Final Report: Quantitative Assessment of Pathogens in Drinking Water

EPA Grant Number: R833002
Title: Quantitative Assessment of Pathogens in Drinking Water
Investigators: Schwab, Kellogg J. , Graczyk, Thaddeus , Halden, Rolf U.
Institution: The Johns Hopkins University
EPA Project Officer: Klieforth, Barbara I
Project Period: August 25, 2006 through August 24, 2009 (Extended to September 30, 2010)
Project Amount: $600,000
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text |  Recipients Lists
Research Category: Drinking Water , Water


A major limiting factor in assessing the human health risk of microbial pathogens in raw and finished drinking water is the lack of robust, efficient methods for concentrating, identifying and quantifying low levels of bacteria, viruses, and protozoa simultaneously, effectively, and rapidly. We have developed a microbial isolation and detection protocol capable of qualitative and quantitative identification of waterborne microbial pathogens by combining the latest high-efficiency filtration technology with rapid and sensitive molecular detection techniques including real-time PCR (PCR), and real-time reverse transcription-PCR (RT-PCR). Following laboratory development and analysis, the sensitivity and specificity of the pathogen recovery was evaluated by spiking and analyzing raw and finished drinking water samples collected from select water resources and distribution systems. In addition, a series of unspiked raw and finished waters was analyzed for pathogenic microorganisms to demonstrate the utility of the approach in real world situations.

Summary/Accomplishments (Outputs/Outcomes):

The project has been summarized in a series of peer-reviewed published papers as outlined in the Publication section of this report. Pathogens capable of causing waterborne diseases include bacteria, protozoa, and viruses. Fecal indicator bacteria are the primary microorganisms used to evaluate microbial water quality based on the analysis of small volume (100 ml) grab samples. Here a tangential flow, hollow fiber ultrafiltration (UF) method was optimized for the recovery of bacteria, protozoan surrogates, and enteric viruses seeded into 100 L dechlorinated drinking water (DW) and surface water (SW) samples. Recovery efficiency was assessed via culture methods and real-time reverse transcription-PCR (RT-PCR). The combined average recovery efficiencies by culture methods for bacteria (E. coli and E. faecalis), viral surrogates (MS2, PRD1, murine norovirus [MNV-1], and poliovirus), and C. perfringens bacterial spores were 58%, 53%, and 64% in DW (n = 6), respectively. In SW (n = 5), the recovery efficiencies by culture methods for bacteria, viral surrogates, and bacterial spores were 95%, 64%, and 36%, respectively. MNV-1 also was analyzed by real-time RT-PCR. Target MNV-1 RNA was detected in all DW (n = 6) and 3 of 5 SW samples. Following UF optimization, the method was applied for the recovery of enteric viruses from 100 L non-seeded DW (n = 6) and SW (n = 6) collected from two separate drinking water treatment plants (DWTP). Target human enteric viruses were not detected in any DWTP samples. Molecular analyses also included an internal standard RNA (hepatitis G virus) for identification of sample inhibition.


This research shows the applicability of a combined UF and real-time RT-PCR method for the concentration, recovery and detection of enteric viruses from both surface and finished drinking water sources.

Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 8 publications 4 publications in selected types All 4 journal articles
Type Citation Project Document Sources
Journal Article Graczyk TK, Sunderland D, Rule AM, da Silva AJ, Moura INS, Tamang L, Girouard AS, Schwab KJ, Breysse PN. Urban feral pigeons (Columba livia) as a source for air- and waterborne contamination with Enterocytozoon bieneusi spores. Applied and Environmental Microbiology 2007;73(13):4357-4358. R833002 (2007)
R833002 (2008)
R833002 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: AEM-Full Text HTML
  • Abstract: AEM
  • Other: AEM-Full Text PDF
  • Journal Article Graczyk TK, Majewska AC, Schwab KJ. The role of birds in dissemination of human waterborne enteropathogens. Trends in Parasitology 2008;24(2):55-59. R833002 (2007)
    R833002 (2008)
    R833002 (Final)
  • Abstract from PubMed
  • Full-text: Science Direct
  • Other: Science Direct PDF
  • Journal Article Young TA, Heidler J, Matos-Perez CR, Sapkota A, Toler T, Gibson KE, Schwab KJ, Halden RU. Ab initio and in situ comparison of caffeine, triclosan, and triclocarbon as indicators of sewage-derived microbes in surface waters. Environmental Science & Technology 2008;42(9):3335-3340. R833002 (2008)
    R833002 (Final)
  • Abstract from PubMed
  • Full-text: Johns Hopkins University PDF
  • Abstract: Environmental Science & Technology-Abstract
  • Supplemental Keywords:

    drinking water, human health, molecular detection, monitoring, real time PCR, pathogens, viruses, protozoa, bacteria, exposure, risk assessment, environmental microbiology, human health
    , RFA, Scientific Discipline, PHYSICAL ASPECTS, INTERNATIONAL COOPERATION, Water, POLLUTANTS/TOXICS, Environmental Chemistry, Environmental Monitoring, Physical Processes, Drinking Water, Environmental Engineering, Microorganisms, pathogens, bacteria, mass spectrometry, exposure, viruses, drinking water monitoring, molecular detection, water quality, drinking water contaminants, human health, pathogenic protozoa

    Progress and Final Reports:

    Original Abstract
  • 2007 Progress Report
  • 2008 Progress Report
  • 2009
  • 2010