Edwardsiella Genomics And Phylogenomics

The genus Edwardsiella, consisting of three species Edwardsiella tarda, Edwardsiella ictaluri and Edwardsiella hoshinae, was firstly described in1965by Ewing et al. to designate a distinct taxa within the family Enterobacteriaceae. E. tarda is the most predominant species of Edwardsiella as it is a common inhabitant of animals including fish, reptiles, amphibians, chickens, other warm-blooded animals and humans. E. tarda is also the etiological agent of edwardsiellosis, characterized by systemic hemorrhagic septicemia, internal abscesses, and skin lesions leading to mass mortality outbreaks in more than20species of freshwater and marine fish, causing devastating economic losses in worldwide aquaculture. E. ictaluri is a notorious fish pathogen causing enteric septicemia exclusively in channel catfish. E. hoshinae is sometimes isolated from animals but its ability to cause disease has not been established and little is known regarding its habitats. In previous work, we have isolated many Edwardsiella strains from different sources. The work will provide useful information for further epidemiological study and genomics study of this pathogen. In this study, we firstly use two high degrees of reproducibility and discriminatory power genotyping methods, including multiple locus sequence typing (MLST) and pulsed field gel electrophoresis typing (PFGE) methods, to detect genetic diversity and intraspecific relationship of E. tarda strains isolated from different hosts and different geographical regions in China. To unravel the genetic properties for habitat adaptation, virulence determinants, invasive nature and multi-drug resistance of E. tarda, we utilized the high-throughput sequencing platform to quickly determine the complete genome sequence of E. tarda EIB202and perform comparative genomic analysis of eight typical Edwardsiella strains. Furthermore, we reconstructed the hypothetical evolutionary pathway to highlight their virulence evolution and niche adaptation mechanisms.By using methods of high degree of reproducibility and discriminatory power, including PFGE method and MLST assays, we identified the distinct phylogenetic profiles of the E. tarda isolates from different sources. The PFGE band patterns of the Spel digested E. tarda genomes were sensitive to classify E. tarda isolates from different hosts (eel, turbot/flounder, and human) and our results revealed that E. tarda isolates from flounder and turbot in Northern China and isolates from eel in Southern China could be distinctly separated into two subgroups. Our results also showed high correlation between MLST method and PFGE profiling. AMOVA statistical analysis showed signifcant difference between flounder/turbot isolates and eel isolates. These data highlighted the intraspecies relationships of E. tarda isolates from different sources in China and indicated potential application of these methods in tracing source of E. tarda infection and enhance understanding of epidemiological relationships among E. tarda isolates of environmental, fish, or human origins.The knowledge about the genetic basis of E. tarda is not very clear and most work focuses on some virulence related genes. We utilized the high-throughput pyrosequencing together with conventional shutgun sequencing method to quickly determine the complete genome sequence of E. tarda EIB202, a highly virulent strain isolated from a diseased turbot in Shandong province. E. tarda EIB202possesses a single chromosome of3,760,463bp, containing3,486predicted protein coding sequences. Strand specific RNA sequencing analysis (ssRNAseq) indicated that the genome contains about600operons of2-to21-gene length, which cover about55%of all EIB202genes. E. tarda EIB202contains a43,703bp conjugative plasmid pEIB202harboring6multi-drug resistant genes and encoding multiple type IV secretion system (T4SS) components.A total of24genomic islands (G1) were discerned to scatter throughout the EIB202chromosome. The Type Ⅲ and Ⅵ secretion systems (T3SS and T6SS) and other invasion/hemagglutinin genes exist in genomic islands, which might contribute to the intracellular colonization of E. tarda in host cells. E. tarda EIB202harbors an array of genes for ferric uptake regulator, ferric reductase, ferrous iron utilization and ferritin protein. Futher ssRNAseq analysis also showed that26ferric uptake related genes were signifeent upreguated in DMEM medium, indicating that these genes contribute to the growth and survival under diverse conditions including intracellular niches. COG analysis reveals that EIB202are enriched in categories of genes involved in nucleotide transport and metabolism, coenzyme metabolism ribosomal structure and biogenesis, signal transduction mechanisms, which is consistent with the high growth rate of this bacterium and adaptated to sea water and marine fish as previously described.In order to investigate genome diversity of Edwardsiella strains from various natural habitats, we used high-throughput sequencing technology to resequence4important Edwardsiella strains isolated from different hosts of the world. Evolutionary parallelism of Edwardsiella lineages provides a model to study evolutionary diversity processes linked to the virulence divergence and niche adaptation of pathogenic microorganisms. Genome-based phylogenetic analysis revealed that eight Edwardsiella strains could be separated into three kinds of genotypes (EdwGI E. tarda, EdwGII E. tarda and E. ictaluri) based on the sequence similarity. The low level of virulence in the EdwGII lineage could be explained by the missing of some important virulence associated gene clusters such as T3SS and T6SS. Moreover, the function comparison analysis of the genes in E. tarda EdwGI and EdwGH strains revealed a high diversity of cell wall/capsule-, regulation/cell signaling-and virulence-related genes, suggesting that this may constitute a genetic basis for the different niche adaptation characteristics and virulence mechanisms of these two E. tarda lineages. Many iron scavenging related genes were under positive selection and showed strong signs of adaptive evolution in the EdwGI lineages. Mutational analysis of these genes really demonstrated their essential roles in virulence and colonization. The loss of the biosynthetic and iron uptake gene clusters, which is specific to the most abundant marine bacteria, might be an important factor restricting the habitats of E. ictaluri species to freshwater fish. Moreover, evolution selection analysis showed that flagellar-and cell wall/capsule-related genes are under an adaptive evolution process in E. ictaluri, which might constitute one of the adaptive traits in this species.Exploration of the genome content of Edwardsiella strains will definitely provide clues enabling us to track and reconstitute the evolutionary events in Edwardsiella species. We proposed hypothetical evolutionary scenarios for the Edwardsiella strains. Edwardsiella ancestral strains were diverged into two major subpopulations, E. tarda EdwGII group and E. tarda EdwGI/E. ictaluri group, which subsequently developed into two distinct clades (E. tarda EdwGI lineage and E. ictaluri) and one nonpathogenic or environmental clade (E. tarda EdwGH lineage).