Survival of Erwinia amylovora in mature apple fruit calyces through the viable but nonculturable (VBNC) state.

AIMS: Survival of Erwinia amylovora, causal agent of fire blight in pome fruits and other rosaceous plants, was monitored inside mature apples calyces under some storage conditions utilized in fruit. METHODS AND RESULTS: Apple fruit calyces inoculated with two E. amylovora strains and their respective GFP-marked strains were maintained at 26 degrees and 5 degrees C, and the effect of copper treatment was assayed at 0.01 and 0.1 mmol l(-1) CuSO4. In nontreated apples at 26 degrees C, part of the population of E. amylovora survived in the 'viable but nonculturable' (VBNC) state, whereas at 5 degrees C the majority of the population retained culturability. In copper-treated apples, the whole population adopted the VBNC state irrespective of temperature. Regardless of temperature, copper and inoculum dose, VBNC cells recovered culturability and pathogenicity in King's B broth or by host plant passage. CONCLUSIONS: Erwinia amylovora survived for at least 35 days in mature apple calyces. Besides, the ability of the pathogen in the VBNC state to regain culturability and pathogenicity suggests that the apple fruit could be a potential carrier of E. amylovora contributing to the spreading of fire blight disease. SIGNIFICANCE AND IMPACT OF THE STUDY: The risk of E. amylovora dissemination through mature fruit transport, although low, has been demonstrated, and should be considered in pest risk assessments.

Exploring diversity among Spanish strains of Erwinia amylovora and possible infection sources.

AIMS: We have examined the intraspecific diversity of a collection of 63 Spanish strains of Erwinia amylovora, isolated from 1995 to 2001, to determine whether or not they could be grouped based on phenotypic or genotypic criteria and to investigate the sources of inoculum for fire blight dissemination in Spain. METHODS AND RESULTS: Several biochemical and molecular techniques, such as miniaturized API 20E, API 50CH, ATB G-5 and API-ZYM tests, BIOLOG metabolic fingerprinting, PCR ribotyping, pulsed-field gel electrophoresis (PFGE), minisatellite-primed PCR (MSP-PCR), random amplified polymorphic DNA (RAPD) analyses and AFLP were used. We report the first identification in Spain of the PFGE pattern Pt1, already described in other European countries, together with Pt3 and Pt4 patterns. Moreover, PFGE, together with MSP-PCR, RAPD analyses and AFLP are, until now, the only techniques that have provided information about the possible infection sources and relationships between the different foci in Spain, with AFLP being the most discriminative. CONCLUSIONS: These techniques have allowed grouping of Spanish strains by their geographical origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results support the hypothesis that some fire blight outbreaks have been caused by the introduction in Spain of infected plant material, or other inoculum sources from different European countries.

First Report of Bacterial Deep Bark Canker of Walnut Caused by Brenneria (Erwinia) rubrifaciens in Europe.

During the summer of 1995 and subsequent years, bark cankers were observed in walnut trees (cv. Hartley grafted on Juglans hindsii) imported from California in 1978 growing in Badajoz, Spain. Two foci were found in an orchard of 200 ha where 80 walnut trees were affected. Cankers were observed on trunks and branches, and dark exudates staining the bark appeared mainly in summer. Isolations were performed from affected tissue using King's B medium, and Brenneria (Erwinia)-like colonies (1) were purified and characterized. Gram reaction, Kovacs' oxidase, O/F metabolism, aesculin hydrolysis, urease activity, and levan production were assayed for five isolates (1). Biochemical characterization was performed by the miniaturized API 20E, API 20NE, and API 50CH systems (BioMérieux, Marcy-l'Etoile, France) as recommended, except for incubations that were made at 25°C for 48 h. Analyses of the cellular fatty acids of selected isolates were performed as described by Sasser (2). They were also tested in indirect enzyme-linked immunosorbent assay (ELISA) using antisera obtained against the reference strain CFBP 1284 and one Spanish isolate. When compared to the reference strain from California, isolates were identified as Brenneria rubrifaciens (1,3) on the basis of physiological and biochemical characteristics, fatty acid profiles, and ELISA. Pathogenicity of two selected Spanish isolates was confirmed using three 2-year-old walnut trees per bacterial isolate by inoculating 108 CFU of each isolate in deep wounds made in the trunk at 40 and 80 cm from the crown. The reference strain and water were also inoculated as controls. Two months later, removal of the outer bark of walnut revealed typical dark lesions in the inner bark at all the inoculation sites on trees inoculated with the Spanish and reference strains, but no external cankers were observed. Four years later, these plants showed internal lesions (20 to 80 cm), from which B. rubrifaciens was reisolated far away from the inoculation site. To our knowledge this is the first report of this bacterium in Europe. References: (1) L. Hauben et al. Syst. Appl. Microbiol 21:384, 1998. (2) M. Sasser. Pages 199-204 in: Methods in Phytobacteriology. Budapest, Hungary, 1990. (3) E. Wilson et al. Phytopathology 57:618, 1966.

An indirect immunofluorescent antibody technique for detection and enumeration of Vibrio vulnificus serovar E (biotype 2): development 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 bacterium.

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

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 degrees 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 degrees C for microcosms at 0.3 and 0.5% and 12 degrees C for microcosms at 1.5 to 3.8%). In the absence of salts, culturability dropped to zero in a few days, without evidence of cellular lysis. Under optimal conditions of salinity and temperature, the bacterium was able to survive in the free-living form for at least 3 years. The presence of a capsule on the bacterial cell seemed to confer an advantage, since the long-term survival rate of opaque variants was significantly higher than that of translucent ones. Long-term-starved cells maintained their infectivity for eels (as determined by both intraperitoneal and immersion challenges) and mice. Examination under the microscope showed that (i) the capsule was maintained, (ii) the cell size decreased, (iii) the rod shape changed to coccuslike along the time of starvation, and (iv) membrane vesicles and extracellular material were occasionally produced. In conclusion, V. vulnificus biotype 2 follows a survival strategy similar to that of biotype 1 of this species in response to starvation conditions in water. Moreover, the aquatic ecosystem is one of its reservoirs.

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

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

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 strains (of serogroup E) are resistant to NIS, and (iii) rough mutants of biotype 2 lacking the O polysaccharide side chain are sensitive to NIS and avirulent for eels.

Phenotypic and genotypic characterization of Vibrio vulnificus: proposal for the substitution of the subspecific taxon biotype for serovar.

The classification of Vibrio vulnificus strains into two biotypes has been maintained on the basis of phenotypic properties and eel virulence. Biotype 2 is virulent for eels, negative for the indole reaction, and serologically homogeneous (serogroup E), whereas strains of biotype 1 are avirulent, indole positive, and serologically heterogeneous. In the present study, we phenotypically and genotypically characterized 21 V. vulnificus isolates, recovered mainly from northern Europe, by comparing them with reference strains of both biotypes to look for new isolates of biotype 2. The results of this work revealed that the majority of isolates virulent for eels presented phenotypic traits previously considered characteristics of biotype 2 and specific ribotypes with HindIII. However, among the new isolates we found (i) a serogroup E strain virulent for eels but indole positive and (ii) one isolate not belonging to serogroup E but pathogenic for eels. Since no biochemical test for specific serogroup can with certainty be associated with eel virulence, we propose to classify V. vulnificus strains into serovars instead of biotypes. Thus, we suggest serovar E as the denomination of those strains previously classified as biotype 2. Finally, the occurrence of serogroup E in eels cultured in Norway and Sweden, as well as from human infections and shrimp, has been demonstrated.

An enzyme-linked immunosorbent assay for detection of Vibrio vulnificus biotype 2: development and field studies.

Vibrio vulnificus biotype 2 is a primary eel pathogen which constitutes a lipopolysaccharide (LPS)-based homogeneous O serogroup within the species. In the present work, we have developed an enzyme-linked immunosorbent assay (ELISA) based on the specificity of LPS for the detection of this pathogen. The ELISA specificity was confirmed after testing 36 biotype 2 strains from laboratory cultures and environmental samples, 31 clinical and environmental biotype 1 isolates, and several strains of Vibrio, Aeromonas, and Yersinia species, including the fish pathogens V. anguillarum, V. furnissii, A. hydrophila, and Y. ruckerii. The detection limits for biotype 2 cells were around 10(4) to 10(5) cells/well, and the immunoassay was also able to detect cells in the nonculturable state. Artificially infected eels and environmental samples were analyzed, and the immunodetection was confirmed by cultural methods (isolation on selective and nonselective media before and after broth enrichment). With this methodology, V. vulnificus biotype 2 was successfully detected in infected eels and asymptomatic carriers, which suggests that eels can act as a reservoir for this pathogen.

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 response to iron restriction. Results from the assays with transferrin suggest that these siderophores could be utilized to sequester iron from Tf, a protein for which no surface receptor was detected in any strain. In summary, the overall data demonstrate that V. damsela expresses siderophore-mediated iron-uptake systems. These systems are probably involved in the survival of the species in the different environments that it can colonize, i.e. water and several vertebrate hosts.

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 fish species also revealed that only biotype 2 strains were virulent.

Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification.

DNA extraction procedures and PCR conditions to detect Vibrio vulnificus cells naturally occurring in oysters were developed. In addition, PCR amplification of V. vulnificus from oysters seeded with biotype 1 cells was demonstrated. By the methods described, V. vulnificus cells on a medium (colistin-polymyxin B-cellobiose agar) selective for this pathogen were detectable in oysters harvested in January and March, containing no culturable cells (< 67 CFU/g), as well as in oysters harvested in May and June, containing culturable cells. It was possible to complete DNA extraction, PCR, and gel electrophoresis within 10 h by using the protocol described for oysters. V. vulnificus biotype 2 cells were also detected in eel tissues that had been infected with this strain and subsequently preserved in formalin. The protocol used for detection of V. vulnificus cells in eels required less than 5 h to complete. Optimum MgCl2 concentrations for the PCR of V. vulnificus from oysters and eels were different, although the same primer pair was used for both. This is the first report on the detection of cells of V. vulnificus naturally present in shellfish and represents a potentially powerful method for monitoring this important human and eel pathogen.

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, and by fish farmers, above all when manipulating eels during epizootic outbreaks.

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) 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-Mr 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).

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 patterns were detected in response to iron stress. Finally, our data suggest that V. vulnificus biotype 2 uses the hydroxamate-type siderophore for removal of iron from transferrin rather than relying on a receptor for this iron-binding protein.

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 of activity, showing marked changes in size and morphology. The rate at which cells became nonculturable was dependent on their physiological age. The capsule seems not to be necessary for the survival of biotype 2 in aquatic environments as a free-living organism. Culturability remained the highest on modified salt water yeast extract agar, which is closer in salt and nutrient composition to ASW than heart infusion agar. Biotype 2 cells recovered culturability on solid media after an increase of incubation temperature from 5 to 25 degrees C. Culturable cells of this bacterium maintained infectivity for either eel or mice, while dormant cells seemed to lose their virulence. The former finding suggests that the aquatic environment is a reservoir and vehicle of transmission of this pathogen.

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 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.

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 of iron-availability on the virulence was investigated in iron-overloaded animals. The iron-treatment before the infection resulted in a significant reduction in the LD50 of virulent strains. This fact demonstrates a positive correlation between iron availability in host fluids and degree of virulence in the species Vibrio damsela.

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.

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 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.