Vibrio Vulnificus In Oysters Research Articles

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

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

Factors affecting the uptake and retention of Vibrio vulnificus in oysters.

Vibrio vulnificus, a bacterium ubiquitous in oysters and coastal water, is capable of causing ailments ranging from gastroenteritis to grievous wound infections or septicemia. The uptake of these bacteria into oysters is often examined in vitro by placing oysters in seawater amended with V. vulnificus. Multiple teams have obtained similar results in studies where laboratory-grown bacteria were observed to be rapidly taken up by oysters but quickly eliminated. This technique, along with suggested modifications, is reviewed here. In contrast, the natural microflora within oysters is notoriously difficult to eliminate via depuration. The reason for the transiency of exogenous bacteria is that those bacteria are competitively excluded by the oyster's preexisting microflora. Evidence of this phenomenon is shown using in vitro oyster studies and a multiyear in situ case study. Depuration of the endogenous oyster bacteria occurs naturally and can also be artificially induced, but both of these events require extreme conditions, natural or otherwise, as explained here. Finally, the "viable but nonculturable" (VBNC) state of Vibrio is discussed. This bacterial torpor can easily be confused with a reduction in bacterial abundance, as bacteria in this state fail to grow on culture media. Thus, oysters collected from colder months may appear to be relatively free of Vibrio but in reality harbor VBNC cells that respond to exogenous bacteria and prevent colonization of oyster matrices. Bacterial-uptake experiments combined with studies involving cell-free spent media are detailed that demonstrate this occurrence, which could explain why the microbial community in oysters does not always mirror that of the surrounding water.

Factors Affecting the Uptake and Retention of Vibrio vulnificus in Oysters

Factors Affecting the Uptake and Retention of Vibrio vulnificus in Oysters

An improved method for quantification of Vibrio vulnificus in oysters

Chromogenic agar was compared with the FDA recommended cellobiose–colistin agar for assessment of Vibrio vulnificus in oysters. A two-step culture confirmation method was also evaluated. The inclusion of CA gave a 33% increase in the detection rate and the two-step culture confirmation eliminated 62.5% of false positives.

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 °C. Cold temperatures (< 20 °C) and slightly elevated temperatures (> 30 °C) substantially increased pressure inactivation of V. vulnificus. For example, a 4-min treatment of 150 MPa at − 2 and 40 °C reduced the counts of V. vulnificus by 4.7 and 2.8 log, respectively, while at 20 °C the same treatment only reduced counts by 0.5 log. Temperatures of − 2 and 1 °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 (≤ 4 min), pressure treatment needs to be conducted at pressure levels of ≥ 250 MPa at − 2 or 1 °C.

Evaluation of postharvest-processed oysters by using PCR-based most-probable-number enumeration of Vibrio vulnificus bacteria.

Postharvest processing (PHP) is used to reduce levels of Vibrio vulnificus in oysters, but process validation is labor-intensive and expensive. Therefore, quantitative PCR was evaluated as a rapid confirmation method for most-probable-number enumeration (QPCR-MPN) of V. vulnificus bacteria in PHP oysters. QPCR-MPN showed excellent correlation (R(2) = 0.97) with standard MPN and increased assay sensitivity and efficiency.

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°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.

Offshore suspension relaying to reduce levels of Vibrio vulnificus in oysters (Crassostrea virginica).

Oysters naturally contaminated with 10(3) to 10(4) most probable numbers (MPN) of Vibrio vulnificus per g were relayed to offshore waters (salinity, 30 to 34 ppt), where they were suspended in racks at a depth of 7.6 m. V. vulnificus counts in oysters were reduced to < 10 MPN/g within 7 to 17 days in five of the six studies. At the end of the studies (17 to 49 days), V. vulnificus levels were reduced further and ranged from a mean of 0.23 to 2.6 MPN/g. Oyster mortalities during relaying were < 6%. The reduction of V. vulnificus in relayed oysters is associated with exposure to high-salinity environments essentially devoid of V. vulnificus. Offshore suspension relaying may be a method that industry can employ to reduce V. vulnificus levels in raw Gulf Coast oysters.

Direct plating procedure for enumerating Vibrio vulnificus in oysters (Crassostrea virginica).

A procedure for enumerating and identifying Vibrio vulnificus in oysters was developed and evaluated. This method consists of growth on a direct plating medium (VVE medium) for isolating the organism from shellfish tissues, followed by biochemical tests for differentiating and identifying presumptively positive isolates. Densities of V. vulnificus are reliably obtained in 2 to 4 days, and as few as 10 culturable cells per 100 g can be identified. The procedure was evaluated by using a DNA probe technique specific for the cytotoxin-hemolysin gene of V. vulnificus and gas chromatographic analysis of the fatty acid contents of positive isolates. Only 3.2 and 0.4% of the isolates gave false-positive and false-negative results, respectively. The average level of recovery on VVE medium for 33 strains, including both clinical and environmental isolates, was 92% of the level of recovery obtained with brain heart infusion agar supplemented with 1% NaCl. The densities of V. vulnificus in oyster homogenates and individual oysters harvested from gulf and Atlantic coastal waters revealed that seasonally high levels occurred. The VVE medium procedure facilitated enumeration of this pathogen in molluscan shellfish and had a distinct advantage over the widely used most-probable-number procedure for V. vulnificus enumeration, which requires 5 to 7 days and often gives improbable and imprecise results.

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.

Lethal cold stress of Vibrio vulnificus in oysters.

Studies were conducted on the survival of Vibrio vulnificus, an estuarine human pathogen, in oyster homogenates held at 4 degrees C. Results indicated a rapid and dramatic decrease in viability not attributable to either cold shock or the oyster homogenate alone but to a combination of the two. Such a decline was not observed with Vibrio parahaemolyticus. Chilled V. vulnificus cells were unable to repair themselves in brain heart infusion broth at 37 degrees C. V. vulnificus cells incubated on whole raw oysters at 0.5 degrees C also exhibited a decline in viability, but of a lesser degree. The effects of various plating media were also investigated. The data reported here suggest that oysters kept on ice are not likely to be a major factor in the epidemiology of V. vulnificus infection. It is further suggested that the standard method of homogenizing oysters for examining bacteriological quality should not be followed because toxic compounds are released from the oysters during this process.