As she walks into her microbiology laboratory at Oregon State University, Kate Field hands her graduate student a Ziploc bag full of tubes of fecal samples. "These are just in from New Zealand," she says with a smile. For just being handed what essentially amounts to a bag of poop, her graduate student seems pretty excited as well. Now, the student's job is to test whether the genetic markers Field has developed can reliably identify what type of animal produced the sample. "Right now they're working pretty well," Field says.
Field is developing a number of PCR-based methods to track fecal contamination culprits. She pulls out a figure of a host clade with dozens of branches to demonstrate the variety of species-specific microbial markers from Bacteroides bacteria, including cat, pig, elk, and human. Her assays look for the presence of particular ribosomal RNA sequences within groups of anaerobic bacteria that live in animal gut (App Environ Microbiol, 71:3184-91, 2005). "These [anaerobes] are the most common in feces, as opposed to E. coli and Enterococci, which are the most easy to grow," Field says.
Field says the technique is superior to the traditional and more time-consuming method of bacterial culture that has dominated the field for 100 years. "I think that most of the labs that are working on MST [microbial source tracking] now are switching to host-specific PCR assays," says Jorge Santo Domingo, a microbiologist with the US Environmental Protection Agency's Office of Research and Development in Cincinnati.
Poop trackers mostly find sources of pollution in water, often in swimming beaches and shellfish beds. When pollutant and microbial levels exceed federal and state limits, scientists and technicians step in to find the source and remediate.
A Florida company, Source Molecular, uses a variation of Field's technique on surface proteins in Enterococci. In a recent project, the company hunted for bacterial hotspots following back-to-back hurricanes in Gainesville. It found that negative controls taken from a pristine stream were lighting up with microbial markers, yet no sewers, homes, or wastewater treatment plants were nearby. "But we know when we have contamination," says Troy Scott, the company's senior research scientist. The company's team fanned out around the site and came upon a large homeless camp near the water, littered with human feces.
Mike Sadowsky at the University of Minnesota has trained a robotic hybridizer to analyze 40,000 E. coli samples from duck and geese feces simultaneously through suppressive subtractive hybridization. In one experiment, the technique tracked 51% of fecal bacteria from two urban Minnesota lakes to geese and ducks (Appl Environ Microbiol, 73:890-6, 2007). Santo Domingo and his colleagues are developing a technique based on fecal metagenomes to develop host-specific gene fragments.
Each technique has its drawbacks in time and cost: Robotic hybridizers, for example, run about $500,000 and the process takes four days, whereas genomic markers are still in early development. Currently no standardized protocols exist to guide poop trackers to the best method, and analyses so far have found "a lot of problems with all of these methods," says Charles Hagedorn, a professor of environmental microbiology at Virginia Tech. In a comparison of seven source-tracking protocols, Hagedorn and his colleagues found most gave false-positive results (Environ Sci Technol, 38:6109-17, 2004).
One year after Hurricane Isabel swept along the North Carolina coast, Hagedorn found himself knee-deep in water at a swimming beach contaminated with high counts of Enterococcus, a bacterial indicator of human feces; nothing in sight could lead him to where the contamination was originating. Hagedorn turned to his real-time sewage-tracking device, a fluorometer strapped to a raft. Optical brighteners in detergent - a sure sign of human sewage - emit at 410 nm and excite at 370 nm. While Hagedorn fed samples to the fluorometer, he was able to follow a plume of sewage runoff back to a particular section of beach. Engineers dug under the beach to determine where the leak was emanating; they found that a sewer line cap had corroded and leaked. "As soon as they dug a hole, it filled up with raw sewage," Hagedorn says.