Development and evaluation of a novel human-specific fecal source tracking marker

Enteric viruses pose a significant health risk to the public through exposure to contaminated environmental waters, costing society billions of dollars to treat millions of excess illnesses annually. Current available methods to monitor human fecal pollution in impacted waters either do not correlate with viral pathogen presence and risk, cross-react with other animal sources, or are not abundant enough in environmental waters to be reliably detected. This dissertation investigates a novel target for a human-specific indicator of fecal pollution, the bacteriophage “crAssphage”. First, a metagenomic evaluation was conducted as an initial step to evaluate the potential of the crAssphage genome for assay development. Results indicated that crAssphage is enriched in sewage and more abundant than other viruses and bacteriophages in sewage, suggesting its high potential as a target for human-specific marker development. Next, end-point PCR primers were designed along the length of the crAssphage genome and screened against human sewage samples and non-human animal fecal samples to assess which genomic regions may be the most useful as source tracking markers. The two best performing primer pairs were adapted to TaqMan qPCR assays. These assays were evaluated head-to-head against two bacterial qPCR human source tracking markers to evaluate their performance. The assays were widespread in geographically diverse human sources and as abundant or more abundant in sewage and impaired environmental water than the bacterial-based assays. Moreover, the assays displayed high human-association. Finally, an initial environmental evaluation was conducted to correlate the crAssphage-based assays to pollution events, culturable bacterial and phage indicators, and molecular bacterial and viral indicators. This study demonstrated the usefulness of the crAssphage assays in a real-world system, validating their high abundance and correlation with pollution events and other indicators of fecal pollution. Ultimately, the research in this dissertation contributes two novel viral-based technologies for detection of human fecal pollution that will enhance management of environmental waters and protect public health through the development of an abundant viral fecal source-tracking marker

Potential probiotic approaches to control Legionella in engineered aquatic ecosystems

Opportunistic pathogens belonging to the genus Legionella are among the most reported waterborne-associated pathogens in industrialized countries. Legionella colonize a variety of engineered aquatic ecosystems and persist in biofilms where they interact with a multitude of other resident microorganisms. In this review, we assess how some of these interactions could be used to develop a biological-driven "probiotic" control approach against Legionella. We focus on: (i) mechanisms limiting the ability of Legionella to establish and replicate within some of their natural protozoan hosts; (ii) exploitative and interference competitive interactions between Legionella and other microorganisms; and (iii) the potential of predatory bacteria and phages against Legionella. This field is still emergent, and we therefore specifically highlight research for future investigations, and propose perspectives on the feasibility and public acceptance of a potential probiotic approach. Keywords: Legionella; antagonism; biofilm; competition; pathogen–host interaction; predation; probiotics; protozoa

Inferring transmission fitness advantage of SARS-CoV-2 variants of concern from wastewater samples using digital PCR, Switzerland, December 2020 through March 2021

BackgroundThroughout the COVID-19 pandemic, SARS-CoV-2 genetic variants of concern (VOCs) have repeatedly and independently arisen. VOCs are characterised by increased transmissibility, increased virulence or reduced neutralisation by antibodies obtained from prior infection or vaccination. Tracking the introduction and transmission of VOCs relies on sequencing, typically whole genome sequencing of clinical samples. Wastewater surveillance is increasingly used to track the introduction and spread of SARS-CoV-2 variants through sequencing approaches.AimHere, we adapt and apply a rapid, high-throughput method for detection and quantification of the relative frequency of two deletions characteristic of the Alpha, Beta, and Gamma VOCs in wastewater.MethodsWe developed drop-off RT-dPCR assays and an associated statistical approach implemented in the R package WWdPCR to analyse temporal dynamics of SARS-CoV-2 signature mutations (spike Δ69-70 and ORF1a Δ3675-3677) in wastewater and quantify transmission fitness advantage of the Alpha VOC.ResultsBased on analysis of Zurich wastewater samples, the estimated transmission fitness advantage of SARS-CoV-2 Alpha based on the spike Δ69-70 was 0.34 (95% confidence interval (CI): 0.30-0.39) and based on ORF1a Δ3675-3677 was 0.53 (95% CI: 0.49-0.57), aligning with the transmission fitness advantage of Alpha estimated by clinical sample sequencing in the surrounding canton of 0.49 (95% CI: 0.38-0.61).ConclusionDigital PCR assays targeting signature mutations in wastewater offer near real-time monitoring of SARS-CoV-2 VOCs and potentially earlier detection and inference on transmission fitness advantage than clinical sequencing. Keywords: B.1.1.7; SARS-CoV-2; digital PCR; drop-off assays; transmission fitness

Early detection and surveillance of SARS-CoV-2 genomic variants in wastewater using COJAC

The continuing emergence of SARS-CoV-2 variants of concern and variants of interest emphasizes the need for early detection and epidemiological surveillance of novel variants. We used genomic sequencing of 122 wastewater samples from three locations in Switzerland to monitor the local spread of B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) variants of SARS-CoV-2 at a population level. We devised a bioinformatics method named COJAC (Co-Occurrence adJusted Analysis and Calling) that uses read pairs carrying multiple variant-specific signature mutations as a robust indicator of low-frequency variants. Application of COJAC revealed that a local outbreak of the Alpha variant in two Swiss cities was observable in wastewater up to 13 d before being first reported in clinical samples. We further confirmed the ability of COJAC to detect emerging variants early for the Delta variant by analysing an additional 1,339 wastewater samples. While sequencing data of single wastewater samples provide limited precision for the quantification of relative prevalence of a variant, we show that replicate and close-meshed longitudinal sequencing allow for robust estimation not only of the local prevalence but also of the transmission fitness advantage of any variant. We conclude that genomic sequencing and our computational analysis can provide population-level estimates of prevalence and fitness of emerging variants from wastewater samples earlier and on the basis of substantially fewer samples than from clinical samples. Our framework is being routinely used in large national projects in Switzerland and the UK.</p

Early detection and surveillance of SARS-CoV-2 genomic variants in wastewater using COJAC

The continuing emergence of SARS-CoV-2 variants of concern and variants of interest emphasizes the need for early detection and epidemiological surveillance of novel variants. We used genomic sequencing of 122 wastewater samples from three locations in Switzerland to monitor the local spread of B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) variants of SARS-CoV-2 at a population level. We devised a bioinformatics method named COJAC (Co-Occurrence adJusted Analysis and Calling) that uses read pairs carrying multiple variant-specific signature mutations as a robust indicator of low-frequency variants. Application of COJAC revealed that a local outbreak of the Alpha variant in two Swiss cities was observable in wastewater up to 13 d before being first reported in clinical samples. We further confirmed the ability of COJAC to detect emerging variants early for the Delta variant by analysing an additional 1,339 wastewater samples. While sequencing data of single wastewater samples provide limited precision for the quantification of relative prevalence of a variant, we show that replicate and close-meshed longitudinal sequencing allow for robust estimation not only of the local prevalence but also of the transmission fitness advantage of any variant. We conclude that genomic sequencing and our computational analysis can provide population-level estimates of prevalence and fitness of emerging variants from wastewater samples earlier and on the basis of substantially fewer samples than from clinical samples. Our framework is being routinely used in large national projects in Switzerland and the UK

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Evaluation of Phi6 Persistence and Suitability as an Enveloped Virus Surrogate

Recent outbreaks involving enveloped viruses, such as Ebola virus, have raised questions regarding the persistence of enveloped viruses in the water environment. Efforts have been made to find enveloped virus surrogates due to challenges investigating viruses that require biosafety-level 3 or 4 handling. In this study, the enveloped bacteriophage Phi6 was evaluated as a surrogate for enveloped waterborne viruses. The persistence of Phi6 was tested in aqueous conditions chosen based on previously published viral persistence studies. Our results demonstrated that the predicted <i>T</i>₉₀ (time for 90% inactivation) of Phi6 under the 12 evaluated conditions varied from 24 min to 117 days depending on temperature, biological activity, and aqueous media composition. Phi6 persistence was then compared with persistence values from other enveloped viruses reported in the literature. The apparent suitability of Phi6 as an enveloped virus surrogate was dependent on the temperature and composition of the media tested. Of evaluated viruses, 33%, including all conditions considered, had <i>T</i>₉₀ values greater than the 95% confidence interval for Phi6. Ultimately, these results highlight the variability of enveloped virus persistence in the environment and the value of working with the virus of interest for environmental persistence studies

Quantitative CrAssphage PCR Assays for Human Fecal Pollution Measurement

Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal-indicator bacteria are used; however, they cannot distinguish human fecal waste from other animal pollution sources. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based on initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against an animal fecal reference library, and crAssphage genetic markers were highly abundant in raw sewage and sewage-impacted water samples. In addition, CPQ_056 and CPQ_064 performance was compared to HF183/BacR287 and HumM2 assays in paired experiments. Findings confirm that viral crAssphage qPCR assays perform at a similar level to well-established bacterial human-associated fecal-source-identification approaches. These new viral-based assays could become important water quality management and research tools