Source Tracking Identifies Origins of Waterborne Pathogens

DNA fingerprinting does detective work

After identifying a particular contaminant in a water body, a water quality specialist's next task is to find or track its source. Once an inexact and uncertain science, source tracking has gained greater precision, with molecular techniques now available to identify the specific source of a contaminant, whether livestock, wildlife or human.

And even more specifically, some source tracking techniques can identify the type of livestock and wildlife, whether cow, pig, goat, geese, deer, racoon, beaver, etc. as the source of fecal contamination in a water source.

The use of source tracking at Sedona Creek demonstrates its potential. Sedona Creek is sporadically closed to swimmers and waders during the summer months due to high coliform bacteria counts. Arizona Department of Environmental Quality officials faulted leaking septic tanks in the canyon. In an effort to determine specifically the contaminant source, Gordan Southam, then a professor of environmental microbiology at Northern Arizona University, conducted DNA testing on water samples taken from various sites in 1998 and 1999.

He found the main culprits were nonhuman critters, with racoons contributing 30 to 35 percent of the coliform bacteria and other animals, including skunks, coyotes, elks, horses and even lamas, contributing about another 50 percent. Humans were responsible for about 16 percent of the bacteria.
Credit for the discriminating detective work is due to source tracking, also called genetic fingerprinting because the actual source of the contaminant is fingerprinted.

Milwaukee Crypto Outbreak Ten Years Ago

April 5 marked the tenth year anniversary of the Cryptosporidium outbreak in Milaukee. The epidemic led to fundamental changes in public health, disease surveillance, and water treatment and testing. Investigators initially focused on farm runoff as a potential source, but genetic analysis revealed that stool samples from infected Milwaukeeans contained a human strain of Cryptosporidium, not a bovine one. The official tally included 403,000 sickened, 44,000 doctor visits, 4,400 hospitalized, more than 100 deaths, 725,000 lost work or school days, $96 million in lost wages and medical expenses and $90 million for a new water purification system.

In applying source tracking or genetic fingerprinting, pure cultures of E.coli (or other enteric pathogens) are isolated from both the receiving water and the suspected sources. For example, the Sedona study involved isolating cultures from Sedona Creek and also from the various possible sources of the contaminant within the watershed, including wildlife, livestock and septic tanks. DNA isolates from the various watershed cultures were then compared to isolates found in the creek to determine the specific sources of pollution.
The use of molecular techniques in source tracking is relatively new, in use for about the last six years. It is an emerging field, with various methods having been developed and more being worked on as research intensifies to expand the potential of the field.

Much of the impetus for developing and applying source tracking techniques came from the U.S. Environmental Protection Agency and its implementation of the total maximum daily load (TMDL) concept. Since source tracking methods are effective tools for determining origins of fecal contamination of water bodies, they can be used to design best management practices to reduce fecal loading.

The molecular approach has distinct advantages over the cultural technique, once the standard method for identifying waterborne pathogens. University of Arizona assistant research scientist Kelly Reynolds says, "We use to rely on growing organism in the laboratory on specific media until we found out that only about one percent of the organism in any environment is actually culturable.

"Molecular techniques look at a much bigger, broader, diverse picture of the microbial community than do cultural techniques. They have enabled us to look at more of the ecosystem instead of only organisms that grow on food we feed them in the laboratory. Molecular methods have definitely taken the forefront."
Reynolds adds, however, it is not an either-or matter, that cultural and molecular techniques are sometimes combined, to take advantage of the benefits of each method. She says, "Typically, people use a variety of methods to make an assessment of the overall picture."

A researcher attempting to determine the source of a pollutant has various source tracking strategies to choose from. One of the more widely used techniques is ribotyping. Ribotyping is able to detect with a high degree of accuracy sources of bacterial contamination, perfectly matching isolates from humans and many different animals.

Each microbial pathogen, including individual strains or subspecies of bacteria, has a unique genetic makeup, and ribotyping determines DNA "fingerprints" of the bacteria or virus. The sample is then matched with E. coli isolates from a contaminated site and potential contaminant sources in the project area to determine a specific source.

Another commonly applied source tracking technique, antibiotic resistance analysis (ARA) relies on patterns of antibiotic resistance of bacteria from human and animal sources. Human fecal bacteria and animal fecal bacteria differ in their resistence to certain antibiotics since humans and farm animals are exposed to different sets of antibiotics. Further, various agricultural species, e.g. cattle, pigs and poultry, each receive different antibiotics. In contrast, wild animals receive relatively little exposure to antibiotics. As a result, their fecal bacteria would not be expected to exhibit substantive antibiotic resistance. ARA's determination of the ability of bacteria to grow in the presence of different antibiotics is a type of fingerprint, used for identifying individual sources of bacterial contamination.

Identifying a pollutant source can be the key to resolving a particular problem. Chuck Gerba, a professor within the UA Soil, Water and Environmental Science Department, says, "An advantage of source tracking is that the source can be identified, and this helps determine a solution to the problem. Before, there was a lot of guess work."

For example, Gerba was involved in study at the Tres Rios wetlands in Phoenix, a constructed facility fed by water released from a wastewater treatment plant. Water leaving the wetlands tested with high levels of E. coli. Whether the high levels were due to human or animal sources was at issue. If the increase traced to a human source, water leaving the wetlands might need further treatment. Using antibiotic resistance analysis and biochemical fingerprinting, Gerba found that although some increase was due to human sources, birds also were a factor.

Gerba says, "We did fingerprinting of the birds and the sewage, and we found it to be about 50 50." The bird percentage increases during the winter months when migrating birds pass through. The situation did not warrant further water treatment.

Gerba says source tracking also has helped to identify causes for diseases of previously unknown origins. For example, he says, "Epidemiological relationships have been established between drinking untreated groundwater and getting ulcers, and nobody would have guessed that 20 yrs ago. They can fingerprint what is in your ulcer and what is in your tap water and find the same bug."

Establishing the source of a pollutant serves other than just public heath purposes. That direct responsibility for an waterborne outbreak can be determined means liability also can be ascertained, thus opening the door to litigation. Gerba warns, "It can become a field day for lawyers."