Transfersomal Phage Cocktail Is an Effective Treatment against Methicillin-Resistant Staphylococcus aureus-Mediated Skin and Soft Tissue Infections

Ethics statement.Experimental protocols were approved by the Institutional Animal Ethics Committee of Panjab University (Chandigarh, India) (approved identification no. IAEC/S/14/129) and performed in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India, regarding animal experimentation. All efforts were made to minimize the suffering of animals.

Bacterial strain.A standard strain of Staphylococcus aureus ATCC 43300 (MRSA) was used in this study. Disk diffusion assays were performed to confirm its resistance to penicillin, methicillin, and oxacillin, followed by determination of the oxacillin MIC value by the broth microdilution assay, as recommended by the Clinical and Laboratory Standards Institute (33). The strain was stored in 60% glycerol at −80°C and, when necessary, was maintained on nutrient agar slants at 4°C.

Bacteriophages.Bacteriophages specific for S. aureus, i.e., phages MR-5 and MR-10, were used. Both phages were isolated previously in our laboratory and were characterized as lytic, double-stranded DNA, tailed phages belonging to the family Myoviridae, order Caudovirales (19, 26, 27).

Bacteriophage concentration and purification.High-titer stocks of each phage were prepared from a single plaque of bacteriophage by the plate lysate method, as described by Sambrook et al. (28). The bacteriophage titers were estimated by the double agar overlay method, as described by Adam (29), and were expressed as PFU per milliliter.

Both phages were concentrated by filtering an initial volume of 400 ml of amplified phage sample on a Millipore Labscale TFF system (Pellicon), fitted with a 10-kDa pore-size polyethersulfone membrane, for a period of 3 to 4 h, until the sample volume reached 25 to 30 ml. The sample was then dialyzed against 10% polyethylene glycol (PEG) 8000 at 4°C for 3 to 4 h, followed by dialysis against fresh phosphate-buffered saline (PBS) (pH 7.2) at 4°C for 24 to 48 h. After dialysis, the sample was centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant was filtered through a 0.45-μm syringe filter into a sterile flask and stored at 4°C. The sample was then assayed to determine the final phage titer achieved after amplification. For long-term storage, the bacteriophages were stored at −70°C in PBS containing 60% glycerol as a preservative. Phages for routine use were stored at 4°C in PBS (pH 7.2).

Preparation of transfersomes.Formulations were prepared using the rotary evaporation technique (30). Various ratios of PC, T-80, and SA were used. PC, T-80, and SA (total mass of 100 mg) were dissolved in 10 ml of a chloroform-methanol mixture (2:1 [vol/vol]) in a beaker at room temperature. The dissolved lipids (10 ml) were transferred into six separate round-bottom flasks of 25 ml each. The solvent was evaporated under high vacuum, with rotation at 100 rpm, at 40°C for 20 min on a Heidolph rotary evaporator. The round-bottom flasks, containing the deposited thin lipid film on their inner walls, were removed from the rotary evaporator and left at room temperature for an additional period of 18 h, to remove traces of solvent in a desiccator under vacuum. Ten milliliters of bacteriophage suspension was added to the thin film at 40°C, and the flasks were rotated for 10 min to detach the film from the glass wall. The suspension was left at room temperature overnight for proper swelling of vesicles and then was sonicated for 30 min in a water bath sonicator (Branson ultrasonic cleaner) prior to further treatment.

Characterization of transfersomes. (i) Morphology and structure of vesicles.The morphological characteristics (shape, uniformity, and structure) of transfersomes were monitored by using an optical microscope (Olympus CH20i) at suitable magnification.

(ii) Average size and polydispersive index.The particle size and polydispersive index of freshly prepared transfersomes were measured by the dynamic light scattering (DLS) technique, using a Beckman Coulter Delsa Nano C instrument, at the Department of Chemistry, Panjab University. Prior to analysis, the samples were diluted 1:10. Each run consisted of two measurements with 70 acquisitions per sample.

(iii) Transmission electron microscopy.The morphology and structure of the phage-loaded transfersomes were determined with transmission electron microscopy (TEM). TEM was performed according to the method of Goodridge et al. (31). Drops of ultracentrifuged phage samples (centrifuged at 100,000 × g for 2 h at 4°C in a Beckman L-80 centrifuge) were dropped onto nitrocellulose-coated grids (diameter, 3 mm; 300 meshes), stained with 2% (wt/vol) potassium phosphotungstate (pH 6.8 to 7.2) for 10 s, and examined with a transmission electron microscope (H-7500; Hitachi, Tokyo, Japan) at 80 kV.

(iv) Entrapment efficiency.The phage entrapment efficiency of transfersomes was determined by the method of Gulati et al. (32), using calcium chloride aggregation. The total phage load was determined by adding Triton X-100 (0.02% [vol/vol]) at a ratio of 1:1 (vol/vol), to disrupt the transfersomes and to release the entrapped phage. The mixture was incubated at room temperature for 1 h and centrifuged at 10,000 × g for 10 min at room temperature, and the total number of phage particles in the supernatant was determined using the plaque assay. Free phage was separated from transfersome-associated phage by the calcium chloride aggregation method. In this method, an aqueous solution of calcium chloride was added to the transfersome dispersion at a final concentration of 20 mM. The mixture was incubated at room temperature for 24 h, the supernatant was collected, and the number of free phages was determined using the plaque assay.

The phage entrapment efficiency was calculated using the following equation: entrapment efficiency = [(E₀ − E₁)/E₁] × 100, where E₀ is the total number of phages, E₁ is the number of free phages, and (E₀ − E₁) represents the number of phage particles entrapped in the transfersome formulation.

Stability and leakage studies.For stability studies, 30-ml plain and phage-loaded transfersome preparations were prepared. The initial sizes of the plain and phage-loaded transfersome formulations were determined. Phage entrapment in phage-loaded transfersomes was also determined.

The two transfersome formulations were distributed in 3 parts (10 ml each), and the glass ampoules were sealed. The sealed ampoules were stored at different temperatures, i.e., 4°C, 37°C, or room temperature (30°C), for a period of at least 3 months. Samples kept at different temperatures were withdrawn periodically from each batch, at regular intervals of 1 week. Phage entrapment and the average size were determined for both formulations (plain and phage-loaded transfersomes) on each occasion.

Animals.Female Wistar rats (4 to 6 weeks of age, weighing 120 to 150 g) were used in the study. The animals were obtained from Central Animal House, Panjab University. The animals were kept in polycarbonate cages, housed at 25 ± 2°C in well-aerated rooms with a 12-h light/12-h dark cycle, and fed standard rodent diet and water ad libitum.

Experimental thigh infections in rats. S. aureus ATCC 43300 was cultivated for 24 h at 37°C in brain heart infusion broth. After 24 h, the cells were pelleted and washed twice with PBS. The bacterial suspension prepared in PBS was adjusted to achieve a cell density corresponding to a range of bacterial inocula (10⁷, 10⁸, or 10⁹ CFU/ml). The number of CFU per milliliter was confirmed by quantitative plate counting. Wistar rats were distributed into four groups, with 24 rats in each group. The posterior portion of the left thigh of each rat was disinfected with 70% alcohol, and 100 μl of a suspension of S. aureus ATCC 43300 was injected intramuscularly into the left thigh muscle. Three groups received different inoculum doses. Animals from the fourth group received the same volume of PBS, injected in the thigh muscle. Four animals from each group were killed on day 1, 3, 5, 7, 10, and 15 after bacterial challenge, and tissue was homogenized, diluted, and plated to determine the bacterial burden.

Phage protection studies.The FPC and TPC, along with a known antibiotic, were evaluated for their ability to resolve MRSA 43300-induced thigh infections in female Wistar rats in two protection studies, i.e., study 1 and study 2. In study 1, a total of five groups, with 33 rats/group, were included. At each time point, three rats were sacrificed and the left and right thighs of each rat were subjected to processing for bacterial load estimation, as described below.

In study 1, the following treatments were given to the groups. In group 1 (infection control), rats were infected with S. aureus ATCC 43300 (10⁷ CFU/ml) by intramuscular injection of inocula (0.1 ml each) in both the left and right thighs. In group 2, rats were infected followed by treatment with two doses of linezolid (10 mg/kg) given orally with a 12-h interval (the first dose was administered 30 min postinfection). In group 3, rats were infected followed by intramuscular administration of plain phage cocktail (FPC), at an MOI of 10, into the thigh tissue 30 min after bacterial challenge. In group 4, rats were infected followed by intramuscular administration of TPC, at an MOI of 10, into the thigh tissue 30 min after bacterial challenge. In group 5, rats were infected followed by intramuscular administration of plain transfersome preparation into the thigh tissue 30 min after bacterial challenge. The progress of infection was monitored on the basis of bacterial loads and phage titers in the different groups.

Three rats from each group were sacrificed on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 15, by cervical dislocation. Left and right thighs of the rats were disinfected with 70% alcohol, and the thigh muscle was dissected. Tissues were homogenized in PBS (pH 7.2), and dilutions of homogenates were plated on nutrient agar. The same homogenates were subjected to centrifugation (15,000 rpm for 20 min at 4°C), followed by filtration of the supernatants through 0.22-μm-pore filters prior to phage titration.

In study 2, the protective effects of immediate and delayed treatment were assessed in terms of death versus survival. A total of seven groups, with six rats/group, were included. The following treatments were given to the groups. In group 1 (infection control), rats were infected with S. aureus ATCC 43300 (10⁹ CFU/ml) in the left thigh muscle. In group 2, rats were infected followed by treatment with two doses of linezolid (10 mg/kg) given orally with a 12-h interval (the first dose was administered 30 min after infection). In group 3, rats were infected followed by intramuscular administration of FPC, at an MOI of 10, into the thigh tissue 30 min after bacterial challenge. In group 4, rats were infected followed by intramuscular administration of TPC, at an MOI of 10, into the thigh tissue 30 min after bacterial challenge. In group 5, rats were infected followed by treatment with two doses of linezolid (10 mg/kg) given orally with a 12 h-interval (the first dose was administered 12 h after infection). In group 6, rats were infected followed by intramuscular administration of FPC, at an MOI of 10, into the thigh tissue 12 h after bacterial challenge. In group 7, rats were infected followed by intramuscular administration of TPC, at an MOI of 10, into the thigh tissue 12 h after bacterial challenge. All animals were monitored daily for death after bacterial challenge.

Statistical analysis.Statistical analysis of the data was performed using Microsoft Excel 2007. Most of the data are expressed as the mean ± standard deviation (SD) of more than two experimental values for every variable. Student's t test was used to compare different variables. P values of <0.05 were considered significant, whereas P values of <0.01 were considered highly significant.