Metabarcoding of Hepatitis E Virus Genotype 3 and Norovirus GII from Wastewater Samples in England Using Nanopore Sequencing.

Norovirus is one of the largest causes of gastroenteritis worldwide, and Hepatitis E virus (HEV) is an emerging pathogen that has become the most dominant cause of acute viral hepatitis in recent years. The presence of norovirus and HEV has been reported within wastewater in many countries previously. Here we used amplicon deep sequencing (metabarcoding) to identify norovirus and HEV strains in wastewater samples from England collected in 2019 and 2020. For HEV, we sequenced a fragment of the RNA-dependent RNA polymerase (RdRp) gene targeting genotype three strains. For norovirus, we sequenced the 5' portion of the major capsid protein gene (VP1) of genogroup II strains. Sequencing of the wastewater samples revealed eight different genotypes of norovirus GII (GII.2, GII.3, GII.4, GII.6, GII.7, GII.9, GII.13 and GII.17). Genotypes GII.3 and GII.4 were the most commonly found. The HEV metabarcoding assay was able to identify HEV genotype 3 strains in some samples with a very low viral concentration determined by RT-qPCR. Analysis showed that most HEV strains found in influent wastewater were typed as G3c and G3e and were likely to have originated from humans or swine. However, the small size of the HEV nested PCR amplicon could cause issues with typing, and so this method is more appropriate for samples with high CTs where methods targeting longer genomic regions are unlikely to be successful. This is the first report of HEV RNA in wastewater in England. This study demonstrates the utility of wastewater sequencing and the need for wider surveillance of norovirus and HEV within host species and environments.

Sea temperature influences accumulation of tetrodotoxin in British bivalve shellfish.

Tetrodotoxin (TTX), a potent neurotoxin mostly associated with pufferfish poisoning, is also found in bivalve shellfish. Recent studies into this emerging food safety threat reported TTX in a few, mainly estuarine, shellfish production areas in some European countries, including the United Kingdom. A pattern in occurrences has started to emerge, however the role of temperature on TTX has not been investigated in detail. Therefore, we conducted a large systematic TTX screening study, encompassing over 3500 bivalve samples collected throughout 2016 from 155 shellfish monitoring sites along the coast of Great Britain. Overall, we found that only 1.1 % of tested samples contained TTX above the reporting limit of 2 µg/kg whole shellfish flesh and these samples all originated from ten shellfish production sites in southern England. Subsequent continuous monitoring of selected areas over a five-year period showed a potential seasonal TTX accumulation in bivalves, starting in June when water temperatures reached around 15 °C. For the first time, satellite-derived data were also applied to investigate temperature differences between sites with and without confirmed presence of TTX in 2016. Although average annual temperatures were similar in both groups, daily mean values were higher in summer and lower in winter at sites where TTX was found. Here, temperature also increased significantly faster during late spring and early summer, the critical period for TTX. Our study supports the hypothesis that temperature is one of the key triggers of events leading to TTX accumulation in European bivalves. However, other factors are also likely to play an important role, including the presence or absence of a de novo biological source, which remains elusive.

Climate warming and increasing Vibrio vulnificus infections in North America.

Vibrio vulnificus is an opportunistic bacterial pathogen, occurring in warm low-salinity waters. V. vulnificus wound infections due to seawater exposure are infrequent but mortality rates are high (~ 18%). Seawater bacterial concentrations are increasing but changing disease pattern assessments or climate change projections are rare. Here, using a 30-year database of V. vulnificus cases for the Eastern USA, changing disease distribution was assessed. An ecological niche model was developed, trained and validated to identify links to oceanographic and climate data. This model was used to predict future disease distribution using data simulated by seven Global Climate Models (GCMs) which belong to the newest Coupled Model Intercomparison Project (CMIP6). Risk was estimated by calculating the total population within 200 km of the disease distribution. Predictions were generated for different "pathways" of global socioeconomic development which incorporate projections of greenhouse gas emissions and demographic change. In Eastern USA between 1988 and 2018, V. vulnificus wound infections increased eightfold (10-80 cases p.a.) and the northern case limit shifted northwards 48 km p.a. By 2041-2060, V. vulnificus infections may expand their current range to encompass major population centres around New York (40.7°N). Combined with a growing and increasingly elderly population, annual case numbers may double. By 2081-2100 V. vulnificus infections may be present in every Eastern USA State under medium-to-high future emissions and warming. The projected expansion of V. vulnificus wound infections stresses the need for increased individual and public health awareness in these areas.

What Whole Genome Sequencing Has Told Us About Pathogenic Vibrios.

When the first microbial genome sequences were published just 20 years ago, our understanding regarding the microbial world changed dramatically. The genomes of the first pathogenic vibrios sequenced, including Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus revealed a functional and phylogenetic diversity previously unimagined as well as a genome structure indelibly shaped by horizontal gene transfer. The initial glimpses into these organisms also revealed a genomic plasticity that allowed these bacteria to thrive in challenging and varied aquatic and marine environments, but critically also a suite of pathogenicity attributes. In this review we outline how our understanding of vibrios has changed over the last two decades with the advent of genomics and advances in bioinformatic and data analysis techniques, it has become possible to provide a more cohesive understanding regarding these bacteria: how these pathogens have evolved and emerged from environmental sources, their evolutionary routes through time and space, how they interact with other bacteria and the human host, as well as initiate disease. We outline novel approaches to the use of whole genome sequencing for this important group of bacteria and how new sequencing technologies may be applied to study these organisms in future studies.

An integrated eco-evolutionary framework to predict population-level responses of climate-sensitive pathogens.

It is critical to gain insight into how climate change impacts evolutionary responses within climate-sensitive pathogen populations, such as increased resilience, opportunistic responses and the emergence of dominant variants from highly variable genomic backgrounds and subsequent global dispersal. This review proposes a framework to support such analysis, by combining genomic evolutionary analysis with climate time-series data in a novel spatiotemporal dataframe for use within machine learning applications, to understand past and future evolutionary pathogen responses to climate change. Recommendations are presented to increase the feasibility of interdisciplinary applications, including the importance of robust spatiotemporal metadata accompanying genome submission to databases. Such workflows will inform accessible public health tools and early-warning systems, to aid decision-making and mitigate future human health threats.

Methodological advances in the detection of biotoxins and pathogens affecting production and consumption of bivalve molluscs in a changing environment.

The production, harvesting and safe consumption of bivalve molluscs can be disrupted by biological hazards that can be divided into three categories: (1) biotoxins produced by naturally occurring phytoplankton that are bioaccumulated by bivalves during filter-feeding, (2) human pathogens also bioaccumulated by bivalves and (3) bivalve pathogens responsible for disease outbreaks. Environmental changes caused by human activities, such as climate change, can further aggravate these challenges. Early detection and accurate quantification of these hazards are key to implementing measures to mitigate their impact on production and safeguard consumers. This review summarises the methods currently used and the technological advances in the detection of biological hazards affecting bivalves, for the screening of known hazards and discovery of new ones.

Establishing a marine monitoring programme to assess antibiotic resistance: A case study from the Gulf Cooperation Council (GCC) region.

The World Health Organization considers antimicrobial resistance as one of the most pressing global issues which poses a fundamental threat to human health, development, and security. Due to demographic and environmental factors, the marine environment of the Gulf Cooperation Council (GCC) region may be particularly susceptible to the threat of antimicrobial resistance. However, there is currently little information on the presence of AMR in the GCC marine environment to inform the design of appropriate targeted surveillance activities. The objective of this study was to develop, implement and conduct a rapid regional baseline monitoring survey of the presence of AMR in the GCC marine environment, through the analysis of seawater collected from high-risk areas across four GCC states: (Bahrain, Oman, Kuwait, and the United Arab Emirates). 560 Escherichia coli strains were analysed as part of this monitoring programme between December 2018 and May 2019. Multi-drug resistance (resistance to three or more structural classes of antimicrobials) was observed in 32.5% of tested isolates. High levels of reduced susceptibility to ampicillin (29.6%), nalidixic acid (27.9%), tetracycline (27.5%), sulfamethoxazole (22.5%) and trimethoprim (22.5%) were observed. Reduced susceptibility to the high priority critically important antimicrobials: azithromycin (9.3%), ceftazidime (12.7%), cefotaxime (12.7%), ciprofloxacin (44.6%), gentamicin (2.7%) and tigecycline (0.5%), was also noted. A subset of 173 isolates was whole genome sequenced, and high carriage rates of qnrS1 (60/173) and bla CTX-M-15 (45/173) were observed, correlating with reduced susceptibility to the fluoroquinolones and third generation cephalosporins, respectively. This study is important because of the resistance patterns observed, the demonstrated utility in applying genomic-based approaches to routine microbiological monitoring, and the overall establishment of a transnational AMR surveillance framework focussed on coastal and marine environments.

Pathogens transported by plastic debris: does this vector pose a risk to aquatic organisms?

Microplastics are small (<5 mm) plastic particles of varying shapes and polymer types that are now widespread global contaminants of marine and freshwater ecosystems. Various estimates suggest that several trillions of microplastic particles are present in our global oceanic system, and that these are readily ingested by a wide range of marine and freshwater species across feeding modes and ecological niches. Here, we present some of the key and pressing issues associated with these globally important contaminants from a microbiological perspective. We discuss the potential mechanisms of pathogen attachment to plastic surfaces. We then describe the ability of pathogens (both human and animal) to form biofilms on microplastics, as well as dispersal of these bacteria, which might lead to their uptake into aquatic species ingesting microplastic particles. Finally, we discuss the role of a changing oceanic system on the potential of microplastic-associated pathogens to cause various disease outcomes using numerous case studies. We set out some key and imperative research questions regarding this globally important issue and present a methodological framework to study how and why plastic-associated pathogens should be addressed.

The Foodborne Transmission of Hepatitis E Virus to Humans.

Globally, Hepatitis E virus (HEV) causes over 20 million cases worldwide. HEV is an emerging and endemic pathogen within economically developed countries, chiefly resulting from infections with genotype 3 (G3) HEV. G3 HEV is known to be a zoonotic pathogen, with a broad host range. The primary source of HEV within more economically developed countries is considered to be pigs, and consumption of pork products is a significant risk factor and known transmission route for the virus to humans. However, other foods have also been implicated in the transmission of HEV to humans. This review consolidates the information available regarding transmission of HEV and looks to identify gaps where further research is required to better understand how HEV is transmitted to humans through food.

Distribution of Tetrodotoxin in Pacific Oysters (Crassostrea gigas).

A potent and heat-stable tetrodotoxin (TTX) has been found to accumulate in various marine bivalve species, including Pacific oysters (Crassostrea gigas), raising a food safety concern. While several studies on geographical occurrence of TTX have been conducted, there is a lack of knowledge about the distribution of the toxin within and between bivalves. We, therefore, measured TTX in the whole flesh, mantle, gills, labial palps, digestive gland, adductor muscle and intravalvular fluid of C. gigas using liquid chromatography-tandem mass spectrometry. Weekly monitoring during summer months revealed the highest TTX concentrations in the digestive gland (up to 242 µg/kg), significantly higher than in other oyster tissues. Intra-population variability of TTX, measured in the whole flesh of each of twenty animals, reached 46% and 32% in the two separate batches, respectively. In addition, an inter-population study was conducted to compare TTX levels at four locations within the oyster production area. TTX concentrations in the whole flesh varied significantly between some of these locations, which was unexplained by the differences in weight of flesh. This is the first study examining TTX distribution in C. gigas and the first confirmation of the preferential accumulation of TTX in oyster digestive gland.

Oceanic Hitchhikers - Assessing Pathogen Risks from Marine Microplastic.

As plastic debris in the environment continues to increase, an emerging concern is the potential for microplastic to act as vectors for pathogen transport. With aquaculture the fastest growing food sector, and microplastic contamination of shellfish increasingly demonstrated, understanding any risk of pathogen transport associated with microplastic is important for this industry. However, there remains a lack of detailed, systematic studies assessing the interactions and potential impacts that the attachment of human and animal pathogens on microplastic may have. Here we synthesise current knowledge regarding these distinct microplastic-associated bacterial communities and microplastic uptake pathways into bivalves, and discuss whether they represent a human and animal health threat, highlighting the outstanding questions critical to our understanding of this potential risk to food safety.

The new tools revolutionizing Vibrio science.

As microbiologists we live in exciting times. A variety of technical and conceptual developments, particularly in the last decade have revolutionized the field of microbiology, redrawing the landscape, and entirely redefining what is possible. Perhaps this paradigm shift is no more apparent than in the study of vibrios. The family Vibrionaceae are almost unique as a group of bacteria to study in microbiology: they are genomically, phylogenetically and functionally diverse yet a distinct group of environmental bacteria encompassing important human and animal pathogens as well as non-pathogenic species such as ecologically critical symbionts. Sensitive to physiochemical stimuli, they are among the fasting replicating bacteria studied, capable of responding almost immediately to favourable environmental conditions such as those afforded by climate warming. Characterized by an unusual double chromosome and frequently carrying numerous cryptic plasmids - their genomes are often pockmarked with insertion elements, transposons, prophages and integrases - paying testament to past genomic promiscuity. With a strong affinity for environmental niches in freshwater and marine systems, they are among the most numerous bacteria present in our oceans, coasts and freshwater environments. As such they offer something for almost anyone interested in microbiology and represent an excellent example of field of microbiology that has benefitted hugely by advances across a gamut of disciplines - not just microbiological - but encompassing genomics, genetics, oceanography, ecological, earth observations sciences and data visualization, among others. We will briefly outline some of the most exciting, innovative and translational scientific advances that are currently being applied to these ecologically, environmentally and clinically important bacteria.

Isolation and characterization of potentially pathogenic Vibrio species in a temperate, higher latitude hotspot.

The recent emergence of Vibrio infections at high latitudes represents a clear human health risk attributable to climate change. Here, we investigate the population dynamics of three Vibrio species: Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae within a British coastal estuarine site, with contrasting salinity and temperature regimes during an intense heatwave event. Water samples were collected weekly through the summer of 2018 and 2019 and filtered using membrane filtration and subsequently grown on selective media. Suspected vibrios were confirmed using a conventional species-specific PCR assay and further analysed for potential pathogenic markers. Results showed that Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae were present at high concentrations throughout both years, with their populations at substantially greater abundances corresponding to conditions of higher water temperatures during the heatwave of 2018 and at lower salinity sites, which is comparable to the results of previous studies. A subset of strains isolated during the extreme heatwave event in 2018 (46 Vibrio parahaemolyticus, 11 Vibrio cholerae and 4 Vibrio vulnificus) were genomically sequenced. Analysis of these 63 sequenced strains revealed a broad phenotypic and genomic diversity of strains circulating in the environment. An analysis of pathogenicity attributes identified a broad array of virulence genes across all three species, including a variety of genes associated with human disease. This study highlights the importance of the need for an increased Vibrio spp. surveillance system in temperate regions and the potential impact warming events such as heatwaves may have on the abundance of potentially pathogenic bacteria in the environment.

Global emergence of environmental non-O1/O139 Vibrio cholerae infections linked with climate change: a neglected research field?

The bacterium Vibrio cholerae is a natural inhabitant of aquatic ecosystems across the planet. V. cholerae serogroups O1 and O139 are responsible for cholera outbreaks in developing countries accounting for 3-5 million infections worldwide and 28.800-130.000 deaths per year according to the World Health Organization. In contrast, V. cholerae serogroups other than O1 and O139, also designated as V. cholerae non-O1/O139 (NOVC), are not associated with epidemic cholera but can cause other illnesses that may range in severity from mild (e.g. gastroenteritis, otitis, etc.) to life-threatening (e.g. necrotizing fasciitis). Although generally neglected, NOVC-related infections are on the rise and represent one of the most striking examples of emerging human diseases linked to climate change. NOVC strains are also believed to potentially contribute to the emergence of new pathogenic strains including strains with epidemic potential as a direct consequence of genetic exchange mechanisms such as horizontal gene transfer and genetic recombination. Besides general features concerning the biology and ecology of NOVC strains and their associated diseases, this review aims to highlight the most relevant aspects related to the emergence and potential threat posed by NOVC strains under a rapidly changing environmental and climatic scenario.

Global Expansion of Pacific Northwest Vibrio parahaemolyticus Sequence Type 36.

We report transcontinental expansion of Vibrio parahaemolyticus sequence type 36 into Lima, Peru. From national collections, we identified 7 isolates from 2 different Pacific Northwest complex lineages that surfaced during 2011-2016. Sequence type 36 is likely established in environmental reservoirs. Systematic surveillance enabled detection of these epidemic isolates.

Corrigendum: Phylogeny of Vibrio vulnificus From the Analysis of the Core-Genome: Implications for Intra-Species Taxonomy.

[This corrects the article DOI: 10.3389/fmicb.2017.02613.].