Multiplex PCR assay for correct identification of the fish pathogenic species of Edwardsiella genus reveals the presence of E. anguillarum in South America in strains previously characterized as E. tarda

Abstract

Aims Develop a species‐specific multiplex PCR to correctly identify Edwardsiella species in routine diagnostic for fish bacterial diseases. Methods and Results The genomes of 62 Edwardsiella spp. isolates available from the National Center for Biotechnology Information (NCBI) database were subjected to taxonomic and pan‐genomic analyses to identify unique regions that could be exploited by species‐specific PCR. The designed primers were tested against isolated Edwardsiella spp. strains, revealing errors in commercial biochemical tests for bacterial classification regarding Edwardsiella species. Conclusion Some of the genomes of Edwardsiella spp. in the NCBI platform were incorrectly classified, which can lead to errors in some research. A functional mPCR was developed to differentiate between phenotypically and genetically ambiguous Edwardsiella, with which, we detected the presence of Edwardsiella anguillarum affecting fish in Brazil. Significance and Impact of the Study This study shows that the misclassification of Edwardsiella spp in Brazil concealed the presence of E. anguillarum in South America. Also, this review of the taxonomic classification of the Edwardsiella genus is a contribution to the field to help researchers with their sequencing and identification of genomes, showing some misclassifications in online databases that must be corrected, as well as developing an easy assay to characterize Edwardsiella species in an end‐point mPCR.

Genome Report of Emergent Fish Pathogen Edwardsiella piscicida Recovered from Pseudoplatystoma corruscans in Brazil

“…A single CFU was subjected to extraction of genomic DNA by use of a PureLink genomic DNA mini kit (Invotrogen, USA), according to the manufacturer's instructions. A multiplex PCR (mPCR) assay was performed to identify the bacterial species, which was characterized as Edwardsiella piscicida ( 6 ). For sequencing, the DNA was prepared using a NEBNext fast DNA fragmentation and library preparation kit (New England Biolabs, Inc.) sequenced on an Illumina HiSeq 2500 platform (2 × 150 bp), and FastQC v0.11.9 ( 7 ) was used to verify the sequencing quality ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc ).…”

Edwardsiella tarda Causing Septicemia in a Wild Crested Ibis (Nipponia nippon)

“…Edwardsiella is a genus of gram-negative, facultative intracellular bacteria that belongs to the family Enterobacteriaceae. It includes five species: Edwardsiella hoshinae , Edwardsiella tarda , Edwardsiella ichtaluri , Edwardsiella piscicida , and Edwardsiella anguillarum (Miniero Davies et al 2018; da Costa et al 2022; Ziarati et al 2022). These bacteria are commonly known as disease agents in aquaculture and are associated with the systemic disease edwardsiellosis (Esteve and Alcaide 2022).…”