Comparison of ribotyping and randomly amplified polymorphic DNA PCR for characterization of Vibrio vulnificus.

A total of 85 isolates of Vibrio vulnificus were characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by randomly amplified polymorphic DNA-PCR (RAPD-PCR) with a 10-mer oligonucleotide primer. The RAPD-PCR results were scanned, and the images were analyzed with a computer program. Ribotype membranes were evaluated visually. Both the ribotyping and the RAPD-PCR results showed that the collection of strains was genetically very heterogeneous. Ribotyping enabled us to differentiate U.S. and Danish strains and V. vulnificus biotypes 1 and 2, while the RAPD-PCR technique was not able to correlate isolates with sources or to differentiate the two biotypes, suggesting that ribotyping is useful for typing V. vulnificus strains whereas RAPD-PCR profiles may subdivide ribotypes. Two Danish clinical biotype 2 strains isolated from fishermen who contracted the infection cleaning eels belonged to the same ribotype as three eel strains (biotype 2), providing further evidence that V. vulnificus biotype 2 is an opportunistic pathogen for humans. One isolate (biotype 2) from Danish coastal waters also showed the same ribotype as the eel strains. This is, to our knowledge, the first time the isolation of V. vulnificus biotype 2 from coastal waters has been described.     

Phenotypic characterization of Vibrio vulnificus biotype 2, a lipopolysaccharide-based homogeneous O serogroup within Vibrio vulnificus.

In this study, we have reevaluated the taxonomic position of biotype 2 of Vibrio vulnificus. For this purpose, we have biochemically and serologically characterized 83 biotype 2 strains from diseased eels, comparing them with 17 biotype 1 strains from different sources. Selected strains were also molecularly analyzed and tested for eel and mouse pathogenicity. Results have shown that biotype 2 (i) is biochemically homogeneous, indole production being the main trait that distinguishes it from biotype 1, (ii) presents small variations in DNA restriction profiles and outer membrane protein patterns, some proteins being immunologically related to outer membrane proteins from biotype 1, (iii) expresses a common lipopolysaccharide (LPS) profile, which is immunologically identical among strains and distinct from that of LPS of tested biotype 1 strains, and (iv) contains at least two high-Mr plasmids. Regarding host range, we have confirmed that both biotypes are pathogenic for mice but only biotype 2 is pathogenic for eels. On the basis of these data, we propose that biotype 2 of V. vulnificus constitutes an LPS-based O serogroup which is phenotypically homogeneous and pathogenic for eels. In this article, the serogroup is designated serogroup E (for eels).     

Intraspecific Differentiation of Vibrio vulnificus Biotypes by Amplified Fragment Length Polymorphism and Ribotyping

The intraspecific genomic relatedness of 80 Vibrio vulnificus isolates, 44 of biotype 1 and 36 of biotype 2, from different geographic origins and sources was evaluated by ribotyping and AFLP (amplified fragment length polymorphism) fingerprinting. Ribopatterns of DNAs digested with KpnI and hybridized with an oligonucleotide complementary to a highly conserved sequence in the 23S rRNA gene revealed up to 19 ribotypes in the species, which were different for the two biotypes. Sixteen different ribotypes were found within biotype 1 strains from clinical and environmental sources, and only three, recovered mainly from diseased eels, were found within biotype 2. Within this biotype, 96% of the strains showed the same ribopattern. The closest similarity was shown by the strains coming from the same eel farm, irrespectively of biotype. AFLP fingerprints obtained by selective PCR amplification of HindIII-TaqI double-restricted DNA fragments exhibited a strain-specific pattern which allowed the finest differentiation of subgroups within the eel-pathogenic isolates sharing the same ribopattern. Both techniques revealed good genetic markers for intraspecific differentiation of V. vulnificus. Ribotyping clearly separated the eel-pathogenic strains from the clinical and environmental isolates, whereas AFLP enabled the monitoring of individual strains and therefore constitutes one of the most discriminative tools for epidemiological and ecological studies.     

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.     

Comparison of outer membrane protein profiles of Vibrio vulnificus biotypes 1 and 2

Abstract The outer membrane proteins of 17 Vibrio vulnificus biotype 2 strains from Japanese and European cels, and 12 biotype 1 strains from clinical and environmental sources have been compared. The overall profile in both biotypes was similar, and a major protein band of molecular mass 36 kDa was detected in the majority of the strain. Differences in the minor bands allowed differentiation of strains from different origins, suggesting that outer membrane protein profiles could be useful as epidemiological markers in the species V. vulnificus . Immunoblotting with antisera to whole cells of selected strains of biotypes 1 and 2 showed a strong antigenic response to outer membrane proteins 66, 60, 48, 46 and 44 kDa; these were common to all strains examined, independent of their biotypes and origins. These results demonstrate the presence of antigenically related outer membrane proteins in both biotypes of V. vulnificus .     

Comparison of the effects of environmental parameters on growth rates of Vibrio vulnificus biotypes I, II, and III by culture and quantitative PCR analysis

Vibrio vulnificus is a natural inhabitant of estuarine waters. The three known biotypes include (i) most human pathogens, (ii) primarily eel pathogens, and (iii) pathogens associated with fish and with human wound infections in Israel. Despite the frequently lethal consequences of V. vulnificus infections, the growth rates of the various biotypes and their response to environmental changes are not well characterized. We compared the specific growth rates (μ) of a representative of each biotype by culture and quantitative PCR (qPCR) analysis in a defined medium under varied pH, temperature, and salinity. Growth rates based on culturable concentrations were always higher than those based on qPCR estimates; however, both enumeration methods yielded comparable results on the influence of environmental factors on growth rates. Temperature (25°C, 30°C, 37°C), pH (7.0, 8.0), and salinity (5 to 40‰) all had significant effects on the μ of each biotype. Temperature had the greatest effect on the μ of biotype 1 (CMCP6), whereas salinity had the greatest effect on the μ of biotypes 2 (ATCC 33147) and 3 (302/99). The biotypes' growth rates varied significantly; biotype 1 grew most rapidly, while biotype 3 grew most slowly. The highest growth rates were achieved at 37°C, pH 7.0, and salinities of 15 to 30‰ (μ = 4.0, 2.9, and 2.4 generations h(-1) for biotypes 1, 2, and 3, respectively). Other strains of the biotypes yielded comparable results, suggesting that the physiological responses of the biotypes are differentially affected by parameters that are highly variable both in estuarine environments and between the free-living and pathogen states of V. vulnificus.     

Protocol for specific isolation of virulent strains of Vibrio vulnificus serovar E (biotype 2) from environmental samples

The eel pathogen Vibrio vulnificus biotype 2 comprises at least three serovars, with serovar E being the only one involved in both epizootics of eel vibriosis and sporadic cases of human infections. The virulent strains of this serovar (VSE) have only been recovered from clinical (mainly eel tissue) sources. The main objective of this work was to design and validate a new protocol for VSE-specific isolation from environmental samples. The key element of the new protocol is the broth used for the first step (saline eel serum broth [SEB]), which contains eel serum as a nutritive and selective component. This approach takes advantage of the ability of VSE cells to grow in eel serum and thus to separate themselves from the pool of competitors. The growth yield in SEB after 8 h of incubation was 1,000 times higher for VSE strains than for their putative competitors (including biotype 1 strains of the species). The selective and differential agar Vibrio vulnificus medium (VVM) was selected from five selective media for the second step because it gave the highest plating efficiency not only for the VSE group but also for other V. vulnificus groups, including biotype 3. The entire protocol was validated by field studies, with alkaline peptone water plus VVM as a control. V. vulnificus was isolated by both protocols, but serovar E was only recovered by the new method described here. All selected serovar E isolates were identified as VSE since they were virulent for both eels and iron-overloaded mice and resisted the bactericidal action of eel and iron-overloaded human sera. In conclusion, this new protocol is a suitable method for the isolation of VSE strains from environmental samples and is recommended for epidemiological studies of the pathogenic serovar E.     

An enzyme-linked immunosorbent assay for detection of Vibrio vulnificus biotype 2: development and field studies.

Vibrio vulnificus biotype 2 is a primary eel pathogen which constitutes a lipopolysaccharide (LPS)-based homogeneous O serogroup within the species. In the present work, we have developed an enzyme-linked immunosorbent assay (ELISA) based on the specificity of LPS for the detection of this pathogen. The ELISA specificity was confirmed after testing 36 biotype 2 strains from laboratory cultures and environmental samples, 31 clinical and environmental biotype 1 isolates, and several strains of Vibrio, Aeromonas, and Yersinia species, including the fish pathogens V. anguillarum, V. furnissii, A. hydrophila, and Y. ruckerii. The detection limits for biotype 2 cells were around 10(4) to 10(5) cells/well, and the immunoassay was also able to detect cells in the nonculturable state. Artificially infected eels and environmental samples were analyzed, and the immunodetection was confirmed by cultural methods (isolation on selective and nonselective media before and after broth enrichment). With this methodology, V. vulnificus biotype 2 was successfully detected in infected eels and asymptomatic carriers, which suggests that eels can act as a reservoir for this pathogen.     

Influence of aquatic microbiota on the survival in water of the human and eel pathogen Vibrio vulnificus serovar E

The eel and human pathogen Vibrio vulnificus serovar E (biotype 2) is seldom isolated from natural waters, although it can survive in sterilized artificial seawater microcosms for years. The main objective of the present study was to investigate whether aquatic microbiota can limit its survival and recovery from water samples. A set of preliminary experiments of survival in microcosms containing natural seawater and water from eel farms showed that the persistence of this pathogen was mainly controlled by grazing, and secondarily by bacterial competition. The bacterial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E (VSE) strains and potential competitors. Competitors included V. vulnificus biotype 1 isolates and strains of selected species that can grow on the selective media designed for V. vulnificus isolation from water samples. Evidences of bacterial competition that was detrimental for VSE recovery were recorded. Thus, some species produced a deleterious effect on VSE strains under starvation, and others were able to use the resources more efficiently under nutrient input. These results suggest that an overgrowth of more efficient competitor bacteria in conventional media used for isolation of V. vulnificus could mask the recovery of VSE strains and explain the scarcity of reports on the isolation of this human and eel pathogen from natural waters.     

Vibrio vulnificus produces quorum sensing signals of the AHL-class

Vibrio vulnificus is an aquatic pathogenic bacterium that can cause vibriosis in humans and fish. The species is subdivided into three biotypes with the fish-virulent strains belonging to biotype 2. The quorum sensing (QS) phenomenon mediated by furanosyl borate diester or autoinducer 2 (AI-2) has been described in human strains of biotype 1, and here we show that the luxS gene which encodes AI-2 is present in all strains of V. vulnificus regardless of origin, biotype or serovar. In this study, we also demonstrate that V. vulnificus produces QS signals of the acylated homoserine lactone (AHL) class (AI-1). AHLs were detected in strains of biotype 1 and 2 from water, fish and human wound infections but not in strains isolated from human septicaemic cases. The AHL compound was identified as N -butanoyl-homoserine-lactone (C₄-HL) by both reporter strains and by HPLC-high-resolution MS. C₄-HL was detected when AHL-positive strains were grown in low-nutrient medium [modified sea water yeast extract (MSWYE)] but not in rich media (tryptic soy broth or brain–heart infusion) and its production was enhanced when blood factors were added to MSWYE. C₄-HL was detected in vivo , in eels infected with AHL-positive biotype 2 strains. No known AHL-related gene was detected by PCR or Southern blot suggesting that AHL-related genes in V. vulnificus are different from those found in other Gram-negative bacteria.     

Population genetics of Vibrio vulnificus: identification of two divisions and a distinct eel-pathogenic clone

Genetic relationships among 62 Vibrio vulnificus strains of different geographical and host origins were analyzed by multilocus enzyme electrophoresis (MLEE), random amplification of polymorphic DNA (RAPD), and sequence analyses of the recA and glnA genes. Out of 15 genetic loci analyzed by MLEE, 11 were polymorphic. Cluster analysis identified 43 distinct electrophoretic types (ETs) separating the V. vulnificus population into two divisions (divisions I and II). One ET (ET 35) included all indole-negative isolates from diseased eels worldwide (biotype 2). A second ET (ET 2) marked all of the strains from Israel isolated from patients who handled St. Peter's fish (biotype 3). RAPD analysis of the 62 V. vulnificus isolates identified 26 different profiles separated into two divisions as well. In general, this subdivision was comparable (but not identical) to that observed by MLEE. Phylogenetic analysis of 543 bp of the recA gene and of 402 bp of the glnA gene also separated the V. vulnificus population into two major divisions in a manner similar to that by MLEE and RAPD. Sequence data again indicated the overall subdivision of the V. vulnificus population into different biotypes. In particular, indole-negative eel-pathogenic isolates (biotype 2) on one hand and the Israeli isolates (biotype 3) on the other tended to cluster together in both gene trees. None of the methods showed an association between distinct clones and human clinical manifestations. Furthermore, except for the Israeli strains, only minor clusters comprising geographically related isolates were observed. In conclusion, all three approaches (MLEE, RAPD, and DNA sequencing) generated comparable but not always equivalent results. The significance of the two divisions (divisions I and II) still remains to be clarified, and a reevaluation of the definition of the biotypes is also needed.     

Polyphyletic origin of Vibrio vulnificus biotype 2 as revealed by sequence-based analysis

A sequence-based analysis of seven housekeeping and virulence-related genes shows that the species Vibrio vulnificus is subdivided into three phylogenetic lineages that do not correspond with the biotypes and that biotype 2 is polyphyletic. These results support the reclassification of biotype 2 as a pathovar that would group the strains with pathogenic potential to develop vibriosis in fish.     

Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification.

DNA extraction procedures and PCR conditions to detect Vibrio vulnificus cells naturally occurring in oysters were developed. In addition, PCR amplification of V. vulnificus from oysters seeded with biotype 1 cells was demonstrated. By the methods described, V. vulnificus cells on a medium (colistin-polymyxin B-cellobiose agar) selective for this pathogen were detectable in oysters harvested in January and March, containing no culturable cells (< 67 CFU/g), as well as in oysters harvested in May and June, containing culturable cells. It was possible to complete DNA extraction, PCR, and gel electrophoresis within 10 h by using the protocol described for oysters. V. vulnificus biotype 2 cells were also detected in eel tissues that had been infected with this strain and subsequently preserved in formalin. The protocol used for detection of V. vulnificus cells in eels required less than 5 h to complete. Optimum MgCl2 concentrations for the PCR of V. vulnificus from oysters and eels were different, although the same primer pair was used for both. This is the first report on the detection of cells of V. vulnificus naturally present in shellfish and represents a potentially powerful method for monitoring this important human and eel pathogen.     

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.     

Ferric-reductase activities in Vibrio vulnificus biotypes 1 and 2

In this paper, the ferric-reductase activities of Vibrio vulnificus were investigated. This species comprises two biotypes pathogenic for humans and eels that are able to express different mechanisms for iron acquisition. All strains of both biotypes used in this study were able to reduce ferric citrate, irrespective of the iron levels in the growth medium. Some variation in the degree of reduction was observed among the strains, with the highest values corresponding to one acapsulated environmental strain of biotype 1. When cell fractions were tested, only those from periplasm and cytoplasm showed reductase activity whereas no activity was detected in membranes. Low temperatures inhibited these activities in both whole cells and cell fractions. At least six bands with ferric-reductase activity were identified in all strains using native polyacrylamide gels. These data demonstrate that the two biotypes of V. vulnificus produce similar ferric-reductases mainly located in the periplasm and cytoplasm and these could be involved in iron acquisition.     

A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid

Strains of Vibrio vulnificus, a marine bacterial species pathogenic for humans and eels, are divided into three biotypes, and those virulent for eels are classified as biotype 2. All biotype 2 strains possess one or more plasmids, which have been shown to harbor the biotype 2-specific DNA sequences. In this study we determined the DNA sequences of three biotype 2 plasmids: pR99 (68.4 kbp) in strain CECT4999 and pC4602-1 (56.6 kb) and pC4602-2 (66.9 kb) in strain CECT4602. Plasmid pC4602-2 showed 92% sequence identity with pR99. Curing of pR99 from strain CECT4999 resulted in loss of resistance to eel serum and virulence for eels but had no effect on the virulence for mice, an animal model, and resistance to human serum. Plasmids pC4602-2 and pR99 could be transferred to the plasmid-cured strain by conjugation in the presence of pC4602-1, which was self-transmissible, and acquisition of pC4602-2 restored the virulence of the cured strain for eels. Therefore, both pR99 and pC4602-2 were virulence plasmids for eels but not mice. A gene in pR99, which encoded a novel protein and had an equivalent in pC4602-2, was further shown to be essential, but not sufficient, for the resistance to eel serum and virulence for eels. There was evidence showing that pC4602-2 may form a cointegrate with pC4602-1. An investigation of six other biotype 2 strains for the presence of various plasmid markers revealed that they all harbored the virulence plasmid and four of them possessed the conjugal plasmid in addition.     

Role of the virulence plasmid pR99 and the metalloprotease Vvp in resistance of Vibrio vulnificus serovar E to eel innate immunity

Vibrio vulnificus biotype 2 serovar E (VSE) is a bacterial pathogen that produces a haemorrhagic septicaemia called vibriosis in eels. Its ability to grow in blood is conferred by a recently described virulence plasmid [Lee CT, Amaro C, Wu KM, Valiente E, Chang YF, Tsai SF, et al. A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid. Journal of Bacteriology, submitted for publication.]. In this study, we analyzed the role of this plasmid together with the role played by the metalloprotease (Vvp) in the interaction between bacteria and eel innate immunity. To this end, we compared and statistically analyzed the differences in resistance to serum and mucus factors (complement, selected antimicrobial peptides, transferrin and lysozyme) and also to phagocytosis/opsonophagocytosis between one VSE strain and its derivatives: a plasmid-cured strain and a vvp-deficient mutant. The wild-type and the metalloprotease-deficient strains were resistant to both the bactericidal action of fresh serum and the phagocytosis and opsonophagocytosis by eel phagocytes, confirming that Vvp is not involved in resistance to eel innate immunity. In contrast, the cured strain was sensitive to both the bactericidal action of eel serum activated by the alternative pathway and phagocytosis/opsonophagocytosis. Since no plasmid-encoded ORF, with homology to known genes, is related to the resistance to innate immunity [Lee CT, Amaro C, Wu KM, Valiente E, Chang YF, Tsai SF, et al. A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid. Journal of Bacteriology, submitted for publication.], this function could be codified by one or more new genes. Further studies are underway to characterize the plasmid-encoded system responsible for V. vulnificus resistance to the innate immune system of eels.     

Role of the metalloprotease Vvp and the virulence plasmid pR99 of Vibrio vulnificus serovar E in surface colonization and fish virulence

The virulence for eels of Vibrio vulnificus biotype 2 serovar E (VSE) is conferred by a plasmid that codifies ability to survive in eel serum and cause septicaemia. To find out whether the plasmid and the selected chromosomal gene vvp plays a role in the initial steps of infection, the VSE strain CECT4999, the cured strain CT218 and the Vvp-deficient mutant CT201 (obtained in this work by allelic exchange) were used in colonization and virulence experiments. The eel avirulent biotype 1 (BT1) strain YJ016, whose genome has been sequenced, was used for comparative purposes. The global results demonstrate that the plasmid does not play a significant role in surface colonization because (i) CECT4999 and CT218 were equally chemoattracted towards and adherent to eel mucus and gills, and (ii) CT218 persisted in gills from bath-infected eels 2 weeks post infection. In contrast, mutation in vvp gene reduced significantly chemoattraction and attachment to eel mucus and gills, as well as virulence degree by immersion challenge. Co-infection experiments by bath with CECT4999 and CT201 confirmed that Vvp was involved in eel colonization and persistence in gills, because CECT4999 was recovered at higher numbers compared with CT201 from both internal organs of moribund fish (ratio 4:1) and gills from survivors (ratio 50:1). Interestingly, YJ016 also showed chemoattraction and attachment to mucus, and complementation of CT201 with BT1- vvp gene restored both activities together with virulence degree by immersion challenge. Additional experiments with algae mucus and purified mucin gave similar results. In conclusion, the protease Vvp of V. vulnificus seems to play an essential role in colonization of mucosal surfaces present in aquatic environments. Among the V. vulnificus strains colonizing fish mucus, only those harbouring the plasmid could survive in blood and cause septicaemia.     

Utilization of hemin and hemoglobin by Vibrio vulnificus biotype 2.

The eel pathogen Vibrio vulnificus biotype 2 is able to use hemoglobin (Hb) and hemin (Hm) to reverse iron limitation. In this stud, the adjuvant effect of both compounds on eel pathogenicity has been evaluated and confirmed. Further, we have studied the heme-iron acquisition mechanism displayed by this bacterium. Whole cells were capable of binding Hb and Hm, independently of (i) iron levels in growth medium and (ii) the presence of polysaccharide capsules on bacterial surface. The Hb- and Hm-binding capacity was retained by the outer membrane protein (OMP) fraction and was abolished after proteolytic digestion of OMP samples. Western blotting (immunoblotting) of denatured OMPs revealed that two major protein bands of 36 and 32 kDa were involved in both Hm and Hb binding. The expression of these proteins was not affected by iron levels. In addition, V. vulnificus biotype 2 produced extracellular proteases, not regulated by iron, that were active against native Hb. In conclusion, the overall data suggest that the eel pathogen V. vulnificus biotype 2 can obtain iron by means of a mechanism which involves a direct interaction between the heme moiety and constitutive OMPs.     

Toxic and enzymatic activities of Vibrio vulnificus biotype 2 with respect to host specificity.

In this work, the enzymatic activities of selected strains of biotypes 1 and 2 of Vabrio vulnificus were analyzed by using conventional methods and the API ZYM system. The toxic activities of extracellular products (ECPs) were further evaluated by in vitro and in vivo experiments. The ECPs of both biotypes (i) showed high-level hydrolytic activities, (ii) displayed cytotoxicity for fish cell lines, and (iii) were lethal for eels. Exotoxins seem to be proteinaceous since heat treatment of ECP samples destroyed their toxicity. Only biotype 2 strains were virulent for cels, suggesting that host specificity must be related to differences in cell surface properties. Infectivity trials with other fish species also revealed that only biotype 2 strains were virulent.