Characterization of a novel broad-spectrum endolysin PlyD4 encoded by a highly conserved prophage found in Aeromonas hydrophila ST251 strains

Aeromonas hydrophila is a zoonotic pathogen that exhibits high level resistance to classic antibiotics and is a heavy burden for aquaculture industry. Lytic enzymes encoded by phages or prophages have shown potential for use against pathogenic bacteria. In this study, an intact prophage (named phAhD4) was identified from A. hydrophila D4. phAhD4 is highly conserved in all 10 published A. hydrophila sequence type (ST) 251 strains and is unique to the ST251 strains. The unique endolysin PlyD4, encoded by phAhD4, was obtained by prokaryotic expression. PlyD4 showed bactericidal activity against a broad range of bacterial species in vitro, including A. hydrophila, Aeromonas veronii, Vibrio parahemolyticus, Pseudomonas aeruginosa, and so on. Synergistically with 5 mmol/L ethylene diamine tetraacetic acid (EDTA), the ratio of the optical density at 600 nm (OD600) of PlyD4 treatment versus the OD600 with no PlyD4 treatment for most tested strains decreased from 1 to 0.1-0.8 within 2 h. PlyD4 exhibited optimal activity at 28 °C and maintained high activity over a wide pH range (pH 6-10). Divalent metal ions conferred significant enhancement to PlyD4 lytic activity at low concentrations (0.1 mmol/L). In vivo, a 4.5 μg dose of PlyD4 protected 75.0% (15/20) of zebrafish in a bacteremia model of A. hydrophila D4 infection. These results indicated that PlyD4 was an effective therapeutic agent against multiple aquaculture-related pathogens. To the best of our knowledge, this study is the first to report on an A. hydrophila prophage endolysin that exerts antibacterial activity against a broad range of pathogens. KEY POINTS: • The prophage phAhD4 is highly conserved in 10 published A. hydrophila ST251 strains. • PlyD4 exerts antibacterial activity against multiple aquaculture-related pathogens. • PlyD4 conferred protection against A. hydrophila infection in a zebrafish model.