Phenotypic and genotypic analysis of Edwardsiella isolates from Taiwan indicates wide variation with a particular reference to Edwardsiella tarda and Edwardsiella anguillarum

Abstract

Edwardsiella spp. is a gram‐negative, facultatively anaerobic, intracellular bacteria threatening the aquaculture industry worldwide. Noticeably, E. tarda is now genotypically classified into three distinct groups (E. tarda, E. piscicida and E. anguillarum), but morphologically, it is unclear due to varying degrees of virulence in different fish hosts. Hence, to reclassify E. tarda, we investigated differences in genotypes, phenotypes and pathogenicity. We collected Edwardsiella isolates from five different counties of Taiwan between 2017 and 2021. At first, gyrB gene was amplified for a phylogenetic tree from 40 isolates from different fish and one reference isolate, BCRC10670, from the human. Thirty‐nine strains clustered into E. anguillarum, 1 strain into E. piscicida and 1 strain into E. tarda from human strain. Second, all isolates were characterized using various phenotypic (API 20E biochemical profiles) and genotypic (pulsed‐field gel electrophoresis [PFGE], and virulence‐related gene detection). SpeI digestion revealed 10 pulsotypes and I‐CeuI into 7 pulsotypes. Virulent genes (citC, gadB, katB, mukF and fimA) confirmed in 35, 31, 28, 37 and 38 isolates, respectively. Finally, in vivo challenge test in milkfish (Chanos chanos) indicated the highest mortality from E. anguillarum. Overall, results revealed unique features with Edwardsiella spp. genotypes and pathogenicity, which are relevant to the host and provide useful insights for future vaccine development.

Comparative genomics of Edwardsiella anguillarum and Edwardsiella piscicida isolated in Taiwan enables the identification of distinctive features and potential virulence factors using Oxford‐Nanopore MinION® sequencing

“…Our study is the first to utilize Nanopore technology to identify aquaculture pathogens at the species level of E. anguillarum and E. piscicida from diseased cultured fish in Taiwan. Moreover, with the application of genome sequencing, this study confirmed the species taxonomy as E. anguillarum or E. piscicida based on the gyr B gene sequence in a previous study (Rahmawaty et al, 2022). The unique findings with regard to the genomes of E. anguillarum and E. piscicida will facilitate the investigation of epidemiology, intraspecies diagnostic methods, vaccine development, and drug treatment for both species.…”

“…In this study, we evaluated two E. anguillarum and E. piscicida strains, which were recently isolated from milkfish ( Chanos chanos ) and Japanese eel ( Anguilla japonica ) in Taiwan. The virulent properties of these two bacteria in milkfish indicated variation in mortality as demonstrated in a recent study (Rahmawaty et al, 2022). Therefore, we sequenced the genome to understand the species identification and compare their genomic characteristics by using Oxford nanopore MinIon technology, which identifies DNA bases by measuring the changes in electrical conductivity generated as DNA strands pass through a biological pore (Lu et al, 2016).…”

“…In this study, we evaluated two E. anguillarum and E. piscicida strains, which were recently isolated from milkfish (Chanos chanos) and Japanese eel (Anguilla japonica) in Taiwan. The virulent properties of these two bacteria in milkfish indicated variation in mortality as demonstrated in a recent study (Rahmawaty et al, 2022).…”

“…This suggests that through horizontal gene transfer, E. anguillarum may have acquired genetic material and/or adapted to its milkfish host or prevailing environmental conditions in Taiwan. In our previous study, we observed that E. anguillarum is more virulent than E. piscicida when inoculated in milkfish (Rahmawaty et al, 2022). A high GC content, ranging from 58 to 59%, was also observed for the two genomes, which is similar to other Edwardsiella genomes, as mentioned in Table S9.…”

“…Edwardsiella anguillarum and E. piscicida were previously isolated from milkfish and Japanese eels (Rahmawaty et al, 2022, Table S1). All Edwardsiella spp.…”