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.

The antimicrobial properties of bacteriophages make them suitable food biopreservatives. However, such applications require the development of strategies that ensure stability of the phage particles during food processing. In this study, we assess the protective effect of encapsulation of the Staphylococcus aureus bacteriophage phiIPLA-RODI in three kinds of nanovesicles (niosomes, liposomes, and transfersomes). All these systems allowed the successful encapsulation of phage phiIPLA-RODI with an efficiency ranged between 62% and 98%, regardless of the concentration of components (like phospholipids and surfactants) used for vesicle formation. Only niosomes containing 30 mg/mL of surfactants exhibited a slightly lower percentage of encapsulation. Regarding particle size distribution, the values determined for niosomes, liposomes, and transfersomes were 0.82 ± 0.09 µm, 1.66 ± 0.21 µm, and 0.55 ± 0.06 µm, respectively. Importantly, bacteriophage infectivity was maintained during storage for 6 months at 4 °C for all three types of nanovesicles, with the exception of liposomes containing a low concentration of components. In addition, we observed that niosomes partially protected the phage particles from low pH. Thus, while free phiIPLA-RODI was not detectable after 60 min of incubation at pH 4.5, titer of phage encapsulated in niosomes decreased only 2 log units. Overall, our results show that encapsulation represents an appropriate procedure to improve stability and, consequently, antimicrobial efficacy of phages for application in the food processing industry.

The use of bacteriophages for killing pathogenic bacteria is a feasible alternative to antibiotics and disinfectants. To obtain the large quantities of phages required for this application, large-scale production of bacteriophages must be optimized. This study aims to define conditions that maximize the phage yield of the virulent and polyvalent staphylococcal bacteriophage vB_SauM-phiIPLA-RODI in broth culture, using the food-grade species Staphylococcus xylosus as the host strain to reduce the risk of growing massive quantities of pathogenic bacteria and therefore, to ensure the safety of the final phage stock. The effect of four variables, namely initial bacterial concentration (5.66–8.40 log10 colony-forming unit (CFU)/mL), initial phage concentration (5–8 log10 plaque-forming unit (PFU)/mL), temperature (21–40 °C) and agitation (20–250 rpm), on phage yield (response) was studied by using response surface methodology (RSM). Successive experimental designs showed that agitation did not significantly impact phage yield, while temperature did have a significant effect, with 38 °C being the optimum for phage propagation. The results allowed the design of a model to describe phage yield as a function of the initial bacterial and phage concentrations at fixed agitation (135 rpm), and optimum temperature (38 °C). The maximum experimental phage yield obtained was 9.3 log10 PFU/mL, while that predicted by the model under the optimized conditions (7.07 log10 CFU/mL initial bacterial population and 6.00 log10 PFU/mL initial phage titer) was 9.25 ± 0.30 log10 PFU/mL, with the desirability of 0.96. This yield is comparable to that obtained when the phage was propagated on the original host, Staphylococcus aureus. Bacteriophage phiIPLA-RODI showed the same host range and very similar biofilm removal ability regardless of the staphylococcal species used for its propagation. The results presented in this study show the suitability of using a food-grade strain of S. xylosus for the propagation of S. aureus infecting phages and the application of RSM to define the optimal propagation conditions.

Bacteriophages are currently considered as a promising alternative to antibiotics and disinfectants. However, the use of phages in different clinical and industrial settings will involve their exposure to other disinfectants. As a result, the outcome of the phage treatment will depend on two aspects derived from such interactions. On the one hand, the susceptibility of the phage to disinfectants at the concentrations used for disinfection and at lower residual concentrations needs to be determined. Additionally, the existence of synergistic or antagonistic interactions between phages and disinfectants would also affect the potential success of phage biocontrol applications. Here, we tested these effects for the antistaphylococcal phage phiIPLA-RODI by using four different disinfectants: benzalkonium chloride, triclosan, chlorhexidine and hydrogen peroxide. Our results highlight the differences between disinfectants regarding their effect on phage survival and antimicrobial properties. For instance, our data suggests that, out of the four disinfectants used, benzalkonium chloride would be the most adequate to use in settings where phages are to be applied. Nonetheless, this preliminary analysis grants the need for further studies with a larger number of disinfectants for the development of a phiIPLA-RODI-based product.