Quantifying the contributions of possible environmental sources (\"sources\") to a specific microbial community (\"sink\") is a classical problem in microbiology known as microbial source tracking (MST). Solving the MST problem will not only help us understand how microbial communities were formed, but also have far-reaching applications in pollution control, public health, and forensics. MST methods generally fall into two categories: target-based methods (focusing on the detection of source-specific indicator species or chemicals); and community-based methods (using community structure to measure similarity between sink samples and potential source environments). As next-generation sequencing becomes a standard community-assessment method in microbiology, numerous community-based computational methods, referred to as MST solvers hereafter have been developed and applied to various real datasets to demonstrate their utility across different contexts. Yet, those MST solvers do not consider microbial interactions and priority effects in microbial communities. Here, we revisit the performance of several representative MST solvers. We show compelling evidence that solving the MST problem using existing MST solvers is impractical when ecological dynamics plays a role in community assembly. In particular, we clearly demonstrate that the presence of either microbial interactions or priority effects will render the MST problem mathematically unsolvable for MST solvers. We further analyze data from fecal microbiota transplantation studies, finding that the state-of-the-art MST solvers fail to identify donors for most of the recipients. Finally, we perform community coalescence experiments to demonstrate that the state-of-the-art MST solvers fail to identify the sources for most of the sinks. Our findings suggest that ecological dynamics imposes fundamental challenges in MST. Interpretation of results of existing MST solvers should be done cautiously.

Manila Bay, a multipurpose body of water located around Metro Manila, Philippines, is progressively deteriorating because of massive pollution. Reports have shown that the bay and its aquatic resources (i.e., seafood) are contaminated with fecal matter and enteric pathogens, posing a threat to public health and industry. This problem raises the need for a microbial source tracking methodology as a part of the rehabilitation efforts in the bay. Bivalve mollusks cultivated in water can serve as sentinel species to detect fecal pollution and can complement water monitoring. With the use of polymerase chain reaction and DNA sequence analysis, this study detected Cryptosporidium spp. in Asian green mussels (Perna viridis) cultivated and harvested in Manila Bay and sold in Bulungan Seafood Market, Parañaque, Philippines, from 2019 to 2021 with an overall occurrence of 8.77% (n = 57). The analysis of the 18S rDNA segment revealed three genotypes from Cryptosporidium-positive samples, namely, Cryptosporidium sp. rat genotype IV (60%), C. galli (20%), and C. meleagridis (20%). These findings suggest fecal pollution in bivalve cultivation sites coming from sewage, nonpoint, and agricultural sources. The presence of C. meleagridis, the third most common cause of human cryptosporidiosis, in mussels poses a threat to human health. Thus, there is a need to establish routine detection and source tracking of Cryptosporidium spp. in Manila Bay and to educate seafood consumers on food safety.

This research investigated the in-situ decay rates of four human wastewater-associated markers (Bacteroides HF183 (HF183), Lachnospiraceae Lachno3 (Lachno3), cross-assembling phage (crAssphage), pepper mild mottle virus (PMMoV) and three enteric viruses (human adenovirus 40/41 (HAdV 40/41), enterovirus (EV) and human norovirus GII (HNoV GII) in two estuarine water environments (Davidson Park (DP) and Hen and Chicken Bay (HCB) in temperate Sydney, NSW, Australia, employing qPCR and RT-qPCR assays. The study also aimed to compare decay rates observed in mesocosms with previously published laboratory microcosms, providing insights into the persistence of markers and viruses in estuarine environments. Results indicated varying decay rates between DP and HCB mesocosms, with HF183 exhibiting relatively faster decay rates compared to other markers and enteric viruses in sunlight and dark mesocosms. In DP mesocosms, HF183 decayed the fastest, contrasting with PMMoV, which exhibited the slowest. Sunlight induced higher decay rates for all markers and viruses in DP mesocosms. In HCB sunlight mesocosms, HF183 nucleic acid decayed most rapidly compared to other markers and enteric viruses. In dark mesocosms, crAssphage showed the fastest decay, while PMMoV decayed at the slowest rate in both sunlight and dark mesocosms. Comparisons with laboratory microcosms revealed faster decay of markers and enteric viruses in laboratory microcosms than the mesocosms, except for crAssphage and HAdV 40/41 in dark, and PMMoV in sunlight mesocosms. The study concludes that decay rates of markers and enteric viruses vary between estuarine mesocosms, emphasizing the impact of sunlight exposure, which was potentially influenced by the elevated turbidity at HCB estuarine waters. The generated decay rates contribute valuable insights for establishing site-specific risk-based thresholds of human wastewater-associated markers.

Different types of inlet water are expected to affect microbial communities of lake ecosystems due to changing environmental conditions and the dispersal of species. However, knowledge of the effects of changes in environmental conditions and export of microbial assemblages on lake ecosystems is limited, especially for glacier-fed lakes. Here, we collected water samples from the surface water of a glacier-fed lake and its two fed streams on the Tibetan Plateau to investigate the importance of glacial and non-glacial streams as sources of diversity for lake bacterial communities. Results showed that the glacial stream was an important source of microorganisms in the studied lake, contributing 45.53% to the total bacterial community in the lake water, while only 19.14% of bacterial community in the lake water was seeded by the non-glacial stream. Bacterial communities were significantly different between the glacier-fed lake and its two fed streams. pH, conductivity, total dissolved solids, water temperature and total nitrogen had a significant effect on bacterial spatial turnover, and together explained 36.2% of the variation of bacterial distribution among habitats. Moreover, bacterial co-occurrence associations tended to be stronger in the lake water than in stream habitats. Collectively, this study may provide an important reference for assessing the contributions of different inlet water sources to glacier-fed lakes.

In settings where humans and animals closely coexist, the introduction of faecal material into unprotected water sources significantly increases the risk of contracting diarrhoeal and zoonotic waterborne diseases. The data were gathered from a survey conducted through interviews at randomly sampled villages; additionally, water samples were collected in randomly selected households and their associated feeder catchments. Molecular techniques were used, specifically qPCR, to run host-specific Bacteroides microbial source tracking (MST) assays for human, cattle, pig, chicken and dog faecal contamination. Unexpectedly, the qPCR assays revealed dogs to be the most prevalent (40.65%) depositor of faecal matter in unprotected surface water, followed by humans (40.63%); this finding was contradictory to survey findings indicating cattle as the leading source. At the household level, dogs (16.67%) and chickens (15.28%) played prominent roles, as was expected. Reflecting on some of the basic daily practices in households, nearly 89.00% of the population was found to store water due to erratic supply, in contrast to 93.23% using an improved water source. Additionally, a significant association was found between water, sanitation and hygiene (WASH) variables and the occurrence of MST markers after performing a bivariate linear regression. However, the inconsistency between the MST results and household surveys suggests pervasive sanitation issues, even in households without domesticated animals.

Identifying primary sources of fecal pollution is important for assessing public health risks and implementing effective remediation strategies. To date, one of the main molecular approaches for identifying sources of fecal pollution relies on detecting molecular markers within bacterial, viral, or mitochondrial nucleic acids, that are indicative of a particular host. With a primary focus on identifying fecal pollution originating from humans, the field of fecal source tracking often places less emphasis on livestock sources, frequently leaving the problem of wildlife fecal pollution unaddressed. In this review, we summarize 55 previously published and validated molecular assays and describe methods for the detection of molecular markers that are indicative of non-human hosts. They cover a range of 15 animal species/groups with a primary focus on domestic animals including cattle, pigs, dogs, and poultry. Among assays associated with wild animals, the majority are designed to detect bird feces, while the availability of assays for detecting feces of other wild animals is limited. Both domestic and wild animals can represent a zoonotic reservoir of human enteropathogens, emphasizing the importance of their role in public health. This review highlights the need to address the complexity of fecal contamination and to include a broader range of animal species into assay validation and marker identification.

Microbial source tracking can determine fecal contamination but requires a relevant, sizable reference library for analysis. We provide a reference library of 100+ fecal microbiome samples relevant to mid-Atlantic United States ecosystems. Included are wild and domesticated fauna, wastewater, and septic samples applicable to Delaware source tracking studies.

The analysis of ancient oral metagenomes from archaeological human and animal samples is largely confounded by contaminant DNA sequences from modern and environmental sources. Existing methods for Microbial Source Tracking (MST) estimate the proportions of environmental sources, but do not perform well on ancient metagenomes. We developed a novel method called decOM for Microbial Source Tracking and classification of ancient and modern metagenomic samples using k-mer matrices.
We analysed a collection of 360 ancient oral, modern oral, sediment/soil and skin metagenomes, using stratified five-fold cross-validation. decOM estimates the contributions of these source environments in ancient oral metagenomic samples with high accuracy, outperforming two state-of-the-art methods for source tracking, FEAST and mSourceTracker.
decOM is a high-accuracy microbial source tracking method, suitable for ancient oral metagenomic data sets. The decOM method is generic and could also be adapted for MST of other ancient and modern types of metagenomes. We anticipate that decOM will be a valuable tool for MST of ancient metagenomic studies. Video Abstract.

Quantitative microbial risk assessment (QMRA) can be used to evaluate health risks associated with recreational beach use. This study developed a site-specific risk assessment using a novel approach that combined quantitative PCR-based measurement of microbial source tracking (MST) genetic markers (human, dog, and gull fecal bacteria) with a QMRA analysis of potential pathogen risk. Water samples (n = 24) from two recreational beaches were collected and analyzed for MST markers as part of a broader Beach Exposure And Child Health Study that examined child behavior interactions with the beach environment. We report here the measurements of fecal bacteria MST markers in the environmental DNA extracts of those samples and a QMRA analysis of potential health risks utilizing the results from the MST measurements in the water samples. Human-specific Bacteroides was enumerated by the HF183 Taqman qPCR assay, gull-specific Catellicoccus was enumerated by the Gull2 qPCR assay, and dog-specific Bacteroides was enumerated by the DogBact qPCR assay. Derived reference pathogen doses, calculated from the MST marker concentrations detected in recreational waters, were used to estimate the risk of gastrointestinal illness for both children and adults. Dose-response equations were used to estimate the probability of the risk of infection (Pinf) per a swimming exposure event. Based on the QMRA simulations presented in this study, the GI risk from swimming or playing in water containing a mixture of human and non-human fecal sources appear to be primarily driven by the human fecal source. However, the estimated median GI health risk for both beaches never exceeded the U.S. EPA risk threshold of 32 illnesses per 1,000 recreation events. Our research suggests that utilizing QMRA together with MST can further extend our understanding of potential recreational bather risk by identifying the source contributing the greatest risk in a particular location, therefore informing beach management responses and decision-making.

This study investigated the decay rates of wastewater-associated markers and enteric viruses in laboratory microcosms mimicking estuarine water environments in temperate Sydney, NSW, Australia using qPCR and RT-qPCR assays. The results demonstrated the reduction in concentrations of Bacteroides HF183, Lachnospiraceae Lachno3, cross-assembly phage (crAssphage), pepper mild mottle virus (PMMoV), human adenovirus (HAdV 40/41), and enterovirus (EV) over a span of 42 days under spring/summer temperatures, presence/absence of microbiota, and different light conditions. The study found that HF183, Lachno3, crAssphage, PMMoV, HAdV 40/41, and EV exhibited varying decay rates depending on the experimental conditions. The average T90 values ranged from a few days to several months, indicating the rapid decay or prolonged persistence of these markers and enteric viruses in the estuarine environment. Furthermore, the study examined the effects of indigenous microbiota and spring/summer temperatures on wastewater-associated markers and enteric viruses decay rates. It was found that the presence of microbiota and temperature significantly influenced the decay rates of HF183 and PMMoV. Additionally, the study compared the effects of artificial sunlight and spring/summer temperatures on marker decay rates. Bacterial markers decayed faster than viral markers, although among viral markers crAssphage decay rates were relatively faster when compared to PMMoV. The exposure to artificial sunlight significantly accelerated the decay rates of bacterial markers, viral markers, and enteric viruses. Temperature also had an impact on the decay rates of Lachno3, crAssphage, and HAdV 40/41. In conclusion, this study provides valuable insights into the decay rates of wastewater-associated markers and enteric viruses under different experimental conditions that mimicked temperate environmental conditions. The findings contribute to our understanding of the fate and persistence of these markers in the environment which is crucial for assessing and managing risks from contamination by untreated human wastewater.