Recovering viable bacteriophages from human milk: implications for sample storage and processing

Aims
Molecular methods have shown bacteriophage diversity in human milk, but this does not reflect viability, particularly within donor human milk which has been subjected to multiple freeze-thaw cycles and heat treatment. Our aim was to isolate bacteriophages from frozen human milk and explore the effects of sterilization and storage on bacteriophage survival.

Methods and results
Twenty frozen human milk samples were chosen for bacteriophages isolation. Analysis showed that these samples stored for 12 months at −20°C did not contain viable bacteriophages. We artificially inoculated 10 human milk and 10 water control samples with Escherichia coli bacteriophage T4 and subjected them to various treatments. We found that ultraviolet-C irradiation significantly inactivated E. coli phage T4 (P < 0.001), with greater inactivation observed in skimmed milk (P < 0.001) than whole milk. In contrast, Holder and High Temperature Short Time pasteurization had no significant effect (P > 0.05). The E. coli bacteriophage T4 was better preserved at 4°C than at room temperature over 48 h. Over 90 days of storage, bacteriophage viability was higher at −80°C (31.6%) than at −20°C (9.4%).

Conclusions
Future work on bacteriophage isolation from human milk should consider the use of fresh samples whenever possible. Where samples are stored prior to analysis this should be at a refrigerated temperature for up to 48 h, and if samples are to be stored longer term, then −80°C is suitable for up to three months and preferable to −20°C, with storage only up to seven days.