Comparative analysis of different preservation techniques for the storage of Staphylococcus phages aimed for the industrial development of phage-based antimicrobial products

Abstract

Bacteriophages have been proven as effective antimicrobial agents in the treatment of infectious diseases and in other biocontrol applications including food preservation and disinfection. The extensive use of bacteriophages requires improved methodologies for medium- and long-term storage as well as for easy shipping. To this aim, we have determined the stability of four Staphylococcus phages (phiIPLA88, phiIPLA35, phiIPLA-RODI and phiIPLA-C1C) with antimicrobial potential at different temperatures (20°C/25°C, 4°C, -20°C, -80°C, -196°C) and during lyophilization (freeze drying) using several stabilizing additives (disaccharides, glycerol, sorbitol and skim milk). Differences between phages were observed at different temperatures (20°C/25°C, 4°C and -20°C), where phages were less stable. At lower temperatures (-80°C and -196°C), all phages showed good viability after 24 months regardless of the stabilizer. Differences between phages were also observed after lyophilization although the addition of skim milk yielded a dry powder with a stable titer after 24 months. As an alternative to facilitate storage and transportation, phage encapsulation has been also explored. Phage phiIPLA-RODI encapsulated in alginate capsules retained high viability when stored at 4°C for 6 months and at 20°C for 1 month. Moreover, the spray-dryer technique allowed obtaining dry powders containing viable encapsulated phages (phiIPLA-RODI and phiIPLA88) in both skim milk and trehalose for 12 months at 4°C. Storage of phages at 20°C was less effective; in fact, phiIPLA88 was stable for at least 12 months in trehalose but not in skim milk, while phiIPLA-RODI was stable only for 6 months in either stabilizer. These results suggest that encapsulated phages might be a suitable way for shipping phages.

Application of Adaptive Evolution to Improve the Stability of Bacteriophages during Storage

“…Although phages may not necessarily be stored at 37 • C, ambient storage conditions are not always constant, and temperatures are likely to fluctuate especially in hot climates where refrigeration is not available. Other factors such as phage type and solute available in a sample may also influence phage stability [49].…”

Pharmacological limitations of phage therapy

“…Phages can also be selected in vivo for improved stability and extended circulation time by e.g. mutation selection, or formulated by encapsulation, lyophilisation, or PEGylation (78)(79)(80)(81)(82). Many other formulation strategies, e.g.…”

Application of Bacteriophages in Nanotechnology

“…Immobilization [ 130 ] or encapsulation [ 202 ] was also showed to be efficient in increasing phage stability. Gonzalez-Menendez showed a comparative analysis of different preservation techniques for the storage of Staphylococcus phages [ 203 ].…”