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Bacterial/Microbial Source Tracking (BST/MST)-
A Review

Soil & Water Conservation Society of Metro Halifax (SWCSMH)

Modified: September 04, 2016                  Soil & Water Conservation Society of Metro Halifax (SWCSMH) Master Homepage

(cf. Mandaville, S.M. 2002)


Contents:




Abstract/Summary

Bacterial Source Tracking (BST) is a new methodology that is being used to determine the sources of fecal bacteria in environmental samples (e.g. from human, livestock, or wildlife origins). BST methodology has been described as having the ability to turn nonpoint sources into point sources. BST is also called Microbiological Source Tracking (MST), fecal source tracking, or fecal typing.

This review concludes that there is no easy, low-cost method for differentiating between human and non-human sources of bacterial contamination at the present time. Quantifying the contribution from different sources is as yet not possible.

The North American research into various identification methodologies is ongoing and it is difficult at the present time to definitively recommend a single one as the most preferable which could be used in perpetuity. Not all methods are being reviewed in this report.

A selection of representative published papers in peer reviewed journals and/or at scientific conferences are herewith included in Section 5: Appendices.

A "toolbox" approach seems warranted where the method chosen depends on the types of fecal sources that one has to deal with (or the questions one needs answered). Obtaining similar results with different BST methods also improves the chances that the source identifications are correct. (Hagedorn, 2002)

The field of bacterial source tracking (BST) continues to evolve rapidly, and researchers see promising developments emerging.

[Img-Prof. Kate Field PhD] One of the challenges is to identify a low-cost, simple methodology. Based on literature survey and consultations, the fecal Bacteroides-Prevotella detection methodology of Prof. Katherine Field PhD of the Dept. of Microbiology, Oregon State University, Corvallis, Oregon appears to be the most economic as well as reliable. Prof. Field’s methodology generally does not require samples of feces of common sources like humans, dogs, ducks, cows. Only the water sample filters are needed for her analyses.

Further, the New York State Department of Health (cf. Email from Prof. Ellen Braun-Howland PhD, June 2001) is in preference of the aforementioned methodology of Prof. Katherine Field. The New York State chose not to go with ribotyping (e.g., Washington State) or antibiotic resistance genes since both those methods require the establishment of large libraries of organisms before one can get any meaningful results. They found it costly and time-intensive.

And based on a timely state-of-the-art Microbiological Source Tracking Workshop organized by the Southern California Coastal Water Research Project Authority (SCCWRP, 2002) in cooperation with several Government stakeholders, Scientist, John Griffith also prefers Prof. Kate Field’s Bacteriodes source ID (pers. comm. February, 25, 2002).

Notwithstanding, there is considerable support for genetic fingerprinting involving isolates of pure cultures of E. coli which is the preferred methodology of the Washington State Department of Ecology among numerous other agencies. Ribotyping is a molecular method first applied to bacterial source detection 11 years ago. Based on discussions with Prof. Mansour Samadpour, I find it quite costly and intensive both in the field as well as in the laboratory work. Dr. Samadpour of the University of Washington is a leading specialist in ribotyping and continues to apply it widely. He is reputed to have the largest library of DNA signatures in North America and has consulted with some regulatory agencies in British Columbia as well).

There was a state-of-the-art Microbiological Source Tracking Workshop organized by the Southern California Coastal Water Research Project Authority (SCCWRP, 2002) in cooperation with the US Environmental Protection Agency, State Water Quality Control Board, and the National Water Research Institute during February 5-7, 2002.

    The goals of the workshop were to:
  1. bring leading researchers together with regulators and other interested parties to discuss the current state of methods used to track microbial contaminants in surface waters, and
  2. begin the process of developing a protocol for a comparison study of microbial source tracking methods. It brought together prominent researchers in the field.
    The SCCWRP member agencies were:
  1. Orange County Sanitation District
  2. City of Los Angeles Bureau of Sanitation
  3. County Sanitation Districts of Los Angeles County
  4. California State Water Resources Control Board
  5. California Regional Water Quality Control Board, Los Angeles Region
  6. City of San Diego Metro Wastewater Dept.
  7. California Regional Water Quality Control Board, San Diego Region
  8. California Regional Water Quality Control Board, Santa Ana Region, and the
  9. U.S. Environmental Protection Agency, Region IX


Introduction


[Img-pathogenic_enteric_bacteria_1.gif] Fecal contamination of aquatic environments afflicts many regions and may carry risks to human health. For example, human fecal pollution may spread dangerous bacterial and viral pathogens, such as hepatitis, while other human pathogens such as Cryptosporidium parvum, Giardia lamblia, Salmonella spp., and E. coli O157:H7 are associated with animal fecal pollution.

Further, contamination of coastal and inland waters by fecal bacteria results in beach closures that suspend recreation and strike a blow to the economies of beach communities.

Often the source of fecal contamination in water cannot be determined. For example, non-point sources such as failing septic systems, overloads at sewage treatment facilities, overflows from sanitary sewage pumping stations, or flows from sewage pipe breaks may all be candidates. In addition, the contribution of bacterial pollution "stored" in sediments and re-suspended during storm events is unknown. In order to adequately assess human health risks and develop watershed management plans, it is necessary to know the sources of fecal contamination.



Erroneous assumptions of fecal sources

Commonly, fecal sources are misinterpreted and more often than not the sources are assumed to be anthropogenic in nature as a result of lack of credible scientific investigations. Following is a full extract from the USEPA’s Nonpoint Source News-Notes:

cf.   DNA Fingerprinting Aids Investigation — Fecal Coliform Sources Traced to Unlikely Suspects. Issue Number: 48, Chapter Name: Technical Notes. Date: 04/97

Tracking the source of fecal coliform contamination may not sound as exciting as reading a good mystery. But recent research into fecal coliform pollution in the southern Chesapeake Bay had more than enough suspense to qualify as a mystery, including several suspects and high tech investigative methods. By systematically tracking clues in the field and using DNA fingerprinting in the lab, researchers at the Virginia Polytechnic Institute and State University appear to have cracked the case.

According to George M. Simmons Jr., Alumni Distinguished Professor at Virginia Tech, tracking the source of fecal coliform contamination was actually a spinoff of other research he was conducting on Virginia's Eastern Shore. It started when Roger Buyrn, a local land owner and clam grower, approached him with the case. Buyrn faced losing his clam business because of contaminated harvest areas in a tidal creek. Harvest area closures can have serious negative impacts on the local economy, and they are becoming commonplace. According to the Division of Shellfish Sanitation in Richmond, Virginia, the number of acres condemned over the last two decades for shellfish harvest in Virginia has increased from 62,272 acres in 1970, to 98,826 acres in 1994. Hoping to avoid condemnation of his own harvest area, Buyrn opened up his land to Simmons and his team of student investigators.

Rounding Up the Usual Suspects

Nonpoint fecal coliform sources may come from human waste, agricultural areas, or wildlife. Although identifiable sources of coliform contamination are not always evident, fecal coliform sources are often linked to faulty on-site waste disposal systems. Few of the inlets in the study area however, were inhabited, intensifying the mystery.

The researchers tracked fecal coliform levels in the field by sampling at land/water interfaces and across marshes or up creeks. Simmons points out that rigorous sampling in creek beds, marshes, and each tiny rivulet that drained to them was the major tool for tracing the fecal coliform signals. Once the general source areas were identified, the study group monitored them extensively.

All along, Simmons and his colleagues believed that the sources would be anthropogenic in nature. ‘It took me about a year to come to grips with the fact that the fecal coliform were not coming from a leaky septic tank or other effluent,’ said Simmons. ‘It got to the point where I was climbing trees to see if I could see any potential sources (i.e., houses), but there weren't any. We all were pretty baffled.’

Breaking the Case

Simmons used DNA fingerprinting of E. Coli to confirm his growing suspicion that the sources were not human. Simmons's group collected fresh fecal samples from raccoon, waterfowl, otter, muskrat, deer, and humans in the area and analyzed and characterized the DNA of coliform found in the samples. The result was a library of more than 200 DNA patterns distributed through more than 700 E. Coli strains. In the process, the researchers developed a DNA dichotomous key and an index of descriptions for the known strains.

Identifying unknown sources was a challenging endeavor. Some E. Coli strains are specific to certain animal species; others may contain several different strains. ‘A total of 88 E. Coli strains from unknown sources were fingerprinted, and of those 88 strains, 58 of them resulted in some degree of [species] identification using the dichotomous key of E. Coli strains from known sources.’ In addition, 22 of the strains closely matched known strains in the existing library. Simmons noted that only eight of the 88 unknown samples collected from the study area could have come from a human source. Comparing E.coli from the samples against the fingerprints of known strains in the DNA library, Simmons traced the sources to deer and raccoon.

Victim's Reprieve

During the winter of 1993, several hundred animals, including deer, raccoon, and muskrat, were removed from the Buyrn property and other nearby areas and by spring 1994, fecal coliform had decreased by one to two orders of magnitude. Threatened areas of the tidal creeks were reopened or escaped closure.

Epilogue

Major funding for this project came from the Virginia Department of Conservation and Recreation, Division of Soil and Water Conservation, and from the Virginia Department of Environmental Quality, Coastal Resources Management Program through a grant from NOAA. In his next case, Simmons hopes to expand the DNA library to see if it is applicable to other parts of the Chesapeake Bay and beyond. As funding becomes available, he would also like to develop libraries for agricultural and urban areas which would also include stormwater runoff. ‘This research to date indicates that field and laboratory methods, alone or in combination, provide a very high likelihood that nonpoint fecal coliform sources can be identified and remediated for the improvement of water quality,’ concludes Simmons."


Microbiological Indicators

Bacterial Pollution Indicators versus Fecal Bacterial Source Indicators

Indicators are used when quantifying possible impacts to water from animal and sewage wastes. They are usually used as a surrogate for more harmful pathogens. It is impossible to try to identify all the enteric pathogens present in the water. The cost is too great and the techniques have not been developed to test for all known pathogens.

There are three important requirements of an indicator. It should be native to the intestinal tract, enter the water with fecal discharge, and be found in the presence of other enteric pathogens. The indicator should normally survive longer than their disease-producing companions. They should be easy to isolate and identify.

Since the early work of Escherich, who identified Bacillus coli (now Escherichia coli) as a dominant bacterium in feces, considerable effort has been expended to identify the dominant bacteria associated with the gastrointestinal tracts of humans and warm-blooded animals.

Total Coliform (TC)

Total coliform was originally used as an indicator of fecal contamination in surface water. Total coliform includes all the aerobic and facultative anaerobic, gram-negative, non-spore forming bacilli that, when incubated at 35oC, can ferment lactose and produce gas within 48 hours. This definition includes the genera Escherichia, Citrobacter, Klebsiella, and Enterobacter. Not all of these organisms inhabit the intestinal tract of warm-blooded animals exclusively.

Fecal Coliform (FC)

The fecal coliform group is a sub-group of total coliform that grow mainly in the intestines of warm-blooded animals. These organisms may be separated from the total coliform group by their ability to grow at elevated temperatures. The most common member of this group is Escherichia coli (an enteric bacteria), but also includes Klebsiella, Enterobacter, and Serratia which can also be found free-living on plants and in soils.

Fecal Streptococci (FS) and Enterococci Groups

Another group of bacteria less numerous in human feces than coliform is fecal streptococci. This group contains a number of species of the genus Streptococcus. Fecal streptococci indicate the presence of fecal contamination by warm-blooded animals. It is not known to multiply in the environment like fecal coliform. At one time S. faecalis and S. faecium were thought to be more human-specific than other Streptococcus species. Other species have been observed in human feces but less frequently. At the same time, S. bovis, S. equinus, and S. avium are not exclusive to animals, although they usually occur at higher densities in animal feces.

Enterococci are a sub-group of the fecal streptococcus group. This group consists of a number of species of Streptococci, S. faecalis, S. faecium, S. gallinarum, and S. avium. The enterococci portion of the fecal streptococcus group is a valuable bacterial indicator for determining the extent of fecal contamination in surface waters. Water quality guidelines based on enterococcal density have been proposed for recreational waters.

Other Bacterial Indicators

The intestines of warm-blooded animals host an incredible variety of bacteria. Most bacteria are a part of the normal intestinal flora. The type and quantity of bacteria species present can vary depending on animal species (Table 1). Testing for specific bacterial species more common to certain animals can give information on possible bacterial sources. Some of the more species-specific microbiological indicators are described below. It is important to remember that bacteria flora can vary among the same species due to diet or location. Also, bacteria can colonize other animals if the environment is hospitable.


Table 1. Numbers of viable bacteria found in the feces of adult animals: logarithm of viable count per gram of feces (Todar, 2002)*
Animal Escherichia coli Clostridium perfringens Streptococci Bacteroides Lactobacilli
Cattle 4.3 2.3 5.3 0 2.4
Sheep 6.5 4.3 6.1 0 3.9
Horses 4.1 0 6.8 0 7.0
Pigs 6.5 3.6 6.4 5.7 8.4
Chickens 6.6 2.4 7.5 0 8.5
           
Rabbits 2.7 0 4.3 8.6 0
Dogs 7.5 8.4 7.6 8.7 4.6
Cats 7.6 7.4 8.3 8.9 8.8
Mice 6.8 0 7.9 8.9 9.1
Humans 6.7 3.2 5.2 9.7 8.8
*Median values from 10 animals


Table 2. Bacteria found in the large intestine of humans (Todar, 2002)
Bacterium Range of Incidence
Bacteroides fragilis 100
Bacteroides melaninogenicus 100
Bacteroides oralis 100
Lactobacillus 20-60
Clostridium perfringens 25-35
   
Clostridium septicum 5-25
Clostridium tetani 1-35
Bifidobacterium bifidum 30-70
Staphylococcus aureus 30-50
Enterococcus faecalis 100
   
Escherichia coli 100
Salmonella enteritidis 3-7
Salmonella typhi 0.00001
Klebsiella sp. 40-80
Enterobacter sp. 40-80
   
Proteus mirabilis 5-55
Pseudomonas aeruginosa 3-11
Peptostreptococcus sp. common
Peptococcus sp. moderate
Methanogens (Archaea) common



Fecal Coliform to Fecal Streptococci Ratios (FC:FS)

The ratio of fecal coliform (FC) to fecal streptococci (FS) concentrations has been used to try to differentiate human from non-human sources of fecal contamination. A ratio of four or greater is considered human fecal contamination, a ratio of less than 0.7 suggests non-human sources. The value of this ratio has been questioned because of variable survival rates of fecal streptococcus group species.  Streptococcus bovis and S. equinus die off rapidly once exposed to aquatic environments, whereas S. faecalis and S. faecium tend to survive longer.

The ratio is also affected by the methods for enumerating fecal streptococci. For these reasons, Standard Methods (Eaton et al., 1995) and the Canadian Council of Ministers of the Environment (CCME, 1993), among other government agencies, do not any longer recommend FC:FS ratios as a method for differentiating between human and non-human sources of fecal contamination.

Peruse also the email from the Ontario Ministry of the Environment (2001).




Bacterial Source Tracking (BST) Methods

BST methods vary in many ways, including costs, level of training and expertise required to implement the method, turnaround time, degree of statistical rigor, and whether a given method is location-specific or database-dependent.

Both molecular (genotype) and biochemical (phenotype) BST methods are under development. DNA fingerprinting has received the greatest publicity, but to date there are at least ten or so different methods described in the scientific literature that show potential. However, BST development is so new that no extensive research comparing BST methods or identifying their relative strengths and weaknesses has yet been completed. Such comparison research has only recently been started, and results should become available over the next few years. At this point it seems reasonable to assume that some combination of BST methods will be needed to provide the most accurate and reliable source identification answers. It is doubtful that any one BST method will emerge as the "best" method for all situations.

Molecular methods may offer the most precise identification of specific types of sources, but are limited by high per-isolate costs, detailed and time-consuming procedures, and are not yet suitable for assaying large numbers of samples in a reasonable time frame. Biochemical methods may or may not be as precise, but are simpler, quicker, less costly, and allow large numbers of samples to be assayed in a short period of time. Perhaps the best approach at this point is to use a biochemical method to determine sources on large numbers of fecal bacterial isolates, and then confirm (validate) both the method and the results by assaying some smaller subset of isolates with a molecular procedure.

Reliability of BST

At present, BST can reliably determine if fecal bacteria are from human or animal sources. If the bacteria are from animal sources, BST can also tell if the animals are livestock or wildlife, but less reliably than the human vs. animal separation. It is unknown at this time if BST can eventually achieve distinctions between different types of livestock (e.g. cattle, horse, swine, poultry, etc.) or wildlife (e.g. deer, waterfowl, raccoon, etc.) or pets (dogs, cats, etc.). Many in the molecular biology community believe such fine differentiation is feasible.

BST methods have been used primarily with E. coli and the fecal streps, and to a lesser degree with bifidobacteria, Bacteroides-Prevotella, and coliphages. While regulatory agencies often prefer E. coli because that is the most widely used indicator standard, there are good reasons to consider other fecal organisms, depending on the situation. For example, E. coli is difficult to detect in composted poultry litters or in high quality biosolids, while the fecal streps are very numerous in both materials.

Also, there is increasing evidence of regrowth of E. coli and fecal streps in tropical and/or subtropical waters and sediments. Under these conditions, other organisms such as the bifidobacteria, Bacteroides-Prevotella, or coliphages may be better organisms for source tracking and perhaps also as better indicators of fecal pollution.

With all BST methodologies (except chemical) it is necessary to first build a library or database of isolates taken from known sources, e.g., human, cow, deer, etc. The size of the library will be partly determined by the number of potential major sources of fecal pollution in the target area. Nobody knows at this point how large a library has to be or how similar libraries from different geographical areas may be. Publications to date indicate that at least a few hundred isolates from each major known source will be needed. Once the known source library has been developed, and correct source identifications are sufficiently high for the desired purpose, then the second step can be taken of comparing fecal isolates of unknown origin against the library to obtain source identification. It is also necessary to regularly recheck the database with known source isolates to be sure that correct source identification remains high. (Hagedorn, 2002)



Well published investigators conducting research in BST (Hagedorn, 2002)



References



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