Spatio-temporal trends in microplastic presence in the sediments of the River Thames catchment (UK).

This study investigated the spatio-temporal variability of microplastics (MPs) in the sediments of the River Thames (UK) catchment over 30 months (July 2019 - Dec 2021). The average MP concentration was 61 items kg-1 d.w., with fragments <1 mm being dominant and polyethylene (PE) the most common polymer. Adjacent land use influenced MP concentrations and types, with industrial sites showing particularly high levels and a prevalence of small beads and industrial polymers. MP concentrations generally decreased after higher winter flows, likely due to sediment rearrangement or winnowing. This study describes the seasonal concentrations and characteristics of MPs present in sediment from the River Thames catchment, and attempts to identify their likely origin. Further, the study provides new insights into the mobility and fate of MPs in riverine settings under varying flow conditions, which is vital given the predicted increases in flooding under various global heating scenarios.

Evaluation of Low-Cost Phage-Based Microbial Source Tracking Tools for Elucidating Human Fecal Contamination Pathways in Kolkata, India.

Phages, such as those infecting Bacteroides spp., have been proven to be reliable indicators of human fecal contamination in microbial source tracking (MST) studies, and the efficacy of these MST markers found to vary geographically. This study reports the application and evaluation of candidate MST methods (phages infecting previously isolated B. fragilis strain GB-124, newly isolated Bacteroides strains (K10, K29, and K33) and recently isolated Kluyvera intermedia strain ASH-08), along with non-source specific somatic coliphages (SOMCPH infecting strain WG-5) and indicator bacteria (Escherichia coli) for identifying fecal contamination pathways in Kolkata, India. Source specificity of the phage-based methods was first tested using 60 known non-human fecal samples from common animals, before being evaluated with 56 known human samples (municipal sewage) collected during both the rainy and dry season. SOMCPH were present in 40-90% of samples from different animal species and in 100% of sewage samples. Phages infecting Bacteroides strain GB-124 were not detected from the majority (95%) of animal samples (except in three porcine samples) and were present in 93 and 71% of the sewage samples in the rainy and dry season (Mean = 1.42 and 1.83 log10PFU/100mL, respectively), though at lower levels than SOMCPH (Mean = 3.27 and 3.02 log10PFU/100mL, respectively). Phages infecting strain ASH-08 were detected in 89 and 96% of the sewage samples in the rainy and dry season, respectively, but were also present in all animal samples tested (except goats). Strains K10, K29, and K30 were not found to be useful MST markers due to low levels of phages and/or co-presence in non-human sources. GB-124 and SOMCPH were subsequently deployed within two low-income neighborhoods to determine the levels and origin of fecal contamination in 110 environmental samples. E. coli, SOMCPH, and phages of GB-124 were detected in 68, 42, and 28% of the samples, respectively. Analyses of 166 wastewater samples from shared community toilets and 21 samples from sewage pumping stations from the same districts showed that SOMCPH were present in 100% and GB-124 phages in 31% of shared toilet samples (Median = 5.59 and <1 log10 PFU/100 mL, respectively), and both SOMCPH and GB-124 phages were detected in 95% of pumping station samples (Median = 5.82 and 4.04 log10 PFU/100 mL, respectively). Our findings suggest that GB-124 and SOMCPH have utility as low-cost fecal indicator tools which can facilitate environmental surveillance of enteric organisms, elucidate human and non-human fecal exposure pathways, and inform interventions to mitigate exposure to fecal contamination in the residential environment of Kolkata, India.

Genome Characterization of a Novel Wastewater Bacteroides fragilis Bacteriophage (vB_BfrS_23) and its Host GB124.

Bacteroides spp. are part of the human intestinal microbiota but can under some circumstances become clinical pathogens. Phages are a potentially valuable therapeutic treatment option for many pathogens, but phage therapy for pathogenic Bacteroides spp. including Bacteroides fragilis is currently limited to three genome-sequenced phages. Here we describe the isolation from sewage wastewater and genome of a lytic phage, vB_BfrS_23, that infects and kills B. fragilis strain GB124. Transmission electron microscopy identified this phage as a member of the Siphoviridae family. The phage is stable when held at temperatures of 4 and 60°C for 1 h. It has a very narrow host range, only infecting one host from a panel of B. fragilis strains (n = 8). Whole-genome sequence analyses of vB_BfrS_23 determined it is double-stranded DNA phage and is circularly permuted, with a genome of 48,011 bp. The genome encodes 73 putative open reading frames. We also sequenced the host bacterium, B. fragilis GB124 (5.1 Mb), which has two plasmids of 43,923 and 4,138 bp. Although this phage is host specific, its isolation together with the detailed characterization of the host B. fragilis GB124 featured in this study represent a useful starting point from which to facilitate the future development of highly specific therapeutic agents. Furthermore, the phage could be a novel tool in determining water (and water reuse) treatment efficacy, and for identifying human fecal transmission pathways within contaminated environmental waters and foodstuffs.

A longitudinal study of the association between domestic contact with livestock and contamination of household point-of-use stored drinking water in rural Siaya County (Kenya).

BACKGROUND: Emerging evidence suggests close domestic proximity of livestock and humans may lead to microbiological contamination of hands, objects, food and water supplies within domestic environments, adversely impacting public health. However, evidence quantifying the relationship between livestock, domestic animals, humans and microbiological contamination of household stored water remains limited. AIM: This longitudinal study aimed to examine the relationship between domestic contact with livestock and domestic animals on microbiological contamination of household Point-of-Use (POU) stored drinking water in rural Kenya and assess the influence of choice of faecal indicator on such associations. METHODOLOGY: A survey was performed in 234 households in Siaya county, Kenya, to observe presence of livestock (cattle, goats, poultry) and domestic animals (cats, dogs) in household compounds, alongside other risk factors for contamination of POU stored drinking water such as sanitation, storage conditions and hygiene practices. Samples from water sources (e.g. piped, spring/wells, boreholes, surface and rainwater) and from POU storage containers were tested for E. coli and intestinal enterococci. Livestock-related risk factors for water contamination were examined through multinomial regression, controlling for confounders. RESULTS: Rainwater was the main POU water source and was found to be highly susceptible to contamination. Multivariate analysis showed greater risk of gross (>100 CFU/100 mL) water contamination (with E. coli) for households where goats were observed, and/or where poultry roosted in proximity to stored household water (relative risk RR = 2.71; p = 0.001 and RR = 2.02; p = 0.012 respectively). Presence of a poultry coop was also associated with elevated intestinal enterococci densities (RR = 4.46; p = 0.001). Associations between contamination and livestock risk factors were thus similar for both bacteria groups, but E. coli counts declined more rapidly following collection from surface waters than enterococci counts (p = 0.024). CONCLUSION: The presence of livestock (particularly goats) and poultry within household compounds increases POU water contamination risk, suggesting the need for improved interventions to address cross-contamination within rural domestic settings. Within Siaya county, more effective community education is needed to raise awareness of POU water quality protection, particularly of rainwater.

Pathogen infection risk to recreational water users, associated with surface waters impacted by de facto and indirect potable reuse activities.

Water deficit, exacerbated by global population increases and climate change, necessitates the investigation of alternative non-traditional water sources to augment existing supplies. Indirect potable reuse (IPR) represents a promising alternative water source in water-stressed regions. Of high concern is the presence of pathogenic microorganisms in wastewater, such as enteric viruses, protozoa and bacteria. Therefore, a greater understanding of the potential impact to human health is required. The aim of this research was to use a quantitative microbial risk assessment (QMRA) approach to calculate the probability of potential pathogen infection risk to the public in surface waters used for a range of recreational activities under scenarios: 1) existing de facto wastewater reuse conditions; 2) after augmentation with conventionally treated wastewater; and 3) after augmentation with reclaimed wastewater from proposed IPR schemes. Forty-four 31 l samples were collected from river sites and a coastal wastewater treatment works from July 2016-May 2017. Concentrations of faecal indicator organisms (enterococci, faecal coliforms, somatic coliphages and Bacteroides phages) determined using culture-based approaches and selected pathogens (adenovirus, Salmonella and Cryptosporidium) determined using molecular approaches (qPCR) were used to inform QMRA. The mean probability of infection from adenovirus under de facto conditions was high (>0.90) for all recreational activities, per single event. The risk of adenovirus and Cryptosporidium infection increased under augmentation scenario (2) (mean probability 0.95-1.00 and 0.01-0.06 per single event, respectively). Adenovirus and Cryptosporidium infection risk decreased under reclaimed water augmentation scenario (3) (mean probability <0.79, excluding swimming, which remained 1.00 and <0.01 per single event, respectively). Pathogen reduction after reclaimed water augmentation in surface waters impacted by de facto reuse, provides important evidence for alternative water supply option selection. As such, this evidence may inform water managers and the public of the potential benefits of IPR and improve acceptance of such practices in the future.

Storm Response of Fluvial Sedimentary Microplastics.

Up to 80% of the plastics in the oceans are believed to have been transferred from river networks. Microplastic contamination of river sediments has been found to be pervasive at the global scale and responsive to periods of flooding. However, the physical controls governing the storage, remobilization and pathways of transfer in fluvial sediments are unknown. This means it is not currently possible to determine the risks posed by microplastics retained within the world's river systems. This problem will be further exacerbated in the future given projected changes to global flood risk and an increased likelihood of fluvial flooding. Using controlled flume experiments we show that the evolution of the sediment bed surface and the flood wave characteristics controls the transition from rivers being 'sinks' to 'sources' of microplastics under flood conditions. By linking bed surface evolution with microplastic transport characteristics we show that similarities exist between granular transport phenomena and the behavior, and hence predictability, of microplastic entrainment during floods. Our findings are significant as they suggest that microplastic release from sediment beds can be managed by altering the timing and magnitude of releases in flow managed systems. As such it may be possible to remediate or remove legacy microplastics in future.

Potential microbial transmission pathways in rural communities using multiple alternative water sources in semi-arid Brazil.

BACKGROUND: Water scarcity combined with high incidences of diarrhoeal disease amongst many rural communities, suggests that the provision of 'safe' water supplies remains a challenge. Subsequent reliance on multi-source water supplies means that microbial transmission pathways may be numerous and complex. OBJECTIVES: This study aimed to identify and elucidate water supply issues and potential microbial transmission pathways at the household level in rural communities in semi-arid Brazil. METHODS: Community and sanitary surveys were applied to 99 households from 10 communities located in four municipalities of Paraiba State, Brazil. Moreover, physicochemical and microbial parameters were investigated throughout the water supply chain. RESULTS: High levels of faecal indicator organisms (FIO) were detected in water from Water Storage Reservoirs (WSR) and from in-house Drinking Water Storage Containers (DWSC). A decrease in microbial water quality was observed between water stored within WSR's and DWSC's, suggesting potential cross-contamination at the household level. Several common practices were observed among rural residents, such as the use of collection buckets left unprotected outside on the ground, that may have also contributed to the observed decrease. Schematic diagrams illustrating the complex water supply chains and potential microbial transmission pathways were developed to facilitate identification of effective intervention strategies. DISCUSSION: Decreases in water supply quality were found to be predominantly caused by cross-contamination within the domestic environment due to several factors, including, a lack of awareness and knowledge of 'safe' collection, handling and storage of water, as well as inadequate sanitation services and/or unhygienic practices. In order to improve public health in such communities, more effort should be directed towards education and training of all stakeholders involved in the water supply chain. This would, certainly, support the successful implementation of further WASH interventions, consequently increasing the likelihood of achieving reduction of excreta-borne diseases.

Assessment of recommended approaches for containment and safe handling of human excreta in emergency settings.

Ebola and cholera treatment centres (ETC and CTC) generate considerable quantities of excreta that can further the transmission of disease amongst patients and health workers. Therefore, approaches for the safe handling, containment and removal of excreta within such settings are needed to minimise the likelihood of onward disease transmission. This study compared the performance and suitability of three chlorine-based approaches (0.5% HTH, NaDCC and NaOCl (domestic bleach)) and three lime-based approaches (10%, 20% and 30% Ca(OH)2). The experiments followed recent recommendations for Ebola Treatment Centres. Three excreta matrices containing either raw municipal wastewater, or raw municipal wastewater plus 10% or 20% (w/v) added faecal sludge, were treated in 14 litre buckets at a ratio of 1:10 (chlorine solutions or lime suspensions: excreta matrix). The effects of mixing versus non-mixing and increasing contact time (10 and 30 mins) were also investigated. Bacterial (faecal coliforms (FC) and intestinal enterococci (IE)) and viral (somatic coliphages (SOMPH), F+specific phages (F+PH) and Bacteroides fragilis phages (GB-124PH)) indicators were used to determine the efficacy of each approach. Lime-based approaches provided greater treatment efficacy than chlorine-based approaches, with lime (30% w/v) demonstrating the greatest efficacy (log reductions values, FC = 4.75, IE = 4.16, SOMPH = 2.85, F+PH = 5.13 and GB124PH = 5.41). There was no statistical difference in efficacy between any of the chlorine-based approaches, and the highest log reduction values were: FC = 2.90, IE = 2.36, SOMPH = 3.01, F+PH = 2.36 and GB124PH = 0.74. No statistical difference was observed with respect to contact time for any of the approaches, and no statistical differences were observed with respect to mixing for the chlorine-based approaches. However, statistically significant increases in the efficacy of some lime-based approaches were observed following mixing. These findings provide evidence and practical advice to inform safe handling and containment of excreta and ensure more effective health protection in future emergency settings.

Global Distribution of Human-Associated Fecal Genetic Markers in Reference Samples from Six Continents.

Numerous bacterial genetic markers are available for the molecular detection of human sources of fecal pollution in environmental waters. However, widespread application is hindered by a lack of knowledge regarding geographical stability, limiting implementation to a small number of well-characterized regions. This study investigates the geographic distribution of five human-associated genetic markers (HF183/BFDrev, HF183/BacR287, BacHum-UCD, BacH, and Lachno2) in municipal wastewaters (raw and treated) from 29 urban and rural wastewater treatment plants (750-4 400 000 population equivalents) from 13 countries spanning six continents. In addition, genetic markers were tested against 280 human and nonhuman fecal samples from domesticated, agricultural and wild animal sources. Findings revealed that all genetic markers are present in consistently high concentrations in raw (median log10 7.2-8.0 marker equivalents (ME) 100 mL-1) and biologically treated wastewater samples (median log10 4.6-6.0 ME 100 mL-1) regardless of location and population. The false positive rates of the various markers in nonhuman fecal samples ranged from 5% to 47%. Results suggest that several genetic markers have considerable potential for measuring human-associated contamination in polluted environmental waters. This will be helpful in water quality monitoring, pollution modeling and health risk assessment (as demonstrated by QMRAcatch) to guide target-oriented water safety management across the globe.

Resolution of habitat-associated ecogenomic signatures in bacteriophage genomes and application to microbial source tracking.

Just as the expansion in genome sequencing has revealed and permitted the exploitation of phylogenetic signals embedded in bacterial genomes, the application of metagenomics has begun to provide similar insights at the ecosystem level for microbial communities. However, little is known regarding this aspect of bacteriophage associated with microbial ecosystems, and if phage encode discernible habitat-associated signals diagnostic of underlying microbiomes. Here we demonstrate that individual phage can encode clear habitat-related 'ecogenomic signatures', based on relative representation of phage-encoded gene homologues in metagenomic data sets. Furthermore, we show the ecogenomic signature encoded by the gut-associated ɸB124-14 can be used to segregate metagenomes according to environmental origin, and distinguish 'contaminated' environmental metagenomes (subject to simulated in silico human faecal pollution) from uncontaminated data sets. This indicates phage-encoded ecological signals likely possess sufficient discriminatory power for use in biotechnological applications, such as development of microbial source tracking tools for monitoring water quality.

The application of bacteriophages as novel indicators of viral pathogens in wastewater treatment systems.

Many wastewater treatment technologies have been shown to remove bacterial pathogens more effectively than viral pathogens and, in aquatic environments, levels of traditional faecal indicator bacteria (FIB) do not appear to correlate consistently with levels of human viral pathogens. There is, therefore, a need for novel viral indicators of faecal pollution and surrogates of viral pathogens, especially given the increasing importance of indirect and direct wastewater reuse. Potential candidates include bacteriophages (phages) and the study described here sought to elucidate the relationship between three groups of phages (somatic coliphages (SOMPH), F-RNA coliphages (F-RNAPH) and human-specific phages infecting B. fragilis (Bf124PH) - enumeration using double layer agar technique) and viral pathogens (human adenovirus (HuAdV) and norovirus (NoV) - enumeration using molecular methods) through full-scale municipal wastewater treatment processes. FIB (faecal coliforms (FC) and intestinal enterococci (ENT) - enumeration using membrane filtration) were also monitored. Samples were collected every fortnight, during a twelve-month period, at each stage of four full-scale wastewater treatment plants (WWTP) in southern England (two activated sludge (AS) and two trickling filter (TF) plants) (n = 360 samples). FIB and SOMPH were consistently found in all samples tested, whereas F-RNAPH, Bf124PH and HuAdV were less frequently detected, especially following AS treatment. The detection rate of NoV was low and consequently discussion of this group of viruses is limited. Concentrations of SOMPH and FIB were statistically higher (p value < 0.05) than concentrations of F-RNAPH, Bf124PH and HuAdV in raw wastewater. FIB were more effectively removed than phages in both systems. Removal rates of HuAdV were similar to those of phages at the secondary treatment stage of both systems. In TF systems, HuAdV were removed at the same rate as F-RNAPH, but at lower rates than SOMPH and Bf124PH. The findings suggest that phages (in particular SOMPH) are better indicators of the fate of viral pathogens in WWTP than existing FIB and that these organisms may have a useful role to play in future sanitation safety planning.

Removal of phages and viral pathogens in a full-scale MBR: Implications for wastewater reuse and potable water.

The aim of this study was to demonstrate how seasonal variability in the removal efficacy of enteric viral pathogens from an MBR-based water recycling system might affect risks to human health if the treated product were to be used for the augmentation of potable water supplies. Samples were taken over a twelve month period (March 2014-February 2015), from nine locations throughout a water recycling plant situated in East London and tested for faecal indicator bacteria (thermotolerant coliforms, intestinal enterococci n = 108), phages (somatic coliphage, F-specific RNA phage and Bacteroides phage (GB-124) n = 108), pathogenic viruses (adenovirus, hepatitis A, norovirus GI/GII n = 48) and a range of physico-chemical parameters (suspended solids, DO, BOD, COD). Thermotolerant coliforms and intestinal enterococci were removed effectively by the water recycling plant throughout the study period. Significant mean log reductions of 3.9-5.6 were also observed for all three phage groups monitored. Concentrations of bacteria and phages did not vary significantly according to season (P < 0.05; Kruskal-Wallis), though recorded levels of norovirus (GI) were significantly higher during autumn/winter months (P = 0.027; Kruskal-Wallis). Log reduction values for norovirus and adenovirus following MBR treatment were 2.3 and 4.4, respectively. However, both adenovirus and norovirus were detected at low levels (2000 and 3240 gene copies/L, respectively) post chlorination in single samples. Whilst phage concentrations did correlate with viral pathogens, the results of this study suggest that phages may not be suitable surrogates, as viral pathogen concentrations varied to a greater degree seasonally than did the phage indicators and were detected on a number of occasions on which phages were not detected (false negative sample results).

Minimizing the Risk of Disease Transmission in Emergency Settings: Novel In Situ Physico-Chemical Disinfection of Pathogen-Laden Hospital Wastewaters.

The operation of a health care facility, such as a cholera or Ebola treatment center in an emergency setting, results in the production of pathogen-laden wastewaters that may potentially lead to onward transmission of the disease. The research presented here evaluated the design and operation of a novel treatment system, successfully used by Médecins Sans Frontières in Haiti to disinfect CTC wastewaters in situ, eliminating the need for road haulage and disposal of the waste to a poorly-managed hazardous waste facility, thereby providing an effective barrier to disease transmission through a novel but simple sanitary intervention. The physico-chemical protocols eventually successfully treated over 600 m3 of wastewater, achieving coagulation/flocculation and disinfection by exposure to high pH (Protocol A) and low pH (Protocol B) environments, using thermotolerant coliforms as a disinfection efficacy index. In Protocol A, the addition of hydrated lime resulted in wastewater disinfection and coagulation/flocculation of suspended solids. In Protocol B, disinfection was achieved by the addition of hydrochloric acid, followed by pH neutralization and coagulation/flocculation of suspended solids using aluminum sulfate. Removal rates achieved were: COD >99%; suspended solids >90%; turbidity >90% and thermotolerant coliforms >99.9%. The proposed approach is the first known successful attempt to disinfect wastewater in a disease outbreak setting without resorting to the alternative, untested, approach of 'super chlorination' which, it has been suggested, may not consistently achieve adequate disinfection. A basic analysis of costs demonstrated a significant saving in reagent costs compared with the less reliable approach of super-chlorination. The proposed approach to in situ sanitation in cholera treatment centers and other disease outbreak settings represents a timely response to a UN call for onsite disinfection of wastewaters generated in such emergencies, and the 'Coalition for Cholera Prevention and Control' recently highlighted the research as meriting serious consideration and further study. Further applications of the method to other emergency settings are being actively explored by the authors through discussion with the World Health Organization with regards to the ongoing Ebola outbreak in West Africa, and with the UK-based NGO Oxfam with regards to excreta-borne disease management in the Philippines and Myanmar, as a component of post-disaster incremental improvements to local sanitation chains.

The application of removal coefficients for viruses in different wastewater treatment processes calculated using stochastic modelling.

This study proposes that calculating and interpreting removal coefficients (K20) for bacteriophages in activated sludge (AS) and trickling filter (TF) systems using stochastic modelling may provide important information that may be used to estimate the removal of phages in such systems using simplified models. In order to achieve this, 14 samples of settled wastewater and post-secondary sedimentation wastewater were collected every 2 weeks, over a 6-month period (May to November), from two AS and two TF systems situated in southern England. Initial results have demonstrated that the removal of somatic coliphages in both AS and TF systems is considerably higher than that of F-RNA coliphages, and that AS more effectively removes both phage groups than TF. The results have also demonstrated that K20 values for phages in AS are higher than in TF, which could be justified by the higher removal rates observed in AS and the models assumed for both systems. The research provides a suggested framework for calculating and predicting removal rates of pathogens and indicator organisms in wastewater treatment systems using simplified models in order to support integrated water and sanitation safety planning approaches to human health risk management.

Bacteriophage removal in a full-scale membrane bioreactor (MBR) - Implications for wastewater reuse.

The aim of this study was to assess the potential removal efficacy of viruses in a full-scale membrane bioreactor (MBR) wastewater reuse system, using a range of indigenous and 'spiked' bacteriophages (phages) of known size and morphology. Samples were taken each week for three months from nine locations at each treatment stage of the water recycling plant (WRP) and tested for a range of microbiological parameters (n = 135). Mean levels of faecal coliforms were reduced to 0.3 CFU/100 ml in the MBR product and were undetected in samples taken after the chlorination stage. A relatively large reduction (5.3 log) in somatic coliphages was also observed following MBR treatment. However, F-specific and human-specific (GB124) phages were less abundant at all stages, and demonstrated log reductions post-MBR of 3.5 and 3.8, respectively. In 'spiking' experiments, suspended 'spiked' phages (MS2 and B-14) displayed post-MBR log reductions of 2.25 and 2.30, respectively. The removal of these suspended phages, which are smaller than the membrane pore size (0.04 µm), also highlights the possible role of the membrane biofilm as an effective additional barrier to virus transmission. The findings from this study of a full-scale MBR system demonstrate that the enumeration of several phage groups may offer a practical and conservative way of assessing the ability of MBR to remove enteric viruses of human health significance. They also suggest that phage removal in MBR systems may be highly variable and may be closely related on the one hand to both the size and morphology of the viruses and, on the other, to whether or not they are attached to solids.

Inactivation of bacteriophage infecting Bacteroides strain GB124 using UV-B radiation.

Ultraviolet-B radiation (280-320 nm) has long been associated with the inactivation of microorganisms in the natural environment. Determination of the environmental inactivation kinetics of specific indicator organisms [used as tools in the field of microbial source tracking (MST)] is fundamental to their successful deployment, particularly in geographic regions subject to high levels of solar radiation. Phage infecting Bacteroides fragilis host strain GB124 (B124 phage) have been demonstrated to be highly specific indicators of human fecal contamination, but to date, little is known about their susceptibility to UV-B radiation. Therefore, B124 phage (n = 7) isolated from municipal wastewater effluent, were irradiated in a controlled laboratory environment using UV-B collimated beam experiments. All B124 phage suspensions possessed highly similar first order log-linear inactivation profiles and the mean fluence required to inactivate phage by 4 - log(10) was 320 mJ cm(-2). These findings suggest that phage infecting GB124 are likely to be inactivated when exposed to the levels of UV-B solar radiation experienced in a variety of environmental settings. As such, this may limit the utility of such methods for determining more remote inputs of fecal contamination in areas subject to high levels of solar radiation.

Genome signature-based dissection of human gut metagenomes to extract subliminal viral sequences.

Bacterial viruses (bacteriophages) have a key role in shaping the development and functional outputs of host microbiomes. Although metagenomic approaches have greatly expanded our understanding of the prokaryotic virosphere, additional tools are required for the phage-oriented dissection of metagenomic data sets, and host-range affiliation of recovered sequences. Here we demonstrate the application of a genome signature-based approach to interrogate conventional whole-community metagenomes and access subliminal, phylogenetically targeted, phage sequences present within. We describe a portion of the biological dark matter extant in the human gut virome, and bring to light a population of potentially gut-specific Bacteroidales-like phage, poorly represented in existing virus like particle-derived viral metagenomes. These predominantly temperate phage were shown to encode functions of direct relevance to human health in the form of antibiotic resistance genes, and provided evidence for the existence of putative 'viral-enterotypes' among this fraction of the human gut virome.

Performance of viruses and bacteriophages for fecal source determination in a multi-laboratory, comparative study.

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10-50 gene copies or plaques × 50 ml(-1)) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40-70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent.

Performance characteristics of qPCR assays targeting human- and ruminant-associated bacteroidetes for microbial source tracking across sixteen countries on six continents.

Numerous quantitative PCR assays for microbial fecal source tracking (MST) have been developed and evaluated in recent years. Widespread application has been hindered by a lack of knowledge regarding the geographical stability and hence applicability of such methods beyond the regional level. This study assessed the performance of five previously reported quantitative PCR assays targeting human-, cattle-, or ruminant-associated Bacteroidetes populations on 280 human and animal fecal samples from 16 countries across six continents. The tested cattle-associated markers were shown to be ruminant-associated. The quantitative distributions of marker concentrations in target and nontarget samples proved to be essential for the assessment of assay performance and were used to establish a new metric for quantitative source-specificity. In general, this study demonstrates that stable target populations required for marker-based MST occur around the globe. Ruminant-associated marker concentrations were strongly correlated with total intestinal Bacteroidetes populations and with each other, indicating that the detected ruminant-associated populations seem to be part of the intestinal core microbiome of ruminants worldwide. Consequently tested ruminant-targeted assays appear to be suitable quantitative MST tools beyond the regional level while the targeted human-associated populations seem to be less prevalent and stable, suggesting potential for improvements in human-targeted methods.

Comparative (meta)genomic analysis and ecological profiling of human gut-specific bacteriophage φB124-14.

Bacteriophage associated with the human gut microbiome are likely to have an important impact on community structure and function, and provide a wealth of biotechnological opportunities. Despite this, knowledge of the ecology and composition of bacteriophage in the gut bacterial community remains poor, with few well characterized gut-associated phage genomes currently available. Here we describe the identification and in-depth (meta)genomic, proteomic, and ecological analysis of a human gut-specific bacteriophage (designated φB124-14). In doing so we illuminate a fraction of the biological dark matter extant in this ecosystem and its surrounding eco-genomic landscape, identifying a novel and uncharted bacteriophage gene-space in this community. φB124-14 infects only a subset of closely related gut-associated Bacteroides fragilis strains, and the circular genome encodes functions previously found to be rare in viral genomes and human gut viral metagenome sequences, including those which potentially confer advantages upon phage and/or host bacteria. Comparative genomic analyses revealed φB124-14 is most closely related to φB40-8, the only other publically available Bacteroides sp. phage genome, whilst comparative metagenomic analysis of both phage failed to identify any homologous sequences in 136 non-human gut metagenomic datasets searched, supporting the human gut-specific nature of this phage. Moreover, a potential geographic variation in the carriage of these and related phage was revealed by analysis of their distribution and prevalence within 151 human gut microbiomes and viromes from Europe, America and Japan. Finally, ecological profiling of φB124-14 and φB40-8, using both gene-centric alignment-driven phylogenetic analyses, as well as alignment-free gene-independent approaches was undertaken. This not only verified the human gut-specific nature of both phage, but also indicated that these phage populate a distinct and unexplored ecological landscape within the human gut microbiome.