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Abstract One Health is a recognition of the shared environment inhabited by humans, animals and plants, and the impact of their interactions on the health of all organisms. The COVID-19 pandemic highlighted the need for a framework of pathogen surveillance in a tractable One Health paradigm to allow timely detection and response to threats to human and animal health. We present case studies centered around the recent global approach to tackle antimicrobial resistance and the current interest in wastewater testing, with the concept of “one sample many analyses” to be further explored as the most appropriate means of initiating this endeavor.

Summary: A recurring trend in evidence scrutinized over the past few decades is that disease outbreaks will become more frequent, intense, and widespread on land and in water, due to climate change. Pathogens and the diseases they inflict represent a major constraint on seafood production and yield, and by extension, food security. The risk(s) for fish and shellfish from disease is a function of pathogen characteristics, biological species identity, and the ambient environmental conditions. A changing climate can adversely influence the host and environment, while augmenting pathogen characteristics simultaneously, thereby favoring disease outbreaks. Herein, we use a series of case studies covering some of the world’s most cultured aquatic species (e.g., salmonids, penaeid shrimp, and oysters), and the pathogens (viral, fungal, bacterial, and parasitic) that afflict them, to illustrate the magnitude of disease-related problems linked to climate change.

Abstract Vibrio parahaemolyticus, Vibrio vulnificus and non‐O1/non‐O139 Vibrio cholerae are the Vibrio spp. of highest relevance for public health in the EU through seafood consumption. Infection with V. parahaemolyticus is associated with the haemolysins thermostable direct haemolysin (TDH) and TDH‐related haemolysin (TRH) and mainly leads to acute gastroenteritis. V. vulnificus infections can lead to sepsis and death in susceptible individuals. V. cholerae non‐O1/non‐O139 can cause mild gastroenteritis or lead to severe infections, including sepsis, in susceptible individuals. The pooled prevalence estimate in seafood is 19.6% (95% CI 13.7–27.4), 6.1% (95% CI 3.0–11.8) and 4.1% (95% CI 2.4–6.9) for V. parahaemolyticus, V. vulnificus and non‐choleragenic V. cholerae, respectively. Approximately one out of five V. parahaemolyticus‐positive samples contain pathogenic strains. A large spectrum of antimicrobial resistances, some of which are intrinsic, has been found in vibrios isolated from seafood or food‐borne infections in Europe. Genes conferring resistance to medically important antimicrobials and associated with mobile genetic elements are increasingly detected in vibrios. Temperature and salinity are the most relevant drivers for Vibrio abundance in the aquatic environment. It is anticipated that the occurrence and levels of the relevant Vibrio spp. in seafood will increase in response to coastal warming and extreme weather events, especially in low‐salinity/brackish waters. While some measures, like high‐pressure processing, irradiation or depuration reduce the levels of Vibrio spp. in seafood, maintaining the cold chain is important to prevent their growth. Available risk assessments addressed V. parahaemolyticus in various types of seafood and V. vulnificus in raw oysters and octopus. A quantitative microbiological risk assessment relevant in an EU context would be V. parahaemolyticus in bivalve molluscs (oysters), evaluating the effect of mitigations, especially in a climate change scenario. Knowledge gaps related to Vibrio spp. in seafood and aquatic environments are identified and future research needs are prioritised.

Tetrodotoxin (TTX) is a potent marine neurotoxin found in several phylogenetically diverse organisms, some of which are sought as seafood. Since 2015, TTX has been reported in bivalve shellfish from several estuarine locations along the Mediterranean and European Atlantic coasts, posing an emerging food safety concern. Although reports on spatial and temporal distribution have increased in recent years, processes leading to TTX accumulation in European bivalves are yet to be described. Here, we explored the hypothesis that the ribbon worm species Cephalothrix simula, known to contain high levels of TTX, could play a role in the trophic transfer of the toxin into shellfish. During a field study at a single location in southern England, we confirmed C. simula DNA in seawater adjacent to trestle-farmed Pacific oysters Magallana gigas (formerly Crassostrea gigas) with a history of TTX occurrence. C. simula DNA in seawater was significantly higher in June and July during the active phase of toxin accumulation compared to periods of either no or continually decreasing TTX concentrations in M. gigas. In addition, C. simula DNA was detected in oyster digestive glands collected on 15 June 2021, the day with the highest recorded C. simula DNA abundance in seawater. These findings show evidence of a relationship between C. simula and TTX occurrence, providing support for the hypothesis that bivalves may acquire TTX through filter-feeding on microscopic life forms of C. simula present in the water column at particular periods each year. Although further evidence is needed to confirm such feeding activity, this study significantly contributes to discussions about the biological source of TTX in European bivalve shellfish.

Abstract 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.

Sewage pollution leads to the contamination of bivalve shellfish by pathogenic microorganisms. Bacterial indicators support the management of risks associated with the consumption of shellfish; however, they often fail to indicate adequately the potential hazard to human health posed by certain human enteric viruses. Bacteriophages have been proposed as alternative indicators that may more effectively predict the presence of enteric viral pathogens. This study explored the relationships between bacterial indicators (Escherichia coli (E. coli), faecal coliforms (FC) and intestinal enterococci (IE)), phages (somatic (SOMPH), F-specific RNA (F + PH) and human-specific Bacteroides GB-124 phages (GB124PH)) and Norovirus (NoV) (GI/GII) in mussels (Mytilus edulis) and their overlying waters. The bioaccumulation of these indicators and Norovirus in shellfish matrices (e.g., flesh, digestive gland) was investigated bimonthly over a 12-month period in an English estuary. The findings revealed a marked seasonality in the distribution of all organisms, with the highest levels occurring during the autumn/winter months. The levels of all phages in shellfish and their overlying waters correlated better with the levels of Norovirus than with those of bacterial indicators. Somatic coliphages were the indicator that exhibited the strongest correlations with NoV (rho = 0.929). This study suggests that relatively low-cost culture-based phage enumeration appears to offer a more accurate indication of the likely presence of Norovirus in mussels than traditional bacterial indicators.

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 blaCTX-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.

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.

It is becoming increasingly clear that the genetic diversity and abundance of antimicrobial resistance (AMR) in non-clinical settings has been underestimated and that the environment plays an integral role in enabling the development of AMR. Due to specific demographic and environmental factors the Gulf Cooperation Council (GCC) region may be particularly susceptible to the threat of AMR, with the marine and aquatic environment potentially playing a specific role in its development and propagation. The demographic factors include rapid population growth, significant international population movements, heavy antibiotic use and insufficient antibiotic stewardship. Environmental factors leading to susceptibility include notable inputs of untreated sewage effluent, high ambient water temperatures, elevated concentrations of heavy metals, and poorly regulated use of antimicrobials in veterinary settings. However, to date there is only a limited understanding of the role that this environment plays in enabling the emergence and propagation AMR in this region. This article provides an overview of the risk associated with AMR in the marine and aquatic environment in the GCC region and proposes a framework for understanding how such environments interact with the wider development and propagation of resistance. It identifies priority actions aligned with the World Health Organisation AMR Global Action Plan and associated national action plans to evaluate the role of marine and aquatic systems relative to the wider factors driving AMR emergence and propagation. The proposed framework and actions to evaluate the role of marine and aquatic environments in driving propagation and emergence of AMR are equally applicable at the regional and national level beyond the GCC. Keywords: AMR, Bacteria, GCC, Emergence, Marine pollution

Non-toxigenic V. parahaemolyticus isolates (tdh−/trh−/T3SS2−) have recently been isolated from patients with gastroenteritis. In this study we report that the larvae of the wax moth (Galleria mellonella) are susceptible to infection by toxigenic or non-toxigenic clinical isolates of V. parahaemolyticus. In comparison larvae inoculated with environmental isolates of V. parahaemolyticus did not succumb to disease. Whole genome sequencing of clinical non-toxigenic isolates revealed the presence of a gene encoding a nudix hydrolase, identified as mutT. A V. parahaemolyticus mutT mutant was unable to kill G. mellonella at 24 h post inoculation, indicating a role of this gene in virulence. Our findings show that G. mellonella is a valuable model for investigating screening of possible virulence genes of V. parahaemolyticus and can provide new insights into mechanisms of virulence of atypical non-toxigenic V. parahaemolyticus. These findings will allow improved genetic tests for the identification of pathogenic V. parahaemolyticus to be developed and will have a significant impact for the scientific community.

Galicia in northwestern Spain has been considered a hotspot for Vibrio parahaemolyticus infections. Infections abruptly emerged in 1998 and, over the next 15 years, were associated with large outbreaks caused by strains belonging to a single clone. We report a recent transition in the epidemiologic pattern in which cases throughout the region have been linked to different and unrelated strains. Global genome-wide phylogenetic analysis revealed that most of the pathogenic strains isolated from infections were associated with globally diverse isolates, indicating frequent episodic introductions from disparate and remote sources. Moreover, we identified that the 2 major switches in the epidemic dynamics of V. parahaemolyticus in the regions, the emergence of cases and an epidemiologic shift in 2015–2016, were associated with the rise of sea surface temperature in coastal areas of Galicia. This association may represent a fundamental contributing factor in the emergence of illness linked to these introduced pathogenic strains.

The Genus Alexandrium is a widespread dinoflagellate marine phytoplankton that is the primary causative organism causing Paralytic Shellfish Poisoning (PSP) intoxications in European waters. EU food safety directives specify that EU Member States must implement a routine monitoring programme to mitigate risks associated with bio-accumulation of biotoxins by bivalve shellfish, such as those produced by Alexandrium. This strategic drive comprises of both direct testing of bivalve flesh for the presence of regulated toxins and an early warning phytoplankton monitoring programme. In the UK the flesh testing moved away from animal bio-assays to analytical chemistry techniques, whereas phytoplankton monitoring methods have seen little technological advancement since implementation. Methods currently utilize light microscopy and manual enumeration of different algal species. These methods although proven are time consuming, reliant on highly trained staff, have high limits of detection (LOD) with low specificity, unable to reliably identify Alexandrium to species level. The implications of these limitations of the techniques mean that in the case of Alexandrium the LOD is also the action limit and as such it is easy to miss positive samples affecting the efficacy of any early warning strategy. This study outlines the development, preliminary method characterisation, validation and trial implementation of an alternative early warning technique, utilizing quantitative PCR to identify water samples containing Alexandrium cells. The approach outlined in this document, showed an improved correlation with flesh toxicity, improved sensitivity, improved throughput compared to traditional light microscopy methods and there was also good correlation with higher cell abundance samples when compared to the light microscopy results. The application of this approach to routine water samples was explored and was found to demonstrate potential as a corroborative method for use during flesh intoxication episodes. This study offers potential for future improvements in the accuracy and sensitivity of phytoplankton monitoring whilst ensuring continuity of public safety, providing cost savings and offering new research opportunities. Keywords: Harmful algal bloom, Biotoxin, Alexandrium, qPCR assay, Monitoring, TaqMan

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.

During summer 2014, a total of 89 Vibrio infections were reported in Sweden and Finland, substantially more yearly infections than previously have been reported in northern Europe. Infections were spread across most coastal counties of Sweden and Finland, but unusually, numerous infections were reported in subarctic regions; cases were reported as far north as 65°N, ≈100 miles (160 km) from the Arctic Circle. Most infections were caused by non-O1/O139 V. cholerae (70 cases, corresponding to 77% of the total, all strains were negative for the cholera toxin gene). An extreme heat wave in northern Scandinavia during summer 2014 led to unprecedented high sea surface temperatures, which appear to have been responsible for the emergence of Vibrio bacteria at these latitudes. The emergence of vibriosis in high-latitude regions requires improved diagnostic detection and clinical awareness of these emerging pathogens.

Vibrio vulnificus (Vv) is a multi-host pathogenic species currently subdivided into three biotypes (Bts). The three Bts are human-pathogens, but only Bt2 is also a fish-pathogen, an ability that is conferred by a transferable virulence-plasmid (pVvbt2). Here we present a phylogenomic analysis from the core genome of 80 Vv strains belonging to the three Bts recovered from a wide range of geographical and ecological sources. We have identified five well-supported phylogenetic groups or lineages (L). L1 comprises a mixture of clinical and environmental Bt1 strains, most of them involved in human clinical cases related to raw seafood ingestion. L2 is formed by a mixture of Bt1 and Bt2 strains from various sources, including diseased fish, and is related to the aquaculture industry. L3 is also linked to the aquaculture industry and includes Bt3 strains exclusively, mostly related to wound infections or secondary septicemia after farmed-fish handling. Lastly, L4 and L5 include a few strains of Bt1 associated with specific geographical areas. The phylogenetic trees for ChrI and II are not congruent to one another, which suggests that inter- and/or intra-chromosomal rearrangements have been produced along Vv evolution. Further, the phylogenetic trees for each chromosome and the virulence plasmid were also not congruent, which also suggests that pVvbt2 has been acquired independently by different clones, probably in fish farms. From all these clones, the one with zoonotic capabilities (Bt2-Serovar E) has successfully spread worldwide. Based on these results, we propose a new updated classification of the species based on phylogenetic lineages rather than on Bts, as well as the inclusion of all Bt2 strains in a pathovar with the particular ability to cause fish vibriosis, for which we suggest the name “piscis.”

ABSTRACT Vibrio parahaemolyticus is the leading cause of seafood-related infections with illnesses undergoing a geographic expansion. In this process of expansion, the most fundamental change has been the transition from infections caused by local strains to the surge of pandemic clonal types. Pandemic clone sequence type 3 (ST3) was the only example of transcontinental spreading until 2012, when ST36 was detected outside the region where it is endemic in the U.S. Pacific Northwest causing infections along the U.S. northeast coast and Spain. Here, we used genome-wide analyses to reconstruct the evolutionary history of the V. parahaemolyticus ST36 clone over the course of its geographic expansion during the previous 25 years. The origin of this lineage was estimated to be in ~1985. By 1995, a new variant emerged in the region and quickly replaced the old clone, which has not been detected since 2000. The new Pacific Northwest (PNW) lineage was responsible for the first cases associated with this clone outside the Pacific Northwest region. After several introductions into the northeast coast, the new PNW clone differentiated into a highly dynamic group that continues to cause illness on the northeast coast of the United States. Surprisingly, the strains detected in Europe in 2012 diverged from this ancestral group around 2000 and have conserved genetic features present only in the old PNW lineage. Recombination was identified as the major driver of diversification, with some preliminary observations suggesting a trend toward a more specialized lifestyle, which may represent a critical element in the expansion of epidemics under scenarios of coastal warming. IMPORTANCE Vibrio parahaemolyticus and Vibrio cholerae represent the only two instances of pandemic expansions of human pathogens originating in the marine environment. However, while the current pandemic of V. cholerae emerged more than 50 years ago, the global expansion of V. parahaemolyticus is a recent phenomenon. These modern expansions provide an exceptional opportunity to study the evolutionary process of these pathogens at first hand and gain an understanding of the mechanisms shaping the epidemic dynamics of these diseases, in particular, the emergence, dispersal, and successful introduction in new regions facilitating global spreading of infections. In this study, we used genomic analysis to examine the evolutionary divergence that has occurred over the course of the most recent transcontinental expansion of a pathogenic Vibrio, the spreading of the V. parahaemolyticus sequence type 36 clone from the region where it is endemic on the Pacific coast of North America to the east coast of the United States and finally to the west coast of Europe.

Vibrio vulnificus is a bacterium responsible for severe gastroenteritis, sepsis and wound infections. Gastroenteritis and sepsis are commonly associated with the consumption of raw oysters, whereas wound infection is often associated with the handling of contaminated fish. Although classical virulence factors of this emerging pathogen are well characterised, there remains a paucity of knowledge regarding the general biology of this species. To investigate the presence of previously unreported virulence factors, we applied whole genome sequencing to a panel of ten V. vulnificus strains with varying virulence potentials. This identified two novel type 6 secretion systems (T6SSs), systems that are known to have a role in bacterial virulence and population dynamics. By utilising a range of molecular techniques and assays we have demonstrated the functionality of one of these T6SSs. Furthermore, we have shown that this system is subject to thermoregulation and is negatively regulated by increasing salinity concentrations. This secretion system was also shown to be involved in the killing of V. vulnificus strains that did not possess this system and a model is proposed as to how this interaction may contribute to population dynamics within V. vulnificus strains. In addition to this intra-species killing, this system also contributes to the killing of inter bacterial species and may have a role in the general composition of Vibrio species in the environment.

The marine nemertean Cephalothrix simula originates from the Pacific Ocean but in recent years has been discovered in northern Europe. The species has been associated with high levels of the marine neurotoxin Tetrodotoxin, traditionally associated with Pufferfish Poisoning. This study reports the first discovery of two organisms of C. simula in the UK, showing the geographical extent of this species is wider than originally described. Species identification was initially conducted morphologically, with confirmation by Cox 1 DNA sequencing. 16S gene sequencing enabled the taxonomic assignment of the microbiome, showing the prevalence of a large number of bacterial genera previously associated with TTX production including Alteromonas, Vibrio and Pseudomonas. LC-MS/MS analysis of the nemertean tissue revealed the presence of multiple analogues of TTX, dominated by the parent TTX, with a total toxin concentration quantified at 54 µg TTX per g of tissue. Pseudomonas luteola isolated from C. simula, together with Vibrio alginolyticus from the native nemertean Tubulanus annulatus, were cultured at low temperature and both found to contain TTX. Overall, this paper confirms the high toxicity of a newly discovered invasive nemertean species with links to toxin-producing marine bacteria and the potential risk to human safety. Further work is required to assess the geographical extent and toxicity range of C. simula along the UK coast in order to properly gauge the potential impacts on the environment and human safety.

Antimicrobial resistance remains a growing and significant concern in human and veterinary medicine. Current laboratory methods for the detection and surveillance of antimicrobial resistant bacteria are limited in their effectiveness and scope. With the rapidly developing field of whole genome sequencing beginning to be utilised in clinical practice, the ability to interrogate sequencing data quickly and easily for the presence of antimicrobial resistance genes will become increasingly important and useful for informing clinical decisions. Additionally, use of such tools will provide insight into the dynamics of antimicrobial resistance genes in metagenomic samples such as those used in environmental monitoring.Here we present the Search Engine for Antimicrobial Resistance (SEAR), a pipeline and web interface for detection of horizontally acquired antimicrobial resistance genes in raw sequencing data. The pipeline provides gene information, abundance estimation and the reconstructed sequence of antimicrobial resistance genes; it also provides web links to additional information on each gene. The pipeline utilises clustering and read mapping to annotate full-length genes relative to a user-defined database. It also uses local alignment of annotated genes to a range of online databases to provide additional information. We demonstrate SEAR's application in the detection and abundance estimation of antimicrobial resistance genes in two novel environmental metagenomes, 32 human faecal microbiome datasets and 126 clinical isolates of Shigella sonnei.We have developed a pipeline that contributes to the improved capacity for antimicrobial resistance detection afforded by next generation sequencing technologies, allowing for rapid detection of antimicrobial resistance genes directly from sequencing data. SEAR uses raw sequencing data via an intuitive interface so can be run rapidly without requiring advanced bioinformatic skills or resources. Finally, we show that SEAR is effective in detecting antimicrobial resistance genes in metagenomic and isolate sequencing data from both environmental metagenomes and sequencing data from clinical isolates.

Tetrodotoxins (TTXs) are traditionally associated with the occurrence of tropical Pufferfish Poisoning. In recent years, however, TTXs have been identified in European bivalve mollusc shellfish, resulting in the need to assess prevalence and risk to shellfish consumers. Following the previous identification of TTXs in shellfish from southern England, this study was designed to assess the wider prevalence of TTXs in shellfish from around the coast of the UK. Samples were collected between 2014 and 2016 and subjected to analysis using HILIC-MS/MS. Results showed the continued presence of toxins in shellfish harvested along the coast of southern England, with the maximum concentration of total TTXs reaching 253 µg/kg. TTX accumulation was detected in Pacific oysters (Crassostrea gigas), native oysters (Ostrea edulis) common mussels (Mytilus edulis) and hard clams (Mercenaria mercenaria), but not found in cockles (Cerastoderma edule), razors (Ensis species) or scallops (Pecten maximus). Whilst the highest concentrations were quantified in samples harvested during the warmer summer months, TTXs were still evident during the winter. An assessment of the potential causative factors did not reveal any links with the phytoplankton species Prorocentrum cordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C.

Between 1996 and 2006, the US Centers for Disease Control reported that the only category of food-borne infections increasing in frequency were those caused by members of the genus Vibrio. The gram-negative bacterium Vibrio vulnificus is a ubiquitous inhabitant of estuarine waters, and is the number one cause of seafood-related deaths in the US. Many V. vulnificus isolates have been studied, and it has been shown that two genetically distinct subtypes, distinguished by 16S rDNA and other gene polymorphisms, are associated predominantly with either environmental or clinical isolation. While local genetic differences between the subtypes have been probed, only the genomes of clinical isolates have so far been completely sequenced. In order to better understand V. vulnificus as an agent of disease and to identify the molecular components of its virulence mechanisms, we have completed whole genome shotgun sequencing of three diverse environmental genotypes using a pyrosequencing approach. V. vulnificus strain JY1305 was sequenced to a depth of 33×, and strains E64MW and JY1701 were sequenced to lesser depth, covering approximately 99.9% of each genome. We have performed a comparative analysis of these sequences against the previously published sequences of three V. vulnificus clinical isolates. We find that the genome of V. vulnificus is dynamic, with 1.27% of genes in the C-genotype genomes not found in the E- genotype genomes. We identified key genes that differentiate between the genomes of the clinical and environmental genotypes. 167 genes were found to be specifically associated with environmental genotypes and 278 genes with clinical genotypes. Genes specific to the clinical strains include components of sialic acid catabolism, mannitol fermentation, and a component of a Type IV secretory pathway VirB4, as well as several other genes with potential significance for human virulence. Genes specific to environmental strains included several that may have implications for the balance between self-preservation under stress and nutritional competence.

BACKGROUND:Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport. METHODOLOGY/PRINCIPAL FINDINGS:To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to > or =15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, bla(TEM-1), tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. CONCLUSIONS:These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.