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

## TL;DR

This study identifies a new, safe virus that can effectively reduce harmful bacteria on fresh produce, making it a potential commercial solution for food safety.

## Contribution

The study introduces and validates a novel lytic phage, vB_AroA_KFSA9, as a safe and effective biocontrol agent for Aeromonas hydrophila on fresh produce.

## Key findings

- vB_AroA_KFSA9 showed high specificity and safety, with no lytic activity against non-target species.
- The phage significantly reduced A. hydrophila on cherry tomatoes and lettuce by over 2 log CFU within 60 minutes.
- Phage stability was confirmed under food industry-relevant stress conditions like pH, temperature, and sanitizers.

## Abstract

Aeromonas hydrophila 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 Aeromonas spp. for food applications. This study characterized and evaluated a novel, safe A. hydrophila-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 A. hydrophila strains, showing no lytic activity against 4 Aeromonas 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 Teseptimavirus within the family Autotranscriptaviridae, 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 A. hydrophila-contaminated cherry tomatoes and lettuce significantly reduced the number of A. hydrophila 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.

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•A novel, lytic vB_AroA_KFSA9 was stable under conditions relevant to fresh produce.•Genomic analysis confirmed the novelty and safety of vB_AroA_KFSA9.•vB_AroA_KFSA9 reduced A. hydrophila significantly on fresh produce.•Bacterial regrowth suggests phage cocktails or hurdle strategies are needed.•vB_AroA_KFSA9 is a biocontrol candidate against A. hydrophila on fresh produce.

A novel, lytic vB_AroA_KFSA9 was stable under conditions relevant to fresh produce.

Genomic analysis confirmed the novelty and safety of vB_AroA_KFSA9.

vB_AroA_KFSA9 reduced A. hydrophila significantly on fresh produce.

Bacterial regrowth suggests phage cocktails or hurdle strategies are needed.

vB_AroA_KFSA9 is a biocontrol candidate against A. hydrophila on fresh produce.