In hot water: effects of climate change on Vibrio-human interactions.

Sea level rise and the anthropogenic warming of the world's oceans is not only an environmental tragedy, but these changes also result in a significant threat to public health. Along with coastal flooding and the encroachment of saltwater farther inland comes an increased risk of human interaction with pathogenic Vibrio species, such as Vibrio cholerae, V. vulnificus and V. parahaemolyticus. This minireview examines the current literature for updates on the climatic changes and practices that impact the location and duration of the presence of Vibrio spp., as well as the infection routes, trends and virulence factors of these highly successful pathogens. Finally, an overview of current treatments and methods for the mitigation of both oral and cutaneous exposures are presented.

A comparison between farmed oysters using floating cages and oysters grown on-bottom reveals more potentially human pathogenic Vibrio in the on-bottom oysters.

Eating raw oysters can come with serious health risks, as oysters can potentially contain bacteria of the Vibrio genus that cause food-borne infections. Vibrio bacteria are concentrated by oysters and, when consumed, infections can result with severe symptoms such as diarrhoea, lesions on the extremities, or even death. Vibrio spp. concentrations are strongly affected by season, location, and other factors such as temperature and salinity. Previous research in North Carolina oysters has been conducted on wild and farmed oysters but not at the same time. Farmed, or aquaculture raised, oysters are considerably different from wild oysters and could possibly pose different health risks. Farmed oysters are handled, raised from seed, and often grown using suspended grow-out systems called 'floating cages'. Therefore, farmed oysters can be grown at the surface of the estuary, while wild oysters typically grow at the bottom of the water column. This project compared the concentrations of Vibrio spp. in suspended, farm-grown oysters and wild oysters at three sites, using a paired approach with farmed and wild oysters sampled in proximity. An important part of this comparison was identifying pathogenicity of the bacteria isolated from the samples. Distinction was made between off- and on-bottom farming. Interestingly, on-bottom oysters had more pathogenic V. vulnificus than off-bottom oysters.

Factors affecting the uptake and retention of Vibrio vulnificus in oysters.

Vibrio vulnificus, a bacterium ubiquitous in oysters and coastal water, is capable of causing ailments ranging from gastroenteritis to grievous wound infections or septicemia. The uptake of these bacteria into oysters is often examined in vitro by placing oysters in seawater amended with V. vulnificus. Multiple teams have obtained similar results in studies where laboratory-grown bacteria were observed to be rapidly taken up by oysters but quickly eliminated. This technique, along with suggested modifications, is reviewed here. In contrast, the natural microflora within oysters is notoriously difficult to eliminate via depuration. The reason for the transiency of exogenous bacteria is that those bacteria are competitively excluded by the oyster's preexisting microflora. Evidence of this phenomenon is shown using in vitro oyster studies and a multiyear in situ case study. Depuration of the endogenous oyster bacteria occurs naturally and can also be artificially induced, but both of these events require extreme conditions, natural or otherwise, as explained here. Finally, the "viable but nonculturable" (VBNC) state of Vibrio is discussed. This bacterial torpor can easily be confused with a reduction in bacterial abundance, as bacteria in this state fail to grow on culture media. Thus, oysters collected from colder months may appear to be relatively free of Vibrio but in reality harbor VBNC cells that respond to exogenous bacteria and prevent colonization of oyster matrices. Bacterial-uptake experiments combined with studies involving cell-free spent media are detailed that demonstrate this occurrence, which could explain why the microbial community in oysters does not always mirror that of the surrounding water.

Increases in the amounts of Vibrio spp. in oysters upon addition of exogenous bacteria.

The bacterial pathogen Vibrio vulnificus is found naturally in brackish coastal waters but can be greatly concentrated by filter-feeding organisms such as shellfish. Numerous experiments in which exogenous V. vulnificus cells are added to oysters in an attempt to measure uptake and depuration have been performed. In nearly all cases, results have shown that laboratory-grown bacteria are rapidly taken up by the oysters but ultimately eliminated, while naturally present Vibrio populations in oysters are resistant to depuration. In this study, oysters harvested during winter months, with low culturable Vibrio concentrations, were incubated in aquaria supplemented with strains of V. vulnificus that were either genotypically or phenotypically distinct from the background bacteria. These exogenous cells were eliminated from the oysters, as previously seen, but other vibrios already inhabiting the oysters responded to the V. vulnificus inoculum by rapidly increasing in number and maintaining a large stable population. The presence of such an oyster-adapted Vibrio population would be expected to prevent colonization by exogenous V. vulnificus cells, thus explaining the rapid depuration of these added bacteria.

Integration of Vibrio vulnificus into marine aggregates and its subsequent uptake by Crassostrea virginica oysters.

Marine aggregates are naturally forming conglomerations of larvacean houses, phytoplankton, microbes, and inorganics adhered together by exocellular polymers. In this study, we show in vitro that the bacterial pathogen Vibrio vulnificus can be concentrated into laboratory-generated aggregates from surrounding water. We further show that environmental (E-genotype) strains exhibit significantly more integration into these aggregates than clinical (C-genotype) strains. Experiments where marine aggregates with attached V. vulnificus cells were fed to oysters (Crassostrea virginica) resulted in greater uptake of both C and E types than nonaggregated controls. When C- and E-genotype strains were cocultured in competitive experiments, the aggregated E-genotype strains exhibited significantly greater uptake by oyster than the C-genotype strains.

The interactions of Vibrio vulnificus and the oyster Crassostrea virginica.

The human bacterial pathogen, Vibrio vulnificus, is found in brackish waters and is concentrated by filter-feeding molluscan shellfish, especially oysters, which inhabit those waters. Ingestion of raw or undercooked oysters containing virulent strains of V. vulnificus can result in rapid septicemia and death in 50 % of victims. This review summarizes the current knowledge of the environmental interactions between these two organisms, including the effects of salinity and temperature on colonization, uptake, and depuration rates of various phenotypes and genotypes of the bacterium, and host-microbe immunological interactions.

Apparent loss of Vibrio vulnificus from North Carolina oysters coincides with a drought-induced increase in salinity.

Despite years of successful isolation of Vibrio vulnificus from estuarine waters, beginning in 2007, it was extremely difficult to culture V. vulnificus from either North Carolina estuarine water or oyster samples. After employing culture-based methods as well as PCR and quantitative PCR for the detection of V. vulnificus, always with negative results, we concluded that this pathogen had become nearly undetectable in the North Carolina estuarine ecosystem. We ensured that the techniques were sound by seeding North Carolina oysters with V. vulnificus and performing the same tests as those previously conducted on unadulterated oysters. V. vulnificus was readily detected in the seeded oysters using both classes of methods. Furthermore, oysters were obtained from the Gulf of Mexico, and V. vulnificus was easily isolated, confirming that the methodology was sound but that the oysters and waters of North Carolina were lacking the V. vulnificus population studied for decades. Strikingly, the apparent loss of detectable V. vulnificus coincided with the most severe drought in the history of North Carolina. The drought continued until the end of 2009, with an elevated water column salinity being observed throughout this period and with V. vulnificus being nearly nonexistent. When salinities returned to normal after the drought abated in 2010, we were again able to routinely isolate V. vulnificus from the water column, although we were still unable to culture it from oysters. We suggest that the oysters were colonized with a more salt-tolerant bacterium during the drought, which displaced V. vulnificus and may be preventing recolonization.

Decadal monitoring reveals an increase in Vibrio spp. concentrations in the Neuse River Estuary, North Carolina, USA.

A decade long study was conducted to investigate the ecological, biological, and temporal conditions that affect concentrations of Vibrio spp. bacteria in a well-studied lagoonal estuary. Water samples collected from the Neuse River Estuary in eastern North Carolina from 2004-2014 (with additional follow-up samples from Fall of 2018) were analyzed to determine Vibrio spp. concentrations, as well as the concentrations of inorganic and organic nutrients, fecal indicator bacteria, phytoplankton biomass, and a wide range of other physio-chemical estuarine parameters. A significant increase in Vibrio spp. was observed to occur in the estuary over the examined period. Strikingly, over this long duration study period, this statistically significant increase in total culturable Vibrio spp. concentrations does not appear to be correlated with changes in salinity, temperature, or dissolved oxygen, the three most commonly cited influential factors that predict estuarine Vibrio spp. abundance. Furthermore, shorter term (~3 years) data on specific Vibrio species (V. vulnificus and V. parahaemolyticus)show that while Vibrio spp. are increasing overall as a genus, the numbers of some key potentially pathogenic species are decreasing as a part of the total population, further supporting the concept that quantification of the entire genus is not a worthwhile use of resources toward predicting levels of specific potentially pathogenic species of public health concern. The significant increase in this concentration of Vibrio spp. in the studied estuary appears to be related to nitrogen and carbon in the system, indicating a continued need for further research.

Different abundance and correlational patterns exist between total and presumed pathogenic Vibrio vulnificus and V. parahaemolyticus in shellfish and waters along the North Carolina coast.

Monitoring of Vibrio vulnificus and V. parahaemolyticus abundance is pertinent due to the ability of these species to cause disease in humans through aquatic vectors. Previously, we performed a multiyear investigation tracking Vibrio spp. levels in five sites along the southeastern North Carolina coast. From February 2013 to October 2015, total V. vulnificus and V. parahaemolyticus abundance was measured in water, oysters and clams. In the current study, pathogenic subpopulations were identified in these isolates using molecular markers, revealing that 5.3% of V. vulnificus isolates possessed the virulence-correlated gene (vcgC), and 1.9% of V. parahaemolyticus isolates harbored one or both of the virulence-associated hemolysin genes (tdh and trh). Total V. parahaemolyticus abundance was not sufficient to predict the abundance of pathogenic subpopulations. Specifically, pathogenic V. parahaemolyticus isolates were more often isolated in cooler waters and were sometimes isolated when no other V. parahaemolyticus strains were detectable. Vibrio vulnificus clinical (C-) genotypes correlated with total V. vulnificus; however, salinity, water depth and total suspended solids influenced C- and E-genotypes differently. Lastly, we documented individual oysters harboring significantly higher V. vulnificus levels for which there was no ecological explanation, a phenomenon that deserves closer attention due to the potentially elevated health hazard associated with these 'hot' shellfish.

Vibrio bacteria in raw oysters: managing risks to human health.

The human-pathogenic marine bacteria Vibrio vulnificus and V. parahaemolyticus are strongly correlated with water temperature, with concentrations increasing as waters warm seasonally. Both of these bacteria can be concentrated in filter-feeding shellfish, especially oysters. Because oysters are often consumed raw, this exposes people to large doses of potentially harmful bacteria. Various models are used to predict the abundance of these bacteria in oysters, which guide shellfish harvest policy meant to reduce human health risk. Vibrio abundance and behaviour varies from site to site, suggesting that location-specific studies are needed to establish targeted risk reduction strategies. Moreover, virulence potential, rather than simple abundance, should be also be included in future modeling efforts.

Managing marine disease emergencies in an era of rapid change.

Infectious marine diseases can decimate populations and are increasing among some taxa due to global change and our increasing reliance on marine environments. Marine diseases become emergencies when significant ecological, economic or social impacts occur. We can prepare for and manage these emergencies through improved surveillance, and the development and iterative refinement of approaches to mitigate disease and its impacts. Improving surveillance requires fast, accurate diagnoses, forecasting disease risk and real-time monitoring of disease-promoting environmental conditions. Diversifying impact mitigation involves increasing host resilience to disease, reducing pathogen abundance and managing environmental factors that facilitate disease. Disease surveillance and mitigation can be adaptive if informed by research advances and catalysed by communication among observers, researchers and decision-makers using information-sharing platforms. Recent increases in the awareness of the threats posed by marine diseases may lead to policy frameworks that facilitate the responses and management that marine disease emergencies require.

The effect of storage time on Vibrio spp. and fecal indicator bacteria in an Isco autosampler.

Monitoring concentrations of bacterial pathogens and indicators of fecal contamination in coastal and estuarine ecosystems is critical to reduce adverse effects to public health. During storm events, particularly hurricanes, floods, Nor'easters, and tropical cyclones, sampling of coastal and estuarine waters is not generally possible due to safety concerns. It is particularly important to monitor waters during these periods as it is at precisely these times that pathogenic bacteria such as Vibrio spp. and fecal indicator bacteria concentrations fluctuate, potentially posing significant risks to public health. Automated samplers, such as the Isco sampler, are commonly used to conduct remote sample collection. Remote sampling is employed during severe storm periods, thereby reducing risk to researchers. Water samples are then stored until conditions are safe enough to retrieve them, typically in less than 21h, to collect the samples. Concerns exist regarding potential "bottle effects", whereby containment of sample might result in altered results. While these effects are well documented in samples being held for 24h or more, there is little data on bottle effects occurring during the first 24h of containment, and less still on the specific effects related to this type of sampling regime. Estuarine water samples were collected in the fall of 2013, placed into an Isco autosampler and subsampled over time to determine the effects of storage within this type of autosampling device. Vibrio spp. and fecal indicator bacteria were quantified using replicated culture-based methods, including Enterolert™ and membrane filtration. The experiments demonstrated no significant impact of storage time when comparing concentrations of total Vibrio spp., Vibrio vulnificus, Vibrio parahaemolyticus, or Enterococcus spp. after storage compared to original concentrations. However, the findings also suggested that increased variability and growth can occur during the middle of the day. Therefore, if at all possible, analysis schedules should be modified to account for this variability, e.g. collection of samples after overnight storage should occur as early in the morning as practicable.

The evaluation of four recent culture-based methods for the isolation and enumeration of Vibrio vulnificus bacteria from oyster meat.

The most common cause of seafood-borne death in the United States is the bacterium Vibrio vulnificus which can be concentrated into high numbers in the tissues of oysters or other shellfish. The ability to quickly, accurately, and inexpensively isolate living strains of this organism from oyster tissues is crucial for effective research on this pathogen. In this report, we evaluate four methods for isolating and quantifying V. vulnificus from oyster tissues, the solid media CPC+ (a refined version of cellobiose-polymyxin B-colistin medium), CHROMagar Vibrio, VVX (Vibrio vulnificus X-gal), and a method termed "Triple plating". Up to 1225 presumptive isolates were detected by each method, and 335 were subjected to molecular typing. The selectivity and sensitivity of each method was examined and VVX was found to be the most accurate method, with each of the other methods being recommended for task-specific uses. CHROMagar Vibrio is recommended for ease of use and relative accuracy, CPC+ is best used to differentiate between clinically associated and environmental strains.

Climate change influences on marine infectious diseases: implications for management and society.

Infectious diseases are common in marine environments, but the effects of a changing climate on marine pathogens are not well understood. Here we review current knowledge about how the climate drives host-pathogen interactions and infectious disease outbreaks. Climate-related impacts on marine diseases are being documented in corals, shellfish, finfish, and humans; these impacts are less clearly linked for other organisms. Oceans and people are inextricably linked, and marine diseases can both directly and indirectly affect human health, livelihoods, and well-being. We recommend an adaptive management approach to better increase the resilience of ocean systems vulnerable to marine diseases in a changing climate. Land-based management methods of quarantining, culling, and vaccinating are not successful in the ocean; therefore, forecasting conditions that lead to outbreaks and designing tools/approaches to influence these conditions may be the best way to manage marine disease.

A new culture-based method for the improved identification of Vibrio vulnificus from environmental samples, reducing the need for molecular confirmation.

Vibrio vulnificus is an opportunistic human pathogen responsible for 95% of seafood related deaths in the US. Monitoring the presence of this bacterium in estuarine waters and shellfish is of medical and economic importance due to its ability to cause severe wound infections and fulminant septicemia. Current methods for isolating V. vulnificus from environmental samples typically employ an initial selective medium which requires subsequent molecular confirmation of presumptive V. vulnificus isolates. Although culture-based methods are accessible and inexpensive, they lack the specificity needed to definitively identify V. vulnificus. The goal of this study was to develop a more accurate, culture-based method for the initial detection of V. vulnificus, thereby decreasing or eliminating the requirement for confirmatory molecular tests. Colony color characteristics of a variety of Vibrio species were determined on three commonly employed media to identify those which present as false-positive isolates for V. vulnificus. We subsequently developed a triple-plating method which utilizes three media in combination to greatly decrease the number of false positive isolates. The number of isolates positively identified as V. vulnificus using the triple-plating method were compared to a typical single-plating method and revealed over a 2-fold increase in ability to accurately predict V. vulnificus isolates. We suggest that this new method will enhance the predictive power of culture-based methods, reduce the cost and time spent on additional detection methods, and may be a valuable alternative when molecular methods are not available or unaffordable.

Mechanistic and statistical models of total Vibrio abundance in the Neuse River Estuary.

Bacteria in the genus Vibrio are ubiquitous to estuarine waters worldwide and are often the dominant genus recovered from these environments. This genus contains several potentially pathogenic species, including Vibrio vulnificus, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio alginolyticus. These bacteria have short generation times, as low as 20-30 min, and can thus respond rapidly to changing environmental conditions. A five-parameter mechanistic model was generated based on environmental processes including hydrodynamics, growth, and death rates of Vibrio bacteria to predict total Vibrio abundance in the Neuse River Estuary of eastern North Carolina. Additionally an improved statistical model was developed using the easily monitored parameters of temperature and salinity. This updated model includes data that covers more than eight years of constant bacterial monitoring, and incorporates extreme weather events such as droughts, storms, and floods. These models can be used to identify days in which bacterial abundance might coincide with increased health risks.

Pyrosequencing-based comparative genome analysis of Vibrio vulnificus environmental isolates.

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.

Interactive effects of cadmium and hypoxia on metabolic responses and bacterial loads of eastern oysters Crassostrea virginica Gmelin.

Pollution by toxic metals including cadmium (Cd) and hypoxia are important stressors in estuaries and coastal waters which may interactively affect sessile benthic organisms, such as oysters. We studied metabolic responses to prolonged hypoxic acclimation (2 weeks at 5% O2) in control and Cd-exposed (30 d at 50 µg L(-1) Cd) oysters Crassostrea virginica, and analyzed the effects of these stressors on abundance of Vibrio spp. in oysters. Hypoxia-acclimated oysters retained normal standard metabolic rates (SMR) at 5% O2, in contrast to a decline of SMR observed during acute hypoxia. However, oysters spent more time actively ventilating in hypoxia than normoxia resulting in enhanced Cd uptake and 2.7-fold higher tissue Cd burdens in hypoxia. Cd exposure led to a significant decrease in tissue glycogen stores, increase in free glucose levels and elevated activity of glycolytic enzymes (hexokinase and aldolase) indicating a greater dependence on carbohydrate catabolism. A compensatory increase in activities of two key mitochondrial enzymes (citrate synthase and cytochrome c oxidase) was found during prolonged hypoxia in control oysters but suppressed in Cd-exposed ones. Cd exposure also resulted in a significant increase in abundance of Vibrio parahaemolyticus and Vibrio vulnificus levels during normoxia and hypoxia, respectively. Overall, Cd- and hypoxia-induced changes in metabolic profile, Cd accumulation and bacterial flora of oysters indicate that these stressors can synergistically impact energy homeostasis, performance and survival of oysters in polluted estuaries and have significant consequences for transfer of Cd and bacterial pathogens to the higher levels of the food chain.

Uptake and depuration of the C- and E-genotypes of Vibrio vulnificus by the Eastern Oyster (Crassostrea virginica).

The human pathogen Vibrio vulnificus is a Gram-negative estuarine bacterium that infect via wounds and ingestion, and is the leading cause of seafood-borne death in the United States. Vibrio vulnificus is part of the naturally occurring flora of both estuaries and estuarine mollusks (especially oysters). Vibrio vulnificus is divided into two genotypes, including a clinically associated C-type, and an environmentally associated E-type that is more rarely involved in septicemia. These two genotypes are found in a nearly even ratio in the aquatic environment, but oysters harvested from those very environments show a V. vulnificus genotype ratio disparity, with 87% of the species being that of the E-genotype. To determine if oysters selectively incorporate E-types over C-types, we placed oysters in water inoculated with either C- or E-type V. vulnificus strains that were phenotypically different from the normal flora and measured the uptake and depuration over a course of 6 days. We found significantly greater uptake, but equally effective depuration, of C-type V. vulnificus in oyster gill tissue, mantle tissue and whole oyster homogenates. Because uptake of the C-genotype was generally greater than the E-genotype, it appears unlikely that simple selective uptake is the cause of E-type V. vulnificus predominating in oysters.