A comparison of the MPN and pour plate methods for estimating shellfish contamination by Escherichia coli.

AIMS: Shellfish production areas are classified for suitability for human consumption using counts of Escherichia coli in shellfish samples. Two alternative laboratory methods are approved in the European Union and UK for measuring E. coli in shellfish samples; the most probable number (MPN) and pour plate methods. These methods have inherently different statistical uncertainty and may give different counts for the same sample. Using two approaches: simulated data and spiking experiments, we investigate the theoretical properties of the two methods to determine their reliability for shellfish waters classification. METHODS AND RESULTS: Assuming a Poisson distribution of E. coli in shellfish samples, we simulate concentrations in 10 000 samples using the MPN and pour plate methods. We show that for higher concentrations of E. coli the pour plate method becomes increasingly more reliable than the MPN method. The MPN method has higher probabilities than pour plate of generating results exceeding shellfish classification thresholds, while conversely having higher probabilities of failing to detect counts that exceed regulatory thresholds. The theoretical analysis also demonstrates that the MPN method can produce genuine extreme outliers, even when E. coli are randomly distributed within the sampled material. A laboratory spiking experiment showed results consistent with the theoretical analysis, suggesting the Poisson assumption used in the theoretical analysis is reasonable. CONCLUSION: The large differences in statistical properties between the pour plate and MPN methods should be taken into consideration in classifying shellfish beds, with the pour plate method being more reliable over the crucial range of E. coli concentrations used to determine class boundaries.

Heterogeneous microgeographic genetic structure of the common cockle (Cerastoderma edule) in the Northeast Atlantic Ocean: biogeographic barriers and environmental factors.

Knowledge of genetic structure at the finest level is essential for the conservation of genetic resources. Despite no visible barriers limiting gene flow, significant genetic structure has been shown in marine species. The common cockle (Cerastoderma edule) is a bivalve of great commercial and ecological value inhabiting the Northeast Atlantic Ocean. Previous population genomics studies demonstrated significant structure both across the Northeast Atlantic, but also within small geographic areas, highlighting the need to investigate fine-scale structuring. Here, we analysed two geographic areas that could represent opposite models of structure for the species: (1) the SW British Isles region, highly fragmented due to biogeographic barriers, and (2) Galicia (NW Spain), a putative homogeneous region. A total of 9250 SNPs genotyped by 2b-RAD on 599 individuals from 22 natural beds were used for the analysis. The entire SNP dataset mostly confirmed previous observations related to genetic diversity and differentiation; however, neutral and divergent SNP outlier datasets enabled disentangling physical barriers from abiotic environmental factors structuring both regions. While Galicia showed a homogeneous structure, the SW British Isles region was split into four reliable genetic regions related to oceanographic features and abiotic factors, such as sea surface salinity and temperature. The information gathered supports specific management policies of cockle resources in SW British and Galician regions also considering their particular socio-economic characteristics; further, these new data will be added to those recently reported in the Northeast Atlantic to define sustainable management actions across the whole distribution range of the species.

Core Community Persistence Despite Dynamic Spatiotemporal Responses in the Associated Bacterial Communities of Farmed Pacific Oysters.

A breakdown in host-bacteria relationships has been associated with the progression of a number of marine diseases and subsequent mortality events. For the Pacific oyster, Crassostrea gigas, summer mortality syndrome (SMS) is one of the biggest constraints to the growth of the sector and is set to expand into temperate systems as ocean temperatures rise. Currently, a lack of understanding of natural spatiotemporal dynamics of the host-bacteria relationship limits our ability to develop microbially based monitoring approaches. Here, we characterised the associated bacterial community of C. gigas, at two Irish oyster farms, unaffected by SMS, over the course of a year. We found C. gigas harboured spatiotemporally variable bacterial communities that were distinct from bacterioplankton in surrounding seawater. Whilst the majority of bacteria-oyster associations were transient and highly variable, we observed clear patterns of stability in the form of a small core consisting of six persistent amplicon sequence variants (ASVs). This core made up a disproportionately large contribution to sample abundance (34 ± 0.14%), despite representing only 0.034% of species richness across the study, and has been associated with healthy oysters in other systems. Overall, our study demonstrates the consistent features of oyster bacterial communities across spatial and temporal scales and provides an ecologically meaningful baseline to track environmental change.

Discovery of the parasite Marteilia cocosarum sp. nov. In common cockle (Cerastoderma edule) fisheries in Wales, UK and its comparison with Marteilia cochillia.

Diseases of bivalve molluscs caused by paramyxid parasites of the genus Marteilia have been linked to mass mortalities and the collapse of commercially important shellfish populations. Until recently, no Marteilia spp. have been detected in common cockle (Cerastoderma edule) populations in the British Isles. Molecular screening of cockles from ten sites on the Welsh coast indicates that a Marteilia parasite is widespread in Welsh C. edule populations, including major fisheries. Phylogenetic analysis of ribosomal DNA (rDNA) gene sequences from this parasite indicates that it is a closely related but different species to Marteilia cochillia, a parasite linked to mass mortality of C. edule fisheries in Spain, and that both are related to Marteilia octospora, for which we provide new rDNA sequence data. Preliminary light and transmission electron microscope (TEM) observations support this conclusion, indicating that the parasite from Wales is located primarily within areas of inflammation in the gills and the connective tissue of the digestive gland, whereas M. cochillia is found mainly within the epithelium of the digestive gland. The impact of infection by the new species, here described as Marteilia cocosarum n. sp., upon Welsh fisheries is currently unknown.

Validation of the flooding dose technique to determine fractional rates of protein synthesis in a model bivalve species, the blue mussel (Mytilus edulis L.).

For the first time, use of the flooding dose technique using (3)H-Phenylalanine is validated for measuring whole-animal and tissue-specific rates of protein synthesis in the blue mussel Mytilus edulis (61mm shell length; 4.0g fresh body mass). Following injection, the phenylalanine-specific radioactivities in the gill, mantle and whole-animal free pools were elevated within one hour and remained elevated and stable for up to 6h following injection of (3)H-phenylalanine into the posterior adductor muscle. Incorporation of (3)H-phenylalanine into body protein was linear over time following injection and the non-significant intercepts for the regressions suggested incorporation into body protein occurred rapidly after injection. These results validate the technique for measuring rates of protein synthesis in mussels. There were no differences in the calculated rates following 1-6h incubation in gill, mantle or whole-animal and fractional rates of protein synthesis from the combined time course data were 9.5±0.8%d(-1) for the gill, 2.5±0.3%d(-1) for the mantle and 2.6±0.3%d(-1) for the whole-animal, respectively (mean values±SEM). The whole-animal absolute rate of protein synthesis was calculated as 18.9±0.6mg protein day(-1). The use of this technique in measuring one of the major components of maintenance metabolism and growth will provide a valuable and convenient tool in furthering our understanding of the protein metabolism and energetics of this keystone marine invertebrate and its ability to adjust and respond to fluctuations, such as that expected as a result of climate change.

Assessment of wastewater derived pollution using viral monitoring in two estuaries.

Human wastewater-derived pollution of the environment is an emerging health risk that increases the number of waterborne and foodborne illnesses globally. To better understand and mitigate such health risks, we investigated the prevalence of faecal indicator bacteria, Escherichia coli, and indicator virus (crAssphage) along with human and animal enteric viruses (adenoviruses, noroviruses, sapoviruses, hepatitis E virus) in shellfish and water samples collected from two shellfish harvesting areas in the UK. Human noroviruses were detected at higher detection rates in oyster and water samples compared to mussels with peaks during the autumn-winter seasons. Human enteric viruses were sporadically detected during the warmer months, suggesting potential introduction by tourists following the relaxation of COVID-19 lockdown measures. Our results suggest that viral indicators are more suitable for risk assessment and source tracking than E. coli. The detection of emerging hepatitis and sapoviruses, support the need for comprehensive viral monitoring in shellfish harvesting areas.

Microcosm experiment investigating climate-induced thermal effects on human virus viability in seawater: qPCR vs capsid integrity for enhanced risk management.

Climate change is intensifying extreme weather events in coastal areas, leading to more frequent discharge of untreated wastewater containing human viruses into coastal waters. This poses a health risk, especially during heatwaves when bathing activity increases. A study examined the survival and viability of seven common wastewater viruses in seawater at different temperatures. Viral genomes were quantified using direct qPCR, whilst viability was assessed using Capsid Integrity qPCR. Results showed that T90 values from direct qPCR were much higher than those from CI-qPCR, suggesting that risk mitigation should be based on viral integrity tests. All viruses remained potentially viable for at least 72 h in environmental seawater and longer in sterile artificial seawater, highlighting the importance of biotic processes in viral inactivation. Viral persistence decreased with increasing temperature. Whilst heatwaves may partially reduce risks from human viral pathogens in coastal waters, they do not eliminate them entirely.

Urban heat mitigation by green and blue infrastructure: Drivers, effectiveness, and future needs.

The combination of urbanization and global warming leads to urban overheating and compounds the frequency and intensity of extreme heat events due to climate change. Yet, the risk of urban overheating can be mitigated by urban green-blue-grey infrastructure (GBGI), such as parks, wetlands, and engineered greening, which have the potential to effectively reduce summer air temperatures. Despite many reviews, the evidence bases on quantified GBGI cooling benefits remains partial and the practical recommendations for implementation are unclear. This systematic literature review synthesizes the evidence base for heat mitigation and related co-benefits, identifies knowledge gaps, and proposes recommendations for their implementation to maximize their benefits. After screening 27,486 papers, 202 were reviewed, based on 51 GBGI types categorized under 10 main divisions. Certain GBGI (green walls, parks, street trees) have been well researched for their urban cooling capabilities. However, several other GBGI have received negligible (zoological garden, golf course, estuary) or minimal (private garden, allotment) attention. The most efficient air cooling was observed in botanical gardens (5.0 ± 3.5°C), wetlands (4.9 ± 3.2°C), green walls (4.1 ± 4.2°C), street trees (3.8 ± 3.1°C), and vegetated balconies (3.8 ± 2.7°C). Under changing climate conditions (2070-2100) with consideration of RCP8.5, there is a shift in climate subtypes, either within the same climate zone (e.g., Dfa to Dfb and Cfb to Cfa) or across other climate zones (e.g., Dfb [continental warm-summer humid] to BSk [dry, cold semi-arid] and Cwa [temperate] to Am [tropical]). These shifts may result in lower efficiency for the current GBGI in the future. Given the importance of multiple services, it is crucial to balance their functionality, cooling performance, and other related co-benefits when planning for the future GBGI. This global GBGI heat mitigation inventory can assist policymakers and urban planners in prioritizing effective interventions to reduce the risk of urban overheating, filling research gaps, and promoting community resilience.

Infection of juvenile edible crabs, Cancer pagurus by a haplosporidian-like parasite.

This study aimed to examine the pathobiology of a haplosporidian-like infection in juvenile (pre-recruit) edible crabs (Cancer pagurus) from two locations in South West Wales, UK. Infected crabs showed no external symptoms of the disease but dissection revealed an infected and hypertrophic antennal gland. Histological examination showed extensive parasitisation of the antennal gland overlying the hepatopancreas. Heavily infected crabs also showed the presence of parasites with morphological similarities to Haplosporidia in the labyrinth of the antennal gland and in the gills. The spread of the infection from the antennal gland to the gills suggests that these parasites are released into the haemolymph. Attempts to characterise the haplosporidian-like organism using several primers previously shown to amplify members of the phylum Haplosporidia failed. The prevalence of infection in juvenile edible crabs varied throughout the sampling period of November 2011 to July 2012 with the lowest level of ca. 15% in November peaking at 70% in March. This parasite may represent a threat to the sustainability of edible crab fisheries in this region if the damage observed in the antennal gland and gills results in host mortality. The identification of these parasites as members of the phylum Haplosporidia based on morphology alone must be seen as tentative in the absence of sequence data.

Use of Capsid Integrity-qPCR for Detecting Viral Capsid Integrity in Wastewater.

Quantifying viruses in wastewater via RT-qPCR provides total genomic data but does not indicate the virus capsid integrity or the potential risk for human infection. Assessing virus capsid integrity in sewage is important for wastewater-based surveillance, since discharged effluent may pose a public health hazard. While integrity assays using cell cultures can provide this information, they require specialised laboratories and expertise. One solution to overcome this limitation is the use of photo-reactive monoazide dyes (e.g., propidium monoazide [PMAxx]) in a capsid integrity-RT-qPCR assay (ci-RT-qPCR). In this study, we tested the efficiency of PMAxx dye at 50 µM and 100 µM concentrations on live and heat-inactivated model viruses commonly detected in wastewater, including adenovirus (AdV), hepatitis A (HAV), influenza A virus (IAV), and norovirus GI (NoV GI). The 100 µM PMAxx dye concentration effectively differentiated live from heat-inactivated viruses for all targets in buffer solution. This method was then applied to wastewater samples (n = 19) for the detection of encapsulated AdV, enterovirus (EV), HAV, IAV, influenza B virus (IBV), NoV GI, NoV GII, and SARS-CoV-2. Samples were negative for AdV, HAV, IAV, and IBV but positive for EV, NoV GI, NoV GII, and SARS-CoV-2. In the PMAxx-treated samples, EV, NoV GI, and NoV GII showed -0.52-1.15, 0.9-1.51, and 0.31-1.69 log reductions in capsid integrity, indicating a high degree of potentially infectious virus in wastewater. In contrast, SARS-CoV-2 was only detected using RT-qPCR but not after PMAxx treatment, indicating the absence of encapsulated and potentially infectious virus. In conclusion, this study demonstrates the utility of PMAxx dyes to evaluate capsid integrity across a diverse range of viruses commonly monitored in wastewater.

Poor air passenger knowledge of COVID-19 symptoms and behaviour undermines strategies aimed at preventing the import of SARS-CoV-2 into the UK.

Air travel mediates transboundary movement of SARS-CoV-2. To prepare for future pandemics, we sought to understand air passenger behaviour and perceived risk during the COVID-19 pandemic. This study of UK adults (n = 2103) quantified knowledge of COVID-19 symptoms, perceived health risk of contracting COVID-19, likelihood of returning to the UK with COVID-19 symptoms, likelihood to obey self-quarantining guidelines, how safe air travellers felt when flying during the pandemic (n = 305), and perceptions towards face covering effectiveness.Overall knowledge of COVID-19 symptoms was poor. Men and younger age groups (18-44) were less informed than women and older age groups (44 +). A significant proportion (21%) of the population would likely travel back to the UK whilst displaying COVID-19 symptoms with many expressing that they would not fully comply with self-isolation guidelines. Overall, males and younger age groups had a reduced perceived personal risk from contracting COVID-19, posing a higher risk of transporting SARS-CoV-2 back to the UK. Poor passenger knowledge and behaviour undermines government guidelines and policies aimed at preventing SARS-CoV-2 entry into the UK. This supports the need for stricter, clearer and more targeted guidelines with point-of-departure viral testing and stricter quarantining upon arrival.

Suitability of aircraft wastewater for pathogen detection and public health surveillance.

International air travel is now widely recognised as one of the primary mechanisms responsible for the transnational movement and global spread of SARS-CoV-2. Monitoring the viral load and novel lineages within human-derived wastewater collected from aircraft and at air transport hubs has been proposed as an effective way to monitor the importation frequency of viral pathogens. The success of this approach, however, is highly dependent on the bathroom and defecation habits of air passengers during their journey. In this study of UK adults (n = 2103), we quantified the likelihood of defecation prior to departure, on the aircraft and upon arrival on both short- and long-haul flights. The results were then used to assess the likelihood of capturing the signal from infected individuals at UK travel hubs. To obtain a representative cross-section of the population, the survey was stratified by geographical region, gender, age, parenting status, and social class. We found that an individual's likelihood to defecate on short-haul flights (< 6 h in duration) was low (< 13 % of the total), but was higher on long-haul flights (< 36 %; > 6 h in duration). This behaviour pattern was higher among males and younger age groups. The maximum likelihood of defecation was prior to departure (< 39 %). Based on known SARS-CoV-2 faecal shedding rates (30-60 %) and an equal probability of infected individuals being on short- (71 % of inbound flights) and long-haul flights (29 %), we estimate that aircraft wastewater is likely to capture ca. 8-14 % of SARS-CoV-2 cases entering the UK. Monte Carlo simulations predicted that SARS-CoV-2 would be present in wastewater on 14 % of short-haul flights and 62 % of long-haul flights under current pandemic conditions. We conclude that aircraft wastewater alone is insufficient to effectively monitor all the transboundary entries of faecal-borne pathogens but can form part of a wider strategy for public heath surveillance at national borders.

Predicting the dispersal of SARS-CoV-2 RNA from the wastewater treatment plant to the coast.

Viral pathogens including SARS-CoV-2 RNA have been detected in wastewater treatment effluent, and untreated sewage overflows, that pose an exposure hazard to humans. We assessed whether SARS-CoV-2 RNA was likely to have been present in detectable quantities in UK rivers and estuaries during the first wave of the Covid-19 pandemic. We simulated realistic viral concentrations parameterised on the Camel and Conwy catchments (UK) and their populations, showing detectable SARS-CoV-2 RNA concentrations for untreated but not for treated loading, but also being contingent on viral decay, hydrology, catchment type/shape, and location. Under mean or low river flow conditions, viral RNA concentrated within the estuaries allowing for viral build-up and caused a lag by up to several weeks between the peak in community infections and the viral peak in the environment. There was an increased hazard posed by SARS-CoV-2 RNA with a T 90 decay rate >24 h, as the estuarine build-up effect increased. High discharge events transported the viral RNA downstream and offshore, increasing the exposure risk to coastal bathing waters and shellfisheries - although dilution in this case reduced viral concentrations well below detectable levels. Our results highlight the sensitivity of exposure to viral pathogens downstream of wastewater treatment, across a range of viral loadings and catchment characteristics - with implications to environmental surveillance.

Genomic survey of edible cockle (Cerastoderma edule) in the Northeast Atlantic: A baseline for sustainable management of its wild resources.

Knowledge on correlations between environmental factors and genome divergence between populations of marine species is crucial for sustainable management of fisheries and wild populations. The edible cockle (Cerastoderma edule) is a marine bivalve distributed along the Northeast Atlantic coast of Europe and is an important resource from both commercial and ecological perspectives. We performed a population genomics screening using 2b-RAD genotyping on 9309 SNPs localized in the cockle's genome on a sample of 536 specimens pertaining to 14 beds in the Northeast Atlantic Ocean to analyse the genetic structure with regard to environmental variables. Larval dispersal modelling considering species behaviour and interannual/interseasonal variation in ocean conditions was carried out as an essential background to which compare genetic information. Cockle populations in the Northeast Atlantic displayed low but significant geographical differentiation between populations (F ST = 0.0240; p < 0.001), albeit not across generations. We identified 742 and 36 outlier SNPs related to divergent and balancing selection in all the geographical scenarios inspected, and sea temperature and salinity were the main environmental correlates suggested. Highly significant linkage disequilibrium was detected at specific genomic regions against the very low values observed across the whole genome. Two main genetic groups were identified, northwards and southwards of French Brittany. Larval dispersal modelling suggested a barrier for larval dispersal linked to the Ushant front that could explain these two genetic clusters. Further genetic subdivision was observed using outlier loci and considering larval advection. The northern group was divided into the Irish/Celtic Seas and the English Channel/North Sea, while the southern group was divided into three subgroups. This information represents the baseline for the management of cockles, designing conservation strategies, founding broodstock for depleted beds and producing suitable seed for aquaculture production.

A comparison of precipitation and filtration-based SARS-CoV-2 recovery methods and the influence of temperature, turbidity, and surfactant load in urban wastewater.

Wastewater-based epidemiology (WBE) has become a complimentary surveillance tool during the SARS-CoV-2 pandemic. Viral concentration methods from wastewater are still being optimised and compared, whilst viral recovery under different wastewater characteristics and storage temperatures remains poorly understood. Using urban wastewater samples, we tested three viral concentration methods; polyethylene glycol precipitation (PEG), ammonium sulphate precipitation (AS), and CP select™ InnovaPrep® (IP) ultrafiltration. We found no major difference in SARS-CoV-2 and faecal indicator virus (crAssphage) recovery from wastewater samples (n = 46) using these methods, PEG slightly (albeit non-significantly), outperformed AS and IP for SARS-CoV-2 detection, as a higher genome copies per litre (gc/l) was recorded for a larger proportion of samples. Next generation sequencing of 8 paired samples revealed non-significant differences in the quality of data between AS and IP, though IP data quality was slightly better and less variable. A controlled experiment assessed the impact of wastewater suspended solids (turbidity; 0-400 NTU), surfactant load (0-200 mg/l), and storage temperature (5-20 °C) on viral recovery using the AS and IP methods. SARS-CoV-2 recoveries were >20% with AS and <10% with IP in turbid samples, whilst viral recoveries for samples with additional surfactant were between 0-18% for AS and 0-5% for IP. Turbidity and sample storage temperature combined had no significant effect on SARS-CoV-2 recovery (p > 0.05), whilst surfactant and storage temperature combined were significant negative correlates (p < 0.001 and p < 0.05, respectively). In conclusion, our results show that choice of methodology had small effect on viral recovery of SARS-CoV-2 and crAssphage in wastewater samples within this study. In contrast, sample turbidity, storage temperature, and surfactant load did affect viral recovery, highlighting the need for careful consideration of the viral concentration methodology used when working with wastewater samples.

Spatial variation of waterborne Escherichia coli - implications for routine water quality monitoring.

Escherichia coli are often used as faecal indicator bacteria (FIB) to provide a measure of microbial pollution in recreational and shellfish harvesting waters. However, although model forecasts for predicting the concentrations of FIB in surface waters are becoming more robust, they suffer from an inconsistency in quantification methods and an understanding of the spatial variation of FIB within a water course. The aim of this study was to investigate the transverse spatial variation in E. coli numbers (as an indicator of faecal pollution) across the estuary of the River Conwy, UK. Water samples were collected from four transverse transects across the estuary. Spatial variation of E. coli was significantly different from one side of the river to the other, although was not correlated with depth or the physiochemical properties of the water. Subsequently, microbial water quality classifications on the two opposite banks suggested very different levels of pollution coming down the river. This work has shown that the side of the river that routine water monitoring samples are taken from can make a significant difference to the classification of microbial water quality. This has important implications for sampling strategies and the use of microbial source tracking (MST) techniques.

Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR.

Wastewater-based epidemiology has become an important tool for the surveillance of SARS-CoV-2 outbreaks. However, the detection of viruses in sewage is challenging and to date there is no standard method available which has been validated for the sensitive detection of SARS-CoV-2. In this paper, we describe a simple concentration method based on polyethylene glycol (PEG) precipitation, followed by RNA extraction and a one-step quantitative reverse transcription PCR (qRT-PCR) for viral detection in wastewater. PEG-based concentration of viruses is a simple procedure which is not limited by the availability of expensive equipment and has reduced risk of disruption to consumable supply chains. The concentration and RNA extraction steps enable 900-1500× concentration of wastewater samples and sufficiently eliminates the majority of organic matter, which could inhibit the subsequent qRT-PCR assay. Due to the high variation in the physico-chemical properties of wastewater samples, we recommend the use of process control viruses to determine the efficiency of each step. This procedure enables the concentration and the extraction the DNA/RNA of different viruses and hence can be used for the surveillance of different viral targets for the comprehensive assessment of viral diseases in a community.

Investigating awareness, fear and control associated with norovirus and other pathogens and pollutants using best-worst scaling.

Pollutants found in the water and air environment represent an ever-growing threat to human health. Contact with some air-, water- and foodborne pathogens (e.g. norovirus) results in gastrointestinal diseases and outbreaks. For future risk mitigation, we aimed to measure people's awareness of waterborne and foodborne norovirus relative to other environment-associated pollutants (e.g. pesticides, bioaerosols, antibiotic resistant bacteria) and well-known risks (e.g. diabetes, dementia, terrorist attack). We used an online survey, which included a best-worst scaling component to elicit personal levels of control and fear prompted by norovirus relative to 15 other risks. There was a negative correlation between levels of fear vs. control for all 16 measured risks. Perceived infection control levels were higher amongst women compared to men and correlated with age and the level of qualification in both groups. Participants who had sought advice regarding the symptoms caused by norovirus appeared to have more control over the risks. Norovirus is associated with high levels of fear, however, the levels of control over it is low compared to other foodborne illnesses, e.g. Salmonella. Addressing this deficit in the public's understanding of how to control exposure to the pathogen in an important health need.

Correction: Farkas et al. Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR. Methods Protoc. 2021, 4, 17.

There was an error in the original article [...].

E. coli Is a Poor End-Product Criterion for Assessing the General Microbial Risk Posed From Consuming Norovirus Contaminated Shellfish.

The fecal indicator organism (FIO) Escherichia coli is frequently used as a general indicator of sewage contamination and for evaluating the success of shellfish cleaning (depuration) processes. To evaluate the robustness of this approach, the accumulation, retention, and depuration of non-pathogenic E. coli, pathogenic E. coli O157:H7 and norovirus GII (NoV GII) RNA were evaluated using a combination of culture-based (E. coli) and molecular methods (E. coli, NoV GII) after exposure of mussels (Mytilus edulis) to water contaminated with human feces. We simulated water contamination after a point-source release from a combined sewer overflow (CSO) where untreated wastewater is released directly into the coastal zone. All three microbiological indicators accumulated rapidly in the mussels, reaching close to maximum concentration within 3 h of exposure, demonstrating that short CSO discharges pose an immediate threat to shellfish harvesting areas. Depuration (72 h) in clean water proved partially successful at removing both pathogenic and non-pathogenic E. coli from shellfish tissue, but failed to eradicate NoV GII RNA. We conclude that current EU standards for evaluating microbiological risk in shellfish are inadequate for protecting consumers against exposure to human norovirus GII found in polluted marine waters.