Biosafety and cellular cytocompatibility assessment of endotoxin-reduced Flavobacterium psychrophilum bacteriophages

Abstract

Flavobacterium psychrophilum causes bacterial cold-water disease in salmonids, leading to substantial losses in juvenile rainbow trout aquaculture. Although bacteriophages represent promising antibiotic alternatives, biosafety concerns persist due to endotoxin contamination originating from Gram-negative hosts. In this study, five lytic phages (FpV30–FpV34) were isolated from trout hatchery effluent using F. psychrophilum ATCC 49418 and characterized by one-step growth, adsorption assays, host range analysis, and transmission electron microscopy (TEM). Phages exhibited adsorption constants between 1.21×10⁻⁹ and 1.58×10⁻⁶ mL·min⁻¹, latent periods of 3–6.5 h, and burst sizes of 11–34.5 particles per cell. Spot tests demonstrated high specificity toward F. psychrophilum, with negligible activity against non-target bacteria. TEM revealed icosahedral capsids (≈75–83 nm) and long tails (≈129–142 nm), consistent with Myoviridae-like morphology. Crude lysates were purified by PEG/NaCl precipitation followed by dialysis, resulting in a ~40-fold increase in phage titer (from 1×10⁸ to 4×10⁹ PFU mL^–1) and a 99.8% reduction in endotoxin levels (from 1.2×10³ to 2.3 EU mL^–1), confirmed by chromogenic LAL assay with interference control. Cellular cytocompatibility assessed in MCF-7 cells revealed that crude preparations reduced viability to 58% at 48 h, whereas endotoxin-reduced phages maintained >90% viability. These findings demonstrate that PEG/NaCl-based purification enables high-titer, low-endotoxin F. psychrophilum phage preparations with favorable cytocompatibility, supporting their biosafe application in aquaculture.