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Vibrio vulnificus is a ubiquitous bacterium found in estuarine and marine environments. It comprises two biotypes: biotype 1 strains are pathogenic in humans, while biotype 2 strains are virulent in eels and opportunistic in humans, although they have been thought to be pathogenic only for eels until recently (2). It has been isolated from a wide variety of marine organisms, such as oysters, crabs, fish, mussels, shellfish, and plankton (10, 17, 18, 36), and from water and sediment (34). It is well known that raw-shellfish consumption increases the risk of human vibrio diseases. Particularly, V. vulnificus can cause fulminant and severe systemic human illness after the consumption of infected raw oysters (30). Mortality rates of up to 60% have been reported in such infections (18). It is remarkably virulent in individuals who are immunocompromised or have liver dysfunction, which results in increased levels of iron in serum (18, 28). Thus, its occurrence in aquatic environments is of concern to shellfish industries and public health agencies.

Because a great number and variety of indigenous bacteria are present in the environment together with some pathogenic Vibrio species, the isolation of V. vulnificus is usually performed by enrichment in alkaline peptone water (APW) or in APW supplemented with polymyxin B. However, this enrichment also increases the number of other bacteria, which requires further inoculation of the enriched sample on selective media (10, 16) such as V. vulnificus agar (7), sodium dodecyl sulfate (SDS)-polymyxin B-sucrose (SPS) agar (21), cellobiose-polymyxin B-colistin (CPC) agar (23), V. vulnificus enumeration agar (24), modified CPC (mCPC) agar (33), and cellobiose-colistin (CC) agar (16). The recovery of V. vulnificus was higher with CPC agar than with thiosulfate-citrate-bile salts-sucrose (TCBS), V. vulnificus enumeration, or SDS-polymyxin-sucrose agar (20, 23, 29, 31, 32). Later, CPC agar was modified (mCPC) by reducing the concentration of colistin (33), which improved the recovery and isolation of V. vulnificus from environmental sources. Recently, a new modification of CPC agar has been described (16).

To identify presumptive V. vulnificus recovered from these media, probes based on the cytolysin gene of V. vulnificus have been developed (11, 25, 37). The possible loss or rearrangement of nonessential genes, such as the cytotoxin-hemolysin gene, can lead to a false-negative result (12). Another method for molecular detection of V. vulnificus based on the 23S rRNA gene (rDNA) using nested PCR has been developed (4). However, certain molecular techniques cannot be used for the routine monitoring of environmental samples because of PCR inhibition (37). In this study, a new selective medium (VVM agar), which improves the recovery of V. vulnificus, and a specific probe (V3VV) have been established. The medium contains electrolytes that improve the recovery of V. vulnificus. The probe based on 16S rRNA sequences was evaluated by dot blot hybridization with PCR-amplified 16S rDNA and then by colony hybridization.

Thirty-eight collection strains, mainly Vibrio strains, were first used to evaluate the VVM medium and the V3VV probe (Table 1). Moreover, 232 Vibrio strains from several environmental origins were used to assess the selectivity of the new medium and the specificity of the probe (Table 2). These strains are available from the LMG collection (Laboratory of Microbiology, Ghent, Belgium). Sixteen V. vulnificus strains, from culture collections, of clinical and environmental origins, were used in the plating efficiency experiments. Sixty-four V. vulnificus strains of clinical and environmental origins (13 and 51 strains, respectively) were used for confirmation of the species specificity studies with the V3VV probe. V. cholerae CECT 658, V. mimicus LMG7896^T, and V. vulnificus NCIMB 2046^T were used to determine the threshold of detection when mixed bacterial suspensions were analyzed.

Selective VVM agar. The potential components of the medium were evaluated according to the differential metabolic features of the genus Vibrio and V. vulnificus (1, 15). After several compositions and growth conditions were assayed, the selective medium chosen had the following composition: D-(+)-cellobiose, 15 g; NaCl, 10 g, yeast extract, 4 g; MgCl₂·6H₂O, 4 g; KCl, 4 g; cresol red, 40 mg; bromothymol blue, 40 mg; polymyxin B, 10⁵ U/liter; colistin methanesulfonate, 10⁵ U/liter; agar, 15 g; distilled water, 1,000 ml. The compounds were dissolved with stirring and boiling. The pH was adjusted to 8.5 with 5 M NaOH after cooling to 50°C. VVM does not require autoclaving. The uninoculated VVM plates are violet-blue. The presumptive identification of V. vulnificus is based on the fermentation of D-(+)-cellobiose, which determines the color of the colonies. The colonies are bright yellow with a yellow diffusion halo. Overnight cultures of each of the 38 collection strains and the 232 Vibrio spp. were plated on nonselective Trypticase soy agar supplemented with 1.5% NaCl (TSA2). Cell suspensions (McFarland standard no. 3) of these cultures were made in sterile phosphate-buffered saline complemented with 1.5% NaCl at pH 7.2 (marine PBS). The specificity of the medium was studied by inoculation of 10 µl of each cell suspension onto a plate of VVM agar. Inoculated plates were incubated at 37°C and examined after 24 and 48 h of incubation. As a control, 10 µl of the same suspension was spotted onto TSA2. Plates were incubated for 24 h at 37°C or at 25°C in the case of Vibrio spp. which do not grow at 37°C (V. costicola, V. logei, V. ordalii, and V. splendidus).

Specific 16S rDNA probe. Full sequences of the 16S rDNAs of 47 Vibrio strains deposited in the EMBL genomic database were compared. Five of them belonged to V. vulnificus. Multiple alignment and visualization of homologies were performed by the methods of Needleman and Wunsch (26) and Devereux et al. (13), respectively. An oligonucleotide of 24 nucleotides (5'-GTC TGC CAG TTT CAA ATG CAG TTC-3') located between positions 618 and 641 of the sequence of the 16S rDNA of Escherichia coli (8) was defined for use as a probe (V3VV). The specificity of V3VV was initially evaluated by comparing its sequences with the sequences of the EMBL database using the FASTA software (13). The oligonucleotide was synthesized and labeled at the 5' end with digoxigenin (Boehringer, Mannheim, Germany). The specificity was evaluated by DNA-DNA hybridization using culture collection strains (Table 1). To that end, the 16S rDNAs of the 38 collection strains were first amplified. A colony from an overnight culture on TSA2 at 37°C was used to extract the DNA template using the Instagene Matrix Kit (Bio-Rad, Hercules, Calif.). PCR was performed using the AmpliTaq DNA polymerase kit (The Perkin-Elmer Corp., Norwalk, Conn.) in accordance with the instructions of the manufacturer. The PCR program was 1 cycle of 95°C for 0.5 min; 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2.5 min; and 1 cycle of 94°C for 1 min, 55°C for 1 min, and 72°C for 3 min. An aliquot of 200 µl of a 1:10 dilution in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) of the amplified DNA was blotted onto nylon membranes (Hybond N; Amersham, Amersham, United Kingdom), using Minifold-I equipment (Schleicher & Schuell, Dassel, Germany) and cross-linked. After the establishment of the conditions of stringency, the hybridization was performed at 68°C as described by Martínez-Picado et al. (22), with minor modifications. The washing steps after hybridization were performed using 2× SSC-0.1% SDS at 68°C and 0.5× SSC-0.1% SDS at room temperature. Detection of probe chemiluminescence was performed with CSPD (Boehringer) as the substrate for alkaline phosphatase. Later, specificity was also tested by colony hybridization using collection and environmental strains (Table 2). To perform colony hybridization, bacteria were grown on nylon membranes (Hybond N⁺; Amersham) deposited onto TSA2 plates and incubated overnight at 37°C. Membranes were processed for hybridization as described above and by Martínez-Picado et al. (22).

Comparison of VVM and other selective agars. The efficiency of recovery on VVM agar was determined by comparing the colony counts of V. vulnificus on this medium with those on TSA2 and TCBS. Moreover, recovery on VVM agar was also compared with that on other selective media: mCPC and CC agars. TCBS (Oxoid) agar was prepared in accordance with the instructions of the manufacturer. The composition and preparation of the CC and mCPC agars have been described elsewhere (16, 33). Overnight cultures of each of the 16 V. vulnificus strains tested were prepared by growing them in Trypticase soy broth supplemented with 1.5% NaCl at 37°C. Tenfold dilutions of the overnight cultures in marine PBS were prepared. An aliquot of 10 µl of each dilution was inoculated in triplicate onto CC, mCPC, VVM, TCBS, and TSA2, and the spots were allowed to be absorbed. TSA2, TCBS, and VVM plates were incubated at 37°C, and mCPC and CC plates were incubated at 40°C as previously recommended (16, 33). Colonies were counted at 24 h and confirmed at 48 h. Efficiency of recovery was calculated by determining plating efficiency as described by Høi et al. (16). This term is defined as the percentage of CFU that can be recovered on a selective medium compared to the CFU encountered on a corresponding reference medium. Plating efficiencies on the different selective media were compared by using one-way analysis of variance with least significant differences between means using Student's t test (Statgraphics Statistical Graphics System; Manugistics, Inc. and Statistical Graphica Corporation; Rockville, Md.).

Recovery of V. vulnificus from mixed bacterial populations. In order to determine the efficiency of recovery and the sensitivity of the medium, V. vulnificus from mixed bacterial cell suspensions was detected. Cultures of V. vulnificus NCIMB 2046^T V. cholerae CECT 658, and V. mimicus LMG 7896^T grown overnight in Trypticase soy broth-1.5% NaCl at 37°C were prepared. Sets of 10-fold dilutions of each culture were obtained in marine PBS. Several cell proportions (1:10, 1:100, and 1:1,000) of V. vulnificus and V. cholerae and of V. vulnificus and V. mimicus were assayed. Aliquots of 100 µl of each mixed suspension were inoculated in duplicate on VVM agar and TSA2. Inoculated plates were incubated at 37°C for 24 h. Colony counts on both media were performed. Yellow (V. vulnificus) and blue (V. cholerae or V. mimicus) colonies on VVM agar were counted.

Recovery of V. vulnificus from spiked mussels. Since the occurrence of virulent strains of V. vulnificus has been related, in some cases, to shellfish consumption, the threshold of detection of this species on VVM agar was tested with mussel samples. Mussels were spiked with a pure culture of V. vulnificus NCIMB 2046^T with a known concentration (3.16 × 10⁵ CFU/ml). The mussels were collected in the Delta de l'Ebre (Spanish Mediterranean coast) and processed as follows. They were aseptically shucked with an autoclaved oyster knife to obtain 50 g of mussel meat, which was homogenized in a sterile blender. The homogenate of mussels was diluted in 50 ml of marine PBS and stirred for 20 min. The resulting suspension was filtered through nylon gauze with a 54-µm pore size to remove thick particles. The filtered suspension was then distributed in 5 aliquots of 5 ml each. An overnight culture of V. vulnificus NCIMB 2046^T was prepared in TSB2 at 37°C, and 10-fold dilutions in marine PBS were prepared to obtain concentrations of 10⁵ to 10² CFU of bacteria per ml. Four aliquots of the filtered suspension of mussels were spiked with 1 ml of each dilution of the V. vulnificus culture. One, the negative control, was not spiked. Thereafter, 10-fold dilutions of the spiked and control suspensions in marine PBS were prepared. An aliquot of 100 µl of each dilution was plated in duplicate on TSA2 and VVM agar plates, which were incubated at 37°C for 24 h. Total and presumptive V. vulnificus (yellow) colony counts were performed. Replica plating was carried out on nylon membranes (Hybond N⁺; Amersham) in order to confirm the detection of V. vulnificus colonies on VVM agar. Later, colony hybridization with the specific digoxigenin-labeled V3VV probe was performed at 68°C as described above. The locations of probe-positive signals on autoradiograms were compared with the positions of yellow colonies on VVM agar.