Efficacy of Vibrio parahaemolyticus depuration in oysters (Crassostrea gigas).

This study investigated the influences of seawater to oyster ratio on depuration for decontaminating V. parahaemolyticus in raw oysters with a goal of identifying the proper ratio of oyster to seawater capable of improving the efficacy of the depuration process. The water to oyster ratios tested in this study ranged from 1.0 to 2.5 L of artificial seawater (ASW) per oyster (40 oysters in 40, 60, 80 and 100 L ASW). The depuration efficacy for purging V. parahaemolyticus from oysters was highest when we applied a 2:1, followed by 1.5:1, 2.5:1, and 1:1 L of ASW/oyster. Further studies of depuration with 2:1 L of ASW/oyster found that the concentration of V. parahaemolyticus in oysters decreased in a nonlinear manner. The depuration curve was fitted to a one phase decay model with a coefficient of determination (R2) of 0.933. The time for a 3 log reduction was 1.75 days with a 95% confidence interval from 1.65 to 1.85 days, which meets the FDA's requirement of larger than a 3.0 log (MPN/g) reduction as a post-harvest process for V. parahaemolyticus control. After 4 days levels in all trials were <100 MPN/g meeting performance standards established by Japan and Canada. Furthermore, the time for a 3.52 log reduction was 3.17 days with a 95% confidence interval from 2.92 to 3.54 days but it took 5 days to reduce levels to <30 MPN/g, which satisfies FDA's requirement as a post-harvest control process (>3.52 log MPN/g reduction) for the purpose of making safety added labeling claims for V. parahaemolyticus.

Characterizing the Adherence Profiles of Virulent Vibrio parahaemolyticus Isolates.

The human pathogen Vibrio parahaemolyticus is a leading cause of seafood-borne illness in the USA, and infections with V. parahaemolyticus typically result from eating raw or undercooked oysters. V. parahaemolyticus has been shown to be highly resistant to oyster depuration, suggesting that the bacterium possesses specific mechanisms or factors for colonizing oysters and persisting during depuration. In this study, we characterized eight different V. parahaemolyticus strains for differences in resistance to oyster depuration, biofilm formation, and motility. While each strain exhibited distinct phenotypes in the various assays, we determined that biofilm formation on abiotic surfaces, such as glass or plastic, does not directly correlate with bacterial retention in oysters during depuration. However, we did observe that the motility phenotype of a strain appeared to be a better indicator for persistence in the oyster. Further studies examining the molecular mechanisms underlying the observed colonization differences by these and other V. parahaemolyticus strains may provide beneficial insights into what critical factors are required for proficient colonization of the Pacific oyster.

Persistence of Vibrio parahaemolyticus in the Pacific oyster, Crassostrea gigas, is a multifactorial process involving pili and flagella but not type III secretion systems or phase variation.

Vibrio parahaemolyticus can resist oyster depuration, suggesting that it possesses specific factors for persistence. We show that type I pili, type IV pili, and both flagellar systems contribute to V. parahaemolyticus persistence in Pacific oysters whereas type III secretion systems and phase variation do not.

Reductions of Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas) by depuration at various temperatures.

Consumption of raw oysters has been linked to several outbreaks of Vibrio parahaemolyticus infection in the United States. This study investigated effects of ice storage and UV-sterilized seawater depuration at various temperatures on reducing V. parahaemolyticus in oysters. Raw Pacific oysters (Crassostrea gigas) were inoculated with a mixed culture of five clinical strains of V. parahaemolyticus (10290, 10292, 10293, BE 98-2029 and 027-1c1) at levels of 104⁻6 MPN/g. Inoculated oysters were either stored in ice or depurated in recirculating artificial seawater at 2, 3, 7, 10, 12.5, and 15 °C for 4-6 days. Holding oysters in ice or depuration of oysters in recirculating seawater at 2 or 3 °C for 4 days did not result in significant reductions (P > 0.05) of V. parahaemolyticus in the oysters. However, depuration at temperatures between 7 and 15 °C reduced V. parahaemolyticus populations in oysters by >3.0 log MPN/g after 5 days with no loss of oysters. Depuration at refrigerated temperatures (7-15 °C) can be applied as a post-harvest treatment for reducing V. parahaemolyticus in Pacific oysters.

Refrigerated seawater depuration for reducing Vibrio parahaemolyticus contamination in pacific oyster (Crassostrea gigas).

The efficacy of refrigerated-seawater depuration for reducing Vibrio parahaemolyticus levels in Pacific oyster (Crassostrea gigas) was investigated. Raw Pacific oysters were inoculated with a mixed culture of five clinical strains of V. parahaemolyticus (10(5) to 10(6) most probable number [MPN] per g) and depurated with refrigerated seawater (5 degrees C) in a laboratory-scale recirculation system equipped with a 15-W gamma UV sterilizer. Depuration with refrigerated seawater for 96 h reduced V. parahaemolyticus populations by >3.0 log MPN/g in oysters harvested in the winter. However, 144 h of depuration at 5 degrees C was required to achieve a 3-log reduction in oysters harvested in the summer. Depuration with refrigerated seawater at 5 degrees C for up to 144 h caused no significant fatality in the Pacific oyster and could be applied as a postharvest treatment to reduce V. parahaemolyticus contamination in Pacific oysters. Further studies are needed to validate the efficacy of the depuration process for reducing naturally accumulated V. parahaemolyticus in oysters.

Deep Neck Infection in Systemic Lupus Erythematosus Patients: Real-World Evidence.

Systemic lupus erythematosus (SLE) might increase deep neck infection (DNI) risk, but evidence supporting this hypothesis is limited. In this retrospective follow-up study, the SLE-DNI association was investigated using data from the Registry for Catastrophic Illness Patients, which is a subset of the Taiwan National Health Insurance Research Database. All patients newly diagnosed as having SLE in 1997-2011 were identified, and every SLE patient was individually matched to four patients without SLE according to sex, age, and socioeconomic status. The study outcome was DNI occurrence. DNI treatment modalities and prognoses in SLE and non-SLE patients, along with the association of steroid dose with DNI risk, were also studied. In total, 17,426 SLE and 69,704 non-SLE patients were enrolled. Cumulative DNI incidence was significantly higher in the SLE cohort than in the non-SLE cohort (p < 0.001). The Cox regression model demonstrated that SLE significantly increased DNI risk (hazard ratio: 4.70; 95% confidence interval: 3.50-6.32, p < 0.001). Moreover, in the sensitivity and subgroup analyses, the effect of SLE on DNI was stable. Relatively few SLE-DNI patients received surgical interventions (15.6% vs. 28.6%, p = 0.033). The between-group differences in tracheostomy use and hospitalisation duration were nonsignificant. In SLE patients, high steroid doses significantly increased DNI incidence (≥3 vs. <3 mg/day = 2.21% vs. 0.52%, p < 0.001). This is the first study demonstrating that SLE increases DNI risk by approximately five times and that high steroid dose increases DNI incidence in SLE patients.

Effects of flash freezing, followed by frozen storage, on reducing Vibrio parahaemolyticus in Pacific raw oysters (Crassostrea gigas).

This study investigated the effects of flash freezing, followed by frozen storage, on reducing Vibrio parahaemolyticus in Pacific raw oysters. Raw Pacific oysters were inoculated with a five-strain cocktail of V. parahaemolyticus at a total level of approximately 3.5 x 10(5) most probable number (MPN) per gram. Inoculated oysters were subjected to an ultralow flash-freezing process (-95.5 degrees C for 12 min) and stored at -10, -20, and -30 degrees C for 6 months. Populations of V. parahaemolyticus in the oysters declined slightly by 0.22 log MPN/g after the freezing process. Subsequent storage of frozen oysters at - 10, -20, and -30 degrees C resulted in considerable reductions of V. parahaemolyticus in the oysters. Storing oysters at -10 degrees C was more effective in inactivating V. parahaemolyticus than was storage at -20 or -30 degrees C. Populations of V. parahaemolyticus in the oysters declined by 2.45, 1.71, and 1.45 log MPN/g after 1 month of storage at -10, -20, and -30 degrees C, respectively, and continued to decline during the storage. The levels of V. parahaemolyticus in oysters were reduced by 4.55, 4.13, and 2.53 log MPN/g after 6 months of storage at -10, -20, and -30 degrees C, respectively. Three process validations, each separated by 1 week and conducted according to the National Shellfish Sanitation Program's postharvest processing validation-verification interim guidance for Vibrio vulnificus and Vibrio parahaemolyticus, confirmed that a process of flash freezing, followed by storage at -21 +/- 2 degrees C for 5 months, was capable of achieving greater than 3.52-log (MPN/g) reductions of V. parahaemolyticus in half-shell Pacific oysters.

Effects of sources of carbon and nitrogen on production of α-glucosidase inhibitor by a newly isolated strain of Bacillus subtilis B2.

This study examined production of α-glucosidase inhibitors by Bacillus subtilis B2 in Luria-Bertani (LB) fermentation with okara, soy powder, starch or pectin as additional source of carbon and nitrogen. All the fermentation broths of B. subtilis B2 exhibited gradual increase in α-glucosidase inhibitory activity during the fermentation process with or without supplemented source of carbon or nitrogen. Addition of okara into the LB medium greatly enhanced the strength (nearly twice as much of that without okara supplement) of α-glucosidase inhibitory activity of fermentation broth. The α-glucosidase inhibitory activity of B. subtilis B2 fermentation broth was positively correlated (p<0.05) with the bacterial populations grown in LB medium containing okara. Glucose and sucrose were not detected in LB medium during the entire fermentation process and were both reduced drastically in media containing okara, soy powder, starch or pectin after 6days of fermentation. The fermented LB medium containing okara by B. subtilis B2 possessed very strong α-glucosidase inhibitory activity and contained little glucose and sucrose, suggesting that fermentation of B. subtilis B2 in LB added with okara might be considered as a strategy for preparing functional foods for diabetic patients.

Metal-Organic Framework Nanocomposite Thin Films with Interfacial Bindings and Self-Standing Robustness for High Water Flux and Enhanced Ion Selectivity.

Metal-organic framework (MOF)-based materials are promising candidates for a range of separation applications. However, the fabrication of self-standing MOF-based thin films remains challenging. Herein, a facile solution casting strategy is developed for fabricating UiO-66 nanocomposite thin films (UiO66TFs) with thicknesses down to ∼400 nm. Nanosizing UiO-66 and incorporating sulfonated polysulfone additives render high dispersity and interfacial bindings between MOFs and polymer matrices, so UiO66TFs are more mechanically robust and thermally stable than their pure-polymer counterparts. Enhanced microporosity with sub-nanometer pore sizes of the self-standing membranes enables the direct translation of UiO-66-based sorption and ion-sieving properties, thus increasing water flux and separation performance (Na2SO4 rejection of 94-96%) under hydraulic pressure-driven processes and eliminating internal concentration polarization in osmotic pressure-driven processes. Enhanced separation performances are achieved with water/Na2SO4 permselectivity of 13.5 L g-1 and high osmotic water permeability up to 1.41 L m-2 h-1 bar-1, providing 3-fold higher water/Na2SO4 permselectivity and 56-fold-higher water flux than polymer membranes for forward osmosis.

Structural characterization and osteogenic bioactivity of a sulfated polysaccharide from pacific abalone (Haliotis discus hannai Ino).

Bone morphogenic protein-2 (BMP-2) is known to promote osteogenesis. To find novel adjuvants to enhance the activity of BMP-2, the present study investigated the structure BMP-2-induced osteogenic activity of a water-soluble polysaccharide from the gonad of pacific abalone (Haliotis discus hannai Ino) named AGSP. Through analysis of aldobiouronic acids released from AGSP, monosaccharide composition comparison of AGSP and its reduced product, and methylation analysis and NMR analysis of AGSP and its desulfated derivative, the main structure residue of AGSP was determined as →3)-GlcA(1→3)-Gal(1→ with sulfated branches comprised of prevelant Gal and minor Glc, and →4)-ß-GlcA(1→2)-α-Man(1→ residue was also found. AGSP possessed a sulfate content of 12.4% with a relative molecular weight of 6.6kDa. AGSP strengthened alkaline phosphatase activity induced by BMP-2 in a dose dependent manner at 10-200µg/mL with 425% enhancement being observed at 200µg/mL, indicating AGSP could be an adjuvant candidate to enhance osteogenic activity of BMP-2.

Characteristics of virulent vibrio parahaemolyticus isolated from Oregon and Washington.

Thirty-four virulent strains of Vibrio parahaemolyticus containing tdh and/or trh genes isolated from Oregon and Washington coastal water were analyzed for O-group antigens and urease activity, and by pulsed-field gel electrophoresis. Six O serotypes (O1, O3, O4, O5, O10, and O11) were identified among the isolates, with the O5 group (19 isolates) being the most prevalent, followed by the 01 group (9 isolates). Nearly all (33 of 34) isolates were capable of producing urease, which reaffirmed the correlation between urease production and virulence factors of V. parahaemolyticus strains isolated from the Pacific Northwest. Pulsed-field gel electrophoresis analysis with NotI and SfiI digestions of the 34 V. parahaemolyticus isolates plus five clinical strains revealed 22 patterns (NlS1 to N20S22), with NIS1 (25.6%) being the most common, followed by N2S2 (10.3%). Nine Oregon isolates were grouped with a 1997 Oregon outbreak strain (027-1C1) with the same serotype (O5), virulence factors (tdh+ and trh+), and genotype (N S 1). Three Washington isolates were found to share the same serotype (O1), virulence factors (tdh' and trh'), and genotype (N2S2) with a 1997 Washington outbreak strain (10293). The repetitive isolation of virulent strains of V. parahaemolyticus identical to clinical strains involved in previous outbreaks indicates potential hazards associated with oyster consumption. These data may be useful in risk assessment of V. parahaemolyticus infections associated with raw oyster consumption in Oregon and Washington.

Characterization of clinical Vibrio parahaemolyticus strains in Zhoushan, China, from 2013 to 2014.

Vibrio parahaemolyticus is recognized as major cause of foodborne illness of global public health concern. This study collected 107 strains of V. parahaemolyticus during active surveillance of diarrheal diseases in hospitals in Zhoushan during 2013 to 2014 and investigated their serotypes, virulence genes (tdh, trh, and orf8), antimicrobial resistance, and genotypes. The dominant serotypes of the 107 clinical strains were O3:K6, O4:K8, and O4:KUT with 87.9% and 3.7% of the strains carrying the virulence genes tdh and trh, respectively. Molecular typing by pulsed-field gel electrophoresis indicated divergence among the clinical strains. Most isolates were sensitive to the common antimicrobial agents used against the Vibrio species except ampicillin. We conclude that continuous surveillance of V. parahaemolyticus in diarrhea patients is a public health priority and is useful for conducting risk assessment of foodborne illnesses caused by V. parahaemolyticus.

Degradation of Histamine by Lactobacillus plantarum Isolated from Miso Products.

Histamine is a toxic chemical and is the causative agent of food poisoning. This foodborne toxin may be degraded by the oxidative deamination activity of certain microorganisms. In this study, we isolated four histamine-degrading Lactobacillus plantarum bacteria from miso products. Among them, L. plantarum D-103 exhibited 100% degradation of histamine in de Man Rogosa Sharpe (MRS) broth containing 50 ppm of histamine after 24 h of incubation at 30°C. The optimal growth, histamine oxidase, and histamine-degrading activity of L. plantarum D-103 were observed in histamine MRS broth at pH 7.0, 3% NaCl, and 30°C. It also exhibited tolerance to broad ranges of pH (4 to 10) and salt concentrations (0 to 12%) in histamine MRS broth. Therefore, the histamine-degrading L. plantarum D-103 might be used as an additive culture to prevent histamine accumulation in miso products during fermentation.

Efficiency of electrolyzed oxidizing water on reducing Listeria monocytogenes contamination on seafood processing gloves.

Food processing gloves are typically used to prevent cross-contamination during food preparation. However, gloves can be contaminated with microorganisms and become a source of contamination. This study investigated the survival of Listeria monocytogenes on gloves and determined the efficacy of electrolyzed oxidizing (EO) water for reducing L. monocytogenes contamination on seafood processing gloves. Three types of reusable gloves (natural rubber latex, natural latex, and nitrile) and two types of disposable gloves (latex and nitrile) were cut into small pieces (4 x 4 cm(2)) and inoculated with 5-strain L. monocytogenes cocktail (5.1 x 10(7) CFU/cm(2)) with and without shrimp meat residue attached to surfaces. L. monocytogenes did not survive well on clean reusable gloves and its populations decreased rapidly to non-detectable levels within 30 min at room temperature. However, high levels of Listeria cells were recovered from clean disposable gloves after 30 min of inoculation. Presence of shrimp meat residue on gloves enhanced the survival of L. monocytogenes. Cells of L. monocytogenes were detected on both reusable and disposal gloves even after 2 h at room temperature. Soaking inoculated gloves in EO water at room temperature for 5 min completely eliminated L. monocytogenes on clean gloves (>4.46 log CFU/cm(2) reductions) and significantly (p<0.05) reduced the contamination on soil-containing gloves when compared with tap water treatment. EO water could be used as a sanitizer to reduce L. monocytogenes contamination on gloves and reduce the possibility of transferring L. monocytogenes from gloves to RTE seafoods.

Effects of electrolyzed oxidizing water on reducing Listeria monocytogenes contamination on seafood processing surfaces.

The effects of electrolyzed oxidizing (EO) water on reducing Listeria monocytogenes contamination on seafood processing surfaces were studied. Chips (5 x 5 cm(2)) of stainless steel sheet (SS), ceramic tile (CT), and floor tile (FT) with and without crabmeat residue on the surface were inoculated with L. monocytogenes and soaked in tap or EO water for 5 min. Viable cells of L. monocytogenes were detected on all chip surfaces with or without crabmeat residue after being held at room temperature for 1 h. Soaking contaminated chips in tap water resulted in small-degree reductions of the organism (0.40-0.66 log cfu/chip on clean surfaces and 0.78-1.33 log cfu/chip on dirty surfaces). Treatments of EO water significantly (p<0.05) reduced L. monocytogenes on clean surfaces (3.73 log on SS, 4.24 log on CT, and 5.12 log on FT). Presence of crabmeat residue on chip surfaces reduced the effectiveness of EO water on inactivating Listeria cells. However, treatments of EO water also resulted in significant reductions of L. monocytogenes on dirty surfaces (2.33 log on SS and CT and 1.52 log on FT) when compared with tap water treatments. The antimicrobial activity of EO water was positively correlated with its chlorine content. High oxidation-reduction potential (ORP) of EO water also contributed significantly to its antimicrobial activity against L. monocytogenes. EO water was more effective than chlorine water on inactivating L. monocytogenes on surfaces and could be used as a chlorine alternative for sanitation purpose. Application of EO water following a thorough cleaning process could greatly reduce L. monocytogenes contamination in seafood processing environments.

Effects of electrolyzed oxidizing water treatment on reducing Vibrio parahaemolyticus and Vibrio vulnificus in raw oysters.

Contamination of Vibrio parahaemolyticus and Vibrio vulnificus in oysters is a food safety concern. This study investigated effects of electrolyzed oxidizing (EO) water treatment on reducing V. parahaemolyticus and V. vulnificus in laboratory-contaminated oysters. EO water exhibited strong antibacterial activity against V. parahaemolyticus and V. vulnificus in pure cultures. Populations of V. parahaemolyticus (8.74 x 10(7) CFU/ml) and V. vulnificus (8.69 x 10(7) CFU/ml) decreased quickly in EO water containing 0.5% NaCl to nondetectable levels (> 6.6 log reductions) within 15 s. Freshly harvested Pacific oysters were inoculated with a five-strain cocktail of V. parahaemolyticus or V. vulnificus at levels of 10(4) and 10(6) most probable number (MPN)/g and treated with EO water (chlorine, 30 ppm; pH 2.82; oxidation-reduction potential, 1131 mV) containing 1% NaCl at room temperature. Reductions of V. parahaemolyticus and V. vulnificus in oysters were determined at 0 (before treatment), 2, 4, 6, and 8 h of treatment. Holding oysters inoculated with V. parahaemolyticus or V. vulnificus in the EO water containing 1% NaCl for 4 to 6 h resulted in significant (P < 0.05) reductions of V. parahaemolyticus and V. vulnificus by 1.13 and 1.05 log MPN/g, respectively. Extended exposure (> 12 h) of oysters in EO water containing high levels of chlorine (> 30 ppm) was found to be detrimental to oysters. EO water could be used as a postharvest treatment to reduce Vibrio contamination in oysters. However, treatment should be limited to 4 to 6 h to avoid death of oysters. Further studies are needed to determine effects of EO water treatment on sensory characteristics of oysters.

Bactericidal effects of wine on Vibrio parahaemolyticus in oysters.

The bactericidal effects of wines on Vibrio parahaemolyticus in oysters were studied to evaluate potential inactivation of V. parahaemolyticus in contaminated oysters by wine consumption. Shucked whole oyster and oyster meat homogenate were inoculated with V. parahaemolyticus and mixed with red or white wine. Survivals of V. parahaemolyticus in inoculated oysters were determined at 7 and 25 degrees C. Populations of V. parahaemolyticus in inoculated whole oysters (5.52 log most probable number [MPN] per g) decreased slightly to 4.90 log MPN/g (a 0.62-log reduction) after 24 h at 7 degrees C but increased to 7.37 log MPN/g over the same period at 25 degrees C. However, the populations in wine-treated whole oysters decreased by >1.7 and >1.9 log MPN/g after 24 h at 7 and 25 degrees C, respectively. Both red and white wines were more effective in inactivating V. parahaemolyticus in oyster meat homogenate than in whole oyster. Populations of V. parahaemolyticus in oyster meat homogenate (7.8 x 10(3) MPN/g) decreased rapidly to nondetectable levels (< 3 MPN/g) after 30 min of mixing with wine at 25 degrees C (a 3.89-log MPN/g reduction). These results suggest that chewing oysters before swallowing when eating raw oysters may result in greater inactivation of V. parahaemolyticus if wine is consumed. More studies are needed to determine the bactericidal effects of wine on V. parahaemolyticus in the complicated stomach environment.

Effect of spatial constraints on Hardy-Weinberg equilibrium.

Panmixia is a key issue in maintaining genetic diversity, which facilitates evolutionary potential during environmental changes. Additionally, conservation biologists suggest the importance of avoiding small or subdivided populations, which are prone to losing genetic diversity. In this paper, computer simulations were performed to the genetic drift of neutral alleles in random mating populations with or without spatial constraints by randomly choosing a mate among the closest neighbours. The results demonstrated that the number of generations required for the neutral allele to become homozygous (Th) varied proportionally to the population size and also strongly correlated with spatial constraints. The average Th for populations of the same size with spatial constraints was approximately one-and-a-half times longer than without constraints. With spatial constraints, homozygous population clusters formed, which reduced local diversity but preserved global diversity. Therefore, panmixia might be harmful in preserving the genetic diversity of an entire population. The results also suggested that the gene flow or gene exchange among the subdivided populations must be carefully processed to restrict diseases transmission or death during transportation and to monitor the genetic diversity. The application of this concept to similar systems, such as information transfer among peers, is also discussed.

Behavior of Salmonella and Listeria monocytogenes in Raw Yellowfin Tuna during Cold Storage.

Behavior of Salmonella and Listeria monocytogenes in raw yellowfin tuna during refrigeration and frozen storage were studied. Growth of Salmonella was inhibited in tuna during refrigerated storage, while L. monocytogenes was able to multiply significantly during refrigerated storage. Populations of Salmonella in tuna were reduced by 1 to 2 log after 12 days of storage at 5-7 °C, regardless levels of contamination. However, populations of L. monocytogenes Scott A, M0507, and SFL0404 in inoculated tuna (104-105 CFU/g) increased by 3.31, 3.56, and 3.98 log CFU/g, respectively, after 12 days of storage at 5-7 °C. Similar increases of L. monocytogenes cells were observed in tuna meat with a lower inoculation level (10²-10³ CFU/g). Populations of Salmonella and L. monocytogenes declined gradually in tuna samples over 84 days (12 weeks) of frozen storage at -18 °C with Salmonella Newport 6962 being decreased to undetectable level (<10 CFU/g) from an initial level of 10³ log CFU/g after 42 days of frozen storage. These results demonstrate that tuna meat intended for raw consumption must be handled properly from farm to table to reduce the risks of foodborne illness caused by Salmonella and L. monocytogenes.