Oyster-to-oyster variability in levels of Vibrio parahaemolyticus

This study examined the variability in the levels of total and pathogenic Vibrio parahaemolyticus in individual oysters. Twenty oysters were collected on three occasions (in June, July, and September 2001) from a site near Mobile Bay, Ala. Ten of these oysters were tested immediately, and 10 were tested after 24 h of storage at 26 degrees C. Levels of total and pathogenic V. parahaemolyticus were determined by alkaline phosphatase-labeled DNA probe procedures targeting the thermolabile hemolysin and thermostable direct hemolysin genes, respectively. Similar V. parahaemolyticus levels (200 to 2,000 CFU/g) were found in nearly 90% of the oysters (for all sampling occasions) prior to storage. The log-transformed densities (means +/- standard deviations) of V. parahaemolyticus in oysters immediately after harvest were 2.90 +/- 0.91, 2.88 +/- 0.36, and 2.47 +/- 0.26 log10 CFU/g for June, July, and September, respectively. After storage for 24 h at 26 degrees C, the mean V. parahaemolyticus densities increased approximately 13- to 26-fold. Before storage, pathogenic V. parahaemolyticus was detected in 40% (10 to 20 CFU/g) of the oysters collected in June and July but was not detected in any oysters collected in September. After storage, pathogenic V. parahaemolyticus was detected in some oysters at levels of > 100 CFU/g. These data should aid in the development of sampling protocols for oyster monitoring programs and in the determination of exposure distributions associated with raw oyster consumption.     

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.     

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.     

温暖月份零售带壳牡蛎中副溶血性弧菌的定量研究

为了解温暖月份零售带壳牡蛎中副溶血性弧菌 (VP)的污染情况 ,2 0 0 3年 4～ 8月在福建省福州和厦门两地共收集带壳牡蛎 113份 ,样品分别来自水产品批发市场 (18% ) ,零售市场 (4 6 % )和饭店 (36 % )。采用Vitek鉴定系统和最可能数法进行VP的定量分析。结果显示 ,带壳牡蛎中VP密度的几何均数为 6 0MPN 10 0g ,4 1 6 %的样品VP密度低于 30MPN 10 0g的最低检出限 ,仅厦门2个样品菌量超过 2 4 0 0 0MPN 10 0g。两个地区、不同采样点和不同月份之间样品VP密度的几何均数差别均有统计学意义 (P <0 0 1)。厦门样品污染菌量高于福州 ;批发市场样品菌量最高 ;5月份样品菌量最高 ,为 14 9MPN 10 0g ,而 6～ 8月样品菌量约为 4 0MPN 10 0g。零售环节带壳牡蛎VP的检出率较高。未来应加强对生食海产品中VP污染状况的监测。     

福建省带壳牡蛎中副溶血性弧菌的市场调查

为了解零售带壳牡蛎中副溶血性弧菌(VP)的污染情况，2003年4月～2004年3月每月在福建省福州和厦门两地收集带壳牡蛎，样品共252份，分别来自水产品批发市场(11％)、零售市场(50％)和饭店(39％)。采用Vitek鉴定系统和最可能数(MPN)法进行VP的定性和定量分析。结果显示，带壳牡蛎VP几何平均密度为46MPN，100g，46％的试样VP密度低于30MPN/100g的最低检出限，仅厦门2个试样菌量超过104MPIN/100g。两个地区、不同采样点和不同季节之间试样vP平均密度差别均有显性。厦门试样菌量高于福州；批发市场试样菌量最高；春季试样菌量(93MPN/100g)高于其它季节(约为40MPN/100g)。研究结果可以用于估计生食牡蛎人群VP的暴露量。     

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.     

Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26 degrees C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r = -0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus-specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.     

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.     

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.     

Response of Vibrio parahaemolyticus 03:K6 to a hot water/cold shock pasteurization process.

Vibrio vulnificus and V. parahaemolyticus are natural inhabitants of estuarine environments world wide. Pathogenic strains of these bacteria are often transmitted to humans through consumption of raw oysters, which flourish in the same estuaries. Previous studies reported the effective use of hot water pasteurization followed by cold shock to eliminate from raw oysters naturally and artificially incurred environmental strains of V. vulnificus and V. parahaemolyticus common to the Gulf of Mexico. The present study focused on the use of the same pasteurization method to reduce a highly process resistant Vibrio strain, V. parahaemolyticus O3:K6 to non-detectable levels. Oysters were artificially contaminated with 10(4) and 10(6) V. parahaemolyticus 03:K6 cfu g(-1) oyster meat. Contaminated oysters were pasteurized between 50 and 52 degrees C for up to 22 min. Samples of processed oysters were enumerated for V. parahaemolyticus O3:K6 at 2-min intervals beginning after the 'come-up time' to achieve an oyster internal temperature of at least 50 degrees C. The D value (D(52)deg C) was 1.3-1.6 min. V. parahaemolyticus O3:K6 proved more process resistant than non-pathogenic environmental strains found in Gulf of Mexico waters. A total processing time of at least 22 min at 52 degrees C was recommended to reduce this bacterium to non-detectable levels (< 3 g(-1) oyster meat).     

Microbiological quality and safety of quahog clams, Mercenaria mercenaria, during refrigeration and at elevated storage temperatures

The effects of storage temperatures and times on the microbiological quality and safety of hard-shelled quahog clams (Mercenaria mercenaria) were examined. Samples were stored at four different incubation temperatures (3.3, 7.2, 10.0, and 12.8 degrees C) for a period of 3 weeks, following their harvest from summer growing waters (> or = 27 degrees C) and winter waters (< or = 4 degrees C). Clams were analyzed for two naturally occurring pathogens, Vibrio parahaemolyticus and Vibrio vulnificus. During the summer, V. parahaemolyticus was isolated from 56% of the stored samples, with the highest concentration, 6,100/g, occurring on day 12 at 12.8 degrees C. Also, during the summer, V. vulnificus was isolated from 11% of the stored samples, with the highest concentration of 1,500/g occurring on day 15 at 7.2 degrees C. No Vibrio spp. were detected during the winter. During summer storage, aerobic mesophilic counts on plate count agar (PCA) containing 2% NaCl ranged from 10(4) to 10(8) CFU/g, and during storage of the winter samples, aerobic mesophilic PCA (with added NaCl) counts ranged from <100 to 10(4) CFU/g. Comparatively, summer storage mesophilic counts on PCA containing no added NaCl ranged from <100 to 10(5) CFU/g, and for the winter samples the range was <100 to 10(2) CFU/g. Coliform and fecal coliform counts ranged from <0.3 to 61.1/g and <0.3 to 24.4/g, respectively. There was no statistical correlation between the length of storage or the temperature of incubation and the presence of V. parahaemolyticus, V. vulnificus, coliforms, or fecal coliforms. However, storage time and incubation temperature affected the PCA counts (P < or = 0.05) in quahog clams.     

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.     

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.     

Occurrence and density of vibrio parahaemolyticus in live edible crustaceans from markets in China

Vibrio parahaemolyticus is a marine bacterium causing foodborne disease. Occurrence of the bacterium was investigated in six species of edible crustaceans available from markets in mainland China. The bacterium was detected in 22 of 45 whole-body, shell, and feces samples, including mitten crabs, which are supposed to be produced in freshwater ponds. The mean densities ranged from 2.8 log CFU/g in mitten crabs (Eriocheir sinensis) to 5.1 CFU/g in giant tiger prawns (Penaeus monodon). In hemolymph and muscle samples collected axenically, V. parahaemolyticus was detected in all of the prawns at a mean density of 2.6 log most probable number (MPN)/g, in two of five striped stone crabs (Charybdis feriatus) at a mean density of 1.1 log MPN/ml, and two of five mangrove mud crabs (Scylla serrata) at a mean density of 1.3 log MPN/ml. When six mitten crabs were collected from two freshwater ponds in China and were examined, V. parahaemolyticus was not detected. It seemed that cross-contamination occurred among live crustaceans at the markets. The results suggest that proper handling, storage, and cooking of these crustaceans will be necessary to lessen the risk of foodborne illness from V. parahaemolyticus.     

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.     

Density of total and pathogenic (tdh+) Vibrio parahaemolyticus in Atlantic and Gulf coast molluscan shellfish at harvest

The densities of total and pathogenic Vibrio parahaemolyticus in 671 samples of molluscan shellfish harvested in 1999 and 2000 from 14 sites in seven Gulf and Atlantic coast states were determined at 2-week intervals over a period of 12 to 16 months in each state. Changes in V. parahaemolyticus densities in shellfish between harvest and sample analysis were minimized with time and temperature controls. Densities were measured by direct plating techniques, and gene probes were used for identification. Total and pathogenic V. parahaemolyticus organisms were identified with probes for the thermolabile direct hemolysin (tlh) gene and the thermostable direct hemolysin (tdh) gene, respectively. An enrichment procedure involving 25 g of shellfish was also used for the recovery of pathogenic V. parahaemolyticus. The densities of V. parahaemolyticus in shellfish from all harvest sites were positively correlated with water temperature. Shellfish from the Gulf Coast typically had higher densities of V. parahaemolyticus than did shellfish harvested from the North Atlantic or mid-Atlantic coast. Vibrio parahaemolyticus counts exceeded 1,000 CFU/g for only 5% of all samples. Pathogenic (tdh+) V. parahaemolyticus was detected in approximately 6% of all samples by both procedures, and 61.5% of populations in the positive samples from the direct plating procedure were at the lower limit of detection (10 CFU/g). The frequency of detection of pathogenic V. parahaemolyticus was significantly related to water temperature and to the density of total V. parahaemolyticus. The failure to detect pathogenic V. parahaemolyticus in shellfish more frequently was attributed to the low numbers and uneven distribution of the organism.     

Growth and Survival Differences of Vibrio vulnificus and Vibrio parahaemolyticus Strains during Cold Storage

ABSTRACT:  Vibrio vulnificus  and  Vibrio parahaemolyticus  are the most common  Vibrio  species associated with seafood illness in the United States. Our study was conducted to determine if strain-to-strain differences exist in the growth and survival of 8 different  V. vulnificus  and  V. parahaemolyticus  strains at low temperatures. By day 10,  V. vulnificus  strain 515-4C2 had significantly higher counts ( P  < 0.05) (1.97 log CFU/g) compared with strains 3315, 1007, 29306 at 5 °C, which reached nondetectable levels. At 8 °C, strain 515-4C2 had significantly higher counts ( P  < 0.05) (2.23 log CFU/mL) compared with 1007, 33815, 541(O) 49C, which reached nondetectable levels. At 10 °C, only  V. vulnificus  strain 33815 reached nondetectable levels. At 5 °C,  V. parahaemolyticus  strain 541(O) 57C had the highest counts (5.28 log CFU/g) by day 10 while strain 33847 had significantly lower counts (3.46 log CFU/g). After 10 d at 8 °C,  V. parahaemolyticus  strain M350A had the highest counts (7.97 log CFU/mL) while strain 541(O) 57C had the lowest counts (4.80 log CFU/mL). At 10 °C,  V. parahaemolyticus  strain NY477 had significantly higher counts ( P  < 0.05) with 8.31 log CFU/mL compared with strain 33847, which had the lowest counts (6.77 log CFU/mL). Our research has shown that various  V. vulnificus  and  V. parahaemolyticus  strains vary in their ability to survive and grow at refrigeration temperatures.     

Populations of Vibrio parahaemolyticus in retail oysters from Florida using two methods

Vibrio parahaemolyticus is a naturally occurring estuarine bacterium that is often associated with gastroenteritis in humans following consumption of raw molluscan shellfish. A number of studies have investigated the environmental distribution of V. parahaemolyticus, but little is known about the levels of this organism during distribution of oysters or at the point of consumption. Duplicate samples of shellstock oysters were collected monthly (September 1997 to May 1998) from the same four restaurants and three wholesale seafood markets in the Gainesville, Fla. area and analyzed for total V. parahaemolyticus densities using two methods: a standard MPN method (BAM-MPN) and a new direct plating procedure (direct-VPAP). Both methods employed an alkaline phosphatase-labeled DNA probe (VPAP) targeting the species-specific thermolabile hemolysin (tlh) gene to confirm suspect colonies as V. parahaemolyticus. The highest monthly geometric mean V. parahaemolyticus density was observed in October of 1997 (approximately 3,000/g) with similarly high values during September and November of 1997. From December 1997 to May 1998 mean densities were generally less than 100/g, falling to approximately 10/g in February and March. A strong correlation (r = 0.78) between the direct-VPAP and BAM-MPN methods for determining V. parahaemolyticus densities in market-level oysters was observed. The direct-VPAP method was more rapid and precise while the BAM-MPN was more sensitive and may better recover stressed cells. The utilization of the VPAP probe for identification of V. parahaemolyticus sharply reduced the labor for either method compared to biochemical identification techniques used in earlier V. parahaemolyticus surveys.     

Inhibitory effect of Melastoma candidum D. Don acetone extract on foodborne pathogenic bacteria survival in food products

Melastoma candidum D. Don, a Taiwanese folk medicinal plant, has high levels of antibacterial and bactericidal activity. Our aim was to determine whether and to what extent an acetone extract of this plant inhibits the growth of foodborne pathogenic bacteria. M. candidum acetone extract had marked inhibitory effect on test bacteria introduced into sliced pork, which was then stored at 4 degrees C. At the end of storage (day 12), the bacterial concentrations dropped by 1.59 to 2.91 log CFU/g compared with the control. In steamed rice stored at 30 degrees C, a 0.2% extract decreased initial (before storage) concentrations of Bacillus cereus from 2.01 log CFU/g to an undetectable level, which remained for at least 24 h. After 72 to 168 h of storage, test bacterial concentrations were reduced by 2.59 to 5.66 log CFU/g. In fresh noodles stored at 30 degrees C, both initial and final bacterial concentrations were decreased. At the end of storage (72 to 168 h), test bacteria concentrations were reduced by 1.85 to 2.88 log CFU/g. Overall, M. candidum acetone extract had an inhibitory effect on foodborne pathogenic bacteria in different food model systems.     

Seasonal distribution of total and pathogenic Vibrio parahaemolyticus in Chesapeake Bay oysters and waters

The objectives of this study were to investigate the seasonal distribution of total and pathogenic Vibrio parahaemolyticus in the Chesapeake Bay oysters and waters, and to determine the degree of association between V. parahaemolyticus densities and selected environmental parameters. Oyster and water samples were collected monthly from three sites in Chesapeake Bay, Maryland from November 2004 through October 2005. During collection of samples, water temperature, salinity, turbidity, dissolved oxygen, pH, chlorophyll a, and fecal coliform levels in oysters were also determined. V. parahaemolyticus levels were enumerated by a quantitative direct-plating method followed by DNA colony hybridization; presence/absence was further determined by overnight broth enrichment followed by either standard colony isolation or real-time PCR. The thermolabile hemolysin (tlh) gene and thermostable direct hemolysin (tdh) gene were targeted for detection of total and pathogenic V. parahaemolyticus, respectively, for both direct plating and enrichment. The thermostable related hemolysin (trh) gene, which is a presumptive pathogenicity marker, was targeted only for the enrichment approach. By direct plating, colonies producing tlh signals were detected in 79% of oyster samples at densities ranging from 1.5x10(1) to 6.0x10(2) CFU/g. Pathogenic V. parahaemolyticus (tdh+) was detected in 3% (level was 10 CFU/g) of oyster samples while no V. parahaemolyticus was detected in water samples. By the enrichment approach with standard colony isolation, 67% of oyster and 55% of water samples (n=33) were positive for total V. parahaemolyticus, and all samples were negative for pathogenic V. parahaemolyticus. In contrast, enrichment followed by real-time PCR detected tlh, tdh and trh in 100%, 20% and 40% of oyster and 100%, 13% and 40% of water enrichments collected from June to October 2005, respectively. V. parahaemolyticus densities in oysters varied seasonally and were found to be positively correlated with water temperature, turbidity, and dissolved oxygen.