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Assessment of Microbial Pathogens in Drinking Water using Molecular Methods Coupled with Solid Phase Cytometry
EPA Grant Number: R833830Title: Assessment of Microbial Pathogens in Drinking Water using Molecular Methods Coupled with Solid Phase Cytometry
Investigators: Pyle, Barry H , Ford, Timothy E.
Institution: Montana State University - Bozeman , Little Big Horn College
EPA Project Officer: Klieforth, Barbara I
Project Period: June 10, 2008 through February 28, 2011
Project Amount: $599,996
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens and Cyanobacteria and Their Toxins in Drinking Water (2007)
Research Category: Drinking Water
Description:
Objective:This proposal describes a laboratory-based research project that involves Montana State University, Bozeman (MSU) and Little Big Horn College (LBHC). The primary objective is to establish novel methods to determine the occurrence of waterborne microbial contaminants, by enumeration of bacteria and intestinal parasites. Target microbial pathogens include Escherichia coli O157:H7, Helicobacter pylori, Legionella pneumophila, Mycobacterium avium, Aeromonas hydrophila, Giardia lamblia, and Cryptosporidium parvum.
Approach:Selected molecular techniques will be combined and optimized to allow for rapid detection and quantification of candidate pathogens in drinking water and source waters using solid phase laser cytometry (SPLC). i.e. the ScanRDI (AES-Chemunex). Pathogen screening will involve fluorescence in situ hybridization (FISH) with multiple probes to detect the target microorganisms. Detection and assessment of individual pathogens will determine viability using direct viable counts such as the Kogure method or fluorescent enzyme substrate labeling, e.g. ChemChrome (AES-Chemunex). Pathogenicity will be assessed by in situ (single cell) amplification of toxin or other virulence factor genes, alone or in combination with fluorescent antibody detection of virulence factors. Procedures will be evaluated using pathogen-free water spiked with target organisms or wastewater, in addition to samples of source waters and household supplies from the Crow Reservation. The screening and individual pathogen procedures will be verified by comparison with standardized methods including culturing, fluorescent antibodies, and other molecular techniques, e.g. real-time quantitative PCR.
Expected Results:The ultimate goal is to establish a suite of assays for each target pathogen that can be performed in one step or in sequence, e.g. initial screening for a range of pathogens followed by more specific detection and assessment of specific organisms according to initial screening results. Novel rapid, specific, sensitive methods for detecting pathogenic bacteria and intestinal parasites, that can be applied locally, throughout the U.S. and beyond, will be developed. Information relevant to source and household water quality and associated health risks on the Crow Reservation will be obtained. Undergraduate students from LBHC will be involved to provide microbiology/laboratory training and research experiences. This will include sampling methods, current water microbiological assessment methods, e.g. coliform testing, along with development and application of novel methods using solid phase laser cytometry in addition to epifluorescence microscopy. Collaboration with science faculty and staff of LBHC the Crow Tribal community and the American Indian Research Opportunities (AIRO) program at MSU will ensure communication of research approaches and results, in addition to respecting tribal sensitivities.
Supplemental Keywords:water, drinking water, groundwater, wastewater, effluent, feedlot, health effects, organisms, pathogens, bacteria, protozoa, biology, microbiology, monitoring, measurement methods, Northwest, Montana, MT, EPA Region 8