SummaryIn recent decades, considerable effort has been devoted to finding microbial source‐tracking (MST) markers that are suitable to assess the health risks of faecally polluted waters, with no universal marker reported so far. In this study, the abundance and prevalence of a crAssphage‐derived DNA marker in wastewaters of human and animal origins were studied by a new qPCR assay with the ultimate aim of assessing its potential as an MST marker. crAssphage showed up to 106 GC/ml in the sewage samples of human origin, in both the total DNA and the viral DNA fraction. In wastewaters containing animal faecal remains, 39% of the samples were negative for the presence of the crAssphage sequence, while those showing positive results (41% of the samples) were at least 1 log10 unit lower than the samples of human origin. Noteworthy, the log10 values of the ratio (R) crAssphage (GC/ml)/Escherichia coli (CFU/ml) varied significantly depending on the human or animal origin (R > 1.5 for human samples and R < −1.5 for animal wastewater samples. This study opens the way for further research to explore if different specific animal variants of crAssphage exist and whether other zones of the crAssphage genome are better suited to source discrimination.

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Given the interest in Bacteroides species as microbial source tracking (MST) markers, and the limited knowledge of the survival of Bacteroides species in the environment, here we examine the survival of Bacteroides fragilis, B. thetaiotaomicron, and environmental species of Bacteroides by use of culture techniques and molecular tools. Two kinds of experiments were performed: (i) on-site experiments, in which bacteria were exposed to changes in the levels of several environmental parameters in a river, and (ii) microcosm assays in the laboratory, with controlled temperatures. On-site experiments showed different survival patterns for the cultivable Bacteroides strains. B. fragilis die-off rate was strongly affected by the combined effect of high temperatures and grazing predators, which were more active under warmer conditions. However, the survival rates of cultivable B. thetaiotaomicron and environmental Bacteroides spp. were more affected by dissolved oxygen (DO) concentration in water. Environmental Bacteroides strains survived longer than either type strain, due to better adaptation to environmental conditions. However, the period of their survival was shorter than that observed for fecal coliforms and enterococci, suggesting Bacteroides species as markers of recent fecal pollution. The total Bacteroides species were detected by molecular techniques throughout the experiment in winter, but they were detected on only two or three days in the summer. This indicates that temperature is the main factor affecting DNA degradation, regardless of species. The use of microcosms in the laboratory also pointed to temperature as the main factor affecting Bacteroides survival, regardless of species. However, the conditions in the laboratory may mask the effects of the environmental factors and their interactions. The observed variability in die-off rate as a function of the species analyzed, the experimental conditions, and the methodology used should be taken into consideration in future persistence studies.

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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.