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.     

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.     

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.     

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

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

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.     

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.     

Transmission to Eels, Portals of Entry, and Putative Reservoirs of Vibrio vulnificus Serovar E (Biotype 2)

Vibrio vulnificus serovar E (formerly biotype 2) is the etiologic agent that is responsible for the main infectious disease affecting farmed eels. Although the pathogen can theoretically use water as a vehicle for disease transmission, it has not been isolated from tank water during epizootics to date. In this work, the mode of transmission of the disease to healthy eels, the portals of entry of the pathogen into fish, and their putative reservoirs have been investigated by means of laboratory and field experiments. Results of the experiments of direct and indirect host-to-host transmission, patch contact challenges, and oral-anal intubations suggest that water is the prime vehicle for disease transmission and that gills are the main portals of entry into the eel body. The pathogen mixed with food can also come into the fish through the gastrointestinal tract and develop the disease. These conclusions were supported by field data obtained during a natural outbreak in which we were able to isolate this microorganism from tank water for the first time. The examination of some survivors from experimental infections by indirect immunofluorescence and scanning electron microscopy showed that V. vulnificus serovar E formed a biofilm-like structure on the eel skin surface. In vitro assays demonstrated that the ability of the pathogen to colonize both hydrophilic and hydrophobic surfaces was inhibited by glucose. The capacity to form biofilms on eel surface could constitute a strategy for surviving between epizootics or outbreaks, and coated survivors could act as reservoirs for the disease.     

Comparative Study of Biological Properties and Electrophoretic Characteristics of Lipopolysaccharide from Eel-Virulent and Eel-A virulent Vibrio vulnificus Strains

In Vibrio vulnificus, virulence for eels is associated with serovar E strains. In this study, we investigated some biological properties of purified lipopolysaccharides (LPSs) from serovar E and non-serovar E strains. Purified LPSs retained their O-polysaccharidic side chains and did not show any differences that could be related to host specificity, except for serological differences.     

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.     

Isolation of Vibrio vulnificus serovar E from aquatic habitats in Taiwan

The existence of strains of Vibrio vulnificus serovar E that are avirulent for eels is reported in this work. These isolates were recovered from water and oysters and differed from eel virulent strains in (i) fermentation and utilization of mannitol, (ii) ribotyping after HindIII digestion, and (iii) susceptibility to eel serum. Lipopolysaccharide of these strains lacked the highest molecular weight immunoreactive bands, which are probably involved in serum resistance.     

Heterogeneity among Isolates of Vibrio vulnificus Recovered from Eels (Anguilla anguilla) in Denmark

The findings of this study demonstrate that Vibrio vulnificus isolates recovered from diseased eels in Denmark are heterogeneous as shown by O serovars, capsule types, ribotyping, phage typing, and plasmid profiling. The study includes 85 V. vulnificus isolates isolated from the gills, intestinal contents, mucus, spleen, and kidneys of eels during five disease outbreaks on two Danish eel farms from 1995 to 1997, along with a collection of 12 V. vulnificus reference strains. The results showed that more than one serovar may be capable of causing disease in eels and that these isolates are genetically heterogenous as shown by ribotyping. Ribotyping also showed that the same isolates may persist in an eel farm and cause recurrent outbreaks. Phage typing did not correlate with ribotyping or serotyping. However, we observed that 26 of 28 isolates, which were not susceptible to any of the phages, showed the same ribotype, O serovar, and capsule type. This suggests that these isolates may possess features that make them resistant to lysis by the phages used in this study. Ninety-three of 97 isolates harbored between one and three high-molecular-weight plasmids which previously had been suggested to be associated with eel virulence. The subdivision of V. vulnificus into two biotypes based on the indole reaction can no longer be supported, since 82 of 97 isolates in this study were indole positive, and a subdivision into serovars appears to be more correct.     

Multiplex PCR assay for detection of Vibrio vulnificus biotype 2 and simultaneous discrimination of serovar E strains

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

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.     

Analysis of Vibrio vulnificus from Market Oysters and Septicemia Cases for Virulence Markers

Representative encapsulated strains of Vibrio vulnificus from market oysters and oyster-associated primary septicemia cases (25 isolates each) were tested in a blinded fashion for potential virulence markers that may distinguish strains from these two sources. These isolates were analyzed for plasmid content, for the presence of a 460-bp amplicon by randomly amplified polymorphic DNA PCR, and for virulence in subcutaneously (s.c.) inoculated, iron-dextran-treated mice. Similar percentages of market oyster and clinical isolates possessed detectable plasmids (24 and 36%, respectively), produced the 460-bp amplicon (45 and 50%, respectively), and were judged to be virulent in the mouse s.c. inoculation-iron-dextran model (88% for each). Therefore, it appears that nearly all V. vulnificus strains in oysters are virulent and that genetic tests for plasmids and specific PCR size amplicons cannot distinguish between fully virulent and less virulent strains or between clinical and environmental isolates. The inability of these methods to distinguish food and clinical V. vulnificus isolates demonstrates the need for alternative subtyping approaches and virulence assays.     

Occurrence of Vibrio vulnificus Biotypes in Danish Marine Environments

During the unusually warm summer in Denmark in 1994, 11 clinical cases of Vibrio vulnificus infection were reported. These reports initiated an investigation of the occurrence of V. vulnificus biotypes in Danish marine environments. Samples of coastal water, sediment, shellfish, and wild fish were analyzed by preenrichment in alkaline peptone water amended with polymyxin B (2.0 × 10⁴ U/liter) followed by streaking onto modified cellobiose-polymyxin B-colistin agar. V. vulnificus-like colonies were tested with a V. vulnificus-specific DNA probe. Low densities of V. vulnificus were detected in water (0.8 to 19 CFU/liter) from June until mid-September and in sediment (0.04 to >11 CFU/g) from July until mid-November. The presence of V. vulnificus was strongly correlated with water temperature. However, we isolated V. vulnificus from water from a mussel farm at a lower temperature than previously reported (7°C). In 1 of the 13 locations studied, V. vulnificus was found in mussels in 7 of 17 samples analyzed; this is the first report of V. vulnificus in European shellfish. V. vulnificus was also isolated from gills, intestinal contents, and mucus from wild fish. Although biotyping of 706 V. vulnificus strains isolated during our investigations revealed that the majority of the strains (99.6%) belonged to biotype 1, biotype 2 was detected in seawater at a low frequency (0.4%). Our findings provide further evidence that seawater can serve as a reservoir and might facilitate spread of V. vulnificus biotype 2 to eels, with subsequent spread to persons handling eels. In conclusion, our data demonstrate that V. vulnificus is ubiquitous in a temperate marine environment and that V. vulnificus biotype 2 is not strictly confined to eels.     

Electrophoretic analysis of heterogeneous lipopolysaccharides from various strains ofVibrio vulnificus biotypes 1 and 2 by silver staining and immunoblotting

Lipopolysaccharides (LPS) of 11 strains of Vibrio vulnificus biotypes 1 and 2, isolated from an eel farm, and of 10 reference strains, were examined by SDS-polyacrylamide gel electrophoresis coupled with silver staining and immunoblotting. LPS samples were obtained from whole-cell lysates, outer membrane fragments, and extracellular products. By silver staining, only a diffuse band of low-molecular weight could be visualized in all cases except for a biotype 1 strain isolated from water. However, immunoblotting with antisera obtained against strains of biotypes 1 and 2 from eels allowed visualization of multiple O-polysaccharide chains. All biotype 2 strains, independently of their origins, belonged to the same serotype and presented the same LPS profile, whereas eel isolates of biotype 1 were serologically identical and different from the rest of tested strains of biotype 1. This is the first report of LPSs with a ladder-like structure in Vibrio vulnificus.     

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 Ι and ΙΙ). 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 Ι and ΙΙ) still remains to be clarified, and a reevaluation of the definition of the biotypes is also needed.     

Genome-Wide SNP-Genotyping Array to Study the Evolution of the Human Pathogen Vibrio vulnificus Biotype 3

Vibrio vulnificus is an aquatic bacterium and an important human pathogen. Strains of V. vulnificus are classified into three different biotypes. The newly emerged biotype 3 has been found to be clonal and restricted to Israel. In the family Vibrionaceae, horizontal gene transfer is the main mechanism responsible for the emergence of new pathogen groups. To better understand the evolution of the bacterium, and in particular to trace the evolution of biotype 3, we performed genome-wide SNP genotyping of 254 clinical and environmental V. vulnificus isolates with worldwide distribution recovered over a 30-year period, representing all phylogeny groups. A custom single-nucleotide polymorphism (SNP) array implemented on the Illumina GoldenGate platform was developed based on 570 SNPs randomly distributed throughout the genome. In general, the genotyping results divided the V. vulnificus species into three main phylogenetic lineages and an additional subgroup, clade B, consisting of environmental and clinical isolates from Israel. Data analysis suggested that 69% of biotype 3 SNPs are similar to SNPs from clade B, indicating that biotype 3 and clade B have a common ancestor. The rest of the biotype 3 SNPs were scattered along the biotype 3 genome, probably representing multiple chromosomal segments that may have been horizontally inserted into the clade B recipient core genome from other phylogroups or bacterial species sharing the same ecological niche. Results emphasize the continuous evolution of V. vulnificus and support the emergence of new pathogenic groups within this species as a recurrent phenomenon. Our findings contribute to a broader understanding of the evolution of this human pathogen.