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Microbial Source Tracking in Aquatic Ecosystems: The State of the Science and an Assessment of Needs

Microbial Source Tracking Overview

Microbial source tracking (MST) is an emerging field that seeks to identify the source of microbial contamination in the environment. The field has been developing rapidly to address growing needs to find the source of fecal pollution contaminating sources of drinking, shellfish and recreational waters. The general approach is based on comparing the similarity of microorganisms collected from aquatic ecosystems to microorganisms collected from known fecal pollution sources in order to make inferences about the likely source of fecal contamination.

Various methods have been developed for microbial source tracking (Simpson et al. 2002; Scott et al. 2002; U.S. EPA 2005). Since the field of MST is evolving rapidly, many new methods are also under investigation. The collection of methods for microbial source tracking has often been referred to as a toolbox, with some methods being more relevant to use than others in certain circumstances. This section provides an overview of some of the more commonly used MST methods.

MST methods can be classified as library-dependent methods or library-independent methods. To date, library-dependent methods have been more widely used in MST studies, although it can be very labour intensive and time consuming to develop the library for these methods. Library-independent methods are increasingly under investigation.

Library-Dependent MST Methods

Library-dependent methods are based upon choosing a fecal indicator microorganism (e.g., Escherichia coli) and establishing a reference library of characteristics of individual isolates of the selected microorganism obtained from known fecal pollution sources. For example, a library could be a database of DNA fingerprints of E. coli isolates obtained from fecal pollution sources such as animal feces, septic tanks or municipal wastewater effluents. The DNA fingerprints of E. coli isolates obtained from aquatic ecosystems ("unknowns") can then be compared to the DNA fingerprints in the library ("knowns") to make inferences about the source of the waterborne E. coli isolates. Sound taxonomic identification of the fecal and waterborne isolates is necessary to ensure similarity comparisons are warranted. A minimum size has not yet been established for libraries, although they need to be sufficiently large to reflect the diversity of isolates in the environment. A targeted sampling approach may be useful for focusing MST efforts (Kuntz et al. 2003).

Sewage treatment secondary clarifying tanks | Photo: Quintin RochfortThe most common fecal indicator microorganisms used in library-dependent methods to date have been Escherichia coli and Enterococcus spp. These bacteria are common inhabitants of warm-blooded animal guts, are relatively easy to isolate and culture in the lab, and are widely used by water quality monitoring programs. The similarity between isolates of a fecal indicator microorganism can be measured by either phenotypic profiling or genotypic fingerprinting methods.

Phenotypic methods use cellular or physiological comparisons between the isolates based on features such as antibiotic resistance (Wiggins 1996; Hagedorn et al. 1999; Harwood et al. 2000) or carbon utilization (Hagedorn et al. 2003). Antibiotic resistance profiling has been the most commonly used phenotypic method to date. For this method, bacterial isolates can be inoculated onto the surface of many agar plates, with each plate containing a different antibiotic mixed into the agar. The isolates are incubated overnight on the agar plates, and their growth is compared to their growth on a control plate (i.e., the same agar without antibiotics). How well each isolate grows on many different agar plates is used to develop a profile of its antibiotic resistance. An antibiotic resistance approach to MST is based on the assumption that bacteria in human and domestic animal guts are exposed to different antibiotics in medical and veterinary treatments, and that these gut bacteria will develop different resistance profiles. Since wildlife species do not receive direct antibiotic treatments, their gut bacteria are typically less resistant to antibiotics.

Genotypic methods use DNA sequence comparisons between the isolates based on approaches like DNA fingerprinting using rep-PCR (Dombek et al. 2000; Johnson et al. 2004), ribotyping (Carson et al. 2001), and amplified fragment length polymorphism (AFLP) (Guan et al. 2002; Leung et al. 2004). In these DNA fingerprinting methods, DNA is extracted from cells of an isolate and different DNA cutting or amplifying techniques can be used to obtain DNA fragments of different sizes. The different sized DNA fragments can then be separated on an electrophoresis gel into a ladder-like pattern of DNA bands that can be visualized and statistically analyzed as a unique DNA fingerprint. Ribotyping and rep-PCR have been the most commonly used DNA fingerprinting techniques to date.

Library-Independent MST Methods

Library-independent methods are based upon detecting host-specific markers to indicate the presence of fecal contamination from a specific human or animal host in the water. Most library-independent methods rely on the polymerase chain reaction (PCR) to detect host-specific markers. These methods do not generally require a cultivation step, although in some cases this is necessary to increase the numbers of microorganisms carrying a host-specific marker gene. For example, cultivation-based methods have been proposed for human-specific markers in Enterococcus (Scott et al. 2005), and cattle and swine markers in E. coli (Khatib et al. 2002, 2003).

Some of the most promising results to date for developing host-specific markers for fecal pollution source tracking involve 16S rDNA markers within the Bacteroidetes family. These anaerobic bacteria comprise a very large portion of the fecal flora in warmblooded animals. Bernhard and Field (2000a) found host-specific 16S rDNA sequences in Bacteroides sp. bacteria in human and cow fecal samples and used a method to track these sequences in coastal waters. Bernhard and Field (2000b) then developed Bacteroides 16S rDNA PCR assays specific to ruminants and humans as culture-independent MST methods. There is now an active research effort to validate and find new Bacteroides sp. host-specific markers (Field 2004). Additional host-specific markers have been proposed based on bacteriophages (Payan et al. 2005) and pathogenic microorganisms such as enteric viruses (Fong et al. 2005) and protozoa like Cryptosporidium sp. (Jiang et al. 2005).

Other Source Tracking Methods

While the field of microbial source tracking is growing rapidly, it should be noted that other methods can also be used to track fecal contamination in aquatic ecosystems. For example, chemical tracers have been used, most commonly to detect chemicals associated with human wastes. As the highest concentration of these chemicals is typically found in wastewater treatment plants, they have been proposed for tracking human fecal waste pollution. For example, fecal sterols and stanols, caffeine, detergents, laundry brighteners, fragrance materials and pharmaceuticals are among chemicals proposed as markers of fecal pollution (Elhmmali et al. 2002; Roser et al. 2003; Glassmeyer et al. 2005). Eukaryotic mitochondrial DNA markers have also been used to discriminate between fecal pollution sources in surface waters (Martellini et al. 2005) as outlined later in this Workshop Report.