Effects of storage on microbial loads of two commercially important shellfish species, Crassostrea virginica and Mercenaria campechiensis.

The effects of storage on the microbial load in two commercially important species of shellfish were examined. Oysters (Crassostrea virginica) were stored as shellstock, shucked meats, and fully processed meats at four temperatures for up to 21 days, and clams (Mercenaria campechiensis) were stored only as shellstock. The concentrations of most microbiological groups of organisms increased with the duration and temperature of storage in both shellfish species, although the increases were significantly lower in claims. Concentrations of Vibrio cholerae rose by approximately 1 log in oysters stored as shellstock after 7 days at 2 degrees C, and Lac+ vibrios increased 2 logs at 8 degrees C. Total counts of bacteria, fungi, coliforms, fecal streptococci, Aeromonas hydrophila, and clostridia were significantly higher in shucked oysters than in those stored as shellstock. Fecal coliforms were statistically the same, but V. cholerae, Vibrio parahaemolyticus, and the Lac+ vibrios were higher in oysters stored as shellstock. The concentrations of all microbial groups were higher in fully processed oysters than in shucked meats, with the exception of the vibrios, which showed no significant difference among the treatments. The results showed that although traditional methods of storing shellfish resulted in an overall increase in the microbial load, vibrio levels increased only in oysters stored as shellstock. Although fecal coliform and total bacterial counts did not correlate with those for vibrios in fresh oysters, strong correlations were observed in oysters stored for 7 days, suggesting that these indicators may be useful in monitoring oyster quality when meats are stored for a limited time as shellstock.     

Effects of temperature abuse on survival of Vibrio vulnificus in oysters.

Opaque and translucent morphotypes of a TnphoA-containing strain of Vibrio vulnificus were fed to oysters, which were subsequently stored at temperatures ranging from 0.5 to 22 degrees C for 10 days. Samples of oysters were homogenized and plated at intervals to determine the cell density of V. vulnificus and total aerobic population of bacteria present. At all temperatures, the numbers of V. vulnificus (both morphotypes) declined over the 10-day study period. The same observation was made with a lower inoculum of V. vulnificus. Identical experiments with shucked oysters showed a more rapid decrease in V. vulnificus. Identical experiments with shucked oysters showed a more rapid decrease in V. vulnificus to levels below limits of detection. Little change in the total bacterial counts was observed in shellstock oysters at any of the test temperatures, whereas incubation at the higher temperatures (17 and 22 degrees C) resulted in large increases in total counts in shucked oysters. These data suggest that temperature abuse of oysters may not be a factor in increasing the public health risk of V. vulnificus through raw oyster consumption. However, the data also suggest that even with proper storage, indigenous levels of V. vulnificus may remain sufficiently higher in shellstock oysters to produce infection in compromised hosts.     

Effects of temperature abuse on survival of Vibrio vulnificus in oysters.

Opaque and translucent morphotypes of a TnphoA-containing strain of Vibrio vulnificus were fed to oysters, which were subsequently stored at temperatures ranging from 0.5 to 22 degrees C for 10 days. Samples of oysters were homogenized and plated at intervals to determine the cell density of V. vulnificus and total aerobic population of bacteria present. At all temperatures, the numbers of V. vulnificus (both morphotypes) declined over the 10-day study period. The same observation was made with a lower inoculum of V. vulnificus. Identical experiments with shucked oysters showed a more rapid decrease in V. vulnificus. Identical experiments with shucked oysters showed a more rapid decrease in V. vulnificus to levels below limits of detection. Little change in the total bacterial counts was observed in shellstock oysters at any of the test temperatures, whereas incubation at the higher temperatures (17 and 22 degrees C) resulted in large increases in total counts in shucked oysters. These data suggest that temperature abuse of oysters may not be a factor in increasing the public health risk of V. vulnificus through raw oyster consumption. However, the data also suggest that even with proper storage, indigenous levels of V. vulnificus may remain sufficiently higher in shellstock oysters to produce infection in compromised hosts.     

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.     

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery.

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.     

Isolation of Vibrio cholerae serotype O1 from the eastern oyster, Crassostrea virginica.

Two strains of Vibrio cholerae serotype O1 Inaba were isolated from eastern oysters, Crassostrea virginica, collected from estuarine waters in Florida during April 1980. The oyster meats and waters from which the oysters were collected had low fecal coliform counts, and the area had no prior evidence of sewage contamination.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish.

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Validation of a green fluorescent protein-labeled strain of Vibrio vulnificus for use in the evaluation of postharvest strategies for handling of raw oysters

In this paper we describe a biological indicator which can be used to study the behavior of Vibrio vulnificus, an important molluscan shellfish-associated human pathogen. A V. vulnificus ATCC 27562 derivative that expresses green fluorescent protein (GFP) and kanamycin resistance was constructed using conjugation. Strain validation was performed by comparing the GFP-expressing strain (Vv-GFP) and the wild-type strain (Vv-WT) with respect to growth characteristics, heat tolerance (45 degrees C), freeze-thaw tolerance (-20(o) and -80 degrees C), acid tolerance (pH 5.0, 4.0, and 3.5), cold storage tolerance (5 degrees C), cold adaptation (15 degrees C), and response to starvation. Levels of recovery were evaluated using nonselective medium (tryptic soy agar containing 2% NaCl) with and without sodium pyruvate. The indicator strain was subsequently used to evaluate the survival of V. vulnificus in oysters exposed to organic acids (citric and acetic acids) and various cooling regimens. In most cases, Vv-GFP was comparable to Vv-WT with respect to growth and survival upon exposure to various biological stressors; when differences between the GFP-expressing and parent strains occurred, they usually disappeared when sodium pyruvate was added to media. When V. vulnificus was inoculated into shellstock oysters, the counts dropped 2 log(10) after 11 to 12 days of refrigerated storage, regardless of the way in which the oysters were initially cooled. Steeper population declines after 12 days of refrigerated storage were observed for both iced and refrigerated products than for slowly cooled product and product held under conservative harvest conditions. By the end of the refrigeration storage study (22 days), the counts of Vv-GFP in iced and refrigerated oysters had reached the limit of detection (10(2) CFU/oyster), but slowly cooled oysters and oysters stored under conservative harvest conditions still contained approximately 10(3) and >10(4) CFU V. vulnificus/oyster by day 22, respectively. The Vv-GFP levels in the oyster meat remained stable for up to 24 h when the meat was exposed to acidic conditions at various pH values. Ease of detection and comparability to the wild-type parent make Vv-GFP a good candidate for use in studying the behavior of V. vulnificus upon exposure to sublethal stressors that might be encountered during postharvest handling of molluscan shellfish.     

Effect of time and temperature on multiplication of Vibrio vulnificus in postharvest Gulf Coast shellstock oysters.

After harvest, shellstock oysters stored under controlled temperatures of 10, 13, and 18 degrees C and at ambient outside air temperature (23 to 34 degrees C) were sampled after 12 and 30 h for Vibrio vulnificus. At 13 degrees C and below, V. vulnificus failed to multiply in the oysters. In oysters held at 18 degrees C for 30 h and under ambient conditions for 12 and 30 h, V. vulnificus numbers were statistically greater (P < 0.05) than those in oysters at harvest. These data indicate that endogenous V. vulnificus can multiply in unchilled shellstock oysters.     

Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.     

Human enteroviruses in oysters and their overlying waters.

The presence of enteroviruses in oysters and oyster-harvesting waters of the Texas Gulf coast was monitored over a period of 10 months. Viruses were detected in water and oyster samples obtained from areas both open and closed to shellfish harvesting. Viruses were detected periodically in waters that met current bacteriological standards for shellfish harvesting. No significant statistical relationship was demonstrated between virus concentration in oysters and the bacteriological and physiochemical quality of water and shellfish. Viruses in water were, however, moderately correlated with total coliforms in water and oysters and with fecal coliforms in oysters. Total coliforms in water were realted to total coliforms in sediment were related only to total coliforms in sediment. Among the physiochemical characteristics of water, turbidity was related statistically to the organic matter content of water and to fecal coliforms in water. There was a marked effect of rainfall on the bacteriological quality of water. Of a total of 44 water samples, 26 yielded virus in concentrations from 4 to 167 plaque-forming units per 100-gallon (ca. 378.5-liter) sample. Of a total of 40 pools of 10 to 12 oysters each, virus was found in 14 pools at a concentration of 6 to 224 plaque-forming units per 100 g of oyster meat. On five occasions, virus was found in water samples when no virus could be detected in oysters harvested from the same sites. This study indicates that current bacteriological standards for determining the safety of shellfish and shellfish-growing waters do no reflect the occurrence of enteroviruses.     

Human enteroviruses in oysters and their overlying waters.

The presence of enteroviruses in oysters and oyster-harvesting waters of the Texas Gulf coast was monitored over a period of 10 months. Viruses were detected in water and oyster samples obtained from areas both open and closed to shellfish harvesting. Viruses were detected periodically in waters that met current bacteriological standards for shellfish harvesting. No significant statistical relationship was demonstrated between virus concentration in oysters and the bacteriological and physiochemical quality of water and shellfish. Viruses in water were, however, moderately correlated with total coliforms in water and oysters and with fecal coliforms in oysters. Total coliforms in water were realted to total coliforms in sediment were related only to total coliforms in sediment. Among the physiochemical characteristics of water, turbidity was related statistically to the organic matter content of water and to fecal coliforms in water. There was a marked effect of rainfall on the bacteriological quality of water. Of a total of 44 water samples, 26 yielded virus in concentrations from 4 to 167 plaque-forming units per 100-gallon (ca. 378.5-liter) sample. Of a total of 40 pools of 10 to 12 oysters each, virus was found in 14 pools at a concentration of 6 to 224 plaque-forming units per 100 g of oyster meat. On five occasions, virus was found in water samples when no virus could be detected in oysters harvested from the same sites. This study indicates that current bacteriological standards for determining the safety of shellfish and shellfish-growing waters do no reflect the occurrence of enteroviruses.     

Survival of Virus in Chilled, Frozen, and Processed Oysters

Samples of whole and shucked Pacific and Olympia oysters, contaminated with 10(4)-plaque-forming units (PFU) of poliovirus Lsc-2ab per ml, were held refrigerated at two temperatures, 5 and - 17.5 C. To study the survival of virus in the oysters under these conditions, samples were assayed for virus content at weekly intervals for as long as 12 weeks. Results indicated that poliovirus would survive in refrigerated oysters for a period varying from 30 to 90 days, depending upon temperature. The survival rate varied from 10 to 13%. To study the extent of the hazard presented by oysters contaminated with virus, samples of whole and shucked Pacific oysters contaminated with 10(4) PFU of poliovirus Lsc-2ab per ml were heat processed in four ways: by stewing, frying, baking, and steaming. Results indicated that virus in oysters withstood these methods of processing. The survival rate varied from 7 to 10% and appeared dependent upon the processing method used. Heat penetration studies showed that the internal temperature in the oyster was not sufficient to inactivate all of the virus present. These results suggest that not only fresh but also refrigerated and cooked oysters can serve as vectors for the dissemination of virus disease if the shellfish are harvested from a polluted area.     

Incidence of Vibrio parahaemolyticus in U.S. coastal waters and oysters.

Oyster and seawater samples were collected seasonally from May 1984 through April 1985 from shellfish-growing areas in Washington, California, Texas, Louisiana, Alabama, Florida, South Carolina, Virginia, and Rhode Island which had been designated as approved or prohibited by the National Shellfish Sanitation Program. Fecal coliforms counts, aerobic plate counts, and Vibrio parahaemolyticus densities were determined for the samples. Mean V. parahaemolyticus density was more than 100 times greater in oysters than in water, whereas density of fecal coliforms was approximately 10 times higher in oysters. Seasonal and geographical distributions of V. parahaemolyticus were related to water temperature, with highest densities in samples collected in the spring and the summer along the Gulf coast. The synthetic DNA probe for thermostable direct hemolysin hybridized with 2 of 50 isolates, 1 of which was positive by the Kanagawa test.     

Incidence of Vibrio parahaemolyticus in U.S. coastal waters and oysters

Oyster and seawater samples were collected seasonally from May 1984 through April 1985 from shellfish-growing areas in Washington, California, Texas, Louisiana, Alabama, Florida, South Carolina, Virginia, and Rhode Island which had been designated as approved or prohibited by the National Shellfish Sanitation Program. Fecal coliforms counts, aerobic plate counts, and Vibrio parahaemolyticus densities were determined for the samples. Mean V. parahaemolyticus density was more than 100 times greater in oysters than in water, whereas density of fecal coliforms was approximately 10 times higher in oysters. Seasonal and geographical distributions of V. parahaemolyticus were related to water temperature, with highest densities in samples collected in the spring and the summer along the Gulf coast. The synthetic DNA probe for thermostable direct hemolysin hybridized with 2 of 50 isolates, 1 of which was positive by the Kanagawa test.     

Ecology, serology, and enterotoxin production of Vibrio cholerae in Chesapeake Bay.

A total of 65 isolates of Vibrio cholerae, serotypes other than O--1, have been recovered from water, sediment, and shellfish samples from the Chesapeake Bay. Isolations were not random, but followed a distinct pattern in which salinity appeared to be a controlling factor in V. cholerae distribution. Water salinity at stations yielding V. cholerae (13 out of 21 stations) was 4 to 17 0/00, whereas the salinity of water at stations from which V. cholerae organisms were not isolated was less than 4 or greater than 17 0/00. From results of statistical analyses, no correlation between incidence of fecal coliforms and V. cholerae could be detected, whereas incidence of Salmonella species, measured concurrently, was clearly correlated with fecal coliforms, with Salmonella isolated only in areas of high fecal coliform levels. A seasonal cycle could not be determined since strains of V. cholerae were detectable at low levels (ca. 1 to 10 cells/liter) throughout the year. Although none of the Chesapeake Bay isolates was agglutinable in V. cholerae O group 1 antiserum, the majority for Y-1 adrenal cells. Furthermore, rabbit ileal loop and mouse lethality tests were also positive for the Chesapeake Bay isolates, with average fluid accumulation in positive ileal loops ranging from 0.21 to 2.11 ml/cm. Serotypes of the strains of V. cholerae recovered from Chesapeake Bay were those of wide geographic distribution. It is concluded from the data assembled to date, that V. cholerae is an autochthonous estuarine bacterial species resident in Chesapeake Bay.     

Pertinence of indicator organisms and sampling variables to Vibrio concentrations.

Vibrio-indicator relationships and effects of day, depth, and tidal levels on the density of vibrios enumerated by the most probable number technique were investigated. Counts of vibrios taken monthly from Apalachicola Bay, Fla., were either negatively correlated or showed no correlation with counts of indicator bacteria (Escherichia coli, enterococci, fecal coliforms, and total coliforms). Water samples collected on two days from the surface and bottom over a complete tidal cycle on each day were analyzed for differences in vibrio concentrations. Concentrations of vibrios in samples taken on different days, in those taken at different depths, and in those taken at different tidal levels were significantly different, indicating that these factors need to be taken into account in health-related studies.     

Vibrio parahaemolyticus and other halophilic vibrios associated with seafood in Hong Kong

The summer prevalence of Vibrio parahaemolyticus and other halophilic vibrios in seafood from Hong Kong markets was investigated. Halophilic vibrios were isolated from all seven types of seafood examined, and comprised 9.1%, 8% and 6.1% of contaminating aerobic heterotrophic bacteria from mussels, clams and oysters respectively. Sucrose-positive vibrios were more common than sucrose-negative varieties. Vibrio alginolyticus was the most frequently isolated species, followed by V. parahaemolyticus, V. harveyi, V. fluvialis, V. vulnificus, V. pelagius, V. campbellii, V. spendidus and V. marinus. Mussels contained the highest concentration of V. parahaemolyticus (4.6 x 10(4)/g); oysters and clams contained 3.4 x 10(4)/g and 6.5 x 10(3)/g respectively. The ubiquity and relatively high concentrations of V. parahaemolyticus and other pathogenic vibrios in shellfish is a potential public health hazard in Hong Kong and other subtropical Asian countries.     

Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink NH microwell plate sandwich hybridization

Outbreak of diseases associated with consumption of raw shellfish especially oysters is a major concern to the seafood industry and public health agencies. A multiplex PCR amplification of targeted gene segments followed by DNA-DNA sandwich hybridization was optimized to detect the etiologic agents. First, a multiplex PCR amplification of hns, spvB, vvh, ctx and tl was developed enabling simultaneous detection of total Salmonella enterica serotype Typhimurium, Vibrio vulnificus, Vibrio cholerae and Vibrio parahaemolyticus from both pure cultures and seeded oysters. Amplicons were then subjected to a colorimetric CovaLink NH microwell plate sandwich hybridization using phosphorylated and biotinlylated oligonucleotide probes, the nucleotide sequences of which were located internal to the amplified DNA. The results from the hybridization with the multiplexed PCR amplified DNA exhibited a high signal/noise ratio ranging between 14.1 and 43.2 measured at 405 nm wavelength. The sensitivity of detection for each pathogen was 10(2) cells/g of oyster tissue homogenate. The results from this study showed that the combination of the multiplex PCR with a colorimetric microwell plate sandwich hybridization assay permits a specific, sensitive, and reproducible system for the detection of the microbial pathogens in shellfish, thereby improving the microbiological safety of shellfish to consumers.