Dual deletion of ugd and phoB Attenuates virulence and enhances vaccine potential in Aeromonas dhakensis

Aeromonas dhakensis, an increasingly prevalent pathogen in tropical areas, presents significant risks to public health and the aquaculture industry. This research evaluates the virulence effects of ugd and phoB gene deletions. The double gene deletion mutant strain ΔugdΔphoB was obtained by further knocking out the ugd gene in the previously constructed single mutant strain ΔphoB. Phenotypic analyses under phosphate-deficient conditions showed marked decreases in both growth and motility for the Δugd and ΔugdΔphoB mutants compared to the wild type. Conversely, under phosphate-rich conditions, growth remained stable, although motility variations were noted. The mutants ΔugdΔphoB and Δugd exhibited diminished resistance to H2O2, ECPase activity, and antimicrobial peptide resistance, alongside reduced adherence to EPC cells. However, the Δugd mutant displayed enhanced biofilm formation. The immunoprotection experiment showed that the protection rate (relative percent survival, RPS) of the Δugd mutant strain was 63.3%, which was significantly higher than the 40.0% of the ΔugdΔphoB mutant strain. Consistently, the Δugd mutant strain also exhibited a more significant degree of virulence attenuation (median lethal dose, LD₅₀ = 2.87 × 10⁸ CFU/mL); in contrast, the LD₅₀ of the ΔugdΔphoB mutant strain was 1.23 × 10⁸ CFU/mL. This indicates that the Δugd mutant strain has greater potential as an attenuated live vaccine candidate due to its stronger immunoprotective efficacy and lower residual virulence. 1. Both ΔugdΔphoB and Δugd mutants show decreased H2O2 resistance, ECPase activity, and antimicrobial peptide resistance. 2. Compared to the ΔugdΔphoB mutant, the Δugd mutant exhibits more significantly reduced virulence. 3. In zebrafish, the Δugd mutant achieves a relative percent survival (RPS) of 63.3% against wild-type challenges, surpassing the ΔugdΔphoB mutant's 40.0% RPS.