Impact/Purpose:

Building Predictive Models for Hazard ID - Prioritization Method Development and Application

Description:

The microbiological water quality standards established by EPA depend on culturing fecal indicator bacteria to predict the risks associated with water usage. For decades this has been the favored approach to microbiological monitoring in spite of the fact that culture-based methods tend to underestimate the densities and diversity of microorganisms in environmental samples (Amann et al., 1995). Relevant to public health is the fact that nonculturable pathogens could be the etiological agents of many waterborne illnesses (Sails et al., 2002). In addition, nonculturable organisms may be better indicators of fecal contamination. Moreover, nonculturable bacterial indicators may be useful in the identification of the fecal sources impacting a particular water system. Identifying the sources responsible for the fecal pollution of a natural waterway is important in order to reduce the fecal bacterial levels and in turn reduce the illnesses associated with recreational activities, and food (e.g., fish) and water consumption (Simpson et al., 2002). Consequently, strict dependence on culturing techniques continues to be a notable roadblock in the path towards understanding the correlation between bacterial indicators of fecal contamination and the hazards associated with fecally impacted waters. Nucleic acid-based approaches can circumvent many of the shortcomings of the culture-based methods. For instance, the possibility of rapidly and simultaneously monitoring hundreds of microorganisms and genes relevant to public and environmental health is now becoming a reality in light of the recent advances in microarray technology (Stahl and Tiedje, 2002). Such a capability will categorically improve microbial risk assessment and the framework used in the development of environmental monitoring and risk management tools.

Record Details:

Record Type:PROJECT

Projected Completion Date:09/30/2008

OMB Category:Other

Record ID: 149130

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Project Information:

Approach :We propose the development of an innovative metagenomics program to enhance our current capabilities for environmental microbial monitoring, risk exposure, risk management, and risk assessment. The general goal of this study is to develop a novel molecular markers based on fecal microbial genomes to better assess the sources of fecal contamination in natural water systems. Assuming that the evolutionary pressure for the selection of host specific populations must involve ecologically driven processes we hypothesize that genes playing a role in host-microbial interactions and cell-cell recognition (e.g., cell surface proteins, toxins, and adhesin) are better markers of host specificity. The primary objective of this study is to discover novel and validate the use of fecal metagenomic sequences as MST host specific markers. By looking at collective genomes we will substantially increase the number of potential genes and microbial species that can be used in the development of molecular microbiological assays (Schloss and Handelsman, 2003). The first phase of this program will focus on constructing fecal metagenomic libraries specific to animals that are known to be relevant pollution sources of watersheds in the United States. The following phases will focus on systematically identifying DNA sequence markers for specific human and bovine fecal sources and testing the specificity of these markers against waters impacted with different pollution levels and pollution sources. We will use genomic subtraction of metagenomic libraries to generate unique sequences from the collective microbial genomes present in animal feces. Due to the complexity of microbial communities, this approach will facilitate the identification of candidate host specific microbial genes. Real-time PCR assays will be developed to test the host specificity of the latter markers and to quantify the target genes in environmental waters impacted with different levels of fecal contamination. A similar approach will be undertaken with samples from ongoing epidemiological studies. Markers that show host specificity will be included on biochips containing sequences specific to fecal indicator bacteria and pathogens and challenged with DNA extracts of different fecal samples and water samples impacted with different levels of pollution. By combining the epidemiological studies with the host specific assays and pathogenic markers, we hope to gain better predictive capability to determine outcomes from scenarios associated with fecally impacted waters.

Relevance :Distinguishing between human and nonhuman fecal contamination in our waterways has never been more important than in recent years in light of the need by the States to comply with deadlines associated with the Total Maximum Daily Loadings program (Simpson et al., 2002). Accurate assessment of the primary sources of fecal pollution is needed in order to calculate the different risks associated with each of the potential sources impacting water systems and to correctly implement and evaluate Best Management Practices (BMP) used to remediate fecally polluted waters. As currently developed, most MST methods depend on markers that have no ecological meaning. The main goal of this study is to develop assays that can be used in source identification, environmental monitoring, and risk assessment. In order to accomplish this goal we will combine a metagenomic-scale approach with high-throughput gene mining (microarrays) and real-time PCR assays. Therefore, this research supports EPA’s GPRA Goal 2 (Clean and Safe Water). The proposed work will provide a rich source of metagenomic information (i.e., novel molecular markers) for sequenced-based analyses aimed at better monitoring of fecal pollution and improving the assessment of the outcome associated with different pollution sources. Consequently, it is anticipated that the results will provide a molecular-based framework to (1) better predict the relationship between microbial water quality and public health risks, (2) determine the impact of different microbial pollution sources on watershed biology, and (3) effectively control or eliminate pollution in our Nation’s watersheds.

Project IDs:

ID Code :IIC-4

Project type :Partner Specific