- Staton, Pamela
- Marshall University Research Corp
- Start date
- End date
- A database of NotI pulse-field gel electrophoresis (PFGE) E. coli DNA profiles isolated from source-known warm-blooded animal feces, to include humans, will be generated in the Huntington, WV region. Analysis of the resulting database (Database 3) will be compared to existing databases including Moorefield, WV/Potomac River(Database 1) and the WV Eastern Panhandle/Berkeley County (Database 2). Although the immediate goal is to create regional database(s) to be used for source tracking fecal pollution in local waterways, it is also desirable to determine if a sufficiently large database could be used for bacterial source tracking in seemingly unrelated geographic areas. Significant savings in time and cost would be realized should it not be necessary to create region-specific databases. To further investigate the feasibility out-of-region BST, existing combined and individual databases will be used to evaluate water samples collected from the Gauley and New Rivers for which no scat-based database exists. In addition, rapid BST methods will be pursued to include a DNA signature sequence approach as an alternative to currently available methods. Such a non-database approach will include microarray technology to identify genomic differences in E. coli isolates by source. Discriminate analysis of E. coli by source at the DNA level would further our knowlege of E. coli's natural host adapability, provide a practical purpose for such information through bacterial source tracking, and bring BST into the scope of any water testing facility through the use of rapid, high-throughput technology.
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- NON-TECHNICAL SUMMARY: Certain waterways in WV located near agricultural industries and areas of dense human populations have been cited for elevated levels of fecal contamination. This water-quality project will build upon current databases developed for microbial source tracking to identify animal and human sources of fecal contamination in our waterways. The purpose of this study is to further expand the E. coli strain DNA database to other regions for the purpose of accurately identifying sources of fecal contamination. New methods of DNA analysis of E. coli by source will be evaluated to compliment, enhance, or replace current methodologies for the purpose of identifying more efficient bacterial source tracking technologies.
APPROACH: Scat sampling, sample processing and PFGE testing and analysis will follow our previously established NotI restriction endonuclease protocol. Upon completion of the Huntington, WV database, specialized software will be utilized to make inter- and intra-database comparisons, characterize the current database(s) for region-specificity and out-of-region applicability, and to provide for an expanded master database. Microarrays will be used to provide clues to E. coli-by-source differences. Microarray-identified regions of interest will be used for targeted characterization of E. coli through rapid, short-read DNA sequencing. Relatively short DNA sequences consistently identifiable by source would then be candidate sequences for primer and probe development. Source-specific primers and probes will be used in quantitative or semi-quantitative methods to include real-time PCR and pyrosequencing. To provide practical information, environmental and spiked water samples will be evaluated for rapid performance, accuracy, reliability and cost-effectiveness. New techniques will be evaluated for their ability to alleviate time-consuming processing and analysis steps currently required for BST. Validation of rapid methods, to include pyrosequencing and real-time PCR, for bacterial source tracking of microbial contaminants, represents an important step toward high performance, high-throughput analysis of E. coli by source, as well as an initial step toward identifying methods that might be utilized for continuous and/or automated microbial monitoring of natural waterways.
PROGRESS: 2003/09 TO 2006/09
Escherichia coli (E. coli), is a bacterium that normally resides in the intestinal tract of warm-blooded animals, including humans. Surface and ground waters become contaminated as a result of domestic animal and wildlife runoff, as well as human-generated fecal contamination from leaky septic systems, straight pipes, storm-sewer overflows, and failing waste treatment facilities, for example. Currently, standard practice requires the enumeration of E. coli (or other microbe) per water sample as an "indicator" that disease-causing pathogens may be present. When counts exceed established limits, it is in the best interest of public safety to locate and resolve the source of fecal pollution. Unfortunately, the origin of fecal contamination may be difficult to identify and thus remediate. E. coli bacterial source tracking (EC-BST), currently still under development, addresses this need by attempting to assign animal-source-designators to E. coli isolates to guide remediation efforts. The Forensic Science Center in previous years has developed five regional databases spread across West Virginia consisting of E. coli DNA fingerprints maintained in each database by host of origin. This year (05-06) we extended our testing to one adjacent state, Ohio, and one distant state, Iowa, utilizing proficiency testing to determine how this EC-BST method performed in various HOME, FOREIGN, and all combinations of West Virginia, Ohio, and Iowa databases. The major purpose of extending our testing to other states was to determine if FOREIGN databases could be used in lieu of HOME databases as this would extend this technology to communities without databases at a considerable cost-savings. This year we exceeded our goal by collecting 1364 samples from Ohio and Iowa and generating 4580 E. coli DNA fingerprints for database inclusion. Based on random proficiency testing, both HOME and FOREIGN databases were shown to have >80% matching efficiency and >80% accuracy rates for a 2-way analysis, i.e., human versus nonhuman classification demonstrating that FOREIGN West Virginia databases can be used in remote regions for source-tracking at these rates. In a 3-way analysis, i.e., human versus wildlife versus domestic animal classification, the matching efficiency and accurate rates of Ohio and Iowa proficiency samples were improved when HOME isolates were included in the test database. One major obstacle we overcame this year was to develop a computer program to manage and perform the vast number of computations associated with the increased number of databases required to evaluate and the significant increase in database isolates. In the past, this had been painstakingly performed manually. This will dramatically assist others who elect to utilize this technology in the future. We continue to develop other supplemental technologies that will add value to our currently standardized testing protocols. One such development is a real-time, PCR procedure that is allowing us direct quantification of E. coli from source waters thus avoiding traditional culture-based testing that is currently in use.
IMPACT: 2003/09 TO 2006/09
Bacterial source tracking methods are generally of two types: library-dependent and non-library-dependent. As our method is library-dependent, a significant number of samples and isolates must be processed to accomplish this task. Our master database now contains a total of 12,791 E. coli DNA fingerprints all of which are cataloged by host source. While plans are underway to use this database for source-tracking E. coli from impacted waters, this technology may also be useful in tracking E. coli to its source in foodborne outbreaks as well. Whether the objective is medical epidemiology, forensic epidemiology, or microbial forensics, each one seeks to track an infectious agent or disease back to its host of origin. Bacterial Source Tracking Project is fundamentally an environmental epidemiology study that has evolved over time from a basic to applied research study to a support service for other communities who need this capability as well. The BST Project demonstrates how basic research can evolve into applied research with a practical use, commercial and public appeal. Adapting molecular technologies to address pressing environmental issues is being demonstrated through a grassroots approach to environmental remediation of fecal pollution of water sources.
- Funding Source
- Nat'l. Inst. of Food and Agriculture
- Project source
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- Project number
- Accession number
- Prevention and Control
- Bacterial Pathogens
- Escherichia coli