Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters

DNAs extracted from Vibrio vulnificus seeded into oyster homogenates were evaluated as templates for the polymerase chain reaction. Several extraction procedures were examined, and it was determined that DNA recovered from cells lysed by guanidine isothiocyanate, extracted with chloroform, and precipitated with ethanol was most suitable for use as a polymerase chain reaction template. The region targeted was a 519-bp portion of the cytotoxin-hemolysin gene of V. vulnificus. This region was amplified only when DNA from this species was present in the homogenate. V. vulnificus seeded into oyster homogenates at an initial level of 10(2) CFU/g of oyster meat was consistently observed after 24 h of incubation in alkaline peptone water.     

Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters.

A 26-mer oligonucleotide specific to Vibrio parahaemolyticus was synthesized from a 1,275-bp thermostable direct hemolysin (tdh) gene. This oligonucleotide probe specifically reacted with DNA from 89 of 95 V. parahaemolyticus isolates but not with DNA from other vibrios or other enteric and nonenteric organisms (n = 48). The probe hybridized with Southern blots of 0.5-kb HindIII-restricted chromosomal DNA fragments from all but five V. parahaemolyticus test isolates. The probe could be used to directly identify V. parahaemolyticus in artificially contaminated food without an isolation step.     

Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters.

A 26-mer oligonucleotide specific to Vibrio parahaemolyticus was synthesized from a 1,275-bp thermostable direct hemolysin (tdh) gene. This oligonucleotide probe specifically reacted with DNA from 89 of 95 V. parahaemolyticus isolates but not with DNA from other vibrios or other enteric and nonenteric organisms (n = 48). The probe hybridized with Southern blots of 0.5-kb HindIII-restricted chromosomal DNA fragments from all but five V. parahaemolyticus test isolates. The probe could be used to directly identify V. parahaemolyticus in artificially contaminated food without an isolation step.     

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.     

Enzyme immunoassay for identification of Vibrio vulnificus in seawater, sediment, and oysters.

Historically, methods used to identify Vibrio vulnificus in environmental samples have been inadequate because isolation and identification procedures are time-consuming and fail to separate V. vulnificus from other bacterial species. We describe an enzyme immunoassay (EIA) and culture techniques which identified V. vulnificus in seawater, sediment, and oysters. The EIA used monoclonal antibody FRBT37 to a species-specific epitope of V. vulnificus. No cross-reactions were observed among 72 non-V. vulnificus strains comprising 34 species and 15 genera. In field trials, the EIA identified correctly 99.7% of 348 biochemically confirmed V. vulnificus isolates. The epitope corresponding to FRBT37 was found in cells lysed by Triton X-100, deionized H2O, and ultrasonication but was not found in culture supernatants, indicating that its location was intracellular. In addition, electron micrographs of V. vulnificus labeled with FRBT37-biotin-avidin-gold showed that epitope FRBT37 reacted with fragments of lysed cells but not whole cells. FRBT37 was expressed when V. vulnificus was cultured in different growth media. The minimum level of detection of the EIA was approximately 2,000 V. vulnificus cells per EIA well. Epitope FRBT37 was labile at 70 degrees C for 30 min. Immunoblot and EIA plate formats reduced assay time and facilitated handling large numbers of test samples.     

Enzyme immunoassay for identification of Vibrio vulnificus in seawater, sediment, and oysters

Historically, methods used to identify Vibrio vulnificus in environmental samples have been inadequate because isolation and identification procedures are time-consuming and fail to separate V. vulnificus from other bacterial species. We describe an enzyme immunoassay (EIA) and culture techniques which identified V. vulnificus in seawater, sediment, and oysters. The EIA used monoclonal antibody FRBT37 to a species-specific epitope of V. vulnificus. No cross-reactions were observed among 72 non-V. vulnificus strains comprising 34 species and 15 genera. In field trials, the EIA identified correctly 99.7% of 348 biochemically confirmed V. vulnificus isolates. The epitope corresponding to FRBT37 was found in cells lysed by Triton X-100, deionized H2O, and ultrasonication but was not found in culture supernatants, indicating that its location was intracellular. In addition, electron micrographs of V. vulnificus labeled with FRBT37-biotin-avidin-gold showed that epitope FRBT37 reacted with fragments of lysed cells but not whole cells. FRBT37 was expressed when V. vulnificus was cultured in different growth media. The minimum level of detection of the EIA was approximately 2,000 V. vulnificus cells per EIA well. Epitope FRBT37 was labile at 70 degrees C for 30 min. Immunoblot and EIA plate formats reduced assay time and facilitated handling large numbers of test samples.     

Real-time PCR analysis of Vibrio vulnificus from oysters

Vibrio vulnificus is an opportunistic human pathogen commonly found in estuarine environments. Infections are associated with raw oyster consumption and can produce rapidly fatal septicemia in susceptible individuals. Standard enumeration of this organism in shellfish or seawater is laborious and inaccurate; therefore, more efficient assays are needed. An oligonucleotide probe derived from the cytolysin gene, vvhA, was previously used for colony hybridizations to enumerate V. vulnificus. However, this method requires overnight growth, and vibrios may lack culturability under certain conditions. In the present study, we targeted the same locus for development of a TaqMan real-time PCR assay. Probe specificity was confirmed by amplification of 28 V. vulnificus templates and by the lack of a PCR product with 22 non-V. vulnificus strains. Detection of V. vulnificus in pure cultures was observed over a 6-log-unit linear range of concentration (10(2) to 10(8) CFU ml(-1)), with a lower limit of 72 fg of genomic DNA micro l of PCR mixture(-1) or the equivalent of six cells. Similar sensitivity was observed in DNA extracted from mixtures of V. vulnificus and V. parahaemolyticus cells. Real-time PCR enumeration of artificially inoculated oyster homogenates correlated well with colony hybridization counts (r(2) = 0.97). Numbers of indigenous V. vulnificus cells in oysters by real-time PCR showed no significant differences from numbers from plate counts with probe (t test; P = 0.43). Viable but nonculturable cells were also enumerated by real-time PCR and confirmed by the BacLight viability assay. These data indicate that real-time PCR can provide sensitive species-specific detection and enumeration of V. vulnificus in seafood.     

Persistence of caliciviruses in artificially contaminated oysters during depuration

The fate of calicivirus in oysters in a 10-day depuration was assessed. The norovirus gene was persistently detected from artificially contaminated oysters during the depuration, whereas feline calicivirus in oysters was promptly eliminated. The prolonged observation of norovirus in oysters implies the existence of a selective retention mechanism for norovirus within oysters.     

Polymerase chain reaction identification of Salmonella serotypes

Seventy-seven Salmonella isolates comprising 61 different serotypes were subjected to polymerase chain reaction (PCR) fingerprinting using two primersets. Primerset L1/G1, amplifying the spacer regions between the 16S and 23S rRNA genes, resulted in simple PCR fingerprints. However, in some cases PCR amplification of different Salmonella serotypes with primerset L1/G1 resulted in identical fingerprint profiles. Fingerprints obtained with the ERIC primerset, that matches the enterobacterial repetitive intergenic consensus sequence, were more complicated but were serotype-specific. Consequently, fingerprinting with the ERIC primerset is applicable for typing Salmonella up to the serotype level. Fingerprinting with the L1 and G1 primers requires an additional treatment of the amplification product for accurate typing of salmonellas. Phage typing is not possible with either primerset.     

Polymerase chain reaction analysis of transgenic plants contaminated byAgrobacterium

Agrobacterium-mediated genetic transformation is a method of choice for the development of transgenic plants. The presence of latentAgrobacterium that multiplies in the plant tissue in spite of antibiotic application confounds the results obtained by polymerase chain reaction (PCR) analysis of putative transgenic plants. The presence ofAgrobacterium can be confirmed by amplification of eitherAgrobacterium chromosomal genes or genes present out of transfer DNA (T-DNA) in the binary vector. However, the transgenic nature ofAgrobacterium-contaminated transgenic plants cannot be confirmed by PCR. Here we report a simple protocol for PCR analysis ofAgrobacterium-contaminated transgenic plants. This protocol is based on denaturation and renaturation of DNA. The contaminating plasmid vector becomes double-stranded after renaturation and is cut by a restriction enzyme having site(s) within the PCR amplicon. As a result, amplification by PCR is not possible. The genomic DNA with a few copies of the transgene remains single-stranded and unaffected by the restriction enzyme, leading to amplification by PCR. This protocol has been successfully tested with 4 different binary vectors and 3Agrobacterium tumefaciens strains: EHA105, LBA4404, and GV3101.     

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.     

Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells.

Vibrio vulnificus is a human pathogen associated with consumption of raw oysters. During the colder months the organism apparently enters a viable but nonculturable state and thus cannot be cultured by ordinary bacteriological methods. For this reason, another means of detecting this bacterium is necessary. In the present study we utilized the polymerase chain reaction (PCR) to detect V. vulnificus DNA, thus eliminating the problem of nonculturability. DNA from both culturable and nonculturable cells of V. vulnificus was amplified by PCR with primers flanking a 340-bp fragment of the cytotoxin-hemolysin gene. As little as 72 pg of DNA from culturable cells and 31 ng of DNA from nonculturable cells could be detected. Fifty cycles of a two-step reaction (30 s [each] at 94 and 65 degrees C) were found to be optimal as well as more time efficient than the three-step PCR. The total procedure from the point of DNA extraction to observation on a gel required less than 8 h. Possible reasons for the difficulties encountered in amplifying DNA from nonculturable cells, e.g., gene rearrangement or loss of the hemolysin gene, are discussed.     

Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells.

Vibrio vulnificus is a human pathogen associated with consumption of raw oysters. During the colder months the organism apparently enters a viable but nonculturable state and thus cannot be cultured by ordinary bacteriological methods. For this reason, another means of detecting this bacterium is necessary. In the present study we utilized the polymerase chain reaction (PCR) to detect V. vulnificus DNA, thus eliminating the problem of nonculturability. DNA from both culturable and nonculturable cells of V. vulnificus was amplified by PCR with primers flanking a 340-bp fragment of the cytotoxin-hemolysin gene. As little as 72 pg of DNA from culturable cells and 31 ng of DNA from nonculturable cells could be detected. Fifty cycles of a two-step reaction (30 s [each] at 94 and 65 degrees C) were found to be optimal as well as more time efficient than the three-step PCR. The total procedure from the point of DNA extraction to observation on a gel required less than 8 h. Possible reasons for the difficulties encountered in amplifying DNA from nonculturable cells, e.g., gene rearrangement or loss of the hemolysin gene, are discussed.