Molecular Indicators Used in the Development of Predictive Models for Microbial Source Tracking

A number of chemical, microbial, and eukaryotic indicators have been proposed as indicators of fecal pollution sources in water bodies. No single one of the indicators tested to date has been able to determine the source of fecal pollution in water. However, the combined use of different indicators has been demonstrated to be the best way of defining predictive models suitable for determining fecal pollution sources. Molecular methods are promising tools that could complement standard microbiological water analysis. In this study, the feasibility of some proposed molecular indicators for microbial source tracking (MST) was compared (names of markers are in parentheses): host-specific Bacteroidetes (HF134, HF183, CF128, and CF193), Bifidobacterium adolescentis (ADO), Bifidobacterium dentium (DEN), the gene esp of Enterococcus faecium, and host-specific mitochondrial DNA associated with humans, cattle, and pigs (Humito, Bomito, and Pomito, respectively). None of the individual molecular markers tested enabled 100% source identification. They should be combined with other markers to raise sensitivity and specificity and increase the number of sources that are identified. MST predictive models using only these molecular markers were developed. The models were evaluated by considering the lowest number of molecular indicators needed to obtain the highest rate of identification of fecal sources. The combined use of three molecular markers (ADO, Bomito, and Pomito) enabled correct identification of 75.7% of the samples, with differentiation between human, swine, bovine, and poultry sources. Discrimination between human and nonhuman fecal pollution was possible using two markers: ADO and Pomito (84.6% correct identification). The percentage of correct identification increased with the number of markers analyzed. The best predictive model for distinguishing human from nonhuman fecal sources was based on 5 molecular markers (HF134, ADO, DEN, Bomito, and Pomito) and provided 90.1% correct classification.Fecal pollution represents a serious public health problem. Pathogens from infected animals and humans can be introduced into the environment through feces and cause health risks, environmental degradation, and economic losses. In recent years, water authorities'' environmental and sanitary regulations have focused on the total fecal load that can be held by a water body and on determining the source of fecal pollution. Accurate and reliable methods for detecting fecal pollution are needed to reduce its occurrence, prevent future spills, decrease economic losses, and take legal measures.Total coliforms, fecal coliforms, enterococci, and Escherichia coli have traditionally been used as microbial fecal indicators in water. These microorganisms are easy to enumerate by cultivation methods. However, they do not identify the source of fecal pollution.Fecal pollution of surface waters comes from point or diffuse sources, including municipal sewage, slaughterhouse wastewater, manure and biosolid disposal, wildlife, and undetermined runoff. Reliable microbial source tracking (MST) methods can provide efficient and rapid fecal source determination and facilitate cost-effective remediation. In recent years, various MST methods have been developed that are based on library-dependent or -independent methods and analyze phenotypic and/or genomic characteristics (39, 54, 55). Library-dependent methods (LDM) require a comprehensive library of isolates from known sources. Isolates from unknown sources are classified by correspondence with those from the library (57). LDM include antibiotic resistance analysis, carbon source utilization, repetitive PCR, and ribotyping. However, the geographic and temporal stability and the numerical methods used for these LDM have been questioned (26, 44). Some cultivation-based methods have already been described, such as the detection of specific enterotoxins of E. coli strains (30, 31, 43), the differentiation and enumeration of sorbitol-fermenting bifidobacteria (10, 37), and the enumeration of phages that infect host-specific Bacteroides spp. (8, 45). Cultivation methods detect only viable bacteria, may give a biased picture of the populations, and thus misrepresent the bacterial diversity (60). The use of PCR-based methods allows the detection of bacteria that are difficult to grow, such as anaerobes, including the genera Bacteroides and Bifidobacterium (5, 9, 14, 63), Rhodococcus coprophilus (51), methanogenic archaeal bacteria (59), and viruses (27). More detailed information on MST methods can be found in several technical reviews (8, 17, 54, 55, 57).Bifidobacterium and Bacteroides have been proposed as possible source-tracking indicators for waterborne fecal pathogens (3, 18, 33, 37, 41, 48). Several Bifidobacterium species are thought to be human host specific, such as Bifidobacterium adolescentis, Bifidobacterium dentium, and Bifidobacterium longum (58). Meanwhile, others have been linked to certain domestic animals (20, 61). A multiplex PCR has been developed to detect human fecal pollution by analyzing the presence of B. dentium and B. adolescentis in water (9). Bacteroidetes markers are mainly based on the definition of host-specific oligonucleotides (for example, to detect human, ruminant, and swine pollution) that are associated with some uncultured populations (5, 15, 32, 46, 47). Geographical differences in host specificity have been observed when these markers are applied in different world regions (1, 2, 11, 21, 22, 24, 40, 42). The detection of the gene esp, which encodes an enterococcal surface protein, has also been proposed as an indicator of human fecal pollution (53). This gene has been associated with the virulence, colonization and biofilm formation found in Enterococcus faecium and Enterococcus faecalis (25). However, recent studies have indicated that the detection of esp may not always be related to human fecal pollution (12, 35). Other MST indicators have been developed for eukaryotic molecular markers. Martellini et al. (38) designed a PCR protocol that targets eukaryotic genetic markers as a fecal source tracking method for differentiating human, porcine, bovine, and ovine fecal pollution. This protocol consists of nested PCRs, based on the amplification of mitochondrial DNA from the host cells. Multiplex and real-time PCR methods for mitochondrial MST indicators have also been developed (4, 13, 52).It has been shown that no single microbial or chemical MST indicator can determine the source of fecal pollution. Therefore, a selection of indicators is required (7, 8, 22, 24). Predictive models to distinguish between human and nonhuman pollution have been developed by combining indicators. These models have achieved a 100% likelihood of success (7, 24, 56). However, they are mostly based on culture-dependent methods, and discernment among different animal sources should be attained. In this study, microbial and eukaryotic molecular markers were compared for use as MST indicators. Potential combinations were also evaluated. Finally, MST predictive models using only these molecular markers were developed using a number of established statistical methods. The models were evaluated by considering the lowest number of molecular indicators needed to obtain the highest rate of discrimination among fecal sources.