Characterisation of a Novel Jumbo Lytic Aeromonas dhakensis Bacteriophage P19 and Its Endolysin

Abstract

Aeromonas dhakensis is an emerging multidrug-resistant (MDR) bacterial pathogen that presents significant threats to public health and aquaculture. This study reports the isolation and comprehensive characterisation of jumbo bacteriophage P19. It is a lytic phage with a long, contractile tail and is placed in the genus Ceceduovirus of the family Straboviridae. P19 possesses a large genome typical of jumbo phages, which likely contributes to its enhanced replication efficiency and adaptability. P19 exhibited strong lytic activity with a high efficiency of plating (EOP) against multiple A. dhakensis strains, a burst size of approximately 50 plaque-forming units (PFU) per cell, and a latent period of 30 min. It remained highly stable across pH 6–10 and temperatures up to 60°C. Genomic analysis revealed a 228 kb genome with 414 open reading frames (ORFs) and absence of genes related to lysogeny, antibiotic resistance or virulence, indicating its potential for safe therapeutic and biocontrol applications. ORF_358 encodes a stable and soluble T4-type lysozyme endolysin (molecular weight 18.8 kDa) belonging to the glycoside hydrolase family 24. Molecular docking analysis demonstrated strong binding affinity (−6.4 kcal/mol) between P19_358 and the peptidoglycan NAG-NAM dimer. Recombinant P19_358 endolysin exhibited 99.98% relative lytic activity against Gram-positive bacterium Bacillus subtilis at a concentration of 75 μg/mL without EDTA pre-treatment; whereas, it showed broad-spectrum antibacterial activity against several Gram-negative pathogens, including A. dhakensis, Shigella flexneri, Salmonella Typhimurium, Pseudomonas aeruginosa and Escherichia coli, with relative activity ranging from 71% to 97% following pre-treatment with 100 mM EDTA. These findings collectively indicate that phage P19 and its endolysin P19_358 possess potent lytic activity and favourable stability profiles, supporting their development as promising antimicrobial agents against MDR Gram-negative pathogens.