Whole‐Genome Single Nucleotide Polymorphism Analysis and High‐Resolution Melting ( <scp>HRM</scp> ) Differentiation of <i>Edwardsiella anguillarum</i> and <i>Edwardsiella piscicida</i>

This study aimed to develop a method to differentiate between two closely related bacterial species within the genus Edwardsiella, namely Edwardsiella anguillarum and Edwardsiella piscicida, which cause Edwardsiellosis in fish aquaculture. Whole-genome single nucleotide polymorphism (SNP) analysis was conducted to assess the genetic relationships among E. piscicida, E. anguillarum and Edwardsiella ictaluri. SNP distance matrices and phylogenetic trees demonstrated that isolates within each species clustered together, confirming genetic SNP distinctiveness. SNP annotation and distance comparisons identified key genes-artI, dnaK, hlyB, ubiB, hutW, ydgD, MCP and fliC-that exhibited significant divergence. We designed specific primers targeting SNPs in these genes and used these primers for real-time PCR and High-Resolution Melting (HRM) analysis. HRM analysis using designed primer pairs revealed distinct melting temperature (Tm) peaks for E. anguillarum and E. piscicida. HRM analysis of 37 isolates that were previously identified as E. tarda based on phenotypic characteristics identified them as either E. anguillarum (35) or E. piscicida (2). These results showed that hlyB was the most reliable target gene for distinguishing between E. anguillarum and E. piscicida. The HRM technique is a fast, cost-effective method for differentiating these closely related bacterial species and provides insights into the genetic variation among different species of Edwardsiella and host-specific strains.