SARS-CoV-2 viral titer measurements in Ontario, Canada wastewaters throughout the COVID-19 pandemic.

During the COVID-19 pandemic, the Province of Ontario, Canada, launched a wastewater surveillance program to monitor SARS-CoV-2, inspired by the early work and successful forecasts of COVID-19 waves in the city of Ottawa, Ontario. This manuscript presents a dataset from January 1, 2021, to March 31, 2023, with RT-qPCR results for SARS-CoV-2 genes and PMMoV from 107 sites across all 34 public health units in Ontario, covering 72% of the province's and 26.2% of Canada's population. Sampling occurred 2-7 times weekly, including geographical coordinates, serviced populations, physico-chemical water characteristics, and flowrates. In doing so, this manuscript ensures data availability and metadata preservation to support future research and epidemic preparedness through detailed analyses and modeling. The dataset has been crucial for public health in tracking disease locally, especially with the rise of the Omicron variant and the decline in clinical testing, highlighting wastewater-based surveillance's role in estimating disease incidence in Ontario.

Polyethylenimine mediated recovery of SARS-CoV-2 and total viral RNA: Impact of aqueous conditions on behaviour and recovery.

Wastewater-based epidemiology (WBE) is an emerging, practical surveillance tool for monitoring community levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, SC2). However, a paucity of data exists regarding SARS-CoV-2 and viral biomarker behaviour in aqueous and wastewater environments. Therefore, there is a pressing need to develop efficient and robust methods that both improve method sensitivity and reduce time and cost. We present a novel method for SARS-CoV-2, Human Coronavirus 229E (229E), and Pepper Mild Mottle Virus (PMMoV) recovery utilizing surface charge-based attraction via the branched cationic polymer, polyethylenimine (PEI). Initially, dose-optimization experiments demonstrated that low concentrations of PEI (0.001% w/v) proved most effective at flocculating suspended viruses and viral material, including additional unbound SC2 viral fragments and/or RNA from raw wastewater. A design-of-experiments (DOE) approach was used to optimize virus and/or viral material aggregation behaviour and recovery across varying aqueous conditions, revealing pH as a major influence on recoverability in this system, combinatorially due to both a reduction in viral material surface charge and increased protonation of PEI-bound amine groups. Overall, this method has shown great promise in significantly improving quantitative viral recovery, providing a straightforward and effective augmentation to standard centrifugation techniques.

Real-time evaluation of signal accuracy in wastewater surveillance of pathogens with high rates of mutation.

Wastewater surveillance of coronavirus disease 2019 (COVID-19) commonly applies reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater over time. In most applications worldwide, maximal sensitivity and specificity of RT-qPCR has been achieved, in part, by monitoring two or more genomic loci of SARS-CoV-2. In Ontario, Canada, the provincial Wastewater Surveillance Initiative reports the average copies of the CDC N1 and N2 loci normalized to the fecal biomarker pepper mild mottle virus. In November 2021, the emergence of the Omicron variant of concern, harboring a C28311T mutation within the CDC N1 probe region, challenged the accuracy of the consensus between the RT-qPCR measurements of the N1 and N2 loci of SARS-CoV-2. In this study, we developed and applied a novel real-time dual loci quality assurance and control framework based on the relative difference between the loci measurements to the City of Ottawa dataset to identify a loss of sensitivity of the N1 assay in the period from July 10, 2022 to January 31, 2023. Further analysis via sequencing and allele-specific RT-qPCR revealed a high proportion of mutations C28312T and A28330G during the study period, both in the City of Ottawa and across the province. It is hypothesized that nucleotide mutations in the probe region, especially A28330G, led to inefficient annealing, resulting in reduction in sensitivity and accuracy of the N1 assay. This study highlights the importance of implementing quality assurance and control criteria to continually evaluate, in near real-time, the accuracy of the signal produced in wastewater surveillance applications that rely on detection of pathogens whose genomes undergo high rates of mutation.

The Effect of Microcystis on the Monitoring of Faecal Indicator Bacteria.

The survival of Escherichia coli (E. coli) bacteria, the most common faecal indicator bacteria (FIB), may be significantly affected by cyanobacteria present during a harmful algal bloom (HAB). Therefore, the effect of Microcystis on the survival of FIB E.coli and coliforms was investigated. Microcosms containing two species of Microcystis (M. aeruginosa and M. smithii) were established and then inoculated with four reference strains of E. coli (ATCC 25922, 8739, 51813, and 11775) to explore the cyanobacteria-bacteria dynamics at a laboratory setting. Monitoring over several days showed normal growth of Microcystis, with or without the presence of E. coli. However, Microcystis was shown to dramatically decrease the survival of E. coli over time. Analysis of microcystin production by Microcystis was found to correlate with loss of E. coli, suggesting a toxic effect of microcystins on E. coli bacteria. This phenomenon was also demonstrated for a natural consortium of E. coli and coliform bacteria by inoculating with contaminated lake water. The results indicate that the use of E. coli as FIB may be greatly compromised in the presence of Microcystis spp. such as during a HAB when associated toxins are produced.

Integrating consumer risk perception and awareness with simulation-based quantitative microbial risk assessment using a coupled systems framework: A case study of private groundwater users in Ontario.

Private well users in Ontario are responsible for ensuring the potability of their own private drinking water source through protective actions (i.e., water treatment, well maintenance, and regular water quality testing). In the absence of regulation and limited surveillance, quantitative microbial risk assessment (QMRA) represents the most practical and robust approach to estimating the human health burden attributable to private wells. For an increasingly accurate estimation, QMRA of private well water should be represented by a coupled model, which includes both the socio-cognitive and physical aspects of private well water contamination and microbial exposure. The objective of the current study was to determine levels of waterborne exposure via well water consumption among three sub-groups (i.e., clusters) of private well users in Ontario and quantify the risk of waterborne acute gastrointestinal illness (AGI) attributed to Giardia, shiga-toxin producing E. coli (STEC) and norovirus from private drinking water sources in Ontario. Baseline simulations were utilized to explore the effect of varying socio-cognitive scenarios on model inputs (i.e., increased awareness, protective actions, aging population). The current study uses a large spatio-temporal groundwater quality dataset and cross-sectional province-wide survey to create socio-cognitive-specific QMRA simulations to estimate the risk of waterborne AGI attributed to three enteric pathogens in private drinking waters source in Ontario. Findings suggest significant differences in the level of exposure among sub-groups of private well users. Private well users within Cluster 3 are characterised by higher levels of exposure and annual illness attributable to STEC, Giardia and norovirus than Clusters 1 and 2. Provincial incidence rates of 520.9 (1522 illness per year), 532.1 (2211 illness per year) and 605.5 (5345 illness per year) cases/100,000 private well users per year were predicted for private well users associated with Clusters 1 through 3. Established models will enable development of necessary tools tailored to specific groups of at-risk well users, allowing for preventative public health management of private groundwater sources.

Classification of sub-populations for quantitative risk assessment based on awareness and perception: A cross-sectional population study of private well users in Ontario.

Private well users in Ontario are responsible for protective actions, including source maintenance, treatment, and submitting samples for laboratory testing. However, low participation rates are reported, thus constituting a public health concern, as risk mitigation behaviours can directly reduce exposure to waterborne pathogens. The current study examined the combined effects of socio-demographic profile, experience(s), and "risk domains" (i.e., awareness, attitudes, risk perceptions and beliefs) on behaviours, and subsequently classified private well users in Ontario based on cognitive factors. A province-wide online survey (n = 1228) was employed to quantify Ontario well owners' awareness, perceptions, and behaviours in relation to their personal groundwater supply and local contamination sources. A scoring protocol for four risk domains was developed. Two-step cluster analysis was used to classify respondents based on individual risk domain scores. Logistic regression was employed to identify key variables associated with cluster membership (i.e., profile analysis). Overall, 1140 survey respondents were included for analyses. Three distinct clusters were identified based on two risk domains; groundwater awareness and source risk perception. Profile analyses indicate "low awareness and source risk perception" (Low A/SRP) members were more likely male, while "low awareness and moderate source risk perception" (Low A/Mod SRP) members were more likely female and bottled water users. Well users characterised as "high awareness and source risk perception" (High A/SRP) were more likely to report higher educational attainment and previous well water testing. Findings illustrate that socio-cognitive clusters and their components (i.e., demographics, awareness, attitudes, perceptions, experiences, and protective actions) are distinct based on the likelihood, frequency, and magnitude of waterborne pathogen exposures (i.e., risk-based). Risk-based clustering, when incorporated into quantitative microbial risk assessment, enables the development of effective risk management and communication initiatives that are demographically focused and tailored to specific sub-groups.

Assessing the risk of acute gastrointestinal illness attributable to three enteric pathogens from contaminated private water wells in Ontario.

The province of Ontario compromises the largest groundwater reliant population in Canada serving approximately 1.6 million individuals. Unlike municipal water systems, private well water is not required to meet water quality regulatory standards and thus source maintenance, treatment and testing remains the responsibility of the well owner. Infections associated with private drinking water systems are rarely documented given their typically sporadic nature, thus the human health effects (e.g., acute gastrointestinal illness (AGI)) on consumers remains relatively unknown, representing a significant gap in water safety management. The current study sought to quantify the risk of waterborne AGI attributed to Giardia, shiga-toxin producing E. coli (STEC) and norovirus from private drinking water sources in Ontario using Monte Carlo simulation-based quantitative microbial risk assessment (QMRA). Findings suggest that consumption of contaminated private well water in Ontario is responsible for approximately 4823 AGI cases annually, with 3464 (71.8%) and 1359 (28.1%) AGI cases predicted to occur in consolidated and unconsolidated aquifers, respectively. By pathogen, waterborne AGI was attributed to norovirus (62%; 2991/4823), Giardia (24.6%; 1186/4823) and STEC (13.4%; 646/4823). The developed QMRA framework was used to assess the potential health impacts of partial and total well water treatment system failure. In the unlikely event of total treatment failure, total mean annual illnesses are predicted to almost double (4217 to 7064 cases per year), highlighting the importance of effective water treatment and comprehensive testing programs in reducing infectious health risks attributable to private well water in Ontario. Study findings indicate significant underreporting of waterborne AGI rates at the provincial level likely biasing public health interventions and programs that are effective in monitoring and minimizing the health risk associated with private well water.

An assessment of total coliforms and associated thresholds as water quality indicators using a large Ontario private drinking water well dataset.

A spatiotemporally static total coliform (TC) concentration threshold of five colony-forming units (CFU) per 100 mL is used in Ontario to determine whether well water is of acceptable quality for drinking. The current study sought to assess the role of TC and associated thresholds as microbial water quality parameters as the authors hypothesized that, since static TC thresholds are not evidence-based, they may not be appropriate for all well water consumers. A dataset containing the microbial water quality information of 795,023 samples (including TC and Escherichia coli (E. coli) counts) collected from 253,136 private wells in Ontario between 2010 and 2017 was used. To accurately assess the relationship between E. coli and non-E. coli TC, "non-E. coli coliform" (NEC) counts were calculated from microbial water quality data and replaced TC throughout analyses. This study analysed NEC and E. coli detection rates to determine differences between the two, and NEC:E. coli concentration ratios to assess links, if any, between NEC and E. coli contamination. Study findings suggest that spatiotemporally static NEC thresholds are not appropriate because seasonal, spatial, and well-specific susceptibility factors are associated with distinct contamination trends. For example, NEC detection rates exhibited bimodality, with summer (29.4 %) and autumn (30.2 %) detection rates being significantly higher (p < 0.05) than winter (21.9 %) and spring (19.9 %). E. coli detection rates also varied seasonally, but peaked in summer rather than autumn. As such, it is recommended that these factors be considered during the development of private well water guidelines and that static thresholds be avoided. Furthermore, the authors propose that, because NEC:E. coli concentration ratios change in the context of the aforementioned factors, they may have a role in inferring groundwater contamination mechanisms, with high ratios being associated with generalized aquifer contamination mechanisms and low ratios with localized contamination mechanisms.

Metagenomics of Wastewater Influent from Wastewater Treatment Facilities across Ontario in the Era of Emerging SARS-CoV-2 Variants of Concern.

We report metagenomic sequencing analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in composite wastewater influent from 10 regions in Ontario, Canada, during the transition between Delta and Omicron variants of concern. The Delta and Omicron BA.1/BA.1.1 and BA.2-defining mutations occurring in various frequencies were reported in the consensus and subconsensus sequences of the composite samples.

Environmental adaptation of E. coli within private groundwater sources in southeastern Ontario: Implications for groundwater quality monitoring and human health.

Groundwater quality monitoring typically employs testing for the presence of E. coli as a fecal indicator of recent ingress of human or animal fecal material. The efficacy of fecal indicator organisms is based on the primary criteria that the organism does not reproduce in the aquatic environment. However, recent studies have reported that E. coli may proliferate (i.e., has adapted to) in the external environment, including soil and surface water. To date, the presence of environmentally-adapted E. coli in groundwater has not been examined. The current study employed Clermont phylotyping and the presence of six accessory genes to identify the likely presence of adapted E. coli in private groundwater sources. E. coli isolates (n = 325) from 76 contaminated private water wells located in a southeastern Ontario watershed were compared with geographically analogous human and animal fecal E. coli isolates (n = 234). Cryptic clades III-V, a well-described environmentally-adapted Escherichia population, were identified in three separate groundwater wells, one of which exclusively comprised this adapted population. Dimensionality reduction (via Principal Component Analysis) was used to develop an "E. coli adaptation model", comprising three distinct components (groundwater, animal feces, human feces) and suggests adaptation occurs frequently in the groundwater environment. Model findings indicate that 23/76 (30.3%) wells had an entirely adapted community. Accordingly, the use of E. coli as a FIO returned a false positive result in these instances, while an additional 23/76 (30.3%) wells exhibited some evidence of adaptation (i.e., not all isolates were adapted) representing an over-estimate of the magnitude (concentration) of contamination. Study findings highlight the need to further characterize environmentally-adapted E. coli in the groundwater environment and the potential implications with respect to water quality policy, legislation and determinants of human health risk both regionally and internationally.

Exploration of E. coli contamination drivers in private drinking water wells: An application of machine learning to a large, multivariable, geo-spatio-temporal dataset.

Groundwater resources are under increasing threats from contamination and overuse, posing direct threats to human and environmental health. The purpose of this study is to better understand drivers of, and relationships between, well and aquifer characteristics, sampling frequencies, and microbiological contamination indicators (specifically E. coli) as a precursor for improving knowledge and tools to assess aquifer vulnerability and well contamination within Ontario, Canada. A dataset with 795, 023 microbiological testing observations over an eight-year period (2010 to 2017) from 253,136 unique wells across Ontario was employed. Variables in this dataset include date and location of test, test results (E. coli concentration), well characteristics (well depth, location), and hydrogeological characteristics (bottom of well stratigraphy, specific capacity). Association rule analysis, univariate and bivariate analyses, regression analyses, and variable discretization techniques were utilized to identify relationships between E. coli concentration and the other variables in the dataset. These relationships can be used to identify drivers of contamination, their relative importance, and therefore potential public health risks associated with the use of private wells in Ontario. Key findings are that: i) bedrock wells completed in sedimentary or igneous rock are more susceptible to contamination events; ii) while shallow wells pose a greater risk to consumers, deep wells are also subject to contamination events and pose a potentially unanticipated risk to health of well users; and, iii) well testing practices are influenced by results of previous tests. Further, while there is a general correlation between months with the greatest testing frequencies and concentrations of E. coli occurring in samples, an offset in this timing is observed in recent years. Testing remains highest in July while peaks in adverse results occur up to three months later. The realization of these trends prompts a need to further explore the bases for such occurrences.

Drinking Water Consumption Patterns among Private Well Users in Ontario: Implications for Exposure Assessment of Waterborne Infection.

Understanding the water consumption patterns within a specific population informs development of increasingly accurate, spatially specific exposure and/or risk assessment of waterborne infection. The current study examined the consumption patterns of private well users in Ontario while considering potentially influential underlying sociodemographics, household characteristics, and experiential factors. A province-wide online survey was circulated between May and August 2018 (n = 1,162). Overall, 81.5% of respondents reported daily well water consumption (i.e., tap water). Results indicate a mean daily well water consumption rate of 1,132 mL/day (SD = 649 mL/day) among well water consumers. Gender was significantly associated with well water consumption, with higher consumption rates found among female respondents. The experience of acute gastrointestinal illness (AGI) symptoms or diagnosis in the past 12 months did not impact the volume of water consumed, suggesting that experiencing previous AGI does not decrease consumption volumes, and therefore exposure over time. Significantly higher rates of well water consumption were found among respondents who reported previous testing or ongoing water treatment. Approximately 45.5% of survey respondents who stated that they do not consume well water selected bottled water as their primary household drinking water supply. Bottled water consumption was also not associated with previous AGI experiences. Findings will inform future quantitative microbial risk assessments associated with private well water use by providing spatially and demographically specific estimates of well water consumption.

Determining binding of polycyclic aromatic hydrocarbons to micelles formed by SDS and SOL using semi-equilibrium dialysis.

Micellar enhanced ultra-filtration (MEUF) is a modified ultrafiltration (UF) method that can remove small molecules that are not effectively removed by UF alone. In this work we used a similar semi-equilibrium dialysis (SED) method to characterize two anionic surfactants, sodium dodecyl sulfate (SDS) and sodium laurate (SOL), as MEUF candidates to remove several polycyclic aromatic hydrocarbon (PAH) compounds. These anionic surfactants have low toxicity and are more biodegradable compared to the fairly toxic and persistent cetyltrimethylammonium bromide (CTABr) and similar cationic surfactants, which is important as MEUF surfactants may be present in the treated effluent. The log binding constants (Log KB) of the PAHs naphthalene, phenanthrene, pyrene and fluorene to SDS ranged from 3.07 to 4.48, which compared well with the previous results for CTABr and indicated that SDS is an excellent candidate for MEUF. The log binding constants for the same PAHs with SOL micelles ranged from 2.11 to 3.53, which suggested that SOL might be adequate for stronger-binding PAH like pyrene and fluorene but less suitable for naphthalene and phenanthrene. The results demonstrated a strong correlation between the Log KB with one micelle type and the Log KB with the other, suggesting a common set of properties and interactions are responsible for the binding. Similar to the previous results for CTABr, a significant correlation between Log KB and Log KOW values indicates that hydrophobic character is the main driving force for PAH binding with these anionic micelles. This may also be useful for predicting MEUF performance for various compound/surfactant combinations where Log KB is not known.

The bioavailability of oil droplets trapped in river gravel by hyporheic flows.

Little is known about the fate of oil spills in rivers. Hyporheic flows of water through river sediments exchange surface and groundwater and create upwelling and downwelling zones that are important for fish spawning and embryo development. Risk assessments of oil spills to rivers do not consider the potential for hyporheic flows to carry oil droplets into sediments and the potential for prolonged exposure of fish to trapped oil. This project assessed whether oil droplets in water flowing through gravel will be trapped and whether hydrocarbons partitioning from trapped oil droplets are bioavailable to fish. Columns packed with gravel were injected with oil-in-water dispersions prepared with light crude, medium crude, diluted bitumens, and heavy fuel oil to generate a series of oil droplet loadings. The concentrations of oil trapped in the gravel increased with oil loading and viscosity. When the columns were perfused with clean water, oil concentrations in column effluents decreased to the detection limit within the first week of water flow, with sporadically higher concentrations associated with oil droplet release. Despite the low concentrations of hydrocarbons measured in column effluent, hydrocarbons were bioavailable to juvenile rainbow trout (Oncorhynchus mykiss) for more than three weeks of water flow, as indicated by strong induction of liver ethoxyresorufin-o-deethylase activity. These findings indicate that ecological risk assessments and spill response should identify and protect areas in rivers sensitive to contaminant trapping.

Examining influential drivers of private well users' perceptions in Ontario: A cross-sectional population study.

Private well users are responsible for managing and maintaining the quality of their drinking water source. Previous studies in Canada have reported low testing rates among well users, a cornerstone of well stewardship behaviours that can prevent the consumption of contaminated groundwater. To improve well stewardship, it is important to understand the interactions between, and the impacts of, various factors that may influence behaviours. Accordingly, the objective of the current study was to investigate the impact of socio-demographics, property characteristics, and experiences with well construction and acute gastrointestinal illness (AGI) (i.e., previous experiences) on levels of awareness, attitudes, risk perceptions, and beliefs (i.e., risk domains) among private well users in Ontario. A link to a province-wide online survey was circulated between May and August 2018 and novel "risk domain" scoring protocols were developed to classify and summarize response data. The survey was undertaken by 1228 respondents, of which 1030 completed the survey in full. Results indicate a low level of waterborne pathogen awareness, with 50.8% of respondents unaware of any groundwater associated pathogens. Respondents' geographic location, gender, and well type were significantly associated with well users' attitudes and perceptions of risk regarding their personal well water supply and the quality and quantity of local groundwater sources. Higher levels of awareness and lower risk perception scores (i.e., lower perceptions of risk) were associated with residential presence during well construction (p < 0.001 and p = 0.017, respectively). Previous case(s) of AGI within the respondent's household were significantly associated with negative attitudes towards their well water (p < 0.001) and higher risk perception scores (p = 0.025) with respect to the quantity of local groundwater sources. Results may be used to identify critical experiential control points (e.g., during well construction or after a physician confirmed AGI diagnosis) and develop improved risk management and communication strategies aimed at private well users.

Effects of Environmentally Relevant Residual Levels of Diluted Bitumen on Wild Fathead Minnows (Pimephales promelas).

Transportation of crude oil across North America's boreal ecozone creates the potential for spills in freshwater where less is known about the sensitivity of resident fish than for marine systems. The sensitivity of wild fathead minnows (FHM) to residual concentrations (ppb range) of the water accommodated fraction (WAF) of diluted bitumen (dilbit) was assessed by exposing them for 21 days followed by a 14 days depuration. Target concentrations were well below detection limits for GC-MS, but were estimated by dilution factor (1:100,000 and 1:1,000,000 WAF:water) to contain less than 0.0003 µg/L of polycyclic aromatic compounds. Confinement and handling stress caused by transfer of wild fish into tanks much smaller than their natural range resulted in mortality and lower body condition among all groups, but interactive effects of oil exposures still resulted in females with smaller cortical alveolar oocytes, and males with larger testicular lobe lumen sizes. Additional studies examining the compounded effects of stress and environmentally relevant oil exposures in wild fishes are needed.

Analysis of a large spatiotemporal groundwater quality dataset, Ontario 2010-2017: Informing human health risk assessment and testing guidance for private drinking water wells.

Approximately 1.5 million individuals in Ontario are supplied by private water wells (private groundwater supplies). Unlike municipal supplies, private well water quality remains unregulated, with owners responsible for testing, treating, and maintaining their own water supplies. The primary goal of this study was to assess the effect of repeat sampling of private well water in Ontario and investigate the efficacy of geographically- and/or temporally specific testing recommendations and health risk assessments. The current study combines the Well Water Information System Dataset and the Well Water Testing Dataset from 2010 to 2017, inclusive. These two large existing province-wide datasets collated over an eight-year period were merged using an integrated spatial fuzzy logic and (next)- nearest neighbour approach. Provincial sampling data from 239,244 wells (702,861 samples) were analyzed for Escherichia coli to study the relationship between sampling frequency and Escherichia coli detection. Dataset variables were delineated based on hydrogeological setting (e.g. aquifer type, overburden depth, well depth, bedrock type) and seasonality to provide an in-depth understanding of Escherichia coli detection in private well water. Findings reveal differences between detection rates in consolidated and unconsolidated aquifers (p = 0.0191), and across seasons (p < 0.0001). The variability associated with Escherichia coli detection rates was explored by estimating sentinel sampling rates for private wells sampled three times, twelve times and twenty-four times per year. As sample size increases on an annual basis, so too does detection rate, highlighting the need to address current testing frequency guidelines. Future health risk assessments for private well water should consider the impact of spatial and temporal factors on the susceptibility of this drinking water source, leading to an increasingly accurate depiction of private well water contamination and the estimated effects on human health.

Developing a toll-like receptor biosensor for Gram-positive bacterial detection and its storage strategies.

The biorecognition ability of hybridized toll-like receptors (TLRs) 2 and 6 proteins on electrode surfaces has been studied. TLR biosensors have been designed to be non-specific to particular bacterial strains but rather to provide broad spectrum detection of cells and toxins containing relevant pathogen-associated molecular patterns (PAMPs). Our electrochemical TLR2/6 biosensors demonstrated selective detection towards Gram-positive bacterial whole-cells and a synthetic diacylated lipopeptide (Pam2CSK4), a PAMP. Responses towards Bacillus licheniformis (B. licheniformis) and Enterococcus hirae (E. hirae) were obtained. The biosensor was able to differentiate signals between B. licheniformis and a Gram-negative bacterial cell (control) as low as 100 CFU mL-1. One challenge in developing protein-based biosensors is to improve the shelf-life of the biosensor chips and preserve the detection activity of the protein molecules, therefore we did our first exploration into storage conditions. The activity of stored biosensors was found to be strongly dependent on storage medium, and that effective 'shelf-life' was obtained makes an important step towards creating robust sensors for real-life applications.

Effects on Trout Alevins of Chronic Exposures to Chemically Dispersed Access Western Blend and Cold Lake Blend Diluted Bitumens.

The present study assessed the chronic toxicity of 2 chemically enhanced water accommodated fractions (CEWAFs) of diluted bitumens (dilbits), Access Western Blend (AWB) and Cold Lake Blend (CLB), to rainbow trout alevins. Chemical dispersion was used to overcome the resistance to dispersion of dilbits and to generate test solutions that contained more and smaller oil droplets for increased partitioning of petroleum hydrocarbons into water. Test solutions were characterized by fluorescence spectroscopy, a rapid and inexpensive analytical tool to compare toxicity endpoints measured by fluorescence (total petroleum hydrocarbons measured by fluorescence [TPH-F]). Cumulative mortality and the prevalence and severity of malformations increased following exposure of alevins to dispersed dilbits. Toxicity curves overlapped for AWB and CLB when expressed as TPH-F and 22- to 24-d median lethal and effect concentrations ranged from 0.36 to 1.5 mg/L. Gene expression in alevins was also altered following exposure to dispersed dilbit, with relative cytochrome P450-1A mRNA levels increasing up to 170-fold for AWB and up to 240-fold for CLB. Access Western Blend and CLB caused similar toxicity to rainbow trout alevins as light to medium conventional crude oils, and rainbow trout alevins were more sensitive than yellow perch, Japanese medaka, and fathead minnow embryos exposed to dispersed AWB and CLB. The present study is the first to assess the embryotoxicity of dilbits to a Canadian freshwater salmonid species. Environ Toxicol Chem 2020;39:1620-1633. © 2020 SETAC.

Comparative genomics of multidrug-resistant Enterococcus spp. isolated from wastewater treatment plants.

BACKGROUND: Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted. RESULTS: VRE isolates, including E. faecalis (n = 24), E. faecium (n = 11), E. casseliflavus (n = 2) and E. gallinarum (n = 2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium (p < 0.001) and E. faecalis (p < 0.001) and with the number of AMR genes in E. faecium (p = 0.005). Genes conferring vancomycin resistance, including vanA and vanM (E. faecium), vanG (E. faecalis), and vanC (E. casseliflavus/E. gallinarum), were detected in 20 genomes. The most prominent functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium, E. faecalis, E. casseliflavus and E. gallinarum, respectively. Virulence genes were more common in E. faecalis and E. faecium, than E. casseliflavus and E. gallinarum. A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium. Phylogenetic analysis demonstrated differential clustering of isolates based on original source but not WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers. CONCLUSIONS: There was no discernible difference between enterococcal genomes from the CAS and BAF WWTPs. E. faecalis and E. faecium have smaller genomes and harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp.