From Cacapon September 2002

Potomac Highlands Watershed School

Can the Poop Detectives solve a Pollution Mystery? 

by David Malakoff

Who done it?

Bacteria after 24 hours growing on a filter.  The blue colonies are fecal coliforms.That’s the question mystery writers have posed for decades. Now, clean water specialists are asking it too. Which animals—from chickens and cows to deer and people—produce the fecal matter that is washing into and polluting the Cacapon and thousands of other rivers around the world? And just as in a novel, the water specialists are calling in some savvy detectives to track down the culprits: an emerging breed of scientific sleuths armed with the latest tools for tracking riverborne fecal pollution back to its source.

Solving this mystery, however, will have major real-world economic impacts. Judges have ordered government officials to develop realistic plans for cleaning up fecal pollution problems in many waterways. But without accurate information on the sources of the problem, money might be wasted attacking the wrong problems. Along the Cacapon, for instance, it isn’t always clear if cows, chickens, people, or wildlife produce the problematic poop that sometimes enters the river—meaning it sometimes isn’t clear if cleanup efforts should focus on fencing livestock from the river, repairing faulty septic systems, or other activities.

To reduce that uncertainty, some microbiologists—let’s call them the Poop Detectives—are developing new methods for tracking down the sources of the potentially harmful bacteria carried in fecal matter. But while many of the methods show promise, the infant science of microbial source tracking (MST) is beset by growing pains. Researchers have yet to agree on standard methods, and there is plenty of debate over the soundness of certain approaches and the interpretation of findings—including data from rivers right here in the Appalachians.

Luckily, help may be on the way. Earlier this year, scientists backed by the Environmental Protection Agency (EPA) released a report that calls for a sustained nationwide study to test, compare, and improve tracking methods. And some of the earliest studies include rivers and wells in West Virginia and Virginia, and could produce findings that will help eventually help the Cacapon Institute solve the Cacapon’s fecal pollution problem, which can occasionally cause its waters to become unsafe for water contact recreation such as swimming and boating.

Stopping the fingerpointing.

For decades, public health officials have measured water quality by monitoring levels of fecal coliforms, bacteria that live in animal guts and survive in the environment when expelled along with feces. Although the coliforms may not cause disease, they can be accompanied by a rogue's gallery of pathogens -- including microbes that cause hepatitis, cholera, and gastrointestinal illnesses. As a result, state officials routinely close swimming areas, wells, and shellfish beds when fecal coliform counts rise above certain levels. In West Virginia, for instance, surface waters are considered unsafe for recreation when fecal coliform counts rise above 400 cells per 100 ml, as sometimes happens along the Cacapon during rainy periods that wash fecal matter into the river.

Unfortunately, rules that regulate fecal counts don’t always keep waterways clean. Although modern sewage treatment has eradicated many of the worst problems, EPA estimates that at least 20,000 miles of streams and coastal waters still carry bacterial loads that exceed health standards. Due to these violations, environmentalists have won a string of court victories in the last decade that require states to set goals for reducing bacteria counts. But efforts to set these targets--called Total Maximum Daily Loads (TMDLs)--often have become exercises in finger-pointing, as farmers, homeowners and wildlife biologists have argued that someone else is to blame.

Desperate to calm such conflicts, TMDL specialists have asked microbiologists to come up with objective ways to pinpoint bacteria sources. Despite sparse funding, a handful of researchers have developed methods that range from fingerprinting the DNA of different species of bacteria to techniques that use viruses that are unique to specific animals to track poop sources.

To date, the most prominent techniques are so-called "library-dependent" methods, which require researchers to match a bacterium found in a waterway to one included in a previously created library of bacteria from known sources. On Page Brook in Clarke County, Virginia, one of the pioneers of source tracking—Charles Hagedorn of Virginia Polytechnic Institute and State University (Virginia Tech) in Blacksburg--used a library-dependent technique called antibiotic resistance analysis (ARA) to track down the sources of pollution in that stream. After the 1997 study, he concluded that cows—not septic systems--were the primary source of the stream's bacteria, prompting county officials to raise funds for fencing projects that have lowered bacterial counts by up to 98%.

The ARA method, developed by Bruce Wiggins of James Madison University in Harrisonburg, Virginia, assumes that the strains of bacteria in people, farm animals, and wildlife have been exposed to specific antibiotics (or none) and therefore show resistance to different kinds. To catalog these signatures, Hagedorn's team first combed the small watershed, collecting poop from major potential sources, such as people, cows, and deer. The researchers then cultured Enterococcus bacteria from the samples, exposed the microbes to a battery of antibiotics, and recorded the results. Later, they found that Enterococcus taken from the stream had signatures that--statistically, at least--matched cataloged signatures produced by cattle-borne bacteria.

Many genetic approaches also require libraries, with researchers seeking to match the unique DNA or RNA profiles of bacteria from waterways with those from bacteria associated with different sources. For example, over the past 5 years, another Virginia Tech scientist, George Simmons, used a genetic technique called pulsed-field gel electrophoresis to fingerprint bacteria sources in Four Mile Run, an urbanized stream near Washington, D.C. Simmons, now retired, found that it carried bacteria that matched those from several hosts, including significant contributions from hard-to-control wild sources such as raccoons and geese.

THE PROBLEM WITH LIBRARIES by W. N. Gillies.

Using DNA to identify criminals based on tissue or blood samples found at a crime scene is very different from using DNA signatures to track intestinal bacteria back to their "host." In the first case, you are looking at tissue or fluids that are actually part of an individual; in the second you are looking at a separate organism that lives in an individual. The underlying assumption here is that certain intestinal bacteria are characteristic of certain species (or, possibly, groups of related species-such as deer, cows, sheep, elk). Researchers are trying to separate intestinal bacteria into "resident" kinds – kinds that "belong there", and "transient" kinds – strains that animals and people pick up on their travels.

In his pioneering work at Virginia Tech in the mid-90s, Dr. George Simmons discovered that individual species in a small geographic area carried many different strains of E. coli (for example, his sample library of known raccoon fecal material from 14 raccoons contained 64 distinct E. coli strains), and that no strains were consistently found in all individuals of a single species. In other words, no single strain jumped out and said "raccoon" or "deer" and he was unable to distinguish raccoons from deer and muskrats in his small study area using DNA alone.

It should surprise no one that animals sharing the same environment also share many of the same bacteria. In 1995, USEPA researcher C.A. Kreader found that she could distinguish human strains of Bacteroides from those found in farm animals, but not from those found in the people’s pets.

The new EPA report, however, cautions that such findings are typically based on small studies and use methods not yet widely replicated. Various source-tracking methods have been applied to fewer than 125 waterways, and reports in peer-reviewed journals are scarce. The report calls for a four-phase national study to find out what really works, starting with basic experiments to see if single laboratories can replicate their own results. The study would end with a real-world test designed to compare different methods in a single, complex watershed.

The report is silent on how much such studies might cost and where funding might come from. The U.S. Geological Survey and Orange County, California, are funding small comparison studies—including one involving streams in West Virginia--and California is considering a multimillion-dollar effort. Virginia, meanwhile, is the first state to require that MST be used in the development of TMDLs, giving researchers a chance to refine their techniques.

New studies could help settle some simmering technical debates over the validity and practicality of several methods. One of the most pressing questions facing MST researchers is whether a library constructed for one study can be used for others (see Box page 3). Intestinal bacteria can vary widely by place, season, and time of day, and even diet can shift the dominant strains found within an individual. Given this variability, some MST researchers expect that they will have to construct new libraries for every new watershed, driving up expenses dramatically.

To avoid that problem, some researchers are exploring techniques that wouldn't require expensive libraries. Microbiologist Katharine Field of Oregon State University in Corvallis, for instance, is looking at using certain Bacteroides bacteria, which may carry host-specific genetic markers that vary little from place to place. Other techniques seek to detect widespread, species-specific antibodies that adhere to shed bacteria.

Fully developing and testing such methods, however, will require researchers to adopt standard methods and share data. And so far, that’s proved to be a stumbling block. But it’s one that is critical to surmount if MST results are to become trusted by the public and policymakers, who must decide how to spend billions of taxpayers dollars on improving water quality.

Here in West Virginia, for instance, the Cacapon Institute has challenged the way some MST data collected in Potomac River tributaries was interpreted by state agriculture officials. They claimed the preliminary data showed that farms along the river weren’t a major player in the waterway’s pollution – but we noted that there really wasn’t yet enough information to support that—or any other—conclusion.

With patience and careful work, however, the new breed of poop detectives could be producing some firm conclusions in the future. And while solving a poop mystery may never be a bestseller, it may prove to be an invaluable tool in cleaning up our nation’s waters.

This article is based on one that originally appeared in Science magazine, March 29, 2002, pp. 2352-2353.

For more information on BST, see the February 5, 2002 U.S. EPA Workshop on Microbial Source Tracking at http://www.sccwrp.org/tools/workshops/source_tracking_agenda.html

and Charles Hagedorn's Bacterial Source Tracking website at Virginia Tech: http://soils1.cses.vt.edu/ch/biol_4684/bst/BST.html.

$ $ $ $ $ $ $ $ Making it Real $ $ $ $ $ $ by W.N. Gillies, Cacapon Institute.

Once you have a Microbial Source Tracking (MST) method that passes scientific muster for reliability, the next step is to use the method to solve real problems. Let’s say you have a stream with a documented bacteria problem -there are many such streams – and you want to determine the dominant reason for contamination —and there are many possible bacterial sources.

Fecal coliform bacteria after 24 hours of growth.  Each blue colony started out as one bacterium.You go into the field and collect a 100-200 ml water sample. That sample represents a single site/date/flow combination and a very small fraction of the water in that stream. You filter a small portion of your sample in the lab, incubate it, and the next day find 50 colonies growing on your filter (picture at left). Each colony grew from a single bacterium. Several—let’s say 10--potential bacterial sources could be combined among the 50 colonies and different combinations might dominate at different times and conditions. How many of those colonies and how many samples, at more than $50 for each colony identified using DNA methods, do you need to run through your MST method before you have reasonable certainty of your answer?

KA-CHING!! The dollars add up very quickly and, in some cases, might be better spent fixing known problems on the ground that reasonable people agree are a likely cause of the problem.

In other cases, however, MST has the potential to help decision-makers save real money. For example, MST might show that fecal contamination of wells in a karst region came from cattle rather than people, and the very expensive "remedy" of a rural sewerage system could be avoided.