Polyclonal emergence of virulent, multidrug-resistant Edwardsiella tarda lineages associated with bacillary necrosis of Pangasius in striped catfish farms

Bacillary necrosis of Pangasius (BNP) is a recurrent bacterial disease that poses a major threat to Vietnam’s striped catfish ( Pangasianodon hypophthalmus ) aquaculture industry. Although BNP has traditionally been attributed to Edwardsiella ictaluri , its etiology remains unresolved due to limitations of species-specific diagnostics. In this study, we investigated acute BNP cases reported at 19 geographically distinct catfish farms across the Mekong River Delta from December 2023 to July 2024. While initial 16S rRNA PCR assays implicated E. ictaluri , whole-genome sequencing of isolates from 15 of 19 farms (79%) confirmed that all strains belonged to Edwardsiella tarda . Experimental challenge in a striped catfish infection model demonstrated that all 15 E. tarda strains were highly virulent, with reisolation of the pathogen fulfilling Koch’s postulates. Phylogenetic analysis identified six distinct clonal lineages, indicating a polyclonal population structure consistent with multiple introductions or diversification of endemic strains. Comparative genomics revealed conserved core genomes within lineages, with diversity in accessory regions driven by prophages and plasmids. Twelve prophages were identified across the genomes, clustering into conserved syntenic pairs and integrating at shared chromosomal hotspots. Antimicrobial susceptibility testing revealed multidrug-resistant phenotypes in multiple lineages, genetically linked to plasmid-borne resistance determinants. Plasmid analyses identified conjugative plasmids with conserved backbones that mediate horizontal transfer of multidrug resistance cassettes, including determinants conferring resistance to β-lactams, enrofloxacin, doxycycline, and trimethoprim-sulfamethoxazole. Collectively, these findings redefined E. tarda as the etiologic agent of recent BNP cases in Vietnam and revealed the genomic features underpinning its virulence and multidrug resistance. IMPORTANCE Bacillary necrosis of Pangasius (BNP) is a serious disease affecting Vietnam’s striped catfish industry, one of the world’s largest freshwater aquaculture industries. For many years, BNP was attributed to Edwardsiella ictaluri , influencing how the disease was diagnosed and treated. In this study, we found that recent BNP outbreaks were instead caused by multiple strains of the related bacterium Edwardsiella tarda . Experimental infections confirmed that these strains can cause severe disease in striped catfish under controlled laboratory conditions. Some strains also carried multidrug resistance plasmids that were experimentally shown to transfer between bacteria, demonstrating a potential mechanism for the spread of antimicrobial resistance in aquaculture systems. We additionally developed a new PCR test that more accurately distinguishes E. tarda from closely related species. Together, these findings improve understanding of BNP and provide new tools for its disease diagnosis, surveillance, and management in aquaculture.