Modeling simulate staphylococcal infection, we tested various infectious doses of the S. aureus SA413 strain and two approaches of administration, intravenous and intraperitoneal. The bacterial inoculum was grown in a liquid nutrient medium to an optical density (OD620) of 0.75 (5 × 10⁹ CFU/ml), and diluted with saline to the desired concentration. The animals were divided into six groups, three mice in each: group 1 — intravenous administration of 5 × 10⁶ CFU/mouse; group 2 — intravenous administration of 5 × 10⁷ CFU/mouse; group 3 — intravenous administration of 5 × 108 CFU/mouse; group 4 — intraperitoneal administration of 5 × 10⁶ CFU/mouse; group 5 — intraperitoneal administration of 5 × 10⁷ CFU/mouse; group 6 — intraperitoneal administration of 5 × 108 CFU/mouse. The volumes of the injected inoculum were 200 μl (intraperitoneal) and 100 μl (intravenous). The animals were monitored for three days to account for deaths. On the third day, bacterial contamination of parenchymal organs and blood was assessed in the surviving animals.

Selection of doses of antimicrobial agents

To assess the therapeutic and minimum inhibitory doses of linezolid and the bacteriophage vB_SauM-515A1, we divided the mice into 6 groups, three mice in each. Two, eight, eighteen, and twenty-four hours after infection, mice were injected with either linezolid (Sigma-Aldrich; USA) at concentrations of 10 mg/kg of animal weight or 40 mg/kg of animal weight, or the vB_ SauM-515A1 bacteriophage at doses of 2 × 10⁵, 2 × 10⁶, and 2 × 10⁷ PFU/mouse. Sterile saline solution was used to dilute the preparations to the necessary concentrations. Control group received saline solution without treatment, similar administration patterns as the test groups. The preparations (200 μl) were injected intraperitoneally. The animals were observed for three days, then euthanized. Parenchymal organs (spleen, kidneys) and blood were collected from them and examined for bacterial contamination and phage content. Blood (1 ml) was sampled from the heart through a puncture into sterile vacuum tubes with sodium heparin (no gel) (Improvacuter; China) designed for blood plasma testing.

Evaluation of the effectiveness of the combined effect of linezolid and bacteriophage

To assess the effectiveness of the combination, we used the antibacterial agents in minimal inhibitory doses. The experiment employed four experimental groups of animals, 12 mice in each, infected with the S. aureus SA413 strain. The infectious dose and the pattern of administration were selected based on the results of preliminary experiments. For the monotherapy stage, the animals received 200 μl of drugs intraperitoneally 2, 8, 18 and 24 hours post-infection. For the combined therapy stage (similar to the monotherapy stage time-wise), the mice were first injected with 200 μl of he antibiotic in one side of the peritoneum, then with 200 μl of the phage in the other side of the peritoneum. The first group of mice was treated with linezolid; the second group was treated with bacteriophage; the third group received the combination of the two; the fourth group (control) was injected with saline solution. Subsequently, three mice from each group were euthanized on the first and second days, and six mice on the third day. Their organs anfd blood were collected and examined for bacterial contamination.

Examination for bacterial contamination bacteriophages in parenchymal organs and blood

The organs were homogenized in sterile mortars, with 1 ml of saline solution added per organ. Next, blood samples and suspension samples were diluted tenfold in saline solution and plated on the Staphylococcagar dense nutrient medium (State Research Center for Applied Microbiology and Biotechnology; Russia). In parallel, we measured bacteriophage content in the suspensions using the Grazia titration method and Staphylococcagar medium (State Research Center for Applied Microbiology and Biotechnology; Russia); the samples made for the purpose were 100 μl serial dilutions. The measurements were done in five technical repetitions.

Data presentation and statistical analysis

For statistical analysis, we used Prism software (GraphPad Software 8; USA). The Shapiro-Wilk test allowed assessing the normalcy of data distribution, and the Student's t-test was used to compare the means between the groups. The differences were considered significant for p < 0.05.

Statement of compliance with ethical standards

All experiments with laboratory animals were approved by the Bioethics Commission of the State Research Center for Applied Microbiology and Biotechnology and conducted in accordance with the Guide for the Care and Use of Laboratory Animals [23].

RESULTS

Staphylococcal infection model

To build an adequate model of staphylococcal infection in laboratory animals, we conducted preliminary studies to select and infecting dose that ensures contamination of parenchymal organs (spleen, kidneys) and blood with the S. aureus strain SA413 on the third day after infection while avoiding animal mortality.

According to the results of those studies, intraperitoneal administration of bacteria at a dose of 5 × 108 CFU/mouse yielded death of all animals on the first day after infection, which is presumably due to the high concentration of the investigated strain in the area of administration and the subsequent toxic shock. Intraperitoneal administration of the bacterial culture at concentrations of 5 × 10⁶ and 5 × 10⁷ CFU/mouse, same as intravenous administration at any of the concentrations studied, left the mice alive three days after the infection. Only the mice that received a dose of 5 × 108 CFU/mouse intravenously exhibited downed motor activity and drowsiness, tousled hair and eyelid hyperemia, which indicate the development of an infectious process.

According to the autopsy, on the third day after intravenous injection of 5 × 10⁸ CFU/mouse the animals had staphylococci in the kidneys (8.9 × 10⁵ – 3.6 × 10⁶ CFU/organ/ml) and a small amount of the pathogen in the spleens (37–52 CFU/ organ/ml) and blood (3.4 × 10² – 1.3 × 10³ CFU/ml). Smaller doses produced either isolated bacterial colonies or none at all, regardless of the method of infection.

Identification of the minimum inhibitory and therapeutic doses of linezolid and bacteriophage against staphylococcal infection

The minimum inhibitory and therapeutic doses of antimicrobial agents were evaluated for two concentrations of linezolid and three variants of bacteriophage doses. Eighteen mice were used for the purpose (fig. 1).

Visual examination showed physical depression, tousled hair, and eyelid hyperemia in mice in the control group and four of the five experimental groups (linezolid 10 mg/kg of animal weight, and all doses of bacteriophage). No animals died through the entire experiment. Animals that received linezolid in the dose of 40 mg/kg of weight did not have the above symptoms.

An autopsy on the third day revealed low spleen contamination (0–25 CFU/organ/ml) in all groups and no bacteria in the blood, with the exception of the control group (0–3 CFU/ml) and the group that received the bacteriophage in the dose of 2 × 10⁵ PFU/mouse. Kidney contamination was the most illustrative indicator. A dose of linezolid 40 mg/kg of animal weight ensured the pathogen was eliminated from the kidneys, indicating this was the therapeutic dose. A dose of 10 mg/kg of animal weight slowed formation of the bacterial colonies by one order of magnitude, made it a minimum inhibitory dose. The doses of bacteriophage 2 × 10⁵ and 2 × 10⁶ PFU/mouse did not deliver results significantly different from those registered in the control group, and were considered ineffective. The dose of bacteriophage 2 × 10⁷ PFU/mouse reduced kidney contamination by one order of magnitude, and was recognized as the minimum inhibitory dose. An amount that could constitute a therapeutic dose was not found.

We detected phage particles only in the kidneys of mice that received a dose of 2 × 10⁷ PFU/mouse (30–70 PFU/organ). There were no bacteriophages found in the blood and spleens of the animals.

Evaluation of the effectiveness of the combined effect of linezolid and bacteriophage

We used 48 mice to assess the combined effect of antimicrobial agents in minimum inhibitory concentrations (linezolid: 10 mg/ kg animal weight; bacteriophage: 2 × 107 PFU/mouse) (fig. 2).

By visual indicators, animals in all groups had the infectious process developing, and their condition was depressed, as described in the previous experiment.

On the first day, we detected no differences in the contamination of parenchymal organs between the monotherapy groups and the control group (fig. 2B). In the combined therapy group, the spleen contamination dropped to almost zero values (p = 0.0014), and that of the kidneys was by one to two orders of magnitude lower than in the control group (p = 0.0318), while blood contamination remained comparable to that in the control group.

The results registered on the second day are shown in fig. 2B. There were insignificant amounts of staphylococci in the spleens of animals of all groups, except those receiving linezolid, where the spleens were clean of the bacteria. Kidney contamination in the bacteriophage group remained at the level of the control group, and in the antibiotic group it significantly decreased by less than an order of magnitude (p = 0.0127); combined therapy pushed the value of this indicator down by two to three orders of magnitude compared to the control group (p = 0.0028). Blood contamination in all groups remained at the level of up to 102 CFU/ml.

On the third day after infection, we registered insignificant amounts of staphylococci in platings from spleen homogenates sampled in all the groups, which points to this organ's ability to independently eliminate the pathogen (:media_2D). Kidney contamination in mice treated with linezolid returned to the level peculiar to the control group, while in mice treated with bacteriophage it remained at that level throughout. In the combined therapy group, the contamination rate was an order of magnitude lower than the control values (p = 0.0079). Blood contamination in all groups remained at the control level.

As for the bacteriophage, its content was insignificant during the entire experiment (20–250 PFU/organ/ml) in the kidneys of the animals that received 2 × 107 PFU/mouse thereof as monotherapy, and in the combined use scenario. We registered no significant differences between the groups. No bacteriophage was detected in the spleens and blood.

DISCUSSION

Combined bacteriophages and antibiotics therapy is, presumably, one of the most promising approaches to the treatment of MDR pathogens. Numerous in vitro studies show promising results, demonstrating the synergistic effect of these agents. However, it is important to conduct in vivo experiments to confirm their effectiveness and practical potential. Animal model studies allow assessing the possibilities and limitations of such therapy in conditions close to those of real-life clinical practice, and enable identification of the aspects that require further study before a full-fledged adoption.

To evaluate the effectiveness of the combined use of the bacteriophage vB_SauM-515A1 and linezolid, we chose a model of systemic infection in BALB/c mice, which aligns with the approaches practiced in similar studies [:lit_24, 25;]. In the preliminary experiments, special attention was paid to the choice of the method of administration, selection of the infecting dose, and establishment of the minimum inhibitory concentrations of active agents. Previously published studies have shown that the infecting dose of S. aureus varies depending on the strain in the range from 106 to 108 CFU/mouse [:lit_24, 26, 27;]. For example, a dose of 106 CFU/mouse was selected for the USA300 strain, known for its high virulence; in this case, the observation period was limited to 24 hours [28]. At the same time, a dose of 108 CFU/mouse used in the studies dedicated to the MDR strains partially killed the animals within 10–24 hours and, in some cases, by the third day [:lit_24, 27;]. We focused on the sequence type 8 SA413 strain, one of the most common and associated with hospital infections worldwide, and found the optimal dose to be 5 × 108 CFU/mouse, administered intravenously. The dose ensured stable organ contamination after three days, thus creating adequate conditions for registration of the effects of therapy. Consistent with the findings reported by other authors, we have established that the results are most reliably reproducible when the injections are intravenous [:lit_27, 29;].

The identified minimum inhibitory concentration of linezolid that does not cause pathogen elimination in monotherapy regimens and, consequently, should be investigated further, is 10 mg/kg of animal weight. This concentration of the antibiotic reduced bacterial contamination minimally, which is also consistent with the results reported by other researchers [10]. The concentration of 40 mg/kg of animal weight completely eliminated bacteria from the kidneys by the third day of the experiment, which is also similar to the data registered by other authors [29]. It should be noted that linezolid and bacteriophage were administered intraperitoneally to avoid vascular damage and the risk of hemorrhages associated with repeated injections. In particular, the effectiveness of this way was demonstrated in staphylococcal infection mice models that have thus received K-like phage ɸSA039 [30].

According to the published data on therapeutic use of bacteriophages in mouse models, the amount of antimicrobial agent varies from 10⁶ to 10¹⁰ PFU/mouse [:lit_27, 31;]. In our study, the minimum inhibitory dose of the bacteriophage was 2 × 10⁷ PFU/mouse. This dose only partially decreased the level of bacterial contamination of kidneys, which underscores the need to use high concentrations of bacteriophages in monotherapy regimens. Moreover, lack of phages in blood and spleen indicates that there probably are some limitations to the system-wide spread of bacteriophages, which once again points to the need for an integrated approach in therapy.

Compared to monotherapy, combined use of linezolid and bacteriophage in minimum inhibitory doses had a more pronounced effect: within the first 24 hours, kidney contamination level decreased by two to three orders of magnitude versus the control values, a fact that backs the synergistic potential of antimicrobial agents. However, by the third day, bacterial contamination damping effect produced by the combination was not as strong as initially, which may indicate the need for a longer course of treatment to achieve the full therapeutic effect.

The evidence of the greater effectiveness of combination therapy compared with monotherapy are consistent with a number of reports covering animal model studies that investigated the effectiveness of the combined use of linezolid and Herelleviridae bacteriophages against other types of infections caused by S. aureus. Previously, it was demonstrated in a mouse model of a diabetic foot staphylococcal infection that a single injection of a Herelleviridae family phage delivers results comparable to those produced by linezolid, and combination therapy was much more effective in stopping the entire infectious process (bacterial load, number of lesions, foot myeloperoxidase activity, and histopathology), as well as accelerating the general tissue healing process [16]. A study that assessed the effectiveness of a linezolid and bacteriophage MR-5 (family Herelleviridae) combination against a skin infection modeled in mice has shown its potency: the agents, taken together, have significantly decreased the bacterial load and, consequently, boosted recovery [17]. There is also a report describing a combined therapy success in a modeled S. aureus infection case after arthroplasty. Mice were implanted with a wire coated with phage (109 PFU/ml) and/or linezolid into the intramedullary canal of the femur, and then inoculated with MRSA. In the group that received wire with a combination of agents, bacterial adhesion was reduced, and the limb's motor functions restored faster [18].

CONCLUSIONS

This study confirmed the promise held by the combined therapy with linezolid and bacteriophage vB_SauM- 515A1 for treatment of systemic infections caused by S. aureus. Minimum inhibitory doses of the antibiotic and the bacteriophage were established to significantly decrease the level of bacterial contamination of parenchymal organs, which indicates a synergistic effect. The results of this study demonstrate that combination therapy is more effective than monotherapy, especially at the early stages, and can help reduce the dosage of the antibiotic, thus minimizing the possible side effects.