National Center for Computational Toxicology

Key Contact: Jorge W. Santo Domingo, Ph. D.; Microbiologist; Office of Research and Development; National Risk Management Research Laboratory; Water Supply and Water Resources Division; Microbial Contaminant Control Branch; 513-569-7085 (ph); 513-569-7328 (fax); santodomingo.jorge@epa.gov

Protection from fecal microbial contamination is one of the most important goals listed in section 303(c) of the Clean Water Act for waters designated for recreation (primary and secondary contact), public water supplies, and propagation of fish and shellfish.. Microbiological impairment of water is assessed by monitoring for the presence of sanitary indicator bacteria such as fecal coliforms and fecal enterococci. These microorganisms are associated with fecal material from humans and other warm blooded animals and their presence in water may also signal the presence of enteric pathogens that could cause illness in exposed persons. Detection of pathogens and tracking them to their sources is a topic of intense interest in view of the current Total Maximum Daily Load requirements. Microbial Source Tracking (MST) is one approach used to determine the sources of fecal pollution and pathogens affecting a water body. While several studies have reported the successful application of MST methods, none of the methods can meet performance expectations in complex water systems. One of the basic problems of MST is the need to rely on the use of culture based methods to assess the primary sources of pollution. Herein, we propose the development of nonculture-based genomic methods for environmental monitoring and risk assessment. The proposed work will focus on the use of a microbial community genome (metagenome) approach to identify novel nucleic acid sequence markers for fecal contamination and source identification. Some of the markers will also be useful in the identification of virulence factors as we predict that the enrichment process will select for genes involved in host-microbial interactions that are commonly associated with bacterial pathogenicity. The basic experimental design will consist of challenging genomic microbial community DNA from different fecal samples in genome subtraction studies to enrich for host specific microbial genes. The specificity of the nucleic acid markers that are identified will be validated using macroarrays, flow cytometry, and conventional PCR. Quantitative PCR assays will also be developed to rapidly detect and quantify these sequences in environmental samples impacted with different levels of fecal contamination. Additional studies will include cross-regional validation of PCR assays and spatial and temporal evaluation of host specific markers in fecally contaminated waters. Markers that show host specificity will also be included on biochips containing other sequences specific to fecal indicator bacteria and pathogens and then challenged against fecally contaminated water samples from ongoing epidemiological studies. The latter effort will provide a unique opportunity to identify microbial markers that correlate well with illnesses and therefore could prove to be useful in better estimating the potential adverse health effects associated with exposure to fecally impacted waters. These molecular markers could also be used in the evaluation of risk management practices designed to prevent, reduce, and eliminate pollution impacting source waters, and as a consequence improve the sanitary condition of surface and underground waters.

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