Genetic distinctions among clinical and environmental strains of Vibrio vulnificus

Vibrio vulnificus causes rare but frequently fatal septicemia associated with raw oyster consumption by persons with underlying hepatic or immune system dysfunction. The virulence potential of environmental reservoirs appears widely distributed, because most strains are virulent in animal models; however, several investigations recently demonstrated genetic divergence among strains from clinical versus environmental origin at independent genetic loci. The present study used PCR to screen DNA polymorphisms in strains from environmental (n = 35) or clinical (n = 33) sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P < 0.01) association was observed for typical "clinical" or "environmental" polymorphism profiles based on strain origin. Most oyster isolates (88%), including all of those with the "environmental" profile, also formed a single rep-PCR genogroup. Clinical isolates within this group did not have the typical "clinical" profile. On the other hand, clinical isolates with the typical polymorphism profile were distributed among multiple rep-PCR genogroups, demonstrating greater genetic diversity than was evident by profiling genetic polymorphisms. Wound isolates were genetically distinct from typical blood isolates by all assays. Strains from an outbreak of wound infections in Israel (biotype 3) were closely related to several U.S. strains by rep-PCR, indicating potential reservoirs of emerging disease. Strains genetically related to blood isolates appeared to be relatively rare in oysters, as only one had the "clinical" polymorphism profile or clustered by rep-PCR. However, this study was not an extensive survey, and more sampling using rep-PCR for sensitive genetic discrimination is needed to determine the virulence potential of environmental reservoirs.     

Identification of environmental Vibrio vulnificus isolates with a DNA probe for the cytotoxin-hemolysin gene

We screened 44 lactose-positive Vibrio strains isolated from the marine environment for homology with a 3.2-kilobase DNA fragment encoding the Vibrio vulnificus cytotoxin-hemolysin gene. All 29 marine isolates identified as V. vulnificus on the basis of numerical taxonomy and DNA-DNA hybridization studies hybridized with the cytotoxin gene probe, as did all V. vulnificus reference strains. Homologous gene sequences were identified in no other lactose-positive marine vibrio isolates nor in 10 other Vibrio species.     

Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish

In this study, we developed a PCR-based rapid detection method for clinically important pathogenic strains of Vibrio vulnificus. Positive amplification of the 504-bp viuB fragment was seen in all 22 clinical isolates tested but only in 8 out of 33 environmental isolates. The combination of the species-specific 205-bp vvh fragment along with viuB in a multiplexed PCR enabled us to confirm the presence of potentially pathogenic strains of V. vulnificus. No amplification of other Vibrio spp. or non-Vibrio bacteria was evidenced, suggesting a high specificity of detection by this method. The sensitivity of detection for both targeted genes was 10 pg of purified DNA, which correlated with 10(3) V. vulnificus CFU in 1 mL of pure culture or 1 g un-enriched seeded oyster tissue homogenate. This sensitivity was improved to 1 CFU per gram of oyster tissue homogenate in overnight-enriched samples. A SYBR Green I based real-time PCR method was also developed that was shown to produce results consistent with the conventional PCR method. Application of the multiplexed real-time PCR to natural oyster tissue homogenates exhibited positive detection of vvh in 51% of the samples collected primarily during the summer months; however, only 15% of vvh positive samples exhibited viuB amplicons. The rapid, sensitive, and specific detection of clinically important pathogenic V. vulnificus in shellfish would be beneficial in reducing illnesses and deaths caused by this pathogen.     

Presence of virulence markers in environmental Vibrio vulnificus strains

Aims

This work aims to demonstrate the presence of several genes and factors associated with virulence in strains isolated from the environment at Pueblo Viejo Lagoon, State of Veracruz, Mexico.

Methods and Results

In this study, we investigated the production of V. vulnificus virulence factors, as cytolysin (haemolysin), RTX toxin, metalloprotease, siderophores, capsular polysaccharide, adhesion structures (like type IV pili), and polar and lateral flagella, involved in swimming and swarming (or, at least, the presence of genes encoding some of them) in 40 strains of V. vulnificus isolated from water and food. The results indicate that strains of environmental origin possess potential virulence characteristics.

Conclusions

Caution should be exercised when consuming raw shellfish (especially by those more susceptible risk groups).

Significance and Impact of the Study

This is the first work focused on the evaluation of V. vulnificus virulence factors in Mexico.     

Identification of environmental Vibrio vulnificus isolates with a DNA probe for the cytotoxin-hemolysin gene.

We screened 44 lactose-positive Vibrio strains isolated from the marine environment for homology with a 3.2-kilobase DNA fragment encoding the Vibrio vulnificus cytotoxin-hemolysin gene. All 29 marine isolates identified as V. vulnificus on the basis of numerical taxonomy and DNA-DNA hybridization studies hybridized with the cytotoxin gene probe, as did all V. vulnificus reference strains. Homologous gene sequences were identified in no other lactose-positive marine vibrio isolates nor in 10 other Vibrio species.     

In situ analysis of nucleic acids in cold-induced nonculturable Vibrio vulnificus.

Low-temperature-induced nonculturable cells of the human pathogenic bacterium Vibrio vulnificus retained significant amounts of nucleic acids for more than 5 months. Upon permeabilization of fixed cells, however, an increasing number of cold-incubated cells released the nucleic acids. This indicates substantial degradation of DNA and RNA in nonculturable cells prior to fixation. Treatment of permeabilized cells with DNase and RNase allowed differential staining of DNA and RNA with the nucleic acid dye 4',6-diamidino-2-phenylindole (DAPI). Epifluorescence microscopy revealed that the could-induced nonculturable populations of V. vulnificus are highly heterogeneous with regard to their nucleic acid content. The fraction of nonculturable cells which maintained DNA and RNA structures decreased gradually during cold incubation. After 5 months at 5 degrees C, less than 0.05% of the cells could be observed to retain DNA and RNA. In parallel with the loss of nucleic acids, an increase in the concentrations of UV-absorbing material in the culture supernatants was observed in nonculturable-cell suspensions. It is hypothesized that there are two phases of the formation of nonculturable cells of V. vulnificus: the first involves a loss of culturability with maintenance of cellular integrity and intact RNA and DNA (and thus possibly viability), and the second is typified by a gradual degradation of nucleic acids, the products of which partly remain inside the cells and partly diffuse into the extracellular space. A small number of nonculturable cells, however, retain DNA and RNA, and thus may be viable despite having reduced culturability.     

Isolation and characterization of a cold-induced nonculturable suppression mutant of Vibrio vulnificus

The viable but nonculturable (VBNC) suppression mutant formed platable cells at low temperature stress after inoculation in artificial seawater (ASW). Suppression subtractive hybridization was used to identify differentially expressed genes among cDNAs of the VBNC suppression mutant and the wild-type Vibrio vulnificus strain. Glutathione S-transferase was identified as a responsive gene of the VBNC suppression mutant in our assay, and was highly expressed from the VBNC suppression mutant at low temperature stress. Culturability tests revealed that the wild-type cells were sensitive to oxidative stress in the hydrogen peroxide (H(2)O(2)) and to 1-chloro-2,4-dinitrobenzene (CDNB) compared with the VBNC suppression mutant cells. Adding glutathione showed that many wild-type V. vulnificus cells maintained culturability in cold ASW. These results suggest that non-nutritional growth inhibitors, such as peroxide that accumulates at low temperatures, influence VBNC in V. vulnificus cells.     

Virulence characteristics of clinical and environmental isolates of Vibrio vulnificus.

Twenty-four randomly selected clinical and environmental Vibrio vulnificus isolates were tested for virulence in iron-overloaded mice (250 mg of iron dextran per kg of body weight). The log10 50% lethal doses of 17 isolates were lower by greater than or equal to 3.5 log10 units in iron-overloaded mice than in control mice. These isolates were classified as virulent. The 50% lethal doses of these virulent isolates were also lower in mice that were immunosuppressed by treatment with cyclophosphamide (150 mg/kg). Four of the seven isolates initially classified as avirulent were virulent in mice that were simultaneously iron overloaded and immunosuppressed. These isolates were classified as moderately virulent. The remaining three isolates were avirulent under all conditions. The incidence of virulent strains among clinical and environmental isolates did not differ. The virulent isolates produced high titers of hemolysin, were resistant to inactivation by serum complement, produced phenolate siderophore, and utilized transferrin-bound iron. The moderately virulent isolates differed from the virulent isolates only in their increased sensitivity to inactivation by serum complement. The avirulent isolates differed from those of the other two classes in their inability to either produce significant amounts of phenolate siderophore or utilize transferrin-bound iron. A modified agar plate diffusion method for transferrin-bound iron utilization was developed to differentiate the two classes of virulent isolates from the avirulent isolates in vitro.     

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 lectin domain of VVH. Nevertheless, two primer sets for polymerase chain reaction could be designed to differentiate the toxin gene of each strain. When 27 V. vulnificus clinical isolates were tested, group 1 strains (9 strains) were shown to react only to the primers designed for vvhA of strain L-180; whereas, the gene of group 2 strains (18 strains) could be amplified with the primers for vvhA of strain CDC B3547. These findings may lead to development of a novel genetic grouping system related to the virulence potential or to the host range.     

Resuscitation of Vibrio vulnificus from the viable but nonculturable state.

Stationary-phase-grown cells of the estuarine bacterium Vibrio vulnificus became nonculturable in nutrient-limited artificial seawater microcosms after 27 days at 5 degrees C. When the nonculturable cells were subjected to temperature upshift by being placed at room temperature, the original bacterial numbers were detectable by plate counts after 3 days, with a corresponding increase in the direct viable counts from 3% to over 80% of the total cell count. No increase in the total cell count was observed during resuscitation, indicating that the plate count increases were not due to growth of a few culturable cells. Chloramphenicol and ampicillin totally inhibited resuscitation of the nonculturable cells when added to samples that had been at room temperature for up to 24 h. After 72 h of resuscitation, the inhibitors had an easily detectable but reduced effect on the resuscitated cells, indicating that protein and peptidoglycan synthesis were still ongoing. Major changes in the morphology of the cells were discovered. Nonculturable cells of V. vulnificus were small cocci (approximately 1.0 micron in diameter). Upon resuscitation, the cells became large rods with a size of mid-log-phase cells (3.0 microns in length). Four days after the cells had become fully resuscitated, the cell size had decreased to approximately 1.5 micron in length and 0.7 micron in width. The cells were able to go through at least two cycles of nonculturability and subsequent resuscitation without changes in the total cell count. This is the first report of resuscitation, without the addition of nutrient, of nonculturable cells, and it is suggested that temperature may be the determining factor in the resuscitation from this survival, or adaptation, state of certain species in estuarine environments.     

Induction,resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio vulnificus in artificial sea water

Vibrio vulnificus, an important food-borne pathogen, is known to enter viable but nonculturable (VBNC) state under low temperature and low nutrition stress conditions. Present study examined the time required for induction of VBNC state and temperature which induces resuscitation of V. vulnificus YJ016. The change in cell morphology and gene expression during VBNC state and in resuscitated cells was also examined. V. vulnificus incubated in artificial sea water at 4 °C entered VBNC state after considerably extended time (70 days). An increase in temperature by 6 °C from the VBNC induction temperature (4 °C) resulted in resuscitation of VBNC cells; however, maximum resuscitation was observed when VBNC cells were held at 23 °C for 24 h. VBNC cells changed their morphology from comma shape to coccoid shape. Two rounds of induction of VBNC and resuscitation were possible with V. vulnificus cells; however, there was progressive reduction in number of resuscitated cells and after 190 days cells failed to resuscitate. Significant up-regulation of genes related to membrane proteins [porinH (10.4-fold), ompU (2.9-fold)], regulatory proteins [envZ (5.6-fold), toxR (4.5-fold), toxS (4.8-fold)], oxidative stress related protein katG (2.3-fold), cell division/maintenance proteins [ftsZ (4.3), mreB (6.5-fold)] and resuscitating promoter factor yeaZ (fourfold) was observed during resuscitation with respect to VBNC state indicating that these genes play a role during resuscitation. Gene expression data presented here would enhance our understanding of resuscitation of V. vulnificus from VBNC state. The results also highlight the importance of maintenance of low temperature during storage of seafood.     

Formation of nonculturable Vibrio vulnificus cells and its relationship to the starvation state.

Entry into the viable but nonculturable state by the human bacterial pathogen Vibrio vulnificus in artificial seawater microcosms was studied. In contrast to the long-term culturability exhibited by cells incubated under these starvation conditions at room temperature, cells exposed to a temperature downshift to 5 degrees C exhibited an immediate decrease in culturability. Cells incubated at low temperature exhibited a morphological change from rods to cocci but demonstrated no reductive division. Of 10 factors studied which might affect the nonculturable response in V. vulnificus, only the physiological age of the cells was found to significantly affect the rate at which cells became nonculturable. The nonculturable response appears to be related to the starvation response, as prestarvation at room temperature for 24 h was found to eliminate the nonculturable response of cells subsequently incubated at 5 degrees C. This observation suggests that the synthesis of starvation proteins may repress the viable but nonculturable program displayed during low-temperature incubation. The possible ecological significance of these findings is discussed.     

Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus

The nonculturable state of Vibrio vulnificus and, for comparison, that of Escherichia coli were studied in artificial-seawater microcosms at 5 degrees C. Total cell counts were monitored by acridine orange epifluorescence, metabolic activity by direct viable counts, and culturability by plate counts on selective and nonselective media. Whereas total counts remained constant, plate counts of V. vulnificus suggested nonculturability by day 24. In contrast, direct viable counts indicated significant cell viability throughout 32 days of incubation. As an indication of the metabolic changes that occurred as cells entered the state of nonrecoverability, membrane fatty acid analyses were performed. At the point of nonculturability of V. vulnificus, the major fatty acid species (C16 and C16:1) had decreased 57% from the T0 level, concomitant with the appearance of several short-chain acids. Although the bacteria were still recoverable, a similar trend was observed with E. coli. Electron microscopy of nonculturable V. vulnificus showed that the cells were rounded and reduced in size and contained fewer ribosomes. Mouse infectivity studies conducted with these cells suggested loss of virulence.     

Formation of nonculturable Vibrio vulnificus cells and its relationship to the starvation state.

Entry into the viable but nonculturable state by the human bacterial pathogen Vibrio vulnificus in artificial seawater microcosms was studied. In contrast to the long-term culturability exhibited by cells incubated under these starvation conditions at room temperature, cells exposed to a temperature downshift to 5 degrees C exhibited an immediate decrease in culturability. Cells incubated at low temperature exhibited a morphological change from rods to cocci but demonstrated no reductive division. Of 10 factors studied which might affect the nonculturable response in V. vulnificus, only the physiological age of the cells was found to significantly affect the rate at which cells became nonculturable. The nonculturable response appears to be related to the starvation response, as prestarvation at room temperature for 24 h was found to eliminate the nonculturable response of cells subsequently incubated at 5 degrees C. This observation suggests that the synthesis of starvation proteins may repress the viable but nonculturable program displayed during low-temperature incubation. The possible ecological significance of these findings is discussed.     

Virulence of Vibrio vulnificus strains from marine environments

Vibrio vulnificus strains isolated from geographically diverse marine sources were compared with clinical isolates for phenotype and in vitro and in vivo production of virulence factors. There were no differences between environmental and clinical strains on the basis of biochemical characteristics or antimicrobial susceptibility patterns. Cytolysin and cytotoxin titers produced by environmental strains were generally comparable to those of clinical strains. Of 29 environmental isolates tested, 25 were pathogenic for mice. These data show that environmental V. vulnificus strains are phenotypically indistinguishable from clinical isolates and that approximately 90% of the environmental strains tested produced in vitro virulence factors and in vivo pathogenicity for mice comparable to those produced by clinical V. vulnificus isolates.     

Pulsed-field gel electrophoresis and ribotype profiles of clinical and environmental Vibrio vulnificus isolates.

Vibrio vulnificus belongs to the autochthonous bacterial flora of warm estuarine waters. It can cause life-threatening extraintestinal disease in persons who have underlying illness and who consume raw shellfish or contact wounds with estuarine water. Currently, very little is known about genetic diversity within this species. In this report, we describe high-level variation in restriction fragment length polymorphism profiles among 53 clinical and 78 environmental isolates, as determined by pulsed-field gel electrophoresis. In contrast, ribotype profiles showed greater similarity. When combined ribotype profiles of clinical and environmental isolates were analyzed, four predominant clusters were observed. Interestingly, a low number (16%) of clinical isolates were found in cluster C, compared with clusters A, B, and D (range, 50 to 83%). In addition, 83% of all Hawaiian isolates were located in a single cluster, indicating a possible relationship between geography and genotype. We also report that spontaneous translucent colonial morphotypes were distinct by both restriction fragment length polymorphism and biochemical profiles, compared with opaque parent strains.     

Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells.

Vibrio vulnificus is a human pathogen associated with consumption of raw oysters. During the colder months the organism apparently enters a viable but nonculturable state and thus cannot be cultured by ordinary bacteriological methods. For this reason, another means of detecting this bacterium is necessary. In the present study we utilized the polymerase chain reaction (PCR) to detect V. vulnificus DNA, thus eliminating the problem of nonculturability. DNA from both culturable and nonculturable cells of V. vulnificus was amplified by PCR with primers flanking a 340-bp fragment of the cytotoxin-hemolysin gene. As little as 72 pg of DNA from culturable cells and 31 ng of DNA from nonculturable cells could be detected. Fifty cycles of a two-step reaction (30 s [each] at 94 and 65 degrees C) were found to be optimal as well as more time efficient than the three-step PCR. The total procedure from the point of DNA extraction to observation on a gel required less than 8 h. Possible reasons for the difficulties encountered in amplifying DNA from nonculturable cells, e.g., gene rearrangement or loss of the hemolysin gene, are discussed.