Real-Time PCR Assays for Quantification and Differentiation of Vibrio vulnificus Strains in Oysters and Water

Vibrio vulnificus is an autochthonous estuarine bacterium and a pathogen that is frequently transmitted via raw shellfish. Septicemia can occur within 24 h; however, isolation and confirmation from water and oysters require days. Real-time PCR assays were developed to detect and differentiate two 16S rRNA variants, types A and B, which were previously associated with environmental sources and clinical fatalities, respectively. Both assays could detect 10² to 10³ V. vulnificus total cells in seeded estuarine water and in oyster homogenates. PCR assays on 11 reference V. vulnificus strains and 22 nontarget species gave expected results (type A or B for V. vulnificus and negative for nontarget species). The relationship between cell number and cycle threshold for the assays was linear (R² = >0.93). The type A/B ratio of Florida clinical isolates was compared to that of isolates from oysters harvested in Florida waters. This ratio was 19:17 in clinical isolates and 5:8 (n = 26) in oysters harvested from restricted sites with poor water quality but was 10:1 (n = 22) in oysters from permitted sites with good water quality. A substantial percentage of isolates from oysters (19.4%) were type AB (both primer sets amplified), but no isolates from overlying waters were type AB. The real-time PCR assays were sensitive, specific, and quantitative in water samples and could also differentiate the strains in oysters without requiring isolation of V. vulnificus and may therefore be useful for rapid detection of the pathogen in shellfish and water, as well as further investigation of its population dynamics.     

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.     

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.     

Multiplex PCR Assay for Detection of Vibrio vulnificus Biotype 2 and Simultaneous Discrimination of Serovar E Strains

In the present work we develop a multiplex PCR assay for the detection and identification of the fish pathogen Vibrio vulnificus biotype 2 with discriminating potential for zoonotic strains (serovar E). The PCR assay allowed the identification of two new biotype 2 serovar E human isolates from culture collections. Finally, the multiplex was successfully applied to both diagnosis and carrier detection in field samples.     

Genetic Distinctions among Clinical and Environmental Strains of Vibrio vulnificus

Vibrio vulnificus causes rare but frequently fatal septicemia associated with raw oyster consumption by persons with underlying hepatic or immune system dysfunction. The virulence potential of environmental reservoirs appears widely distributed, because most strains are virulent in animal models; however, several investigations recently demonstrated genetic divergence among strains from clinical versus environmental origin at independent genetic loci. The present study used PCR to screen DNA polymorphisms in strains from environmental (n = 35) or clinical (n = 33) sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P < 0.01) association was observed for typical “clinical” or “environmental” polymorphism profiles based on strain origin. Most oyster isolates (88%), including all of those with the “environmental” profile, also formed a single rep-PCR genogroup. Clinical isolates within this group did not have the typical “clinical” profile. On the other hand, clinical isolates with the typical polymorphism profile were distributed among multiple rep-PCR genogroups, demonstrating greater genetic diversity than was evident by profiling genetic polymorphisms. Wound isolates were genetically distinct from typical blood isolates by all assays. Strains from an outbreak of wound infections in Israel (biotype 3) were closely related to several U.S. strains by rep-PCR, indicating potential reservoirs of emerging disease. Strains genetically related to blood isolates appeared to be relatively rare in oysters, as only one had the “clinical” polymorphism profile or clustered by rep-PCR. However, this study was not an extensive survey, and more sampling using rep-PCR for sensitive genetic discrimination is needed to determine the virulence potential of environmental reservoirs.     

Genetic Characterization of Vibrio vulnificus Strains from Tilapia Aquaculture in Bangladesh

Outbreaks of Vibrio vulnificus wound infections in Israel were previously attributed to tilapia aquaculture. In this study, V. vulnificus was frequently isolated from coastal but not freshwater aquaculture in Bangladesh. Phylogenetic analyses showed that strains from Bangladesh differed remarkably from isolates commonly recovered elsewhere from fish or oysters and were more closely related to strains of clinical origin.Vibrio vulnificus causes severe wound infections and life-threatening septicemia (mortality, >50%), primarily in patients with underlying chronic diseases (10, 19, 23) and primarily from raw oyster consumption (21). This Gram-negative halophile is readily recovered from oysters (27, 35, 43) and fish (14) and was initially classified into two biotypes (BTs) based on growth characteristics and serology (5, 18, 39). Most human isolates are BT1, while BT2 is usually associated with diseased eels (1, 39). An outbreak of wound infections from aquacultured tilapia in Israel (6) revealed a new biotype (BT3). Phenotypic assays do not consistently distinguish biotypes (33), but genetic analyses have helped resolve relationships (20). A 10-locus multilocus sequence typing (MLST) scheme (8, 9) and a similar analysis of 6 loci (13) segregated V. vulnificus strains into two clusters. BT1 strains were in both clusters, while BT2 segregated into a single cluster and BT3 was a genetic mosaic of the two lineages. Significant associations were observed between MLST clusters and strain origin: most clinical strains (BT1) were in one cluster, and the other cluster was comprised mostly of environmental strains (some BT1 and all BT2). Clinical isolates were also associated with a unique genomic island (13).The relationship between genetic lineages and virulence has not been determined, and confirmed virulence genes are universally present in V. vulnificus strains from both clinical and environmental origins (19, 23). However, segregation of several polymorphic alleles agreed with the MLST analysis and correlated genotype with either clinical or environmental strain origin. Alleles include 16S rRNA loci (15, 26, 42), a virulence-correlated gene (vcg) locus (31, 41, 42), and repetitive sequence in the CPS operon (12). DiversiLab repetitive extrageneic palindromic (rep-PCR) analysis also confirmed these genetic distinctions and showed greater diversity among clinical strains (12).Wound infections associated with tilapia in Israel implicated aquaculture as a potential source of V. vulnificus in human disease (6, 40). Tilapia aquaculture is increasing rapidly, as shown by a 2.8-fold increase in tons produced from 1998 to 2007 (Food and Agriculture Organization; http://www.fao.org/fishery/statistics/en). Therefore, presence of V. vulnificus in tilapia aquaculture was examined in Bangladesh, a region that supports both coastal and freshwater sources of industrial-scale aquaculture. V. vulnificus strains were recovered from market fish, netted fish, and water samples, and the phylogenetic relationship among strains was examined relative to clinical and environmental reference strains collected elsewhere.     

Thermal factors influencing detection of Vibrio vulnificus using real-time PCR

Five thermal factors, including initial denaturation temperature, cycling denaturation temperature, annealing temperature, extension temperature and the temperature at which the intensity of the fluorescent signal is read, were evaluated for their effects on the detection of Vibrio vulnificus via real-time PCR. Fluorescent signal detection after extension was set between the Tm value of the primer-dimers (79 degrees C) and that of the PCR target amplicons (84 degrees C). This effectively eliminated the overestimation of the yield of PCR amplicons due to the presence of primer-dimers which otherwise led to erroneously lower Ct values (1.91+/-0.22 cycles lower). The annealing and extension steps were combined to convert a three-step PCR to a two-step PCR. This consisted of initial denaturation at 95 degrees C for 3 min, cycling denaturation at 94 degrees C for 15 s and a combined annealing and extension step at 60 degrees C for 5 s in each PCR cycle. One genomic target per real-time PCR reaction was detected with the simplified two-step PCR.     

A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus

AIM: To establish a simple multiplex polymerase chain reaction (PCR) that will identify Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. METHODS AND RESULTS: A total of 429 Vibrio spp. from various origins were tested with the novel primers targeting toxR. The reverse primers were all designed to be species specific, while the forward primer was universal. The primers correctly identified all the V. parahaemolyticus, V. cholerae and V. vulnificus isolates tested. CONCLUSIONS: The toxR multiplex PCR works well when the initial colony morphology is known. If not, Vibrio alginolyticus might represent a diagnostic obstacle. SIGNIFICANCE AND IMPACT OF THE STUDY: The method provides a fast and reliable way of identifying the main Vibrio spp. involved in food-borne disease. The method could prove very useful for laboratories working with identification of these Vibrio spp.     

多重PCR方法检测霍乱弧菌的研究

霍乱弧菌是霍乱的病原体,可以分为O1群、O139群和非O1/非O139群。O1群和O139群霍乱弧菌产生的霍乱肠毒素(也称霍乱毒素)是产生霍乱的主要原因,也只有O1群和O139群霍乱弧菌可引起霍乱。其他群的霍乱弧菌毒性不高,但在食品中也不允许被检出。实验以霍乱胶原酶基因和霍乱毒素基因为目的基因,试图建立一种PCR方法对霍乱弧菌进行检测研究,结果表明此方法可以用于食品中的霍乱弧菌检测。     

Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR

Ethidium bromide monoazide (EMA) was utilized to selectively allow the real-time PCR (RT-PCR) amplification of a targeted DNA sequence in viable but not dead cells of Vibrio vulnificus. The optimized light exposure time to achieve cross-linking of DNA by the EMA in dead cells and to photolyse the free EMA in solution was at least 15 min. The use of 3.0 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable cells of V. vulnificus. The minimum amount of EMA to completely inhibit the RT-PCR amplification of DNA derived from heat-killed cells was 2.5 microg/ml. Amplification of DNA from dead cells in a mixture with viable cells was successfully inhibited by 2.5 microg/ml of EMA, whereas the DNA from viable cells present was successfully amplified by RT-PCR.     

水产品中创伤弧菌和副溶血弧菌双重PCR检测方法的建立

目的 建立一种同步检测创伤弧菌和副溶血弧菌的双重PCR方法。方法 选择副溶血弧菌tlh基因和创伤弧菌vvhA基因作为靶序列各设计一对引物。用合成的引物对副溶血弧菌和创伤弧菌进行双重PCR扩增,确定特异性和最低检出限。然后用此方法对53株副溶血弧菌和7株创伤弧菌进行检测。结果 确定了双重PCR检测创伤弧菌和副溶血弧菌的最优反应条件,其中退火温度为60 ℃,方法具有较好的特异性。对副溶血弧菌的最低限为1.0×102 CFU/mL,创伤弧菌最低限为4.2×104 CFU/mL。双重PCR对分离株检测符合率达100%。结论 建立的双重PCR方法简便、快速、特异性好,可同时检测副溶血弧菌和创伤弧菌,为水产品中病原菌的基层检测提供解决方案。     

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus

Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species‐specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co‐existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost‐effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.     

Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish

In this study, we developed a PCR-based rapid detection method for clinically important pathogenic strains of Vibrio vulnificus. Positive amplification of the 504-bp viuB fragment was seen in all 22 clinical isolates tested but only in 8 out of 33 environmental isolates. The combination of the species-specific 205-bp vvh fragment along with viuB in a multiplexed PCR enabled us to confirm the presence of potentially pathogenic strains of V. vulnificus. No amplification of other Vibrio spp. or non-Vibrio bacteria was evidenced, suggesting a high specificity of detection by this method. The sensitivity of detection for both targeted genes was 10 pg of purified DNA, which correlated with 10(3) V. vulnificus CFU in 1 mL of pure culture or 1 g un-enriched seeded oyster tissue homogenate. This sensitivity was improved to 1 CFU per gram of oyster tissue homogenate in overnight-enriched samples. A SYBR Green I based real-time PCR method was also developed that was shown to produce results consistent with the conventional PCR method. Application of the multiplexed real-time PCR to natural oyster tissue homogenates exhibited positive detection of vvh in 51% of the samples collected primarily during the summer months; however, only 15% of vvh positive samples exhibited viuB amplicons. The rapid, sensitive, and specific detection of clinically important pathogenic V. vulnificus in shellfish would be beneficial in reducing illnesses and deaths caused by this pathogen.     

Adaptive Response to Cold Temperatures in Vibrio vulnificus

The effectiveness of rapid chilling or freezing of oysters to reduce Vibrio vulnificus levels in shellfish may be compromised by product handling procedures that permit cold adaptation. When a V. vulnificus culture was shifted from 35°C to 6°C conditions, it underwent transition to a non-culturable state. Cells adapted to 15°C prior to change to 6°C condition, however, remain viable and culturable. In addition, cultures adapted to 15°C were able to survive better upon freezing at −78°C compared with cultures frozen directly from 35°C. Inhibition of protein synthesis by addition of chloramphenicol in a V. vulnificus culture immediately prior to the exposure to the adaptive temperature eliminated inducible cold tolerance. These results suggest that cold-adaptive “protective” proteins may enhance survival and tolerance at cold temperatures. In addition, removal of iron from the growth medium by adding 2,2′-Dipyridyl prior to cold adaptation decreased the viability by approximately 2 logarithm levels. This suggests that iron plays an important role in adaptation at cold temperatures. Analysis of total cellular proteins on an SDS polyacrylamide gel electrophoresis, labeled with ³⁵S-methionine during exposure at 15°C, showed elevated expressions of a 6-kDa and a 40-kDa protein and decreased expression of an 80-kDa protein. These results suggest that, for V. vulnificus, survival and tolerance at cold temperatures could be due to the expression of cold-adaptive proteins other than previously documented major cold shock proteins such as CS7.4 and CsdA. In this study, for the first time we have shown that exposure to an intermediate cold temperature (15°C) causes a cold adaptive response, helping this pathogen remain in culturable state when exposed to a much colder temperature (6°C). This adaptive nature to cold temperatures could be important for shellfish industry efforts to reduce the risk of V. vulnificus infection from consuming raw oysters. Received: 30 July 1998 / Accepted: 1 October 1998     

Pulsed-Field Gel Electrophoresis Analysis of Vibrio vulnificus Strains Isolated from Taiwan and the United States

Vibrio vulnificus is a marine bacterium that causes human wound infections and septicemia with a high mortality rate. V. vulnificus strains from different clinical and environmental sources or geographic regions have been successfully characterized by ribotyping and several other methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminative method, but previous studies suggested that it was not suitable for examining the correlation of V. vulnificus strains from different origins. We employed PFGE to determine its efficacy for characterizing V. vulnificus strains from different geographic regions, characterizing a total of 153 strains from clinical and environmental origins from the United States and Taiwan after SfiI or NotI digestion. V. vulnificus strains showed a high intraspecific diversity by PFGE after SfiI or NotI digestion, and about 12% of the strains could not be typed by the use of either of these enzymes. For PFGE with SfiI digestion, most of the clinical and environmental strains from the United States were grouped into cluster A, while the strains from Taiwan were grouped into other clusters. Clinical strains from the United States showed a higher level of genetic homogeneity than clinical strains from Taiwan, and environmental strains from both regions showed a similarly high level of heterogeneity. PFGE with NotI digestion was useful for studying the correlation of clinical strains from the United States and Taiwan, but it was not suitable for analyzing environmental strains. The results showed that PFGE with SfiI digestion may be used to characterize V. vulnificus strains from distant geographic regions, with NotI being a recommended alternative enzyme.     

Identification of DNA Sequences Specific for Vibrio vulnificus Biotype 2 Strains by Suppression Subtractive Hybridization

Vibrio vulnificus can be divided into three biotypes, and only biotype 2, which is further divided into serovars, contains eel-virulent strains. We compared the genomic DNA of a biotype 2 serovar E isolate (tester) with the genomic DNAs of three biotype 1 strains by suppression subtractive hybridization and then tested the distribution of the tester-specific DNA sequences in a wide collection of bacterial strains. In this way we identified three plasmid-borne DNA sequences that were specific for biotype 2 strains irrespective of the serovar and three chromosomal DNA sequences that were specific for serovar E biotype 2 strains. These sequences have potential for use in the diagnosis of eel vibriosis caused by V. vulnificus and in the detection of biotype 2 serovar E strains.     

Molecular Typing of Environmental and Clinical Strains of Vibrio vulnificus Isolated in the Northeastern USA

Vibrio vulnificus is a ubiquitous marine bacterium that is responsible for infections and some seafood-related illnesses and deaths in the United States, mainly in individuals with compromised health status in the Gulf of Mexico region. Most phylogenetic studies focus on V. vulnificus strains isolated in the southern United States, but almost no genetic data are available on northeastern bacterial isolates of clinical or environmental origin. Our goal in this study was to examine the genetic diversity of environmental strains isolated from commercially-produced oysters and in clinical strains of known pathogenicity in northeastern United States. We conducted analyses of a total of eighty-three strains of V. vulnificus, including 18 clinical strains known to be pathogenic. A polyphasic, molecular-typing approach was carried out, based upon established biotypes, vcg, CPS, 16S rRNA types and three other genes possibly associated with virulence (arylsulfatase A, mtlABC, and nanA). An established Multi Locus Sequence Typing (MLST) method was also performed. Phylogenetic analyses of these markers and MLST results produced similar patterns of clustering of strains into two main lineages (we categorized as ‘LI’ and ‘LII’), with clinical and environmental strains clustering together in both lineages. Lineage LII was comprised primarily but not entirely of clinical bacterial isolates. Putative virulence markers were present in both clinical and environmental strains. These results suggest that some northeastern environmental strains of V. vulnificus are phylogenetically close to clinical strains and probably are capable of virulence. Further studies are necessary to assess the risk of human illness from consuming raw oysters harvested in the northeastern US.