I iiiled Slides r.ii\imiiim-iil;il Prnleclinn Ajicno Office of Research jiikI l)o\ olopmoiiI National Hxposure Research Laboratory Research Abstract Government Performance Results Act (GPRA) Goal #2 Annual Performance Measure #29 Significant Research Findings: Temporal and Spatial Variability of Fecal Indicator Bacteria: Implications for the Application of MST Methodologies to Differentiate Sources of Fecal Contamination Scientific The Clean Water Act (CWA) was enacted to restore and maintain the chemical, Problem and physical, and biological integrity of the nation's waters in order to support "the Policy Issues protection and propagation of fish, shellfish, and wildlife and recreation in and on the water." Section 303(d) establishes total maximum daily loads (TMDL) as a process for allocating and reducing the discharge of pollutants that impair a water body. Because fecal contamination is one of the major causes of water quality impairments, methods to detect pathogens and pathogen indicators (and track them to their sources) are needed to allocate TMDLs and reduce the sources of contamination. Microbial source tracking (MST) is used to determine the sources of the fecal contamination in impaired water bodies. Temporal variability in the gastrointestinal flora of animals can produce low source identification rates when applying MST methods. Understanding how bacterial species and genotypes vary over time is highly relevant when the fecal material used to create a source library is collected under different seasonal conditions than the environmental sample. To date, very few studies have addressed how the temporal stability of fecal indicators impacts the application and utility of MST. Research The objective was to identify and compare the temporal and spatial variability of Approach fecal indicator bacteria from a specific host in manure and water samples, and evaluate the implications of such variability on microbial source tracking approaches and applications. Escherichia coli and Enterococcus sp. are the two fecal indicator bacteria that were used by the EPA Office of Water to establish the current national ambient water quality criteria for assessing microbial contamination. We selected Enterococcus as the model fecal indicator, given the supposedly high specificity of some of the species of this genus to the host organism. Cattle were chosen as the model host organism because of the documented impact that cattle have on impairment of surface waters. The study sites are located at a farm where cattle have unrestricted access to the stream. Enterococci were isolated monthly from water and manure samples using membrane-Enterococcus Indoxyl-(3-D-Glucoside agar (mEI). A library of over 1,500 isolates was developed. Each isolate was identified using a multiplex PCR (polymerase chain reaction) procedure that targets the genus and the species- specific gene superoxide dismutase. Bacteroides markers were also applied to differentiate human from cattle fecal contamination. ------- Results and Impact One of the limitations most MST studies have is the inability to understand the natural variability of bacterial fecal indicator levels. Our results indicate that to increase the validity of MST studies, the experimental approach should consider: (1) Conducting a temporal variability assessment when constructing source libraries and designing the sampling scheme of the source material. (We observed that Enterococcus species isolated from cattle manure, which were also observed in the stream samples, exhibited a high degree of seasonal variability, and in some cases, were absent during certain seasons.) (2) Determining the survival of a selected bacterial fecal indicator in the environmental matrix of interest. (From 11 Enterococcus species, two were found only in cattle manure, but were not recovered in stream water; this makes such species unreliable indicators of cattle fecal contamination in surface waters since they do not survive in this environment.) (3) Addressing the possibility of finding high background concentrations of fecal indicator bacteria due to wildlife and non point human sources. (Results showed that a human-DNA marker was identified in stream areas not visibly impacted by humans, and a cattle marker was identified in the water upstream from the cattle-impacted area.) In addition, the results indicate that use of cluster analysis seems to be a good approach to identify groups of enterococci species that are specific to a location or source, and suggests that a community fingerprint rather than an individual species could be as an effective MST approach. The culture-dependent vs. the culture independent technique applied in this study differ greatly in terms of cost effectiveness and turn-around time of results. Time availability (when the results are expected or needed) and funding support (largely for laboratory consumables) are two important considerations. Library-independent approaches are generally much faster and possibly less expensive than library-dependent approaches. However, more research is needed for field validation and sensitivity testing of available DNA markers. Research Collaboration and Research Products This research was performed in collaboration with the laboratory of Dr. Charlene Jackson from the U.S. Department of Agriculture-Agricultural Research Service and USEPA Region 4 through the Regional Research Partnership Program. In addition, statistical analysis was performed by the University of Georgia Statistical Consulting Services class. Examples of recent publications from this study include: Molina, M., Maimes, J., Fisher, J., Johnson, B., and Jackson, C. "Temporal Variability Of Enterococci Species In Streams Impacted By Cattle Fecal Contamination." Presented at American Society for Microbiology Annual Meeting, Atlanta, GA, June 05-09, 2005. Molina, M., and Frick, W.E. "Polymerase Chain Reaction (PCR) Technology In Visual Beach." Presented at National Beaches Conference, San Diego, CA, October 13-15, 2004. Frick, W. E., and Molina, M. "Visual Beach: Software For Achieving Beach Aesthetic And Public Health Protection." Presented at National Beaches Conference, San Diego, CA, October 13-15, 2004. Johnson, B., and Molina, M. "Using DNA to Identify Sources of Pathogen Contamination." Presented at the 2004 EPA Science Forum. Washington, D.C., June 9-11, 2004. Future Research The next phase of this research includes the comparison of DNA fingerprints of the most important enterococci species identified in the statistical analysis to determine the temporal variability of the species at the genomic level and whether a specific pattern can be used as a fecal indicator of cattle contamination in impaired streams. In addition, culture-independent markers of poultry, cattle and ------- human fecal contamination will be temporally and spatially tested under various environmental conditions. Contacts for Questions and inquiries can be directed to: Additional Marirosa Molina, Ph.D. Information jj.S. EPA, Office of Research and Development National Exposure Research Laboratory 960 College Station Road Athens, GA 30605 Phone: 706/355-8113 E-mail: molina.marirosa@epa.gov Funding for this project was through the U.S. EPA's Office of Research and Development, National Exposure Research Laboratory, and the work was conducted by the Ecosystems Research Division. ------- |