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

PLoS One. 2018 Oct 11;13(10):e0205728. doi: 10.1371/journal.pone.0205728. eCollection 2018.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Infective Agents / metabolism*
  • Capsules
  • Freeze Drying
  • Humans
  • Industrial Development
  • Staphylococcal Infections / therapy
  • Staphylococcus Phages / metabolism*
  • Staphylococcus aureus / virology
  • Staphylococcus epidermidis / virology
  • Temperature
  • Virology / methods

Substances

  • Anti-Infective Agents
  • Capsules

Grants and funding

This research study was supported by grants AGL2015-65673-R (Ministry of Science and Innovation, Spain), GRUPIN14-139 (Program of Science, Technology and Innovation 2013-2017 and FEDER EU funds, Principado de Asturias, Spain). L.F. is a “Marie Curie Clarin-Cofund” postdoctoral fellow. PG, BM, and AR are members of the FWO Vlaanderen funded “Phagebiotics” research community (WO.016.14) and the bacteriophage network FAGOMA II. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.