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1. Sakai  T, Kanai  K, Osatomi  K, Yoshikoshi  K,     ( 2003 )

Identification of a 19.3-kDa protein in MRHA-positive Edwardsiella tarda: putative fimbrial major subunit.

FEMS microbiology letters 226 (1)
PMID : 13129618  :   DOI  :   10.1016/S0378-1097(03)00608-6    
Abstract >>
The hemagglutinating properties of Edwardsiella tarda isolated from fish were investigated. Hemagglutination of E. tarda was not inhibited by D-mannose but was strongly inhibited by fetuin and N-acetylneuraminic acid. Extraction of hemagglutinating activity from bacterial cells was achieved using n-octyl-beta-D-thioglucoside (NOTG), and the NOTG extracts were fractionated by sucrose density gradient ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the fractions revealed that a 19.3-kDa protein band appeared in the fractions exhibiting highest hemagglutinating activity. In an immunoblot analysis of NOTG extracts from 18 strains of E. tarda, the 19.3-kDa protein was detected only in the extracts possessing hemagglutinating activity. The predicted amino acid sequence of a 534-bp gene encoding the 19.3-kDa protein was identical to fimbrial subunit (FimA) of E. tarda by FASTA homology search. These findings suggest that fimbriae are implicated in the hemagglutination of E. tarda.
KeywordMeSH Terms
2. Srinivasa Rao  PS, Yamada  Y, Leung  KY,     ( 2003 )

A major catalase (KatB) that is required for resistance to H2O2 and phagocyte-mediated killing in Edwardsiella tarda.

Microbiology (Reading, England) 149 (Pt 9)
PMID : 12949187  :   DOI  :   10.1099/mic.0.26478-0    
Abstract >>
Edwardsiella tarda causes haemorrhagic septicaemia in fish and gastro- and extra-intestinal infections in animals including humans. Resistance to phagocyte-mediated killing is one of the virulence factors of Ed. tarda. The authors' previous studies using TnphoA transposon mutagenesis indicated that katB mutants derived from the strain PPD130/91 are at least 1.6 log higher in LD50 values than the wild-type strain. These findings suggest the involvement of catalase (KatB) in Ed. tarda pathogenesis. In this study, experiments were conducted to characterize the contribution of KatB to Ed. tarda infection. Zymographic analyses indicated that the 22 Ed. tarda strains examined expressed three different types of catalase-peroxidases (Kat1-3) based on their mobility in non-denaturing polyacrylamide gels. KatB (Kat1), the major catalase enzyme, was expressed in eight out of 22 Ed. tarda strains, and was commonly found in virulent strains except AL9379. AL9379 has a mutated katB, which has a base substitution and a deletion that translate into stop codons in the catalase gene. KatB produced by PPD130/91 was located in both periplasmic and cytoplasmic fractions and was constitutively expressed in various growth phases. Kinetics studies indicated that the catalase provided resistance to H2O2- and phagocyte-mediated killing. Infection kinetics studies of katB mutant 34 in gourami fish demonstrated its inability to survive and replicate in phagocyte-rich organs and this prevented the dissemination of infections when compared to the wild-type. Complementation of catalase mutants restored the production of catalase, and led to an increase in the resistance to H2O2- and phagocyte-mediated killing, and a decrease in LD50 values. This study has identified and characterized a major catalase gene (katB) that is required for resistance to H2O2- and phagocyte-mediated killing in Ed. tarda. The results also suggest that catalase may play a role as a virulence factor in Ed. tarda pathogenesis.
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3. Srinivasa Rao  PS, Lim  TM, Leung  KY,     ( 2003 )

Functional genomics approach to the identification of virulence genes involved in Edwardsiella tarda pathogenesis.

Infection and immunity 71 (3)
PMID : 12595451  :   DOI  :   10.1128/iai.71.3.1343-1351.2003     PMC  :   PMC148833    
Abstract >>
Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also of gastro- and extraintestinal infections in humans. Here, we report the identification of 14 virulence genes of pathogenic E. tarda that are essential for disseminated infection, via a genome-wide analysis. We screened 490 alkaline phosphatase fusion mutants from a library of 450,000 TnphoA transconjugants derived from strain PPD130/91, using fish as an infection model. Compared to the wild type, 15 mutants showed significant decreases in virulence. Six mutants had insertions in the known virulence-related genes, namely, fimA, gadB, katB, pstS, pstC, and ssrB. Some mutants corresponded to known genes (astA, isor, and ompS2) that had not been previously shown to be involved in pathogenesis, and three had insertions in two novel genes. In vivo infection kinetics experiments confirmed the inability of these attenuated mutants to proliferate and cause fatal infection in fish. Screening for the presence of the above-described virulence genes in six virulent and seven avirulent strains of E. tarda indicated that seven of the genes were specific to pathogenic E. tarda. The genes identified here may be used to develop vaccines and diagnostic kits as well as for further studying the pathogenesis of E. tarda and other pathogenic bacteria.
KeywordMeSH Terms
Genome, Bacterial
4. Tan  YP, Lin  Q, Wang  XH, Joshi  S, Hew  CL, Leung  KY,     ( 2002 )

Comparative proteomic analysis of extracellular proteins of Edwardsiella tarda.

Infection and immunity 70 (11)
PMID : 12379732  :   DOI  :   10.1128/iai.70.11.6475-6480.2002     PMC  :   PMC130330    
Abstract >>
A comparison of extracellular proteins of virulent and avirulent Edwardsiella tarda strains revealed several major, virulent-strain-specific proteins. Proteomic analysis identified two of the proteins in the virulent strain PPD130/91 as flagellin and SseB, which are virulence factors in bacterial pathogens. PCR amplification and DNA sequencing confirmed the presence of the genes that encode these proteins. Our results clearly demonstrated the potency of the proteomic approach in identifying virulence factors.
KeywordMeSH Terms
Escherichia coli Proteins
Proteomics
5. Srinivasa Rao  PS, Lim  TM, Leung  KY,     ( 2001 )

Opsonized virulent Edwardsiella tarda strains are able to adhere to and survive and replicate within fish phagocytes but fail to stimulate reactive oxygen intermediates.

Infection and immunity 69 (9)
PMID : 11500445  :   DOI  :   10.1128/iai.69.9.5689-5697.2001     PMC  :   PMC98685    
Abstract >>
Edwardsiella tarda is responsible for hemorrhagic septicemia (edwardsiellosis) in fish and also causes diseases in higher vertebrates such as birds, reptiles, and mammals, including humans. Interactions of E. tarda with blue gourami phagocytes were studied by light microscopy as well as by adherence, intracellular replication, and superoxide anion assays. Both nonopsonized virulent (PPD130/91 and AL9379) and avirulent (PPD125/87 and PPD76/87) bacteria could adhere to and survive and replicate within phagocytes, while only opsonized virulent strains replicated within the phagocytes. Furthermore, only avirulent E. tarda elicited a higher rate of production of reactive oxygen intermediates (ROIs) by phagocytes, indicating that they were unable to avoid and/or resist reactive oxygen radical-based killing by the fish phagocytes. TnphoA transposon mutagenesis was used to construct a library of 200 alkaline phosphatase (PhoA+) fusion mutants from a total of 182,000 transconjugants derived from E. tarda PPD130/91. Five of these mutants induced more ROI production in phagocytes than the wild-type strain. Two mutants had lower replication ability inside phagocytes and moderately higher 50% lethal dose values than the wild-type strain. Sequence analysis revealed that three of these mutants had insertions at sequences having homology to PhoS, dipeptidase, and a surface polymer ligase of lipid A core proteins of other pathogens. These three independent mutations might have changed the cell surface characteristics of the bacteria, which in turn induced phagocytes to produce increased ROIs. Sequences from two other mutants had no homology to known genes, indicating that they may be novel genes for antiphagocytic killing. The present study showed that there are differences in the interactions of virulent and avirulent E. tarda organisms with fish phagocytes and PhoA+ fusion mutants that could be used successfully to identify virulence genes. The information elucidated here would help in the development of suitable strategies to combat the disease caused by E. tarda.
KeywordMeSH Terms
Perciformes
6. Zheng  J, Li  N, Tan  YP, Sivaraman  J, Mok  YK, Mo  ZL, Leung  KY,     ( 2007 )

EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD.

Microbiology (Reading, England) 153 (Pt 6)
PMID : 17526852  :   DOI  :   10.1099/mic.0.2006/004952-0    
Abstract >>
Edwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the 'translocon' of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by coils. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of DeltaescC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB-LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.
KeywordMeSH Terms
7. Salerno  A, Delétoile  A, Lefevre  M, Ciznar  I, Krovacek  K, Grimont  P, Brisse  S,     ( 2007 )

Recombining population structure of Plesiomonas shigelloides (Enterobacteriaceae) revealed by multilocus sequence typing.

Journal of bacteriology 189 (21)
PMID : 17693512  :   DOI  :   10.1128/JB.00796-07     PMC  :   PMC2168737    
Abstract >>
Plesiomonas shigelloides is an emerging pathogen that is widespread in the aquatic environment and is responsible for intestinal diseases and extraintestinal infections in humans and other animals. Virtually nothing is known about its genetic diversity, population structure, and evolution, which severely limits epidemiological control. We addressed these questions by developing a multilocus sequence typing (MLST) system based on five genes (fusA, leuS, pyrG, recG, and rpoB) and analyzing 77 epidemiologically unrelated strains from several countries and several ecological sources. The phylogenetic position of P. shigelloides within family Enterobacteriaceae was precisely defined by phylogenetic analysis of the same gene portions in other family members. Within P. shigelloides, high levels of nucleotide diversity (average percentage of nucleotide differences between strains, 1.49%) and genotypic diversity (64 distinct sequence types; Simpson's index, 99.7%) were found, with no salient internal phylogenetic structure. We estimated that homologous recombination in housekeeping genes affects P. shigelloides alleles and nucleotides 7 and 77 times more frequently than mutation, respectively. These ratios are similar to those observed in the naturally transformable species Streptococcus pneumoniae with a high rate of recombination. In contrast, recombination within Salmonella enterica, Escherichia coli, and Yersinia enterocolitica was much less frequent. P. shigelloides thus stands out among members of the Enterobacteriaceae. Its high rate of recombination results in a lack of association between genomic background and O and H antigenic factors, as observed for the 51 serotypes found in our sample. Given its robustness and discriminatory power, we recommend MLST as a reference method for population biology studies and epidemiological tracking of P. shigelloides strains.
KeywordMeSH Terms
8. Pham  HN, Ohkusu  K, Mishima  N, Noda  M, Monir Shah  M, Sun  X, Hayashi  M, Ezaki  T,     ( 2007 )

Phylogeny and species identification of the family Enterobacteriaceae based on dnaJ sequences.

Diagnostic microbiology and infectious disease 58 (2)
PMID : 17368802  :   DOI  :   10.1016/j.diagmicrobio.2006.12.019    
Abstract >>
Phylogenetic relations within the family Enterobacteriaceae were analyzed using partial dnaJ sequences of 165 strains belonging to 93 species from 27 enterobacterial genera. The dnaJ phylogeny was in relative agreement with that constructed by 16S rDNA sequences, but more monophyletic groups were obtained from the dnaJ tree than from the 16S rDNA tree. The degree of divergence of the dnaJ gene was approximately 6 times greater than that of 16S rDNA. Also, the dnaJ gene showed the most discriminatory power in comparison with tuf and atpD genes, facilitating clear differentiation of any 2 enterobacterial species by dnaJ sequence analysis. The application of dnaJ sequences to the identification was confirmed by assigning 72 clinical isolates to the correct enterobacterial species. Our data indicate that analysis of the dnaJ gene sequences can be used as a powerful marker for phylogenetic study and identification at the species level of the family Enterobacteriaceae.
KeywordMeSH Terms
HSP40 Heat-Shock Proteins
Phylogeny
9. Han  HJ, Kim  DH, Lee  DC, Kim  SM, Park  SI,     ( 2006 )

Pathogenicity of Edwardsiella tarda to olive flounder, Paralichthys olivaceus (Temminck & Schlegel).

Journal of fish diseases 29 (10)
PMID : 17026669  :   DOI  :   10.1111/j.1365-2761.2006.00754.x    
Abstract >>
The LD50 and cytotoxic and enzymatic activities of both cells and extracellular products (ECPs) of eight Edwardsiella tarda strains were determined and their bacterial superoxide dismutase gene (sodB) and catalase gene (katB) were sequenced. Strains were also examined for their ability to resist the immune responses of olive flounder, Paralichthys olivaceus. LD50 values of strains (FSW910410, KE1, 2, 3, 4, 5 and 6) in olive flounder ranged between 10(2.5) and 10(5.3) cfu (colony forming units) per fish. Unlike the avirulent strain SU100 (LD50>or=10(7)), all pathogenic strains were able to survive in flounder serum and head kidney leucocytes (except for KE2). The virulent strains possessed type I sodB and katB, whereas SU100 had type II sodB but not katB. However, there was no difference between avirulent and virulent strains in haemolytic and cytotoxic activities. The results of this study demonstrated that the ability of E. tarda to resist complement activity and phagocytosis is conferred by its superoxide dismutase and catalase, which thus play an essential role in the pathogenicity of this bacterium. In addition genotyping of sodB and kat B proved to be a very useful tool to distinguish virulent from avirulent strains.
KeywordMeSH Terms
Flounder
10. Delmas  J, Breysse  F, Devulder  G, Flandrois  JP, Chomarat  M,     ( 2006 )

Rapid identification of Enterobacteriaceae by sequencing DNA gyrase subunit B encoding gene.

Diagnostic microbiology and infectious disease 55 (4)
PMID : 16626902  :   DOI  :   10.1016/j.diagmicrobio.2006.02.003    
Abstract >>
Real-time polymerase chain reaction and sequencing were used to characterize a 506-bp-long DNA fragment internal to the gyrB gene (gyrBint). The sequences obtained from 32 Enterobacteriaceae-type strains and those available in the Genbank nucleotide sequence database (n = 24) were used as a database to identify 240 clinical enterobacteria isolates. Sequence analysis of the gyrBint fragment of 240 strains showed that gyrBint constitutes a discriminative target sequence to differentiate between Enterobacteriaceae species. Comparison of these identifications with those obtained by phenotypic methods (Vitek 1 system and/or Rapid ID 32E; bioM?rieux, Marcy l'Etoile, France) revealed discrepancies essentially with genera Citrobacter and Enterobacter. Most of the strains identified as Enterobacter cloacae by phenotypic methods were identified as Enterobacter hormaechei strains by gyrBint sequencing. The direct sequencing of gyrBint would be useful as a complementary tool in the identification of clinical Enterobacteriaceae isolates.
KeywordMeSH Terms
11. Zheng  J, Tung  SL, Leung  KY,     ( 2005 )

Regulation of a type III and a putative secretion system in Edwardsiella tarda by EsrC is under the control of a two-component system, EsrA-EsrB.

Infection and immunity 73 (7)
PMID : 15972502  :   DOI  :   10.1128/IAI.73.7.4127-4137.2005     PMC  :   PMC1168592    
Abstract >>
Edwardsiella tarda is a gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and gastro- and extraintestinal infections in humans. A type III secretion system (TTSS) and a putative secretion system (EVP) have been found to play important roles in E. tarda pathogenesis. Our previous studies suggested that the TTSS and EVP gene clusters were regulated by a two-component system of EsrA-EsrB. In the present study, we characterized another regulator, EsrC, which showed significant sequence similarity to the AraC family of transcriptional regulators. Mutants with in-frame deletions of esrC increased the 50% lethal doses in blue gourami fish, reduced extracellular protein production, and failed to aggregate. Complementation of esrC restored these three phenotypes. Two-dimensional gel electrophoresis showed that EsrC regulated the expression of secreted proteins encoded by the TTSS (such as EseB and EseD) and EVP (EvpC) gene clusters. The expression of esrC required a functional two-component system of EsrA-EsrB. EsrC in turn regulated the expression of selected genes encoded in TTSS (such as the transcriptional unit of orf29and orf30, but not esaC) and genes encoded in the EVP gene cluster. The present study sheds light on the regulation of these two key virulence-associated secretion systems and provides greater insight into the pathogenic mechanisms of this bacterium.
KeywordMeSH Terms
12. Morohoshi  T, Inaba  T, Kato  N, Kanai  K, Ikeda  T,     ( 2004 )

Identification of quorum-sensing signal molecules and the LuxRI homologs in fish pathogen Edwardsiella tarda.

Journal of bioscience and bioengineering 98 (4)
PMID : 16233705  :   DOI  :   10.1016/S1389-1723(04)00281-6    
Abstract >>
Edwardsiella tarda is a gram-negative bacterium that causes septicaemia in fish and serious damage to the aquaculture industry. The virulence factors of this pathogen and control mechanisms of the expression of virulence genes have not yet been clearly elucidated. A number of gram-negative pathogenic bacteria have a quorum-sensing system. These bacteria produce N-acyl-L-homoserine lactone (AHL) that they use them as a quorum-sensing signal molecule. In this study, we found that E. tarda isolated from deceased flounder produces AHLs. Thin layer chromatography analysis indicated that the two kinds of AHL produced by E. tarda seemed to be N-hexanoyl-L-homoserine lactone (C6-HSL) and N-heptanoyl-L-homoserine lactone (C7-HSL). We have cloned and sequenced the quorum-sensing genes, luxI homolog (edwI) and luxR homolog (edwR). EdwI and EdwR showed high identity with CarI/CarR and ExpI/ExpR from Erwinia carotovora, respectively. SDS-PAGE analysis of extracellular proteins revealed that the expression of the 55-kDa protein, which was reported as a virulent-strain-specific protein, is controlled by AHLs. These results suggest that some virulence factors are regulated by the quorum-sensing system in E. tarda.
KeywordMeSH Terms
13. Verjan  N, Hirono  I, Aoki  T,     ( 2005 )

Genetic loci of major antigenic protein genes of Edwardsiella tarda.

Applied and environmental microbiology 71 (9)
PMID : 16151172  :   DOI  :   10.1128/AEM.71.9.5654-5658.2005     PMC  :   PMC1214691    
Abstract >>
Seven antigenic proteins of Edwardsiella tarda were identified by using a rabbit polyclonal antiserum. Four of these proteins also reacted with a Japanese flounder antiserum. The amino acid sequences had identity to lipoproteins, periplasmic proteins, and exported and secreted proteins with roles in transport of metabolites across the cell membrane, stress response, and motility. These genes and their products are useful for developing DNA or recombinant subunit vaccines to control edwardsiellosis.
KeywordMeSH Terms
14. Liu  Y, Oshima  S, Kurohara  K, Ohnishi  K, Kawai  K,     ( 2005 )

Vaccine efficacy of recombinant GAPDH of Edwardsiella tarda against Edwardsiellosis.

Microbiology and immunology 49 (7)
PMID : 16034203  :   DOI  :   10.1111/j.1348-0421.2005.tb03652.x    
Abstract >>
Thirty-seven kilodalton glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Edwardsiella tarda was suggested to be an effective vaccine candidate against E. tarda infection in previous research. For developing a vaccine, obtaining GAPDH in large quantities is necessary. In this study, we determined the complete nucleotide sequence of the gene that encodes GAPDH of E. tarda, and overexpressed the GAPDH of E. tarda by using the Escherichia coli expression system. We immunized Japanese flounder with recombinant GAPDH (rGAPDH) and evaluated its vaccine efficacy. Our results showed that rGAPDH effectively protected Japanese flounder from experimental E. tarda infection, and will contribute to the development of a vaccine against E. tarda.
KeywordMeSH Terms
15. Tan  YP, Zheng  J, Tung  SL, Rosenshine  I, Leung  KY,     ( 2005 )

Role of type III secretion in Edwardsiella tarda virulence.

Microbiology (Reading, England) 151 (Pt 7)
PMID : 16000720  :   DOI  :   10.1099/mic.0.28005-0    
Abstract >>
Edwardsiella tarda is a Gram-negative enteric bacterium affecting both animals and humans. Recently, a type III secretion system (TTSS) was found in Ed. tarda. Such systems are generally used by bacterial pathogens to deliver virulence factors into host cells to subvert normal cell functions. Genome-walking was performed from the eseB and esrB genes (homologues of Salmonella sseB and ssrB, respectively) identified in previous studies, to determine the sequences of the TTSS. Thirty-five ORFs were identified which encode the TTSS apparatus, chaperones, effectors and regulators. Mutants affected in genes representing each category were generated and found to have decreased survival and growth in fish phagocytes. LD(50) values of the mutants were increased by at least 10-fold in comparison to those of the wild-type strain. The adherence and invasion rates of the esrA and esrB mutants were enhanced while those of the other mutants remained similar to the wild-type. The eseC and eseD mutants showed slight autoaggregation in Dulbecco's Modified Eagle Medium, whereas the rest of the mutants failed to autoaggregate. Regulation of the TTSS was found to involve the two-component regulatory system esrA-esrB. This study showed that the TTSS is important for Ed. tarda pathogenesis. An understanding of this system will provide greater insight into the virulence mechanisms of this bacterial pathogen.
KeywordMeSH Terms
16. Hill  JE, Penny  SL, Crowell  KG, Goh  SH, Hemmingsen  SM,     ( 2004 )

cpnDB: a chaperonin sequence database.

Genome research 14 (8)
PMID : 15289485  :   DOI  :   10.1101/gr.2649204     PMC  :   PMC509277    
Abstract >>
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.
KeywordMeSH Terms
17. Rao  PS, Yamada  Y, Tan  YP, Leung  KY,     ( 2004 )

Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis.

Molecular microbiology 53 (2)
PMID : 15228535  :   DOI  :   10.1111/j.1365-2958.2004.04123.x    
Abstract >>
Edwardsiella tarda is an important cause of haemorrhagic septicaemia in fish and also of gastro- and extraintestinal infections in humans. Using a combination of comparative proteomics and TnphoA mutagenesis, we have identified five proteins that may contribute to E. tarda PPD130/91 pathogenesis. Lowered protein secretion, impaired autoaggregation and the absence of six proteins were observed only in three highly attenuated mutants when cultured in Dulbecco's modified eagle medium (DMEM). Five out of six proteins could be identified by their mass spectra. Three proteins were identified as putative effector proteins (EseB, EseC and EseD) that are homologous to SseB, SseC and SseD of a type III secretion system (TTSS) in Salmonella species. The other two were EvpA and EvpC, homologous to Eip20 and Eip18 in Edwardsiella ictaluri. The complete sequencing and homology studies of evpA-H indicate that similar gene clusters are widely distributed in other pathogens such as Escherichia, Salmonella, Vibrio and Yersinia species with unknown functions. Insertional inactivation and deletion of evpB or evpC led to lower replication rates in gourami phagocytes, and reduced protein secretion and virulence in blue gourami. Complementation of these deletion mutants showed partial recovery in the above three phenotypes, indicating that these genes are vital for E. tarda pathogenesis. The transport of the EvpC protein may not use the TTSS in E. tarda. The expression of EvpA and EvpC as well as EseB, EseC and EseD was temperature dependent (suppressed at 37 degrees C), and disruption of esrB affected their expression. The present study identifies two possible secretion systems (TTSS and Evp) that are vital for E. tarda pathogenesis.
KeywordMeSH Terms
Proteomics
18. Maiti  B, Shetty  M, Shekar  M, Karunasagar  I, Karunasagar  I,     ( 2011 )

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

Microbiological research 167 (1)
PMID : 21482086  :   DOI  :   10.1016/j.micres.2011.02.002    
Abstract >>
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 ~38kDa. In Western blot the native protein showed expression at ~36kDa molecular weight which was within the range of major outer membrane proteins (36-44kDa) 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.
KeywordMeSH Terms
19. Li  GY, Li  J, Xiao  P, Guo  YH, Mo  ZL,     ( 2011 )

Detection of type III secretion gene as an indicator for pathogenic Edwardsiella tarda.

Letters in applied microbiology 52 (3)
PMID : 21219368  :   DOI  :   10.1111/j.1472-765X.2010.02984.x    
Abstract >>
To differentiate pathogenic and nonpathogenic Edwardsiella tarda strains based on the detection of type III secretion system (T3SS) gene using polymerase chain reaction (PCR). Primers were designed to amplify Edw. tarda T3SS component gene esaV, catalase gene katB, haemolysin gene hlyA and 16S rRNA gene as an internal positive control. Genomic DNAs were extracted using a commercial isolation kit from 36 Edw. tarda strains consisting of 18 pathogenic and 18 nonpathogenic strains, and 50 ng of each DNA was used as the template for PCR amplification. PCR was performed with a thermocycler (TaKaRa TP600) in a 25-�gl volume. Products of esaV were detected in all pathogenic strains, but not in nonpathogenic strains; katB was detected in all pathogenic strains and one of nonpathogenic strains; hlyA was not detected in any strains. The detection of esaV gene can be used for the assessment of pathogenic Edw. tarda strains. The strategy using T3SS gene as the virulence indicator provides a useful tool for the clinical assessment of pathogenic Edw. tarda strains and prediction of edwardsiellosis risk in fish culture environments.
KeywordMeSH Terms
20. Wang  X, Wang  Q, Yang  M, Xiao  J, Liu  Q, Wu  H, Zhang  Y,     ( 2011 )

QseBC controls flagellar motility, fimbrial hemagglutination and intracellular virulence in fish pathogen Edwardsiella tarda.

Fish & shellfish immunology 30 (3)
PMID : 21288493  :   DOI  :   10.1016/j.fsi.2011.01.019     DOI  :   10.1016/j.fsi.2011.01.019    
Abstract >>
The inter-kingdom communication with the mammalian hosts mediated by autoinducer-3 (AI-3)/epinephrine (Epi)/norepinephrine (NE), and transduced by two-component systems QseBC has recently been described. As a fish pathogen and opportunistic pathogen for human beings, Edwardsiella tarda develops surface structures such as flagellar and fimbriae to cause different hemagglutination phenotypes and serotypes and initiate pathogen-host recognition and invasion process. E. tarda survives within macrophages in fish using type III secretion system (TTSS). Here, the genes of E. tarda two-component system, qseB and qseC, were found to be co-transcribed. Phylogenetic analysis indicated that evolution of QseC strongly correlated to different host niches. Compared with the wild type and their complemented strains, �GqseB and �GqseC mutants exhibited significant impaired flagellar motilities. Mammalian Epi was able to stimuli the flagellar motility in E. tarda via QseBC. Hemagglutination caused by fimbriae was induced in �GqseB but repressed in �GqseC. Disruption of qseB or qseC down-regulated the intracellular expressions of TTSS elements EseB and EsaC, and impaired their intracellular survival capabilities as well as in vivo competitive abilities. Furthermore, in vitro tests indicated that expression of EseB was induced by Epi via QseBC. Our results revealed that the QseBC system modified the virulence-related surface structures (flagellum, fimbriae and secretion system) and that hormone might stimulate the virulence of the pathogen in fish.
KeywordMeSH Terms
Fimbriae, Bacterial
Hemagglutination
Fimbriae, Bacterial
Hemagglutination
21. Wang  X, Wang  Q, Yang  M, Xiao  J, Liu  Q, Wu  H, Zhang  Y,     ( 2011 )

QseBC controls flagellar motility, fimbrial hemagglutination and intracellular virulence in fish pathogen Edwardsiella tarda.

Fish & shellfish immunology 30 (3)
PMID : 21288493  :   DOI  :   10.1016/j.fsi.2011.01.019     DOI  :   10.1016/j.fsi.2011.01.019    
Abstract >>
The inter-kingdom communication with the mammalian hosts mediated by autoinducer-3 (AI-3)/epinephrine (Epi)/norepinephrine (NE), and transduced by two-component systems QseBC has recently been described. As a fish pathogen and opportunistic pathogen for human beings, Edwardsiella tarda develops surface structures such as flagellar and fimbriae to cause different hemagglutination phenotypes and serotypes and initiate pathogen-host recognition and invasion process. E. tarda survives within macrophages in fish using type III secretion system (TTSS). Here, the genes of E. tarda two-component system, qseB and qseC, were found to be co-transcribed. Phylogenetic analysis indicated that evolution of QseC strongly correlated to different host niches. Compared with the wild type and their complemented strains, �GqseB and �GqseC mutants exhibited significant impaired flagellar motilities. Mammalian Epi was able to stimuli the flagellar motility in E. tarda via QseBC. Hemagglutination caused by fimbriae was induced in �GqseB but repressed in �GqseC. Disruption of qseB or qseC down-regulated the intracellular expressions of TTSS elements EseB and EsaC, and impaired their intracellular survival capabilities as well as in vivo competitive abilities. Furthermore, in vitro tests indicated that expression of EseB was induced by Epi via QseBC. Our results revealed that the QseBC system modified the virulence-related surface structures (flagellum, fimbriae and secretion system) and that hormone might stimulate the virulence of the pathogen in fish.
KeywordMeSH Terms
Fimbriae, Bacterial
Hemagglutination
Fimbriae, Bacterial
Hemagglutination
22. Sun  Y, Liu  CS, Sun  L,     ( 2011 )

Comparative study of the immune effect of an Edwardsiella tarda antigen in two forms: subunit vaccine vs DNA vaccine.

Vaccine 29 (11)
PMID : 21255681  :   DOI  :   10.1016/j.vaccine.2011.01.013    
Abstract >>
Edwardsiella tarda is a Gram-negative bacterial pathogen and the etiological agent of a systematic fish disease called edwardsiellosis, which affects a wide range of marine and freshwater fish. E. tarda vaccines in various forms have been reported by a number of research groups; however, comparative studies on the immune mechanisms of these vaccines are lacking. In this report, we identified a new E. tarda vaccine candidate, Eta2, and analyzed in a comparative manner the immune response induced by Eta2 in two different forms: purified recombinant subunit vaccine and DNA vaccine. Eta2 is a protein of 178 residues and shares high levels of sequence identities with the OmpH family of outer membrane protein chaperones of several bacterial species. Recombinant Eta2 (rEta2) purified from Escherichia coli was highly protective against E. tarda challenge in a Japanese flounder (Paralichthys olivaceus) model and produced relative percent of survival rates of 83% and 78%, respectively, at 4- and 8-week post-vaccination (p.v.). Eta2 as a DNA vaccine in the form of plasmid pCEta2 also induced strong protective immunity at 4- and 8-week p.v. Immunological analysis indicated that (i) rEta2 and pCEta2 enhanced head kidney macrophage activation at 1- and, for pCEta2, 7-day p.v.; (ii) rEta2 and pCEta2 induced similar patterns of serum antibody production, however, the antibodies induced by rEta2 were of much higher levels and afforded stronger passive immunoprotection upon na?ve flounder than those induced by pCEta2; (iii) both rEta2 and pCEta2 upregulated the expression of specific and nonspecific immune factors which include, in the case of pCEta2 but not rEta2, interferon, interferon-induced Mx protein, and CD8�\; however, the induction patterns effected by rEta2 and pCEta2 were different. While high levels of interleukin 1�] (IL-1�]), natural killer cell enhancing factor, Mx, MHC I�\, and IgM inductions were observed in pCEta2-vaccinated fish, only IL-1�], complement C3, and IgM inductions were highly induced in rEta2-vaccinated fish. Taken together, these results indicate that both rEta2 and pCEta2 induce specific and nonspecific immunities, however, pCEta2 induces both B cell and T cell responses, whereas rEta2 induces mainly humoral response.
KeywordMeSH Terms
23. Kim  MS, Jun  LJ, Shin  SB, Park  MA, Jung  SH, Kim  K, Moon  KH, Jeong  HD,     ( 2010 )

Mutations in the gyrB, parC, and parE genes of quinolone-resistant isolates and mutants of Edwardsiella tarda.

Journal of microbiology and biotechnology 20 (12)
PMID : 21193831  :  
Abstract >>
The full length genes gyrB (2,415 bp), parC (2,277 bp), and parE (1,896 bp) in Edwardsiella tarda were cloned by PCR with degenerate primers based on the sequence of the respective quinolone resistance-determining region (QRDR), followed by elongation of 5' and 3' ends using cassette ligation-mediated PCR (CLMP). Analysis of the cloned genes revealed open reading frames (ORFs) encoding proteins of 804 (GyrB), 758 (ParC), and 631 (ParE) amino acids with conserved gyrase/topoisomerase features and motifs important for enzymatic function. The ORFs were preceded by putative promoters, ribosome binding sites, and inverted repeats with the potential to form cruciform structures for binding of DNA-binding proteins. When comparing the deduced amino acid sequences of E. tarda GyrB, ParC, and ParE with those of the corresponding proteins in other bacteria, they were found to be most closely related to Escherichia coli GyrB (87.6% identity), Klebsiella pneumoniae ParC (78.8% identity) and Salmonella typhimurium ParE (89.5% identity), respectively. The two topoisomerase genes, parC and parE, were found to be contiguous on the E. tarda chromosome. All 18 quinoloneresistant isolates obtained from Korea thus far did not contain subunit alternations apart from a substitution in GyrA (Ser83��Arg). However, an alteration in the QRDR of ParC (Ser84��Ile) following an amino acid substitution in GyrA (Asp87��Gly) was detected in E. tarda mutants selected in vitro at 8 microng/ml ciprofloxacin (CIP). A mutant with a GyrB (Ser464��Leu) and GyrA (Asp87��Gly) substitution did not show a significant increase in the minimum inhibitory concentration (MIC) of CIP. None of the in vitro mutants exhibited mutations in parE. Thus, gyrA and parC should be considered to be the primary and secondary targets, respectively, of quinolones in E. tarda.
KeywordMeSH Terms
Drug Resistance, Bacterial
24. Chakraborty  S, Li  M, Chatterjee  C, Sivaraman  J, Leung  KY, Mok  YK,     ( 2010 )

Temperature and Mg2+ sensing by a novel PhoP-PhoQ two-component system for regulation of virulence in Edwardsiella tarda.

The Journal of biological chemistry 285 (50)
PMID : 20937832  :   DOI  :   10.1074/jbc.M110.179150     PMC  :   PMC2998078    
Abstract >>
The PhoP-PhoQ two-component system is commonly used by bacteria to sense environmental factors. Here we show that the PhoP-PhoQ system of Edwardsiella tarda detects changes in environmental temperature and Mg(2+) concentration as well as regulates the type III and VI secretion systems through direct activation of esrB. Protein secretion is activated from 23 to 35 �XC or at low Mg(2+) concentrations, but it is suppressed at or below 20 �XC, at or above 37 �XC, or at high Mg(2+) concentrations. The effects of temperature and Mg(2+) concentration are additive. The PhoQ sensor domain has a low T(m) of 37.9 �XC, and it detects temperatures through a conformational change of its secondary structure. Mutation of specific Pro or Thr residues increased the stability of the PhoQ sensor drastically, altering its temperature-sensing ability. The PhoQ sensor detects Mg(2+) concentration through the direct binding of Mg(2+) to a cluster of acidic residues (DDDSAD) and through changes that likely affect its tertiary structure. Here, we describe for the first time the use of PhoP-PhoQ as a temperature sensor for bacterial virulence control.
KeywordMeSH Terms
25. Jobichen  C, Chakraborty  S, Li  M, Zheng  J, Joseph  L, Mok  YK, Leung  KY, Sivaraman  J,     ( 2010 )

Structural basis for the secretion of EvpC: a key type VI secretion system protein from Edwardsiella tarda.

PloS one 5 (9)
PMID : 20886112  :   DOI  :   10.1371/journal.pone.0012910     PMC  :   PMC2944823    
Abstract >>
The recently identified type VI secretion system (T6SS) is implicated in the virulence of many gram-negative bacteria. Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also gastro- and extra-intestinal infections in humans. The E. tardavirulent protein (EVP) gene cluster encodes a conserved T6SS which contains 16 open reading frames. EvpC is one of the three major EVP secreted proteins and shares high sequence similarity with Hcp1, a key T6SS virulence factor from Pseudomonas aeruginosa. EvpC contributes to the virulence of E. tarda by playing an essential role in functional T6SS. Here, we report the crystal structure of EvpC from E. tarda PPD130/91 at a 2.8 ? resolution, along with functional studies of the protein. EvpC has a �]-barrel domain with extended loops. The �]-barrel consists of 11 anti-parallel �]-strands with an �\-helix located on one side. In solution, EvpC exists as a dimer at low concentration and as a hexamer at higher concentration. In the crystal, the symmetry related EvpC molecules form hexameric rings which stack together to form a tube similar to Hcp1. Structure based mutagenesis revealed that N-terminal negatively charged residues, Asp4, Glu15 and Glu26, and C-terminal positively charged residues, Lys161, Lys162 and Lys163, played crucial roles in the secretion of EvpC. Moreover, the localization study indicates the presence of wild type EvpC in cytoplasm, periplasm and secreted fractions, whereas the N-terminal and C-terminal mutants were found mostly in the periplasmic region and was completely absent in the secreted fraction. Results reported here provide insight into the structure, assembly and function of EvpC. Further, these findings can be extended to other EvpC homologs for understanding the mechanism of T6SS and targeting T6SS mediated virulence in gram-negative pathogens.
KeywordMeSH Terms
26. Sun  Y, Liu  CS, Sun  L,     ( 2010 )

Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain.

Vaccine 28 (40)
PMID : 20673823  :   DOI  :   10.1016/j.vaccine.2010.07.050    
Abstract >>
Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of na?ve fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen.
KeywordMeSH Terms
27. Jiao  XD, Zhang  M, Cheng  S, Sun  L,     ( 2010 )

Analysis of Edwardsiella tarda DegP, a serine protease and a protective immunogen.

Fish & shellfish immunology 28 (4)
PMID : 20060910  :   DOI  :   10.1016/j.fsi.2010.01.004    
Abstract >>
Edwardsiella tarda is a severe aquaculture pathogen with a broad host range that includes humans, animal, and fish. A gene (degP(Et)) encoding a DegP homologue was cloned from TX01, a pathogenic E. tarda strain isolated from diseased fish. DegP(Et) shares high sequence identities with the DegP proteins of several bacterial species. Functional analyses showed that degP(Et) could complement the temperature-sensitive phenotype of an Escherichia coli degP null mutant. Expression of degP(Et) in TX01 was modulated by growth phase and temperature, the latter possibly through the action of the sigma(E)-like factor. Overexpression of degP(Et) (i) enhanced the ability of TX01 to disseminate in fish blood at the advanced stage of infection, (ii) heightened the activity of type 2 autoinducer, and (iii) increased the expression of luxS and the genes encoding components of the virulence-associated type III secretion system. Recombinant DegP(Et) purified from E. coli was a serine protease that exhibited maximum activity at 40 degrees C and pH8.0. The proteolytic activity of recombinant DegP(Et) depended on the catalytic triad and the PDZ domains. Immunoprotective analyses showed that purified recombinant DegP(Et) was a protective immunogen that could induce the production of specific serum antibodies and elicit strong protective immunity in fish vaccinated with DegP(Et).
KeywordMeSH Terms
28. Jiao  XD, Zhang  M, Hu  YH, Sun  L,     ( 2009 )

Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens.

Vaccine 27 (38)
PMID : 19596416  :   DOI  :   10.1016/j.vaccine.2009.06.071    
Abstract >>
Edwardsiella tarda is an opportunistic pathogen that can infect humans, animal, and fish. Two E. tarda antigens, Eta6 and FliC, which are homologues to an ecotin precursor and the FliC flagellin, respectively, were identified by in vivo-induced antigen technology from a pathogenic E. tarda strain isolated from diseased fish. When used as a subunit vaccine, purified recombinant Eta6 was moderately protective against lethal challenge of E. tarda in a Japanese flounder model, whereas purified recombinant FliC showed no apparent immunoprotectivity. Similarly, DNA vaccines based on eta6 and fliC in the form of plasmids pEta6 and pFliC induced, respectively, moderate and marginal protection against E. tarda infection. To improve the vaccine efficacy of eta6, a chimeric DNA vaccine, pCE6, was constructed, which encodes Eta6 fused in-frame to FliC. pCE6 was found to induce significantly higher level of protection than pEta6. Likewise, another chimeric DNA vaccine, pCE18, which expresses FliC fused to a previously identified E. tarda antigen Et18, elicited significantly stronger protective immunity than the DNA vaccine based on et18 alone. Fish immunized with pEta6 and pCE6 produced specific serum antibodies and exhibited significantly enhanced expression of the genes encoding elements that are involved in both innate and adaptive immune responses. Furthermore, the induction magnitudes of most of these genes were significantly higher in pCE6-vaccinated fish than in pEta6-vaccinated fish.
KeywordMeSH Terms
29. Wang  X, Wang  Q, Xiao  J, Liu  Q, Wu  H, Xu  L, Zhang  Y,     ( 2009 )

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

Fish & shellfish immunology 27 (3)
PMID : 19563898  :   DOI  :   10.1016/j.fsi.2009.06.013    
Abstract >>
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.
KeywordMeSH Terms
30. Sakai  T, Matsuyama  T, Sano  M, Iida  T,     ( 2009 )

Identification of novel putative virulence factors, adhesin AIDA and type VI secretion system, in atypical strains of fish pathogenic Edwardsiella tarda by genomic subtractive hybridization.

Microbiology and immunology 53 (3)
PMID : 19302523  :   DOI  :   10.1111/j.1348-0421.2009.00108.x    
Abstract >>
Edwardsiella tarda, which is known to be the causative agent of edwardsiellosis in freshwater and marine fish, has two motility phenotypes. Typical strains exhibiting motility are isolated mainly from freshwater fish and Japanese flounder. Atypical strains exhibiting non-motility are isolated mainly from marine fish, with the exception of Japanese flounder. Subtractive hybridization was performed to identify genomic differences between these two phenotypes. Two fragments which showed homology to potential virulence factors were isolated from atypical strains: the autotransporter adhesin AIDA and a component of T6SS. We analysed DNA sequences of about 5 kbp containing these fragments and identified two partial ORF, and ORF encoding for other components of T6SS. The predicted amino acid sequences showed remarkably low homology to components of T6SS reported in the typical E. tarda strain PPD130/91. Furthermore, the organization of these ORF was different from the gene cluster of the typical E. tarda strain. AIDA and T6SS may therefore be associated with different pathogenicity in typical and atypical E. tarda hosts.
KeywordMeSH Terms
31. Wang  F, Zhang  M, Hu  YH, Zhang  WW, Sun  L,     ( 2009 )

Regulation of the Edwardsiella tarda hemolysin gene and luxS by EthR.

Journal of microbiology and biotechnology 19 (8)
PMID : 19734713  :  
Abstract >>
Edwardsiella tarda is a pathogen with a broad host range that includes human and animals. The E. tarda hemolysin (Eth) system, which comprises EthA and EthB, is a noted virulence element that is widely distributed in pathogenic isolates of E. tarda. Previous study has shown that the expression of ethB is regulated by iron, which suggests the possibility that the ferric uptake regulator (Fur) is involved in the regulation of ethB. The work presented in this report supports the previous findings and demonstrates that ethB expression was decreased under conditions when the E. tarda Fur (Fur(Et)) was overproduced, and enhanced when FurEt was inactivated. We also identified a second ethB regulator, EthR, which is a transcription regulator of the GntR family. EthR represses ethB expression by direct interaction with the ethB promoter region. In addition to ethB, EthR also modulates, but positively, luxS expression and AI-2 production by binding to the luxS promoter region. The expression of ethR itself is subject to negative autoregulation; interference with this regulation by overexpressing ethR during the process of infection caused (i) drastic changes in ethB and luxS expressions, (ii) vitiation in the tissue dissemination and survival ability of the bacterium, and (iii) significant attenuation of the overall bacterial virulence. These results not only provide new insights into the regulation mechanisms of the Eth hemolysin and LuxS/AI-2 quorum sensing systems but also highlight the importance of these systems in bacterial virulence.
KeywordMeSH Terms
32. Morohoshi  T, Yokoyama  Y, Ouchi  M, Kato  N, Ikeda  T,     ( 2009 )

Motility and the expression of the flagellin protein FliC are negatively regulated by quorum sensing in Edwardsiella tarda.

Journal of bioscience and bioengineering 108 (4)
PMID : 19716521  :   DOI  :   10.1016/j.jbiosc.2009.04.006    
Abstract >>
Edwardsiella tarda is a gram-negative bacterial pathogen of fish and animals. A number of gram-negative bacteria have quorum-sensing systems and produce N-acylhomoserine lactone (AHL) that they use as a quorum-sensing signaling molecule. We have already reported that E. tarda NUF251 produces AHLs and has the AHL-synthase gene, edwI. Inactivation of NUF251 edwI induces expression of an approximately 45 kDa extracellular protein, identified as a flagellin encoded by FliC. Mutation of edwI also changes the motility pattern of NUF251 from a radial expansion pattern to concentric rings. The addition of exogenous AHL was capable of restoring normal motility to NUF251 edwI mutants. These results demonstrate that quorum sensing negatively regulates motility and expression of the FliC protein.
KeywordMeSH Terms
33. Xiao  J, Wang  Q, Liu  Q, Xu  L, Wang  X, Wu  H, Zhang  Y,     ( 2009 )

Characterization of Edwardsiella tarda rpoS: effect on serum resistance, chondroitinase activity, biofilm formation, and autoinducer synthetases expression.

Applied microbiology and biotechnology 83 (1)
PMID : 19283379  :   DOI  :   10.1007/s00253-009-1924-9    
Abstract >>
In this study, rpoS gene was identified from Edwardsiella tarda EIB202 and its functional role was analyzed by using an in-frame deletion mutant rpoS and the complemental strain rpoS (+). Compared with the wild type and rpoS (+), rpoS was impaired in terms of the ability to survive under oxidative stress and nutrient starvation, as well as the resistance to 50% serum of Scophthalmus maximus in 3 h, demonstrating essential roles of RpoS in stress adaptation. The rpoS mutant also displayed markedly increased chondroitinase activity and biofilm formation. Real-time polymerase chain reaction revealed that the expression level of quorum sensing autoinducer synthetase genes luxS and edwI was increased by 3.7- and 2.5-fold in the rpoS mutant strain. Those results suggested that rpoS might be involved in the negative or positive regulation of chondroitinase and biofilm formation, or quorum sensing networks in E. tarda, respectively. Although there were no obvious differences between the wild-type and the rpoS mutant in adherence of epithelioma papulosum cyprini (EPC) cell and in the lethality on fish model, rpoS deletion leads to the drastically reduced capacity for E. tarda to internalize in EPC cells, indicating that RpoS was, while not the main, the factor required for the virulence network of E. tarda.
KeywordMeSH Terms
Blood Bactericidal Activity
Quorum Sensing
34. Zhang  M, Sun  K, Sun  L,     ( 2008 )

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain.

Microbiology (Reading, England) 154 (Pt 7)
PMID : 18599834  :   DOI  :   10.1099/mic.0.2008/017343-0    
Abstract >>
Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (AI-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the AI-2 synthase was cloned from TX1 and designated luxS(Et)(.) LuxS(Et) was able to complement the AI-2 mutant phenotype of Escherichia coli strain DH5alpha. Expression of luxS(Et) correlated with AI-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced AI-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of AI-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous AI-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the AI-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/AI-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
35. Wang  F, Cheng  S, Sun  K, Sun  L,     ( 2008 )

Molecular analysis of the fur (ferric uptake regulator) gene of a pathogenic Edwardsiella tarda strain.

Journal of microbiology (Seoul, Korea) 46 (3)
PMID : 18604507  :   DOI  :   10.1007/s12275-008-0038-x    
Abstract >>
The gene encoding the Edwardsiella tarda ferric uptake regulator (Fur(Et)) was cloned from a pathogenic E. tarda strain isolated from diseased fish. Fur(Et) shares 90% overall sequence identity with the Escherichia coli Fur (Fur(Ec)) and was able to complement the mutant phenotype of a fur (Ec)-defective E. coli strain. Mutational analysis indicated that C92S and C95S mutations inactivated Fur(Et) whereas E112K mutation resulted in a superactive Fur(Et) variant. Fur(Et) negatively regulated its own expression; interruption of this regulation impaired bacterial growth, altered the production of certain outer membrane proteins, and attenuated bacterial virulence.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
36. Lan  J, Zhang  XH, Wang  Y, Chen  J, Han  Y,     ( 2008 )

Isolation of an unusual strain of Edwardsiella tarda from turbot and establish a PCR detection technique with the gyrB gene.

Journal of applied microbiology 105 (3)
PMID : 18341558  :   DOI  :   10.1111/j.1365-2672.2008.03779.x    
Abstract >>
The aim of this study was to report an unusual Edwardsiella tarda and develop an effective method to identify this bacterium. During the spring and summer of 2006, an epizootic occurred among cultured turbot (Scophthalmus maximus) in Qingdao, China. A gram-negative, rod-shaped bacterium (designated as LTB-4) was isolated from the infected fish, and was proved to be virulent to turbot. Based on the 16S rDNA sequencing and phenotypic tests, the bacterial pathogen was identified as E. tarda. Unlike those commonly described E. tarda strains, no flagellate was observed. Partial gyrB genes were amplified from E. tarda using the universal primers of gyrB genes and sequenced. The polymerase chain reaction (PCR) primers for the gyrB gene were designed specific to E. tarda. It revealed positive amplification of the gyrB fragment in E. tarda, whereas other bacterial species were negative. In addition, the technique enabled the recognition of E. tarda from diseased fish. The isolate was identified as E. tarda without flagellate and an effective method was developed to identify E. tarda based on using the gyrB gene as a taxonomic marker. The unusual E. tarda was first reported in China and the PCR allowed the rapid and sensitive detection of E. tarda.
KeywordMeSH Terms
37. Akinbowale  OL, Peng  H, Barton  MD,     ( 2007 )

Diversity of tetracycline resistance genes in bacteria from aquaculture sources in Australia.

Journal of applied microbiology 103 (5)
PMID : 17953612  :   DOI  :   10.1111/j.1365-2672.2007.03445.x    
Abstract >>
To determine the genetic determinants responsible for tetracycline resistance in oxytetracycline resistant bacteria from aquaculture sources in Australia. Twenty of 104 (19%) isolates tested were resistant to oxytetracycline (MIC > or = 16 microg ml(-1)). Using polymerase chain reaction (PCR) amplification, one or more tet genes were detected in 15/20 (75%) isolates tested, but none were found in 5/20 (25%). tetM (50%) was the most common determinant, followed by tetE (45%), tetA (35%) and tetD (15%). Five of 12 oxytetracycline resistant isolates studied were able to transfer their R-plasmid to Escherichia coli recipients of chicken, pig and human origin. tetA, tetD and tetM were found to be transferred while tetE was not transferred. Southern hybridization and PCR were used to confirm transfer of determinants. Bacterial isolates from aquaculture sources in Australia harbour a variety of tetracycline resistance genes, which can be transferred to other bacteria of different origin. Bacteria from aquaculture sources in Australia contribute to the resistance gene pool reservoir. The in vitro transfer of tetracycline R-plasmid from aquatic bacteria to E. coli isolates from various sources is an indication of the potential public health risk associated with these resistance determinants.
KeywordMeSH Terms
Aquaculture
Genes, MDR
Water Microbiology
38. Shao  S, Lai  Q, Liu  Q, Wu  H, Xiao  J, Shao  Z, Wang  Q, Zhang  Y,     ( 2015 )

Phylogenomics characterization of a highly virulent Edwardsiella strain ET080813(T) encoding two distinct T3SS and three T6SS gene clusters: Propose a novel species as Edwardsiella anguillarum sp. nov.

Systematic and applied microbiology 38 (1)
PMID : 25466920  :   DOI  :   10.1016/j.syapm.2014.10.008    
Abstract >>
As important zoonotic organisms causing infections in humans, Edwardsiella bacteria are also notorious leading fish pathogens haunting worldwide aquaculture industries. However, the taxa are now widely recognized to be misclassified, which hurdled the understanding of the epidemiology and development of effective diagnostics and vaccines. Currently the genus Edwardsiella consists of three species Edwardsiella tarda, E. ictaluri, and E. hoshinae. Previous phylogenomic analysis revealed that E. tarda strains display two major highly divergent genomic types (genotypes), EdwGI and EdwGII, and the former represents a genotype of fish-pathogenic isolates and being recently proposed as a novel species E. piscicida, sp. nov. Here multiple phylogenetic analyses and the genome-level comparisons of EdwGI strains disclose that the phylogroup strains from diseased eel formed an obviously distinct cluster that could be equated with a new species status. The phylogenetic evidence for the new species assignment was also supported by corresponding DNA-DNA hybridization estimation values and by phenotypic characteristics. Interestingly, further comparative genomics reveals that these strains have acquired the locus of enterocyte effacement (LEE) genes and as a result these bacteria contain at least 2 sets of distinct T3SS and 3 sets of T6SS gene clusters, respectively. It is therefore proposed that the phylogroup strains from diseased eel should be classified as Edwardisella anguillarum sp. nov., and the type strain is ET080813(T) (=DSM27202(T)=CCUG 64215(T)=CCTCC AB2013118(T)=MCCC 1K00238(T)). These findings will contribute to development of species-specific control measures against Edwardsiella bacterium in aquatic animals, while also shedding light on the pathogenesis evolution in Edwardsiella bacterium.
KeywordMeSH Terms
DDH
Edwardsiella anguillarum sp. nov.
MLST
Phylogenomics
Taxonomy
DDH
Edwardsiella anguillarum sp. nov.
MLST
Phylogenomics
Taxonomy
39. Xie  HX, Lu  JF, Zhou  Y, Yi  J, Yu  XJ, Leung  KY, Nie  P,     ( 2015 )

Identification and functional characterization of the novel Edwardsiella tarda effector EseJ.

Infection and immunity 83 (4)
PMID : 25667268  :   DOI  :   10.1128/IAI.02566-14     PMC  :   PMC4363441    
Abstract >>
Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and gastro- and extraintestinal infections in humans. The type III secretion system (T3SS) of E. tarda has been identified as a key virulence factor that contributes to pathogenesis in fish. However, little is known about the associated effectors translocated by this T3SS. In this study, by comparing the profile of secreted proteins of the wild-type PPD130/91 and its T3SS ATPase �GesaN mutant, we identified a new effector by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. This effector consists of 1,359 amino acids, sharing high sequence similarity with Orf29/30 of E. tarda strain EIB202, and is renamed EseJ. The secretion and translocation of EseJ depend on the T3SS. A �GeseJ mutant strain adheres to epithelioma papillosum of carp (EPC) cells 3 to 5 times more extensively than the wild-type strain does. EseJ inhibits bacterial adhesion to EPC cells from within bacterial cells. Importantly, the �GeseJ mutant strain does not replicate efficiently in EPC cells and fails to replicate in J774A.1 macrophages. In infected J774A.1 macrophages, the �GeseJ mutant elicits higher production of reactive oxygen species than wild-type E. tarda. The replication defect is consistent with the attenuation of the �GeseJ mutant in the blue gourami fish model: the 50% lethal dose (LD50) of the �GeseJ mutant is 2.34 times greater than that of the wild type, and the �GeseJ mutant is less competitive than the wild type in mixed infection. Thus, EseJ represents a novel effector that contributes to virulence by reducing bacterial adhesion to EPC cells and facilitating intracellular bacterial replication.
KeywordMeSH Terms
40. Yang  W, Wang  L, Zhang  L, Qu  J, Wang  Q, Zhang  Y,     ( 2015 )

An invasive and low virulent Edwardsiella tarda esrB mutant promising as live attenuated vaccine in aquaculture.

Applied microbiology and biotechnology 99 (4)
PMID : 25431010  :   DOI  :   10.1007/s00253-014-6214-5    
Abstract >>
Edwardsiella tarda is a leading fish pathogen haunting worldwide aquaculture industry. In E. tarda, two-component system EsrA-EsrB positively regulates type III and VI secretion systems (T3SS and T6SS) and negatively regulates hemolysin EthA, which has been demonstrated to be essential for the invasion processes in fish. In order to develop a live attenuated vaccine (LAV) with high invasiveness to be practically and economically used as immersion-administered vaccine in aquaculture, here, we generated a random mutation library of esrB sequences by error-prone PCR and introduced them into the E. tarda esrB deletion mutant. The mutant YWZ47 with significantly increased hemolytic activity and low T3SS and T6SS secretion was screened. Phenotypes including extracellular protein profiles, invasion in macrophages, lethality toward fish, and infection kinetics were investigated in the wild-type strain EIB202 and the mutants �GesrB, �GT3SS, �GT6SS, �GT3SS/�GT6SS, and YWZ47. Compared to the documented LAV strain �GesrB, YWZ47 showed higher invasive capability and low in vivo virulence toward fish. Significantly higher relative percent survival (RPS) could be generated in turbot (Scophthalmus maximus) against the challenge of the wild-type EIB202 when inoculated through immersion route, and the RPS was comparable with that of �GesrB through intraperitoneal (i.p.) injection inoculation. Two mutated points, K167M and H197L, were found by sequence analysis of EsrBYWZ47 variant. These structural modifications underpin the variations in the regulatory functions of the mutant and wild-type EsrB. This study promoted understanding of virulence regulation by EsrB in E. tarda and presented a promising candidate of invasive attenuated vaccine used in aquaculture industries.
KeywordMeSH Terms
Mutation
41. Griffin  MJ, Ware  C, Quiniou  SM, Steadman  JM, Gaunt  PS, Khoo  LH, Soto  E,     ( 2014 )

Edwardsiella piscicida identified in the Southeastern USA by gyrB sequence, species-specific and repetitive sequence-mediated PCR.

Diseases of aquatic organisms 108 (1)
PMID : 24492051  :   DOI  :   10.3354/dao02687     DOI  :   10.3354/dao02687    
Abstract >>
A new Edwardsiella taxon was recently described from fishes of Europe and Asia. Phenotypically similar to E. tarda, extensive genetic and phenotypic characterization determined this new strain does not belong to any established Edwardsiella taxa, leading to the adoption of a new taxon, E. piscicida. Concurrent research in the USA also identified 2 genetically distinct taxa within the group of organisms traditionally classified as E. tarda. Comparisons of gyrB sequences between US isolates and E. piscicida from Europe and Asia identified several US isolates with >99.6% similarity to the gyrB sequence of the E. piscicida type strain (ET883) but <87% similarity to the E. tarda type strain (ATCC #15947). A discriminatory PCR was developed for the identification of E. tarda and 2 genetic variants of E. piscicida (E. piscicida and E. piscicida-like species). Using these PCR assays, a survey was conducted of 44 archived bacterial specimens from disease case submissions to the Aquatic Research and Diagnostic Laboratory (Stoneville, MS, USA) between 2007 and 2012. All 44 isolates, originally identified phenotypically and biochemically as E. tarda, were identified as E. piscicida by PCR. Repetitive sequence-mediated PCR (rep-PCR) analysis of these archived specimens suggests they are largely homogenous, similar to what has been observed for E. ictaluri. The gyrB sequence data, coupled with the E. piscicida specific-PCR and rep-PCR data, confirms that E. piscicida has been isolated from fish disease cases in the southeastern USA. Moreover, our survey data suggests E. piscicida may be more prevalent in catfish aquaculture than E. tarda.
KeywordMeSH Terms
42. Griffin  MJ, Ware  C, Quiniou  SM, Steadman  JM, Gaunt  PS, Khoo  LH, Soto  E,     ( 2014 )

Edwardsiella piscicida identified in the Southeastern USA by gyrB sequence, species-specific and repetitive sequence-mediated PCR.

Diseases of aquatic organisms 108 (1)
PMID : 24492051  :   DOI  :   10.3354/dao02687     DOI  :   10.3354/dao02687    
Abstract >>
A new Edwardsiella taxon was recently described from fishes of Europe and Asia. Phenotypically similar to E. tarda, extensive genetic and phenotypic characterization determined this new strain does not belong to any established Edwardsiella taxa, leading to the adoption of a new taxon, E. piscicida. Concurrent research in the USA also identified 2 genetically distinct taxa within the group of organisms traditionally classified as E. tarda. Comparisons of gyrB sequences between US isolates and E. piscicida from Europe and Asia identified several US isolates with >99.6% similarity to the gyrB sequence of the E. piscicida type strain (ET883) but <87% similarity to the E. tarda type strain (ATCC #15947). A discriminatory PCR was developed for the identification of E. tarda and 2 genetic variants of E. piscicida (E. piscicida and E. piscicida-like species). Using these PCR assays, a survey was conducted of 44 archived bacterial specimens from disease case submissions to the Aquatic Research and Diagnostic Laboratory (Stoneville, MS, USA) between 2007 and 2012. All 44 isolates, originally identified phenotypically and biochemically as E. tarda, were identified as E. piscicida by PCR. Repetitive sequence-mediated PCR (rep-PCR) analysis of these archived specimens suggests they are largely homogenous, similar to what has been observed for E. ictaluri. The gyrB sequence data, coupled with the E. piscicida specific-PCR and rep-PCR data, confirms that E. piscicida has been isolated from fish disease cases in the southeastern USA. Moreover, our survey data suggests E. piscicida may be more prevalent in catfish aquaculture than E. tarda.
KeywordMeSH Terms
43. Shetty  M, Maiti  B, Venugopal  MN, Karunasagar  I, Karunasagar  I,     ( 2014 )

First isolation and characterization of Edwardsiella tarda from diseased striped catfish, Pangasianodon hypophthalmus (Sauvage).

Journal of fish diseases 37 (3)
PMID : 24344765  :   DOI  :   10.1111/jfd.12039    
Abstract >>
N/A
KeywordMeSH Terms
Edwardsiella tarda
Pangasianodon hypophthalmus
enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction
insertion sequence
moribund catfish
pathogenicity assay
phylogenetic analysis
β-lactamase
Edwardsiella tarda
Pangasianodon hypophthalmus
enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction
insertion sequence
moribund catfish
pathogenicity assay
phylogenetic analysis
β-lactamase
Catfishes
44. Yang  M, Lv  Y, Xiao  J, Wu  H, Zheng  H, Liu  Q, Zhang  Y, Wang  Q,     ( 2012 )

Edwardsiella comparative phylogenomics reveal the new intra/inter-species taxonomic relationships, virulence evolution and niche adaptation mechanisms.

PloS one 7 (5)
PMID : 22590641  :   DOI  :   10.1371/journal.pone.0036987     PMC  :   PMC3349661    
Abstract >>
Edwardsiella bacteria are leading fish pathogens causing huge losses to aquaculture industries worldwide. E. tarda is a broad-host range pathogen that infects more than 20 species of fish and other animals including humans while E. ictaluri is host-adapted to channel catfish causing enteric septicemia of catfish (ESC). Thus, these two species consist of a useful comparative system for studying the intricacies of pathogen evolution. Here we present for the first time the phylogenomic comparisons of 8 genomes of E. tarda and E. ictaluri isolates. Genome-based phylogenetic analysis revealed that E. tarda could be separate into two kinds of genotypes (genotype I, EdwGI and genotype II, EdwGII) based on the sequence similarity. E. tarda strains of EdwGI were clustered together with the E. ictaluri lineage and showed low sequence conservation to E. tarda strains of EdwGII. Multilocus sequence analysis (MLSA) of 48 distinct Edwardsiella strains also supports the new taxonomic relationship of the lineages. We identified the type III and VI secretion systems (T3SS and T6SS) as well as iron scavenging related genes that fulfilled the criteria of a key evolutionary factor likely facilitating the virulence evolution and adaptation to a broad range of hosts in EdwGI E. tarda. The surface structure-related genes may underlie the adaptive evolution of E. ictaluri in the host specification processes. Virulence and competition assays of the null mutants of the representative genes experimentally confirmed their contributive roles in the evolution/niche adaptive processes. We also reconstructed the hypothetical evolutionary pathway to highlight the virulence evolution and niche adaptation mechanisms of Edwardsiella. This study may facilitate the development of diagnostics, vaccines, and therapeutics for this under-studied pathogen.
KeywordMeSH Terms
Edwardsiella ictaluri
Edwardsiella tarda
Evolution, Molecular
Genome, Bacterial
Genotype
Phylogeny
45. Sun  Y, Zheng  WJ, Hu  YH, Sun  BG, Sun  L,     ( 2012 )

Edwardsiella tarda Eta1, an in vivo-induced antigen that is involved in host infection.

Infection and immunity 80 (8)
PMID : 22585967  :   DOI  :   10.1128/IAI.00063-12     PMC  :   PMC3434574    
Abstract >>
Edwardsiella tarda, a Gram-negative bacterium, is a severe fish pathogen that can also infect humans. In this study, we identified, via in vivo-induced antigen technology, an E. tarda antigen, Eta1, and analyzed its function in a Japanese flounder (Paralichthys olivaceus) model. Eta1 is composed of 226 residues and shares homology with putative bacterial adhesins. Quantitative real-time reverse transcriptase (RT)-PCR analysis indicated that when cultured in vitro, eta1 expression was growth phase dependent and reached maximum at mid-logarithmic phase. During infection of flounder lymphocytes, eta1 expression was drastically increased at the early stage of infection. Compared to the wild type, the eta1-defective mutant, TXeta1, was unaffected in growth but exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, and impaired ability to invade flounder lymphocytes and to block the immune response of host cells. The lost virulence of TXeta1 was restored when a functional eta1 gene was reintroduced into the strain. Western blot and immunodetection analyses showed that Eta1 is localized to the outer membrane and exposed on the surface of E. tarda and that recombinant Eta1 (rEta1) was able to interact with flounder lymphocytes. Consistent with these observations, antibody blocking of Eta1 inhibited E. tarda infection at the cellular level. Furthermore, when used as a subunit vaccine, rEta1 induced strong protective immunity in flounder against lethal E. tarda challenge. Taken together, these results indicate that Eta1 is an in vivo-induced antigen that mediates pathogen-host interaction and, as a result, is required for optimal bacterial infection.
KeywordMeSH Terms
Flounder
46. Abayneh  T, Colquhoun  DJ, Sørum  H,     ( 2012 )

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

Veterinary microbiology 158 (3��4��)
PMID : 22464156  :   DOI  :   10.1016/j.vetmic.2012.03.006     DOI  :   10.1016/j.vetmic.2012.03.006    
Abstract >>
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.
KeywordMeSH Terms
Genetic Variation
Phylogeny
Genetic Variation
Phylogeny
47. Abayneh  T, Colquhoun  DJ, Sørum  H,     ( 2012 )

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

Veterinary microbiology 158 (3��4��)
PMID : 22464156  :   DOI  :   10.1016/j.vetmic.2012.03.006     DOI  :   10.1016/j.vetmic.2012.03.006    
Abstract >>
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.
KeywordMeSH Terms
Genetic Variation
Phylogeny
Genetic Variation
Phylogeny
48. Yu  JE, Cho  MY, Kim  JW, Kang  HY,     ( 2012 )

Large antibiotic-resistance plasmid of Edwardsiella tarda contributes to virulence in fish.

Microbial pathogenesis 52 (5)
PMID : 22342431  :   DOI  :   10.1016/j.micpath.2012.01.006    
Abstract >>
Edwardsiella tarda, an enteric gram negative bacterium, infects a wide range of fish and causes a systemic fish disease called edwardsiellosis. E. tarda CK41, isolated from Japanese flounder diagnosed with edwardsiellosis, has exhibited a high degree of resistance to multiple antibiotics, including kanamycin, tetracycline, streptomycin, among others. As the bacterial antibiotic-resistance genes are usually contained in plasmids, we hypothesized that E. tarda CK41 may harbor one or more plasmids for antibiotic resistance. We showed the existence of plasmids in E. tarda CK41, and the size of the plasmid, designated as pCK41, was estimated to be approximately 70 kb. Escherichia coli DH5�\ transformed by the pCK41 plasmid exhibited an antibiotic-resistance phenotype against kanamycin (30 �gg/mL), tetracycline (30 �gg/mL), and streptomycin (10 �gg/mL), indicating the existence of at least 3 antibiotic-resistance genes in pCK41. Through a procedure for pCK41 plasmid curing, a plasmid-cured strain, designated as E. tarda CK108, was identified, which was unable to grow in the presence of either kanamycin or tetracycline. As virulence-associated genes are occasionally encoded in bacterial plasmids, we examined the virulence of E. tarda CK108 in Japanese flounder. The virulence of plasmid-cured E. tarda CK108 was lower (survival rate 80%) than that of CK41 (20%), indicating the existence of virulence-associated genes in pCK41. The strain also appeared to be attenuated in both goldfish and zebrafish pathogenesis models. To analyze genes for antibiotic resistance and virulence in pCK41, the entire nucleotide sequences of pCK41 were determined (GenBank accession number: HQ332785). A total of 84 open reading frames (ORFs) were annotated. The pCK41 plasmid consists of potential virulence genes, transposases, plasmid maintenance genes, antibiotic-resistance genes (including kanamycin, tetracycline, and streptomycin), conjugal transfer genes, and unknown ORFs. These results suggest that pCK41 is a virulence plasmid of substantial importance in the E. tarda pathogenesis to fish.
KeywordMeSH Terms
Drug Resistance, Bacterial
49. Chakraborty  S, Sivaraman  J, Leung  KY, Mok  YK,     ( 2011 )

Two-component PhoB-PhoR regulatory system and ferric uptake regulator sense phosphate and iron to control virulence genes in type III and VI secretion systems of Edwardsiella tarda.

The Journal of biological chemistry 286 (45)
PMID : 21953460  :   DOI  :   10.1074/jbc.M111.295188     PMC  :   PMC3234765    
Abstract >>
Inorganic phosphate (P(i)) and iron are essential nutrients that are depleted by vertebrates as a protective mechanism against bacterial infection. This depletion, however, is sensed by some pathogens as a signal to turn on the expression of virulence genes. Here, we show that the PhoB-PhoR two-component system senses changes in P(i) concentration, whereas the ferric uptake regulator (Fur) senses changes in iron concentration in Edwardsiella tarda PPD130/91 to regulate the expression of type III and VI secretion systems (T3SS and T6SS) through an E. tarda secretion regulator, EsrC. In sensing low P(i) concentration, PhoB-PhoR autoregulates and activates the phosphate-specific transport operon, pstSCAB-phoU, by binding directly to the Pho box in the promoters of phoB and pstS. PhoB also binds with EsrC simultaneously on the promoter of an E. tarda virulence protein, evpA, to regulate directly the transcription of genes from T6SS. In addition, PhoB requires and interacts with PhoU to activate esrC and suppress fur indirectly through unidentified regulators. Fur, on the other hand, senses high iron concentration and binds directly to the Fur box in the promoter of evpP to inhibit EsrC binding to the same region. In addition, Fur suppresses transcription of phoB, pstSCAB-phoU, and esrC indirectly via unidentified regulators, suggesting negative cross-talk with the Pho regulon. Physical interactions exist between Fur and PhoU and between Fur and EsrC. Our findings suggest that T3SS and T6SS may carry out distinct roles in the pathogenicity of E. tarda by responding to different environmental factors.
KeywordMeSH Terms
Bacterial Secretion Systems
50.     ( 1997 )

Iron-regulated haemolysin gene from Edwardsiella tarda.

Molecular microbiology 24 (4)
PMID : 9194711  :   DOI  :   10.1046/j.1365-2958.1997.3971760.x    
Abstract >>
We have cloned and sequenced the haemolysin gene locus from Edwardsiella tarda (ETH). This region encoded two open reading frames, designated ethA and ethB. ethA is the haemolysin gene consisting of 4782bp encoding a product of 165.3 kDa and ethB is an activation/secretion protein gene of 1677bp that encodes a product of 61.9 kDa. There were two putative ferric uptake regulator (Fur) binding sites on the 5' upstream region of the ethB gene overlapping the promoter region and ribosome-binding site. The haemolysin produced by the cloned gene was secreted by Escherichia coli. The deduced amino acid sequences of the ethA and ethB genes were found to be homologous to those of the haemolysin and activation/secretion proteins of Haemophilus ducreyi, Proteus mirabilis, and Serratia marcescens. E. coli carrying the ethA gene but not the ethB gene completely lost haemolytic activity, although the ethA gene was transcribed. The protein expressed by E. coli carrying a recombinant plasmid which encoded the ethA gene had haemagglutination activity. The EthB protein was necessary for activation of EthA protein (haemolysin). The ethA and ethB genes were very prevalent in haemolytic E. tarda strains isolated from diseased fish. Transcription of the ethB gene was regulated by iron. The ethA and ethB genes were transcribed independently.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
51.     ( 1996 )

Molecular cloning, characterization, and sequencing of the hemolysin gene from Edwardsiella tarda.

Archives of microbiology 165 (1)
PMID : 8639026  :  
Abstract >>
Hemolysis is a major symptom of diseased eels infected by Edwardsiella tarda. The hemolysin gene of E. tarda strain ET16 was cloned into plasmid pSK and expressed in Escherichia coli. The mol. mass of the functional beta-hemolysin was estimated to be approximately 34 kDa by gel filtration and by SDS-PAGE followed by in situ hemolysin activity analysis. The cloned fragment containing the beta-hemolysin locus from E. tarda strain ET16 expressed in E. coli was estimated to be 5.3 kb in length; the deduced gene product was identical in mol. mass and properties to the extracellular products of E. tarda strain ET16. The presence of EcoRI and XbaI sites within the beta-hemolysin gene of E. tarda was determined from the loss of hemolytic activity in subclones. Analysis of the DNA sequence of a 2,436-bp HaeIII-HindIII fragment that included EcoRI and XbaI sites revealed three ORFs organized as an operon that encoded three predicted polypeptides of 15,874, 7,055, and 34,804 Da. A 34-kDa polypeptide expressing hemolytic activity in cell lysates of the clone DH5 alpha(pETH3E) is very likely the beta-hemolysin encoded by the third ORF. The observation that hemolytic activity appeared in the culture medium of E. tarda, but not in that of E. coli strain DH5 alpha(pETH3E) indicates the existence of a mechanism for transporting the hemolysin across the cell envelope in E. tarda that is different from that of E. coli.
KeywordMeSH Terms
Genes, Bacterial
52. Harms  E, Hsu  JH, Subrahmanyam  CS, Umbarger  HE,     ( 1985 )

Comparison of the regulatory regions of ilvGEDA operons from several enteric organisms.

Journal of bacteriology 164 (1)
PMID : 3900037  :   PMC  :   PMC214231    
Abstract >>
The nucleotide sequence preceding the ilvGEDA operon has been examined and compared in five enteric organisms. The sequence in Escherichia coli B was identical to the earlier-described strain K-12 sequence. The sequences of Salmonella typhimurium and Klebsiella aerogenes were remarkably similar to that of E. coli and identical in that part of the leader region that specified the putative 32-amino-acid peptide. Thus, identical secondary structures could be postulated for the leaders of all three organisms, and regulation of operon expression could be like that postulated earlier for E. coli. Different secondary structures had to be postulated for the leader transcripts of Edwardsiella tarda and Serratia marcescens. Control of attenuation of the operon in these organisms by the level of leucyl tRNA could be explained only if ribosome stalling occurred at a single leucine codon. In both organisms, that single leucine codon is the rarely used CUA rather than the CUG that is in E. coli, S. typhimurium, and K. aerogenes.
KeywordMeSH Terms
Genes, Regulator
Operon
53. Miniero Davies  Y, Xavier de Oliveira  MG, Paulo Vieira Cunha  M, Soares Franco  L, Pulecio Santos  SL, Zanolli Moreno  L, Túlio de Moura Gomes  V, Zanolli Sato  MI, Schiavo Nardi  M, Micke Moreno  A, Becker Saidenberg  A, Rose Marques de Sá  L, Knöbl  T,     ( 2018 )

Edwardsiella tarda outbreak affecting fishes and aquatic birds in Brazil.

The Veterinary quarterly 38 (1)
PMID : 30668277  :   DOI  :   10.1080/01652176.2018.1540070    
Abstract >>
Edwardsiella tarda infections are frequent causes of severe outbreaks in the fish farming industry besides representing possible zoonotic risks. However, naturally occurring outbreaks that affect various species besides fishes are seldom described. To report an outbreak of acute mortality caused by E. tarda affecting multiple species that inhabited a natural pond in the state of S?o Paulo, Brazil. Three adult tilapias, three Mallard ducks and one Snow egret were necropsied and subjected to further microbiological tests. Gross and microscopic lesions were documented. The antibiotic susceptibility and phylogenetic similarities among fish and avian strains were also determined. The E. tarda species was confirmed through MALDI-TOF, partial sodB sequencing and phylogenetic analysis. Macroscopical findings between the three species included intestinal dilatation, mucosal hyperaemia and mucous to liquid contents. Common histopathology findings included acute enteritis, increased number of intraepithelial lymphocytes with bacteria adhered to the intestinal epithelium and lymphoid depletion in the spleen. E. tarda was isolated from several organs from all affected species. The phylogeny employing amplified fragment length polymorphism (AFLP) of eleven strains revealed high similarity (>90%) among the isolates regardless of the affected species or sampled organs. Ten isolates of E. tarda showed susceptibility to all tested antibiotics. E. tarda was identified as the cause of death of the species examined. Further studies would be necessary to determine the virulence of these strains and the possible risks regarding public health.
KeywordMeSH Terms
birds
edwardsiellosis
enterobacteria
fishes
public health
birds
edwardsiellosis
enterobacteria
fishes
public health
54.     ( 2013 )

Attempt to develop live attenuated bacterial vaccines by selecting resistance to gossypol, proflavine hemisulfate, novobiocin, or ciprofloxacin.

Vaccine 31 (18)
PMID : 23499519  :   DOI  :   10.1016/j.vaccine.2013.03.004    
Abstract >>
In an attempt to develop attenuated bacteria as potential live vaccines, four chemicals (gossypol, proflavine hemisulfate, novobiocin, and ciprofloxacin) were used to modify the following four genera of bacteria through chemical-resistance strategy: (1) Aeromonas hydrophila (9 isolates); (2) Edwardsiella tarda (9 isolates); (3) Streptococcus iniae (9 isolates); and (4) S. agalactiae (11 isolates). All bacteria used in this study were able to develop high resistance to gossypol. However, only some bacteria were able to develop resistance to proflavine hemisulfate, novobiocin, or ciprofloxacin. When the virulence of resistant bacteria was tested in tilapia or catfish, none of the gossypol-resistant isolate was attenuated, whereas majority of the proflavine hemisulfate-resistant isolates were attenuated. However, all proflavine hemisulfate-attenuated bacteria failed to provide significant protection to fish. Eight novobiocin- or ciprofloxacin-resistant Gram-positive bacteria (S. agalactiae and S. inaie) were found to be attenuated. However, none of them offered protection higher than 70%. Of seven attenuated novobiocin- or ciprofloxacin-resistant Gram-negative isolates (A. hydrophila and E. tarda), only one (novobiocin-resistant E. tarda 30305) was found to safe and highly efficacious. When E. tarda 30305-novo vaccinated Nile tilapia were challenged by its virulent E. tarda 30305, relative percent of survival of vaccinated fish at 14- and 28-days post vaccination (dpv) was 100% and 92%, respectively. Similarly, E. tarda 30305-novo offered 100% protection to channel catfish against challenges with virulent parent isolate E. tarda 30305 at both 14- and 28-dpv. Our results suggest that the development of live attenuated bacterial vaccines that are safe and efficacious is challenging, although it is feasible.
KeywordMeSH Terms
Drug Resistance, Bacterial
55.     ( 2013 )

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

Veterinary microbiology 165 (3��4��)
PMID : 23623688  :   DOI  :   10.1016/j.vetmic.2013.03.027     DOI  :   10.1016/j.vetmic.2013.03.027    
Abstract >>
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.
KeywordMeSH Terms
Genetic Variation
Phylogeny
Genetic Variation
Phylogeny
56.     ( 2013 )

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

Veterinary microbiology 165 (3��4��)
PMID : 23623688  :   DOI  :   10.1016/j.vetmic.2013.03.027     DOI  :   10.1016/j.vetmic.2013.03.027    
Abstract >>
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.
KeywordMeSH Terms
Genetic Variation
Phylogeny
Genetic Variation
Phylogeny
57. Reichley  SR, Ware  C, Steadman  J, Gaunt  PS, García  JC, LaFrentz  BR, Thachil  A, Waldbieser  GC, Stine  CB, Buján  N, Arias  CR, Loch  T, Welch  TJ, Cipriano  RC, Greenway  TE, Khoo  LH, Wise  DJ, Lawrence  ML, Griffin  MJ,     ( 2017 )

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.

Journal of clinical microbiology 55 (12)
PMID : 28978684  :   DOI  :   10.1128/JCM.00970-17     PMC  :   PMC5703813    
Abstract >>
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.
KeywordMeSH Terms
Edwardsiella
FAME
MALDI-TOF
aquaculture
gyrB
multiplex PCR
rep-PCR
sequencing
sodB
Edwardsiella
FAME
MALDI-TOF
aquaculture
gyrB
multiplex PCR
rep-PCR
sequencing
sodB
Genotype
Phenotype

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