Impact/Purpose:

The goal of this research project is to evaluate and compare methods that rapidly (less than 2 hours) measure fecal contamination of water with respect to accuracy, specificity, and ease of use.

Description:

Current methodology for determining fecal contamination of drinking water sources and recreational waters rely on the time-consuming process of bacterial multiplication and require at least 24 hours from the time of sampling to the possible determination that the water is unsafe for recreational use. Often it is feared that by the time the contamination is detected, significant exposures have already occurred. New methods are needed that would allow near real-time determination of contamination, such that public notifications could be made and exposrues avoided. The purpose of this task is to evaluate several new approaches of rapidly (less than 2 hours) measuring the presence of fecal contamination in recreational water. These approaches include the use of fluorescently- antibodies-paramagnetic bead complex, which would adsorb to specific fecal bacteria. The fluorescent labelled bacteria could then be detected using flow cytometers and lasers detection, or fiber optic systems. Other approaches make use of highly specific molecular amplification of bacterial nucleic acids or immunoassays on cells immobilized on filters (Task 7002). Each of these approaches will be applied to current indicators of fecal contamination, such as E. coli or enterococci, and also to other fecal bacteria, such as Bacteroides. The output of this research will be one or more methods able to measure water quality in a very short time interval. These methods will be used to measure water quality of bathing beach waters during a joint NERL/NHEERL epidemiological study in the summers of 2002-05 (Task 9656).

Record Details:

Record Type:PROJECT

Start Date:09/01/2000

Completion Date:09/01/2003

Record ID: 56169

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Project Information:

Progress :Several methodologies for the rapid microbiological analysis of recreational waters are being developed and evaluated in this task. All methodologies from this task which are deemed sufficiently mature will be used in an epidemiological bathing beach study due to begin in earnst this summer, 2003.

A real-time PCR method has been developed for the detection and quantitation of the enterococci group of bacteria. These bacteria will be the target group for all of the methods from this task (in addition, some methods may also be adapted to measure bacteria in the Bacteroides group). Real-time PCR involves the amplification of group-specific genetic sequences and the almost simultaneous detection of these amplified sequences, allowing one to quantify the number of target cells in the sample. Water samples are passed through a member filter to concentrate the bacteria and any inhibitory compounds are removed in an extraction procedure. This method was evaluated in the pilot epidemiological study in the summer of 2002 and is ready to be used this summer.

An antibody-based method employing a benchtop flow cytometer is also beeing developed in cooperation with the manufacturer of the cytometer (Advanced Analytical Technologies, Inc.) This method uses fluorescent antibodies that specifically react with several members of the enterococci group. Once labelled and thus fluorescent, the cells are passed, one at a time, through a small orifice and past a fluorescence detector, where the individual cells are then counted. Concentration protocols have been developed utilizing either immunomagnetic beads or centrifugation. Background fluorescence caused most likely by algal pigments interfere with the detection phase of the method. Extraction protocols to eliminate this background fluorescence are currently being developed. This method will be ready for field implementation this summer for the bathing beach study.

The IAG with the US NAVY has resulted in one instrument ready for field evaluation this summer. The Raptor System is an fluorescent antibody-based method that uses fiber optics for detection of target cells. Target cells are captured with specific antibodies and then labelled with fluorescent antibodies specific to other molecules on the cell surface. The binding of these fluorescent antibodies are then detected after optical excitation. This process is all completed in a small instrument, designed for field use. This method will also be used in the bathing beach study this summer.

The IAG with the US ARMY has yet to produce a method suitable for implementation in the bathing beach study.

Relevance :The U.S. Environmental Protection Agency is implementing an action plan for beaches and recreational waters that calls for the improvement of methods to measure recreational water quality and to re-define the relationship between gastrointestinal illness and the quality of bathing waters as measured with new rapid methods. The significance of this research will be that it will lead to better, scientifically defensible standards for surface waters and more meaningful monitoring approaches that will enable fecal contamination to be detected and beaches closed before significant exposures have taken place. This capability will be most useful to state and local public health authorities responsible for protecting the public from exposure to unsafe recreational waters.

Clients :Office of Water

Project IDs:

ID Code :5436

Project type :OMIS