Effects of a commercial heat-shock process on Vibrio vulnificus in the American oyster, Crassostrea virginica, harvested from the Gulf Coast

Oysters (Crassostrea virginica) harvested from the Gulf Coast, containing 10(2) to 10(4) most probable number (MPN) per gram of Vibrio vulnificus, were subjected to a commercial heat-shock process. After 1 to 4 min at internal oyster meat temperatures exceeding 50 degrees C, shellstock oysters were shucked, chilled, washed, and packed. V. vulnificus and total bacterial levels in Gulf Coast oysters were significantly reduced from 1 to 4 logs in the finished product. Similar reductions were not observed in shellstock oysters that were subject to conventional processing. Under the National Shellfish Sanitation Program, heat shocking is an acceptable process to use to assist in the shucking of shellstock. This research revealed that the heat-shock process may also serve to significantly reduce V. vulnificus in summer Gulf Coast oysters.     

Vibrio vulnificus load reduction in oysters after combined exposure to Vibrio vulnificus--specific bacteriophage and to an oyster extract component

Oysters infected with Vibrio vulnificus can present a serious health risk to diabetic, immunocompromised, and iron-deficient individuals. Numerous studies have been conducted with the goal of eliminating this organism from raw oysters. We utilized two natural oyster-associated components: pooled Vibrio vulnificus-specific bacteriophage and an extract of the eastern oyster (Crassostrea virginica) that contains an antimicrobial component we named anti-Vibrio vulnificus factor, which is bactericidal for V. vulnificus. Although each component alone can reduce V. vulnificus numbers independently, the simultaneous use of both components in an in vitro system successfully more effectively reduced V. vulnificus bacterial loads.     

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.     

Temperature Effects on the Depuration of Vibrio parahaemolyticus and Vibrio vulnificus from the American Oyster (Crassostrea virginica)

ABSTRACT:  This study investigated temperature effects on depuration for reducing Vibrio parahaemolyticus and Vibrio vulnificus in American oyster ( Crassostrea virginica ). Raw oysters were inoculated with 5-strain cocktail of V. parahaemolyticus or V. vulnificus to levels of 10⁴ to 10⁵ MPN (most probable number)/g and depurated in artificial seawater (ASW) at 22, 15, 10, and 5 °C. Depuration of oysters at 22 °C had limited effects on reducing V. parahaemolyticus or V. vulnificus in the oysters. Populations of V. parahaemolyticus and V. vulnificus were reduced by 1.2 and 2.0 log MPN/g, respectively, after 48 h of depuration at 22 °C. Decreasing water temperature to 15 °C increased the efficacy of depuration in reducing V. parahaemolyticus and V. vulnificus in oysters. Reductions of V. parahaemolyticus and V. vulnificus in oysters increased to 2.1 and 2.9 log MPN/g, respectively, after 48 h of depuration at 15 °C. However, depurations at 10 and 5 °C were less effective than at 15 °C in reducing the Vibrio spp. in oysters. Extended depuration at 15 °C for 96 h increased reductions of V. parahaemolyticus and V. vulnificus in oysters to 2.6 and 3.3 log MPN/g, respectively.     

Low temperature pasteurization to reduce the risk of vibrio infections from raw shell-stock oysters

Vibrio vulnificus and V. parahaemolyticus are natural inhabitants of estuarine environments and may be transmitted to humans by ingestion of raw oysters. This study focused on the use of low temperature pasteurization, to reduce these Vibrio spp. to nondetectable levels, thus reducing the risk of infection associated with raw oyster consumption. Artificially inoculated V. vulnificus and V. parahaemolyticus and naturally-contaminated V. vulnificus in live oysters were pasteurized at 50%deg;C for up to 15min. Samples of processed and unprocessed oysters were enumerated for V. vulnificus, V. parahaemolyticus, and aerobic spoilage bacteria for 0-14 days. Low temperature pasteurization was effective in reducing these pathogens from > 100000 to non-detectable levels in less than 10min of processing. Spoilage bacteria were reduced by 2-3 logs, thus increasing the shelf-life for up to 7 days beyond live unprocessed oysters. Vibrio vulnificus in control oysters was reduced by 102 during ice storage alone. Following pasteurization and during a temperature storage abuse study (24h at 22°C), V. vulnificus was not recovered. During this storage period spoilage bacteria exceeded 1 million/g oyster meat.     

NOVEL NONTHERMAL METHODS TO REDUCE VIBRIO VULNIFICUS IN RAW OYSTERS

Vibrio vulnificus is a foodborne pathogen associated with consumption of raw oyster. No scientific data is available on postharvest treatments of oyster by ultrasound, ozone, and organic acids. This study was designed to investigate the effects of these treatments on inactivation of V. vulnificus naturally present in the in-shell or half-shelled oysters. In in-shell oysters, these treatments were not effective in reducing the number of this pathogen. Half-shelled oysters treated with ultrasound, and ozone in 2% saline for 30 min had 1 and 1.5 log less V. vulnificus, respectively (p<0.05). Treatment of half-shelled oysters by 50 and 100% lemon juice, 5% citric acid, 10% citric acid, or vinegar for 30 min resulted in a significant reduction (2–4 log) in the numbers of V. vulnificus (p<0.05). Although these methods significantly reduced the population of V. vulnificus in raw oysters, they were not able to reduce the numbers of this pathogen to acceptable level (<3 MPN/g).     

Ice immersion as a postharvest treatment of oysters for the reduction of Vibrio vulnificus

Vibrio vulnificus produces serious illnesses that are commonly associated with shellfish consumption, particularly raw oysters. Ingestion can result in fatal septicemia in susceptible individuals with hepatitis, cirrhosis, immune dysfunction, diabetes, or hemochromatosis (metabolic iron overload). Therefore, postharvest treatments to reduce vibrio levels in oysters have been recommended. In this study, rapid chilling by immersion of unwashed whole oysters in ice for 3 h was assessed as a postharvest treatment for reduction of V. vulnificus. Treated oysters were subsequently refrigerated at 45 degrees F (7.2 degrees C), whereas control oysters were not iced but were maintained at 45 degrees F throughout the study. Homogenized meats were monitored for total heterotrophic aerobic bacteria, V. vulnificus, and fecal coliform content before and after treatment over a 2-week period. V. vulnificus was enumerated by DNA probe hybridization of colonies from standard plate counts on nonselective medium, and recovery was compared for several media. Loss of plating efficiency was observed on standard selective and differential media compared with nonselective agars. Numbers of V. vulnificus generally declined in treated samples compared with controls; however, increases in total heterotrophic bacteria and fecal coliforms were also observed in treated samples at some time points. This study does not support the use of ice immersion as a postharvest method because of the relatively small declines in V. vulnificus numbers and the possibility of concomitant increases in fecal coliform and total bacterial contamination.     

Conditions for a 5-log reduction of Vibrio vulnificus in oysters through high hydrostatic pressure treatment

Vibrio vulnificus is frequently associated with oysters, and since oysters are typically consumed raw on a half shell, they can pose a threat to public health due to ingestion of this pathogenic marine microorganism. Oysters should be processed to reduce the number of this pathogen. High pressure processing is gaining more and more acceptance among oyster processors due to its ability to shuck oysters while keeping the fresh-like characteristics of oysters. Nine strains of V. vulnificus were tested for their sensitivities to high pressure. The most pressure-resistant strain of V. vulnificus, MLT 403, was selected and used in the subsequent experiments to represent a worst case scenario for evaluation of the processing parameters for inactivation of V. vulnificus in oysters. To evaluate the effect of temperature on pressure inactivation of V. vulnificus, oyster meats were inoculated with V. vulnificus MLT 403 and incubated at room temperature for 24 h. Oyster meats were then blended and treated at 150 MPa for 4 min, and 200 MPa for 1 min. Pressure treatments were carried out at -2, 1, 5, 10, 20, 30, 40, and 45 degrees C. Cold temperatures (<20 degrees C) and slightly elevated temperatures (>30 degrees C) substantially increased pressure inactivation of V. vulnificus. For example, a 4-min treatment of 150 MPa at -2 and 40 degrees C reduced the counts of V. vulnificus by 4.7 and 2.8 log, respectively, while at 20 degrees C the same treatment only reduced counts by 0.5 log. Temperatures of -2 and 1 degrees C were used to determine the effect of pressure level, temperature, and treatment time on the inactivation of V. vulnificus infected to live oysters through feeding. To achieve a >5-log reduction in the counts of V. vulnificus in a relatively short treatment time (or=250 MPa at -2 or 1 degrees C.     

Depuration of Oysters (Crassostrea gigas) Contaminated with Vibrio parahaemolyticus and Vibrio vulnificus with UV Light and Chlorinated Seawater

The efficacy of depuration using UV light and chlorinated seawater for decontaminating Vibrio parahaemolyticus and Vibrio vulnificus from oysters was investigated. Oysters were contaminated with a five-strain cocktail of V. parahaemolyticus or V. vulnificus to levels of 10(4) to 10(5) CFU ml(-1) for bioaccumulation. The depuration was conducted in a closed system in which 350 liters of seawater was recirculated at a rate of 7 liters/min for 48 h at room temperature. Counts of V. parahaemolyticus or V. vulnificus were determined at 0, 6, 18, 24, and 48 h. Three treatments were conducted: T1, control treatment; T2, UV treatment; and T3, UV plus chlorine treatment. After 48 h of depuration of V. parahaemolyticus, T3 reduced the count by 3.1 log most probable number (MPN) g(-1) and T2 reduced the count by 2.4 log MPN g(-1), while T1 reduced the count by only 2.0 log MPN g(-1). After 48 h of depuration of V. vulnificus, T2 and T3 were efficient, reducing the counts by 2.5 and 2.4 log MPN g(-1), respectively, while T1 reduced the count by only 1.4 log MPN g(-1). The UV light plus chlorine treatment was more efficient for controlling V. parahaemolyticus in oysters. Both UV light and UV light plus chlorine were efficient for V. vulnificus. The present study is the first report showing the efficacy of depuration systems for decontaminating V. parahaemolyticus and V. vulnificus in oysters cultivated on the Brazilian coast. This study provides information on processes that can contribute to controlling and preventing such microorganisms in oysters and could be used for effective postharvest treatment by restaurants and small producers of oysters on the coast of Brazil.     

Inactivation of vibrio parahaemolyticus and vibrio vulnificus in phosphate-buffered saline and in inoculated whole oysters by high-pressure processing

Inactivation studies for Vibrio parahaemolyticus TX-2103 (serotype O3:K6) and Vibrio vulnificus MO-624 (clinical isolate) were conducted in phosphate-buffered saline (PBS) and in inoculated oysters under high-pressure processing conditions. V. parahaemolyticus was more resistant than V. vulnificus in PBS at all pressures and times. A 6-log reduction of V. parahaemolyticus and V. vulnificus in PBS at 241 MPa required 11 and 5 min, respectively, which included a 3-min pressure come-up time. A 4.5-log reduction of V. parahaemolyticus in oysters at 345 MPa required 7.7 min, which included a 6.7-min pressure come-up time. More than a 5.4-log reduction of V. vulnificus in oysters at 345 MPa occurred during the 6-min pressure come-up time. Both V. parahaemolyticus and V. vulnificus in PBS and in oysters were reduced to nondetectable numbers at 586 MPa during the 8- and 7-min pressure come-up times, respectively.     

Sensitivity of Vibrio species in phosphate-buffered saline and in oysters to high-pressure processing

Multiple strains of Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio cholerae non-O1 were tested in phosphate-buffered saline for their sensitivity to high-pressure processing (HPP). Variability in sensitivity among strains was observed for all species; this variability decreased at higher pressures. V. vulnificus was the species that was most sensitive to treatment at 200 MPa (decimal reduction time [D] = 26 s), and V. cholerae was the species that was most resistant to treatment at 200 MPa (D = 149 s). The O3:K6 serotype of V. parahaemolyticus was more resistant to pressure than other serotypes of V. parahaemolyticus were. The results of studies involving V. vulnificus naturally occurring in oysters revealed that a pressure treatment of 250 MPa for 120 s achieved a > 5-log reduction in the levels of this bacterium. V. parahaemolyticus serotype O3:K6 in oysters required a pressure of 300 MPa for 180 s for a comparable 5-log reduction. When properly applied, HPP can be effective in improving the safety of shellfish with respect to Vibrio spp.     

Effectiveness of icing as a postharvest treatment for control of Vibrio vulnificus and Vibrio parahaemolyticus in the eastern oyster (Crassostrea virginica)

The focus of this research was to investigate the efficacy of icing as a postharvest treatment for reduction of the levels of Vibrio vulnificus and Vibrio parahaemolyticus in commercial quantities of shellstock oysters. The experiments were conducted in June and August of 2006 and consisted of the following treatments: (i) on-board icing immediately after harvest; (ii) dockside icing approximately 1 to 2 h prior to shipment; and (iii) no icing (control). Changes in the levels of pathogenic Vibrio spp. during wholesale and retail handling for 2 weeks postharvest were also monitored. On-board icing achieved temperature reductions in all sacks in accordance with the National Shellfish Sanitation Program standard, but dockside icing did not meet this standard. Based on one-way analysis of variance, the only statistically significant relationship between Vibrio levels and treatment occurred for samples harvested in August; in this case, the levels of V. vulnificus in the noniced oysters were significantly higher (P < 0.05) than were the levels in the samples iced on-board. When analyzing counts over the 14-day storage period, using factorial analysis, there were statistically significant differences in V. vulnificus and V. parahaemolyticus levels by sample date and/or treatment (P < 0.05), but these relationships were not consistent. Treated (iced) oysters had significantly higher gaping (approximately 20%) after 1 week in cold storage than did noniced oysters (approximately 10%) and gaping increased significantly by day 14 of commercial storage. On-board and dockside icing did not predictably reduce the levels of V. vulnificus or V. parahaemolyticus in oysters, and icing negatively impacted oyster survival during subsequent cold storage.     

Effect of Chlorinated Wash Water on Vibrio cholerae in Oyster Meats

Live oysters exposed to seawater artificially contaminated with Vibrio cholerue, El Tor, Inaba, in a closed system for 18 hr were harvested, shucked, sampled and placed into blower tanks containing 25 and 50 ppm chlorine for 5- and 10-min intervals in each tank. Regardless of chlorine concentrations encountered during the blowing process, V. cholerae MPNs of oyster meats sampled after the prescribed contact period were essentially equivalent to those of oysters sampled at the end of the 18-hr exposure period. Chlorination of the water used in blower tanks did not eliminate V. cholerae, El Tor, Inaba, organisms from oyster meats.     

Vibrio vulnificus and Vibrio parahaemolyticus in U.S. retail shell oysters: a national survey from June 1998 to July 1999.

From June 1998 to July 1999, 370 lots of oysters in the shell were sampled at 275 different establishments (71%, restaurants or oyster bars; 27%, retail seafood markets: and 2%, wholesale seafood markets) in coastal and inland markets throughout the United States. The oysters were harvested from the Gulf (49%). Pacific (14%), Mid-Atlantic (18%), and North Atlantic (11%) Coasts of the United States and from Canada (8%). Densities of Vibrio vulnificus and Vibrio parahaemolyticus were determined using a modification of the most probable number (MPN) techniques described in the Food and Drug Administration's Bacteriological Analytical Manual. DNA probes and enzyme immunoassay were used to identify suspect isolates and to determine the presence of the thermostable direct hemolysin gene associated with pathogenicity of V. parahaemolyticus. Densities of both V. vulnifcus and V. parahaemolyticus in market oysters from all harvest regions followed a seasonal distribution, with highest densities in the summer. Highest densities of both organisms were observed in oysters harvested from the Gulf Coast, where densities often exceeded 10,000 MPN/g. The majority (78%) of lots harvested in the North Atlantic, Pacific, and Canadian Coasts had V. vulnificus densities below the detectable level of 0.2 MPN/g; none exceeded 100 MPN/g. V. parahaemolyticus densities were greater than those of V. vulnificus in lots from these same areas, with some lots exceeding 1,000 MPN/g for V. parahaemolyticus. Some lots from the Mid-Atlantic states exceeded 10,000 MPN/g for both V. vulnificus and V. parahaemolyicus. Overall, there was a significant correlation between V. vulificus and V. parahaemolyticus densities (r = 0.72, n = 202, P < 0.0001), but neither density correlated with salinity. Storage time significantly affected the V. vulnificus (10% decrease per day) and V. parahaemolyticus (7% decrease per day) densities in market oysters. The thermostable direct hemolysin gene associated with V parahaemolyticus virulence was detected in 9 of 3,429 (0.3%) V. parahaemolyticus cultures and in 8 of 198 (4.0%) lots of oysters. These data can be used to estimate the exposure of raw oyster consumers to V. vulnificus and V. parahaemolyticus.     

Fate of Staphylococcus aureus, Salmonella enterica serovar typhimurium, and vibrio vulnificus in raw oysters treated with chitosan

The fate of Staphylococcus aureus, Salmonella enterica serovar Typhimurium, and Vibrio vulnificus in oysters treated with chitosan was investigated. Three concentrations (0.5, 1.0, and 2.0%) of chitosan in 0.5% hydrochloric acid were prepared and coated onto raw oysters, which were then stored at 4 degrees C for 12 days. Untreated oysters and oysters coated with 0.5% hydrochloric acid without chitosan were used as controls. S. aureus cells were most sensitive to 2.0% chitosan followed by 0.5 and 1.0%. In general, chitosan treatment of oysters produced a decline in the population of S. aureus by 1 to 4 log CFU/ml compared with the untreated control. Chitosan treatment had no influence on the reduction of Salmonella Typhimurium over the 12-day storage period; inhibition of Salmonella Typhimurium growth was similar in both the control samples and the chitosan-treated samples. However, time of storage had a major effect on the survival of Salmonella Typhimurium on oysters. Neither time nor chitosan concentration had a significant effect on the growth of V. vulnificus during storage. All treatments were similar in inhibiting V. vulnificus growth.     

High salinity relay as a postharvest processing strategy to reduce vibrio vulnificus levels in Chesapeake Bay oysters (Crassostrea virginica)

In 2009 the U.S. Food and Drug Administration (FDA) announced its intention to implement postharvest processing (PHP) methods to eliminate Vibrio vulnificus from oysters intended for the raw, half-shell market that are harvested from the Gulf of Mexico during warmer months. FDA-approved PHP methods can be expensive and may be associated with unfavorable responses from some consumers. A relatively unexplored PHP method that uses relaying to high salinity waters could be an alternative strategy, considering that high salinities appear to negatively affect the survival of V. vulnificus. During relay, however, oysters may be exposed to rapid and large salinity increases that could cause increased mortality. In this study, the effectiveness of high salinity relay to reduce V. vulnificus to <30 most probable number (MPN) per g and the impact on oyster mortality were assessed in the lower Chesapeake Bay. Two relay experiments were performed during the summer and fall of 2010. Oysters collected from three grow-out sites, a low salinity site (14 to 15 practical salinity units [psu]) and two moderate salinity sites (22 to 25 psu), were relayed directly to a high salinity site (≥30 psu) on Virginia's Eastern Shore. Oysters were assayed for V. vulnificus and Vibrio parahaemolyticus (another Vibrio species of concern) densities at time 0 prior to relay and after 7 and 14 days of relay, using the FDA MPN enrichment method combined with detection by real-time PCR. After 14 days, both V. vulnificus and V. parahaemolyticus densities were ≤0.8 MPN/g, and decreases of 2 to 3 log in V. vulnificus densities were observed. Oyster mortalities were low (≤4%) even for oysters from the low salinity harvest site, which experienced a salinity increase of approximately 15 psu. Results, although preliminary and requiring formal validation and economic analysis, suggest that high salinity relay could be an effective PHP method.     

Levels of Vibrio vulnificus and organoleptic quality of raw shellstock oysters (Crassostrea virginica) maintained at different storage temperatures.

Temperature abuse during raw oyster harvesting and storage may allow for the multiplication of natural spoilage flora as well as microbial pathogens, thus posing a potential health threat to susceptible consumers and compromising product quality. The objective of this study was to provide a scientific basis for determining whether different refrigeration and abuse temperatures for raw oysters would result in a spoiled product before it became unsafe. Raw shellstock oysters (Crassostrea virginica) purchased from a commercial Virginia processor were subjected to different temperature abuse conditions (7, 13, and 21 degrees C) over a 10-day storage period. Salinity, pH, halophilic plate count (HPC), total culturable Vibrio counts, and culturable Vibrio vulnificus counts were determined at each abuse condition. V. vulnificus isolates were confirmed by a specific enzyme-linked immunosorbent assay. Olfactory analysis was performed to determine consumer acceptability of the oysters at each abuse stage. The pH of the oysters decreased over time in each storage condition. The HPC increased 2 to 4 logs for all storage conditions, while olfactory acceptance decreased over time. V. vulnificus levels increased over time, reaching 10(5) to 10(6) CFU/g by day 6. The length of storage had a greater effect on the bacterial counts and olfactory acceptance of the oysters (P < 0.05) over time than did the storage temperature (P < 0.05).     

Extension of Shelf-Life of Oysters by Pasteurization in Flexible Pouches

Combination of chlorination, packaging in flexible pouches, heating, cooling and cold storage could extend shelf-life of oysters from 2 wk to 3 months. Freshly shucked oysters were first blown (or washed) in aerated chlorinated water, drained and vacuum sealed in flexible pouches. Pouched oysters were heated in two successive water baths for appropriate thermal treatment. After cooling in ice water, the products were stored at 0.5°C. Sensory tests performed immediately after pasteurization indicated that the pasteurized oysters were similar to freshly shucked oysters. Both aerobic and facultatively anaerobic bacterial counts were drastically decreased after pasteurization. Chemical changes during treatment and storage were determined and are discussed.     

Effects of refrigeration and alcohol on the load of Aeromonas hydrophila in oysters

Members of the bacterial genus Aeromonas are widely distributed throughout the environment and are readily cultured from a variety of foods. One member of this genus, Aeromonas hydrophila, has been reputed to be a significant cause of gastrointestinal disease. In this study, we examined the effects of refrigeration and alcohol on the level of A. hydrophila in oysters. Specifically, vodka was examined because it is used by the food service industry in preparation of Oysters Romanoff. One set of oysters was shucked on receipt, whereas others were refrigerated intact for 7 days at 5 degrees C. The oysters were blended and the numbers of A. hydrophila present determined using starch ampicillin agar. Oysters were also shucked and placed on the half shell with 5 ml of vodka for 10 min. The oysters were then washed and presumptive A. hydrophila levels determined in both the washate and homogenate. On the day of purchase, the average number of presumptive A. hydrophila found was 7.6 x 10(4) CFU/g of oyster meat. After 7 days of refrigeration, the average number had increased to 3.2 x 10(5) CFU/g of oyster meat. In the oysters treated with vodka, the average number of A. hydrophila present internally was 9.9 x 10(4) with high numbers (10(3) to 10(4)) isolated from the oyster surface. From these data, it is clear that refrigeration and alcohol treatment are not sufficient to reduce loads of A. hydrophila in or on oysters.