Validation of Rapid Methods for Enumeration of Markers for Human Sewage Contamination in Recreational Waters

202 pages

English

Découvre YouScribe en t'inscrivant gratuitement

Je m'inscris

Validation of Rapid Methods for Enumeration of Markers for Human Sewage Contamination in Recreational Waters , livre ebook

IWA Publishing - J. Harwood Valerie

Découvre YouScribe en t'inscrivant gratuitement

Je m'inscris

Obtenez un accès à la bibliothèque pour le consulter en ligne
En savoir plus

202 pages

English

Obtenez un accès à la bibliothèque pour le consulter en ligne
En savoir plus

A propos
Informations
Extrait

Description

Rapid methods of water quality assessment are essential for informing the public and officials of sewage-polluted water in a timely manner. Since fecal indicator bacteria can be contributed to the water by many sources, not all of which have equal risk for human health, rapid, quantitative methods that can discriminate human from non-human fecal contamination are informative for risk assessment and to identify problems in infrastructure or land use that contribute to degradation of water quality. This work compared standard, membrane filtration-based measures of the fecal indicator bacteria E. coli and enterococci to qPCR methods in terms of quantitative correlation and consistency of performance in various types of waters. Furthermore, the genes of two human-associated microorganisms, one from the bacterial group Bacteroidales and one from human polyomaviruses JCV and BKV, were used to quantitatively assess the presence of human sewage using qPCR in various types of subtropical waters in Florida, including fresh, brackish and salt waters. The qPCR methods performed well across the range of water types, although highly tannic water types (e.g. swamp-fed river) were more likely to be subject to inhibition than lake, estuarine or marine waters. Clean-up of inhibited samples by chemical methods produced highly variable results, therefore dilution of template material proved to be a better option for relieving inhibition. Concentrations of qPCR targets for which culture-dependent methods exist were generally in agreement when culture and qPCR methods were compared.Comparison of three different sample processing methods showed that the EPA CE and PE methods were superior to the USF methods for bacterial targets, but not for HPyVs. The EPA CE method had less sample-sample variability than the PE method, but also had a higher detection limit, making it potentially less sensitive than the PE method.

Sujets

Sciences et techniques

Informations

Publié par	IWA Publishing
Date de parution	29 juin 2011
Nombre de lectures	0
EAN13	9781780400020
Langue	English
Poids de l'ouvrage	3 Mo

Informations légales : prix de location à la page 0,1680€. Cette information est donnée uniquement à titre indicatif conformément à la législation en vigueur.

Extrait

PATH3C09 VALIDATION OFRAPIDMETHODSFORENUMERATION OFMARKERS FOR HUMANSEWAGECONTAMINATION IN RECREATIONALWATERSby: Valerie J. Harwood, Ph.D. Katrina V. Gordon, Ph.D. Christopher Staley, B.S. University of South Florida 2011

The Water Environment Research Foundation, a not-for-profit organization, funds and manages water quality research for its subscribers through a diverse public-private partnership between municipal utilities, corporations, academia, industry, and the federal government. WERF subscribers include municipal and regional water and wastewater utilities, industrial corporations, environmental engineering firms, and others that share a commitment to cost-effective water quality solutions. WERF is dedicated to advancing science and technology addressing water quality issues as they impact water resources, the atmosphere, the lands, and quality of life. For more information, contact: Water Environment Research Foundation 635 Slaters Lane, Suite G-110 Alexandria, VA 22314-1177 Tel: (571) 384-2100 Fax: (703) 299-0742 www.werf.org werf@werf.org This report was co-published by the following organization. IWA Publishing Alliance House, 12 Caxton Street London SW1H 0QS, United Kingdom Tel: +44 (0) 20 7654 5500 Fax: +44 (0) 20 7654 5555 www.iwapublishing.com publications@iwap.co.uk © Copyright 2011 by the Water Environment Research Foundation. All rights reserved. Permission to copy must be obtained from the Water Environment Research Foundation. Library of Congress Catalog Card Number: 2011920209 Printed in the United States of America IWAP ISBN: 978-1-78040-002-0/1-78040-002-0 This report was prepared by the organization(s) named below as an account of work sponsored by the Water Environment Research Foundation (WERF). Neither WERF, members of WERF, the organization(s) named below, nor any person acting on their behalf: (a) makes any warranty, express or implied, with respect to the use of any information, apparatus, method, or process disclosed in this report or that such use may not infringe on privately owned rights; or (b) assumes any liabilities with respect to the use of, or for damages resulting from the use of, any information, apparatus, method, or process disclosed in this report. University of South Florida This document was reviewed by a panel of independent experts selected by WERF. Mention of trade names or commercial products or services does not constitute endorsement or recommendations for use. Similarly, omission of products or trade names indicates nothing concerning WERF's positions regarding product effectiveness or applicability.

ACKNOWLEDGMENTS Thanks are extended to the following individuals and organizations: Rhonda Kranz (WERF) for her help with grant management, Gary Toranzos, Ph.D. (University of Puerto Rico), Patricia Anderson (Florida Department of Health), and David Whiting (Florida Department of Environmental Protection) for input on research strategies and priorities, and Theresa Tran at USF for sample collection and analytical help. The Florida Department of Environmental Protection (FL DEP), the Florida Stormwater Association Education Foundation (FSAEF) and the Water Environment Research Foundation (WERF) co-sponsored this research and each has rights to the content in this report. Research Team Principal Investigator: Valerie J. Harwood, Ph.D. University of South Florida, Department of Integrative Biology Project Team: Katrina V. Gordon, Ph.D. Christopher Staley, B.S. University of South Florida, Department of Integrative Biology Technical Review CommitteePatricia Anderson, P.E., M.S. Florida Department of HealthBart Bibler, P.E. Ferrate Treatment Technologies, LLCSharon Nappier, MSPH, Ph.D. U.S. Environmental Protection Agency, Office of WaterDon Stoeckel, Ph.D. Battelle Memorial InstituteGary Toranzos, Ph.D. University of Puerto RicoWERF Waterborne Pathogens and Human Health Challenge Issue Area Team

Shari Barash U.S. Environmental Protection AgencyBart Bibler, P.E. Ferrate Treatment Technologies, LLCBarbara Biggs Metro Wastewater Reclamation District of Denver Gail Boyd, M.S. Independent ConsultantMichael Casteel, Ph.D. San Francisco Public Utilities Commission

Validation of Rapid Methods for Enumeration of Markers for Human Sewage Contamination in Recreational

iii

Christobel Ferguson, Ph.D., GAICD Ecowise EnvironmentalThomas C. Granato, Ph.D. Metropolitan Water Reclamation District of Greater ChicagoJames A. Hodges, CPEng Watercare Services Limited, Auckland, New ZealandPaul W. Keohan Boston Water and Sewer CommissionNorman E. LeBlanc Hampton Roads Sanitation DistrictKeith Linn Retired from Northeast Ohio Regional Sewer DistrictKaren Pallansch, P.E., BCEE Alexandria Sanitation AuthorityGeeta Rijal, Ph.D. The Metropolitan Water Reclamation District of Greater ChicagoPeter Tennant, P.E. Ohio River Valley Water Sanitation CommissionGary Toranzos, Ph.D. University of Puerto Rico

Water Environment research Foundation Staff

Director of ResearchProgram Director

Daniel M. Woltering, Ph.D. Rhonda Kranz, M.S.

ABSTRACT AND BENEFITS Abstract: Fecal indicator bacteria (fecal coliforms,Escherichia coli, enterococci) are used as surrogates to indicate fecal contamination and increased public health risk. They can be contributed to the water by many sources, not all of which pose equal risks for human health, so there is a need to determine the source of contamination in order to evaluate public health risk and to remediate the waterbody. Fecal indicator bacteria have historically been enumerated via culture-based methods. Rapid, quantitative methods that can discriminate human from non-human fecal contamination have recently been developed and are informative for risk assessment and to identify problems in infrastructure or land use that contribute to degradation of water quality. These new methods can inform the public and officials of sewage-polluted water in a more timely manner than culture-based methods. This study compared standard, membrane filtration culture-based measures of the fecal indicator bacteriaE. coliand enterococci to rapid quantitative polymerase chain reaction (qPCR) methods in terms of correlation and consistency of performance in various types of waters. Furthermore, the genes of two human-associated microorganisms, one from the bacterial group Bacteroidalesand one from human polyomaviruses JCV and BKV, were used to quantitatively assess the presence of human sewage using qPCR in various types of subtropical waters in Florida, including fresh, brackish and salt waters. The qPCR methods performed well across the range of water types, although highly tannic water types (e.g swamp-fed river or blackwater) were more likely to be subject to inhibition than lake, estuarine or marine waters. Inhibition occurs when the expected qPCR signal is diminished by certain chemicals in the sample. Clean-up of inhibited samples by chemical methods produced highly variable results, therefore dilution of template material proved to be a better option for relieving inhibition. Concentrations of specific genes amplified by qPCR (targets) for which culture-dependent methods exist were generally in agreement when culture and qPCR methods were compared. A tradeoff between sensitivity and specificity was noted for the qPCR assays for human-associatedBacteroidales(more sensitive) and human polyomaviruses (more specific), suggesting they should be used together whenever possible. The assessment of human-health risk should be considered strongest when both markers are present and probable when one of the two markers is detected. Comparison of three different sample processing methods showed that the U.S. Environmental Protection Agency crude extract (CE) and purified extract (PE) methods were superior to the University of South Florida (USF) methods for bacterial targets, but not for human polyomavirus HPyVs. The U.S. EPA CE method had a higher detection limit than the PE method, but had greater precision and is more cost effective, therefore it was chosen for processing samples for bacterial qPCR targets.

Validation of Rapid Methods for Enumeration of Markers for Human Sewage Contamination in Recreational

Benefits: ♦Provides a systematic approach to insure that any new indicator for “pathogens” that is chosen as a regulatory tool has been validated across the range of water types that commonly occur in the U.S., including subtropical waters.

♦Rigorously validates methods for rapid detection of human pollution sources, which are among the most dangerous for human health.

♦Provides standard operating procedures for cutting-edge qPCR methods that can be followed by regulatory agency staff, utility managers and other water sector professionals.

♦Informs the regulatory effort to devise new criteria for rapid analysis of fecal indicator bacteria in subtropical waters.

♦Helps establish the linkage between old and new indicator organism paradigms, and the connection between indicator testing and public health protection.

Keywords:Enterococci,E. coli,microbial source tracking, qPCR, water quality.

TABLE OF CONTENTS Acknowledgments.......................................................................................................................... iii Abstract and Benefits .......................................................................................................................v List of Tables ...................................................................................................................................x List of Figures ............................................................................................................................... xii List of Acronyms ......................................................................................................................... xiii Executive Summary ...................................................................................................................ES-1 1.0 Introduction1-......1.............................................................................................................. 1.1 Background .......................................................................................................... 1-1 1.2 Project Description............................................................................................... 1-1 1.3 Literature Review of Human-Associated MST Targets ...................................... 1-2 2.0 Study Methodology........................................................................................................ 2-1 2.1 Literature Review................................................................................................ 2-1 2.2 qPCR Assays........................................................................................................ 2-4 2.2.1 Generation of Standard Curves ................................................................ 2-4 2.2.2 qPCR Reaction Chemistry ....................................................................... 2-4 2.2.3 qPCR Cycling Conditions and Instrument Response .............................. 2-6 2.3 Sample Collection ................................................................................................ 2-6 2.3.1 Sewage Sample Collection ...................................................................... 2-6 2.3.2 Ambient Water Sample Collection .......................................................... 2-7 2.4 Sample Filtration.................................................................................................. 2-7 2.4.1 Filtration for Culture-Based Enumeration of Fecal Indicator Bacteria ... 2-7 2.4.2 USF Method for qPCR Membrane Filtration .......................................... 2-7 2.4.3 EPA Method for qPCR Membrane Filtration .......................................... 2-7 2.5 DNA Extraction ................................................................................................... 2-7 2.5.1 USF Method of DNA Extraction ............................................................. 2-8 2.5.2 EPA Method of DNA Extraction – Crude Extract................................... 2-8 2.5.3 EPA Method of DNA Extraction – Purified Extract ............................... 2-8 3.0 Validation of QPCR Methods in the Laboratory........................................................ 3-1 3.1 Introduction.......................................................................................................... 3-1 3.1.1 Specific Task Goals ................................................................................. 3-1 3.2 Establishment of Method Performance Criteria .................................................. 3-1 3.2.1 Materials and Methods – Standard Curve Generation for Human-Specific Targets .......................................................................... 3-1 3.2.2 Evaluation of Validation Standard Curve Performance Criteria ............. 3-2 3.2.3 Interpretation of Validation Standard Curve Criteria .............................. 3-3 3.2.4 Validation Standard Curves for General Fecal Pollution qPCR Targets ... 3-4 3.3 Method Limits of Detection................................................................................. 3-6 3.3.1 Materials and Methods - Preparation of Sewage DNA Dilution Series .. 3-6 3.3.2 Method Detection Limits ......................................................................... 3-6 3.4 Method Specificity............................................................................................... 3-9 3.4.1 Materials and Methods – Composite Sample Preparation ....................... 3-9

Validation of Rapid Methods for Enumeration of Markers for Human Sewage Contamination in Recreational

vii

4.0

5.0

6.0

3.5

3.4.2 Human-Specific Assay Specificity Results ............................................. 3-9 Conclusions........................................................................................................ 3-11

Method Performance in Ambient and Sewage-Spiked Surface Waters................... 4-1 4.1 Introduction and Methods .................................................................................... 4-1 4.2 Human-Specific Assay Performance in Ambient Water ..................................... 4-2 4.2.1 Pre-Test Results to Determine Background Indicator Bacteria and Human-Specific Target Concentrations................................................... 4-2 4.2.2 Human-Specific Assay Detection and Quantification Limits in Ambient Water......................................................................................... 4-3 4.2.3 Inhibition in Ambient Waters .................................................................. 4-6 4.3 General Fecal Pollution Assay Performance in Ambient Water ......................... 4-8 4.3.1 Background Concentrations of General Fecal Pollution Targets in Ambient Water......................................................................................... 4-8 4.3.2 Limits of Detection of Quantification of General Fecal Pollution Targets in Ambient Water........................................................................ 4-9 4.4 Amplification of Negative Control Samples...................................................... 4-12 4.5 Conclusions........................................................................................................ 4-13

Persistence of Human-Associated MST Markers on a Simulated Tidally Influenced Estuarine Beach After a Contamination Event........................................................... 5-1 5.1 Introduction.......................................................................................................... 5-1 5.1.1 Materials and Methods – Mesocosm Construction and Sampling........... 5-1 5.2 Persistence of MST Markers Compared to Culturable Indicator Bacteria .......... 5-2 5.3 Conclusions.......................................................................................................... 5-8

Field Testing and Relationships of MST Markers with Standard Fecal Indicators.. 6-1 6.1 Introduction.......................................................................................................... 6-1 6.1.1 Materials and Methods............................................................................. 6-1 6.2 Determining Concentrations of Conventional Indicators .................................... 6-2 6.3 Determining Concentrations of Human-Associated and General Water Quality MST Markers. ...................................................................................................... 6-3 6.3.1 Concentration of Human Polyomavirus (HPyVs) in Ambient Water Samples. ............................................................................................................... 6-3 6.3.2 Determining Concentration of Human-AssociatedBacteroidales(HF183) in Ambient Water Samples. ..................................................................... 6-5 6.3.3 Determining Concentration ofE. coliin Ambient Water Samples Using qPCR. ............................................................................................ 6-7 6.3.4 Determining Concentration of Enterococci in Ambient Water Samples Using qPCR. ............................................................................................ 6-9 6.3.5 Concentration of GeneralBacteroidales in Ambient Water Samples Using qPCR. .......................................................................................... 6-12 6.4 Conclusions from Comparison. ......................................................................... 6-13

viii

7.0 Additional Testing of USF vs EPA Sample Processing for the Detection of HPyVs and HF183 in Artificially Contaminated Samples......................................... 7-1 7.1 Introduction.......................................................................................................... 7-1 7.1.1 Materials and Methods............................................................................. 7-1 7.2 Determining Concentrations of Conventional Indicators .................................... 7-1 7.3 Determining Concentrations of MST Targets (HPyVs and HF183) ................... 7-2 7.4 Conclusions from Comparison. ........................................................................... 7-4 8.0 Study Conclusions and Recommendations.................................................................. 8-1 8.1 Validation of qPCR Methods in the Laboratory .................................................. 8-1 8.2 Method Performance in Ambient and Sewage-Spiked Surface Waters .............. 8-1 8.3 Persistence of Human-Associated MST Markers on a Simulated Tidally Influenced Estuarine Beach After a Contamination Event .................................. 8-2 8.4 Field Testing and Relationship of MST Markers with Standard FIBs and Comparison of Three Sample Processing Methods ............................................. 8-2 8.5 Wrap-Up .............................................................................................................. 8-3 ® Appendix A: Human Polyomavirus (HPyVs) in Water by TaqMan Quantitative Polymerase Chain Reaction (qPCR) Assay ...................................................................................................... A-1

® Appendix B: Human-AssociatedBacteroidesQuantitative Polymerasein Water by TaqMan ChainReaction(qPCR)Assay.......................................................................................................B-1

Appendix C: ABI 7500 Real Time PCR System Operation ........................................................C-1

Appendix D: Microbial Source Tracking Markers for Detection of Human Sewage and Fecal Contamination in Environmental Waters: Relationships to Pathogens and Human Health Outcomes: A Literature Review ................................................................................................. D-1

References....................................................................................................................................R-1

Validation of Rapid Methods for Enumeration of Markers for Human Sewage Contamination in Recreational