Biosafety improvement of aquaculture-associated bacteriophage preparations via endotoxin reduction: implications for Flavobacterium psychrophilum control

The use of bacteriophages specific to Flavobacterium psychrophilum in aquaculture has shown promising results in controlling infections and reducing fish mortality. However, the biosafety and cytocompatibility of these phage preparations, especially under long-term exposure conditions, remain largely unexplored. In this study, bacteriophages specific to F. psychrophilum were isolated and characterized. To enhance biosafety, the phages were subjected to endotoxin reduction procedures. Endotoxin levels and phage titers were quantitatively determined before and after purification. Cytocompatibility was evaluated using the MTT assay on the mammalian cell line MCF-7 over a 120-hour exposure period. The purification process reduced endotoxin levels by 97.3%, while phage titers were preserved or increased, indicating that biological activity was maintained. Time-dependent analyses revealed that crude phage preparations caused a decrease in cell viability, with statistically significant reductions observed at 72, 96, and 120 hours (p < 0.05). In contrast, no difference in cell viability was observed in cells treated with purified phage preparations compared to the control group (p > 0.05). These findings demonstrate that the cytotoxic profile of phage preparations is determined by endotoxin content and prolonged exposure duration. This study highlights that endotoxin removal is critically important for ensuring cytocompatibility and demonstrates that endotoxin content in phage preparations used in aquaculture may lead to reduced cell viability in mammalian cells under long-term exposure conditions.