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

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.

Assessment of Sep1virus interaction with stationary cultures by transcriptional and flow cytometry studies.

The majority of phage infection studies are performed in bacteria that are growing exponentially, although in nature, phages usually interact also with non-replicating cells. These stationary-phase cells differ from exponential cells morphologically, physiologically and metabolically. The interaction of a Sep1virus with Staphylococcus epidermidis stationary and exponential phase cells was explored. Phage SEP1 efficiently infected both cell culture states, without the addition of any fresh nutrients to stationary cultures. Phage-host interactions, analysed by flow cytometry, showed stationary-phase cells response to phage immediately after SEP1 addition. Quantitative PCR experiments corroborate that phage genes are expressed within 5 min of contact with stationary phase cells. The increase of host RNA polymerase transcripts in stationary cells suggests that SEP1 infection leads to the upregulation of host machinery fundamental for completion of its lytic life cycle. SEP1 infection and replication process highlights its potential clinical interest targeting stationary phase cells.