Strains of Vibrio vuln~flcus biotype 2 were recovered from diseased European eels Angujlla anguilla during 2 epizootics These stralns were biochemically and serologically identical to the ATCC 33149 strain, which was lsolated from the Japanese eel Angujlla japonica. The isolates of biotype 2 from European eels were more virulent for elvers (LD50 < 10' cfu) than the Japanese strain used as control (LDS0 = 104 cfu). All strains of biotype 2 were able to adhere to human and fish cell lines and were also strongly cytotoxic. The extracellu1a1-products (ECPs) of the Japanese strain exhibited lipolytic, phospholipolytic, proteolytic, and haernolyt~c activities, whereas the ECPs of the Spanish isolates displayed only proteolytic activity. Strains of biotype 1 were recovered from tank water and healthy eels during the second outbreak. These isolates were biochemically similar to the type strain ATCC 27562, and serologically distinct from the isolates of biotype 2. Strains of biotype 1 produced proteases, haemolysins, and cytotoxins and showed specific adhesion to human cells, but they were non-pathogenic for eels. The strains of biotype 2 were more virulent for mice (LDS" = 10' to 106cfu) than the strains of biotype 1 (LDS(, = 10%fu), suggesting that this eel pathogen may also represent a health hazard problem for fish farmers.

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

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Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu 2؉ was inoculated with 10 7 CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/ DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.

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 the disease is dependent on temperature and water salinity, thus suggesting a method to reduce the risk of epizootics and that of infection 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 M r s, 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, and by fish farmers, above all when manipulating eels during epizootic outbreaks.

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 (509% lethal dose, about 105 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.

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 observable capsular materials. No differences in plasmid profiles were detected between the two cell types so that plasmids do not seem to be implicated in the mechanism of phase shift of biotype 2 strains. No apparent difference in outer membrane protein and lipopolysaccharide patterns could be observed between the cell types. Both isogenic morphotypes were able to grow in eel serum and minimal medium supplemented with ethylenediamine di(O-hydroxyphenyl-acetic acid) or transferrin. Therefore, the presence of capsule was not required for the acquisition of iron from iron chelators or for resistance to serum bactericidal action. Both morphotypes were highly virulent for elvers, although the 50% lethal dose for translucent cells was higher than that for the corresponding opaque cells. The latter observation, together with the overall data, suggests that the production of capsular materials by biotype 2 of V. vulnificus is not essential for the development of vibriosis in eels, at least when cells are injected intraperitoneally.