Impact/Purpose:

1) Develop rapid TaqMan PCR methods for the detection and quantification of total enterococci and specific groups or species of Enterococcous in water and determine, in a laboratory setting, the efficacy of the methods. 2) Use TaqMan methods to determine the occurrence of total and specific enterococci groups in fresh and marine recreational water samples and compare results with those of standard culture based methods.

Description:

The current methods for measuring the quality of recreational waters were developed in the 1970's and were recommended to the States by EPA in 1986. These methods detect and quantify Escherichia coli and enterococci, two bacteria that are consistently associated with fecal wastes from warm-blooded animals. Both of these water quality indicators have been shown to be linearly related to swimming associated illness, i.e., as the water quality indicator densities increase, the illness rate increases. The concentrations of enterococci were correlated with illness in both fresh and marine waters, while E. coli concentrations correlated with illness rates in fresh water only. Because of the relationship of these microbial indicators to swimming-associated illness, they have been recommended as monitoring tools for recreational waters. There are, however, some problems associated with the use of these indicators. Results of testing water samples are not available until 24 hours after the samples are taken. If the water is not in compliance with the standard, the risk of exposure to enteric pathogens has potentially occurred long before the quality of the water was identified as being hazardous. This shortcoming can be eliminated by developing a rapid method (same day results, ideally in less than two hours) for the quantitative detection of indicator organisms. Such a method, using the fluorigenic 5' nuclease (TaqMan) PCR assay, will be developed and evaluated for the detection, identification and quantification of enterococci in fresh and marine recreational water samples.

Record Details:

Record Type:PROJECT

Start Date:10/01/1998

Completion Date:09/01/2002

Record ID: 56194

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Project Information:

Progress :A genus specific TaqMan probe and primer set for Enterococcus has been identified in the literature. A species specific TaqMan probe and primer set has been developed for E. faecalis. Group specific TaqMan probe and primer sets have been developed for E. faecium, E. durans, E.hirae, and E.dispar and for E. casseliflavus and E. gallinarum. A rapid method for the extraction of nucleic acids from water sample filtrates has been developed. An interagency agreement is in place to provide recreational water samples for the in-house research.

Santo Domingo, JW, Tia Newby, Sherie Brown, and Steve Harmon. Evaluation of the use of different antibiotics in the direct viable count method to detect viable fecal enterococci. Presentation at 100th General Meeting of the American Society for Microbiology, May 2000.

Brown, S, JW Santo Domingo, SD Siefring, and RA Haugland. 2001. Use of TaqMan to enumerate Enterococcus faecalis in water. Presentation at 101st General Meeting of the American Society for Microbiology, May 2001.

Siefring, SD, JW Santo Domingo, N Brinkman, and RA Haugland. 2001. Detection of fecal enterococci using a real time PCR method. Presentation at 101st General Meeting of the American Society for Microbiology, May 2001.

Santo Domingo, JW, SD Siefring, and RA Haugland. Real-time PCR method to detect Enterococcus faecalis in water. Manuscript submitted for review.



Relevance : Development of a new rapid water quality method for recreational water, which is a high priority research area for the Agency, will meet the needs stated in GPRA (Goal 2, Objective 3, Subobjective 3.3), the Clean Water Action Plan, and the Beach Action Plan. Use of the rapid method for compliance monitoring will facilitate the detection of contaminated recreational water in a timely manner and allow the beach managers to alert the public about the potential health hazards before exposure to unsafe water can occur.

Clients :Office of Water

Project IDs:

ID Code :7001

Project type :OMIS