Construction and Evaluation of Recombinant Attenuated Edwardsiella piscicida Vaccine (RAEV) Vector System Encoding Ichthyophthirius multifiliis (Ich) Antigen IAG52B.

We have successfully designed and constructed a RAEV vector system with regulated-delayed attenuation in vivo attributes that synthesizes Ichthyophthirius multifiliis (Ich) protective antigen IAG52B to enable vaccination of fish susceptible to edwardsiellosis and white spot disease. The first feature of this vaccine delivery system is an Edwardsiella piscicida strain carrying genomic deletions of asdA. AsdA is an enzyme necessary for the synthesis of diaminopimelic acid (DAP), which is an essential component of the peptidoglycan layer of the cell wall of Gram-negative bacteria. asdA mutant strains have obligate growth requirements for DAP in the medium or a plasmid vector with the wild-type asdA gene enabling synthesis of DAP. This balanced-lethal plasmid vector-host system in E. piscicida enables as a second feature the synthesis of recombinant antigens to induce protective immunity against fish pathogens. Recombinant protective antigen IAG52B from the fish pathogen I. multifiliis was synthesized by RAEV strains harboring the AsdA+ plasmid pG8R8029. The third feature of this vaccine strain is a regulated-delayed attenuation in vivo phenotype that is based on the replacement of an arabinose-regulated araC ParaBAD cassette for the promoters of the fur and crp genes of E. piscicida such that the expression of these genes is dependent on arabinose provided during growth. Thus, following colonization, the Fur and Crp proteins stop being synthesized due to the lack of arabinose and attenuation is progressively achieved in vivo to prevent generation of diseases symptoms. Our vaccine strain χ16022 with the genotype ΔasdA10 ΔPfur170::TT araC ParaBADfur ΔPcrp68::TT araC ParaBADcrp contains the AsdA+ plasmid, pG8R8029, which encodes the IAG52B antigen. Vaccine strain χ16022(pG8R8029) is attenuated and induces systemic and mucosal IgM titer against E. piscicida and Ich in zebrafish. In addition, transcript levels of tnf-α, il-1ß, il-6 and il-8 were significantly increased in different tissues of vaccinated zebrafish compared to unimmunized fish. Zebrafish vaccinated with χ16022(pG8R8029) showed 60% survival upon intracoelomic (i.c.) challenge with a lethal dose of virulent E. piscicida strain J118. Our RAEV system could be used as a generalized vaccine-vector system to protect teleost fish against multiple bacterial, viral and parasitic infectious diseases.

Characterization of a Novel Conjugative Plasmid in Edwardsiella piscicida Strain MS-18-199.

Edwardsiella piscicida is a pathogenic bacterium responsible for significant losses in important wild and cultured fish species. E. piscicida strain MS-18-199 recovered from a diseased hybrid catfish from East Mississippi and showed resistance to florfenicol, chloramphenicol, oxytetracycline, doxycycline, erythromycin, tetracycline, azitromycin, spectinomycin, sulfonamide, and bacitracin. To explore the mechanisms of resistance in E. piscicida strain MS-18-199, genomic DNA was extracted and subjected to whole genome sequencing (WGS) using a combination of long (Oxford Nanopore) and short (Illumina) reads. The genome of strain MS-18-199 revealed a novel plasmid named pEPMS-18199. The 117,448 bp plasmid contains several antimicrobial resistance (AMR) elements/genes, including florfenicol efflux pump (floR), tetracycline efflux pump (tetA), tetracycline repressor protein (tetR), sulfonamide resistance (sul2), aminoglycoside O-phosphotransferase aph(6)-Id (strB), and aminoglycoside O-phosphotransferase aph(3)-Ib (strA). Two genes, arsA and arsD, that encode protein components related to transport/resistance to arsenic were also found in pEPMS-18199. In addition, pEPMS-18199 carried twelve conjugative transfer genes (tra), eight transposases and insertion elements, two plasmid stability proteins, two replication proteins, and three partitioning proteins (par system). Results from mobilization and stability experiments revealed that pEPMS-18199 is highly stable in the host cell and could be transferred to Escherichia coli and Edwardsiella ictaluri by conjugation. To our knowledge, this is the first detection of a multidrug resistance (MDR) conjugative plasmid in E. piscicida in the United States. Careful tracking of this plasmid in the aquaculture system is warranted. Knowledge regarding the molecular mechanisms of AMR in aquaculture is important for antimicrobial stewardship.

Population genetic and evolution analysis of controversial genus Edwardsiella by multilocus sequence typing.

At present, the genus Edwardsiella compiles five species: E. tarda, E. hoshinae, E. ictaluri, E. piscicida and E. anguillarum. Some species of this genus such us E. ictaluri and E. piscicida are important pathogens of numerous fish species. With the description of the two latter species, the phylogeny of Edwardsiella became more complicated. With the aim to clarify the relationships among all species in the genus, a multilocus sequence typing (MLST) approach was developed and applied to characterize 56 isolates and 6 reference strains belonging to the five Edwardsiella species. Moreover, several analyses based on the MLST scheme were performed to investigate the evolution within the genus, as well as the influence of recombination and mutation in the speciation. Edwardsiella isolates presented a high genetic variability reflected in the fourteen sequence types (ST) represented by a single isolates out of eighteen total ST. Mutation events were considerably more frequent than recombination, although both approximately equal influenced the genetic diversification. However, the speciation among species occurred mostly by recombination. Edwardsiella genus displays a non-clonal population structure with some degree of geographical isolation followed by a population expansion of E. piscicida. A database from this study was created and hosted on pubmlst.org (http://pubmlst.org/edwardsiella/).

Genetic studies to re-affiliate Edwardsiella tarda fish isolates to Edwardsiella piscicida and Edwardsiella anguillarum species.

Until 2012, the genus Edwardsiella was composed by three species Edwardsiella tarda, Edwardsiella hoshinae and Edwardsiella ictaluri. In 2013, Edwardsiella piscicida, compiling fish pathogenic strains previously identified as E. tarda was described, and more recently a new species isolated from diseased eel was reported, namely Edwardsiella anguillarum. The incorporation of these species into the genus makes necessary a revision of the taxonomic position of the isolates previously identified as E. tarda. Using AFLP technique, MLSA studies and in silico DNA-DNA hybridization, 46 of 49 E. tarda isolates were re-assigned as E. piscicida and 2 as E. anguillarum, whereas it was confirmed previous classification of the Edwardsiella types and reference strains used. The study of the taxonomic resolution of the genes 16S rRNA, adk, atpD, dnaJ, glnA, hsp60, tuf as well as the possible combinations among housekeeping genes, showed that the gene dnaJ was the more resolutive. In conclusion, the use of molecular techniques is necessary to accurately identify Edwardsiella isolates, especially when differentiating new species from E. tarda.

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

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.

Edwardsiella piscicida sp. nov., a novel species pathogenic to fish.

AIMS: This study describes a novel species within the genus Edwardsiella based on phenotypic and genetic characterization of fish pathogenic Edwardsiella isolates previously identified as E. tarda. METHODS AND RESULTS: Phenotypic characterization, DNA-DNA hybridization and phylogenetic analysis of representative Edwardsiella isolates from fish previously identified as E. tarda were conducted and compared with E. tarda type strain (ATCC 15947(T)). Phenotypically, strains from fish grow with pin-point colonies producing slight ß-haemolysis under the colony. In contrast to the E. tarda type strain, fish strains did not [corrected] degrade ß-methyl-D-glucoside [corrected] (with the exception of NCIMB 2034), citric acid and L-proline. [corrected]. With the exception of strain ETK01, all fish strains were highly pathogenic to zebra fish, while ATCC 15947(T) and NCIMB 2034 were nonpathogenic. DNA-DNA hybridization (DDH) levels between representative fish isolates and the E. tarda type strain ranged from 15 to 43·6%, while NCIMB 2034 hybridised with the type strain at the level of 63·2%. DDH values between the various fish isolates ranged from 68·2 to 93·9% defining a new and separate DNA hybridization group differing from the E. tarda type strain consistent with the findings of phylogenetic analysis, in which the fish isolates comprised a separate clade. CONCLUSIONS: Phenotypical and genetic characterizations demonstrated that Edwardsiella isolates from fish described in this study do not belong to the species E. tarda or any of the previously established taxa within the genus Edwardsiella. The fish related strains studied here (excluding NCIMB 2034) represent, therefore, a novel species within the genus Edwardsiella for which we propose the name Edwardsiella piscicida sp. nov, with strain ET883(T) (NCIMB 14824(T) = CCUG 62929) as the type strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The current finding will improve the diagnosis, understanding of the epidemiology and in establishment of effective control measures against this serious fish pathogen.

Unusual food-borne pathogens. Listeria monocytogenes, Aeromonas, Plesiomonas, and Edwardsiella species.

Although these four groups of organisms are perceived as infrequent food-borne pathogens or of dubious significance, increasing epidemiologic data indicate that L. monocytogenes is an emerging cause of infections, particularly gastroenteritis. Furthermore, if data are ever generated that prove that most fecal isolates of Aeromonas are involved in bacterial diarrhea, then aeromonads will become recognized as important food-borne pathogens. For Plesiomonas and Edwardsiella, recognition of possible involvement in food-borne disease requires detailed medical histories, including foreign travel, contact with pets or animals, and food consumption histories.