Efficacy of a bivalent vaccine against eel diseases caused by Vibrio vulnificus after its administration by four different routes

Vulnivaccine, a vaccine against vibriosis caused by Vibrio vulnificus serovar E (formerly biotype 2), confers acceptable levels of protection to eels after its administration by prolonged immersion in three doses. Recently, a new pathogenic serovar, named serovar A, has been isolated from vaccinated eels in a Spanish freshwater eel farm. The main objective of this work was to design a bivalent vaccine, and to study its effectiveness against the two pathogenic serovars. With this aim, eels weighing around 20 g were immunised with the bivalent vaccine by oral and anal intubation, intraperitoneal injection (i.p.) and prolonged immersion. The overall results indicated that: (i) the new vaccine …

The lipopolysaccharide O side chain of Vibrio vulnificus serogroup E is a virulence determinant for eels

Vibrio vulnificus is a gram-negative bacterium capable of producing septicemic infections in eels and immunocompromised humans. Two biotypes are classically recognized, with the virulence for eels being specific to strains belonging to biotype 2, which constitutes a homogeneous lipopolysaccharide (LPS)-based O serogroup (which we have designated serogroup E). In the present study we demonstrated that the O side chain of this LPS determines the selective virulence of biotype 2 for eels: (i) biotype 1 strains (which do not belong to serogroup E) are destroyed by the bactericidal action of nonimmune eel serum (NIS) through activation of the alternative pathway of complement, (ii) biotype 2 str…

Microbial and histopathological study of the vibriosis caused by Vibrio vulnificus serovar E in eels: The metalloprotease Vvp is not an essential lesional factor

Vibrio vulnificus biotype 2 serovar E (Bt2-serE) is a zoonotic pathogen that causes a haemorrhagic septicaemia in eels, called warm water vibriosis. The main objective of the present work was to study the onset of the eel vibriosis from the microbiological and histopathological viewpoint, as well as to ascertain the role of the protease Vvp as a lesional factor by comparing the histopathological lesions caused by the wild strain and its vvp deficient derivative. The wild-type strain was observed to attach to the gills, where it multiplied following saturation dynamics, subsequently invading the blood stream and reaching the internal organs. Here it reached population sizes that are notably …

Characterization of PAMP/PRR interactions in European eel (Anguilla anguilla) macrophage-like primary cell cultures

The eel (Anguilla anguilla) has been identified as a vulnerable species with stocks dramatically declining over the past decade. In an effort to support the species from overfishing of wild stocks increased interest in eel aquaculture has been notable. In order to expand the scarce knowledge concerning the biology of this species significant research efforts are required in several fields of biology. The development of cell culture systems to study the immune response is a key step towards an increased understanding of the immune response and to develop resources to support further study in this threatened species. Macrophages are one of the most important effector cells of the innate immun…

MARTX Toxin in the Zoonotic Serovar of Vibrio vulnificus Triggers an Early Cytokine Storm in Mice

Vibrio vulnificus biotype 2-serovar E is a zoonotic clonal complex that can cause death by sepsis in humans and fish. Unlike other biotypes, Bt2 produces a unique type of MARTXVv (Multifunctional-Autoprocessive-Repeats-in-Toxin; RtxA13), which is encoded by a gene duplicated in the pVvBt2 plasmid and chromosome II. In this work, we analyzed the activity of this toxin and its role in human sepsis by performing in vitro, ex vivo, and in vivo assays. First, we demonstrated that the ACD domain, present exclusively in this toxin variant, effectively has an actin-cross-linking activity. Second, we determined that the whole toxin caused death of human endotheliocytes and monocytes by lysis and apo…

The Effect of the Environmental Temperature on the Adaptation to Host in the Zoonotic Pathogen Vibrio vulnificus

Vibrio vulnificus is a zoonotic pathogen that lives in temperate, tropical and subtropical aquatic ecosystems whose geographical distribution is expanding due to global warming. The species is genetically variable and only the strains that belong to the zoonotic clonal-complex can cause vibriosis in both humans and fish (being its main host the eel). Interestingly, the severity of the vibriosis in the eel and the human depends largely on the water temperature (highly virulent at 28°C, avirulent at 20°C or below) and on the iron content in the blood, respectively. The objective of this work was to unravel the role of temperature in the adaptation to the host through a transcriptomic and phen…

Domain organization and evolution of multifunctional autoprocessing repeats-in-toxin (MARTX) toxin in Vibrio vulnificus.

ABSTRACT The objective of this study was to analyze multifunctional autoprocessing repeats-in-toxin (MARTX) toxin domain organization within the aquatic species Vibrio vulnificus as well as to study the evolution of the rtxA1 gene. The species is subdivided into three biotypes that differ in host range and geographical distribution. We have found three different types (I, II, and III) of V. vulnificus MARTX (MARTX Vv ) toxins with common domains (an autocatalytic cysteine protease domain [CPD], an α / β-hydrolase domain, and a domain resembling that of the LifA protein of Escherichia coli O127:H6 E2348/69 [Efa/LifA]) and specific domains (a Rho-GTPase inactivation domain [RID], a domain of …

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) ex…

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

ABSTRACT 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.

Protocol for Specific Isolation of Virulent Strains of Vibrio vulnificus Serovar E (Biotype 2) from Environmental Samples

ABSTRACT 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 an…

Metagenomics of the Mucosal Microbiota of European Eels

ABSTRACT European eels are an economically important and threatened species that are prone to rapid collapse in farm conditions. Using metagenomics, we show that the eel mucosal microbiota has specific features distinguishing it from the surrounding aquatic community. This is a first step in dissecting the resident microbiota of this critical barrier that may have implications for maintenance of healthy eel populations.

Siderophores and related outer membrane proteins in Vibrio spp. which are potential pathogens of fish and shellfish

. A total of eight reference strains and 43 environmental isolates of Vibrio species that are potential fish pathogens, were assayed for the production and utilization of siderophores. Chemical and biological assays indicated that all species produced phenolate compounds and only some strains of V. cholerae non-O1, V. parahaemolyticus and V. fluvialis produced hydroxamates. Bioassays indicated that all species produced compounds that stimulated the growth of the homologous and the heterologous species in low-iron media. The catechol-type siderophores produced may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants. However, the chromatogr…

Evidence that water transmits Vibrio vulnificus biotype 2 infections to eels

Vibrio vulnificus biotype 2 is classically considered an obligate eel pathogen. However, it has recently been associated with one human septicemic case. In this paper, the opportunistic behavior of this pathogen is discussed. The bacterium can survive alone in brackish water or attached to eel surfaces for at least 14 days. It is able to spread through water and infect healthy eels by using skin as a portal of entry. These results suggest that water and infected eels may act as reservoirs of infection. A capsule seems to be essential for waterborne infectivity, which would explain why cells recovered from naturally diseased eels give rise to pure cultures of opaque colonies. The spread of t…

Phenotypic and genotypic characterization of a new fish-virulent Vibrio vulnificus serovar that lacks potential to infect humans.

Vibrio vulnificus is a bacterial species that is virulent for humans and fish. Human isolates are classified into biotypes 1 and 3 (BT1 and BT3) and fish isolates into biotype 2 (BT2). However, a few human infections caused by BT2 isolates have been reported worldwide (zoonosis). These BT2 human isolates belong to serovar E (SerE), which is also present in diseased fish. The aim of the present work was to characterize a new BT2 serovar [serovar A (SerA)], which emerged in the European fish-farming industry in 2000, by means of phenotypic, serological and genetic [plasmid profiling, ribotyping and random amplified polymorphic DNA (RAPD)] methodologies. The results confirmed that SerA constit…

Host–pathogen interactions in Vibrio vulnificus: responses of monocytes and vascular endothelial cells to live bacteria

ABSTRACT  Aim: To demonstrate that Vibrio vulnificus, a sepsis-related aquatic pathogen, can provoke a strong pro-inflammatory reaction in blood-associated target cells. Materials & methods: We selected two strains of the two main phylogenetic lineages, two human cell lines, monocytes and vascular endothelial cells and designed an in vitro infection model simulating early septicemia. Results: Both strains caused a strong cell-specific pro-inflammatory response and produced a high degree of cell damage that ended with death by lysis (endothelial cells) or apoptosis/lysis (monocytes). The interaction with endothelial cells was stronger than expected and significantly different for both l…

Identification of DNA sequences specific for Vibrio vulnificus biotype 2 strains by suppression subtractive hybridization.

ABSTRACT 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…

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) CEC…

First record of Vibrio vulnificus biotype 2 from diseased European eel, Anguilla anguilla L.

Presence of a capsule in Vibrio vulnificus biotype 2 and its relationship to virulence for eels

Strains of Vibrio vulnificus biotype 2, isolated from internal organs of diseased European eels as pure cultures of opaque cells, together with some reference strains from Japanese eels, were used in this study. Spontaneous translucent-phase variants were obtained from the corresponding parent strains and compared for a variety of phenotypic traits related to virulence for eels. The rate of colony dissociation from opaque to translucent cells was higher (around 10(-2)) than that observed for translucent to opaque cells (10(-3) to 10(-4)). Electron microscopy with ruthenium red revealed the presence of a capsule of variable thickness on opaque cells, whereas translucent-type colonies had no …

Iron-binding compounds and related outer membrane proteins in Vibrio cholerae non-O1 strains from aquatic environments

A total of 156 strains of Vibrio cholerae non-O1 from aquatic origins were examined for the presence of iron uptake mechanisms and compared with O1 strains and other Vibrio species. All non-O1 strains were able to grow in iron-limiting conditions, with MICs of ethylenediaminedi (O-hydroxyphenylacetic acid) ranging from 20 microM to 2 mM. The production of siderophores was demonstrated by growth in chrome azurol S agar and cross-feeding assays. All strains produced phenolate-type compounds, as assessed by the chemical tests and by bioassays with Salmonella typhimurium enb-7. Some of the strains also promoted the growth of S. typhimurium enb-1 (which can use only enterobactin as a siderophore…

Susceptibility of Nile tilapia (Oreochromis niloticus) to vibriosis due to Vibrio vulnificus biotype 2 (serovar E)

The present study documents the susceptibility of Nile tilapia to the experimental vibriosis caused by Vibrio vulnificus biotype 2 (serovar E) using a reference strain (Spanish Collection of Type Cultures, CECT 4604) selected for its high degree of virulence for eels. The biotype 1 of this species is one of the usual organisms involved in epizootics occurred in tilapia. After intraperitoneal injection, the selected strain developed a haemorrhagic septicaemia similar to eel vibriosis with a LD50 four log units lower than that exhibited by the type strain of the species, which belongs to the biotype 1. The results obtained in waterborne and intubation challenges indicated that water and feed …

Biochemical and toxigenic properties of Vibrio furnissii isolated from a European eel farm

Abstract The present study describes for the first time the isolation of Vibrio furnissii strains from a European eel culture system which are pathogenic for eels ( Anguilla anguilla ) (LD 50 dose, 10 6 cfu/fish). Biochemical characterization of the isolates was performed by API 20E system and by classical numerical taxonomy. Growth at 4 °C, and with 6–8% (w/v) NaCl in a broth medium were necessary to differentiate Vibrio furnissii from Aeromonas species when the API 20E system was used. Virulence for eels of V. furnissii isolates was demonstrated by intraperitoneal injection of living cells. The extracellular products (ECPs) produced by V. furnissii were lethal to elvers and induced some o…

Effectiveness of different vaccine formulations against vibriosis caused by Vibrio vulnificus serovar E (biotype 2) in European eels Anguilla anguilla

Vibriosis due to Vibrio vulnificus serovar E (biotype 2) is one of the main causes of mortality in European eels cultured in Europe. The main objective of this study was to develop a vaccine and a vaccination procedure against this pathogen. With this aim, we tested several vaccine formulations (inactivated whole-cells with and without toxoids‹inactivated extracellular products‹from capsulated and uncapsulated strains, attenuated live vaccines and purified lipopolysaccharide [LPS]) on eels maintained under controlled laboratory conditions using different delivery routes (injection and immersion). To study the immune response we estimated antibody titers and bactericidal/bacteriostatic activ…

Vibrio vulnificus biotype 2, pathogenic for eels, is also an opportunistic pathogen for humans

We report that the eel pathogen Vibrio vulnificus biotype 2 is also an opportunistic pathogen for humans. Results from a detailed comparative study using reference strains of both biotypes revealed that the clinical strain ATCC 33817, originally isolated from a human leg wound and classified as V. vulnificus (no reference on its biotype is noted), belongs to biotype 2 of the species. As a biotype 2 strain, it is negative for indole and pathogenic for eels and mice, harbors two plasmids of high MrS, and belongs to serogroup E, recently proposed as characteristic of biotype 2 strains. In consequence, appropriate measures must be taken by consumers, particularly by those running a health risk,…

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

ABSTRACT 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…

Role of iron in the pathogenicity of Vibrio damsela for fish and mammals.

The ability to obtain iron of 14 isolates of Vibrio damsela with different degrees of virulence for mice and turbot (Scophthalmus maximus) has been evaluated in artificial and natural iron-restricted environments. All strains were capable of utilizing haemoglobin (Hb) and ferric ammonium citrate (FAC) as the sole iron sources in vitro. However, only virulent V. damsela strains were able to resist the bacteriostatic and bactericidal effects of human and turbot sera, their growth being enhanced by the addition of Hb and FAC. The inhibitory effect of these sera on the growth of the non-pathogenic strain (ATCC 35083), however, was reversed by heat treatment (56 degrees C for 60 min). The role o…

Survival of fish-virulent strains ofPhotobacterium damselaesubsp.damselaein seawater under starvation conditions

The survival of fish-virulent strains of Photobacterium damselae subsp. damselae in seawater microcosms, with and without sediment, was investigated. The strains survived as culturable bacteria at 14 and 22 degrees C for at least 1 year, and infectivity for fish was maintained. At 5 degrees C, cells lost culturability on solid media, but this was recovered when the temperature was increased to 22 degrees C. Finally, morphological changes in the bacterium (rod to coccus), and production of vesicles and extracellular material were observed during the time of starvation. The overall results suggest that seawater and sediment can act as reservoirs for these virulent strains.

Vibrio vulnificus serovar A: an emerging pathogen in European anguilliculture.

The spread of the emerging pathogen Vibrio vulnificus biotype 2 serovar A in Danish anguilliculture is reported. Serovar A was originally isolated in a Spanish eel farm in 2000 and occurred in Denmark in the summer of 2004, affecting eels of 5-10 g body weight cultured in fresh water. The Danish eels showed clinical signs different from those reported for Spanish eels, such as severe haemorrhages in the head and gill region with necrosis of the soft tissues. Danish isolates were biochemically and serologically identical to Spanish serovar A strains and also highly virulent for eels by both intraperitoneal injection and immersion challenges. Vaccination with Vulnivaccine, a vaccine against V…

In vitro study of antimicrobial activity on Klebsiella Pneumoniae biofilms in endotracheal tubes

Effective treatment approaches for biofilms in endotracheal tubes (ETTs) are lacking. In this study, we evaluated the in vitro effects of five antimicrobials against biofilms formed by Klebsiella pneumoniae in ETTs. K. pneumoniae was added to minimal mucin medium prior to inoculation in microtiter plates containing ETT fragments. Biofilm susceptibility was assessed by crystal violet staining. At 24 h, the antimicrobials significantly reduced biofilm formation. At 48 h, all of the antimicrobial agents exhibited significant reductions in biofilm formation, even at concentrations above the minimum inhibitory concentration (MIC). Tigecycline and fosfomycin showed the greatest inhibition capacit…

Potentially human-virulent Vibrio vulnificus isolates from diseased great pompano (Trachinotus goodei).

Vibrio vulnificus is an opportunistic human pathogen responsible for the majority of seafood-associated deaths worldwide and is also a relevant fish pathogen for the aquaculture industry. In addition to infections in aquatic livestock, V. vulnificus also represents a risk to aquarium animals. For the first time, this work describes an important mortality outbreak in Trachinotus goodei in a zoo aquarium, with the isolation of Vibrio vulnificus (Vv) from the internal organs of the diseased fish. The isolates were identified by MALDI-TOF MS, serotyped and characterized by pulsed-field gel electrophoresis (PFGE). Although the isolates from great pompanos did not belong to pathovar piscis (forme…

Pathogenicity of live bacteria and extracellular products of motileAeromonasisolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD50 105·4‐107·6 cfu fish‐1) but no Aer. sobria, Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 108·4 cfu fish‐1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD50 1·0–3·2 μg (g fish)‐1) reproducing the symptoms associated with natur…

Comparative study of biological properties and electrophoretic characteristics of lipopolysaccharide from eel-virulent and eel-A virulent Vibrio vulnificus strains.

ABSTRACT 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.

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 …

Siderophore-mediated iron acquisition mechanisms in Vibrio vulnificus biotype 2

Vibrio vulnificus biotype 2 is a primary pathogen for eels and, as has recently been suggested, an opportunistic pathogen for humans. In this study we have investigated the ability of V. vulnificus biotype 2 to obtain iron by siderophore-mediated mechanisms and evaluated the importance of free iron in vibriosis. The virulence degree for eels was dependent on iron availability from host fluids, as was revealed by a reduction in the 50% lethal dose for iron-overloaded eels. This biotype produced both phenolate- and hydroxamate-type siderophores of an unknown nature and two new outer membrane proteins of around 84 and 72 kDa in response to iron starvation. No alterations in lipopolysaccharide …

Draft Genome Sequence of Environmental Bacterium Vibrio vulnificus CladeA-yb158

ABSTRACT We report the genome sequence of the environmental Vibrio vulnificus biotype 1_cladeA. This draft genome of the CladeA-yb158 strain, isolated in Israel, represents this newly emerged clonal group that contains both clinical and environmental strains.

Evidence that water transmits the disease caused by the fish pathogen Photobacterium damselae subsp. damselae

The transmission through water of the disease caused by the fish pathogen, Photobacterium damselae subsp. damselae, as well as the role of the skin mucus in the initial steps of the infection, have been studied. All tested strains resisted the bactericidal activity of the mucus and showed an ability to adhere to it, but only those virulent by the intraperitoneal route were infective through water. Moribund fishes showed the typical signs of the disease: haemorrhaged areas on the body surface and ulcerative lesions with mucus degradation. These results suggest that the pathogen can be transmitted to fish through water and use the skin as a portal of entry.

Evaluation of genotypic and phenotypic methods to distinguish clinical from environmental Vibrio vulnificus strains.

ABSTRACT Vibrio vulnificus is a heterogeneous bacterial species that comprises virulent and avirulent strains from environmental and clinical sources that have been grouped into three biotypes. To validate the typing methods proposed to distinguish clinical from environmental isolates, we performed phenotypic (API 20E, API 20NE, and BIOLOG tests) and genetic (ribotyping and DNA polymorphism at several loci) studies with a large strain collection representing different biotypes, origins, and host ranges. No phenotypic method was useful for biotyping or grouping strains with regard to the origin of an isolate, and only the BIOLOG system was reliable for identifying the strains at the species …

Plasmid diversity in Vibrio vulnificus biotypes.

Vibrio vulnificus is a heterogeneous bacterial species that can be virulent for humans and fish. Virulence in fish seems to rely on a recently described plasmid that can be transmitted between strains, aided by a conjugative plasmid. The main objective of this work was to analyse the plasmid content of a wide collection of strains from the three biotypes of the species, as well as to identify putative conjugative and virulence plasmids by means of Southern hybridization with specific probes and sequence analysis of selected gene markers. We found 28 different plasmid profiles in a total of 112 strains, which were relatively biotype- or serovar-specific. Biotype 1 lacked high-molecular-mass…

MARTX ofVibrio vulnificusbiotype 2 is a virulence and survival factor

Vibrio vulnificus biotype 2 is a polyphyletic group whose virulence for fish relies on a plasmid. This plasmid contains an rtxA gene duplicated in the small chromosome that encodes a MARTX (Multifunctional, Autoprocessing Repeats-in-Toxin) unique within the species in domain structure (MARTX type III). To discover the role of this toxin in the fitness of this biotype in the fish-farming environment, single- and double-knockout mutants were isolated from a zoonotic strain and analysed in a series of in vivo and in vitro experiments with eel, fish cell lines and amoebae isolated from gills. Mice, murine and human cell lines were also assayed for comparative purposes. The results suggest that …

Serum antibodies to Vibrio vulnificus biotype 3 lipopolysaccharide and susceptibility to disease caused by the homologous V. vulnificus biotype

SUMMARYIn 1996 an outbreak of severe soft tissue infections caused byVibrio vulnificusunexpectedly erupted in fish consumers in Israel with relatively little morbidity in fish farmers. To test the hypothesis that recurrent exposure of fishermen to the virulent strain may have provided protection against severe or symptomatic disease, we investigated the association between the immune response toV. vulnificusbiotype 3 lipopolysaccharide (BT3 LPS) and disease susceptibility in fish farmers and fish consumers. Serum samples were tested for IgA and IgG of anti-BT3 LPS in fishermen and fish consumers who suffered fromV. vulnificusBT3 infections and their matched controls. Pre-existing levels of …

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

ABSTRACT 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 anim…

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…

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 …

Early steps in the European eel (Anguilla anguilla)–Vibrio vulnificus interaction in the gills: Role of the RtxA13 toxin

Vibrio vulnificus is an aquatic gram-negative bacterium that causes a systemic disease in eels called warm-water vibriosis. Natural disease occurs via water born infection; bacteria attach to the gills (the main portal of entry) and spread to the internal organs through the bloodstream, provoking host death by haemorrhagic septicaemia. V.vulnificus produces a toxin called RtxA13 that hypothetically interferes with the eel immune system facilitating bacterial invasion and subsequent death by septic shock. The aim of this work was to study the early steps of warm-water vibriosis by analysing the expression of three marker mRNA transcripts related to pathogen recognition (tlr2 and tlr5) and in…

A Method of Transposon Insertion Sequencing in Comprehensively Identifying Vibrio vulnificus Genes Required for Growth in Human Serum

One of the most powerful approaches to detect the loci that enable a pathogen to cause disease is the creation of a high-density transposon mutant library by transposon insertion sequencing (TIS) and the screening of the library using an adequate in vivo and/or ex vivo model of the disease. Here we describe the procedure for detection of the putative loci required for a septicemic pathogen to cause sepsis in humans by using TIS plus an ex vivo model of septicaemia: to grow the pathogen in fresh and inactivated human serum. We selected V. vulnificus because it is a highly invasive pathogen capable of spreading from an infection site to the bloodstream, causing sepsis and death in less than 2…

A method to diagnose the carrier state of Vibrio vulnificus serovar E in eels: Development and field studies

Abstract The pathogen Vibrio vulnificus serovar E (VSE) has been related to both human infections and to epizootics causing high mortality in brackish water eel farms. To control the spread of the eel vibriosis and prevent VSE transmission to humans we designed and tested a protocol to detect carriers, which involves isolating the pathogen. To identify the organs where VSE persists in survivors we infected eels with different degrees of immunity against the pathogen (non-immune [NI], immune [I, eels vaccinated 1 year before] and freshly vaccinated [V]) by bath challenge. Then, we followed the pathogen survival in selected external and internal organs for 72 h post-infection. VSE was isolate…

Physico-chemical and bacteriological parameters in a hypereutrophic lagoon (Albufera Lake, Valencia, Spain).

Several physico-chemical parameters related to water quality, as well as saprophytic and public health-related heterotrophic bacterial groups were studied in a hypereutrophic lake (Albufera, Valencia) at different seasons. Total microscopic and viable counts were compared, and, together with faecal indicators, were determined in water samples from different sites. Heterotrophic bacteria grown on nonselective medium were identified to genus level and a diversity index was calculated. Pseudomonas-Alcaligenes was the most frequently isolated group from all sampling sites along the study, following by Moraxella, Acinetobacter, Vibrio and Aeromonas. The relationships between all parameters were …

pilF polymorphism-based PCR to distinguish Vibrio vulnificus strains potentially dangerous to public health

ABSTRACT Vibrio vulnificus is a heterogeneous species that comprises strains virulent and avirulent for humans and fish, and it is grouped into three biotypes. In this report, we describe a PCR-based methodology that allows both the species identification and discrimination of those isolates that could be considered dangerous to public health. Discrimination is based on the amplification of a variable region located within the gene pilF , which seems to be associated with potential human pathogenicity, regardless of the biotype of the strain.

Vibriosis in aquaculture. 16th EAFP Conference, Tampere, Finland, 4th September 2013

Aquaculture in brackish and marine water is growing worldwide (FAO, 2014), including new cultured species. Various Vibrio infections occur, and cause significant problems, in fish, crustacean and shellfish (Austin & Austin, 2007. Vibrio anguillarum, V. salmonicida, V. ordalii and V. vulnificus are among the pathogens that lead to the biggest losses in aquaculture all over the world (Toranzo et al. 2005, Sandlund et al., 2010; Sitjà-Bobadilla et al. 2014).

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 inf…

Surface and virulence properties of environmental Vibrio cholerae non-O1 from Albufera Lake (Valencia, Spain).

A total of 140 environmental Vibrio cholerae non-O1 isolates, together with several culture collection strains from both environmental and clinical sources, were studied in relation to hemagglutination, surface hydrophobicity, and the enzymatic, hemolytic, cytotoxic, and enterotoxic activities of their extracellular products. A total of 78 and 62% of the strains produced hemagglutinins and exohemagglutinins, respectively. Four different hemagglutinating and two exohemagglutinating activities were found by using eight sugars in the inhibition assays. Cell-bound mannose-sensitive hemagglutination was detected mainly in chicken blood, whereas fucose-sensitive hemagglutination was recorded only…

Efficacy of oral reimmunisation after immersion vaccination against Vibrio vulnificus in farmed European eels

Abstract Vulnivaccine, a licensed vaccine against Vibrio vulnificus , protects eels against vibriosis after vaccination by triple prolonged immersion at glass eel stage. Protection lasts for at least 6 months, but later, protection decreases, and eels can suffer stress-related vibriosis. The main objective of this work was to design an oral vaccine that can be used for reimmunisation at any developmental eel stage. With this aim, the efficacy of Vulnivaccine mixed with food as an oral booster was tested in an eel farm. The protection and the immune response (innate and acquired) in serum, mucus (from skin and intestine) and bile were evaluated in reimmunised and control animals (non-reimmun…

Impact of analytic provenance in genome analysis

Background Many computational methods are available for assembly and annotation of newly sequenced microbial genomes. However, when new genomes are reported in the literature, there is frequently very little critical analysis of choices made during the sequence assembly and gene annotation stages. These choices have a direct impact on the biologically relevant products of a genomic analysis - for instance identification of common and differentiating regions among genomes in a comparison, or identification of enriched gene functional categories in a specific strain. Here, we examine the outcomes of different assembly and analysis steps in typical workflows in a comparison among strains of Vi…

Replicating phages in the epidermal mucosa of the eel (Anguilla anguilla)

In this work, we used the eel (Anguilla anguilla) as an animal model to test the hypothesis of Barr et al. (2013a,b) about the putative role of the epidermal mucosa as a phage enrichment layer. To this end, we analyzed the microbial content of the skin mucus of wild and farmed eels by using a metagenomic approach. We found a great abundance of replicating phage genomes (concatemers) in all the samples. They were assembled in four complete genomes of three Myovirus and one Podovirus. We also found evidences that ΦKZ and Podovirus phages could be part of the resident microbiota associated to the eel mucosal surface and persist on them over the time. Moreover, the viral abundance estimated by …

The emergence of Vibrio pathogens in Europe : ecology, evolution, and pathogenesis (Paris, 11-12th March 2015)

Global change has caused a worldwide increase in reports of Vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals. In Europe, higher prevalence of human infections followed regional climatic trends with outbreaks occurring during episodes of unusually warm weather. Similar patterns were also observed in Vibrio-associated diseases affecting marine organisms such as fish, bivalves and corals. Basic knowledge is still lacking on the ecology and evolutionary biology of these bacteria as well as on their virulence mechanisms. Current limitations in experimental systems to study infection and the lack of diagnostic tools still prevent a better understanding of Vibrio…

Host-Nonspecific Iron Acquisition Systems and Virulence in the Zoonotic Serovar of Vibrio vulnificus

ABSTRACT The zoonotic serovar of Vibrio vulnificus (known as biotype 2 serovar E) is the etiological agent of human and fish vibriosis. The aim of the present work was to discover the role of the vulnibactin- and hemin-dependent iron acquisition systems in the pathogenicity of this zoonotic serovar under the hypothesis that both are host-nonspecific virulence factors. To this end, we selected three genes for three outer membrane receptors ( vuuA , a receptor for ferric vulnibactin, and hupA and hutR , two hemin receptors), obtained single and multiple mutants as well as complemented strains, and tested them in a series of in vitro and in vivo assays, using eels and mice as animal models. Th…

Method for Specific Identification of the Emerging Zoonotic Pathogen Vibrio vulnificus Lineage 3 (Formerly Biotype 3).

ABSTRACT Vibrio vulnificus is a zoonotic pathogen that is spreading worldwide due to global warming. Lineage 3 (L3; formerly biotype 3) includes the strains of the species with the unique ability to cause fish farm-linked outbreaks of septicemia. The L3 strains emerged recently and are particularly virulent and difficult to identify. Here, we describe a newly developed PCR method based on a comparative genomic study useful for both rapid identification and epidemiological studies of this interesting emerging group. The comparative genomic analysis also revealed the presence of a genetic duplication in the L3 strains that could be related to the unique ability of this lineage to produce sept…

Siderophore production by environmental strains ofSalmonellaspecies

Iron uptake mechanisms were investigated in different species of Salmonella isolated from environmental waters. All strains examined were able to grow in the presence of high concentrations (10 mM) of the iron chelator EDDA. All strains excreted phenolate and hydroxamate siderophores, as assessed by bioassays and chemical tests. Bioassays with different indicator strains showed that all Salmonella strains can cross-feed other Enterobacteria, as well as mutants of Salmonella typhimurium deficient in the Enterobactin system, suggesting that this siderophore may be produced by the environmental Salmonella strains. The siderophore aerobactin may also be produced by one of the strains, according…

An Enriched European Eel Transcriptome Sheds Light upon Host-Pathogen Interactions with Vibrio vulnificus.

Infectious diseases are one of the principal bottlenecks for the European eel recovery. The aim of this study was to develop a new molecular tool to be used in host-pathogen interaction experiments in the eel. To this end, we first stimulated adult eels with different pathogen- associated molecular patterns (PAMPs), extracted RNA from the immune-related tissues and sequenced the transcriptome.We obtained more than 2x106 reads that were assembled and annotated into 45,067 new descriptions with a notable representation of novel transcripts related with pathogen recognition, signal transduction and the immune response. Then, we designed a DNA-microarray that was used to analyze the early immun…

Draft genome sequences of Vibrio vulnificus strains recovered from moribund tilapia

Potentially zoonotic Vibrio vulnificus strains were isolated from vibriosis outbreaks occurring on eastern Mediterranean tilapia farms between 2016 and 2019. In this work, the draft genome sequences of three representative isolates are presented.

Vibrio vulnificus biotype 2 serovar E gne but not galE is essential for lipopolysaccharide biosynthesis and virulence

ABSTRACT This work aimed to establish the role of gne (encoding UDP-GalNAc 4-epimerase activity) and galE (encoding UDP-Gal-4-epimerase activity) in the biosynthesis of surface polysaccharides, as well as in the virulence for eels and humans of the zoonotic serovar of Vibrio vulnificus biotype 2, serovar E. DNA sequence data revealed that gne and galE are quite homologous within this species (≥90% homology). Mutation in gne of strain CECT4999 increased the surface hydrophobicity, produced deep alterations in the outer membrane architecture, and resulted in noticeable increases in the sensitivity to microcidal peptides (MP), to eel and human sera, and to phagocytosis/opsonophagocytosis. Furt…

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

Summary 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 bacte- rial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E…

Role of iron, capsule, and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for mice

The virulence mechanisms of Vibrio vulnificus biotype 2 have been studied and compared with those of biotype 1 in mice as the experimental animals. Biotype 2 isolates from European eels were as virulent for mice as biotype 1 strains (50% lethal dose, about 10(5) CFU per mouse); a septicemic infection developed in less than 24 h. These strains had several properties in common with biotype 1 organisms including capsule expression, uptake of various iron sources, and production of exoproteins, whose role in mouse virulence has been demonstrated. We also discuss the implication of biotype 2 strains in human infections.

Isolation and characterization of Vibrio parahaemolyticus causing infection in Iberian toothcarp Aphanius iberus

High mortality among laboratory cultured Iberian toothcarp Aphanius iberus occurred in February 1997 in Valencia (Spain). The main signs of the disease were external haemorrhage and tail rot. Bacteria isolated from internal organs of infected fish were biochemically homogeneous and identified as Vibrio parahaemolyticus. The bacteria were haemolytic against erythrocytes from eel Anguilla anguilla, amberjack Seriola dumerili, toothcarp A. iberus and humans, and were Kanagawa-phenomenon-negative. Infectivity tests showed that the virulence for A. iberus was dependent on salinity. Finally, all strains were virulent for amberjack and eel.

Wild eel microbiome reveals that skin mucus of fish could be a natural niche for aquatic mucosal pathogen evolution

Background Fish skin mucosal surfaces (SMS) are quite similar in composition and function to some mammalian MS and, in consequence, could constitute an adequate niche for the evolution of mucosal aquatic pathogens in natural environments. We aimed to test this hypothesis by searching for metagenomic and genomic evidences in the SMS-microbiome of a model fish species (Anguilla Anguilla or eel), from different ecosystems (four natural environments of different water salinity and one eel farm) as well as the water microbiome (W-microbiome) surrounding the host. Results Remarkably, potentially pathogenic Vibrio monopolized wild eel SMS-microbiome from natural ecosystems, Vibrio anguillarum/Vibr…

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

The outer membrane proteins of 17 Vibrio vulnificus biotype 2 strains from Japanese and European eels, 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 strains. 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, 4…

Spontaneous Quinolone Resistance in the Zoonotic Serovar of Vibrio vulnificus

ABSTRACT This work demonstrates that Vibrio vulnificus biotype 2, serovar E, an eel pathogen able to infect humans, can become resistant to quinolone by specific mutations in gyrA (substitution of isoleucine for serine at position 83) and to some fluoroquinolones by additional mutations in parC (substitution of lysine for serine at position 85). Thus, to avoid the selection of resistant strains that are potentially pathogenic for humans, antibiotics other than quinolones must be used to treat vibriosis on farms.

Corrigendum: Phylogeny of Vibrio vulnificus From the Analysis of the Core-Genome: Implications for Intra-Species Taxonomy

Vibrio vulnificus (Vv) is a multi-host pathogenic species currently subdivided into three biotypes (Bts). The three Bts are human-pathogens, but only Bt2 is also a fish-pathogen, an ability that is conferred by a transferable virulence-plasmid (pVvbt2). Here we present a phylogenomic analysis from the core genome of 80 Vv strains belonging to the three Bts recovered from a wide range of geographical and ecological sources. We have identified five well-supported phylogenetic groups or lineages (L). LI comprises a mixture of clinical and environmental Bt1 strains, most of them involved in human clinical cases related to raw seafood ingestion. LII is linked to the aquaculture industry and incl…

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 th…

Characterization of R-plasmids in environmental isolates ofsalmonella: Host range and stability

Four environmental isolates ofSalmonella, resistant to several drugs, were examined for plasmid carriage with four different plasmid DNA isolation procedures. The method of Birnboim and Doly gave the best results. Three of the strains possessed a single plasmid with molecular weights of 60 (kanamycin resistant), 44.5 (kanamcin resistant), and 23.4 Md (ampicillin and amoxicillin resistant); the other strain (resistant to tetracycline) harbored two plasmids of 69.8 and 2.2 Md. The 69.8 Md was the one responsible for resistance. All plasmids were fi−, and the 44.5 Md Kcr plasmid synthesized a sex pilus type F. Some properties related to the dissemination of R-plasmids, such as host range, tran…

Serological and molecular characteristics of Vibrio vulnificus biotype 3: evidence for high clonality.

Vibrio vulnificus biotype 3 has been implicated as the causative pathogen of an ongoing disease outbreak that erupted in Israel in 1996. Recent work based on multi-locus sequence typing (MLST) showed that V. vulnificus biotype 3 is genetically homogeneous. The aim of this study was to investigate the existence of subpopulations within this homogeneous biotype by characterizing the surface antigens and analysing the sequence diversity of selected outer-membrane protein (OMP)-encoding genes. Rabbit antisera were prepared against biotype 1, 2 and 3 strains. The results of the slide-agglutination test, dot-blot assay (using fresh and boiled cells), and immunoblotting of lipopolysaccharides (LPS…

The cytotoxin-hemolysin genes of human and eel pathogenic Vibrio vulnificus strains: comparison of nucleotide sequences and application to the genetic grouping.

Vibrio vulnificus can be divided into two groups on the basis of pathogenesis. Group 1 is pathogenic only to humans, whereas group 2 is pathogenic to eels and occasionally to humans. Although both groups produce a 50-kDa cytotoxin-hemolysin (V. vulnificus hemolysin; VVH), the toxins are different. In the present study, the nucleotide sequence of the toxin gene (vvhA ) of strain CDC B3547 (a group 2 strain) was determined, and the deduced amino acid sequence was compared to that of strain L-180 (a group 1 strain). The nucleotide sequence of vvhA of strain CDC B3547 was about 96% identical with that of strain L-180, which results in a difference of 3 amino acid residues in the C-terminal lect…

Effect of low temperature on starvation-survival of the eel pathogen Vibrio vulnificus biotype 2

At present, no reports exist on the isolation of the eel pathogen Vibrio vulnificus biotype 2 from water samples. Nevertheless, it has recently been demonstrated that this biotype can use water as a route of infection. In the present study, the survival of this pathogen in artificial seawater (ASW) microcosms at different temperatures (25 and 5 degrees C) was investigated during a 50-day period, with biotype 1 as a control, V. vulnificus biotype 2 was able to survive in the culturable state in ASW at 25 degrees C in the free-living form, at least for 50 days, entering into the nonculturable state when exposed to low temperature. In this state, this microorganism survived with reduced rates …

Additional file 1: Table S1. of Wild eel microbiome reveals that skin mucus of fish could be a natural niche for aquatic mucosal pathogen evolution

Metagenomes used to detect MGE. Table S2. General data for each metagenome and alpha diversity. Table S3. Contigs with MGE detected using the methodology described in Fig. 1. Figure S1. Sampling points, location and description. Figure S2. From nature to the laboratory: skin mucus sampling from wild eels and DNA extraction. Figure S3. Mobile genetic elements (MGE) detection workflow diagram. Figure S4. %GC content profiles of the eel’s SMS- and W-metagenomes. Figure S5. Wild eel’s versus farmed eel’s SMS metagenomes. Figure S6. V. metoecus M12v BLAST atlas. Figure S7. Schematic representation of VPI-2 in M12v. Figure S8. Main bacterial genera detected in eel’s SMS- and W-metagenomes. Figure…

Isolation of a new serovar of Vibrio vulnificus pathogenic for eels cultured in freshwater farms

The present work reports the isolation of a new serovar of Vibrio vulnificus pathogenic for eels cultured in freshwater farms. This serovar, named Serovar A, produced two outbreaks of low morbidity and mortality in one Spanish eel farm in September 2000 and March 2001. The vibriosis affected healthy eels, weighting between 10 and 100 g, that had been vaccinated against the serovar E of the species (formerly biotype 2) at least 6 months before. Some of the diseased eels showed symptoms different to those reported for the classical vibriosis due to serovar E. These symptoms included bleeding ventral ulcers together with haemorrhagic intestine. Bath experimental challenges demonstrated that (i…

Isolation of a hemin and hemoglobin binding outer membrane protein of Vibrio vulnificus biotype 2 (serogroup E)

The eel pathogen Vibrio vulnificus biotype 2 (serogroup E) is able to use hemin (Hm) or hemoglobin (Hb) as the sole iron source for growth in vitro and in vivo. The mechanism of heme-iron acquisition in this bacterium requires a direct interaction through binding sites on the bacterial surface (constitutive outer membrane proteins). Using affinity chromatography techniques, a unique protein of around 36.5 kDa was isolated from cell envelopes of E86 strain regardless of the affinity ligand used, hemoglobin or hemin. This protein was purified from both iron-enriched and iron-restricted grown cells. These results support the hypothesis that in this pathogen Hm- and Hb-iron acquisition is media…

The kinetics of antibody production in mucus and serum of European eel (Anguilla anguilla L.) after vaccination against Vibrio vulnificus: development of a new method for antibody quantification in skin mucus

Abstract Vibrio vulnificus serovar E, a bacterial pathogen for eels cultured in intensive systems, is transmitted through water and enters into new hosts mainly via gills. The main objective of this work was to study the kinetics of antibody production to V. vulnificus in serum and mucus and their relationship with protection after vaccination. To quantify local mucus antibodies, a new ‘in situ’ dot blot immunoassay using image analysis has been developed. This assay was applied to measure antibody production in the skin zone next to the gills. We found that (i) the immune response in mucus was faster (peak at days 3–4) and shorter in duration (titres significantly elevated up to day 5 and …

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) ce…

Corrigendum: Phylogeny of Vibrio vulnificus From the Analysis of the Core-Genome: Implications for Intra-Species Taxonomy

Iron and Fur in the life cycle of the zoonotic pathogenVibrio vulnificus

Summary In this study, we aimed to analyze the global response to iron in the broad-range host pathogen Vibrio vulnificus under the hypothesis that iron is one of the main signals triggering survival mechanisms both inside and outside its hosts. To this end, we selected a strain from the main zoonotic clonal-complex, obtained a mutant in the ferric-uptake-regulator (Fur), and analyzed their transcriptomic profiles in both iron-excess and iron-poor conditions by using a strain-specific microarray platform. Among the genes differentially expressed, we identified around 250 as putatively involved in virulence and survival-related mechanisms. Then, we designed and performed a series of in vivo …

First description of non-motile Yersinia ruckeri serovar I strains causing disease in rainbow trout, Oncorhynchus mykiss (Walbaum), cultured in Spain.

Yersinia ruckeri, the causal agent of enteric redmouth (ERM) disease, was isolated from epizootics that occurred in different Spanish rainbow trout, Oncorhynchus mykiss (Walbaum), farms in which vaccination against ERM had been performed. In all episodes, the most pronounced clinical signs exhibited by affected fish were severe haemorrhages in the mouth, eyes and around the vent. The isolates were identified as Y. ruckeri serovar I by 16S rRNA sequencing together with serological tests. They lacked motility and lipase activity and thus belonged to biotype 2, and were highly virulent for juvenile rainbow trout, both by intraperitoneal injection (from 3.1 x 10(2) to 6.3 x 10(3) cfu per fish) …

pilF polymorphism-based real-time PCR to distinguish Vibrio vulnificus strains of human health relevance

The Gram-negative bacterium Vibrio vulnificus is a common inhabitant of estuarine environments. Globally, V. vulnificus is a significant foodborne pathogen capable of causing necrotizing wound infections and primary septicemia, and is a leading cause of seafood-related mortality. Unfortunately, molecular methods for the detection and enumeration of pathogenic V. vulnificus are hampered by the genetically diverse nature of this pathogen, the range of different biotypes capable of infecting humans and aquatic animals, and the fact that V. vulnificus contains pathogenic as well as non-pathogenic variants. Here we report an alternative approach utilizing the development of a real-time PCR assay…

Vaccination of market-size eels against vibriosis due to Vibrio vulnificus serovar E

Vaccination with Vulnivaccine at eel farms has been previously shown to protect cultured eels against vibriosis caused by Vibrio vulnificus serovar E for more than 1 year. The reported protocol included an initial vaccination by triple prolonged immersion at the glass-eel stage together with one optional oral booster at the elver stage. However, eels at the market-size stage (around 150 g body weight) can suffer stress-related vibriosis after handling and transport to the selling facilities, which implies a serious risk for consumer health. The main objective of this work was therefore to develop an effective re-vaccination procedure, useful for preventing stress-related vibriosis and zoono…

Phylogeny and life cycle of the zoonotic pathogen Vibrio vulnificus

Vibrio vulnificus is a zoonotic pathogen able to cause diseases in humans and fish that occasionally result in sepsis and death. Most reviews about this pathogen (including those related to its ecology) are clearly biased towards its role as a human pathogen, emphasizing its relationship with oysters as its main reservoir, the role of the known virulence factors as well as the clinic and the epidemiology of the human disease. This review tries to give to the reader a wider vision of the biology of this pathogen covering aspects related to its phylogeny and evolution and filling the gaps in our understanding of the general strategies that V. vulnificus uses to survive outside and inside its …

Adaptation to host inVibrio vulnificus, a zoonotic pathogen that causes septicemia in fish and humans

Vibrio vulnificus is a siderophilic pathogen spreading due to global warming. The zoonotic strains constitute a clonal-complex related to fish farms that are distributed worldwide. In this study, we applied a transcriptomic and single gene approach and discover that the zoonotic strains bypassed the iron requirement of the species thanks to the acquisition of two iron-regulated outer membrane proteins (IROMPs) involved in resistance to fish innate immunity. Both proteins have been acquired by horizontal gene transfer and are contributing to the successful spreading of this clonal-complex. We have also discovered that the zoonotic strains express a virulent phenotype in the blood of its main…

Immunogenic antigens of the eel pathogen Vibrio vulnificus serovar E.

Abstract The immunogenic antigens of Vibrio vulnificus serovar E were investigated in the eel. Fish were vaccinated by immersion with Vulnivaccine (V), revaccinated 2 years later by intraperitoneal injection (RV) and bath infected 15 days post-revaccination (RVI). The specific immune response in serum was followed in all groups, and selected sera were used for immunostaining of surface (SA) and extracellular antigens (ECA). Bacteria were grown in iron-rich (TSB and MSWYE) and iron-poor media (TSB and MSWYE plus human transferrin (TSB-T and MSWYE-T)) as well as eel serum (ES), and their SA and ECA were extracted and electrophoretically analysed. Cells grown in MSWYE-T and ES presented the sa…

Field testing of a vaccine against eel diseases caused by Vibrio vulnificus

The field results of a vaccination programme against Vibrio vulnificus serovar E (biotype 2) in a Spanish eel farm are reported. A total of 9.5 million glass eels were vaccinated from January 1998 to March 2000 by prolonged immersion followed by 2 subsequent reimmunisations after 12 to 14 and 24 to 28 d, respectively. The acquired protection and the immune response against serovar E were estimated over a period of 6 mo after vaccination. A similar vaccination schedule was conducted with elvers in a Danish eel farm. In this case, the acquired protection and the immune response against serovar E and the new eel-pathogenic serovars, recently described in Denmark, were evaluated over a short te…

Comprehensive identification of Vibrio vulnificus genes required for growth in human serum.

ABSTRACT Vibrio vulnificus can be a highly invasive pathogen capable of spreading from an infection site to the bloodstream, causing sepsis and death. To survive and proliferate in blood, the pathogen requires mechanisms to overcome the innate immune defenses and metabolic limitations of this host niche. We created a high-density transposon mutant library in YJ016, a strain representative of the most virulent V. vulnificus lineage (or phylogroup) and used transposon insertion sequencing (TIS) screens to identify loci that enable the pathogen to survive and proliferate in human serum. Initially, genes underrepresented for insertions were used to estimate the V. vulnificus essential gene set;…

Eel immune response to Vibrio vulnificus infection. Host-pathogen relationship

The European eel (Anguilla anguilla), has experienced a dangerous decline in recruitment, yield and stock over the last 30 years and this decline is likely to continue into the future. Several major threats are responsible for this situation, including overfishing of glass eels for consumption, new infections by introduced pathogens, dams and blocking of migration routes. The lack of knowledge concerning the biology of this species represents a handicap to the conservation and recovery of the population. Nevertheless,the eel farming industry is increasing, therefore it could be considered one of the major risks for wild eel stock perpetuation, which is threatened principally by the lack of …

A multiplex PCR for the detection of Vibrio vulnificus hazardous to human and/or animal health from seafood

Vibrio vulnificus is a zoonotic pathogen linked to aquaculture that is spreading due to climate change. The pathogen can be transmitted to humans and animals by ingestion of raw shellfish or seafood feed, respectively. The aim of this work was to design and test a new procedure to detect V. vulnificus hazardous to human and/or animal health in food/feed samples. For this purpose, we combined a pre-enrichment step with multiplex PCR using primers for the species and for human and animal virulence markers. In vitro assays with mixed DNA from different Vibrio species and Vibrio cultures showed that the new protocol was 100 % specific with a detection limit of 10 cfu/mL. The protocol was succes…

Isolation of Vibrio vulnificus Serovar E from Aquatic Habitats in Taiwan

ABSTRACT 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 Hin dIII 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.

O-Serogrouping and surface components ofAeromonas hydrophilaandAeromonas jandaeipathogenic for eels

The relationship between virulence, O-serogroup, and some cell-surface features (self-pelleting [SP] and precipitation after boiling [PAB], profile of lipopolysaccharides [LPSs] and outer membrane proteins [OMPs]) was investigated in strains of the pathogenic species Aeromonas hydrophila and A. jandaei isolated from eels. Virulent strains of A. hydrophila reacted mostly with O:19 antiserum, and those of A. jandaei reacted with O:4, O:11, O:15 and O:29 antisera (Guinée and Jansen system). Regarding the PAB and LPS profiles two groups could be distinguished; (i) five PAB+ strains of serotype O:19 that possessed a homogeneous O polysaccharide side chain and (ii) thirteen PAB- strains antigenic…

A comparative epizootiologic study of the two fish-pathogenic serovars ofVibrio vulnificusbiotype 2

Vibrio vulnificus biotype 2 is subdivided into two main serovars, serovar E, able to infect fish and humans, and serovar A, only virulent for fish. Serovar E emerged in 1976 as the causative agent of a haemorrhagic septicaemia (warm-water vibriosis) affecting eels cultured in brackish water. Serovar A emerged in 2000 in freshwater-cultured eels vaccinated against serovar E, causing warm-water vibriosis with fish showing a haemorrhagic intestine as the main differential sign. The aim of the present work was to compare the disease caused by both serovars in terms of transmission routes, portals of entry and host range. Results of bath, patch-contact and oral-anal challenges demonstrated that …

Novel host-specific iron acquisition system in the zoonotic pathogenVibrio vulnificus

Summary Vibrio vulnificus is a marine bacterium associated with human and fish (mainly farmed eels) diseases globally known as vibriosis. The ability to infect and overcome eel innate immunity relies on a virulence plasmid (pVvbt2) specific for biotype 2 (Bt2) strains. In the present study, we demonstrated that pVvbt2 encodes a host-specific iron acquisition system that depends on an outer membrane receptor for eel transferrin called Vep20. The inactivation of vep20 did not affect either bacterial growth in human plasma or virulence for mice, while bacterial growth in eel blood/plasma was abolished and virulence for eels was significantly impaired. Furthermore, vep20 is an iron-regulated ge…

Evaluation of the API 20E system for identification and discrimination of Vibrio vulnificus biotypes 1 and 2

Effects of salinity and temperature on long-term survival of the eel pathogen Vibrio vulnificus biotype 2 (serovar E)

ABSTRACT Vibrio vulnificus biotype 2 (serovar E) is a primary eel pathogen. In this study, we performed long-term survival experiments to investigate whether the aquatic ecosystem can be a reservoir for this bacterium. We have used microcosms containing water of different salinities (ranging from 0.3 to 3.8%) maintained at three temperatures (12, 25, and 30°C). Temperature and salinity significantly affected long-term survival: (i) the optimal salinity for survival was 1.5%; (ii) lower salinities reduced survival, although they were nonlethal; and (ii) the optimal temperature for survival was dependent on the salinity (25°C for microcosms at 0.3 and 0.5% and 12°C for microcosms at 1.5 to 3.…

Polyphyletic Origin of Vibrio vulnificus Biotype 2 as Revealed by Sequence-Based Analysis ▿ †

ABSTRACT 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.

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 …

Toxicity of the extracellular products ofVibrio damsela isolated from diseased fish

In this work we analyzed the pathogenic in vivo and in vitro activities for both fish and mammals of extracellular products (ECP) of several isolates of Vibrio damsela implicated in disease problems in marine culture. The ECP from all the strains were strongly lethal for fish (LD50 ranging from 0.06 to 3.7 μg protein/g fish) and mice (LD50 ranging from 0.02 to 0.43 μg protein/g mouse), causing death between 4 and 72 h after inoculation. These ECP samples possessed low proteolytic activity without production of caseinase, gelatinase, or elastase. However, most of them showed remarkable phospholipase and hemolytic activity for sheep, human, and turbot red blood cells. In addition, all the ECP…

An indirect immunofluorescent antibody technique for detection and enumeration of Vibrio vulnificus serovar E (biotype 2): delevopment and applications

The applications of an indirect fluorescent antibody technique (IFAT), developed to detect and enumerate the pathogenic bacterium Vibrio vulnificus serovar E from water and clinical samples, are described. This technique proved accurate for detecting V. vulnificus, even under starvation conditions and in the non-culturable state, and could differentiate this species from other bacteria which share the same habitats. The IFAT was successfully used to diagnose vibriosis from naturally- and artificially-infected eels. The overall data suggest that applying this technique properly in environmental and epidemiological/epizootiological studies could significantly increase our knowledge of this ba…

Vibrio harveyi causes disease in seahorse, Hippocampus sp.

A mass mortality among cultured seahorses, Hippocampus kuda and Hippocampus sp., occurred in spring 1998 in Tenerife, Spain. Seahorses were held together with tropical shrimps, Stenopus hispidus, in glass aquaria supplied with 1000 L of sea water at 25 °C. The water supply was conducted between different tanks that contained various marine species, such as octopus, Octopus vulgaris, star®sh, Asterias rubens, sea-urchin, Paracentrotus lividu, greater weever, Trachinus draco, grouper, Epinephelus guaz and Canarian shrimp, Lismata amboiens. None of these species was affected, including the shrimps that shared aquaria with the seahorses. Mortalities of seahorses were very high (more than 90%), …

Electrophoretic analysis of heterogeneous lipopolysaccharides from various strains of Vibrio 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,…

R plasmids in environmental Vibrio cholerae non-O1 strains.

The occurrence of drug resistance and its plasmid-mediated transferability was investigated in 140 environmental strains of Vibrio cholerae non-O1 and 6 strains of Vibrio cholerae, both O1 and non-O1, of clinical origin. Of the 146 strains tested, 93% were resistant to at least one drug and 74% were resistant to two or more antibiotics. The O1 strains were susceptible to all antibiotics used. A total of 26 of 28 selected resistant wild strains carried R plasmids that were transferable by intraspecific and intergeneric matings. The most common transmissible R factor determined resistance to ampicillin, amoxicillin, and sulfanilamide (30%), followed by resistance to ampicillin and amoxicillin…

High affinity iron-uptake systems in Vibrio damsela: role in the acquisition of iron from transferrin

In this work, the high affinity iron-acquisition systems displayed by virulent and avirulent strains of Vibrio damsela have been investigated. This species is an autochthonous member of marine ecosystems that can behave as an opportunistic pathogen for fish and mammals. All strains tested (i) were able to grow under the restricted conditions imposed by the iron chelators transferrin (Tf) and EDDHA, (ii) secreted siderophores of hydroxamic type, other than aerobactin and desferal, that were able to stimulate the growth of the auxotroph mutant Arthrobacter flavescens JG9, and (iii) expressed common iron-regulated outer membrane proteins (IROMPs). No change in LPS patterns was observed in resp…

The Fish Pathogen Vibrio vulnificus Biotype 2: Epidemiology, Phylogeny, and Virulence Factors Involved in Warm-Water Vibriosis

ABSTRACT Vibrio vulnificus biotype 2 is the etiological agent of warm-water vibriosis, a disease that affects eels and other teleosts, especially in fish farms. Biotype 2 is polyphyletic and probably emerged from aquatic bacteria by acquisition of a transferable virulence plasmid that encodes resistance to innate immunity of eels and other teleosts. Interestingly, biotype 2 comprises a zoonotic clonal complex designated as serovar E that has extended worldwide. One of the most interesting virulence factors produced by serovar E is RtxA1 3 , a multifunctional protein that acts as a lethal factor for fish, an invasion factor for mice, and a survival factor outside the host. Two practically id…

The widespread presence of a family of fish virulence plasmids in Vibrio vulnificus stresses its relevance as a zoonotic pathogen linked to fish farms

Vibrio vulnificus is a pathogen of public health concern that causes either primary septicemia after ingestion of raw shellfish or secondary septicemia after wound exposure to seawater. In consequence, shellfish and seawater are considered its main reservoirs. However, there is one aspect of its biology that is systematically overlooked: its association with fish in its natural environment. This association led in 1975 to the emergence of a zoonotic clade within phylogenetic lineage 2 following successive outbreaks of vibriosis in farmed eels. Although this clade is now worldwide distributed, no new zoonotic clades were subsequently reported. In this work, we have performed phylogenetic, ge…

The widespread presence of a family of fish virulence plasmids in Vibrio vulnificus stresses its relevance as a zoonotic pathogen linked to fish farms

Vibrio vulnificus is a pathogen of public health concern that causes either primary septicemia after ingestion of raw shellfish or secondary septicemia after wound exposure to seawater. In consequence, shellfish and seawater are considered its main reservoirs. However, there is one aspect of its biology that is systematically overlooked: its association with fish in its natural environment. This association led in 1975 to the emergence of a zoonotic clade within phylogenetic lineage 2 following successive outbreaks of vibriosis in farmed eels. Although this clade is now worldwide distributed, no new zoonotic clades were subsequently reported. In this work, we have performed phylogenetic, ge…

Additional file 2: Table S1. of Wild eel microbiome reveals that skin mucus of fish could be a natural niche for aquatic mucosal pathogen evolution

Abundance and comparison of functions in water and skin-mucus surface microbiomes. (XLSX 646 kb)