OREGON STATE UNIVERSITY

Test developed to determine source of fecal coliform

01/31/2002

CORVALLIS - A microbiologist at Oregon State University has developed a test for the source of fecal contamination of water that is both faster and far more specific than existing tests, which rely on culturing fecal coliform bacteria.

Fecal contamination of water supplies is a growing worldwide problem, and finding the source of fecal contamination in waterways is essential - regulators and officials can't really begin controlling water pollution until they know where it comes from. One of the drawbacks with the standard fecal coliform tests is that, while they do confirm whether there is contamination, they don't differentiate between sources.

Is the source from cattle manure washing into the waterway from adjoining fields, or is it human waste spilled from a city's sewage plant or percolating through the groundwater from a failed septic system? A lot rides on the answer to that question, and in this example either the dairy industry, city officials or a specific rural resident might face serious regulatory pressure. But without knowing the source, it is hard for environmental officials to control the problem.

A test developed by Katharine Field, an OSU assistant professor specializing in environmental microbiology, molecular ecology and antibiotic resistance, shows promise of being able to make that determination, and in far less time than the standard tests. Field's work has been supported by research funds from Oregon Sea Grant.

Instead of focusing on fecal coliform, it looks for different bacteria associated with feces.

Field chose a group of anaerobic bacteria, bacteria that grow without oxygen, called Bacteroides-Prevotella, associated with feces. Then, using a process called polymerase chain reaction (PCR,) she creates millions of copies of a particular gene. Using those genes, she looks for specific markers, gene sequences unique to particular species.

"These bacteria are more diverse than coliforms, and there are more genetic markers," she said.

By isolating the genetic markers for different species, she can build a "library" of such indicators to compare with marker genes in samples taken from polluted water. The comparison should reveal the source. Field is working with the Tillamook Bay National Estuary Project to see if her tests can show exactly where these fecal markers are turning up, and when.

"A lot of ecological and water quality studies have been done there, and it has a relatively small population in a relatively confined area," qualities that make it ideal for the experiment, she said.

In another recent case, Field was approached to identify the source of polluted well-water on a Eugene-area farm. There were two possible suspects: composted human sewage that had been sprayed on a nearby field, and a chicken-farming operation.

The tests came back negative for a human source, Field said. Because she had not yet found a Bacteroides-Prevotella marker for chickens, she could not say definitely that chickens were the cause. As a result, she has gone back to the site to acquire the material that will allow her to find the genetic primer and make that distinction.

The Bacteroides-Prevotella test also seems to be much faster than other fecal source tests, another factor that could lead to its success. According to Field, other methods require growing large numbers of bacteria, testing them, then doing a fairly complex statistical analysis. The turnaround time for these approaches takes from two weeks to a month, she said.

For the BP test, which is still under development, the turnaround is about a week.

"It should take a day, but life is never that easy," she said.

While a patent application on the test works its way through the process, Field continues to work on the project. She wants to standardize the procedure so that a simple kit can be developed containing everything needed to perform the test. She also wants to expand the "library" of genetic markers, eventually to include all the animals likely to be the source of contamination, everything from cows and pigs to other ruminants, marine mammals, and even dogs and cats. Dogs, she noted, are a major issue for urban non-point fecal pollution, and a recent article in The Scientist magazine suggested that flushable cat litter may be a culprit in a die-off of sea otters in Northern California.

"There are a lot of species out there," she said.

Russell T. Hill, an associate professor at the University of Maryland's Center of Marine Biotechnology who reviewed Field's work, said the expansion of this work could be enormously important for efforts to control contamination of rivers and the ocean.

"If Katharine is successful, Bacteroides could emerge as the most useful single indicator of fecal pollution," he said. "We have known the limitations of fecal coliform counting for many decades. It will be nice to have a solution at last."