Novel crAssphage marker genes ascertain sewage pollution in a recreational lake receiving urban stormwater runoff

Ahmed, Warish; Payyappat, Sudhi; Cassidy, Michele; Besley, C.H.; Power, Kaye

doi:10.1016/j.watres.2018.08.049

Cited by 90 publications

(78 citation statements)

References 39 publications

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“…Our results would be in line with previous articles that have detected crAssphage via qPCR in fecal samples from cats, and wastewater derived from cattle, pigs and poultry (36,37). Since these studies were PCR-based, they could not discard the possibility of a cross-reaction of the PCR to some similar genomic regions of other related phages.…”

Section: Assembly Of An Uncultured Crassphage From Canine Fecal Metagsupporting

confidence: 91%

“…Since the initial screening of CrAssphage via PCR in several animal cohorts was negative, it has been regarded as a potential marker for human fecal pollution, to distinguish the origin of the pollution from other animal sources (34,35). Using this methods, dog fecal samples have already been screened for CrAssphage, with negative results (35,36). However, our results suggest that uncultured crAssphage can be an abundant member of dog fecal metagenome.…”

Section: Assembly Of An Uncultured Crassphage From Canine Fecal Metagmentioning

confidence: 90%

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Shallow metagenomics with Nanopore sequencing in canine fecal microbiota improved bacterial taxonomy and identified an uncultured CrAssphage

Cuscó¹,

Salas²,

Torre³

et al. 2019

Preprint

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Long-read metagenomics -using single-molecule sequencers-has the potential to assembly entire genomes, even from complex metagenomics datasets. Using long-read metagenomics with Nanopore sequencing in pooled samples, we aim to improve the individual taxonomic profiles obtained with V4 16S rRNA massive sequencing and to assemble the fecal metagenome of healthy dogs.Fecal samples from healthy dogs were sequenced individually using V4 16S rRNA gene and in pools using a shallow metagenomics approach with Nanopore sequencing. Longread metagenomics allowed us refining the V4 16S rRNA results up to the species level and determining the main bacterial species inhabiting on fecal microbiota of our cohort of healthy dogs. Among them, the most abundant were Fusobacterium varium; Megamonas hypermegale; Bacteroides vulgatus; Blautia hansenii; Clostridium perfringens; and Clostridoides difficile. Once performed the metagenome assembly, one contig was suggested to be circular and hit to an uncultured crAssphage.To conclude, shallow long-read metagenomics with pooled samples using MinION allowed characterizing the dog fecal microbiota at lower taxonomic level, such as bacterial species. The assembly of the reads retrieved a contig that represents a circular draft genome of an uncultured CrAssphage from dog fecal samples that is one of the most abundant bacteriophages in the human gut.

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Section: Assembly Of An Uncultured Crassphage From Canine Fecal Metagsupporting

confidence: 91%

Section: Assembly Of An Uncultured Crassphage From Canine Fecal Metagmentioning

confidence: 90%

Shallow metagenomics with Nanopore sequencing in canine fecal microbiota improved bacterial taxonomy and identified an uncultured CrAssphage

Cuscó¹,

Salas²,

Torre³

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…The HF183 marker was consistently detected with human pathogen NoV (96% positive agreement in San Diego River and 72% positive agreement in Tourmaline Creek). Ahmed et al, 2018c examined the extent of sewage contamination in an urban recreational lake located in Sydney, Australia that receives wet weather overflows using two human feces-associated crAssphage marker genes (CPQ_056 and CPQ_064). The concentrations of both markers were higher (CPQ_056 ranging from 3.40 to 7.62 log 10 GC/L Table 1 Prevalence and concentrations (log 10 GC/L) of sewage and animal-associated marker genes in stormwater runoff and outfall samples.…”

Section: Mst Marker Genes In Stormwatermentioning

confidence: 99%

A review on microbial contaminants in stormwater runoff and outfalls: Potential health risks and mitigation strategies

Ahmed

Hamilton

Toze

et al. 2019

Science of The Total Environment

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119

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Editor: Patricia HoldenDemands on global water supplies are increasing in response to the need to provide more food, water, and energy for a rapidly growing population. These water stressors are exacerbated by climate change, as well as the growth and urbanisation of industry and commerce. Consequently, urban water authorities around the globe are exploring alternative water sources to meet ever-increasing demands. These alternative sources are primarily treated sewage, stormwater, and groundwater. Stormwater including roof-harvested rainwater has been considered as an alternative water source for both potable and non-potable uses. One of the most significant issues concerning alternative water reuse is the public health risk associated with chemical and microbial contaminants. Several studies to date have quantified fecal indicators and pathogens in stormwater. Microbial source tracking (MST) approaches have also been used to determine the sources of fecal contamination in stormwater and receiving waters. This review paper summarizes occurrence and concentrations of fecal indicators, pathogens, and MST marker genes in urban stormwater. A section of the review highlights the removal of fecal indicators and pathogens through water sensitive urban design (WSUD) or Best Management Practices (BMPs). We also discuss approaches for assessing and mitigating health risks associated with stormwater, including a summary of existing quantitative microbial risk assessment (QMRA) models for potable and non-potable reuse of stormwater. Finally, the most critical research gaps are identified for formulating risk management strategies.Crown

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“…Thus, their presence in water bodies indicates fecal contamination has occurred from specific host groups. Most commonly used MST tools identify or quantify a DNA sequence(s) or marker gene(s) from the host-associated microorganisms by PCR or quantitative PCR (qPCR) assays (3)(4)(5)(6).…”

mentioning

confidence: 99%

“…Urbanization and extreme weather events such as floods and wet/dry weather overflows result in an increased amount of sewage being discharged into water bodies (3,(25)(26)(27). Sewage-contaminated water bodies are potential public health hazards and can cause severe illnesses to humans due to the presence of infectious pathogens (28).…”

mentioning

confidence: 99%

Host Specificity and Sensitivity of Established and Novel Sewage-Associated Marker Genes in Human and Nonhuman Fecal Samples

Ahmed

Gyawali

Feng

et al. 2019

Appl Environ Microbiol

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Microbial source tracking (MST) methods measure fecal contamination levels and identify possible sources using quantitative PCR (qPCR) that targets host-associated fecal microorganisms. To date, most established MST assays for human sources, especially bacterial markers, have shown some nonhuman host cross-reactions. Recently developed assays, such as the crAssphage CPQ_056, Lachnospiraceae Lachno3, and Bacteroides BacV6-21, have more limited information on host sensitivity and host specificity for human or sewage sources, particularly in countries other than the United States. In this study, we rigorously evaluated six sewage-associated MST assays (i.e., Bacteroides HF183, human adenovirus [HAdV], human polyomavirus [HPyV], crAssphage CPQ_056, Lachno3, and BacV6-21) to show advantages and disadvantages of their applications for MST. A total of 29 human and 3 sewage samples and 360 nonhuman fecal samples across 14 hosts collected from a subtropical region of Australia were tested for marker host specificity, host sensitivity, and concentrations. All sewage samples were positive for all six marker genes tested in this study. Bacterial markers were more prevalent than viral markers in human feces. Testing against animal hosts showed human feces (or sewage)associated marker gene specificity was HAdV (1.00) Ͼ HPyV (0.99) Ͼ crAssphage CPQ_056 (0.98) Ͼ HF183 (0.96) Ͼ Lachno3 (0.95) Ͼ BacV6-21 (0.90), with marker concentrations in some animal fecal samples being 3 to 5 orders of magnitude lower than those in sewage. When considering host specificity, sensitivity, and concentrations in source samples, the HF183, Lachno3, and crAssphage CPQ_056 tests were the most suitable assays in this study for sewage contamination tracking in subtropical waters of Australia. IMPORTANCE Large financial investments are required to remediate fecal contamination sources in waterways, and accurate results from field studies are crucial to build confidence in MST approaches. Host specificity and sensitivity are two main performance characteristics for consideration when choosing MST assays. Ongoing efforts for marker assay validation will improve interpretation of results and could shed light on patterns of occurrence in nontarget hosts that might explain the underlying drivers of cross-reaction of certain markers. For field applications, caution should be taken to choose appropriate MST marker genes and assays based on available host specificity and sensitivity data and background knowledge of the contaminating sources in the study area. Since many waterborne pathogens are viruses, employing both viral and bacterial markers in investigations could provide insight into contamination dynamics and ecological behavior in the environment. Therefore, combined usage of marker assays is recommended for more accurate and informative sewage contamination detection and fecal source resolution. KEYWORDS microbial source trackingCitation Ahmed W, Gyawali P, Feng S, McLellan SL. 2019. Host specificity and sensitivity of established and novel sewageassoc...

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Novel crAssphage marker genes ascertain sewage pollution in a recreational lake receiving urban stormwater runoff

Cited by 90 publications

References 39 publications

Shallow metagenomics with Nanopore sequencing in canine fecal microbiota improved bacterial taxonomy and identified an uncultured CrAssphage

Shallow metagenomics with Nanopore sequencing in canine fecal microbiota improved bacterial taxonomy and identified an uncultured CrAssphage

A review on microbial contaminants in stormwater runoff and outfalls: Potential health risks and mitigation strategies

Host Specificity and Sensitivity of Established and Novel Sewage-Associated Marker Genes in Human and Nonhuman Fecal Samples

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