. Our goal is that this model can be used to predict the oocyst removal during riverbank filtration. To
do this, we are in the process of providing: (1) improved characterization of the properties of C. parvum oo-
cysts related to transport in porous media; (2) improved understanding of the mechanisms of oocyst removal in
porous media; (3) including special features of riverbank filtration in alluvial valley aquifers; and (4) incor-
porating the improved characterization and mechanistic understanding into a two-dimensional model of mi-
crobe transport during riverbank filtration.
Approach
The experiments are being conducted in a stagnation point flow apparatus, flow-through columns, an inter-
mediate-scale two-dimensional aquifer tank (5 m length, 0.5 m height, 10 cm width). The stagnation point flow
experiments are being used to examine the effects of the surface charge heterogeneity and the DLVO secon-
dary minimum on oocyst attachment and release. The flow-through column experiments are being used to ex-
plore the effects of grain-scale heterogeneities on oocyst transport and to provide data for modeling of the
intermediate-scale tank experiments. The tank experiments are being used to examine random physical and
geochemical heterogeneities above the grain scale. The porous media being used in the column and tank ex-
periments are designed to simulate the complex and variable stratigraphies and geochemical gradients encoun-
tered in alluvial valley aquifers.
The following tasks are being conducted to achieve the project objectives and goals: (1) stagnation point
flow and column experiments to test the effect of microbe size on attachment to porous media, velocity en-
hancement, and straining; (2) column and tank experiments to test the effect of grain size on attachment to po-
rous media, velocity enhancement, and straining; (3) stagnation point flow, column, and tank experiments to
test the effect of geochemical heterogeneity on attachment to porous media; and (4) stagnation point flow and
column experiments to test the effect of flow rate on oocyst deposition in the DLVO secondary minimum and
release.
Preliminary Findings
We have conducted column experiments and a tank experiment examining the effects of physical and geo-
chemical heterogeneity on oocyst transport. The tank was filled with a heterogeneous porous medium consist-
ing of sands of 5 grain sizes and 12 ferric oxyhydroxide surface coverages. Oocyst transport in each of the
porous media was tested in column experiments. Oocysts (formalin-inactivated) and polystyrene latex micro-
spheres (4.6 |am diameter) were injected into the tank and monitored over 5 days. Physical heterogeneity (the
difference in hydraulic conductivity as a result of grain size) was much more important than geochemical het-
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Research on Microorganisms in Drinking Water Progress Review Workshop
erogeneity in controlling oocyst transport. Micro spheres broke through at the same time as the oocysts, but the
microspheres were removed much more rapidly than the oocysts.
In column experiments, we showed that straining is contributing to the removal of oocysts in fine-grained
sands. Straining was demonstrated by comparing the transport of polystyrene latex microspheres (0.32 to
4.1 |am) to oocyst transport. Removal was consistent for microspheres from 0.32 to 1.9 jam and increased sig-
nificantly for 4.1 |am microspheres (see Figure 1).
Stagnation point flow experiments have been conducted to explore the effect of ionic strength on oocyst
deposition. The dynamics of oocyst deposition in these experiments—the oocysts come into contact with the
glass deposition surface, but do not remain in the position of first contact—clearly indicate that the oocysts are
depositing in the secondary minimum of the DLVO potential energy profile.
Significance of Findings
The physical and geochemical heterogeneity experiments show that oocyst transport modeling can focus
on variations in the hydraulic conductivity of porous media. Geochemical heterogeneity is far less important
because oocysts deposit equally well on unfavorable and favorable surfaces. The straining and secondary mini-
mum experiments show that: (1) straining must be considered in oocyst transport modeling; (2) oocyst deposi-
tion depends on solution ionic strength; and (3) oocyst deposition is reversible.
Next Steps
The next steps in this research project will focus on column experiments to explore the effects of microbe
size, grain size, and flow rate on oocyst deposition, release, and velocity enhancement.
Figure 1.
0.00
1000 2000 3000
Time (seconds)
4000
Breakthrough of carboxyl-modified polystyrene latex microspheres through a column
filled with quartz sand (d50 of 0.210 mm) as a function of time. Microsphere concentration
normalized to the influent microsphere concentration (C/C0). Diameters of the micro-
spheres shown above the breakthrough curves. The experiments were conducted at a pH
of 5.6-5.8, an ionic strength of 1 mM, and a flow rate of 2 mL min"1.
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Research on Microorganisms in Drinking Water Progress Review Workshop
NCER Calendar of Events
September 2003
Title: The U.S. Environmental Protection Agency / U.S. Geological Survey Meeting on
Cryptosporidium Removal by Bank Filtration
Date: Tuesday, September 9 - Wednesday, September 10, 2003
Location: U.S. Geological Survey National Center
Main Auditorium
12210 Sunrise Valley Drive
Reston, VA 20192
See Logistics below for information regarding hotel reservations at the
Sheraton Reston Hotel, Reston, VA.
Please make your reservations by Wednesday, August 27, 2003.
Contact: Angela Page, (202) 564-5172 or page.angelad@epa.gov
Philip Berger (202) 564-5255 or berger.philip@epa.gov and
Tina Conley (202) 564-3209 or conley.tina@epa.gov
Purpose: The U.S. Environmental Protection Agency (EPA) Office of Research and
Development, National Center for Environmental Research (NCER) and the
EPA's Office of Water together with the U.S. Geological Survey (USGS) are
sponsoring a meeting to discuss the research being conducted on
cryptosporidium removal by bank filtration. The USEPA/USGS Meeting on
Cryptosporidium Removal by Bank Filtration will be held on September 9-10,
2003 at the USGS facility in Reston, VA. The meeting will consist of a series of
plenary sessions where EPA, NCER's Science To Achieve Results (STAR) grant-
ees, USGS, U.S. Department of Agriculture, university and state researchers will
present their research. This public meeting is open to all who are interested in
hearing about the research in this exciting area.
Please mark your calendars now and make your hotel reservations by August 27.
Registration: Fill out the Registration Form at:
http://www.scgcorp.eom/epachildhealth2003/registration.asphttp://www.scgcorp.
com/USEPA_USGS/registration.asp
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Research on Microorganisms in Drinking Water Progress Review Workshop
The U.S. Environmental Protection Agency/U.S. Geological Survey Meeting
on Cryptosporidium Removal by Bank Filtration
U.S. Geological Survey National Center
12201 Sunrise Valley Drive
Reston, VA 20192
September 9-10, 2003
AGENDA
Tuesday, September 9,2003
10:00 - 10:15 a.m. Welcome
James LaBaugh, USGS Office of Ground Water
10:15 - 10:30 a.m. Overview of the U.S. EPA's Office of Research and Development and The Science
To Achieve Results (STAR) Program
Cynthia Nolt-Helms, EPA, Office of Research and Development
10:30 — 10:45 a.m. Overview Presentation From the U.S. EPA's Office of Ground Water and Drinking
Water
Dan Schmelling, EPA, Office of Ground Water and Drinking Water
10:45 - 11:00 a.m. Overview of the U.S. EPA's Water Security Program
Regan Murray, EPA, Homeland Security Research Center
11:00 - 2:00 p.m. Field Studies of Cryptosporidium, Surrogate and Indicator Transport in
Saturated Porous Media
Moderator: Glenn Patterson, USGS, Reston, VA
11:00 - 11:25 a.m. Initial Results From the Rio Grande Bank Filtration Site
Presented by Rick Langford, University of Texas, El Paso, TX,
STAR Grant
11:25 - 11:50 a.m. Using Riverbank Filtration To Improve Water Quality
Presented by Ed Bouwer, The Johns Hopkins University,
Baltimore, MD, STAR Grant
11:50-12:50 p.m. Lunch
12:50 - 1:15 p.m. Bank Filtration Studies at the City of Lincoln, Nebraska
Wellfield
Research conducted by Jason Vogel, USGS; Presented by
Philip Berger, EPA, Washington, DC
1:15-1:40 p.m. Assessment of the Microbial Removal Capabilities of Riverbank
Filtration
Presented by Robin Collins, New England Water Treatment
Technology Assistance Center at the University of New
Hampshire, Durham, NH, Funded by EPA
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Research on Microorganisms in Drinking Water Progress Review Workshop
1:40-2:00 p.m. General Discussion
2:00-2:15 p.m. Break
2:15 - 3:45 p.m. Laboratory and Simulation Studies of Cryptosporidium, Surrogate and Indicator
Transport in Saturated Porous Media
Moderator: Ingrid Verstraeten, USGS, Baltimore, MD
2:15 - 2:40 p.m. Effect of Heterogeneity on Transport of Cryptosporidium
parvum in Saturated Porous Media
Presented by Joe Ryan, University of Colorado, Boulder, CO,
STAR Grant
2:40 - 3:05 p.m. Cryptosporidium Transport in Porous Media
Presented by Scott Bradford, USDA Salinity Laboratory,
Riverside, CA
3:05 — 3:30 p.m. Streamline-Based Simulation of Cryptosporidium Transport
in Riverbank Filtration
Presented by Reed Maxwell, Lawrence Livermore National
Laboratory, Livermore, CA
3:30-3:45 p.m. General Discussion
3:45-4:00 p.m. Break
4:00 - 5:00 p.m. Estimating Cryptosporidium Removal and Health Effects
Moderator: John Grace, Maryland Department of the Environment, Baltimore, MD
4:00 — 4:25 p.m. Cryptosporidium Removal at the Louisville, Kentucky Wellfield
Presented by Steve Hubbs, Louisville Water Company,
Louisville, KY
4:25 - 4:50 p.m. Serological Monitoring of Pathogen Occurrence
Presented by Floyd Frost, Lovelace Respiratory Research
Institute, Albuquerque, NM, STAR Grant
4:50-5:00 p.m. General Discussion
5:00 p.m. Adjournment
Wednesday, September 10,2003
9:00 - 11:45 a.m. Ground Water Flow, Heat Flow, and Environmental Tracer Studies at Bank
Filtration Sites
Moderator: Tom Grubbs, EPA, Washington, DC
9:00 - 9:25 a.m. Infiltration Rate Variability and Ground Water Flow at the
Cincinnati Wellfield
Presented by Bill Gollnitz, Greater Cincinnati Water Works,
Cincinnati, OH
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Research on Microorganisms in Drinking Water Progress Review Workshop
9:25 — 9:50 a.m. Application of Different Tracers To Evaluate the Flow Regime
at Riverbank Filtration Sites in Berlin Germany
Presented by Gudrun Massmann, Free University of Berlin,
Berlin, Germany
9:50 - 10:15 a.m. Diatom Proteins as a Surface Water Indicator in Ground Water
Presented by Tim Reilly, USGS, Trenton, NJ
10:15-10:30 a.m. Break
10:30 - 10:55 a.m. Inclined Well Studies at the Cincinnati Wellfield
Presented by Bruce Whitteberry, Greater Cincinnati Water
Works, Cincinnati, OH
10:55 - 11:20 a.m. Heat as a Tracer at Sonoma County Bank Filtration Site
Presented by Jim Constantz, USGS, Menlo Park, CA (via
PlaceWare)
11:20 - 11:45 a.m. General Discussion
11:45- 12:45 p.m. Lunch
12:45- 3:55 p.m.
Moderator: Mike Finn, EPA, Washington, DC
12:45 — 1:10 p.m. Cryptosporidium Transport in Soil-Aquifer Treatment
Presented by William Blanford, Louisiana State University,
Baton Rouge, LA, STAR Grant (to University of Arizona)
1:10 — 1:35 p.m. Cryptosporidium Transport in Unsaturated Flow
Presented by Christophe Darnault, Environmental Engineering
&
Technology, Inc., Newport News, VA
1:35 - 2:00 p.m. Comparison of Batch and Flow Experimental Data on Retention
of Manure-Borne Cryptosporidium parvum Oocysts in Soils
Presented by Yakov Pachepsky, USDA, Beltsville, MD
2:00-2:15 p.m. Break
2:15 - 2:40 p.m. Release of Cryptosporidium and Giardia Dairy Manure Due
to Flowing Water
Presented by Scott Bradford, USDA Salinity Laboratory,
Riverside, CA
2:40 - 3:05 p.m. Unsaturated Zone Processes in the Sonoma County Recharge
Basins
Presented by Jim Constantz, USGS, Menlo Park, CA (via
PlaceWare)
3:05 — 3:30 p.m. One-Dimensional Variably Saturated Microbial Transport
Simulations
Presented by Bart Faulkner, EPA, Ada, OK
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Research on Microorganisms in Drinking Water Progress Review Workshop
3:30-3:55 p.m. General Discussion
3:55-4:10 p.m. Break
4:10 - 4:50 p.m. Cryptosporidium Removal by Bank Filtration Summary, Regulations and Caveats
Moderator: Ronald Payer, U.S. Department of Agriculture, Beltsville, MD
4:10 - 4:35 p.m. Cryptosporidium Removal by Bank Filtration Summary,
Regulations, and Caveats
Presented by Philip Berger, EPA, Washington, DC
4:35 - 4:50 p.m. General Discussion and Closing Remarks
4:50 p.m. Adjournment
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