2Microbial source tracking (MST) includes a group of methodologies that are aimed at identifying, and in some cases quantifying, the dominant source(s) of fecal contamination in resource waters, including drinking, ground, recreational, and wildlife habitat waters. MST methods can be grouped into two major types. Library-dependent methods are culture based and rely on isolate-by-isolate typing of bacteria cultured from various fecal sources and from water samples These isolates are matched to their corresponding source categories by direct subtype matching (41,70) or by statistical means (23,37,40,41,80,83,102). In contrast, library-independent methods frequently are based on sample-level detection of a specific, hostassociated genetic marker in a DNA extract by PCR (6,11,26,88). Analyses of certain chemicals associated with sewage, including fecal sterols (29,30,47), optical brighteners (29,30,68), and host mitochondrial DNA (67), have also been utilized for what can be more broadly termed fecal source tracking; however, in this review we compare the performance of only fecal source tracking studies in which the target(s) is microbial.Mounting pressure to determine the origin of nonpoint source fecal pollution, as exemplified by the U.S. Environmental Protection Agency's total maximum daily load program, has led to a steady increase in manuscript submissions and grant applications that include MST approaches. At the same time, resource managers concerned with water quality and regulatory pressures struggle with the choice of methodology in the face of requirements for immediate application. Although there has been significant progress in the MST field over the last 10 years, variability among performance measurements and validation approaches in laboratory and field studies has led to a body of literature that is very difficult to interpret, both for scientists and for end users (99).In this review, we first consider the development and validation of MST methods in a historical context to describe the lessons learned in early studies. Next, uniform performance characteristics are introduced to allow comparison of method performance across MST studies (Tables 1 and 2), and this is followed by a discussion of considerations for field study design and implementation. DEVELOPMENT OF EVALUATION AND VALIDATION STRATEGIESLibrary-dependent MST owes its origins to the studies of Escherichia coli population biology (14, 71, 77) and antimicrobial resistance studies conducted 15 to 25 years ago (4,5,19,54,58). Many of the basic premises in early MST studies, such as the assumption that host-specific genetic or phenotypic characteristics are influenced by selective pressure (79, 102), reflected this understanding of population biology. Validation of isolate-by-isolate (generally library-dependent) and samplelevel (generally library-independent) classification methods is based on various performance criteria, some of which are approach specific and some of which are common to all approaches. Many of these validation strategi...

Fecal indicator bacteria (FIB), commonly used to regulate sanitary water quality, cannot discriminate among sources of contamination. The use of alternative quantitative PCR (qPCR) methods for monitoring fecal contamination or microbial source tracking requires an understanding of relationships with cultivated FIB, as contamination ages under various conditions in the environment. In this study, the decay rates of three Bacteroidales 16S rRNA gene markers (AllBac for general contamination and qHF183 and BacHum for human-associated contamination) were compared with the decay rate of cultivated Escherichia coli in river water microcosms spiked with human wastewater. The following five sets of microcosms were monitored over 11 days: control, artificial sunlight, sediment exposure, reduced temperature, and no autochthonous predation. Decay was characterized by estimation of the time needed to produce a 2-log reduction (t 99 ). No treatmentassociated differences in the decay of the 4 targets were evident except with reduced predation, where E. coli, qHF183, and BacHum markers had lower levels of decay by day 3. However, there were substantial targetassociated differences. Decay curves for the AllBac marker indicated a larger persistent population than those of the other targets. Exposure to sunlight, sediment, and reduced predation resulted in more rapid decay of the human-associated markers relative to cultivable E. coli, but there were no differences in t 99 values among the 4 targets under control conditions or at reduced temperatures. Further evaluation of epidemiological relationships will be needed in order to relate the markers directly to health risk. These findings suggest that the tested human-associated markers can complement E. coli as indicators of the human impact on sanitary water quality under the constrained conditions described in this paper.Recreational water quality standards for freshwater are commonly based on the cultivable concentration of the fecal indicator bacterium (FIB) Escherichia coli (42). Epidemiological studies demonstrated a correlation between E. coli concentration and rates of gastrointestinal illness among swimmers (19,32,46), and water quality criteria based on E. coli have been established by the U.S. Environmental Protection Agency for the protection of human health (41). However, E. coli are found in many hosts, both human and nonhuman, that carry different cohorts of human-pathogenic microorganisms (13), and E. coli types have limited host specificity (4,16,17). E. coli also have been shown to reproduce in the environment under some conditions (7,12,23,37,49). These limitations of E. coli as an indicator of public health risk are well recognized (34) and may result in revision of U.S. recreational water quality criteria (44).In the pursuit of alternate assessment strategies, researchers in the field of microbial source tracking (MST) have developed and applied host-associated molecular markers of fecal contamination. Prominent among candidate MST protocols are those that det...

Microbial source tracking (MST) uses various approaches to classify fecal-indicator microorganisms to source hosts. Reproducibility, accuracy, and robustness of seven phenotypic and genotypic MST protocols were evaluated by use of Escherichia coli from an eight-host library of known-source isolates and a separate, blinded challenge library. In reproducibility tests, measuring each protocol's ability to reclassify blinded replicates, only one (pulsed-field gel electrophoresis; PFGE) correctly classified all test replicates to host species; three protocols classified 48-62% correctly, and the remaining three classified fewer than 25% correctly. In accuracy tests, measuring each protocol's ability to correctly classify new isolates, ribotyping with EcoRI and PvuII approached 100% correctclassification but only 6% of isolates were classified; four of the other six protocols (antibiotic resistance analysis, PFGE, and two repetitive-element PCR protocols) achieved better than random accuracy rates when 30-100% of challenge isolates were classified. In robustness tests, measuring each protocol's ability to recognize isolates from nonlibrary