Variation of extracellular proteases produced by Vibrio vulnificus clinical isolates: genetic diversity of the metalloprotease gene (vvp), and serine protease secretion by vvp-negative strains

Vibrio vulnificus is a causative agent of septicemia or wound infection in human and eel; however, the genetic variation between human and eel isolates has been reported. In the present study, the difference in the vvp gene encoding a tissue-damaging metalloprotease was investigated. The gene of strain E86 from a diseased eel (type B vvp) was 95.2% identical with that of strain L-180 from human blood (type A vvp). PCR using oligonucleotide primers designed to differentiate two types of the gene showed that eel avirulent strains (9 isolates) commonly carry type A vvp, whereas eel virulent strains (18 isolates) revealed significant genetic variation. The vvp genes from 12 strains including strain E86 were placed on type B while those from 3 strains were on type A. Other strains were found to be vvp-negative, but PAGE and amino acid sequencing analysis showed that they secreted a serine protease (VVA0302) instead of the metalloprotease. This protease is an orthologue of a toxic protease from Vibrio parahaemolyticus, a human pathogen causing wound infection as well as gastroenteritis. These findings suggest that, in addition to metalloprotease, the extracellular serine protease may contribute to pathogenicity of V. vulnificus.     

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.     

Demonstration of a common antigen in sonicated cells for identification of Vibrio vulnificus serotypes.

Ouchterlony immunodiffusion of sonicated Vibrio vulnificus cells illustrated a single major precipitation line with antiserum prepared from whole cells of the same species. Antigenic analysis by two-dimensional immunoelectrophoresis verified the presence of a single dominant precipitation line. Tandem two-dimensional immunoelectrophoretic analyses of V. vulnificus antigens from various strains revealed one fused precipitation line of identity. No fused precipitation lines were seen with other Vibrio species tested. This dominant antigen, designated VVA, was not dialyzable, lost antigenicity by heating at 100 degrees C but not at 70 degrees C, and was precipitated by 70%, but not by 50%, saturated ammonium sulfate. VVA was not present in concentrated (20X) spent culture medium. VVA is possibly an intracellular protein specific to V. vulnificus species and may be useful in the serological identification of this important human pathogen.     

Comparative genome analysis of Vibrio vulnificus, a marine pathogen

The halophile Vibrio vulnificus is an etiologic agent of human mortality from seafood-borne infections. We applied whole-genome sequencing and comparative analysis to investigate the evolution of this pathogen. The genome of biotype 1 strain, V. vulnificus YJ016, was sequenced and includes two chromosomes of estimated 3377 kbp and 1857 kbp in size, and a plasmid of 48,508 bp. A super-integron (SI) was identified, and the SI region spans 139 kbp and contains 188 gene cassettes. In contrast to non-SI sequences, the captured gene cassettes are unique for any given Vibrio species and are highly variable among V. vulnificus strains. Multiple rearrangements were found when comparing the 5.3-Mbp V. vulnificus YJ016 genome and the 4.0-Mbp V. cholerae El Tor N16961 genome. The organization of gene clusters of capsular polysaccharide, iron metabolism, and RTX toxin showed distinct genetic features of V. vulnificus and V. cholerae. The content of the V. vulnificus genome contained gene duplications and evidence of horizontal transfer, allowing for genetic diversity and function in the marine environment. The genomic information obtained in this study can be applied to monitoring vibrio infections and identifying virulence genes in V. vulnificus.     

Identification of the emerging pathogen Vibrio vulnificus biotype 3 by commercially available phenotypic methods

Identification of the emerging pathogen Vibrio vulnificus biotype 3 has become a challenge for clinical laboratories in the last few years. In this study, the abilities of five commercial systems to identify this new species have been evaluated for the first time, using a unique collection of strains. Fifty-one well-documented wild strains of V. vulnificus biotype 3 were processed using API 20 NE, GNI+ Vitek 1 cards, ID-GNB Vitek 2 cards, Neg Combo 20 Microscan panels, and NMIC/ID-5 BD Phoenix panels. The numbers of strains identified as V. vulnificus by ID-GNB, NMIC/ID-5, and GNI+ were 50 (98.0%), 46 (90.2%), and 7 (13.7%), respectively. Neg Combo 20 Microscan panels and API 20 NE were unable to identify any of the strains of this emerging pathogen to the species level and mostly misidentifies them as other species of the Vibrionaceae family. Data on the phenotypic pattern of V. vulnificus biotype 3 when processed in all five systems as presented here could help clinical laboratories in identifying this new pathogen.     

Detection and differentiation of Vibrio vulnificus in seawater and plankton of a coastal zone of the Mediterranean Sea

Vibrio vulnificus, a human and animal pathogen, is present in low numbers in the Mediterranean Sea. Seawater and plankton samples were collected from a marine coastal zone of the Straits of Messina in the Mediterranean Sea (Italy) in order to investigate V. vulnificus as free-living (>0.2 microm) and associated with small (>64 microm) and large plankton (>200 microm) utilizing cultural and molecular techniques. Characteristic colonies, grown on thiosulfate, citrate, bile salts and sucrose agar plates, were identified using a biochemical protocol system. A PCR assay was used to confirm isolates and to directly detect V. vulnificus in environmental concentrated samples. Specific primers were used to target the structural cytotoxin/hemolysin gene and the variable regions of 16S rRNA species-specific for V. vulnificus. In addition, a tri-primer PCR of 16S rRNA was used for the differentiation of V. vulnificus strains. Direct detection in marine samples was more frequent than isolation of culturable forms. All isolates were assigned to V. vulnificus biotype 1, 16S rRNA type B. These results confirm the low incidence of V. vulnificus in Mediterranean coastal waters. The isolation of cultivable forms is limited to the warmest months. 16S rRNA primers were the most sensitive molecular tool as they allowed detection of V. vulnificus in 79.1% of samples. Due to the low incidence of V. vulnificus in the Mediterranean coastal environment, its detection requires a molecular approach. The occurrence of V. vulnificus as plankton-associated confirms the role of plankton as a potential reservoir for this pathogen.     

Vibrio vulnificus typing based on simple sequence repeats: insights into the biotype 3 group

Vibrio vulnificus is an opportunistic, highly invasive human pathogen with worldwide distribution. V. vulnificus strains are commonly divided into three biochemical groups (biotypes), most members of which are pathogenic. Simple sequence repeats (SSR) provide a source of high-level genomic polymorphism used in bacterial typing. Here, we describe the use of variations in mutable SSR loci for accurate and rapid genotyping of V. vulnificus. An in silico screen of the genomes of two V. vulnificus strains revealed thousands of SSR tracts. Twelve SSR with core motifs longer than 5 bp in a panel of 32 characterized and 56 other V. vulnificus isolates, including both clinical and environmental isolates from all three biotypes, were tested for polymorphism. All tested SSR were polymorphic, and diversity indices ranged from 0.17 to 0.90, allowing a high degree of discrimination among isolates (27 of 32 characterized isolates). Genetic analysis of the SSR data resulted in the clear distinction of isolates that belong to the highly virulent biotype 3 group. Despite the clonal nature of this new group, SSR analysis demonstrated high-level discriminatory power within the biotype 3 group, as opposed to other molecular methods that failed to differentiate these isolates. Thus, SSR are suitable for rapid typing and classification of V. vulnificus strains by high-throughput capillary electrophoresis methods. SSR (>/=5 bp) by their nature enable the identification of variations occurring on a small scale and, therefore, may provide new insights into the newly emerged biotype 3 group of V. vulnificus and may be used as an efficient tool in epidemiological studies.     

Procaspase-3 activation by a metalloprotease secreted from Vibrio vulnificus

Vibrio vulnificus is a marine bacterium and a human pathogen capable of causing wound infection and septicemia. We previously showed that the metalloprotease vEP secreted by V. vulnificus activates prothrombin in vitro. To further investigate the ability of vEP to activate other zymogens, we used a mutant form of procaspase-3 which lacks the native cleavage sites as a zymogen. The mutant zymogen was activated by vEP to yield a mature enzyme with a maximum increase in caspase-3 activity of approximately 14-fold in a time-dependent manner. However, the increase started to decline with prolonged incubation and with higher protease concentration as a result of further cleavage of the mature enzyme. Western blot analysis revealed a band of approximately 17 kDa for the cleavage product, which corresponded with the change in caspase-3 activity. The activated procaspase-3 by vEP was also able to cleave poly(ADP-ribose) polymerase in a cell-free system, and was inhibited by Ac-DEVD-CHO, a potent caspase-3 inhibitor. The results presented are the first to demonstrate the in vitro activation of one of the crucial enzymes involved in cell death by a bacterial extracellular metalloprotease.     

Extracellular phospholipase A2 and lysophospholipase produced by Vibrio vulnificus.

Phospholipase A2 and lysophospholipase activities were detected in the culture supernatant fluids of a virulent strain of Vibrio vulnificus. The phospholipase A2 was inactivated by heating at 56 degrees C for 30 min, had an apparent molecular weight of greater than or equal to 80,000 (estimated by gel filtration with Sephadex G-75), and a pI of ca. 5.0. Phospholipid hydrolysis was unaffected by Ca2+ or ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid and was optimal at pH 5.0 to 5.5. The lysophospholipase was not affected by heating at 56 degrees C for 30 min but was inactivated at 100 degrees C and had an apparent molecular weight of greater than or equal to 80,000 and a pI of ca. 4.0. The enzymes were detected coincidentally with a previously described extracellular cytolysin of V. vulnificus; however, they were physically separable from the toxin (which did not possess phospholipase A, C, or D activity) by gel filtration with Sephadex G-75.     

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

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 (> or =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. Furthermore, significant attenuation of virulence for eels and mice was observed. By contrast, mutation in galE did not alter the cellular surface, did not increase the sensitivity to MP, serum, or phagocytosis, and did not affect the virulence for fish and mice. The change in the attenuated-virulence phenotype produced by a mutation in gne was correlated with the loss of the O-antigen lipopolysaccharide (LPS), while the capsule was maintained. Complementation of a gne-deficient mutant restored the LPS structure together with the whole virulence phenotype. In conclusion, gne, but not galE, is essential for LPS biosynthesis and virulence in the zoonotic serovar of V. vulnificus biotype 2.     

Essential Role for Estrogen in Protection against Vibrio vulnificus-Induced Endotoxic Shock

Little is known about the underlying mechanisms that result in a sexually dimorphic response to Vibrio vulnificus endotoxic shock. V. vulnificus is a gram-negative bacterium, considered one of the most invasive and rapidly fatal human pathogens known. However, 85% of individuals that develop endotoxic shock from V. vulnificus are males. Using the rat, we have developed a model for V. vulnificus endotoxic shock that mimics the sexually dimorphic response in humans. Gonadectomy in females results in increased mortality, and estrogen replacement results in decreased mortality in both gonadectomized males and females. These results demonstrate that estrogen is providing protection against V. vulnificus lipopolysaccharide-induced endotoxic shock.     

Capsular polysaccharide-protein conjugate vaccines of carbotype 1 Vibrio vulnificus: construction, immunogenicity, and protective efficacy in a murine model.

Vibrio vulnificus causes septicemia and wound infections in immunocompromised humans. The capsular polysaccharide of Vibrio vulnificus (VvPS) is critical for virulence. We synthesized conjugate vaccines of carbotype 1 VvPS under conditions and in formulations suitable for human use. Purified VvPS was conjugated to tetanus toxoid (TT) or to inactivated V. vulnificus cytolysin or elastase by two different schemes. All conjugates elicited elevated anticapsular immunoglobulin G (IgG) and IgM and antiprotein IgG responses in mice compared with saline placebo. The conjugates prepared through caboxyl activation of VvPS (VvPS-TTa, VvPS-cytolysin, and VvPS-elastase) were more immunogenic than the one prepared through hydroxyl activation (VvPS-TTb). The protective efficacy of conjugated and unconjugated formulations of VvPS and that of protein carriers were evaluated in a mouse septicemia model. Eighty percent of mice actively immunized with VvPS-TTa vaccine survived challenge with carbotype 1 V. vulnificus, while VvPS-cytolysin and VvPS-elastase conjugates conferred 44 and 40% protection, respectively. Control mice immunized with VvPS, cytolysin, or elastase alone, or saline only, showed 70 to 100% mortality. VvPS-TTa vaccine is nontoxic, immunogenic, and protective in mice.     

Evidence that mortality from Vibrio vulnificus infection results from single strains among heterogeneous populations in shellfish.

Vibrio vulnificus is the leading cause of food-related mortality reported in the state of Florida. It is normal microflora in marine environments, where seawater and molluscan shellfish are the primary vectors of V. vulnificus disease. Risk correlates with seasonally high numbers of V. vulnificus bacteria during the summer months. Currently, the infectious dose for humans, as well as whether the disease is caused by single or multiple strains found in molluscan shellfish, is unknown. In this work, we studied pulsed-field gel electrophoresis profiles of V. vulnificus strains isolated from blood and oysters associated with V. vulnificus disease. Results showed that ca. 10(3) V. vulnificus bacteria/gram of oyster and higher concentrations were associated with human infections and that a single V. vulnificus strain, evidenced by pulsed-field gel electrophoresis profiles, was isolated from human tissues.     

Differential complement activation and susceptibility to human serum bactericidal action by Vibrio species

The ability of Vibrio vulnificus to resist human serum bactericidal action and to activate human complement was compared with similar cultures of Vibrio cholerae and Vibrio parahaemolyticus. Both V. vulnificus and V. parahaemolyticus had similar survival rates in sera and were much more resistant to killing than was V. cholerae. In contrast, V. vulnificus activated significantly less serum complement than did V. cholerae and V. parahaemolyticus. The relative ability of V. vulnificus to survive in serum and activate less complement than other Vibrio spp. tested may be related to its ability to cause chronic tissue infections and septicemias.     

Influence of temperature, salinity and pH on the growth of environmental Aeromonas and Vibrio species isolated from Mai Po and the Inner Deep Bay Nature Reserve Ramsar Site of Hong Kong

Four environmental bacterial isolates including Aeromonas hydrophila MP-3, A. salmonicida MP-4, Vibrio vulnificus MP-2 and V. cholerae MP-1 isolated from sediment and water of Mai Po Nature Reserve of Hong Kong were examined for their responses to temperature, pH and salinity under laboratory conditions in this study. V. cholerae MP-1 was found to resist vibriostatic agent O/129 at concentration of 10 microg/ml. In addition, bacterial growth under test conditions was measured and the results were fitted into the Gompertz model to obtain important parameters related to bacterial growth, lag time (lambda), specific growth rate (mu(m)), and maximum biomass (A) for comparison. V. cholerae MP-1 did not show any apparent growth at 15 degrees C, but was adapted to a much wider environmental pH from 5.2 to 9.2 for growth while V. vulnificus MP-2 was more sensitive to pH changes yielding the highest biomass at pH 6.2. A. salmonicida MP-4 was surprisingly tolerant to salinity as high as 60.0 per thousand NaCl and grew almost equally well as under conditions of other treatments. All four bacterial isolates showed a wide spectrum of plasticity to the environmental conditions and they pose a potential threat to public health and animal health.     

Ability of Vibrio vulnificus to obtain iron from hemoglobin-haptoglobin complexes.

It has been suggested that the normal serum protein, haptoglobin (Hp), serves a bacteriostatic role by binding free hemoglobin (Hm), thus making heme iron unavailable for bacterial growth. Previous studies showed that, unlike Escherichia coli, Vibrio vulnificus was able to overcome this Hp-blocking effect. We report here a study on the iron-withholding property of the three major human Hp phenotypes, Hp 1, 2, and 2-1. Results of experiments with human serum showed that V. vulnificus C7184 was able to obtain iron from Hm bound to Hp types 1 and 2, but not that bound to Hp 2-1. E. coli 2395-80, on the other hand, was unable to overcome the blocking effect of any Hp phenotype. Using purified Hp 1, we also demonstrated that, although V. vulnificus was unable to grow in a deferrated medium without an additional iron source, it was able to grow with the addition of the Hm-Hp complex.     

A highly homologous 68 kbp plasmid found in Vibrio vulnificus strains virulent for eels

Vibrio vulnificus serovar E (biotype 2) strains are virulent for eels and have also been reported to cause illness in humans. Studies on the plasmid content of serovar E strains revealed the existence of a plasmid of approximately 70 kbp present in most of these strains. In this study we characterized the 70 kbp plasmids of seven biotype 2 strains isolated from seawater, diseased eels and wound infections in humans. We determined the exact size of the high molecular weight plasmids to be 68 kbp. A comparison of the plasmids of the seven strains by restriction length polymorphism and hybridization analysis showed them to be almost identical.     

Identification of Vibrio vulnificus O serovars with antilipopolysaccharide monoclonal antibody.

A serotyping scheme for Vibrio vulnificus predicated on the detection of lipopolysaccharide (LPS) antigens is proposed. The serovar O typing scheme used to type V. vulnificus employs polyclonal antisera raised in rabbits immunized with heat-killed whole-cell vaccines. Polyclonal typing sera produced in this manner cross-react with heterologous strains. Affinity purification of polyclonal antisera with LPS affinity columns resolved some of these cross-reactions; however, affinity-purified polyclonal antisera still showed cross-reactions that were nonreciprocal. On the basis of the serological patterns that were obtained with affinity-purified polyclonal antisera, V. vulnificus strains were selected as vaccine strains for production of monoclonal antibody. Spleen cells harvested from BALB/c mice immunized with formalin-killed V. vulnificus cells were fused with SP2/O-Ag 14 myeloma cells. Hybridomas were screened by using LPS and whole-cell enzyme-linked immunosorbent assay to identify clones secreting LPS-specific antibodies. Monoclonal antibodies identified five LPS serological varieties of V. vulnificus and a single serovar each for Vibrio damsela and Vibrio hollisae. No cross-reactions between V. vulnificus and V. hollisae or V. damsela were observed.