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In this study we use a machine learning software (Ichnaea) to generate predictive models for water samples with different concentrations of fecal contamination (point source, moderate and low). We applied several MST methods (host-specific Bacteroides phages, mitochondrial DNA genetic markers, Bifidobacterium adolescentis and Bifidobacterium dentium markers, and bifidobacterial host-specific qPCR), and general indicators (Escherichia colt, enterococci and somatic coliphages) to evaluate the source of contamination in the samples. The results provided data to the Ichnaea software, that evaluated the performance of each method in the different scenarios and determined the source of the contamination. Almost all MST methods in this study determined correctly the origin of fecal contamination at point source and in moderate concentration samples. When the dilution of the fecal pollution increased (below 3 log(10) CPU E. coli/100 ml) some of these indicators (bifidobacterial host-specific qPCR, some mitochondrial markers or B. dentium marker) were not suitable because their concentrations decreased below the detection limit. Using the data from source point samples, the software Ichnaea produced models for waters with low levels of fecal pollution. These models included some MST methods, on the basis of their best performance, that were used to determine the source of pollution in this area. Regardless the methods selected, that could vary depending on the scenario, inductive machine learning methods are a promising tool in MST studies and may represent a leap forward in solving MST cases.

Bifidobacterium spp. belong to the commensal intestinal microbiota of warm-blooded animals. Some strains of Bifidobacterium show host specificity and have thus been proposed as host-specific targets to determine the origin of fecal pollution. Most strains have been used in microbial-source-tracking (MST) studies based on culture-dependent methods. Although some of these approaches have proved very useful, the low prevalence of culturable Bifidobacterium strains in the environment means that molecular culture-independent procedures could provide practical applications for MST. Reported here is a set of common primers and four Bifidobacterium sp. host-associated (human, cattle, pig, and poultry) probes for quantitative-PCR (qPCR) assessment of fecal source tracking. This set was tested using 25 water samples of diverse origin: urban sewage samples, wastewater from four abattoirs (porcine, bovine, and poultry), and water from a river with a low pollution load. The selected sequences showed a high degree of host specificity. There were no cross-reactions between the qPCR assays specific for each origin and samples from different fecal origins. On the basis of the findings, it was concluded that the host-specific qPCRs are sufficiently robust to be applied in environmental MST studies.

Microbial source tracking (MST) methods allow the identification of specific faecal sources. The aim is to detect the sources of faecal pollution in a water body to allow targeted, efficient and cost-effective remediation efforts in the catchment. Bacteriophages infecting selected host strains of Bacteroides species are used as markers to track faecal contaminants in water. By using a suitable Bacteroides host from a given faecal origin, it is possible to specifically detect bacteriophages of this faecal origin. It can thus be used to detect specific phages of Bacteroides for MST. With this objective, we isolated several Bacteroides strains from pig, cow and poultry faeces by applying a previously optimized methodology used to isolate the host strains from humans. The isolated strains belonged to Bacteroides fragilis and Bacteroides thetaiotaomicron. These strains, like most Bacteroides species, detected phages of the Siphoviridae morphology. Using the newly isolated host strains for phage enumeration in a range of samples, we showed that these detect phages in faecal sources that coincide with their own origin (70-100% of the samples), and show no detection or very low percentages of detection of phages from other animal origins (from 0 to 20% of the samples). Only strains isolated from pig wastewater detected phages in 50% of human sewage samples. Nevertheless, those strains detecting phages from faecal origins other than their own detected fewer phages (2-3 log₁₀ pfu·100 ml⁻¹) than the phages detected by the specific strain of the same origin. On the basis of our results, we propose that faecal source tracking with phages infecting specific Bacteroides host strains is a useful method for MST. In addition, the method presented here is feasible in laboratories equipped with only basic microbiological equipment, it is more rapid and cost-effective than other procedures and it does not require highly qualified staff.

Bacteroides spp. have been proposed as indicators of fecal contamination in microbial source tracking (MST) methodologies. The aim of this study was to develop new qPCR assays that target host‐specific Bacteroidal 16S ribosomal RNA genes, to determine the source of fecal contamination in water. Denaturing gradient gel electrophoresis (DGGE) was used to select for host‐specific bands of Bacteroides associated with a fecal pollution source and later to design four qPCR host‐specific assays. A set of common primers for Bacteroides spp., four different Bacteroides spp. host‐associated hydrolysis probes (human, cattle, pig, and poultry), and one hydrolysis probe for the Bacteroides genus were designed. This set of qPCR assays together with other previously developed Bacteroidetes MST targets were used to analyze water samples with fecal contamination from the four sources studied. The host‐specific Bacteroides qPCRs designed for human (HMprobeBac), pig (PGprobeBac), and poultry (PLprobeBac) were highly specific for its sources (1.0, 0.97, and 1.0, respectively) although its sensitivity was lower (0.45, 0.50, and 0.73, respectively). The cattle‐specific qPCR was totally unspecific and was discarded for future experiments. When compared to previously designed assays, the human and pig qPCRs showed better accuracies (0.86 and 0.84) than their counterparts HF183 and Pig‐2‐Bac (0.38 and 0.65). Thus, the newly designed human, pig, and poultry qPCR assays outperform other methods developed until date and may be useful for source tracking purposes.

Microbial source tracking (MST) methods allow the identification of specific faecal sources. The aim is to detect the sources of faecal pollution in a water body to allow targeted, efficient and cost-effective remediation efforts in the catchment. Bacteriophages infecting selected host strains of Bacteroides species are used as markers to track faecal contaminants in water. By using a suitable Bacteroides host from a given faecal origin, it is possible to specifically detect bacteriophages of this faecal origin. It can thus be used to detect specific phages of Bacteroides for MST. With this objective, we isolated several Bacteroides strains from pig, cow and poultry faeces by applying a previously optimized methodology used to isolate the host strains from humans. The isolated strains belonged to Bacteroides fragilis and Bacteroides thetaiotaomicron. These strains, like most Bacteroides species, detected phages of the Siphoviridae morphology. Using the newly isolated host strains for phage enumeration in a range of samples, we showed that these detect phages in faecal sources that coincide with their own origin (70-100% of the samples), and show no detection or very low percentages of detection of phages from other animal origins (from 0 to 20% of the samples). Only strains isolated from pig wastewater detected phages in 50% of human sewage samples. Nevertheless, those strains detecting phages from faecal origins other than their own detected fewer phages (2-3 log₁₀ pfu·100 ml⁻¹) than the phages detected by the specific strain of the same origin. On the basis of our results, we propose that faecal source tracking with phages infecting specific Bacteroides host strains is a useful method for MST. In addition, the method presented here is feasible in laboratories equipped with only basic microbiological equipment, it is more rapid and cost-effective than other procedures and it does not require highly qualified staff.