Biological properties of Staphylococcus virus ΦSA012 for phage therapy

Abstract

Staphylococcus virus ΦSA012 has a wide host range and efficient lytic activity. Here, we assessed the biological stability of ΦSA012 against temperature, freeze-thawing, and pH to clinically apply the phage. In addition, inoculation of ΦSA012 through i.p. and i.v. injections into mice revealed that phages were reached the limit of detection in serum and accumulated notably spleens without inflammation at 48 h post-inoculation. Furthermore, inoculation of ΦSA012 through s.c. injections in mice significantly induced IgG, which possesses neutralizing activity against ΦSA012 and other Staphylococcus viruses, ΦSA039 and ΦMR003, but not Pseudomonas viruses ΦS12-3 and ΦR18 or Escherichia viruses T1, T4, and T7 in vitro. Immunoelectron microscopic analysis showed that purified anti-phage IgG recognizes the long-tail fiber of staphylococcus viruses. Although S. aureus inoculation resulted in a 25% survival rate in a mouse i.p. model, ΦSA012 inoculation (i.p.) improved the survival rate to 75%; however, the survival rate of ΦSA012-immunized mice decreased to less than non-immunized mice with phage i.v. injection at a MOI of 100. These results indicated that ΦSA012 possesses promise for use against staphylococcal infections but we should carefully address the appropriate dose and periods of phage administration. Our findings facilitate understandings of staphylococcus viruses for phage therapy.

Figure 1

Phylogenetic analysis using the whole-genome sequence of ΦSA012. The phylogenetic tree was constructed by VICTOR using 16 whole-genome sequences of all Staphylococcus K-virus master species registered by the International Committee on the Taxonomy of Viruses (ICTV). The red circle indicates ΦSA012 and the blue circle indicates Staphylococcus virus K. S, G, and F after the phage names refer to species, genus, and family clusters, respectively. Bootstrap values are shown on the branch nodes, and the scale bar represents a distance of 0.03 substitutions per site.

Figure 2

EoP of ΦSA012 against Staphylococcus spp. strains and S. aureus clinical isolates. SA003 was assigned as the control host against ΦSA012. EoP values are indicated as fold changes in the effect on SA003 and are presented as means ± SD (n = 3).

Figure 3

ΦSA012 stability against several environmental conditions. (A) Phage stability against storage conditions at room temperature and 4 °C. ΦSA012 infectivity is indicated as fold changes compared to day 0 and is presented as means ± SD (n = 3). (B) Environmental resistance of ΦSA012 against freeze–thaw cycles. The titer of ΦSA012 is indicated as the fold change compared to cycle 0 and is presented as means ± SD (n = 3). (C) Phage stability at temperatures of 37 °C, 56 °C, and 60 °C. The titer of ΦSA012 is indicated as the fold change compared to 0 h and is presented as means ± SD (n = 3). (D) Phage stability against different pH ranges. The titer of ΦSA012 is indicated as the fold change compared to pH 7 and is presented as means ± SD (n = 3). (E and F) Effects of serum on the plaque-forming activity of ΦSA012. The titer of ΦSA012 is indicated as the fold change compared to 0% FBS and is presented as means ± SD (n = 3). (G) Effects of other phage particles on the plaque-forming activity of ΦSA012. The titer of ΦSA012 is indicated as the fold change compared to non-treated ΦSA012 and is presented as means ± SD (n = 3). The significance compared to the control under each condition was analyzed by Tukey’s test based on one-way ANOVA: *p < 0.05, **p < 0.01.

Figure 4

Pharmacokinetics and side effects of ΦSA012 in mice. (A) Pharmacokinetics of ΦSA012 administered with i.p. and i.v. injections into mice. Phage titers in the serum were monitored from 3 to 48 hpi for i.p. and from 0.25 hpi to 48 hpi for i.v. injections. The titers of ΦSA012 in serum samples are presented as means ± SD (n = 3). 10² pfu/mL is the limit of detection of the phage in the plaque assay. (B) Accumulation of ΦSA012 in organs at 48 hpi after i.p. injection. The titers of ΦSA012 in organ homogenates are presented as means ± SD (n = 3). (C) Accumulation of ΦSA012 in organs at 48 hpi after i.v. injection. The titers of ΦSA012 in organs homogenates are presented as means ± SD (n = 3). ND means that plaques were not detected, indicating values less than 1.2 × 10² pfu/g in the heart, 8.9 × 10¹ pfu/g in the lung, 4.5 × 10² pfu/g in the kidney, and 8.7 × 10¹ pfu/g in the intestine. (D) Histopathology and pathological changes of organs in ΦSA012-inoculated mice (i.p.). The spleens, hearts, kidneys, livers, lungs, and intestines were stained with hematoxylin and eosin. The number in each picture represents magnification.

Figure 5

Immune response in mice to ΦSA012. (A) Time course of ΦSA012 immunization in mice by s.c. injection. (B) Concentrations of IgG and IgM in serum samples from immunized mice measured using ELISA. Data are presented as means ± SD (n = 4). Significance against 0 dpi was analyzed by Tukey’s test based on one-way ANOVA: *p < 0.05, **p < 0.01. (C) Concentrations of IgE in serum samples from immunized mice measured using ELISA. Data are presented as means ± SD (n = 4). (D) Anti-phage antibody responses against ΦSA012. Serum samples from mice immunized with s.c. injections or inoculated with i.p. injections of ΦSA012 were tested for phage-specific IgG responses by ELISA. Data are presented as means ± SD (n = 4). PBS was added as a control instead of serum samples in the phage ELISA. Significance among each group was analyzed by Tukey’s test based on one-way ANOVA: *p < 0.05, **p < 0.01. (E) Neutralization activity of purified anti-phage IgG against ΦSA012. The titers of ΦSA012 are indicated as fold changes against the control and presented as means ± SD (n = 3). Significance against control was analyzed by Tukey’s test based on one-way ANOVA: *p < 0.05, **p < 0.01. (F) Lytic curves of SA003 growing in the presence of ΦSA012 treated with purified anti-phage IgG were obtained by monitoring the OD₅₉₀ until 24 hpi. The individual points in each lytic curve are presented as means ± SD (n = 4). (G and H) Neutralization activity of purified anti-phage IgG against ΦSA039 and ΦMR003. The titers of phages are indicated as fold changes against the control and presented as means ± SD (n = 3). Significance against the control was analyzed by Tukey’s test based on one-way ANOVA: *p < 0.05, **p < 0.01. (I) Neutralization activity of purified anti-phage IgG against Pseudomonas phages ΦS12-3 and ΦR18 and Escherichia phages T1, T4, and T7. The titers of phages are indicated as fold changes against the control and presented as means ± SD (n = 3). Significance against the control was analyzed by t test. (J–L) Immunoelectron micrographs of ΦSA012, ΦSA039, and ΦMR003 stained with a gold-conjugated secondary antibody. White bars represent 100 nm. The size of the gold particles is 12 nm and black spots in the images represent gold particles conjugated to the secondary antibody.

Figure 6

Therapeutic effect of ΦSA012 in a mouse infection model. (A) Survival rates of mice inoculated with SA003 through i.p. injections without ΦSA012 treatment (i.p. or i.v.). ΦSA012 was inoculated to mice i.p. or i.v. (n = 12 in the control group, n = 10 at MOI of 100 in the i.p. group, n = 10 at MOI of 100 in the i.v. group, n = 4 at MOI of 1 in the i.p. group). In every experiment, ΦSA012 or SM buffer was injected into mice 15 min after SA003 inoculation. (B) Survival rates of mice inoculated with SA003 through i.p. injection with anti-phage antibody (1 µg/mL or 10 µg/mL)-pretreated ΦSA012 treatment and ΦSA012 treatment in immunized mice (n = 4 in each group). ΦSA012 was injected into the mice 15 min after SA003 inoculation. (C) Neutralization activity of purified anti-phage IgG against ΦSA012 in vitro (equivalent to MOI of 100 in the mouse infection model). Titers of ΦSA012 are indicated as fold changes against the control and presented as means ± SD (n = 3). Significance against the control was analyzed by Tukey’s test based on one-way ANOVA: *p < 0.05, **p < 0.01.