Comprehensive investigation of a novel and safe lytic phage vB_AroA_KFSA9 as a commercial candidate for biocontrol of Aeromonas hydrophila on fresh produce

<i>Aeromonas hydrophila</i> is an emerging foodborne pathogen, particularly prevalent in fresh produce, posing a risk to public health. Despite its significance, no commercial phage products have yet been developed to target <i>Aeromonas</i> spp. for food applications. This study characterized and evaluated a novel, safe <i>A. hydrophila</i>-specific phage, vB_AroA_KFSA9 as a commercial biocontrol candidate for application on fresh produce. vB_AroA_KFSA9, isolated from a poultry processing plant, exhibited high specificity only against nine <i>A. hydrophila</i> strains, showing no lytic activity against 4 <i>Aeromonas</i> species, 3 probiotic species, and 19 species of foodborne pathogen. Comprehensive genomic analysis confirmed the safety of the phage by revealing no undesirable genes associated with antibiotic resistance, bacterial virulence, or lysogeny. Furthermore, it identified vB_AroA_KFSA9 as a novel member of the genus <i>Teseptimavirus</i> within the family <i>Autotranscriptaviridae</i>, distinguished from related phages by a divergent tail fiber and unique endonuclease-associated genes. The phage remained stable even under food industry-relevant stress conditions, including pHs 4-10, 4-40 °C, and common sanitizers (acetic acid, citric acid, and hydrogen peroxide). The phage exhibited excellent bactericidal effects, achieving complete cell lysis within 3 h, even at a low multiplicity of infection (MOI) of 0.01. Applying vB_AroA_KFSA9 on <i>A. hydrophila</i>-contaminated cherry tomatoes and lettuce significantly reduced the number of <i>A. hydrophila</i> by ∼2.79 log CFU/cherry tomato and ∼2.45 log CFU/4 cm² lettuce in 60 min, using an MOI of 1. These findings demonstrate vB_AroA_KFSA9 as a promising biocontrol candidate for future commercial application in the fresh produce chain.