Quantitative Assessment of Pathogens in Drinking WaterEPA 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 will develop 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 quantitative PCR (qPCR), quantitative reverse transcription-PCR (qRT-PCR), fluorescent in situ hybridization (FISH) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The sensitivity and specificity of the proposed pathogen recovery and detection approach will be directly compared to current USEPA methods via spiking and analysis of raw and finished drinking water samples collected from various water resources and distribution systems. Following method validation, a series of unspiked raw or finished waters (including waters from distribution systems), will be monitored for pathogenic microorganisms to demonstrate the utility of the approach in real world situations.
Commercially available hollow fiber ultrafilter (UF) membranes will be assessed with respect to both microbe collection efficiency and subsequent microbe recoverability using a suite of viruses, bacteria and protozoa. An existing unit for bench-scale testing will be used for these evaluations. The unit is comprised of two parallel channels that allow two membranes to be tested simultaneously. Recovered microorganisms will be enumerated by both the molecular methods outlined above and existing infectivity assays. Following selection of suitable membranes, a scaled-up UF system will be employed to recover spiked microorganisms from large volumes of raw and finished drinking water. As part of the method evaluation, split samples will be analyzed using current USEPA methods for viruses (1MDS filtration and BGM cell culture analysis), protozoa (method 1623) and bacteria (EPA E. coli and enterococci methods 1603 and 1600 respectively). Additional method comparison experiments will be conducted using unaltered natural water samples.
Three critical needs of microbial drinking water risk assessment will be addressed: (1) adaptation of tangential-flow ultrafiltration is expected to result in a robust, universal microbial recovery system allowing for improved virus and protozoa recovery (and subsequent detection); (2) the new approach will move away from reliance on bacterial indicators as the only determinants of microbial water quality; (3) the study is expected to yield quantitative molecular detection protocols. We will demonstrate that, through the use of appropriate controls and standards, established protocols for qRT-PCR/qPCR and FISH can be adopted to yield robust environmental diagnostics. These methods have the potential to be applied widely by drinking water agencies and regulators for years to come. Concurrently, we will optimize proteomic mass spectrometry for the global detection of microorganisms, i.e., for monitoring of all microbial pathogens, irrespective of whether their presence is anticipated or not, and for determination of the infectious nature of isolated microorganisms via detection of target proteins in cell fractions representing intact cells obtained by physical screening. By combining an efficient membrane filtration recovery method with advanced molecular detection protocols, this study will provide a tool for obtaining quantitative data on human exposures to pathogenic viruses, protozoa and bacteria present in raw and finished drinking water. As such, the study will enable regulatory agencies to make better-informed risk management decisions.