Identification, Genotyping And Whole Genome Sequencing Of Edwardsiella Tarda From Asian Swamp Eel(Monopterus Albus)

We isolated a strain of bacteria from Asian swamp eel which was named as ASE201307 in this study. It was identified as Edwardsiella tarda based on Phenotypic and molecular method. We amplified 16 S rDNA, gyr B gene, dna K gene, pyr G gene and pho R gene to conduct MLSA(Multi-Locus Sequence Analysis) study. Based on MLSA study, we identified ASE201307 as a new E. tarda genotype. Whole genome of ASE201307 was sequenced for further study and comparative genomics analysis was also conducted between ASE201307 and other E. tarda strains isolated from different hosts and locations. The main results are as follows:1. The clinical symptoms of diseased Asian swamp eel included cutaneous hemorrhage, visceral hemorrhage and anal swollen. A strain of bacteria named ASE201307 was isolated from liver, kidney and spleen. ASE201307 isolates were Gram-negative. The comparison results showed that the sequence of ASE201307 was most similar(99%) to those of the members of E. tarda. ASE201307 clustered with Edwardsiella in phylogenetic trees. Biochemically, the strain was very similar to known E. tarda strains, such as positive for indole production, H2 S production and gas from glucose. ASE201307 was proved to be the pathogen of this disease by experimental infection. The LD50 was calculated as 1.0×106 CFU/mL which suggested its high virulence. ASE201307 was sensitive to potentiated sulphonamide, florfenicol, ciprofloxacin and nitrofurantoin, resistant to penicillin and amoxicillin which were classed as β-lactam antibiotics. E. tarda has been reported to produce β-lactamase, an enzyme that associated with the drug resistance to β-lactam antibiotics.2. We amplified 16 S rDNA, gyrB gene, dna K gene, pyrG gene and pho R gene of ASE201307 and downloaded these five housekeeping genes of ten E. tarda strains and one Edwardsiella ictaluri strain isolated from different hosts and locations from NCBI. MLSA was conducted. Evolutionary trees of individual gene loci and concatenated gene were similar in topological structure.ASE201307 clustered with the ATCC reference strains, while other E. tarda strains isolated from other fish species clustered with E. ictaluri strain. This result suggested that ASE201307 was a new genotype. Some researchers divided E. tarda isolated from fish in China into two groups, flatfish isolates in Northern China and eel isolates in Southern China. The study of ASE201307 added a new E. tarda group isolated from fish in China, Asian swamp eel isolates in Central China.3. We sequenced the whole genome of ASE201307 and selected some reference strains to conduct comparative genomics analysis. ASE201307 possessed a single chromosome of 3.68 M and the GC content was 57.09%. KEEG analysis revealed that ASE201307 was enriched in categories of genes involved in environmental information processing, genetic information processing and metabolism. COG analysis revealed that ASE201307 was enriched in categories of genes involved in carbohydrate, amino acid and coenzyme metabolism. GO analysis revealed that ASE201307 was enriched in categories of genes involved in cellular and metabolic process. Comparative genomics analysis which included two-dimension collinearity analysis, SNP, SV, heat map of dispensable genes and phylogenetic analysis based on whole genome sequence revealed ASE201307 was highly similar to E. tarda FL95.01 and reference strain ATCC15947. Considering the taxonomic viewpoint proposed by Yuanxing Zhang, we divided E. tarda into three groups, freshwater group, migratory group and marine group.