Novel T3SS effector EseK in Edwardsiella piscicida is chaperoned by EscH and EscS to express virulence.

Bacterium usually utilises type III secretion systems (T3SS) to deliver effectors directly into host cells with the aids of chaperones. Hence, it is very important to identify bacterial T3SS effectors and chaperones for better understanding of host-pathogen interactions. Edwardsiella piscicida is an invasive enteric bacterium, which infects a wide range of hosts from fish to human. Given E. piscicida encodes a functional T3SS to promote infection, very few T3SS effectors and chaperones have been identified in this bacterium so far. Here, we reported that EseK is a new T3SS effector protein translocated by E. piscicida. Bioinformatic analysis indicated that escH and escS encode two putative class I T3SS chaperones. Further investigation indicated that EscH and EscS can enhance the secretion and translocation of EseK. EscH directly binds EseK through undetermined binding domains, whereas EscS binds EseK via its N-terminal α-helix. We also found that EseK has an N-terminal chaperone-binding domain, which binds EscH and EscS to form a ternary complex. Zebrafish infection experiments showed that EseK and its chaperones EscH and EscS are necessary for bacterial colonisation in zebrafish. This work identified a new T3SS effector, EseK, and its two T3SS chaperones, EscH and EscS, in E. piscicida, which enriches our knowledge of bacterial T3SS effector-chaperone interaction and contributes to our understanding of bacterial pathogenesis.

Comparative Phenotypic and Genotypic Analysis of Edwardsiella Isolates from Different Hosts and Geographic Origins, with Emphasis on Isolates Formerly Classified as E. tarda, and Evaluation of Diagnostic Methods.

Edwardsiella spp. are responsible for significant losses in important wild and cultured fish species worldwide. Recent phylogenomic investigations have determined that bacteria historically classified as Edwardsiella tarda actually represent three genetically distinct yet phenotypically ambiguous taxa with various degrees of pathogenicity in different hosts. Previous recognition of these taxa was hampered by the lack of a distinguishing phenotypic character. Commercial test panel configurations are relatively constant over time, and as new species are defined, appropriate discriminatory tests may not be present in current test panel arrangements. While phenobiochemical tests fail to discriminate between these taxa, data presented here revealed discriminatory peaks for each Edwardsiella species using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) methodology, suggesting that MALDI-TOF can offer rapid, reliable identification in line with current systematic classifications. Furthermore, a multiplex PCR assay was validated for rapid molecular differentiation of the Edwardsiella spp. affecting fish. Moreover, the limitations of relying on partial 16S rRNA for discrimination of Edwardsiella spp. and advantages of employing alternative single-copy genes gyrB and sodB for molecular identification and classification of Edwardsiella were demonstrated. Last, sodB sequencing confirmed that isolates previously defined as typical motile fish-pathogenic E. tarda are synonymous with Edwardsiella piscicida, while atypical nonmotile fish-pathogenic E. tarda isolates are equivalent to Edwardsiella anguillarum Fish-nonpathogenic E. tarda isolates are consistent with E. tarda as it is currently defined. These analyses help deconvolute the scientific literature regarding these organisms and provide baseline information to better facilitate proper taxonomic assignment and minimize erroneous identifications of Edwardsiella isolates in clinical and research settings.

On the synonymy of Edwardsiellatarda Ewing and McWhorter 1965 (Approved Lists 1980) and Edwardsiellaanguillimortifera (Hoshina 1962) Sakazaki and Tamura 1975 (Approved Lists 1980), an old problem in need of a solution.

Edwardsiella tarda Ewing and McWhorter 1965 (Approved Lists 1980) and Edwardsiella anguillimortifera (Hoshina 1962) Sakazaki and Tamura 1975 (Approved Lists 1980) are known to be synonyms that share the same nomenclatural type. Edwardsiella tarda Ewing and McWhorter 1965 (Approved Lists 1980) is the nomenclatural type of the genus Edwardsiella Ewing and McWhorter 1965 (Approved Lists 1980). The question of the priority of the epithet anguillimortifera Hoshina 1962 over the epithet tarda Ewing and McWhorter 1965 has been raised in the past, and a Request for an Opinion to conserve the epithet tarda Ewing and McWhorter 1965 in Edwardsiella tarda Ewing and McWhorter 1965 (Approved Lists 1980) was published but later withdrawn. Close examination of the wording of the International Code of Nomenclature of Prokayotes indicates that there may be problems associated with the nomenclatural type of Edwardsiella anguillimortifera (Hoshina 1962) Sakazaki and Tamura 1975 (Approved Lists 1980). Additional issues also arise with the recognition of homonyms under other codes of nomenclature.

Phenotypic traits, virulence-associated gene profile and genetic relatedness of Edwardsiella tarda isolates from Japanese eel Anguilla japonica in Korea.

UNLABELLED: Edwardsiella tarda is the predominant bacterium in farm-cultured eel in Korea. Here, we evaluated the heterogeneity of 37 E. tarda isolates derived from Japanese eel with various origins (olive flounder, common carp and ornamental fish) between 2003 and 2010. Regardless of origins, the biochemical characteristics of E. tarda isolates were homogenous except hydrogen sulfide production, citrate utilization and mannitol fermentation. Based on the phylogenetic analysis of 16S rRNA, E. tarda isolates could be classified into two subgroups and displayed a close relation with Edwardsiella ictaluri and Edwardsiella hosinae lineages, suggesting that the subgroup I has been a predominant type in the Jeonnam and Jeonbuk provinces. I-CeuI-based pulsed-field gel electrophoresis (PFGE) typing showed that the isolates from Japanese eels belonged to 11 pulsotypes, indicating that the presence of highly genomic diversity. Additionally, two isolates, ET-060 and ET-191, showed a high frequency of virulence genes (100%) and caused 90% and 60% mortality in Japanese eel, respectively. This finding suggests a substantial congruence of virulence gene profiles and pathogenicity. Our results demonstrate that the intraspecific diversity within E. tarda strains from Japanese eel has been in prior existence. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on the biochemical characteristics, the phylogenetic property of the 16S rRNA gene and PFGE types of Edwardsiella tarda, we could identify the intraspecific diversity of isolates from Japanese eel, Anguilla japonica in Korea. In addition, this study describes the strong congruence of virulence-related genes and pathogenicity, suggesting that the virulence profile may be useful tool for prediction of pathogenicity.

A multiplex PCR for the simultaneous detection of Tenacibaculum maritimum and Edwardsiella tarda in aquaculture.

A specific and sensitive multiplex PCR (mPCR) method was developed as a useful tool for the simultaneous detection of two important flatfish pathogens in marine aquaculture, Tenacibaculum maritimum and Edwardsiella tarda. In fish tissues, the average detection limit for these mPCR-amplified organisms was 2 × 10 5 ± 0.2 CFU/g and 4 × 10 5 ± 0.3 CFU/g, respectively. These values are similar or even lower than those previously obtained using the corresponding single PCR. Moreover, mPCR did not produce any nonspecific amplification products when tested against 36 taxonomically related and unrelated strains belonging to 33 different bacterial species. Large amounts of DNA from one of the target bacterial species in the presence of low amounts from the other did not have a significant effect on the amplification sensitivity of the latter.

Comparative genomics reveals that a fish pathogenic bacterium Edwardsiella tarda has acquired the locus of enterocyte effacement (LEE) through horizontal gene transfer.

BACKGROUND: Edwardsiella tarda is an enterobacterium which causes edwardsiellosis, a fatal disease of cultured fishes such as red sea bream, eel, and flounder. Preventing the occurrence of E. tarda infection has thus been an important issue in aquaculture. E. tarda has been isolated from other animals and from many environments; however, the relationship between the genotype and evolutionary process of this pathogen is not fully understood. To clarify this relationship, we sequenced and compared the genomes of pathogenic and non-pathogenic E. tarda strains isolated from fish, human, and eel pond using next-generation sequencing technology. RESULTS: Eight strains of E. tarda were sequenced with high accuracy (>99.9%) with coverages from 50- to 400-fold. The obtained reads were mapped to a public reference genome. By comparing single nucleotide and insertion/deletion polymorphisms, we found that an attenuated strain of E. tarda had a loss-of-function mutation in a gene related to the type III secretion system (T3SS), suggesting that this gene is involved in the virulence of E. tarda. A comprehensive gene comparison indicated that fish pathogenic strains possessed a type VI secretion system (T6SS) and pilus assembly genes in addition to the T3SS. Moreover, we found that an E. tarda strain isolated from red sea bream harbored two pathogenicity islands of T3SS and T6SS, which were absent in other strains. In particular, this T3SS was homologous to the locus of enterocyte effacement (LEE) in enteropathogenic and enterohemorrhagic Escherichia coli. Evolutionary analysis suggested that this locus, here named Et-LEE (E. tarda LEE), was introgressed into the E. tarda genome through horizontal transfer. CONCLUSIONS: We found significant differences in the presence/absence of virulence-related genes among E. tarda strains, reflecting their evolutionary relationship. In particular, a single genotype previously proposed for fish-pathogenic strains may be further divided into two subgroups. Furthermore, the current study demonstrated, for the first time, that a fish pathogenic bacterium carried a LEE-like pathogenicity island which was previously reported only in zoonotic pathogenic enterobacteria. These findings will contribute to the exploration of strain-specific drug targets against E. tarda in aquafarms, while also shedding light on the evolution of pathogenesis in enterobacteria.

Comparative analysis of Edwardsiella isolates from fish in the eastern United States identifies two distinct genetic taxa amongst organisms phenotypically classified as E. tarda.

Edwardsiella tarda, a Gram-negative member of the family Enterobacteriaceae, has been implicated in significant losses in aquaculture facilities worldwide. Here, we assessed the intra-specific variability of E. tarda isolates from 4 different fish species in the eastern United States. Repetitive sequence mediated PCR (rep-PCR) using 4 different primer sets (ERIC I & II, ERIC II, BOX, and GTG5) and multi-locus sequence analysis of 16S SSU rDNA, groEl, gyrA, gyrB, pho, pgi, pgm, and rpoA gene fragments identified two distinct genotypes of E. tarda (DNA group I; DNA group II). Isolates that fell into DNA group II demonstrated more similarity to E. ictaluri than DNA group I, which contained the reference E. tarda strain (ATCC #15947). Conventional PCR analysis using published E. tarda-specific primer sets yielded variable results, with several primer sets producing no observable amplification of target DNA from some isolates. Fluorometric determination of G+C content demonstrated 56.4% G+C content for DNA group I, 60.2% for DNA group II, and 58.4% for E. ictaluri. Surprisingly, these isolates were indistinguishable using conventional biochemical techniques, with all isolates demonstrating phenotypic characteristics consistent with E. tarda. Analysis using two commercial test kits identified multiple phenotypes, although no single metabolic characteristic could reliably discriminate between genetic groups. Additionally, anti-microbial susceptibility and fatty acid profiles did not demonstrate remarkable differences between groups. The significant genetic variation (<90% similarity at gyrA, gyrB, pho, phi and pgm; <40% similarity by rep-PCR) between these groups suggests organisms from DNA group II may represent an unrecognized, genetically distinct taxa of Edwardsiella that is phenotypically indistinguishable from E. tarda.

Environmental isolates P1SW and V3SW as a bivalent vaccine induce effective cross-protection against Edwardsiella tarda and Vibrio anguillarum .

Edwardsiella tarda and Vibrio anguillarum are severe fish pathogens. In this study, we aimed at selecting avirulent environmental isolates with application potential in the prevention of E. tarda- and V. anguillarum-associated diseases. For this purpose, we selected and analyzed 2 seawater isolates, P1SW and V3SW, belonging to the genera Pseudomonas and Vibrio, respectively. When administered to turbot Scophthalmus maximus via immersion and oral feeding, P1SW and V3SW at a dose of 2 × 108 colony-forming units caused no mortality, but both strains were able to disseminate into internal organs in a transient, time-dependent manner. When turbot were immunized with P1SW, V3SW, or P1SW plus V3SW (named P1V3) via immersion plus oral routes, the latter with vaccines embedded in sodium alginate microspheres, moderate protection against E. tarda and V. anguillarum was induced by V3SW, and moderate protection against E. tarda was induced by P1SW. Compared to P1SW and V3SW, P1V3 elicited a significantly stronger protection against both E. tarda and V. anguillarum. Immunological analysis showed that (1) P1SW, V3SW, and especially P1V3 activated head kidney macrophages, (2) P1V3 induced significantly higher levels of serum antibodies against E. tarda and V. anguillarum than P1SW and V3SW, and (3) P1V3-induced antibodies were able to bind E. tarda and V. anguillarum and enhance serum bactericidal activity. These results indicate that P1V3 as a naturally delivered vaccine elicited a humoral immune response against both E. tarda and V. anguillarum and, as a result, was cross-protective against E. tarda and V. anguillarum infection.

Pathogenesis of and strategies for preventing Edwardsiella tarda infection in fish.

Edwardsiella tarda is one of the serious fish pathogens, infecting both cultured and wild fish species. Research on edwardsiellosis has revealed that E. tarda has a broad host range and geographic distribution, and contains important virulence factors that enhance bacterial survival and pathogenesis in hosts. Although recent progress in edwardsiellosis research has enabled the development of numerous, highly effective vaccine candidates, these efforts have not been translated into a commercialized vaccine. The present review aims to provide an overview of the identification, pathology, diagnosis and virulence factors of E. tarda in fish, and describe recent strategies for developing vaccines against edwardsiellosis. The hope is that this presentation will be useful not only from the standpoint of understanding the pathogenesis of E. tarda, but also from the perspective of facilitating the development of effective vaccines.

Multi-locus Sequence Analysis (MLSA) of Edwardsiella tarda isolates from fish.

Edwardsiella tarda is an enteric fish pathogen that has caused significant economic losses in a range of fish species residing in diverse ecological conditions. Several molecular methods relying on DNA fingerprinting (RAPD, RFLP and ERIC-PCR) and the gyrB gene marker have been used to characterize E. tarda isolates. However, all had drawbacks in resolving power and reproducibility. The present study was aimed at developing a novel Multi-locus Sequence Analysis (MLSA) scheme for genetic characterization of E. tarda isolates originating from multiple sources. MLSA has been described as an effective molecular tool with superior discriminatory power and reproducibility for exploring intra-species genetic diversity of several bacterial species. Nucleotide sequence fragments of eight protein coding housekeeping genes (gyrB, mdh, adk, dnaK, phoR, metG, pyrG and aroE2) were obtained from 23 fish pathogenic E. tarda isolates of different geographical origins, one human isolate and 3 reference strains. The phylogenetic relationships between isolates in individual gene analyses were not consistent, although some common patterns were apparent. Phylogenetic analysis based on concatenated sequences of seven gene loci, however, buffered the conflicting phylogenetic signals and resolved isolates according to their geographical origin and/or fish host. The MLSA revealed two major genetically diverging clusters in E. tarda isolates examined, one cluster representing isolates from fish and the other representing (in the main) human isolates, with E. ictaluri cluster situated in between. The results suggest, therefore, that the fish pathogenic E. tarda isolates may have been previously misclassified and probably represent one or more as yet unrecognized taxa within the genus Edwardsiella. The MLSA described here was robust enough in discriminating E. tarda isolates not only with respect to their geographical origins but also within different hosts from the same geographical location, high-lighting its potential application in tracing the source of infection and understand the epidemiological relationships among isolates of environmental, fish, other domestic animals or human origins.

Genetic relationships of Edwardsiella strains isolated in China aquaculture revealed by rep-PCR genomic fingerprinting and investigation of Edwardsiella virulence genes.

AIMS: The aim of this study was to classify Edwardsiella strains isolated from China aquaculture based on biochemical and molecular methods. METHODS AND RESULTS: In this study, biochemical characterization of 19 Edwardsiella tarda isolates and two Edwardsiella ictaluri isolates was performed with API 20E system. Other pathogenicity-related phenotypes such as haemagglutination, haemolytic activities and lethality to fish were also examined in these strains. As it was difficult to categorize the subgroups of Edw. tarda according to their origins or phenotypic properties, three PCR-based methods, i.e. PCR amplification of virulence genes, Enterobacterial repetitive intergenic consensus-PCR and BOX-PCR, were carried out to further resolve the relatedness of the Edw. tarda isolates. As a result, all Edw. tarda isolates could be generally grouped into pathogenic and nonpathogenic branches before being classified into strain-specific or origin-specific clades. CONCLUSIONS: Biochemical characterization was sensitive for interspecific typing, while PCR-based approaches permitted a more accurate discrimination for intraspecific typing resulting in pathogenic and nonpathogenic clusters and further more delicate clades for Edwardsiella. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-based genomic fingerprinting to study the relatedness and trace the pathogenicity of the Edwardsiella strains will be helpful in investigating the virulence factors of Edwardsiella and in the development of vaccines and diagnostics for edwardsiellosis.

Phenotypic diversity of Edwardsiella tarda isolated from different origins.

AIMS: To evaluate the diversity of phenotypic characteristics among isolates of Edwardsiella tarda from various origins. METHODS AND RESULTS: A total of 10 E. tarda strains were investigated on biological characteristics including flagella formation, bacterial motility, biofilm formation, extracellular protein and plasmid profiles. All the E. tarda strains (including two previous recognized as nonflagellation strains) were proven to have an average of 1-7 peritrichous flagella with the precise number positively correlated with motility and biofilm formation. All the E. tarda strains exhibited similar protein profiles except ET2034, LMG2793 and ET080814, which lacked the three major bands of approximately 18, 21 and 55 kDa. E. tarda with the same geographic location shared similar plasmid profiles. CONCLUSIONS: Edwardsiella tarda strains exhibited diversities in phenotypic characteristics that may be linked to differences in geographic location or host origin. In addition, the number of flagella is essential for bacterial motility and biofilm formation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report demonstrating the difference in flagella formation between E. tarda strains, which may broaden the understanding of flagellation trait at intra-species level. Furthermore, evaluation of virulence-associated characteristics can provide useful information for unveiling the diverse pathogenic mechanisms of E. tarda.

Identification of Edwardsiella ictaluri and E. tarda by species-specific polymerase chain reaction targeted to the upstream region of the fimbrial gene.

Phylogenetic analysis of nine strains of Edwardsiella ictaluri and eight strains of E. tarda (six typical motile strains and two atypical nonmotile strains) isolated from diseased fish was performed using the upstream region of the fimbrial gene cluster. Strains of E. ictaluri and E. tarda were significantly clustered into separate groups. Moreover, atypical E. tarda strains were clustered into a different group from the other strains. Three polymerase chain reaction (PCR) primer sets for differential detection of E. ictaluri as well as typical and atypical E. tarda were developed from the respective characteristic sequences. Strains of E. ictaluri, typical E. tarda, and atypical E. tarda were specifically detected by PCR using each primer set. No amplifications were observed after the use of these three primer sets with 25 other bacterial species, including fish pathogens. In addition, the three primer sets were able to detect the DNA of each target species from fish kidney and liver artificially infected with E. ictaluri or E. tarda.

Phenotypic and genetic characterization of Edwardsiella tarda isolated from pond sediments.

The extent of genotypic and phenotypic diversity of Edwardsiella tarda isolated from pond sediment was assessed by SDS-PAGE, Plasmid Profiling and ERIC-PCR. SDS-PAGE of whole cell protein extracts reveals 20-23 discrete bands with molecular wt of 14-110 kDa. Several bands with molecular weight range of 38-83 kDa were present in all the isolates. Numerical analysis of protein electrophoregram delineated the isolates into four clusters. Two different types of plasmids having molecular mass of 23 kDa and 29 kDa were obtained by plasmid profiling. About 51% of the isolates carried both the plasmids. ERIC-PCR generates 3-7 bands with molecular mass of 14-1013 bp. Numerical analysis differentiated the ERIC pattern into 5 clusters at 60% similarly level. It was concluded that out of three methods ERIC-PCR was found to be more sensitive for intraspecific typing of E. tarda and can be used as a potential tool for epidemiological studies in the future.

A 12-year-old boy with acute gastroenteritis caused by Edwardsiella tarda O4:H4.

A 12-year-old boy was brought to the hospital with a 3-week history of watery diarrhea mixed with mucus and colicky abdominal pain. Stool culture identified Edwardsiella tarda O4: H4, and no other pathogenic bacteria were detected. Acute gastroenteritis caused by Edwardsiella tarda O4: H4 was diagnosed. This bacterium was shown to be sensitive to ampicillin hydrate. When this antibiotic was administered, the condition of the patient improved within a week. The patient had a history of eating raw shrimp and fish while traveling with his parents.

Genotyping of Edwardsiella tarda isolated from freshwater fish culture system.

The applicability of PCR-RFLP of 16S rDNA and conventional phenotypic methods for differentiation of Edwardsiella tarda associated in freshwater fish culture system was studied. In this study, by conventional biochemical tests and antibiotic resistant patterns 2 and 14 groups were obtained. But these methods failed to discriminate the isolates habitat wise. However, PCR-RFLP of 16S rDNA was found to be specific to detect habitat-specific isolates. All the fish isolates belonging to particular genotypes were found only in fish, not in water or sediment. Some of the genotypes were exclusively present in water and sediment. This study indicates the prevalence of site-specific genotypes in freshwater ecosystems. Molecular method is found to be superior to discriminate the E. tarda habitat wise to conventional typing methods.

Characterization of Edwardsiella tarda strains isolated from turbot, Psetta maxima (L.).

The biochemical, serological and molecular characteristics of a group of 21 Edwardsiella tarda strains isolated from turbot, Psetta maxima, in two different areas of Europe were analysed and compared with a total of 13 strains of this bacterial species with different geographical and host origins. All the turbot isolates were biochemically identical to the E. tarda strains included as reference. The use of different techniques including microagglutination, dot blot and Western blot of lipopolysaccharides allowed us to determine that all the turbot isolates constitute an homogeneous and distinctive serological group. Genetic analysis by randomly amplified polymorphic DNA (RAPD) analysis demonstrated that although the E. tarda strains from turbot were compiled in a unique group using the primers P3 and P6, two clonal lineages could be detected when oligonucleotides P4 and P5 were employed.

Liver abscess caused by Edwardsiella tarda biogroup 1 and identification of its epidemiological triad by ribotyping.

Two clinical isolates and an environmental isolate of Edwardsiella tarda biogroup 1 (ETB1), recovered from liver pus, the stool specimen and from the pond water of the village of the patient, diagnosed to have liver abscess, were found to be identical by protein fingerprinting and ribotyping. It can be construed that the pond water served as the source of infection. The epidemiological triad of the agent (ETB1), host (the patient) and environment (pond water) was thus established. This is the first report in which the triad for extraintestinal Edwardsiellosis caused by ETB1 has been identified. This also constitutes the first report of typing of ETB1 strains by SDS-PAGE and ribotyping.

Structural characterization of the O-polysaccharide antigen of Edwardsiella tarda MT 108.

Edwardsiella tarda, a Gram-negative bacterium, is an important cause of hemorrhagic septicemia in fish and also of gastro- and extraintestinal infections in humans. The lipopolysaccharide produced by the fish pathogenic strain E. tarda MT 108 was isolated and the structure of its antigenic O-polysaccharide component determined by the application of chemical analyses, high-resolution 1D and 2D nuclear magnetic resonance spectroscopy, and mass spectrometry. The polysaccharide was found to be a polymer of a repeating pentasaccharide unit composed of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), 2-acetamido-2-deoxy-D-galactose (D-GalNAc), D-galactose (D-Gal), L-rhamnose (L-Rha), D-galacturonic acid (D-GalA) and (2S,3R)-threonine (1:1:1:1:1:1) having the structure: [structure: see text].