Isolating pathogenic multidrug-resistant Aeromonas hydrophila from diseased fish and assessing the effectiveness of a novel lytic Aeromonas veronii bacteriophage (AVP3) for biocontrol.

The increasing trend of antimicrobial resistance (AMR) pathogens in aquaculture makes it is imperative to find control measures for AMR pathogens causing high economic losses in aquaculture. In the present study, a multidrug resistance (MDR) Aeromonas hydrophila bacterium was isolated from kidney samples of diseased carp originating from a fish farm in Awankot, Rupnagar, Punjab, India. Moribund-infected fish exhibited large irregular hemorrhages on the external body surfaces, exophthalmia and fin-rot-like lesions. Phenotypic characterization using Rimler-Shotts (RS) media showed characteristic yellow color colonies and beta hemolysis on sheep blood agar. Genotyping using species-specific primers for the rpoB and gyrB genes characterized the isolate as A. hydrophila. The Multiple Antibiotic Resistance (MAR) index analysis showed that the isolated A. hydrophila had an MAR score of 0.29 signifying its resistance to more than three antibiotics, which underscores the need of finding treatment methods for MDR A. hydrophila isolates causing disease in aquaculture. Bacteriophages are considered a better eco-friendly alternative to antibiotics because of their inherent properties of not causing drug residues and resistance. Of the 13 phages tested, the Aeromonas veronii phage designated as AVP3, initially isolated against Aeromonas veronii, showed lytic activity against the MDR A. hydrophila isolated from diseased carp in this study. In addition, it also showed the lytic activity against Aeromonas spp. And A. caviae indicating that it had lytic properties against a wide host range within the Aeromonas species. This finding points to the potential efficacy of bacteriophages in mitigating pathogenic infections in aquaculture.

Identification of novel vaccine candidates in the whole-cell Aeromonas hydrophila biofilm vaccine through reverse vaccinology approach.

Biofilm vaccine has been recognised as one of the successful strategy to reduce the Aeromonas hydrophila infection in fish. But, the vaccine contains the protective and non-protective proteins, which may lead to show altered heterologous adaptive immunity response. Moreover, cross protection and effectiveness of previously developed biofilm vaccine was not tested against different geographical A. hydrophila isolates. Therefore, in the present study, whole-cell A. hydrophila biofilm vaccine was evaluated in rohu, vaccinated group showed increased antibody titer and protection against the different geographical A. hydrophila isolates namely KAH1 and AAH2 with 78.9% and 84.2% relative percentage survival, respectively. In addition, by using the immune sera of biofilm vaccinated group, a total of six protective proteins were detected using western blot assay. Further, the same proteins were identified by nano LC-MS/MS method, a total of fourteen candidate proteins showing the immunogenic property including highly expressed OMP's tolC, bamA, lamb, AH4AK4_2542, AHGSH82_029580 were identified as potential vaccine candidates. The STRING analysis revealed that, top candidate proteins identified may potentially interact with other intracellular proteins; involved in ribosomal and (tricarboxylic acid) TCA pathway. Importantly, all the selected vaccine candidate proteins contain the B-cell epitope region. Finally, the present study concludes that, whole-cell A. hydrophila biofilm vaccine able to protect the fish against the different geographical A. hydrophila isolates. Further, through reverse vaccinology approach, a total of fourteen proteins were identified as potential vaccine candidates against A. hydrophila pathogen.