Revealing the rfb gene as a critical virulence determinant enabling vaccine development for hypervirulent Aeromonas dhakensis

Aeromonas dhakensis is an emerging pathogen causing bacteremia in a wide range of aquatic animals and humans, but its pathogenesis remains unclear. The hypervirulent A. dhakensis strain C4-1 was recovered from a fatal septicemia outbreak among farmed Siamese crocodiles in 2016. This study aimed to uncover the key virulence factors of C4-1 via whole-genome analysis and evaluate the vaccine potential of attenuated mutants. Predicted virulence-associated genes of C4-1 were predominantly associated with membrane function, metabolic adaptation, and environmental responses. Through comparative genomic analysis against other 17 aquatic-animal-derived A. dhakensis isolates, rfb ( ctg _00167) was selected as a major virulence-related candidate. We constructed an in-frame rfb deletion mutant (Δ rfb ) and phenotypic assays demonstrated that rfb deletion significantly impaired bacterial growth, biofilm formation, swimming motility, and hemolytic activity. In the zebrafish infection model, Δ rfb exhibited virulence attenuation, showing alleviated pathological lesions, prolonged host survival, and a 33.3-fold higher LD 50 relative to wild-type C4-1. Immunization with Δ rfb produced a 57.69% relative percent survival upon C4-1 challenge and reshaped host immune-gene profiles, including elevated pro-inflammatory-cytokine and mucosal-gene expression together with altered IgM transcript abundance. Transcriptomic analysis identified 611 differentially expressed genes in Δ rfb , which were enriched in lipopolysaccharide biosynthesis, outer-membrane biogenesis, flagellar assembly, chemotaxis, β-lactam resistance, and central-metabolism pathways. In conclusion, these findings demonstrate that the rfb gene affects the pathogenicity of A. dhakensis and provides preliminary experimental evidence for Δ rfb as an experimental live-attenuated vaccine candidate for aquaculture, offering insights into Aeromonas pathogenesis and supporting rfb as a candidate target for aquatic animal disease control strategies.