Rapid serological identification of Vibrio vulnificus by anti-H coagglutination.

Staphylococcus aureus Cowan 1 cells were armed with anti-flagellar (anti-H) antibody produced in rabbits immunized with flagellar core protein prepared from Vibrio vulnificus. This reagent was assessed by coagglutination for its capacity to agglutinate and identify V. vulnificus. A species-specific H antigen is expressed in the core proteins of the polar flagella of V. vulnificus. Of 435 V. vulnificus isolates identified bacteriologically, 432 (99.3%) were agglutinated in the slide test within 2 min after the addition of the anti-V. vulnificus H coagglutination reagent. Other than Vibrio pelagius, the reagent did not agglutinate 19 heterologous Vibrio spp. tested, including 290 V. cholerae, 22 V. mimicus, 395 V. parahaemolyticus, and 16 V. fluvialis isolates recovered from seafood and the marine environment. The serological resolution of the coagglutination reaction was enhanced if the organism under test was suspended in 0.1 M Tris buffer-0.1 mM EDTA-1.0% Triton X-100 (TET) for 24 h before serological examination. The TET buffer also increased the sensitivity of the coagglutination reaction 100-fold over that for isolates suspended in 0.3% formalinized phosphate-buffered saline before testing. The anti-H coagglutination test is a rapid, serologically specific, and inexpensive procedure for identifying V. vulnificus one step beyond primary isolation.     

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.     

Distribution of Vibrio vulnificus and other lactose-fermenting vibrios in the marine environment

During the summer of 1981, 3,887 sucrose-negative vibrios were isolated from seawater, sediment, plankton, and animal samples taken from 80 sites from Miami, Fla., to Portland, Maine. Of these, 4.2% were able to ferment lactose. The lactose-positive strains isolated from the various samples correlated positively with pH and turbidity of the water, vibrios in the sediment and oysters, and total bacterial counts in oysters. Negative correlations were obtained for water salinity. Numerical taxonomy was performed on 95 of the lactose-fermenting environmental isolates and 23 reference strains. Five clusters resulted, with the major cluster containing 33 of the environmental isolates and all of the Vibrio vulnificus reference strains. The 33 isolates, which produced an acid reaction in lactose broth within hours of initial inoculation, represented 20% of all lactose-fermenting vibrios studied. These isolates were nearly identical phenotypically to clinical strains of V. vulnificus studied by the Centers for Disease Control, Atlanta, Ga., and by our laboratory, and their identification was confirmed by DNA-DNA hybridization studies. V. vulnificus was isolated from all sample types and from Miami to Cape Cod, Mass., and comparison of the environmental parameters of the eight subsites yielding this species with those of all 80 subsites revealed no significant differences. The majority of the isolates were obtained from animals, with clams providing most (84%) of these. On injection into mice, 82% of the V. vulnificus isolates resulted in death. Members of the remaining four clusters contained strains which differed from V. vulnificus in such phenotypic traits as luminescence and in urease or H(2)S production. None of the other reference cultures, including nine other Vibrio species, were contained in the remaining clusters, and these isolates could not be identified. Most of these were also lethal for mice. Phenotypic differences, potential pathogenicity, and geographic distribution of the five clusters were examined. It is concluded that V. vulnificus is a ubiquitous organism, both geographically and in a variety of environmental sources, although it occurs in relatively low numbers. The public health significance of this organism and of the other unidentified lactose-fermenting Vibrio species is discussed.     

Occurrence of Vibrio parahaemolyticus, V. cholerae, and V. vulnificus in Norwegian Blue Mussels (Mytilus edulis)

Vibrio parahaemolyticus, V. cholerae, and V. vulnificus were isolated from 10.3%, 1.0%, and 0.1% of 885 blue mussel samples, respectively. Four of the samples contained trh(+) V. parahaemolyticus, while no tdh-positive isolates were detected. The V. cholerae isolates were non-O:1/non-O:139 serotypes and were ctxA negative.     

Identification of Vibrio spp. (other than V. vulnificus) recovered on CPC agar from marine natural samples.

Two hundred and eighty four presumptive but not confirmed Vibrio vulnificus isolates grown on cellobiose-polymixin B-colistin agar (CPC) at 40 degrees C, recovered from sea water samples from Valencia, Spain, during a microbiological survey for V. vulnificus, were phenotypically identified. Most of the isolates (91%) corresponded to Vibrio species. V. harveyi (24%) and V. splendidus(19%) were the most abundant species identified, followed by V. navarrensis (13%), V. alginolyticus (8%) and V. parahaemolyticus (5%). The ability to grow on CPC agar and ferment cellobiose of several V. vulnificus strains from different origins and serovars, including reference strains, was tested. Most serovar E isolates and 25% of non-serovar E isolates could not grow on CPC agar.     

Occurrence of pathogenic vibrios in coastal areas of France

AIMS: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. METHODS AND RESULTS: Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains. CONCLUSIONS: The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.     

Identification of environmental Vibrio vulnificus isolates with a DNA probe for the cytotoxin-hemolysin gene

We screened 44 lactose-positive Vibrio strains isolated from the marine environment for homology with a 3.2-kilobase DNA fragment encoding the Vibrio vulnificus cytotoxin-hemolysin gene. All 29 marine isolates identified as V. vulnificus on the basis of numerical taxonomy and DNA-DNA hybridization studies hybridized with the cytotoxin gene probe, as did all V. vulnificus reference strains. Homologous gene sequences were identified in no other lactose-positive marine vibrio isolates nor in 10 other Vibrio species.     

Identification of environmental Vibrio vulnificus isolates with a DNA probe for the cytotoxin-hemolysin gene.

We screened 44 lactose-positive Vibrio strains isolated from the marine environment for homology with a 3.2-kilobase DNA fragment encoding the Vibrio vulnificus cytotoxin-hemolysin gene. All 29 marine isolates identified as V. vulnificus on the basis of numerical taxonomy and DNA-DNA hybridization studies hybridized with the cytotoxin gene probe, as did all V. vulnificus reference strains. Homologous gene sequences were identified in no other lactose-positive marine vibrio isolates nor in 10 other Vibrio species.     

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.     

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.     

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.     

Sandwich enzyme-linked immunosorbent assay for Vibrio vulnificus hemolysin to detect V. vulnificus in environmental specimens.

Vibrio vulnificus hemolysin, purified by quantitative isoelectric focusing, was used to prepare rabbit and goat anti-hemolysin. The resulting antibodies were used as capture and detector antibody reagents in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect V. vulnificus in environmental samples. By this technique, 4 laboratory-maintained V. vulnificus strains and 33 environmental V. vulnificus isolates were detected. Also, the technique distinguished five other Vibrio species from V. vulnificus, and when it was used in combination with colistin-polymyxin-cellobiose agar, 31 non-V. vulnificus isolated were excluded. This sandwich ELISA compared favorably with the current Food and Drug Administration standard immunoassay in confirming presumptive V. vulnificus colonies from environmental specimens: oysters, sediment, and seawater. Among 340 presumptive V. vulnificus colonies, the sandwich ELISA detected 95% of the confirmed V. vulnificus colonies. Equally important, the technique correctly distinguished 99% of the non-V. vulnificus colonies. The sandwich ELISA offers time-saving and labor-saving advantages over the currently accepted immunoassay.     

Real-time PCR detection of Vibrio vulnificus in oysters: comparison of oligonucleotide primers and probes targeting vvhA

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (C(T)) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 10(3) V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 x 10(3) CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.     

Ecology of Vibrio vulnificus in estuarine waters of eastern North Carolina

While several studies on the ecology of Vibrio vulnificus in Gulf Coast environments have been reported, there is little information on the distribution of this pathogen in East Coast waters. Thus, we conducted a multiyear study on the ecology of V. vulnificus in estuarine waters of the eastern United States, employing extensive multiple regression analyses to reveal the major environmental factors controlling the presence of this pathogen, and of Vibrio spp., in these environments. Monthly field samplings were conducted between July 2000 and April 2002 at six different estuarine sites along the eastern coast of North Carolina. At each site, water samples were taken and nine physicochemical parameters were measured. V. vulnificus isolates, along with estuarine bacteria, Vibrio spp., Escherichia coli organisms, and total coliforms, were enumerated in samples from each site by using selective media. During the last 6 months of the study, sediment samples were also analyzed for the presence of vibrios, including V. vulnificus. Isolates were confirmed as V. vulnificus by using hemolysin gene PCR or colony hybridization. V. vulnificus was isolated only when water temperatures were between 15 and 27 degrees C, and its presence correlated with water temperature and dissolved oxygen and vibrio levels. Levels of V. vulnificus in sediments were low, and no evidence for an overwintering in this environment was found. Multiple regression analysis indicated that vibrio levels were controlled primarily by temperature, turbidity, and levels of dissolved oxygen, estuarine bacteria, and coliforms. Water temperature accounted for most of the variability in the concentrations of both V. vulnificus (47%) and Vibrio spp. (48%).     

Virulence of Vibrio vulnificus strains from marine environments

Vibrio vulnificus strains isolated from geographically diverse marine sources were compared with clinical isolates for phenotype and in vitro and in vivo production of virulence factors. There were no differences between environmental and clinical strains on the basis of biochemical characteristics or antimicrobial susceptibility patterns. Cytolysin and cytotoxin titers produced by environmental strains were generally comparable to those of clinical strains. Of 29 environmental isolates tested, 25 were pathogenic for mice. These data show that environmental V. vulnificus strains are phenotypically indistinguishable from clinical isolates and that approximately 90% of the environmental strains tested produced in vitro virulence factors and in vivo pathogenicity for mice comparable to those produced by clinical V. vulnificus isolates.     

Virulence of Vibrio vulnificus strains from marine environments.

Vibrio vulnificus strains isolated from geographically diverse marine sources were compared with clinical isolates for phenotype and in vitro and in vivo production of virulence factors. There were no differences between environmental and clinical strains on the basis of biochemical characteristics or antimicrobial susceptibility patterns. Cytolysin and cytotoxin titers produced by environmental strains were generally comparable to those of clinical strains. Of 29 environmental isolates tested, 25 were pathogenic for mice. These data show that environmental V. vulnificus strains are phenotypically indistinguishable from clinical isolates and that approximately 90% of the environmental strains tested produced in vitro virulence factors and in vivo pathogenicity for mice comparable to those produced by clinical V. vulnificus isolates.     

First description of nonmotile Vibrio vulnificus strains virulent for eels

Nonmotile Vibrio vulnificus strains were isolated as pure cultures from body ulcers and internal organs of wild diseased European eels caught in a Mediterranean freshwater coastal lagoon. All 54 V. vulnificus isolates were nonmotile, indole-, ornithine decarboxilase-, mannitol- and cellobiose-positive, developed the opaque variant in culture, belonged to the O-antigenic serovar A and were highly virulent for eels by both intraperitoneal injection and immersion challenges. The nonmotile phenotype found in our V. vulnificus isolates was stable: nonmotile cells were always recovered from experimentally infected eels; no variation in the immobility of the V. vulnificus cells was observed for repeated subculture by daily passages on solid media, at different temperatures or incubation times and with or without magnesium sulfate. Many of the fla genes of Vibrio were present in the genome of the nonmotile strains (flaCDE and flaFBA for flagellins and flaH for the distal capping protein), although we observed by transmission electron microscopy that these V. vulnificus strains always lacked the polar flagellum. This is the first report on the existence of nonmotile wild-type V. vulnificus strains.     

Detection of free-living and plankton-bound vibrios in coastal waters of the Adriatic Sea (Italy) and study of their pathogenicity-associated properties

Culturable vibrios were isolated from water and plankton fractions collected during an 18-month sampling study performed along the north-central coast of the Adriatic Sea (Italy). Unculturable Vibrio vulnificus and V. parahaemolyticus were detected in plankton fractions by polymerase chain reaction amplification of DNA sequences for cytotoxin-haemolysin and thermolabile haemolysin respectively. The presence of V. parahaemolyticus, V. vulnificus and V. cholerae virulence genes and the expression of pathogenicity-associated traits were analysed in all isolates. The results showed the spreading of these properties among the environmental isolates and confirm the need of both monitoring the presence of vibrios in coastal areas and studying their pathogenicity potential in order to properly protect human health.     

香港养殖海鲷弧菌致病菌药物敏感性及耐药质粒研究

从发病海鲷(Sparus sarba)中共分离到51株弧菌(\%Vibrio)\%,经API20E细菌快速鉴定系统及Alsina和Blanch关键生理生化特性分析鉴定为7个种,它们分别是：溶藻胶弧菌(\%V.alginolyticus)(24株),创伤弧菌(V.vulnificus)(12株)和副溶血弧菌(V.parahaemolyticus)(7株),火神弧菌(V.logei)(4株),远洋弧菌Ⅱ菌(V.pelagius Ⅱ)(2株),河弧菌(V.fluvialis)(1株)和地中海弧菌(V.mediterranei)(1株)\%。其中3种优势菌溶藻胶弧菌创伤弧菌和副溶血弧菌证实对海鲷有致病性。另外采用平板稀释法检测了51株菌对16种抗菌素的敏感性。发现所有菌株对ceftriaxone,链霉素,萘啶酮酸和利福霉素敏感,几乎所有菌株对ceftazidime, netilimicin,氯霉素和sulfamethoxazole敏感.大部分菌株对氨苄青霉素 (60.8%),cefuroxime(667%),丁胺卡那霉素(55%),卡那霉素(588%)和三甲氧苄氨嘧啶(765%)等具有较强的耐药性。通过对菌株中所含有的耐药质粒进行分析,发现15株菌株含有1～4个质粒,分子量范围为9～123kb之间,对12株既含有较大分子量质粒又具有耐药性的菌株进行了质粒转化试验,结果其中9株菌的质粒具有转化能力,转化率为１０－１１～１０－９,表明所分离的菌株的抗药性是由于细菌染色体相关突变造成的。