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…

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…

Plant-associated microbiota as a source of antagonistic bacteria against the phytopathogen Erwinia amylovora.

Control of bacterial plant diseases is a major concern, as they affect economically important species and spread easily, such as the case of fire blight of rosaceous caused by Erwinia amylovora. In the search for alternatives to the use of agrochemicals and antibiotics, this work presents a screening of natural bacterial antagonists of this relevant and devastating phytopathogen. We recovered bacterial isolates from different plant tissues and geographical origins and then selected those with the strongest ability to reduce fire blight symptoms ex vivo and remarkable in vitro antagonistic activity against E. amylovora. None of them elicited a hypersensitivity reaction in tobacco leaves, mos…

Brenneria quercina and Serratia spp. isolated from Spanish oak trees Molecular characterization and development of PCR primers

Brenneria quercina has been reported as one of the causal agents of oak decline in Spain. To investigate the bacterial variability of this pathogen from different Spanish oak forests, a collection of 38 bacterial isolates from seven geographic locations and from different oak species was analysed by sequencing 16S rDNA and rep-PCR fingerprinting. All Spanish isolates of B. quercina were grouped by rep-PCR into a homogenous cluster that differed significantly from B. quercina reference strains from California. 16S rDNA analysis revealed that 34 out of 38 isolates were Brenneria. However, four isolates belonged to the genus Serratia, suggesting that this bacterium could cause cankers in oak t…

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…

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 …

Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

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 Cu2+ was inoculated with 107 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 chlori…

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…

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,…

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…

Bacteriophage-Based Bacterial Wilt Biocontrol for an Environmentally Sustainable Agriculture

Bacterial wilt diseases caused by Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii subsp. indonesiensis (former R. solanacearum species complex) are among the most important plant diseases worldwide, severely affecting a high number of crops and ornamentals. Difficulties of bacterial wilt control by non-biological methods are related to effectiveness, bacterial resistance and environmental impact. Alternatively, a great many biocontrol strategies have been carried out, with the advantage of being environmentally friendly. Advances in bacterial wilt biocontrol include an increasing interest in bacteriophage-based treatments as a promising re-emerging strategy. Bacteriophages agai…

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.

Innovative Approaches Using Lichen Enriched Media to Improve Isolation and Culturability of Lichen Associated Bacteria

Lichens, self-supporting mutualistic associations between a fungal partner and one or more photosynthetic partners, also harbor non-photosynthetic bacteria. The diversity and contribution of these bacteria to the functioning of lichen symbiosis have recently begun to be studied, often by culture-independent techniques due to difficulties in their isolation and culture. However, culturing as yet unculturable lichenic bacteria is critical to unravel their potential functional roles in lichen symbiogenesis, to explore and exploit their biotechnological potential and for the description of new taxa. Our objective was to improve the recovery of lichen associated bacteria by developing novel isol…

Erwinia amylovoracatalases KatA and KatG are virulence factors and delay the starvation-induced viable but non-culturable (VBNC) response

The life cycle of the plant pathogen Erwinia amylovora comprises periods inside and outside the host in which it faces oxidative stress caused by hydrogen peroxide (H2 O2 ) and other compounds. The sources of this stress are plant defences, other microorganisms and/or exposure to starvation or other environmental challenges. However, the functional roles of H2 O2 -neutralizing enzymes, such as catalases, during plant-pathogen interactions and/or under starvation conditions in phytopathogens of the family Erwiniaceae or closely related families have not yet been investigated. In this work, the contribution of E. amylovora catalases KatA and KatG to virulence and survival in non-host environm…

Influence of native microbiota on survival of Ralstonia solanacearum phylotype II in river water microcosms.

ABSTRACT Ralstonia solanacearum phylotype II biovar 2 causes bacterial wilt in solanaceous hosts, producing severe economic losses worldwide. Waterways can be major dissemination routes of this pathogen, which is able to survive for long periods in sterilized water. However, little is known about its survival in natural water when other microorganisms, such as bacteriophages, other bacteria, and protozoa, are present. This study looks into the fate of a Spanish strain of R. solanacearum inoculated in water microcosms from a Spanish river, containing different microbiota fractions, at 24°C and 14°C, for a month. At both temperatures, R. solanacearum densities remained constant at the initial…

Erwinia amylovora psychrotrophic adaptations: evidence of pathogenic potential and survival at temperate and low environmental temperatures

The fire blight pathogenErwinia amylovoracan be considered a psychrotrophic bacterial species since it can grow at temperatures ranging from 4 °C to 37 °C, with an optimum of 28 °C. In many plant pathogens the expression of virulence determinants is restricted to a certain range of temperatures. In the case ofE. amylovora,temperatures above 18 °C are required for blossom blight epidemics under field conditions. Moreover, this bacterium is able to infect a variety of host tissues/organs apart from flowers, but it is still unknown how environmental temperatures, especially those below 18 °C, affect the pathogen ability to cause fire blight disease symptoms in such tissues/organs. There is als…

Survival of Erwinia amylovora in rain water at low temperatures

E.G. Biosca , R.D. Santander, M. Ordax, E. Marco-Noales, B. Aguila, A. Flores and M.M. Lopez 1 Universidad de Valencia, Departamento de Microbiologia y Ecologia, Avenida Dr. Moliner 50, 46100, Burjassot, Valencia, Spain. 2 Instituto Valenciano de Investigaciones Agrarias (IVIA), Centro de Proteccion Vegetal y Biotecnologia, Carretera Moncada – Naquera, km 4.5, 46113, Moncada,Valencia, Spain. 3 Universidad de Valencia, Servicio Central de Soporte a la Investigacion Experimental, Avenida Dr. Moliner 50, 46100, Burjassot, Valencia, Spain.

Micromón València (Universitat de València)

En Julio de 2017 se creó la red SWI@Spain, auspiciada por el grupo de Docencia y Difusión de la Microbiología (DDM) de la Sociedad Española de Microbiología (SEM), para desarrollar la iniciativa internacional Small World Initiative (SWI) en la península ibérica. En la Universitat de València (UV) se constituyó entonces el grupo de Innovación Docente en Microbiología (IDM) para implementar el proyecto a nivel local. Avalados por el Servei de Formació Permanent i Innovació Educativa (SFPIE) de la UV, el grupo ha llevado a cabo diferentes iniciativas relacionadas con el objetivo fundamental del proyecto: divulgar la problemática actual relacionada con el uso inadecuado de antibióticos, el incr…

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…

Cellular, physiological, and molecular adaptive responses of Erwinia amylovora to starvation.

Erwinia amylovora causes fire blight, a destructive disease of rosaceous plants distributed worldwide. This bacterium is a nonobligate pathogen able to survive outside the host under starvation conditions, allowing its spread by various means such as rainwater. We studied E. amylovora responses to starvation using water microcosms to mimic natural oligotrophy. Initially, survivability under optimal (28 °C) and suboptimal (20 °C) growth temperatures was compared. Starvation induced a loss of culturability much more pronounced at 28 °C than at 20 °C. Natural water microcosms at 20 °C were then used to characterize cellular, physiological, and molecular starvation responses of E. amylovora. Ch…

Exploring new roles for the rpoS gene in the survival and virulence of the fire blight pathogen Erwinia amylovora

Erwinia amylovora causes fire blight in economically important plants of the family Rosaceae. This bacterial pathogen spends part of its life cycle coping with starvation and other fluctuating environmental conditions. In many Gram-negative bacteria, starvation and other stress responses are regulated by the sigma factor RpoS. We obtained an E. amylovora rpoS mutant to explore the role of this gene in starvation responses and its potential implication in other processes not yet studied in this pathogen. Results showed that E. amylovora needs rpoS to develop normal starvation survival and viable but nonculturable (VBNC) responses. Furthermore, this gene contributed to stationary phase cross-…

Seasonal Variation of Ralstonia solanacearum Biovar 2 Populations in a Spanish River: Recovery of Stressed Cells at Low Temperatures

ABSTRACT The presence of Ralstonia solanacearum biovar 2 in the watercourses of European countries is increasing, but little is known about its ecology in aquatic habitats. The detection of this pathogen in 2000 in one Spanish river led us to study its population density at different locations on the river over a period of 3 years. During 2000 and 2001, the pathogen was recovered at low densities (10 to 80 CFU/ml) by direct plating on modified SMSA agar from water samples at 14°C or higher, but its isolation was usually unsuccessful at temperatures below 9°C. To monitor the pathogen's abundance in winter, we used two liquid selective media for enrichment (at 29 and 35°C) and compared them b…

Implementation of Antibiotic Discovery by Student Crowdsourcing in the Valencian Community Through a Service Learning Strategy

© 2020 Maicas, Fouz, Figàs-Segura, Zueco, Rico, Navarro, Carbó, Segura-García and Biosca.

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° and 5°C, and the effect of copper treatment was assayed at 0·01 and 0·1 mmol l−1 CuSO4. In nontreated apples at 26°C, part of the population of E. amylovora survived in the ‘viable but nonculturable’ (VBNC) state, whereas at 5°C the majority of the population retained culturability. In copper-treated apples, the whole population adopted the VBNC stat…

Survival ofRalstonia solanacearum Biovar 2 in River Water: Influence of Water Microbiota

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.

Exopolysaccharides favor the survival of Erwinia amylovora under copper stress through different strategies.

Erwinia amylovora causes fire blight, a destructive disease of rosaceous plants very difficult to control. We demonstrated that copper, employed to control plant diseases, induces the "viable-but-nonculturable" (VBNC) state in E. amylovora. Moreover, it was previously reported that copper increases production of its main exopolysaccharide (EPS), amylovoran. In this work, the copper-complexing ability of amylovoran and levan, other major EPS of E. amylovora, was demonstrated. Following this, EPS-deficient mutants were used to determine the role of these EPSs in survival of this bacterium in AB mineral medium with copper, compared to their wild type strain and AB without copper. Tot…

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…

Medfly Ceratitis capitata as Potential Vector for Fire Blight Pathogen Erwinia amylovora: Survival and Transmission

Monitoring the ability of bacterial plant pathogens to survive in insects is required for elucidating unknown aspects of their epidemiology and for designing appropriate control strategies. Erwinia amylovora is a plant pathogenic bacterium that causes fire blight, a devastating disease in apple and pear commercial orchards. Studies on fire blight spread by insects have mainly focused on pollinating agents, such as honeybees. However, the Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae), one of the most damaging fruit pests worldwide, is also common in pome fruit orchards. The main objective of the study was to investigate whether E. amylovora can survive and be tra…

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 PFG…

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 …

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…

Current Technologies for Pseudomonas spp. And Ralstonia solanacearum Detection and Molecular Typing

Standard protocols for detection of phytopathogenic Pseudomonas spp. and Ralstonia solanacearum in plant material, soil, water or other sources, often still rely on the isolation of bacterial colonies on appropriate media, and/or on the use of serological techniques. However, over the last several years, molecular techniques, mainly based on PCR methods after extraction of nucleic acids from samples, have improved enough to allow a more rapid, reliable detection of these bacteria. When maximum accuracy is required the use of multiple techniques in an integrated approach is advised. Other promising technologies like flow cytometry, electronic nose or microarrays are emerging due to the need …

Survival strategies and pathogenicity of Ralstonia solanacearum phylotype II subjected to prolonged starvation in environmental water microcosms

Survival strategies exhibited over 4 years by Ralstonia solanacearum phylotype (ph) II biovar (bv) 2 in environmental water microcosms were examined. The bacterium is a devastating phytopathogen whose ph II bv 2 causes bacterial wilt in solanaceous crops and ornamental plants. Outbreaks of the disease may originate from dissemination of the pathogen in watercourses, where it has to cope with prolonged nutrient limitation. To ascertain the effect of long-term starvation on survival and pathogenicity of R. solanacearum in natural water microcosms, survival experiments were conducted. Microcosms were prepared from different sterile river water samples, inoculated separately with two European s…

Biocontrol of the Major Plant Pathogen Ralstonia solanacearum in Irrigation Water and Host Plants by Novel Waterborne Lytic Bacteriophages

Three new lytic bacteriophages were found to effectively control the pathogen Ralstonia solanacearum, a quarantine bacterium in many countries, and causative agent of bacterial wilt, one of the most important vascular plant diseases. Bacterial wilt management has been carried out with fluctuating effects, suggesting the need to find alternative treatments. In this work, three lytic phages were isolated from environmental water from geographically distant regions in Spain. They proved to specifically infect a collection of R. solanacearum strains, and some of the closely related pathogenic species Ralstonia pseudosolanacearum, without affecting non-target environmental bacteria, and were abl…

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.…

From the roots to the stem: unveiling pear root colonization and infection pathways by Erwinia amylovora

ABSTRACT Fire blight caused by Erwinia amylovora affects pome fruit worldwide, generating serious economic losses. Despite the abundant literature on E. amylovora infection mechanisms of aerial plant organs, root infection routes remain virtually unexplored. Assessing these infection pathways is necessary for a full understanding of the pathogen's ecology. Using the pathosystem Pyrus communis–E. amylovora and different experimental approaches including a green fluorescent protein transformant (GFP1) and epifluorescence microscopy (EFM) and laser confocal scanning microscopy (LCSM), we demonstrated the pathogen's ability to infect, colonize and invade pear roots and cause characteristic fire…

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…

Characterization ofBrenneria sp. from Poplar Cankers in Spain

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,…

Innovation in bacteriophage-based biocontrol of the plant pathogen ralstonia solanacearum through irrigation water

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…