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OWML: Microbial Source Tracking

Identifying Hot Spots of Fecal Contamination

Identifying the presence of specific pathogens can help understand sources of fecal contamination.  Because viruses are often species-specific, monitoring for human-specific viruses (enteric viruses), such as adenovirus and enterovirus, can provide direct evidence of a human source.  Two of the most common methods for examining water for enteric viruses are cell culture and quantitative polymerase chain reaction (qPCR).  The cell culture method provides information on the infectivity of the viruses, but it is expensive and time consuming, requiring several weeks for confirmed positive results.  The qPCR method is semi-quantitative, more rapid, and less expensive than the cell culture method; however qPCR is a molecular method that detects both infective and noninfective viruses.  Monitoring for bacterial and protozoan pathogens provides less information than viruses on the source of contamination, but still provides information on human health risk.  Many bacterial and protozoan pathogens are zoonotic, meaning that they could be transferred from animals to humans and cause disease in humans.  These include E. coli O157:H7 and specific types of Cryptosporidium, Salmonella, or Campylobacter.  However, Shigella spp. are exclusively transferred from humans to humans.

Chemicals found in human wastewater can be used to discriminate between human and animal fecal sources. For a suite of 78 wastewater chemicals detected in a national study (Glassmeyer and others, 2005), there was a higher frequency of detection in wastewater effluents than in water samples collected upstream. Chemical concentrations decreased downstream as distance from the outfall increased. Specifically, the distinct changes in concentrations of the fragrance chemicals ethyl citrate, galaxolide, and tonalide between the upstream, effluent, and two downstream sites suggest that they may be good indicator chemicals for human wastewater discharge.

Microbial source tracking (MST)
Microbial source tracking (MST) is a term used for the process of identifying the source of fecal contamination in water or sediment.  MST techniques are based on the concept that various warm-blooded animal intestinal systems have different selective pressures caused by differences in diet and physiology that select for specific microbial populations.  MST attempts to categorize the microbial populations by host animal through identifying some unique genetic (DNA sequence) or phenotypic (observable characteristic or expression, such as antibiotic resistance) trait.  Despite some initial success using MST techniques, most of the methods are still experimental (Griffith and others, 2003) and no single MST method is ideal (Field and Samadpour, 2007). 

MST researchers have been moving away from library-dependent methods to library-independent methods.  Library-dependent methods were shown to include high source misclassification rates and higher costs in building large known-source libraries for a particular area (Stewart and others, 2003).    A commonly-used library-independent method is the use of host-associated markers.  For this method, a targeted genetic sequence of DNA is copied or amplified by the polymerase chain reaction (PCR) into an amount that can be characterized.   The use of host-associated markers has gained favor among MST researchers because of improved accuracy and reduced cost and time to perform over library-dependent methods.   In addition, PCR methods have the potential to be sensitive, quantitative (qPCR), and automated (Stewart and others, 2003; Santo Domingo and others, 2007).

Host-specific markers have been identified from different groups of fecal-origin bacteria, often from the genus Bacteroides, a bacterium abundant in the gut of warm-blooded animals.  The following list describes the different MST markers that have been utilized in the OWML:

  • Two general Bacteroides marker present in most warm-blooded animals (AllBac: Layton and others, 2006; GenBac: Siefring and others, 2008)
  • Two human-associated markers (HF 183: Bernhard and Field, 2000; BacHum: Kildare and others, 2007)
  • A ruminant-associated marker (BoBac; Layton and others, 2006)
  • A gull-associated marker from the species Catellicoccus marimammalium (Gull2; Lu and others, 2008)
  • A dog-associated marker (BacCan: Kildare and others, 2007)
  • A chicken-associated marker from Brevibacterium  sp. bacteria (LA-35; Weidhaas and others, 2010)
  • Two Canada goose-associated markers (CGOF1 and CGOF2; Fremaux and others, 2010)

Utilization of MST markers to characterize different sources of fecal contamination has the potential to be a powerful tool in identifying major contributors to water-quality deficiencies (Santo Domingo and others, 2007; Balleste and others, 2010).  Given that the application of MST markers is a relatively new tool, broad generalizations should not be made with these results.  The potential for regional variability in MST marker results demonstrates the need for local validation in a given study area (Gawler and others, 2007)

The specific uses and limitations of MST results should be noted when applying these tools.  While the presence of a source-associated MST marker indicates that fecal contributions have been made by that animal, a negative result for the MST marker does not necessarily signify the absence of fecal contributions from that host animal.  Analysis of MST markers by the quantitative polymerase chain reaction (qPCR) allows for an assessment of the level of these markers in a given water sample.  However, interpretation of results for quantification will require future studies documenting the relative survival of the markers in environmental waters (Field and Samadpour, 2007), the relative abundance of markers in source fecal material, and ultimately, the public-health significance of marker quantity results.  Additionally, at this point in the state of MST science, it is not possible to determine the exact proportion of fecal contamination contributed by the various host-animal sources in a given watershed, water segment, or water sample.  Analysis by qPCR does allow for relative comparisons of the MST marker for a specific host animal, i.e. MST marker levels can be compared both spatially and temporally under varying weather and hydrologic conditions.  These comparisons may provide the means to identify trends among the fecal source contributions.


Bernhard, A.E., and Field, K.G., 2000, A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA: Applied and Environmental Microbiology, v. 66, no. 10, p. 4571–4574.

Boehm, A.B., Fuhrman, J.A., Morse, R.D., Grant, S.B., 2003, Tiered approach for identification of a human fecal pollution source at a recreational beach: Environmental Science and Technology, v. 37, p. 673–580.

Field, K.S., and Samadpour, M., 2007, Fecal source tracking, the indicator paradigm, and managing water quality: Water Research, v. 41, p. 3517–3538.

Francy, D.S., Bertke, E.E., Finnegan, D.P., Kephart, C.M., Sheets, R.A., Rhoades, J., and Stumpe, L., 2006, Use of spatial sampling and microbial source-tracking tools for understanding fecal contamination at two Lake Erie beaches, U.S. Geological Survey Scientific Investigations Report 2006–5298, 29 p.

Fremaux, B., Boa, T., and Yost, C.K., 2010, Quantitative real-time PCR assays for sensitive detection of Canada goose-specific fecal pollution in water samples: Applied and Environmental Microbiology, v. 76, no. 14, p. 4886–4889.

Glassmeyer, S.T., Furlong, E.T., Kolpin, D.W., Cahill, J.D., Zaugg, S.D., Werner, S.L., Meyer, M.T., and Cryak, D.D., 2005, Transport of chemical and microbial compounds from known wastewater discharges—potential for use as indicators of human fecal contamination: Environmental Science and technology, v. 39, no. 14, p. 5157–5169.

Griffin, J.F., Weisberg, S.B., and McGee, C.D., 2003, Evaluation of microbial source tracking methods using mixed fecal sources in aqueous test samples: Journal of Water and Health, v. 1, no. 4, p. 141–151.

Kildare, B.J., Leutenegger, C.M., McSwain, B.S., Bambic, D.G., Rajal, V.B., and Wuertz, S., 2007, 16S rRNA-based assays for quantitative detection of universal, human-, cow-, and dog-specific fecal Bacteroidales: A Bayesian approach: Water Research, v. 41, p. 3701–3715.

Layton, Alice, McKay, L., Williams, D., Garrett, V., Gentry, R., and Sayler, G., 2006, Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water: Applied and Environmental Microbiology, v. 72, no. 6, p. 4214–4224.

Lu, J., Santo Domingo, J., Shanks, O.C., 2007, Identification of chicken-specific microbial sequences using a metagenomic approach: Water Research, v. 41, p. 3561–3574. 

Lu, J., Santo Domingo, J.W., Lamendella, R., Edge, T., and Hill, S., 2008, Phylogenetic diversity and molecular detection of bacteria in gull feces: Applied and Environmental Microbiology, v. 74, no. 13, p. 3969–3976.

Santo Domingo, J.W., Bambic, D.G., Edge, T.A., and Wuertz, S., 2007, Quo vadis source tracking? Towards a strategic framework for environmental monitoring of fecal pollution: Water Research, v. 41, no. 16, p. 3639–3552.

Siefring S., Varma M., Atikovic E., Wymer L., and Haugland R., 2007, Improved real-time PCR assays for the detection of fecal indicator bacteria in surface waters with different instrument and reagent systems: Journal of Water and Health, v. 6, no. 2, p. 225–237.

Stewart, J.R., Ellender, R.D., Gooch, J.A., Jiang, S., Myoda, S.P., and Weisberg, S.B., 2003, Recommendations for microbial source tracking lessons learned from a methods comparison study: Journal of Water and health, v. 1, no. 4, p. 225–231.

Weidhaas, J.L., Macbeth, T.W., Olsen, R.L., Sadowsky, M.J., Norat, D., and Harwood, V.J., 2010, Identification of a Brevibacterium marker gene specific to poultry litter and development of a quantitative PCR assay: Journal of Applied Microbiology, v. 109, p. 334–347.