Intraspecific diversity of the marine fish pathogen Tenacibaculum maritimum as determined by randomly amplified polymorphic DNA-PCR

AIM: The aim of the present study was to evaluate the intraspecific genetic variability within Tenacibaculum maritimum strains isolated from different species of marine fish. METHODS AND RESULTS: Twenty-nine strains isolated from five different fish species and three reference strains were characterized by randomly amplified polymorphic DNA (RAPD) method. Cluster analysis of RAPD-PCR profiles showed that the strains, regardless of the oligonucleotide primer employed (P2 and P6), were separated into two main groups that strongly correlated with the host species and/or O-serotypes described for this pathogen. One group composed all strains isolated from sole (Solea senegalensis and S. solea) and gilthead seabream (Sparus aurata), and the other compiled the T. maritimum isolates from yellowtail (Seriola quinqueradiata), Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus). An important exception was observed in the RAPD patterns of the reference strains, which were included in different genetic groups depending on the primer employed. CONCLUSIONS: The results obtained demonstrated genetic variability within the T. maritimum isolated from different marine fish. Such genetic variability proved to be strongly associated with the host and/or serogroups described for this pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD analysis constitutes a valuable molecular technique for epidemiological studies of T. maritimum. Interestingly, this is the first report of intraspecific differentiation and characterization of T. maritimum strains isolated from cultured fish.     

Intraspecific genetic variability of Edwardsiella tarda strains from cultured turbot

Edwardsiella tarda is an enterobacterial fish pathogen that causes mortality in various fish species worldwide. In this study, we analyzed the intraspecific variability in a collection of E. tarda strains isolated from turbot. To do this we employed 4 polymerase chain reaction (PCR)-based methods: (1) random amplified polymorphic DNA (RAPD), (2) enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), (3) repetitive extragenic palindromic-PCR (REP-PCR) and (4) BOX-PCR. E. tarda isolates from different hosts were also included for comparison. E. tarda strains from turbot showed high molecular homogeneity when RAPD (primers P3 and P6), ERIC-PCR and BOX-PCR were employed. However, with regard to the REP-PCR and RAPD (primers P4 and P5) techniques, different genetic groups could be established within these isolates using either technique. The 2 RAPD types presented an 85% similarity, while those obtained with REP-PCR showed 74% similarity. Based on the results obtained, although a high genetic homogeneity was found in turbot isolates, the RAPD test (with primers P4 and P5) and REP-PCR were capable of discrimination within these strains, and they are therefore considered the most appropriate typing methods for studies of edwardsiellosis in turbot.     

Complete genome sequence and immunoproteomic analyses of the bacterial fish pathogen Streptococcus parauberis

Although Streptococcus parauberis is known as a bacterial pathogen associated with bovine udder mastitis, it has recently become one of the major causative agents of olive flounder (Paralichthys olivaceus) streptococcosis in northeast Asia, causing massive mortality resulting in severe economic losses. S. parauberis contains two serotypes, and it is likely that capsular polysaccharide antigens serve to differentiate the serotypes. In the present study, the complete genome sequence of S. parauberis (serotype I) was determined using the GS-FLX system to investigate its phylogeny, virulence factors, and antigenic proteins. S. parauberis possesses a single chromosome of 2,143,887 bp containing 1,868 predicted coding sequences (CDSs), with an average GC content of 35.6%. Whole-genome dot plot analysis and phylogenetic analysis of a 60-kDa chaperonin-encoding gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding gene showed that the strain was evolutionarily closely related to Streptococcus uberis. S. parauberis antigenic proteins were analyzed using an immunoproteomic technique. Twenty-one antigenic protein spots were identified in S. parauberis, by reaction with an antiserum obtained from S. parauberis-challenged olive flounder. This work provides the foundation needed to understand more clearly the relationship between pathogen and host and develops new approaches toward prophylactic and therapeutic strategies to deal with streptococcosis in fish. The work also provides a better understanding of the physiology and evolution of a significant representative of the Streptococcaceae.     

Dormancy as a survival strategy of the fish pathogen Streptococcus parauberis in the marine environment

The fate of Streptococcus parauberis in seawater and sediment microcosms at different temperatures (6 and 22 degrees C) was investigated by comparing the survival dynamics of 2 strains of this bacterial species, isolated respectively from diseased turbot and cattle. The turbot and the bovine isolate showed similar survival kinetics, remaining culturable for approximately 1 mo in water and 6 mo in sediment. A slight influence of temperature on the stability of the cells was observed, in that the number of culturable cells was about 1 log10 unit higher at 6 than at 22 degrees C. During the starvation period, the metabolic activity of the cells, after suffering a strong reduction during the first 12 d, stabilized at levels ranging from 20 to 40% of the initial values. However, in all the microcosms, the acridine orange (AO) and 4',6-diamidino-2-phenilindole (DAPI) counts remained at about 10(5) cells ml(-1) throughout the experimental period, even when cells became undetectable by standard plate count methods. The addition of fresh medium to microcosms containing nonculturable cells induced the return to culturability of S. parauberis strains. On the basis of these results, it seems that S. parauberis has the ability to enter into a viable but nonculturable (VBNC) state. Dormant cells of the turbot isolate maintained their infectivity and pathogenic potential for fish.     

Intestinal colonization potential of turbot (Scophthalmus maximus)- and dab (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum.

Of more than 400 bacteria isolated from turbot (Scophthalmus maximus), 89 have previously been shown to inhibit the in vitro growth of the fish pathogen Vibrio anguillarum. The aim of the present study was to investigate the potential of seven of these strains, as well as of intestinal isolates (four strains) from a closely related fish, dab (Limanda limanda), for colonizing farmed turbot as a means of protecting the host from infection by V. anguillarum. In addition, the inhibitory effect of these strains on the pathogen was further studied. Colonization potential was measured by the capacity of the strains to adhere to and grow in turbot intestinal mucus. These parameters were also used to investigate the potential of V. anguillarum to amplify in the turbot intestinal tract. Because of the observed rapid growth of V. anguillarum in intestinal mucus, it can be proposed that the intestinal tract is a site for V. anguillarum multiplication. Strains isolated from the intestine showed greater capacity for adhesion to and growth in fish intestinal mucus than did the pathogen and the skin mucus isolates. All of the isolates released metabolites into the culture medium that had inhibitory effects against V. anguillarum. The results are discussed with emphasis on administering bacteria of host origin to farmed turbot in order to control V. anguillarum-induced disease.     

Intestinal colonization potential of turbot (Scophthalmus maximus)- and dab (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum.

Of more than 400 bacteria isolated from turbot (Scophthalmus maximus), 89 have previously been shown to inhibit the in vitro growth of the fish pathogen Vibrio anguillarum. The aim of the present study was to investigate the potential of seven of these strains, as well as of intestinal isolates (four strains) from a closely related fish, dab (Limanda limanda), for colonizing farmed turbot as a means of protecting the host from infection by V. anguillarum. In addition, the inhibitory effect of these strains on the pathogen was further studied. Colonization potential was measured by the capacity of the strains to adhere to and grow in turbot intestinal mucus. These parameters were also used to investigate the potential of V. anguillarum to amplify in the turbot intestinal tract. Because of the observed rapid growth of V. anguillarum in intestinal mucus, it can be proposed that the intestinal tract is a site for V. anguillarum multiplication. Strains isolated from the intestine showed greater capacity for adhesion to and growth in fish intestinal mucus than did the pathogen and the skin mucus isolates. All of the isolates released metabolites into the culture medium that had inhibitory effects against V. anguillarum. The results are discussed with emphasis on administering bacteria of host origin to farmed turbot in order to control V. anguillarum-induced disease.     

Host range susceptibility of Enterococcus sp. strains isolated from diseased turbot: possible routes of infection.

Experiments were conducted to assess the pathogenicity of Enterococcus sp. strains isolated from diseased turbot for several fish species (turbot, salmon, trout, and seabream), as well as for mice. The intraperitoneal injection assays indicated that the tested strains showed host specificity for turbot, with a high degree of virulence (50% lethal dose of 10(4) cells per g of fish). The Spanish Enterococcus sp. isolates were nonpathogenic for the other fish species studied and for mice. The possible routes of infection were determined by bath exposure (with and without prior abrasion of the skin) and by intragastric inoculations with food and feces contaminated with the pathogen. The bath challenges indicated that the Enterococcus isolates were able to overcome the defense mechanisms present on the surface of the turbot only if the skin was abraded prior to the exposure. The antibacterial activities of components of a glycoprotein nature present in the turbot skin mucus are probably responsible in part for the resistance in noninjured fish to infection. On the other hand, we demonstrated the capacity of this pathogen to overcome adverse conditions in the stomachs of fish when associated with food or fecal material, since it is able to establish an infective state and to produce mortalities after 16 to 20 days postingestion. From all of these findings, we can conclude that horizontal transmissions through water and the fecal-oral route are the main avenues of infection of turbot streptococcosis.     

Molecular differentiation of Renibacterium salmoninarum isolates from worldwide locations

Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences.     

Use of RAPD to investigate the epidemiology of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> infection in Malaysian hospitals

Summary Randomly ampli.ed polymorphic DNA (RAPD) with four different decamer oligonucleotide primers was performed on 50 clinical Staphylococcus aureus isolates obtained from di.erent hospitals in Malaysia. All the four primers generated polymorphisms in all 50 isolates of S. aureus studied, revealing DNA markers with sizes ranging from 100 to 7000 bp. The dendrogram generated from the RAPD analysis revealed two major groups (Groups I-II) with three clusters each in one group. S. aureus strains isolated from the same hospital were found to be genetically closely related and most of them were placed in the same cluster. In addition RAPD di.erentiated between MRSA and non-MRSA based on the clustering, where all MRSA and non-MRSA were placed in their respective clusters. The RAPD analysis showed that there could be four to .ve clones of S. aureus spreading around Malaysia, of which two clones may be MRSA. The overall genetic distances ranged from 0.088235 to 0.954545 among the isolates. This technique was found to be a simple and e.ective method for epidemiological investigation. Because of these e.cient features, this technique may have more general application for the study of S. aureus infections in hospitals and the community.     

Determination of 16S ribosomal RNA gene copy number in Streptococcus uberis, S. agalactiae, S. dysgalactiae and S. parauberis

Abstract Species-specific oligonucleotide probes and a universal oligonucleotide probe derived from sequences of 16S rRNA were hybridised to chromosomal DNA from Streptococcus agalactiae, S. dysgalactiae, S. parauberis and S. uberis following digestion with Eco RI. Due to the presence of a unique Eco RI site in each 16S rRNA gene, the number of hybridised fragments was indicative of the number of 16S rRNA genes. Southern hybridisation indicated six 16S rRNA genes in ten isolates of S. agalactiae , five genes in ten isolates of S. uberis , five genes in six isolates and six in another isolate of S. dysgalactiae , and six genes in four isolates of S. parauberis . For a fifth isolate of S. parauberis , six 16S rRNA genes were indicated by the universal probe but only five when hybridised to the species-specific probe, indicating sequence variation (microheterogeneity) within the probe target region.     

Phenotypic characteristics of Streptococcus iniae and Streptococcus parauberis isolated from olive flounder (Paralichthys olivaceus)

The etiological agents of streptococcosis were isolated from diseased olive flounder collected on the Jeju island of Korea. A total of 151 bacterial isolates were collected between 2003 and 2006. The isolates were examined using various phenotypic and proteomic analyses, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and glycoprotein assays. In addition, isolates were grown on blood agar to assess hemolytic activity, and biochemical assays were performed using the API20 Strep kit. Our results revealed that all isolates were nonmotile, Gram-positive cocci that displayed negative catalase and oxidase activities. Multiplex PCR assays revealed that 43% and 57% of the isolates were Streptococcus iniae and Streptococcus parauberis , respectively. These results were consistent with those of the SDS-PAGE and immunoblot analyses using whole-cell lysates of bacterial isolates. Significant differences were observed with respect to the Voges–Proskauer, pyrrodonyl arylamidase, alkaline phosphatase, and hemolytic activities of the S. iniae and S. parauberis isolates. Isolates of S. iniae displayed uniform profiles in the immunoblot and glycoprotein assays; however, immunoblot assays of S. parauberis isolates (using a chicken IgY antibody raised against a homologous isolate) revealed three distinct antigenic profiles. Our findings suggest that S. parauberis and S. iniae are endemic pathogens responsible for the development of streptococcosis in olive flounder.     

Multiplex PCR assay for detection of bacterial pathogens associated with warm-water Streptococcosis in fish

A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish (Streptococcus iniae, Streptococcus difficilis, Streptococcus parauberis, and Lactococcus garvieae). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The sensitivity of the multiplex PCR using purified DNA was 25 pg for S. iniae, 12.5 pg for S. difficilis, 50 pg for S. parauberis, and 30 pg for L. garvieae. The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish. Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.     

Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals

Members of most Chryseobacterium species occur in aquatic environments or food products, while strains of some other species are pathogenic to humans and animals. A collection of 52 Chryseobacterium sp. strains isolated from diseased fish, one frog isolate and 22 reference strains were included in a polyphasic taxonomy study. Fourteen clusters of strains were delineated following the comparison of whole-cell protein profiles. Most of these clusters were confirmed when the phenotypic and RAPD profiles and the 16S rRNA gene sequences were compared. Fatty acid composition helped differentiate the Chryseobacterium strains from members of related genera. None of the fish isolates could be allocated to the two species previously reported from fish but two isolates belonged to C. joostei, while the frog isolate was identified as Elizabethkingia meningoseptica, a human pathogen previously included in the genus Chryseobacterium. Three clusters grouping from 3 to 13 isolates will probably constitute the core of new Chryseobacterium species but all other isolates occupied separate or uncertain positions in the genus. This study further demonstrated the overall high similarity displayed by most Chryseobacterium strains whatever the technique used and the resulting difficulty in delineating new species in the genus. Members of this bacterial group should be considered potential emergent pathogens in various fish and frog species, farming conditions and geographical areas.     

Evaluation of a RAPD-based typing scheme in a molecular epidemiology study of Vibrio cholerae O1, Brazil

AIMS: To evaluate the utility of random amplification of polymorphic DNA (RAPD) technique for routine practice in public health laboratories for epidemiological studies of Vibrio cholerae O1 isolates. MATERIALS AND RESULTS: Seventy-nine strains were examined by PCR for the toxin genes (ctx A, zot and ace), virulence-associated genes (tcp A and tox T) and RAPD sequences. Except for one strain (no. 1123) from the Amazonas State, all the strains analysed carried the genes ctx A, zot, ace, tcp A and tox T. RAPD fingerprinting revealed variability but no correlation with serotype, biotype or geographical origin of the isolates was found. CONCLUSION: A standardized RAPD method does not enable the establishment of a pattern data bank for the identification of V. cholerae O1 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The simplicity and discriminative capacity of this technique make it useful for detecting genetic diversity among micro-organisms from a defined group or for outbreak investigation.     

Identification of Yersinia pestis as the causative organism of plague in India as determined by 16S rDNA sequencing and RAPD-based genomic fingerprinting

Eighteen isolates of bacteria obtained from the sputum of pneumonic plague patients and from the liver and spleen of rodents from the plague-affected areas of India during 1994-1995 when analyzed by 16S rDNA analysis clearly demonstrated that all 18 isolates exhibit an average similarity of 98.5% with the genus Yersinia and 99.1% with Yersinia pestis, thus identifying the isolates as Y. pestis. The isolates from the human plague patients were found to be genetically more homogeneous compared to the isolates from the rodents which were more heterogeneous. An epidemiological linkage among the rodents and human patients is also indicated by 16S rDNA analysis, which suggests that only a sub-population of the rodents was probably the source of the infectious pathogen to the humans initiating the outbreak of the epidemic. The results of the randomly amplified DNA polymorphisms (RAPD)-based DNA fingerprinting are in agreement with the above conclusions.     

Mapping of DNA Sex-Specific Markers and Genes Related to Sex Differentiation in Turbot (Scophthalmus maximus)

Production of all-female populations in turbot can increase farmer's benefits since sexual dimorphism in growth in this species is among the highest within marine fish, turbot females reaching commercial size 3-6?months earlier than males. Puberty in males occurs earlier than in females, which additionally slows their growth. Thus, elucidating the mechanisms of sex determination and gonad differentiation is a relevant goal for turbot production. A ZZ/ZW sex determination mechanism has been suggested for this species, and four sex-related quantitative trait loci (QTL) were detected, the major one located in linkage group (LG) 5 and the three minor ones in LG6, LG8, and LG21. In the present work, we carried out a linkage analysis for several sex-related markers: (1) three anonymous sex-associated RAPD and (2) several candidate genes related to sex determination and gonad differentiation in other species (Sox3, Sox6, Sox8, Sox9, Sox17, Sox19, Amh, Dmrta2, Cyp19a, Cyp19b). We focused our attention on their co-localization with the major and minor sex-related QTL trying to approach to the master sex-determining gene of this species. Previously described growth-related QTL were also considered since the association observed between growth and sex determination in fish. Amh, Dmrta2, and one RAPD were located in LG5, while Sox9 and Sox17 (LG21), Cyp19b (LG6), and a second RAPD (LG8) co-mapped with suggestive sex-related QTL, thus supporting further analyses on these genes to elucidate the genetic basis of this relevant trait for turbot farming.     

Diversity among clinical isolates of Histoplasma capsulatum detected by polymerase chain reaction with arbitrary primers.

Clinical isolates of the fungal respiratory and systemic pathogen Histoplasma capsulatum have been placed in several different classes by using genomic restriction fragment length polymorphisms (RFLPs), but in general have not been distinguished further. We report here that a polymerase chain reaction (PCR)-based DNA fingerprinting method that has been termed arbitrary primer or random amplified polymorphic DNA (RAPD) PCR can distinguish among isolates in a single RFLP class. In this method, arbitrarily chosen oligonucleotides are used to prime DNA synthesis from genomic sites that they fortuitously match, or almost match, to generate strain-specific arrays of DNA fragments. Each of 29 isolates of RFLP class 2, the group endemic in the American Midwest, was distinguished by using just three arbitrary primers. In contrast, laboratory-derived S and E colony morphology variants of two strains were not distinguished from their R parents by using 18 such primers. Thus, the clinical isolates of H. capsulatum are quite diverse, but their genomes remain stable during laboratory culture. These outcomes suggest new possibilities for epidemiological analysis and studies of fungal populations in infected hosts.     

A sex-associated sequence identified by RAPD screening in gynogenetic individuals of turbot (Scophthalmus maximus)

Understanding the genetic basis of sex determination mechanisms is essential for improving the productivity of farmed aquaculture fish species like turbot (Scophthalmus maximus). In culture conditions turbot males grow slower than females starting from eight months post-hatch, and this differential growth rate is maintained until sexual maturation is reached, being mature females almost twice as big as males of the same age. The goal of this study was to identify sex-specific DNA markers in turbot using comparative random amplified polymorphism DNA (RAPD) profiles in males and females to get new insights of the genetic architecture related to sex determination. In order to do this, we analyzed 540 commercial 10-mer RAPD primers in male and female pools of a gynogenetic family because of its higher inbreeding, which facilitates the detection of associations across the genome. Two sex-linked RAPD markers were identified in the female pool and one in the male pool. After the analysis of the three markers on individual samples of each pool and also in unrelated individuals, only one RAPD showed significant association with females. This marker was isolated, cloned and sequenced, containing two sequences, a microsatellite (SEX01) and a minisatellite (SEX02), which were mapped in the turbot reference map. From this map position, through a comparative mapping approach, we identified Foxl2, a relevant gene related to initial steps of sex differentiation, and Wnt4, a gene related with ovarian development, close to the microsatellite and minisatellite markers, respectively. The position of Foxl2 and Wnt4 was confirmed by linkage mapping in the reference turbot map.     

Diversity of lactic acid bacteria associated with fish and the fish farm environment, established by amplified rRNA gene restriction analysis

Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae, several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis, L. garvieae, Lactococcus l., S. iniae, S. dysgalactiae, S. parauberis, S. agalactiae, Carnobacterium spp., the Enterococcus "faecium" group, a heterogeneous Enterococcus-like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus, V. salmoninarum, and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately.     

Genetic Relatedness among Environmental, Clinical, and Diseased-Eel Vibrio vulnificus Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates.