Tarda Isolates Research Articles

Phylogenetic investigation of Edwardsiella tarda with multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) typing methods

Edwardsiella tarda is the etiologic agent of Edwardsiellosis, a devastating fish disease prevailing in worldwide aquaculture industries. Here we developed the SpeI-based pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) assays to investigate the genetic diversity of Edwardsiella isolates obtained from aquaculture farms and to get insights into the epidemiological relationships of E. tarda isolates in China. The results showed a strong congruence of clustering profiles obtained by PFGE and MLST methods. This investigation also indicated distinct phylogenetic profiles of E. tarda isolates from different hosts and different geographical regions in China with the following conclusions: i) E. tarda isolates collected from flatfish (turbot or flounder) in Northern China were phylogenetically distinct from the eel isolates from Southern China, and ii) E. tarda isolates from humans and fish (except strain DT from Oscar fish) were clustered into two distinct clades. dnaK-based phylogenetic analysis of the 67 Edwardsiella strains from all over the world further confirmed the above inference as well as the previous findings that there is misclassification in Edwardsiella species and Edwardsiella ictaluri is close to fish isolates of E. tarda while showing distant phylogenetic relationship to human isolates of E. tarda. Our data highlighted the intraspecies relationships of E. tarda and indicated potential application of PFGE and MLST methods in tracing source of infection and enhancing understanding of epidemiological relationships among isolates of various origins.

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.

Characterization of the unusual non-thiosulfate-reducing Edwardsiella tarda isolated from eel (Anguilla japonica) farms

Unusual Edwardsiella tarda, that do not produce hydrogen sulfide (H2S) from thiosulfate, was isolated from diseased eels (Anguilla japonica) in varying farming sites. In this study, we examined the biochemical characteristics of H2S production from the bacterium E. tarda. Diseased eels, along with pond water, were sampled from eight eel farms. All isolates were used for biochemical characterization and 16S rRNA and fimbrial gene sequencing. The phs gene of some isolates were also sequenced. H2S production was analyzed from either thiosulfate (S2O32−)- or sulfite (SO3−)-containing media. DNA analysis identified all isolates as E. tarda. Of the 17 E. tarda isolates, 11 were unable to reduce thiosulfate to H2S, while all isolates produced H2S from sulfite. In addition, non-thiosulfate-reducing E. tarda were isolated from four of the eight sampled eel farms. In media containing sulfate (SO42−) and in media without thiosulfate or sulfite, no H2S production was observed. In addition, glucose and galactose appeared to repress thiosulfate reduction in all strains, whereas these different carbon sources did not affect sulfite reduction. This indicates that thiosulfate and sulfite reduction pathways do not overlap in E. tarda. For this reason, careful attention should be made when identifying or differentiating non-thiosulfate-reducing E. tarda using H2S production characteristics.

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 Edwardseilla. 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

The aim of this study was to classify Edwardsiella strains isolated from China aquaculture based on biochemical and molecular methods. 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. 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. 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.

Intraspecific genetic variability of Edwardsiella tarda strains from cultured turbot

Edwardsiella tarda is an enterobacterial fish pathogen that causes mortality in various fish species worldwide. In this study, we analyzed the intraspecific variability in a collection of E. tarda strains isolated from turbot. To do this we employed 4 polymerase chain reaction (PCR)-based methods: (1) random amplified polymorphic DNA (RAPD), (2) enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), (3) repetitive extragenic palindromic-PCR (REP-PCR) and (4) BOX-PCR. E. tarda isolates from different hosts were also included for comparison. E. tarda strains from turbot showed high molecular homogeneity when RAPD (primers P3 and P6), ERIC-PCR and BOX-PCR were employed. However, with regard to the REP-PCR and RAPD (primers P4 and P5) techniques, different genetic groups could be established within these isolates using either technique. The 2 RAPD types presented an 85% similarity, while those obtained with REP-PCR showed 74% similarity. Based on the results obtained, although a high genetic homogeneity was found in turbot isolates, the RAPD test (with primers P4 and P5) and REP-PCR were capable of discrimination within these strains, and they are therefore considered the most appropriate typing methods for studies of edwardsiellosis in turbot.

Phenotypic diversity of Edwardsiella tarda isolated from different origins

To evaluate the diversity of phenotypic characteristics among isolates of Edwardsiella tarda from various origins. 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. 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. 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.

Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda, a potential vaccine candidate for fish, common carp

Outer membrane protein A (OmpA) is a component of the outer membrane of Edwardsiella tarda and is wildly distributed in Enterobacteriaceae family. The gene encoding the OmpA protein was cloned from E. tarda and expressed in Escherichia coli M15 cells. The recombinant OmpA protein containing His 6 residues was estimated to have a molecular weight of ∼38 kDa. In Western blot the native protein showed expression at ∼36 kDa molecular weight which was within the range of major outer membrane proteins (36–44 kDa) observed in this study. All E. tarda isolates tested harbored the ompA gene and the antibody raised to this protein was seen to cross react with other Gram negative bacteria. The OmpA protein characterized in this study was observed to be highly immunogenic in both rabbit and fish. In Enzyme linked immunosorbent assay, rabbit antisera showed an antibody titer of 1: 128,000. Common carp vaccinated with recombinant OmpA protein elicited high antibody production and immunized fish showed a relative percentage survival of 54.3 on challenge.

Pathogenic Potential of Edwardsiella tarda Strains Isolated from Turbot

In this study, pathogenicity of Edwardsiella tarda isolates from turbot Scophthalmus maximus as well as other strains from different hosts were tested against turbot, sole Solea senegalensis and sea bass Dicentrarchus labrax. In addition, the influence of challenge route and temperature in the pathogenic potential of E. tarda strains from turbot were also examined. The results obtained showed that E. tarda did not have host specificity in virulence and that the turbot isolates were highly virulent regardless of the inoculation route and temperature. Moreover, inoculation experiments performed in mice suggest that these isolates are virulent for mammals. All these findings suggest that this emergent turbot pathogen constitutes an important risk factor for the marine aquaculture.

Characterization of Edwardsiella tarda Isolated from Farm-Cultured Eels, Anguilla japonica, in the Republic of Korea

We surveyed the occurrence of edwardsiellosis on eel farms and investigated the characteristics of Edwardsiella tarda isolated from farm-cultured eels in the Republic of Korea. The occurrence rate of edwardsiellosis was 72% in the investigated samples. Among the edwardsiellosis cases, 46% were found to be mixed infections, with parasites and other kinds of bacteria. Some of the biochemical characteristics of the E. tarda isolates were different from those of the previously reported E. tarda isolated from several kinds of fish from different countries, especially in terms of hydrogen sulfide and indole production. The E. tarda isolated from the eels in the Republic of Korea had the characteristics of two biogroups, the wild-type biogroup and biogroup 1. The enzymatic activity of the E. tarda showed similar patterns to previously reported E. tarda strains and ATCC strains. This is the first it has been reported that E. tarda isolated from farm-cultured eels had some different biochemical characteristics from those of previously reported E. tarda isolated from several kinds of fish.

Edwardsiella tarda T6SS component evpP is regulated by esrB and iron, and plays essential roles in the invasion of fish

Edwardsiella tarda is a gram-negative pathogen for hemorrhagic septicemia in a broad range of hosts. The type VI secretion system (T6SS) has recently been dissected in E. tarda to secrete EvpC, EvpI and a novel effector protein EvpP. In this study, sequencing and genetic alignments showed that evpP genes from different E. tarda isolates were highly similar and an evpP homolog was also found in Aeromonas hydrophila 0865 isolated from a diseased eel, suggesting the possible lateral gene transfer of evpP or the whole T6SS gene island. With reporter strains carrying gfp gene fused to the evpP promoter region, flow cytometric analysis revealed that transcription of evpP was positively regulated by either the two-component system EsrA–EsrB in E. tarda or the iron concentration in media. Compared with the parental strain, in-frame deletion of evpP in E. tarda EIB202 led to the significantly increased 50% lethal doses in zebrafish ( Danio rerio) and Japanese flounder ( Paralichthys olivaceus), decreased hemolytic activities, failure to adhere to mucus and reduced serum resistance, and complementation of an intact evpP gene restored these phenotypes in the evpP mutant. Investigation of infection kinetics indicated that the evpP deletion mutant was unable to proliferate in vivo, particularly in immune organs of fish. Moreover, the evpP deletion mutant exhibited incapacity to internalize in EPC cell model in vitro, demonstrating that EvpP in T6SS plays critical roles for invasion mechanism of E. tarda and merits as potential target for attenuated live vaccine construction.

Isolation and identification of fish pathogenEdwardsiella tardafrom mariculture in China

The causative agent was isolated from diseased turbots (Scophthalmus maximus) stricken by a high-mortality outbreak of bacterial septicaemia occurring in a mariculture farm in Yantai, a northern coastal city of China. Seven pure isolates, namely EH-15, EH-103, EH-107, EH-202, EH-203, EH-305 and EH-306, belonged to Edwardsiella tarda. The phenotypic features of the cultures were analysed extensively. Three of the isolates showed high 16S rDNA sequence similarities with E. tarda sequence (GenBank accession no. EF467289). However, unlike the E. tarda ATCC 15947, all the isolates, except EH-15, contained a novel large plasmid sized about 23.7 kb. Furthermore, pathogenicity of the isolates was addressed by experimental challenges with fish models. The isolates exhibited strong virulence to swordtail fish with LD50 ranging between 3.8 × 103 and 3.8 × 105 CFU g−1, and EH-202 displaying the lowest LD50 value among them. Antibiotic susceptibilities of E. tarda isolates were assayed. Compared with E. tarda ATCC 15947, the isolates displayed strong resistance to chloramphenicol, and the probable dominant chloramphenicol resistance determinant was cat III. Depicting the main biological properties of turbot-borne E. tarda strains in China, the study provided useful information for further unveiling their pathogenic mechanisms.

The macrophage chemotactic activity of Edwardsiella tarda extracellular products

The chemoattractant capabilities of Edwardsiella tarda extracellular products (ECP) were investigated from two isolates, the virulent FL6-60 parent and less virulent RET-04 mutant. Chemotaxis and chemokinesis were assayed in vitro using blind well chambers with peritoneal macrophages obtained from Nile tilapia, Oreochromis niloticus, 5 days following squalene injection. Non-purified ECP derived from both isolates stimulated predominantly chemokinetic migration of macrophages. Additionally, the ECP were semi-purified by high pressure liquid chromatography. The FL6-60 parent ECP yielded higher molecular weight components than did the ECP from the RET-04 mutant. The chemotactic activity of the macrophages for both the FL6-60 parent and RET-04 mutant semi-purified ECP was increased over the non-purified ECP and overall migration was primarily chemotactic. Exposure to ECP derived from virulent and less virulent E. tarda isolates promoted chemokinetic movement of macrophages that may be involved in inflammatory responses of Nile tilapia to E. tarda infection.

Isolation and characterization of outer membrane proteins of Edwardsiella tarda and its application in immunoassays

Edwardsiella tarda is an important cause for hemorrhagic septicemia in fish and also for gastro intestinal infections in humans. Sodium lauryl sarcosinate extraction method was used for the isolation of outer membrane proteins (OMPs) from 16 isolates of E. tarda, recovered from snakeheads (Ophiocephalus punctatus). Sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles of OMPs from E. tarda isolates had one major and common protein band of 44 kilodalton (kDa) and 9 other variable protein bands. Rabbit polyclonal antibodies (RPAbs) against OMPs of E. tarda ET-7 detected mainly one common 44 kDa protein in all isolates by western blotting. RPAbs strongly reacted with all isolates of E. tarda by indirect enzyme linked immunosorbent assay (ELISA). These polyclonal antibodies showed no cross reactivity with OMPs or whole cells of other selected aquatic bacteria, i.e., Escherichia coli, Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio cholerae and Flavobacterium ferrugineum. This study indicated that RPAbs against OMPs is useful for detection of E. tarda by ELISA.

Base changes in the fliC gene of Edwardsiella tarda: possible effects on flagellation and motility

Edwardsiella tarda is a broad host-range pathogen infecting both animals and humans. E. tarda isolates from red sea bream Pagrus major are non-motile, whereas isolates from Japanese eel Anguilla japonica and Japanese flounder Paralichthys olivaceus are motile with peritrichous flagella. We compared the fliC gene coding for flagellin (FliC) in motile and non-motile E. tarda strains isolated from diseased fish. Twenty-two amino acid residues differed in the predicted FliC amino acid sequences between non-motile and motile strains. There were no significant differences either in the upstream sequences regulating transcription of the fliC gene or in the fliC transcript levels between motile and non-motile strains. The predicted secondary structure of FliC in non-motile E. tarda differed from that of motile strains, and the modeled data suggested that the secondary structure may be the important factor responsible for non-flagellation in the non-motile strains.

Occurrence of Edwardsiella tarda in wild European eels Anguilla anguilla from Mediterranean Spain

Pure cultures of Edwarsiella tarda were isolated from body ulcers and internal organs of wild European eels caught in a Mediterranean freshwater coastal lagoon (Albufera Lake, Valencia, Spain) over a 1 yr period. Overall, the E. tarda isolation rate from wild eels was 9%, but this increased to 22.8% in diseased individuals. All 22 E. tarda isolates belonged to the 'wild-type' biogroup of the species and were virulent for eels (lethal dose that kills 50% of exposed individuals [LD50 dose]: 10(4.85) to 10(6.83) CFU ind.(-1)), and therefore represented the aetiological agent of the haemorrhagic disease observed in wild European eels. The E. tarda isolates and E. tarda CECT 894T type strain were biochemically and serologically related and resistant to macrolides, antifolates, and glycopeptides, but only the isolates from wild eels were resistant to clindamicyn. This study is the first description of edwardsiellosis in a wild European eel population, and alerts us to the presence of E. tarda in natural wetland environments in Mediterranean Europe.

Intra- and interspecific phenotypic characteristics of fish-pathogenic Edwardsiella ictaluri and E. tarda

Intra- and interspecific characteristics of fish-pathogenic Edwardsiella ictaluri, and E. tarda were determined by numerical analysis of gel electrophoresed protein profiles, fatty acid methyl esters (FAMEs) and immunoblotting. The 18 E. ictaluri isolates revealed a high degree of homogeneity (70% similarity or higher) in their protein profiles and 95% similarity in their FAME, while the nine E. tarda isolates revealed 30% similarity in their protein profiles and 95% similarity in their FAME. Immunoblots probed for antigenic epitopes with goat antiserum produced against E. ictaluri and E. tarda, respectively, revealed that E. ictaluri were more homogeneous compared with the E. tarda isolates. Overall, there was a considerable degree of relatedness between the two species. Our findings suggest that phenotypically E. ictaluri represents a clonal bacterial population structure compared with the less monomorphic E. tarda.

Analysis of 16S-23S intergenic spacer regions of the rRNA operons in Edwardsiella ictaluri and Edwardsiella tarda isolates from fish

To analyse interspecies and intraspecies differences based on the 16S-23S rRNA intergenic spacer region (ISR) sequences of the fish pathogens Edwardsiella ictaluri and Edwardsiella tarda. The 16S-23S rRNA spacer regions of 19 Edw. ictaluri and four Edw. tarda isolates from four geographical regions were amplified by PCR with primers complementary to conserved sequences within the flanking 16S-23S rRNA coding sequences. Two products were generated from all isolates, without interspecies or intraspecific size polymorphisms. Sequence analysis of the amplified fragments revealed a smaller ISR of 350 bp, which contained a gene for tRNA(Glu), and a larger ISR of 441 bp, which contained genes for tRNA(Ile) and tRNA(Ala). The sequences of the smaller ISR of different Edw. ictaluri isolates were essentially identical to each other. Partial sequences of larger ISR from several Edw. ictaluri isolates also revealed no differences from the one complete Edw. ictaluri large ISR sequence obtained. The sequences of the smaller ISR of Edw. tarda were 97% identical to the Edw. ictaluri smaller ISR and the larger ISR were 96-98% identical to the Edw. ictaluri larger ISR sequence. The Edw. tarda isolates displayed limited ISR sequence heterogeneity, with > or =97% sequence identity among isolates for both small and large ISR. There is a high degree of size and sequence similarity of 16S-23S ISR both among isolates within Edw. ictaluri and Edw. tarda species and between the two species. Our results confirm a close genetic relationship between Edw. ictaluri and Edw. tarda and the relative homogeneity of Edw. ictaluri isolates compared with Edw. tarda isolates. Because no differences were found in ISR sequences among Edw. ictaluri isolates, sequence analysis of the ISR will not be useful to distinguish isolates of Edw. ictaluri. However, we identified restriction sites that differ between ISR sequences of Edw. ictaluri and Edw. tarda, which will be useful in distinguishing the two species.

Serotype and Siderophore Production of Edwardsiella tarda Isolated from Water at Eel Farms

Appearance of Edwardsiella tarda were monitored in pond water culturing eel Anguilla japonica in Kagoshima prefecture. E. tarda were isolated at almost all sampling times. Serotype and siderophore production of the E. tarda isolates, which are involved with its pathogenesis, were determined. Out of 370 isolates, 194 isolates (52%) were classified into serotype A and 246 isolates (66%) produced siderophore. Ninety-one percent of the isolates that were classified into serotype A produced siderophore;72%of those that produced siderophore were classified into serotype A. There was no cross linkage between serotype and siderophore production in E. tarda.

Detection of tetracycline-resistance determinants by multiplex polymerase chain reaction in Edwardsiella tarda isolated from fish farms in Korea

To determine and discriminate the types of tetracycline (Tc)-resistance determinants ( tet), we developed a multiplex polymerase chain reaction (PCR). With minimized numbers of primers derived from the variable and conserved regions of six different types of tet genes, tet(A)–(E) and (G), the multiplex PCR was sensitive and specific enough to discriminate the various types of tet genes, even multiple tet genes in an individual resistant isolate, by the different sizes of the resulting PCR products. Each of 20 Tc-resistant Edwardsiella tarda ( Ed. tarda) isolates from diseased fish from aquatic farms in Korea carrying either one or two tet genes of types (A), (D), (B), or (G) gave PCR products of the appropriate lengths. Among the four types of tet genes found in Ed. tarda, two types, tet(A) and (D), were always present on mobile plasmids, and the other two types, tet(B) and (G), were located on the nonmobile nucleic acids. This is the first time that either of these two genes, type (B) or (G), have been found in Ed. tarda isolates. The two most common types of Tc-resistance determinant were, tet(A) and (D), occurring in 55% and 45% of total Tc-resistant Ed. tarda isolates, respectively.