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.     

Presence of virulence markers in environmental Vibrio vulnificus strains

Aims

This work aims to demonstrate the presence of several genes and factors associated with virulence in strains isolated from the environment at Pueblo Viejo Lagoon, State of Veracruz, Mexico.

Methods and Results

In this study, we investigated the production of V. vulnificus virulence factors, as cytolysin (haemolysin), RTX toxin, metalloprotease, siderophores, capsular polysaccharide, adhesion structures (like type IV pili), and polar and lateral flagella, involved in swimming and swarming (or, at least, the presence of genes encoding some of them) in 40 strains of V. vulnificus isolated from water and food. The results indicate that strains of environmental origin possess potential virulence characteristics.

Conclusions

Caution should be exercised when consuming raw shellfish (especially by those more susceptible risk groups).

Significance and Impact of the Study

This is the first work focused on the evaluation of V. vulnificus virulence factors in Mexico.     

Antimicrobial susceptibilities of Vibrio parahaemolyticus and Vibrio vulnificus isolates from Louisiana Gulf and retail raw oysters

The antimicrobial susceptibilities of 168 Vibrio parahaemolyticus and 151 Vibrio vulnificus isolates recovered from 82 Louisiana Gulf and retail oysters in 2005 and 2006 were determined. Overall, the two vibrios remained susceptible to the majority of antimicrobials tested; reduced susceptibility was detected only in V. parahaemolyticus for ampicillin (81%; MIC > or = 16 microg/ml). Additionally, V. parahaemolyticus displayed significantly higher MICs for cefotaxime, ciprofloxacin, and tetracycline than V. vulnificus.     

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.     

Uptake and clearance of Vibrio vulnificus from Gulf coast oysters (Crassostrea virginica).

Oysters collected in late winter, when they were free of Vibrio vulnificus, were exposed in the organism in the laboratory. The oysters effectively concentrated the bacteria from seawater, but when the inoculum was removed, the bacteria were rapidly cleared from the oyster tissues. These results suggest that V. vulnificus may be found in oysters as a result of filtration of the bacteria from seawater rather than active multiplication of the bacteria in the oysters.     

Methods for isolation and confirmation of Vibrio vulnificus from oysters and environmental sources: a review

The gram-negative bacterium Vibrio vulnificus is a natural inhabitant of estuarine waters and poses a significant health threat to humans who suffer from immune disorders, liver disease, or hemochromatosis (iron overload). V. vulnificus enters human hosts via wound infections or consumption of raw shellfish (primarily oysters), and infections frequently progress to septicemia and death in susceptible individuals. Prevalence in waters and shellfish is not correlated with fecal indicator organisms; therefore, species-specific detection and enumeration of V. vulnificus in the environment has become a priority for agencies that are responsible for shellfish safety. The many selective-differential media developed for isolation of Vibrio spp., and specifically for V. vulnificus detection, are reviewed here; however, none of the media developed to date combines the sensitivity to low numbers with the specificity necessary to inhibit growth of other organisms. Therefore, immunological and molecular protocols are needed for confirmation of the identity of the organism and are discussed in detail. Methods under development that hold promise for rapid, accurate, and sensitive detection and enumeration of the organism include multiplex and real-time PCR. Developing technologies that have proven useful for detection and investigation of other pathogens such as biosensors, spectroscopy and microarrays may provide the next generation of tools for investigation of the prevalence and ecology of V. vulnificus.     

Uptake and clearance of Vibrio vulnificus from Gulf coast oysters (Crassostrea virginica)

Oysters collected in late winter, when they were free of Vibrio vulnificus, were exposed in the organism in the laboratory. The oysters effectively concentrated the bacteria from seawater, but when the inoculum was removed, the bacteria were rapidly cleared from the oyster tissues. These results suggest that V. vulnificus may be found in oysters as a result of filtration of the bacteria from seawater rather than active multiplication of the bacteria in the oysters.     

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.     

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.     

Factors and markers of virulence in Escherichia coli from human septicemia

A lethal and necrotic factor which causes cell multinucleation in HeLa cell cultures has previously been shown to be coded by the Vir plasmid of Escherichia coli. Using an absorbed rabbit antiserum which neutralized the Vir toxic properties, we have compared the SDS-PAGE immunoblots from laboratory and field strains which either produce or do not produce Vir toxicity. A single band of 110 kDa was found to be specifically associated with vir toxicity in E. coli strains. This antiserum also recognized the 115 kDa protein band which was previously identified as the cytotoxic necretozing factor (CNF) of certain E. coli strains. These results suggest that the toxin coded by the Vir plasmid is a protein of 110 kDa distinct from, but immunologically related to CNF.     

创伤弧菌致病性及其毒力因子研究进展

创伤弧菌是一种嗜盐性的革兰阴性弧菌,存在于河海交界之处。自1964年首次被美国CDC分离出后得到了研究者的广泛关注,它与霍乱弧菌、副溶血性弧菌合称为致人类感染的三大弧菌。创伤弧菌可引起肠胃感染、伤口感染及原发性败血症等疾病,其感染存在发病急、病死率高等特点,给公共卫生造成了沉重负担。其中,多种毒力因子在创伤弧菌的致病中起到了至关重要的作用,如溶细胞素、金属蛋白酶、铁载体、荚膜多糖等。因此,本文对创伤弧菌的生物学特性、致病情况及相关毒力因子进行了详细介绍,以期为病原微生物的诊断、预防和治疗提供新的启示。     

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.     

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.     

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.     

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 (10² to 10⁸ CFU ml⁻¹), with a lower limit of 72 fg of genomic DNA μl 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 (r² = 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.     

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.     

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.