Exploring the Feasibility of 16S rRNA Short Amplicon Sequencing-Based Microbiota Analysis for Microbiological Safety Assessment of Raw Oyster.

16S rRNA short amplicon sequencing-based microbiota profiling has been thought of and suggested as a feasible method to assess food safety. However, even if a comprehensive microbial information can be obtained by microbiota profiling, it would not be necessarily sufficient for all circumstances. To prove this, the feasibility of the most widely used V3-V4 amplicon sequencing method for food safety assessment was examined here. We designed a pathogen (Vibrio parahaemolyticus) contamination and/or V. parahaemolyticus-specific phage treatment model of raw oysters under improper storage temperature and monitored their microbial structure changes. The samples stored at refrigerator temperature (negative control, NC) and those that were stored at room temperature without any treatment (no treatment, NT) were included as control groups. The profiling results revealed that no statistical difference exists between the NT group and the pathogen spiked- and/or phage treated-groups even when the bacterial composition was compared at the possible lowest-rank taxa, family/genus level. In the beta-diversity analysis, all the samples except the NC group formed one distinct cluster. Notably, the samples with pathogen and/or phage addition did not form each cluster even though the enumerated number of V. parahaemolyticus in those samples were extremely different. These discrepant results indicate that the feasibility of 16S rRNA short amplicon sequencing should not be overgeneralized in microbiological safety assessment of food samples, such as raw oyster.

Host and Water Microbiota Are Differentially Linked to Potential Human Pathogen Accumulation in Oysters.

Oysters play an important role in coastal ecology and are a globally popular seafood source. However, their filter-feeding lifestyle enables coastal pathogens, toxins, and pollutants to accumulate in their tissues, potentially endangering human health. While pathogen concentrations in coastal waters are often linked to environmental conditions and runoff events, these do not always correlate with pathogen concentrations in oysters. Additional factors related to the microbial ecology of pathogenic bacteria and their relationship with oyster hosts likely play a role in accumulation but are poorly understood. In this study, we investigated whether microbial communities in water and oysters were linked to accumulation of Vibrio parahaemolyticus, Vibrio vulnificus, or fecal indicator bacteria. Site-specific environmental conditions significantly influenced microbial communities and potential pathogen concentrations in water. Oyster microbial communities, however, exhibited less variability in microbial community diversity and accumulation of target bacteria overall and were less impacted by environmental differences between sites. Instead, changes in specific microbial taxa in oyster and water samples, particularly in oyster digestive glands, were linked to elevated levels of potential pathogens. For example, increased levels of V. parahaemolyticus were associated with higher relative abundances of cyanobacteria, which could represent an environmental vector for Vibrio spp. transport, and with decreased relative abundance of Mycoplasma and other key members of the oyster digestive gland microbiota. These findings suggest that host and microbial factors, in addition to environmental variables, may influence pathogen accumulation in oysters. IMPORTANCE Bacteria in the marine environment cause thousands of human illnesses annually. Bivalves are a popular seafood source and are important in coastal ecology, but their ability to concentrate pathogens from the water can cause human illness, threatening seafood safety and security. To predict and prevent disease, it is critical to understand what causes pathogenic bacteria to accumulate in bivalves. In this study, we examined how environmental factors and host and water microbial communities were linked to potential human pathogen accumulation in oysters. Oyster microbial communities were more stable than water communities, and both contained the highest concentrations of Vibrio parahaemolyticus at sites with warmer temperatures and lower salinities. High oyster V. parahaemolyticus concentrations corresponded with abundant cyanobacteria, a potential vector for transmission, and a decrease in potentially beneficial oyster microbes. Our study suggests that poorly understood factors, including host and water microbiota, likely play a role in pathogen distribution and pathogen transmission.

Microbiological quality, antibiotic resistant bacteria and relevant resistance genes in ready-to-eat Pacific oysters (Magallana gigas).

Oysters are a highly valued seafood but can endanger public health, if they are eaten raw or barely cooked. We evaluated the microbiological quality of Pacific oysters (Magallana gigas) by international standard methods in four groups (each with four to five animals) acquired from supermarkets and directly from a farm producer. Most of the groups presented satisfactory microbiological quality. In two groups of oysters, 'questionable' or 'unsatisfactory' quality was observed for the coagulase-positive Staphylococcus parameter. Culture-based methods did not detect Salmonella spp. or enteropathogenic Vibrio spp., but Vibrio alginolyticus, a potential foodborne pathogen, was identified by molecular analysis. Fifty strains, belonging to 19 species, were isolated in antibiotic-supplemented media, and their antibiotic susceptibility profile was evaluated. Genes coding for ß-lactamases were searched by PCR in bacteria showing resistance phenotype. Decreased susceptibility or resistance to distinct antibiotics were observed for bacteria from depurated and non-depurated oysters. The blaTEM gene was identified in Escherichia fergusonii and Shigella dysenteriae strains, which showed multidrug-resistant phenotypes. The possibility that oysters might be a source of antibiotic-resistant bacteria/antibiotic resistance genes is of great concern and highlights the need for stricter controls and preventative measures to mitigate and counteract the dissemination of antibiotic resistance across the food chain.

Environmental Factors Influencing Occurrence of Vibrio parahaemolyticus and Vibrio vulnificus.

Incidence of vibriosis is rising globally, with evidence that changing climatic conditions are influencing environmental factors that enhance growth of pathogenic Vibrio spp. in aquatic ecosystems. To determine the impact of environmental factors on occurrence of pathogenic Vibrio spp., samples were collected in the Chesapeake Bay, Maryland, during 2009 to 2012 and 2019 to 2022. Genetic markers for Vibrio vulnificus (vvhA) and Vibrio parahaemolyticus (tlh, tdh, and trh) were enumerated by direct plating and DNA colony hybridization. Results confirmed seasonality and environmental parameters as predictors. Water temperature showed a linear correlation with vvhA and tlh, and two critical thresholds were observed, an initial increase in detectable numbers (>15°C) and a second increase when maximum counts were recorded (>25°C). Temperature and pathogenic V. parahaemolyticus (tdh and trh) were not strongly correlated; however, the evidence showed that these organisms persist in oyster and sediment at colder temperatures. Salinity (10 to 15 ppt), total chlorophyll a (5 to 25 µg/L), dissolved oxygen (5 to 10 mg/L), and pH (8) were associated with increased abundance of vvhA and tlh. Importantly, a long-term increase in Vibrio spp. numbers was observed in water samples between the two collection periods, specifically at Tangier Sound (lower bay), with the evidence suggesting an extended seasonality for these bacteria in the area. Notably, tlh showed a mean positive increase that was ca. 3-fold overall, with the most significant increase observed during the fall. In conclusion, vibriosis continues to be a risk in the Chesapeake Bay region. A predictive intelligence system to assist decision makers, with respect to climate and human health, is warranted. IMPORTANCE The genus Vibrio includes pathogenic species that are naturally occurring in marine and estuarine environments globally. Routine monitoring for Vibrio species and environmental parameters influencing their incidence is critical to provide a warning system for the public when the risk of infection is high. In this study, occurrence of Vibrio parahaemolyticus and Vibrio vulnificus, both potential human pathogens, in Chesapeake Bay water, oysters, and sediment samples collected over a 13-year period was analyzed. The results provide a confirmation of environmental predictors for these bacteria, notably temperature, salinity, and total chlorophyll a, and their seasonality of occurrence. New findings refine environmental parameter thresholds of culturable Vibrio species and document a long-term increase in Vibrio populations in the Chesapeake Bay. This study provides a valuable foundation for development of predicative risk intelligence models for Vibrio incidence during climate change.

Microbiological Quality of Oysters and Mussels Along Its Market Supply Chain.

Oysters and mussels are known vectors of foodborne pathogens because of their immobile and filter-feeding nature leading to the accumulation of biological particles in their tissues. Accumulated bacteria which comes from the culture environment and unsanitary handling can cause food poisoning if these shellfish are consumed raw or partially processed. This study determined the incidence of bacterial pathogen contamination along the different channels of the oyster and mussel supply chain through a time-distribution simulation analysis. First, the route of the fresh bivalve products from a local farm to its market was established through interviews. From the data gathered, a simulation experiment was conducted following the observed time-temperature conditions and the actual bulk packaging material used by the traders. The presence of target pathogens Escherichia coli, Salmonella spp., Vibrio parahaemolyticus, and Vibrio cholerae were detected using standard conventional culture techniques. Initial E. coli counts in both mussels and oysters were higher than the safety limit of 330 MPN in 100 g tissue. Interestingly, E. coli counts in mussels decreased after 6 h and maintained low numbers after more than 24 h postharvest. Counts in oysters however increased to 1000 MPN in 100 g tissue. V. parahaemolyticus in mussels and oysters showed a gradual increase in counts with increasing holding time albeit in numbers that are lower than the safety limit of 1000 cfu g-1 tissue. Qualitative detection of Salmonella and V. cholerae showed the presence of both pathogens in all the sampling points. All four pathogens were also detected in the culture waters and in the sediment. Results of the study showed that the culture environment and the handling practices contribute greatly to the pathogen contamination in oysters and mussels.

Antibacterial Mechanism of Shikonin Against Vibrio vulnificus and Its Healing Potential on Infected Mice with Full-Thickness Excised Skin.

Shikonin has anticancer, anti-inflammatory, and wound healing activities. Vibrio vulnificus is an important marine foodborne pathogen with a high fatality rate and rapid pathogenesis that can infect humans through ingestion and wounds. In this study, the antibacterial activity and possible antibacterial mechanism of shikonin against V. vulnificus were investigated. In addition, the ability of shikonin to control V. vulnificus infection in both pathways was assessed by artificially contaminated oysters and full-thickness excised skin-infected mice. Shikonin treatment can cause abnormal cell membrane function, as evidenced by hyperpolarization of the cell membrane, significant decreased intracellular ATP concentration (p < 0.05), significant increased intracellular reactive oxygen species and malondialdehyde content (p < 0.05), decreased cell membrane integrity, and changes in cell morphology. Shikonin at 40 and 80 µg/mL reduced bacterial numbers in shikonin-contaminated oysters by 3.58 and 2.18 log colony-forming unit (CFU)/mL. Shikonin can promote wound healing in mice infected with V. vulnificus by promoting the formation of granulation tissue, hair follicles, and sebaceous glands, promoting epithelial cell regeneration and epidermal growth factor production. These findings suggest that shikonin has a strong inactivation effect on V. vulnificus and can be used in food production and wound healing to effectively control V. vulnificus and reduce the number of diseases associated with it.

A Genome-Scale Metabolic Model of Marine Heterotroph Vibrio splendidus Strain 1A01.

While Vibrio splendidus is best known as an opportunistic pathogen in oysters, Vibrio splendidus strain 1A01 was first identified as an early colonizer of synthetic chitin particles incubated in seawater. To gain a better understanding of its metabolism, a genome-scale metabolic model (GSMM) of V. splendidus 1A01 was reconstructed. GSMMs enable us to simulate all metabolic reactions in a bacterial cell using flux balance analysis. A draft model was built using an automated pipeline from BioCyc. Manual curation was then performed based on experimental data, in part by gap-filling metabolic pathways and tailoring the model's biomass reaction to V. splendidus 1A01. The challenges of building a metabolic model for a marine microorganism like V. splendidus 1A01 are described. IMPORTANCE A genome-scale metabolic model of V. splendidus 1A01 was reconstructed in this work. We offer solutions to the technical problems associated with model reconstruction for a marine bacterial strain like V. splendidus 1A01, which arise largely from the high salt concentration found in both seawater and culture media that simulate seawater.

Screening of marine lactic acid bacteria for Vibrio parahaemolyticus inhibition and application to depuration in Pacific oysters (Crassostrea gigas).

AIMS: This study aims to assess the use of marine lactic acid bacteria (LAB) to reduce Vibrio parahaemolyticus levels during oyster depuration process. METHODS AND RESULTS: The inhibitory effect of 30 marine LAB strains against V. parahaemolyticus strains was evaluated by in vitro assays. A total of three positive strains (Latilactobacillus sakei SF1583, Lactococcus lactis SF1945, and Vagococcus fluvialis CD264) were selected for V. parahaemolyticus levels reduction during oyster depuration. Pacific oysters Crassostrea gigas were artificially and independently contaminated by four GFP-labelled V. parahaemolyticus strains (IFVp201, IFVp69, IFVp195, and LMG2850T) at 105 CFU ml-1 and then exposed by balneation to 106 CFU ml-1 of each LAB strains during 24 h, at 19°C. Quantification of V. parahaemolyticus in haemolymph by flow cytometry revealed variations in natural depuration of the different V. parahaemolyticus strains alone. Furthermore, the addition of LABs improved up to 1-log bacteria ml-1 the reduction of IFVp201 concentration in comparison to the control condition. CONCLUSIONS: Although further optimizations of procedure are needed, addition of marine LABs during oyster depuration may be an interesting strategy to reduce V. parahaemolyticus levels in Crassostrea gigas.

Deep learning-based ensemble modeling of Vibrio parahaemolyticus concentration in marine environment.

Vibrio parahaemolyticus (V.p) is a marine pathogenic bacterium that poses a high risk to human health and shellfish industry, yet an effective regional-scale nowcasting model for managing the risk remains lacking. This study presents the first regional-scale model for nowcasting the level of V.p in oysters in the marine environment by developing an ensemble modeling approach. The ensemble modeling approach involves the integration of genetic programming (GP) and deep artificial neural networks (DNN)-based modeling. The new approach was demonstrated by developing three GP-DNN ensemble models for predicting the V.p level in North Carolina, New Hampshire, and the combined region. Specifically, GP was employed to establish nonlinear functions between the V.p level and antecedent conditions of environmental variables. The nonlinear GP functions and current conditions of individual environmental variables were then utilized as inputs into a DNN model, forming a GP-DNN ensemble model. Modeling results indicated that the GP-DNN ensemble models were capable of predicting the V.p level with the correlation coefficient of 0.91, 0.90, and 0.80 for North Carolina, New Hampshire, and the combined region, respectively, demonstrating the impact of distinct environmental conditions in the local areas on accuracy of the combined regional-scale model. Sensitivity analysis results showed that sea surface temperature and sea surface salinity are the two most important environmental predictors for the abundance of V.p in oysters, followed by water level, pH, chlorophyll-a, and turbidity. The findings suggested that the GP-DNN ensemble models could be utilized as effective predictive tools for mitigating the V.p risk.

Growth Rates of Vibrio parahaemolyticus Sequence Type 36 Strains in Live Oysters and in Culture Medium.

The pathogenic marine bacterium Vibrio parahaemolyticus can cause seafood-related gastroenteritis via the consumption of raw or undercooked seafood. Infections originating from relatively cool waters in the northeast United States are typically rare, but recently, this region has shown an increase in infections attributed to the ecological introduction of pathogenic sequence type 36 (ST36) strains, which are endemic to the cool waters of the Pacific Northwest. A 2005 risk assessment performed by the Food and Drug Administration (FDA) modeled the postharvest growth of V. parahaemolyticus in oysters as a function of air temperature and the length of time the oysters remained unrefrigerated. This model, while useful, has raised questions about strain growth differences in oyster tissue and whether invasive pathogenic strains exhibit different growth rates than nonclinical strains, particularly at lower temperatures. To investigate this question, live eastern oysters were injected with ST36 clinical strains and non-ST36 nonclinical strains, and growth rates were measured using the most probable number (MPN) enumeration. The presence of V. parahaemolyticus was confirmed using PCR by targeting the thermolabile hemolysin gene (tlh), thermostable direct hemolysin (tdh), tdh-related hemolysin (trh), and a pathogenesis-related protein (prp). The growth rates of the ST36 strains were compared to the FDA model and several other data sets of V. parahaemolyticus growth in naturally inoculated oysters harvested from the Chesapeake Bay. Our data indicate that the growth rates from most studies fall within the mean of the FDA model, but with slightly higher growth at lower temperatures for ST36 strains injected into live oysters. These data suggest that further investigations of ST36 growth capability in oysters at temperatures previously thought unsuitably low for Vibrio growth are warranted. IMPORTANCE Vibrio parahaemolyticus is the leading cause of seafood-related gastroenteritis in the United States, with an estimated 45,000 cases per year. Most individuals who suffer from vibriosis consume raw or undercooked seafood, including oysters. While gastroenteritis vibriosis is usually self-limiting and treatable, V. parahaemolyticus infections are a stressor on the growing aquaculture industry. Much effort has been placed on modeling the growth of Vibrio cells in oysters in order to aid oyster growers in designing harvesting best practices and ultimately, to protect the consumer. However, ecological invasions of nonnative bacterial strains make modeling their growth complicated, as these strains are not accounted for in current models. The National Shellfish Sanitation Program (NSSP) considers 10°C (50°F) a temperature too low to enable Vibrio growth, where 15°C is considered a cutoff temperature for optimal Vibrio growth, with temperatures approaching 20°C supporting higher growth rates. However, invasive strains may be native to cooler waters. This research aimed to understand strain growth in live oysters by measuring growth rates when oysters containing ST36 strains, which may be endemic to the U.S. Pacific Northwest, were exposed to multiple temperatures postharvest. Our results will be used to aid future model development and harvesting best practices for the aquaculture industry.

Vibrio ostreae sp. nov., a novel gut bacterium isolated from a Yellow Sea oyster.

A Gram-stain-negative, oxidase- and catalase-positive, facultative anaerobic motile bacterium, designated strain OG9-811T, was isolated from the gut of an oyster collected in the Yellow Sea, Republic of Korea. The strain grew at 10-37 °C, pH 6.0-9.0 and with 0.5-10% (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain OG9-811T affiliated with the genus Vibrio, with the highest sequence similarity of 98.2% to Vibrio coralliilyticus ATCC BAA-450T followed by Vibrio variabilis R-40492T (98.0 %), Vibrio hepatarius LMG 20362T (97.7 %) and Vibrio neptunius LMG 20536T (97.6 %); other relatives were Vibrio tritonius JCM 16456T (97.4 %), Vibrio fluvialis NBRC 103150T (97.0 %) and Vibrio furnissii CIP 102972T (97.0 %). The complete genome of strain OG9-811T comprised two chromosomes of a total 4 807 684 bp and the G+C content was 50.2 %. Results of analysis based on the whole genome sequence showed the distinctiveness of strain OG9-811T. The average nucleotide identity (ANI) values between strain OG9-811T and the closest strains V. coralliilyticus ATCC BAA-450T, V. variabilis R-40492T, V. hepatarius LMG 20362T, V. neptunius KCTC 12702T , V. tritonius JCM 16456T, V. fluvialis ATCC 33809T and V. furnissi CIP 102972T were 73.0, 72.6, 73.3, 73.0, 72.7, 78.5 and 77.8 %, respectively, while the digital DNA-DNA hybridization values between strain OG9-811T and the above closely related strains were 20.8, 21.2, 20.8, 21.7, 20.7, 23.2 and 22.4 %, respectively. The major fatty acids of strain OG9-811T were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), summed feature 8 (C18:1 ω6c and/or C18:1 ω7c) and C16:0. The polar lipids contained phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Strain OG9-811T contained Q-8 as a quinone. On the basis of polyphasic taxonomic characteristics, strain OG9-811T is considered to represent a novel species, for which the name Vibrio ostreae sp. nov. is proposed. The type strain is OG9-811T (=KCTC 72623T=GDMCC 1.2610T).

The effect of off-bottom versus on-bottom oyster culture on total and pathogenic Vibrio spp. abundances in oyster tissue, water and sediment samples.

Varying culture methods are commonly used for eastern oyster, Crassostrea virginica, aquaculture in the Northeast United States. Vibrio vulnificus and V. parahaemolyticus, two human pathogenic bacteria species, accumulate in this edible, filter feeding shellfish. This study examined the use of two methods in an intertidal area (oysters cultured in trays and in bags on sediment) and two methods in a subtidal area (oysters cultured in trays and loose on the sediment) in Massachusetts over the growing season in 2015. Abundance of total V. vulnificus along with total and pathogenic (tdh+/trh+) V. parahaemolyticus were determined in oysters, sediment and water using real-time PCR. Temperature, salinity, turbidity and chlorophyll were continually measured every 15 min at each location. There were significantly higher abundances of total and pathogenic V. parahaemolyticus in on-bottom cultured oysters, while significantly higher abundances of V. vulnificus were identified in oysters from off-bottom culture in a subtidal location in Duxbury Bay, MA. In an intertidal location, Wellfleet Bay, MA, significantly higher abundances of total and tdh+V. parahaemolyticus were found in off-bottom oysters, but significantly higher abundances of V. vulnificus and trh+V. parahaemolyticus were found in on-bottom oysters. Spearman's correlation indicated that temperature is positively associated with concentrations of Vibrio spp. in oysters, water and sediment, but positive correlations between salinity and Vibrio spp. was also observed. Conversely, turbidity had a negative effect on Vibrio spp. concentrations in all sample types. There was no observed relationship inferred between chlorophyll and Vibrio spp. abundances in oysters, water or sediment.

Depuration of live oysters to reduce Vibrio parahaemolyticus and Vibrio vulnificus: A review of ecology and processing parameters.

Consumption of raw oysters, whether wild-caught or aquacultured, may increase health risks for humans. Vibrio vulnificus and Vibrio parahaemolyticus are two potentially pathogenic bacteria that can be concentrated in oysters during filter feeding. As Vibrio abundance increases in coastal waters worldwide, ingesting raw oysters contaminated with V. vulnificus and V. parahaemolyticus can possibly result in human illness and death in susceptible individuals. Depuration is a postharvest processing method that maintains oyster viability while they filter clean salt water that either continuously flows through a holding tank or is recirculated and replenished periodically. This process can reduce endogenous bacteria, including coliforms, thus providing a safer, live oyster product for human consumption; however, depuration of Vibrios has presented challenges. When considering the difficulty of removing endogenous Vibrios in oysters, a more standardized framework of effective depuration parameters is needed. Understanding Vibrio ecology and its relation to certain depuration parameters could help optimize the process for the reduction of Vibrio. In the past, researchers have manipulated key depuration parameters like depuration processing time, water salinity, water temperature, and water flow rate and explored the use of processing additives to enhance disinfection in oysters. In summation, depuration processing from 4 to 6 days, low temperature, high salinity, and flowing water effectively reduced V. vulnificus and V. parahaemolyticus in live oysters. This review aims to emphasize trends among the results of these past works and provide suggestions for future oyster depuration studies.

Development of Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay To Detect Hemolysin Gene of Vibrio vulnificus in Oysters.

ABSTRACT: Vibrio vulnificus inhabits estuarine waters around the world and can cause severe infections in people who eat contaminated raw or undercooked oysters. Although current detection methods are sensitive and specific, there are continuous demands for the development of rapid and accurate methods without a trained operator and equipment in the field conditions. Herein, we developed a simple and rapid method by detecting the hemolysin (vvh) gene of V. vulnificus by using recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD). The RPA-LFD could detect 100 fg of DNA (P < 0.05) and 20 CFU of V. vulnificus per reaction within 30 min (P < 0.01) and showed the result with incubation temperature ranges from 30 to 45°C (P < 0.001). The test was specific only to V. vulnificus and was not responsive to 10 other closely related Vibrio species and 18 foodborne pathogenic bacteria. Compared with PCR, quantitative PCR, and colony hybridization assays by using naturally contaminated oyster samples, our RPA-LFD showed the same detection ability as quantitative PCR assay. Therefore, the current RPA-LFD would be a valuable tool to detect V. vulnificus in oysters, especially in field conditions.

Sewage Promotes Vibrio vulnificus Growth and Alters Gene Transcription in Vibrio vulnificus CMCP6.

Vibrio vulnificus is a naturally occurring, potentially lethal pathogen found in coastal waters, fish, and shellfish. Sewage spills in coastal waters occur when infrastructure fails due to severe storms or age, and may affect bacterial populations by altering nutrient levels. This study investigated effects of sewage on clonal and natural V. vulnificus populations in microcosms. Addition of 1% sewage to estuarine water caused the density of a pure culture of V. vulnificus CMCP6 and a natural V. vulnificus population to increase significantly, by two to three orders of magnitude, whether measured by quantitative PCR (qPCR) or culture and in batch and continuous cultures. Changes in the transcription of six virulence- and survival-associated genes in response to sewage were assessed using continuous culture. Exposure to sewage affected transcription of genes that may be associated with virulence, i.e., it modulated the oxidative stress response by altering superoxide dismutase transcription, significantly increasing sodB transcription while repressing sodA. Sewage also repressed transcription of nptA, which encodes a sodium-phosphate cotransporter. Sewage had no effect on sodC transcription or the putative virulence-associated genes hupA or wza. The effects of environmentally relevant levels of sewage on V. vulnificus populations and gene transcription suggest that sewage spills that impact warm coastal waters could lead to an increased risk of V. vulnificus infections. IMPORTANCE Vibrio vulnificus infections have profound impacts such as limb amputation and death for individuals with predisposing conditions. The warming climate is contributing to rising V. vulnificus prevalence in waters that were previously too cold to support high levels of the pathogen. Climate change is also expected to increase precipitation in many regions, which puts more pressure on wastewater infrastructure and will result in more frequent sewage spills. The finding that 1% wastewater in estuarine water leads to 100 to over 1,000-fold greater V. vulnificus concentrations suggests that human exposure to oysters and estuarine water could have greater health impacts in the future. Further, wastewater had a significant effect on gene transcription and has the potential to affect virulence during the initial environment-to-host transition.

Effects of curcumin-mediated photodynamic treatment on lipid degradation of oysters during refrigerated storage.

BACKGROUND: Oyster's lipid degradation leads to a decrease in edible and nutritional value. Curcumin-mediated photodynamic treatment (PDT) is an innovative non-thermal technology, although evaluation of the oyster's lipid degradation has been scarce. In the present study, we investigated peroxide value, thiobarbituric acid reactive substance, triacylglycerol and free fatty acids to evaluate the effect of curcumin-mediated PDT on lipid degradation of oysters during refrigerated storage. RESULTS: The results showed that curcumin-mediated PDT could delay oyster's lipid degradation. Next, the activities of enzymes were detected to determine the mechanisms behind the effects of curcumin-mediated PDT. It was revealed that the activities of lipase, phospholipase A2 (PLA2 ), phospholipase C (PLC), phospholipase D (PLD) and lipoxygenase (LOX) were significantly inhibited after curcumin-mediated PDT (P < 0.05). Furthermore, 16 s rRNA analysis established that the relative abundances of Pseudoalteromonas and Psychrilyobacter were reduced by 51.58% and 43.82%, respectively, after curcumin-mediated PDT. CONCLUSION: Curcumin-mediated PDT could delay oyster's lipid degradation by inhibiting the activities of lipase, PLA2 , PLC, PLD and LOX, as well as by changing the oyster's microbial composition, reducing the relative abundance of Pseudoalteromonas and Psychrilyobacter. © 2021 Society of Chemical Industry.

Microbiological Quality and Heavy Metal Concentrations in Slipper Oyster (Crassostrea iredalei) Cultured in Major Growing Areas in Capiz Province, Western Visayas, Philippines: Compliance with International Shellfish Safety and Sanitation Standards.

ABSTRACT: The increasing demand for slipper oyster (Crassostrea iredalei) has propelled farmers to expand oyster cultivation areas in the Philippines, chiefly for local consumption and feasibly for export overseas. As filter feeders, oysters can accumulate pathogens from the surrounding waters, and these pathogens can cause foodborne diseases in consumers. Therefore, oyster farming areas must be monitored for microbiological quality and heavy metal concentrations. In the present study, the microbiological quality of oysters and their growing waters in the major oyster farming areas of the Cogon and Palina Rivers and Cabugao Bay (in Roxas City and the Municipality of Ivisan, respectively, Capiz Province, Western Visayas, Philippines) were examined monthly during the wet (May to October) and dry (November to April) seasons over 12 months. Regardless of the sampling period, high levels of fecal coliforms in the water and Escherichia coli in oysters were found, clearly illustrating that these oyster growing areas would meet only the class B standard under the European Union classification system and would be considered "prohibited" for growing oysters under the U.S. classification system. Although Salmonella was occasionally detected in oysters, Vibrio cholerae was not detected and Vibrio parahaemolyticus was within acceptable limits. The heavy metal concentrations in oyster meat were also determined during the wet (July) and dry (March) seasons. Zinc and copper were the most abundant metals detected, and concentrations of lead, cadmium, mercury, and chromium were below the regulatory limits set by the European Union and the U.S. Food and Drug Administration. These oyster culture areas should be rehabilitated immediately to improve the microbiological quality of the oysters. Oysters harvested from these sites must be depurated or relayed to ensure quality and safety.

Microbiomes of an oyster are shaped by metabolism and environment.

Microbiomes can both influence and be influenced by metabolism, but this relationship remains unexplored for invertebrates. We examined the relationship between microbiome and metabolism in response to climate change using oysters as a model marine invertebrate. Oysters form economies and ecosystems across the globe, yet are vulnerable to climate change. Nine genetic lineages of the oyster Saccostrea glomerata were exposed to ambient and elevated temperature and PCO2 treatments. The metabolic rate (MR) and metabolic by-products of extracellular pH and CO2 were measured. The oyster-associated bacterial community in haemolymph was characterised using 16 s rRNA gene sequencing. We found a significant negative relationship between MR and bacterial richness. Bacterial community composition was also significantly influenced by MR, extracellular CO2 and extracellular pH. The effects of extracellular CO2 depended on genotype, and the effects of extracellular pH depended on CO2 and temperature treatments. Changes in MR aligned with a shift in the relative abundance of 152 Amplicon Sequencing Variants (ASVs), with 113 negatively correlated with MR. Some spirochaete ASVs showed positive relationships with MR. We have identified a clear relationship between host metabolism and the microbiome in oysters. Altering this relationship will likely have consequences for the 12 billion USD oyster economy.

Ecological diversification reveals routes of pathogen emergence in endemic Vibrio vulnificus populations.

Pathogen emergence is a complex phenomenon that, despite its public health relevance, remains poorly understood. Vibrio vulnificus, an emergent human pathogen, can cause a deadly septicaemia with over 50% mortality rate. To date, the ecological drivers that lead to the emergence of clinical strains and the unique genetic traits that allow these clones to colonize the human host remain mostly unknown. We recently surveyed a large estuary in eastern Florida, where outbreaks of the disease frequently occur, and found endemic populations of the bacterium. We established two sampling sites and observed strong correlations between location and pathogenic potential. One site is significantly enriched with strains that belong to one phylogenomic cluster (C1) in which the majority of clinical strains belong. Interestingly, strains isolated from this site exhibit phenotypic traits associated with clinical outcomes, whereas strains from the second site belong to a cluster that rarely causes disease in humans (C2). Analyses of C1 genomes indicate unique genetic markers in the form of clinical-associated alleles with a potential role in virulence. Finally, metagenomic and physicochemical analyses of the sampling sites indicate that this marked cluster distribution and genetic traits are strongly associated with distinct biotic and abiotic factors (e.g., salinity, nutrients, or biodiversity), revealing how ecosystems generate selective pressures that facilitate the emergence of specific strains with pathogenic potential in a population. This knowledge can be applied to assess the risk of pathogen emergence from environmental sources and integrated toward the development of novel strategies for the prevention of future outbreaks.

Prevalence, typing and antimicrobial resistance of Salmonella isolates from commercial shellfish in the North coast of Morocco.

Salmonellosis is one of the most common foodborne illnesses in the world. The irrational use of antibiotics in medicine and in animal nutrition has greatly favored the emergence and spread of resistant strains of non-typhoid Salmonella. This study aims the determination of the prevalence of Salmonella in bivalve mollusks in Northern Morocco, as well as the molecular typing and antibiotic susceptibility testing of the strains isolated from positive samples. In total, 150 samples from shellfish composed of mussels (Mytilus galloprovincialis), clams (Callista chione and Ruditapes descussatus) and oysters (Magallana gigas). Isolated Salmonella were characterized by Molecular techniques PCR, MLST and MLVA, phylogenetically grouped by MLSA, and susceptibilities were determined for 30 antimicrobial drugs using microdilution method by the BD Phoenix Automated Microbiology System. Prevalence of Salmonella enterica subsp. enterica was 12.67%, grouped in four serovars identified as Chester, Hadar, Typhimurium and Kentucky. Five different MLST STs (sequence types) were detected, ST1954 being the most common, which was mostly found in Chester isolates. Forty-two percent of the isolates showed resistance to more than one antibiotic, especially trimethoprim, sulfa drugs, quinolones and ß-lactam. There was a marked change in the serovars and antimicrobial resistance profiles of the Salmonella isolates in this study compared to those in previous studies.